Up-converting reporters for biological and other assays

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OTHER PUBLICATIONS McFarlane, “Dual Wavelength Visible Upconversion Laser,” Bethune et al., “Atoms in carbon cages: the Structure and Appl. Phys. Letts. (1989) 54:2301-2302. McFarlane, “Violet Cw Neodymium Upconversion Laser,” properties of endohedral fullerences”, Nature (1993) Appl. Phys. Letts. (1988). 54: 1300–1302.

Beverloo et al., “Preparation and microscopic visualization Mukkala et al., “The Synthesis and Use of Activate N-ben of multicolor luminescent immunophosphors', Cytometry Zyl Derivatives of Diethylenetriaminetetraacetic Acids: Alternative Reagents for Labeling of Antibodies with Metal (1992) 13:561-570. Ions,” Anal. Bio. (1989) 176:319–325. Beverloo et al., “Inorganic phosphors as new luminescent Nguyen et al., “Blue-green (450-nm) Upconversion Tm": labels for immunocytochemistry and time-resolved microS Ylf Laser." Applied Optics (1989) 28:3553–3555. copy,” Cytometry (1990) 11:784-792. P.A. Santa Cruz, et al., “Quim, Nova” (1983) 6:149-151. B.J. Tromberg et al. “Proc. SPIE-INT" Soc. Opt. Eng., Rich and Pinow, Exploring the Ultimate Efficiency in Infra (1991) 1427:101–108. red-to-Visible Coverting Phosphors Activated with Er and Camus et al., “Two-photon absorption spectroscopy in Sensitized with Yb, J. Appl. Phys. (1972) 43:2357-2365. ytterbium”, J. Phys. B. Atom. Molec. Phys. (1978) Schindele and Renzoni, “Ultra Fluors: New Fluorophores

Campiglia, A.D. et al., “Utilization of an Inorganic Phosphor for Immunological Applications”, J. Clin. Immun. (1990)

as a Reference Signal in Solid-Surface Room Temperature Seveus et al., “Time-Resolved Fluorescence Imaging of Phosphorimetry”, Anal. Chem. (1988) vol. Europium chelate Label in Immunohistochemistry and in 60(g):2165-2167. Situ Hybridization,” Cytometry (1992) 13:329-338. D.C. Yeh et al., “J. Appl. Phys”., (1988) 63:4644–4650. Silversmith et al., “Green Infrared-Pumped Erbium Upcon D.C. Yeh et al., “Phys. Rev. B", (1989) 39:80–90. version Laser,” J. Opt. Soc. Am. (1982) 3:128-12. Diamandis and Christopoulos, Detection of Lanthanide Che Smart et al., “Cw Room Temperature Upconversion Lasing lates and Multiple Labeling Strategies Based on Time-re at Blue, Green and Red Wavelengths in Infrared-Pumped solved Fluorescence, in Nonisotopic DNA Probe Tech Pr-doped Fluoride Fibre.” Electronics Letters (1991) niques, Academic Press (1992) 263-274. 27:1307-1309.

D.R. Tallant et al., J. Chem. Phys., (1975) 63:2074–2085. Soini and Kojola, “Time-resolved Fluorometer for Lan Eichstein et al., “Laser-excited Time-resolved Solid-phase thanide Cachelates-a New Generation of Nonisotopic Fluoroimmunoassays with the New Europium Chelate 4, Immunoassays,” Clin Chem. (1983) 29/1:65–68. 7-bis (chlorosulfophenyl)-1, 10-phenanthroline-2, Soules and Hoffman, “Luminescent Materials (Phosphors).” 9-dicarboxylc Acid as Label.” Anal. Chem. (1988) Encyclopedia of Chemical Technology, (1981) Third Edi

Evangelista et al., “Enzyme-amplified Lanthanide Lumines tion, 14:527–545.

Tiffany, “Fluorometry, Nephelometry, and Turbidimetry,” cence for Enzyme Detection in Bioanalytical ASSays,” Anal. Textbook of Clinical Chemistry, (1986) 78-90. Biol. (1991) 137:213–224. Voller, “The Enzyme Linked Immunosorbent Assay Gudgin Templeton et al., “Time Resolved Fluorescence (ELISA),” in Diagnostic Horizons, (1978) 2:1:1-7. Detection of Enzyme-amplified Lanthanide Luminescence Xu and Hemmila, “Co-fluorescence Enhancement System for Nucleic Acid Hybridization Assays.” Clin. Chem. (1991) Based on Pivaloyltrifluoroacetone and Yttrium for the

Hemmila et al., “Europium as a Label in Time-resolved Simultaneous Detection of Europium, Terbium, Samarium Immunofluorometric Assays,” Anal. Biol. (1984) and Dysprosium,” Anal. Chimica Acta. (1992) 256:9-16. 137:335-343. Wojciechowski, et al., “Infrared-to-Blue Up-coverting Johnson et al., “Infrared-to-visible Conversion by Rare-e- Phosphor.” Electron Technology (1978) 11:3:31-47. arth Ions in Crystals,” J. Appl. Phys. (1972) 43:3. Moser, K., et al., “Infrared Spectral Distribution of Photo Johnston and Wright, “Trace Analysis of Nonfluorescent conductivity and Up-conversion in GaP Light Emitting Ions by Associate Clustering with a Fluorescent Probe.” Diodes,” J. Appl Phys, (1985) 57:12:5438–5444. Anal. Chem. (1979) 51:1774–1780. Tanabe, S., et al., “Up-conversion Fluorescences of Te0 Kano et al., NaLnF: YB", Er", (Ln:Y.Gd.La): Efficient and Ga-O-Based Oxide Glasses Containing Er", "Journa Green-emitting Infrared-excited Phosphors, J. Electro of Non-Crystalline Solids, (1990) 122:79-82. chem. Soc. (1972) 119:1561–1564. Franz, K. A., et al., “Luminescent Materials.” Ullman's Leif and Vallarino, “Rare-earth Chelates as Fluorescent Encyclopedia of Industrial Chemistry, 5th Edition, Marks in Cell Separation and Analysis,” Cell Separation A15:519-558.

Science and Technology (1991) 3:41-58. “TransFluoSpheres Fluorescent Microspheres-A Break Lenth and Macfarlane, Lasers, Optics & Photonics News, through in Microsphere Technology,” Molecular Probes, (1992) 3:8–15. 114-115.

Louge et al., “Optical Fiber Measurements of Particle Veloc Wright, W.H., et al. “High-Sensitivity Immunoassay Using ity Using Laser-induced Phosphorescence, Applied Optics A Novel Upconverting Phosphor Reporter, SRI Interna (1991) 30:1976-1981. tional.

Lovgren et al., “Detection of Lanthanide Chelates by Tim Wollenberger, L.V. et al. “Detection of DNA Using Upcon e-resolved Fluorescence, in Nonisotopic DNA Probe Tech verting Phosphor Reporter Probes,” SRI International. niques” Academic Press (1992) 227–261. Riris, H. et al., “A Compact Upconverting Phosphor Detec Manashirov et al., “Effect of the Purity of Initial Substances tion Sytem for Wick Assays, SRI International. on Luminescence Intensity of Erbium in Anti-Stroke Lumi Mufti, N.A., et al., “Design and Manufacture of Capillary nophores,” Chemical Abstracts, (1989) 110:457 Abstract Wicks for Ultrasensitive Detection of Antigenic and Nucleic No. 3075OB. Acid Analytes,” SRI International.

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Visible Light Out = Negative Sample

Infroned

Cooted n Fiber Opti Phosphor Der UpTIC

Target linked

Step 1: Mix Antigen Step 2: Interogate with Coated Phosphors Antibody Coated Probe Competitive Homogeneous Assay

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ADD ANTIGEN

COATED

PHOSPHOR

PHOSPHOR

ANTIGEN

EMITTED LIGHT

FOCAL (HIGH

INTENSITY

EXCITATION)

EXCITATION LIGHT

OF LOW INTENSITY

CAPTURE

ATSURFACE

EMITTED LIGHT

S OF HIGH

EXCITATION LIGHT

OF LOW INTENSITY

COMPETITIVE HOMOGENOUS

ANTIGEN CAPTURE ASSAY

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Phosphor particles phosphor particles In the presence of analyte, Sharply convergent the antibody coated excitation light beam phosphors will form a large enough complex to drop to the bottom of the well

Homogeneous Immunoprecipitation ASSOy

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FIG 51A F3

10 x 10 Gridded Array of 980 nm Diode Losers

Aqueous

Sample Flow

10 x 10 Gridded Arroy

F5 /2 ZZZZZZZV MYZZYZZZY

M777S7/7ZVV of Photodiode Detectors

Individual

Diode Laser in Array

Aqueous

Silicon Chip Sample Flow F9 Support Matrix is a Y as a YLY at A as a Za a as a L as a as an 2 a Y as a

10-25 um Polymer film

Overloy Used as

Individual Photodiode Capture Surface Detector in Array

Antibody Immund or Nudeic Acid Conjugated Antigen Capture Probe Bonded to 0.1-0.5 um F7 Film Overlay PhoSchor

FIG. 31B

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UP-CONVERTING REPORTERS FOR Signal, and accurate measurement with precision radiometric BIOLOGICAL AND OTHER ASSAYS instruments (Scintillation and gamma counters) or with

CROSS REFERENCE TO RELATED

inexpensive and Sensitive autoradiographic techniques.

APPLICATION

However, radioisotopic labels also have Several.

5 disadvantages, Such as: potential health hazards, difficulty in

This application is a continuation of application Ser. No. disposal, Special licensing requirements, and instability 08962,673 filed Nov. 3, 1997, now abandoned which is a (radioactive decay and radiolysis). Further, the fact that continuation-in-part of application Ser. No. 08/482,203 filed radioisotopic labels typically do not produce a strong (i.e., Jun. 7, 1995, now U.S. Pat. No. 5,698,397 which is a non-Cerenkov) signal in the ultraViolet, infrared, or visible division of application Ser. No. 08/416,023 filed Mar. 30, portions of the electromagnetic spectrum makes radioiso 1995, now U.S. Pat. No. 5,674,698, which is a continuation topes generally unsuitable as labels for applications, Such as in-part of application Ser. No. 08/381,006 (now abandoned), microScopy, image spectroscopy, and flow cytometry, that filed Jan. 30, 1995, which is a continuation of Ser. No. employ optical methods for detection. 07/946,068 filed Sep. 14, 1992 (now abandoned). This For these and other reasons, the fields of clinical application also claims priority benefits to provisional appli 15 chemistry, water and air monitoring, and biomedical cation Ser. No. 60/037,392 filed Feb. 7, 1997. Each of these research have Sought alternative detectable labels that do not applications is specifically incorporated herein by reference. require radioisotopes. Examples of Such non-radioactive BACKGROUND OF THE INVENTION labels include: (1) enzymes that catalyze conversion of a chromogenic Substrate to an insoluble, colored product (e.g.,

The invention relates generally to detectable labels and alkaline phosphatase, beta-galactosidase, horseradish compositions useful in assay methods for detecting Soluble, peroxidase) or catalyze a reaction that yields a fluorescent or Suspended, or particulate Substances or analytes Such as luminescent product (e.g., luciferase) (Beck and Koster proteins, carbohydrates, nucleic acids, bacteria, Viruses, and (1990) Anal. Chem. 62:2258; Durrant, I. (1990) Nature 346: eukaryotic cells and more Specifically relates to composi 297, Analytical Applications of Bioluminescence and tions and methods that include luminescent (phosphorescent 25 Chemiluminescence (1984) Kricka et al. (Eds.) Academic or fluorescent) labels. Press, London), and (2) direct fluorescent labels (e.g., fluo Methods for detecting specific macromolecular species, rescein isothiocyanate, rhodamine, Cascade blue), which Such as proteins, drugs, and polynucleotides, have proven to absorb electromagnetic energy in a particular absorption be very valuable analytical techniques in biology and wavelength spectrum and Subsequently emit visible light at medicine, particularly for characterizing the molecular com one or more longer (i.e., less energetic) wavelengths. position of normal and abnormal tissue samples and genetic Using enzymes and phosphorescent/fluorescent or colo material. Many different types of Such detection methods are rimetric detectable labels offers the Significant advantage of widely used in biomedical research and clinical laboratory Signal amplification, Since a single enzyme molecule typi medicine. Examples of Such detection methods inclide: cally has a persistent capacity to catalyze the transformation immunoassays, immunochemical Staining for microscopy, 35 of a chromogenic substrate into detectable product. With fluorescence-activated cell Sorting (FACS), nucleic acid appropriate reaction conditions and incubation time, a Single hybridization, water Sampling, air Sampling, and others. enzyme molecule can produce a large amount of product, Typically, a detection method employs at least one ana and hence yield considerable Signal amplification. However, lytical reagent that binds to a Specific target macromolecular detection methods that employ enzymes as labels disadvan Species and produces a detectable Signal. These analytical 40 tageously require additional procedures and reagents in reagents typically have two components: (1) a probe order to provide a proper concentration of Substrate under macro molecule, for example, an antibody or conditions Suitable for the production and detection of the oligonucleotide, that can bind a target macromolecule with colored product. Further, detection methods that rely on a high degree of specificity and affinity, and (2) a detectable enzyme labels typically require prolonged time intervals for label, Such as a radioisotope or covalently-linked fluorescent 45 generating detectable quantities of product, and also gener dye molecule. In general, the binding properties of the probe ate an insoluble product that is not attached to the probe macromolecule define the Specificity of the detection molecule.

method, and the detectability of the associated label deter An additional disadvantage of enzyme labels is the dif mines the sensitivity of the detection method. The sensitivity ficulty of detecting multiple target Species with enzyme of detection is in turn related to both the type of label 50 labeled probes. It is problematic to optimize reaction con employed and the quality and type of equipment available to ditions and development time(s) for two or more discrete detect it. enzyme label Species and, moreover, there is often consid For example, radioimmunoassays (RIA) have been erable spectral overlap in the chromophore end products among the most Sensitive and Specific analytical methods which makes discrimination of the reaction products diffi used for detecting and quantitating biological macromol 55 cult.

ecules. Radioimmunoassay techniques have been used to Fluorescent labels do not offer the Signal amplification detect and measure minute quantities of Specific analytes, advantage of enzyme labels, nonetheless, fluorescent labels Such as polypeptides, drugs, Steroid hormones, possess significant advantages which have resulted in their polynucleotides, metabolites, and tumor markers, in biologi widespread adoption in immunocytochemistry. Fluorescent cal Samples. Radioimmunoassay methods employ immuno 60 labels typically are Small organic dye molecules, Such as globulins labeled with one or more radioisotopes as the fluorescein, Texas Red, or rhodamine, which can be readily analytical reagent. Radiation (alpha, beta, or gamma) pro conjugated to probe molecules, Such as immunoglobulins or duced by decay of the attached radioisotope label Serves as Staph. aureus Protein A. The fluorescent molecules the Signal which can be detected and quantitated by various (fluorophores) can be detected by illumination with light of radiometric methods. 65 an appropriate excitation frequency and the resultant Spec Radioisotopic labels possess Several advantages, Such as: tral emissions can be detected by electro-optical Sensors or very high Sensitivity of detection, Very low background light microScopy.

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A wide variety of fluorescent dyes are available and offer region is not well-Suited for detection by the human eye, a Selection of excitation and emission spectra. It is possible hampering the use of phycobiliprotein and cyanine labels in to Select fluorophores having emission spectra that are optical fluorescence microscopy, (2) cyanines, Sufficiently different So as to permit multitarget detection and phycobiliproteins, and the coupled accessory molecules discrimination with multiple probes, wherein each probe (e.g., Azure A) are organic molecules Susceptible to pho Species is linked to a different fluorophore. Because the tobleaching and undergoing undesirable chemical interac Spectra of fluorophores can be discriminated on the basis of tions with other reagents, and (3) emitted radiation is down both narrow band excitation and selective detection of converted, i.e., of longer wavelength(s) than the absorbed emission Spectra, two or more distinct target Species can be excitation radiation. For example, AZure A absorbs at 632 detected and resolved (Titus et al. (1982) J. Immunol. nm and emits at 645 nm, and allophycocyanin absorbs at 645 Methods 50: 193; Nederlof et al. (1989) Cytometry 10:20; nm and emits at 655 nm, and therefore autofluorescence and Ploem, J. S. (1971) Ann. NY Acad. Sci. 177:414). background noise from Scattered excitation light is not Unfortunately, detection methods which employ fluores eliminated.

cent labels are of limited Sensitivity for a variety of reasons. Another alternative class of fluorophore that has been First, with conventional fluorophores it is difficult to dis 15 proposed are the down-converting luminescent lanthanide criminate Specific fluorescent Signals from nonspecific back chelates (Soini and Lovgren (1987) CRC Crit. Rev. Anal. ground Signals. Most common fluorophores are aromatic Chem. 18: 105; Leif et al. (1977) Clin. Chem. 23: 1492; organic molecules which have broad absorption and emis Soini and Hemmila (1979) Clin. Chem. 25: 353; Seveus et Sion Spectra, with the emission maximum red-shifted al. (1992) Cytometry 13: 329). Down-converting lanthanide 50-100 nm to a longer wavelength than the excitation (i.e., chelates are inorganic phosphors which possess a large absorption) wavelength. Typically, both the absorption and downward Stokes shift (i.e., emission maxima is typically at emission bands are located in the UV/visible portion of the least 100 nm greater than absorption maxima) which aids in Spectrum. Further, the lifetime of the fluorescence emission the discrimination of Signal from Scattered excitation light. is usually short, on the order of 1 to 100 ns. Unfortunately, Lanthanide phosphors possess emission lifetimes that are these general characteristics of organic dye fluorescence are 25 Sufficiently long (i.e., greater than 1 mus) to permit their use also applicable to background Signals which are contributed in time-gated detection methods which can reduce, but not by other reagents (e.g., fixative or Serum), or autofluores totally eliminate, noise caused by shorter-lived autofluores cence or the sample itself (Jongkind et al. (1982) Exp. Cell cence and Scattered excitation light. Further, lanthanide Res. 138: 409; Aubin, J. E. (1979) J. Histochem. Cytochem. phosphors possess narrow-band emission, which facilitates 27: 36). Autofluorescence of optical lenses and reflected wavelength discrimination against background noise and excitation light are additional Sources of background noise Scattered excitation light, particularly when a laser excita in the visible spectrum (Beverloo et al. (1991) Cytometry 11: tion source is utilized (Reichstein et al. (1988) Anal. Chem. 784; Beverloo et al. (1992) Cytometry 13: 561). Therefore, 60: 1069). Recently, enzyme-amplified lanthanide lumines the limit of detection of Specific fluorescent signal from cence using down-converting lanthanide chelates has been typical fluorophores is limited by the Significant background 35 proposed as a fluorescent labeling technique (Evangelista et noise contributed by nonspecific fluorescence and reflected al. (1991) Anal. Biochem. 197: 213; Gudgin-Templeton et excitation light. al. (1991) Clin Chem. 37:1506).

A Second problem of organic dye fluorophores that limits Until recently, down-converting lanthanide phosphors Sensitivity is photolytic decomposition of the dye molecule have had the Significant disadvantage that their quantum (i.e., photobleaching). Thus, even in situations where back 40 efficiency in aqueous (oxygenated) Solutions is So low as to ground noise is relatively low, it is often not possible to render them unsuitable for cytochemical Staining. Beverloo integrate a weak fluorescent Signal over a long detection et al. (op.cit.) have described a particular down-converting time, Since the dye molecules decompose as a function of lanthanide phosphor (yttrium oxysulfide activated with incident irradiation in the UV and near-UV bands. europium) that produces a signal in aqueous Solutions which However, because fluorescent labels are attractive for 45 can be detected by time-resolved methods. Seveus et al. various applications, Several alternative fluorophores having (op.cit.) have used down-converting europium chelates in advantageous properties for Sensitive detection have been conjunction with time-resolved fluorescence microscopy to proposed. One approach has been to employ organic dyes reject the Signal from prompt fluorescence and thereby comprising a phycobiliprotein acceptor molecule dye that reduce autofluorescence.

emits in the far red or near infrared region of the Spectrum 50 However, the down-converting lanthanide phosphor of where nonspecific fluorescent noise is reduced. Phycobilip Beverloo et al. and the europium chelate of Seveus et al. roteins are used in conjunction with accessory molecules require excitation wavelength maxima that are in the ultra that effect a large Stokes shift via energy transfer mecha Violet range, and thus produce Significant Sample autofluo nisms (U.S. Pat. No. 4,666,862; Oi et al. (1982) J. Cell. Biol. rescence and background noise (e.g., Serum and/or fixative 93: 891). Phycobiliprotein labels reduce the degree of spec 55 fluorescence, excitation light Scattering and refraction, etc.) tral overlap between excitation frequencies and emission that must be rejected (e.g., by fillers or time-gated signal frequencies. An alternative approach has been to use cyanine rejection). Further, excitation with ultraviolet irradiation dyes which absorb in the yellow or red region and emit in the damageS nucleic acids and other biological macromolecules, red or far red where autofluorescence is reduced (Mujumbar posing Serious problems for immunocytochemical applica et al. (1989) Cytometry 10: 11). 60 tions where it is desirable to preserve the viability of living However, with both the phycobiliproteins and the cyanine cells and retain cellular structures (e.g., FACS, cyto dyes the emission frequencies are red-shifted (i.e., frequency architectural microscopy).

downshifted) and emission lifetimes are short, therefore Laser Scanning fluorescence microScopy has been used background autofluorescence is not completely eliminated for two-photon excitation of a UV-excitable fluorescent as a noise Source. More importantly perhaps, phycobilipro 65 organic dye, Hoechst 33258, using a stream of Strongly teins and cyanine dyes possess Several distinct disadvan focused laser pulses (Denk et al. (1990) Science 248: 73). tages: (1) emission in the red, far red, and near infrared The organic fluorphore used by Denk et al. was significantly

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S 6 photobleached by the intense, highly focused laser light at least Some wavelengths in the reporter's emission band. during the course of imaging. Motsenbocker et al. (EP 476 The laser light is preferably focused to a Small region in the 556) describes a method to increase luminol chemilumines Sample, and light emanating from that region is collected cence by adding a dye catalyst that absorbs long wavelength and directed to the detector. An electrical Signal representing radiation (deep red light) and Subsequently reacts with the intensity of light in the emission band provides a molecular oxygen to generate an oxidant which can itself measure of the amount of reporter present. Depending on the react with luminol and produce oxidized luminol which detector's Spectral response, it may be necessary to provide emits blue light. Gavrilovic (U.S. Pat. No. 5,166,948) dis a filter to block the excitation light. closes a method and apparatus for optical pumping of Simultaneous detection of multiple reporters is possible, infrared pump light to a visible or ultraViolet emission light at least where the reporters have different excitation bands or having a wavelength shorter than the pump light (i.e., different emission bands. Where the excitation bands differ, up-converted emission). multiple laser diodes emitting at respective appropriate Thus, there exists a significant need in the art for labels multiplexer orareother wavelengths combined using a wavelength division

Suitable techniques, Such as frequency and detection methods that permit Sensitive optical and/or labeling, frequency modulation, and lock-in detector device. spectroscopic detection of specific label signal(s) with If the emission bands are different

(whether or not the essentially total rejection of nonspecific background noise, and which are compatible with intact viable cells and bands is separated and Sent to multiple different excitation bands are different), light in the emission detectors. If the aqueous or airborne environments. emission bands overlap, a single detector may be used, but The references discussed herein are provided solely for other detection techniques are used. One example is to use their disclosure prior to the filing date of the present appli time multiplexing techniques So that only one reporter is cation. Nothing herein is to be construed as an admission emitting at a given time. Alternatively, the different laser that the inventors are not entitled to antedate Such disclosure diodes can be modulated at different characteristic frequen by virtue of prior invention. cies and lock-in detection performed. SUMMARY OF THE INVENTION 25 Detection methods and detection apparatus of the present invention enable the ultrasensitive detection of

The present invention provides labels, detection methods, up-converting phosphors and up-converting organic dyes by and detection apparatus which permit ultrasensitive detec exploiting what is essentially the total absence of back tion of cells, biological macromolecules, and other analytes, ground noise (e.g., autofluorescence, Serum/fixative which can be used for multiple target detection and target fluorescence, excitation light Scatter) that are advantageous discrimination. The up-converting labels of the invention characteristics of up-converting labels. Some embodiments permit essentially total rejection of non-specific background of the invention utilize time-gated detection and/or autofluorescence and are characterized by excitation and wavelength-gated detection for optimizing detection emitted wavelengths that are typically in the infrared or Sensitivity, discriminating multiple samples, and/or detect Visible portions of the Spectrum, respectively, and thus avoid 35 ing multiple probes on a Single Sample. Phase-Sensitive the potentially damaging effects of ultraViolet radiation. The detection can also be used to provide discrimination between up-converting labels of the invention convert long Signal(s) attributable to an up-converting phosphor and wavelength excitation radiation (e.g., near-IR) to emitted background noise (e.g. autofluorescence) which has a dif radiation, which is generally about one-half to one-third the ferent phase shift.

wavelength of the excitation wavelength. Since background 40 Up-converting organic dyes, Such as red-absorbing dyes, fluorescence in the Visible range is negligible if near-IR also can be used in an alternate embodiment that converts excitation wavelengths are used, the use of up-converting the photons absorbed by the dye into a transient Voltage that labels provides essentially background-free detection of can be measured using electrodes and conventional elec Signal. tronic circuitry. After having undergone two-photon absorp In brief, the invention provides the use of luminescent 45 tion the dye is ionized by additional photons from the light materials that are capable of multiphoton excitation and Source (e.g., a laser) leading to short-lived molecular ions have upshifted emission spectra. In one embodiment of the whose presence can be detected and quantified by measuring invention, up-converting phosphors (i.e., which absorb mul the transient photoconductivity following the excitation irra tiple photons in a low frequency band and emit in a higher diation. In this embodiment, resonant multiphoton ioniza frequency band) are used as labels which can be linked to 50 tion is used to provide a quantitative measurement of the one or more probes, Such as an immunoglobulin, number and/or concentration of dye molecules in a Sample. polynucleotide, Streptavidin, Protein A, receptor ligand, or Furthermore, essentially all photoions formed in the irradi other probe molecule. In an another embodiment, ated Sample contribute to the Signal, whereas photons are up-converting organic dyes Serve as the label. The organic emitted isotopically and only a fraction can be collected dye labels and phosphor labels of the invention are highly 55 using optics. Measurement of the transient photocurrent compatible with automated diagnostic testing, microscopic effectively transfers the conversion of photons into an elec imaging applications, and coded particle detection, among tronic Signal that is readily measured with relatively simple many other applications. and inexpensive Sensors Such as electrodes. The nature of the invention provides considerable flex In Some embodiments, the present invention utilizes one ibility in the apparatus for carrying out the methods. AS a 60 or more optical laser Sources for generating excitation general matter, the excitation Source may be any convenient illumination of one or more discrete frequency(ies). In light Source, including inexpensive near-infrared laser certain variations of the invention, laser irradiation of an diodes or light-emitting diodes (LEDs), and the detector up-converting label can modify the immediate molecular may be any convenient detector, Such as a photodiode. In the environment through laser-induced photochemical pro case of a single reporter, the apparatus includes a laser diode 65 ceSSes involving either direct absorption or energy transfer; capable of emitting light at one or more wavelengths in the Such spatially-controlled deposition of energy can be used to reporter's excitation band and a detector that is Sensitive to produce localized damage and/or to probe the chemical

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environment of a defined location. In Such embodiments, the hybridization reactions, including hybridization kinetics and up-converting label can preferably act as a photophysical thermodynamic stability of hybridized polynucleotides. catalyst. The invention also provides methods, up-converting The invention provides methods for producing targeted labels, and compositions of labeled binding reagents for damage (e.g., catalysis) in chemical or biological materials, performing fluorescence-activated cell Sorting (FACS) by wherein a probe is employed to localize a linked flow cytometry using excitation radiation that is in the up-converting label to a position near a targeted biological infrared portion of the Spectrum and does not significantly structure that is bound by the probe. The localized damage cells. This provides a significant advantage over up-converting label is excited by one or more excitation present FACS methods which rely on excitation illumination wavelengths and emits at a shorter wavelength which may in the ultraViolet portion of the Spectrum, including wave be directly cytotoxic or genotoxic (e.g., by producing free lengths which are known to produce DNA lesions and radicals Such as Superoxide, and/or by generating thymine damage cells.

thymine dimers), or which may induce a local photolytic The invention also provides compositions comprising at chemical reaction to produce reactive chemical Species in least one fluorescent organic dye molecule attached to an the immediate vicinity of the label, and hence in the vicinity 15 inorganic up-converting phosphor. The fluorescent organic of the targeted biological material. Thus, targeting probes dye molecule is Selected from the group consisting of: labeled with one or more up-converting labels (e.g., an rhodamines, cyanines, Xanthenes, acridines, oxazines, up-converting inorganic phosphor) may be used to produce porphyrins, and phthalocyanines, and may optionally be targeted damage to biological Structures, Such as cells, complexed with a heavy metal. The fluorescent organic dye tissues, neoplasms, vasculature, or other anatomical or his may be adsorbed to the inorganic up-converting phosphor tological Structures. crystal and/or may be covalently attached to a coated Embodiments of the present invention also include inorganic up-converting phosphor, a derivatized vitrocer up-converting phosphors which can also be excited by an amic up-converting phosphor, or a microencapsulated inor electron beam or other beam of energetic radiation of ganic up-converting phosphor. Frequently, covalent conju Sufficient energy and are cathodoluminescent. Such 25 gation between the up-converting inorganic phosphor electron-stimulated labels afford novel advantages in elimi particles and proteins (e.g., avidin, immunoglobulin) can be nating background in ultrasensitive biomolecule detection accomplished with hetrobifunctional crosslinkers. methods. Typically, Stimulation of the up-converting phos BRIEF DESCRIPTION OF THE DRAWINGS phor with at least two electrons is employed to generate a visible-light or UV band emission. FIG. 1 is an optical and electronic block diagram illus The invention also provides for the Simultaneous detec trating representative apparatus for performing diagnostics tion of multiple target Species by exploiting the multiphoton on a Sample according to the present invention; excitation and Subsequent background-free fluorescence FIG. 2A shows apparatus for implementing phase Sensi detection of Several up-converting phosphors or tive detection in the context of a single channel; up-converting dyes. In one embodiment, Several phosphors 35 FIG. 2B shows apparatus where first and second laser dyes are Selected which have overlapping absorption bands diodes are modulated by Signals from waveform generators, which allow simultaneous excitation at one wavelength (or FIG. 3 shows apparatus for performing gated detection; in a narrow bandwidth), but which vary in emission char FIG. 4 shows an apparatus for performing diagnostics on acteristics Such that each probe-label Species is endowed a Sample using first and Second reporters excitation bands with a distinguishable fluorescent "fingerprint.” By using 40 centered at lambda 1 and lambda 2, respectively, and having various methods and devices, the presence and concentra overlapping emission bands near lambda 3, tion of each of the phosphors or dyes can be determined. FIG. 5A, 5B, 5C show schematically energy state transi The invention also provides biochemical assay methods tions in multi-photon excitation Schemes.

for determining the presence and concentration of one or FIG. 6 shows a miniaturized instrument using a hand-held more analytes, typically in Solution. The assay methods 45 probe, employ compositions of probes labeled with up-converting FIG. 7A shows the use of a charge-coupled device (CCD) phosphors and/or up-converting dyes and apparatus for array used to detect emissions from a large plurality of magnetically and/or optically trapping particles that com binding Sites, prise the analyte and the labeled probe. In one embodiment, a Sandwich assay is performed, wherein an immobilized 50 lensFIG. 7B shows the CCD array used in conjunction with a array;

probe, immobilized on a particle, binds to a predetermined FIG. 8 shows an embodiment using optical trapping, analyte, producing-an immobilization of the bound analyte on the particle; a Second probe, labeled with an tionFIG. of 9 ShowS Schematically dye coating and encapsula an up-converting phosphor particle;

up-converting label can then bind to the bound analyte to produce a bound Sandwich complex containing an 55 FIG. 10 shows schematically an apparatus for determin up-converting label bound to a particle. By combining ing particle Velocity and hydrodynamic or aerodynamic different probe-label combinations, particles of various properties of a target;

sizes, colors, and/or shapes with distinct immobilized probe FIG. 11 is a phosphor emission spectrum of Sodium (S), and/or various excitation wavelengths, it is possible to yttrium fluoride-ytterbium/erbium up-converting phosphor perform multiple assays essentially simultaneously or con 60 with an excitation laser Source at a wavelength maximum of temporaneously. This multipleX advantage affords detection 977.2 mm; emission maximum is about 541.0 nm, and quantitation of multiple analyte species in a single FIG. 12 is an excitation scan of the sodium yttrium Sample. The assay methods are also useful for monitoring fluoride-ytterbium/erbium phosphor excitation Spectrum, the progreSS of a reaction, Such as a physical, chemical, with emission collection window set at 541.0 nm, biochemical, or immunological reaction, including binding 65 FIG. 13 is a time-decay measurement of the phosphor reactions. For example, the invention may be used to moni luminescence at 541.0 nm after termination of excitation tor the progreSS of ligand-binding reactions, polynucleotide illumination for sodium yttrium fluoride-ytterbium/erbium;

Page 38

FIG. 14 shows the phosphor emission intensity as a FIG. 32 is an emission Spectrum for up-conversion from function of excitation illumination intensity for a Sodium neodymium chelated in EDTA.

yttrium fluoride-ytterbium/erbium phosphor; DESCRIPTION OF SPECIFIC EMBODIMENTS FIG. 15 shows effective single-photon phosphorescence Definitions cross-section for 0.3 um particles of Na(YosYboEros) F. Unless defined otherwise, all technical and Scientific following excitation with 200 Wem' at 970 nm. terms used herein have the same meaning as commonly FIG. 16 shows size-dependence of phosphorescence understood by one of ordinary skill in the art to which this cross-section for Na(YosYboEroos)F particles. invention belongs. Although any methods and materials FIG. 17A shows a fluorescence Scan of an up-converting Similar or equivalent to those described herein can be used phosphor reporter in Hepes-buffered Saline induced by exci in the practice or testing of the present invention, the tation with a 970-nm laser Source; preferred methods and materials are described. For purposes of the present invention, the following terms are defined

FIG. 17B shows a fluorescence spectrum scan of an below.

up-converting phosphor reporter coated with Streptavidin in

Hepes-buffered saline induced by excitation with a 970-nm 15 thatASproduces, used herein, “label” refers to a chemical Substituent under appropriate excitation conditions, a laser Source; detectable optical Signal. The optical Signal produced by an FIG. 18A shows an excitation spectrum scan of an excited label is typically electromagnetic radiation in the up-converting phosphor reporter in Hepes-buffered Saline near-infrared, visible, or ultraViolet portions of the Spectrum. with monochromatic detection of emission at 541 nm, The labels of the invention are generally up-converting FIG. 18B shows an excitation spectrum scan of an labels, which means that the chemical Substituent typically up-converting phosphor reporter coated with Streptavidin in absorbs at least two photons at an excitation frequency and Hepes-buffered saline with monochromatic detection of Subsequently emits electromagnetic energy at an emission emission at 541 nm, frequency higher than the excitation frequency. Thus, there FIG. 19 shows the integrated signal obtained from is generally a significant Stokes shift between the original Samples of(YosYboosEroos)O2S showing the relationship 25 excitation frequency and the final emission frequency. A between phosphor concentration and up-converted Signal; label is generally attached to a probe to Serve as a reporter FIG. 20 shows schematically one embodiment of an that indicates the presence and/or location of probe. The Sandwich immunoassay for detecting an analyte in a Solution invention encompasses organic and inorganic up-converting by binding the analyte (e.g., an antigen target) to a biotiny labels, but preferably employs up-converting inorganic lan lated antibody and to an immobilized antibody, wherein the thanide phosphors as labels. Thus, a typical label of the analyte forms a Sandwich complex immobilized on a Solid invention is a Submicron-size up-converting lanthanide Substrate Superparamagnetic microbead; and phosphor particle. The label can alternatively comprise a FIG. 21 shows schematically the detection and discrimi lanthanide

AS used ion in a chelate or cage compound.

herein, a "probe' refers to a binding component nation of two cell Surface antigens with Specific antibodies 35 labeled with two phosphors with distinct phosphorescence which binds preferentially to one or more targets (e.g., antigenic epitopes, polynucleotide Sequences, macromo characteristics.

lecular

FIG. 22 shows a Schematic of an apparatus for phase crimination receptors) with an affinity Sufficient to permit dis Sensitive detection. of labeled probe bound to target from nonspe cifically bound labeled probe (i.e., background). Generally,

FIG. 23 show a Schematic of a competitive homogeneous 40 the probe-target binding is a non-covalent interaction with a assay using phosphors as labels and fiber optic illumination binding affinity (KD) of at least about 1x10''', prefer at a capture Surface. ably with at least about 1x107M, and more preferably with FIG. 24 show a Schematic of a competitive homogeneous an affinity of at least about 1x10M or greater. Antibodies antigen capture assay using phosphors as labels and a typically have a binding affinity for cognate antigen of about convergent illumination beam focused on the capture Sur 45 1x10'M' or more. For example but not limitation, probes face. of the invention include: antibodies, polypeptide hormones, FIG. 25 shows a Schematic of a homogeneous immuno polynucleotides, Streptavidin, Staphlyococcus aureus pro precipitation assay using phosphors as labels and a conver tein A, receptor ligands (e.g., Steroid or polypeptide gent illumination beam focused on the capture Surface hormones), leucine Zipper polypeptides, lectins, antigens wherein the capture Surface collects immunoprecipitates. 50 (polypeptide, carbohydrate, nucleic acid, and hapten FIG. 26 shows a block diagram of one embodiment of epitopes), and others.

apparatus for carrying out the present invention on a Sample AS used herein, a “probe-label conjugate' and a "labeled using a microscope. probe' refer to a combination comprising a label attached to FIG. 27 is a block diagram of a microtiter plate reader for 55 a probe. In certain embodiments, more than one label use with the present invention. Substituent may be attached to a probe. Alternatively, in FIG. 28 is an illustration of the data for upconverting Some embodiments more than one probe may be attached to a label (e.g., multiple antibody molecules may be attached to phosphors in three test wells. a Submicron-size inorganic up-converting phosphor bead). FIG. 29 is a schematic view of a second embodiment of a hand-held probe for carrying out the present invention. 60 label to aattachment

Various chemistries can be employed to link a probe, including, but not limited to, the formation

FIG. 30 illustrates a three channel configuration using of covalent bonds, hydrogen bonds, ionic bonds, electro interference filters. Static interactions, and Surface tension (phase boundary) FIG. 31A is an illustration of an embodiment of the interactions. Attachment of label can also involve incorpo invention in which a diode laser array F1 and a detector ration of the label into or onto microSpheres, microparticles, array F2 are combined in a single device. 65 immunobeads, and Superparamagnetic magnetic beads FIG. 31B is a detailed view of a small section of the (PolySciences, Inc., Warrington, Pa.; Bangs Laboratories, device shown in FIG. 31A. Inc.,979 Keystone Way, Carmel, Ind. 46032). For example,

Page 39

inorganic up-converting phosphor particles can be encapsu telomere repeat TTAGGG or an Alu repetitive sequence) for lated in microSpheres that are composed of polymer material specific hybridization, a transcribed RNA (e.g., from an SP6 that is essentially transparent or translucent in the wave cloning vector insert), or a polyamide nucleic acid (Nielsen length range(s) of the excitation and emitted electromagnetic et al. (1991) Science 254: 1497). Various target polynucle radiation (U.S. Pat. No. 5,132,242, incorporated herein by otides may be detected by hybridization of a labeled probe reference). Such microspheres can be functionalized by polynucleotide to the target sequence(s). For example but Surface derivatization with one or more reactive groups (e.g., not limitation, target polynucleotides may be: genomic carboxylate, amino, hydroxylate, or polyatcrolein) for cova Sequences (e.g., structural genes, chromosomal repeated lent attachment to a probe, Such as a protein. Probe-label Sequences, regulatory Sequences, etc.), RNA (e.g., mRNA, conjugates can also comprise a phosphor chelate. hnRNA, rRNA, etc.), pathogen sequences (e.g., viral or AS used herein, the term "target” and “target analyte” mycoplasmal DNA or RNA sequences), or transgene refer to the object(s) that is/are assayed for by the methods Sequences.

of the invention. For example but not limitation, targets can “Specific hybridization” is defined herein as the formation comprise polypeptides (e.g., hCGH, insulin, albumin), glyco of hybrids between a probe polynucleotide and a target proteins (e.g., immunoglobulins, thrombomodulin, gamma - 15 polynucleotide, wherein the probe polynucleotide preferen glutamyltranspeptidase; Goodspeed et al. (1989) Gene 76: tially hybridizes to the target DNA such that, for example, at 1), lipoproteins, Viruses, microorganisms (e.g., pathogenic least one discrete band can be identified on a Southern blot bacteria, yeasts), polynucleotides (e.g., cellular genomic of DNA prepared from eukaryotic cells that contain the DNA, RNA in a fixed histological specimen for in situ target polynucleotide Sequence, and/or a probe polynucle hybridization, DNA or RNA immobilized on a nylon or otide in an intact nucleus localizes to a discrete chromo nitrocellulose membrane, viral DNA or RNA in a tissue or Somal location characteristic of a unique or repetitive biological fluid), and pharmaceuticals (i.e., prescribed or Sequence. In Some instances, a target Sequence may be over-the-counter drugs listed in the Physicians Drug Refer present in more than one target polynucleotide species (e.g., ence and/or Merck Manual, or illegal Substances Such as a particular target Sequence may occur in multiple members intoxicants or anabolic Steroids). 25 of a gene family or in a known repetitive sequence). It is As used herein, the term “antibody” refers to a protein evident that optimal hybridization conditions will vary consisting of one or more polypeptides Substantially depending upon the sequence composition and length(s) of encoded by immunoglobulin genes. The recognized immu the targeting polynucleotide(s) and target(s), and the experi noglobulin genes include the kappa, lambda, alpha, gamma mental method Selected by the practitioner. Various guide (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant lines may be used to Select appropriate hybridization con region genes, as well as the myriad immunoglobulin Vari ditions (see, Maniatis et al., Molecular Cloning: A able region genes. Full-length immunoglobulin “light Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, chains” (about 25Kd or 214 amino acids) are encoded by a N.Y. and Berger and Kimmel, Methods in Enzymology, variable region gene at the NH2-terminus (about 110 amino Volume 152 Guide to Molecular Cloning Techniques (1987), acids) and a kappa or lambda constant region gene at the 35 Academic Press, Inc., San Diego, Calif., Dunn et al. (1989) COOH-terminus. Full-length immunoglobulin “heavy J. Biol. Chem. 264: 13057 and Goodspeed et al. (1989) Gene chains” (about 50 Kd or 446 amino acids), are similarly 76: 1.

encoded by a variable region gene (about 116 amino acids) As used herein, the term “label excitation wavelength” and one of the other aforementioned constant region genes, refers to an electromagnetic radiation wavelength that, when e.g., gamma (encoding about 330 amino acids). One form of 40 absorbed by an up-converting label, produces a detectable immunoglobulin constitutes the basic Structural unit of an fluorescent emission from the up-converting label, wherein antibody. This form is a tetramer and consists of two the fluorescent emission is of a shorter wavelength (i.e., identical pairs of immunoglobulin chains, each pair having higher frequency radiation) that the label excitation wave one light and one heavy chain. In each pair, the light and length. AS used herein, the term "label emission wave heavy chain variable regions are together responsible for 45 length” refers to a wavelength that is emitted from an binding to an antigen, and the constant regions are respon up-converting label Subsequent to, or contemporaneously sible for the antibody effector functions. In addition to with, illumination of the up-converting label with one or antibodies, immunoaiobulins may exist in a variety of other more excitation wavelengths, label emission wavelengths of forms including, for example, Fv, Fab, and F(ab')2, as well up-converting labels are shorter (i.e., higher frequency as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., 50 radiation) than the corresponding excitation wavelengths. Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Both label excitation wavelengths and label emission wave Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879–5883 lengths are characteristic to individual up-converting label (1988) and Bird et al., Science, 242,423-426 (1988)). (See, Species, and are readily determined by performing simple generally, Hood et al., “Immunology’, Benjamin, N.Y., 2nd excitation and emission Scans.

ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 55 Invention Overview (1986)). Thus, not all immunoglobulins are antibodies. (See, The Subject invention encompasses fluorescent labels that U.S. Ser. No. 07/634.278, which is incorporated herein by are excited by an excitation wavelength and Subsequently reference, and Co et al. (1991) Proc. Natl. Acad. Sci. emit electromagnetic radiation at up-shifted frequencies (U.S.A.) 88: 2869, which is incorporated herein by (i.e., at higher frequencies than the excitation radiation). reference). 60 In accordance with the present invention, labels compris AS used herein, "probe polynucleotide' refers to a poly ing up-converting inorganic phosphors and/or up-converting nucleotide that specifically hybridizes to a predetermined organic dyes are provided for various applications. The target polynucleotide. For example but not limitation, a up-converting labels of the invention may be attached to one probe polynucleotide may be a portion of a cDNA corre or more probe(s) to serve as a reporter (i.e., a detectable sponding to a particular mRNA sequence, a portion of a 65 marker) of the location of the probe(s). The up-converting genomic clone, a Synthetic oligonucleotide having Sufficient labels can be attached to various probes, Such as antibodies, Sequence homology to a known target Sequence (e.g., a Streptavidin, protein A, polypeptide ligands of cellular

Page 40

receptors, polynucleotide probes, drugs, antigens, toxins, function of particle size. In Some applications, Such as those and others. Attachment of the up-converting label to the requiring highly Sensitive detection of Small phosphor probe can be accomplished using various linkage particles, infrared laser diodes are preferably Selected as an chemistries, depending upon the nature of the Specific probe. excitation Source.

For example but not limitation, microcrystalline Although the properties of the up-converting phosphors up-converting lanthanide phosphor particles may be coated will be described in detail in a later section, it is useful to with a polycarboxylic acid (e.g., Addition XW 330, Hoechst, outline the basic mechanisms involved. Up-conversion has Frankfurt, Germany) during milling and various proteins been found to occur in certain materials containing rare (e.g., immunoglobulin, Streptavidin or protein A) can be earth ions in certain crystal materials. For example, ytter physically adsorbed to the Surface of the phosphor particle bium and erbium act as an activator couple in a phosphor (Beverloo et al. (1991) op.cit., which is incorporated herein host material such as bariumn-yttrium-fluoride. The ytter by reference). Alternatively, various inorganic phosphor bium ions act as the absorber, and transfer energy non coating techniques can be employed including, but not ritdiatively to excite the erbium ions. The emission is thus limited to: Spray drying, plasma deposition, and derivatiza characteristic of the erbium ion’s energy levels. tion with functional groups (e.g., -COOH, -NH2, 15 Up-Converting Microcrvstalline Phosphors -CONH2) attached by a silane coupling agent to -SiOH Although the invention can be practiced with a variety of moieties coated on the phosphor particle or incorporated into up-converting inorganic phosphors, it is believed that the a vitroceramic phosphor particle comprising Silicon oxide(s) preferred embodiment(s) employ one or more phosphors and up-converting phosphor compositions. Vitroceramic derived from one of several different phosphor host phosphor particles can be aminated with, for example, materials, each doped with at least one activator couple. aminopropyltriethoxysilane for the purpose of attaching Suitable phosphor host materials include: Sodium yttrium amino groups to the vitroceramic Surface on linker fluoride (NaYF), lanthanum fluoride (LaF), lanthanum molecules, however other omega-functionalized Silanes can oxysulfide, yttrium oxysulfide, yttrium fluoride (YF), be Substituted to attach alternative functional groups. yttrium gallate, yttrium aluminum garnet, gadolinium fluo Probes, Such as proteins or polynucleotides may then be 25 ride (GdF), barium yttrium fluoride (BaYFs, BaYF), and directly attached to the vitroceramic phosphor by covalent gadolinium oxysulfide. Suitable activator couples are linkage, for example through Siloxane bonds or through selected from: ytterbium/erbium, ytterbium/thulium, and carbon-carbon bonds to linker-molecules (e.g., organofunc ytterbium/holmium. Other activator couples suitable for tional silylating agents) that are covalently bonded to or up-conversion may also be used. By combination of these adsorbed to the Surface of a phosphor particle. Covalent host materials with the activator couples, at least three conjugation between the up-converting inorganic phosphor phosphors with at least three different emission spectra (red, particles and proteins (e.g., avidin, immunoglobulin) can be green, and blue Nisible light) are provided. Generally, the accomplished with homobifunctional, or preferably absorber is ytterbium and the emitting center can be selected heterobifunctional, crosslinkers. For example, Surface from: erbium, holmium, terbium, and thulium; however, Silanization of the phosphors with tri(ethoxy)thiopropyl 35 other up-converting phosphors of the invention may contain Silane leaves a phosphor Surface with a thiol finctionality to other absorbers and/or emitters. The molar ratio of absorber: which a protein (e.g., antibody) or any compound containing emitting center is typically at least about 1:1, more usually a primary amine can be grafted using conventional at least about 3:1 to 5:1, preferably at least about 8:1 to 10:1, N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) chem more preferably at least about 11:1 to 20:1, and typically less istry (Weltman et al. (1983). Other silanization and cross 40 than about 250:1, usually less than about 100:1, and more linking methods compatible with the inorganic phosphors usually less than about 50:1 to 25:1, although various ratios may be used at the discretion of the practitioner. may be Selected by the practitioner on the basis of desired Microcrystalline up-converting phosphor particles are characteristics (e.g., chemical properties, manufacturing typically Smaller than about 2 microns in diameter, prefer efficiency, absorption cross-section, excitation and emission ably less than about 1 micron in diameter (i.e., Submicron), 45 wavelengths, quantum efficiency, or other considerations). and more preferably are 0.1 to 0.3 microns or less in The ratio(s) chosen will generally also depend upon the diameter. It is generally most preferred that the phosphor particular absorber-emitter couple(s) selected, and can be particles are as Small as possible while retaining Sufficient calculated from reference values in accordance with the quantum conversion efficiency to produce a detectable Sig desired characteristics.

nal; however, for any particular application, the size of the 50 The optimum ratio of absorber (e.g., ytterbium) to the phosphor particle(s) to be used should be selected at the emitting center (e.g., erbium, thulium, or holmium) varies, discretion of the practitioner. For instance, Some applica depending upon the Specific absorberemitter couple. For tions (e.g., detection of a non-abundant cell Surface antigen) example, the absorber:emitter ratio for Yb:Er couples is may require a highly Sensitive phosphor label that need not typically in the range of about 20:1 to about 100:1, whereas be Small but must have high conversion efficiency and/or 55 the absorber:emitter ratio for Yb:Tm and Yb:Ho couples is absorption cross-section, while other applications (e.g., typically in the range of about 500:1 to about 2000:1. These detection of an abundant nuclear antigen in a permeablized different ratios are attributable to the different matching cell) may require a very Small phosphor particle that can energy levels of the Er, Tm, or Ho with respect to the Yb readily diffuse and penetrate Subcellular structures, but level in the crystal. For most applications, up-converting which need not have high conversion efficiency. Therefore, 60 phosphors may conveniently comprise about 10-30% Yb the optimal size of inorganic phosphor particle is application and either: about 1-2% Er, about 0.1-0.05% Ho, or about dependent and is Selected by the practitioner on the basis of 0.1-0.05% Tm, although other formulations may be quantum efficiency data for the various phosphors of the employed.

invention. Such conversion efficiency data may be obtained Some embodiments of the invention employ inorganic from avaliable Sources (e.g., handbooks and published 65 phosphors that are optimally excited by infrared radiation of references) or may be obtained by generating a standard about 950 to 1000 nm, preferably about 960 to 980 nm. For ization curve measuring quantum conversion efficiency as a example but not limitation, a microcrystalline inorganic

Page 41

phosphor of the formula YF:YbooEroo exhibits a lumi istics (e.g., manufacturing efficiency, ease of derivatization, neScence intensity maximum at an excitation wavelength of etc.); Such low efficiency up-converting phosphors are pref about 980 nm. Inorganic phosphors of the invention typi erably excited with laser illumination at a frequency at or cally have emission maxima that are in the Visible range. For near (i.e., within about 25 to 75 nm) an absorption maximum example, Specific activator couples have characteristic emis of the material. The fact that no other light is generated in the Sion Spectra: ytterbium-erbium couples have emission System other than from the up-converting phosphor allows maxima in the red or green portions of the visible Spectrum, for extremely Sensitive signal detection, particularly when depending upon the phosphor host, ytterbium-holmium intense laser illumination is used as the Source of excitation couples generally emit maximally in the green portion, radiation. Thus, the unique property of up-conversion of ytterbium-thulium typically have an emission maximum in photon energy by up-converting phosphors makes possible the blue range, and ytterbiumterbium usually emit maxi the detection of very Small particles of microcrystalline mally in the green range. For example, Yo so YbooEroo F2 inorganic phosphors. For practical implementation of phos emits maximally in the green portion of the Spectrum. phors as ultrasensitive reporters, particularly as intracellular Although up-converting inorganic phosphor crystals of reporters, it is essential that the grain size of the phosphor be various formulae are Suitable for use in the invention, the 15 as Small as practicable (typically less than about 0.3 to 0.1 following formulae, provided for example and not to limit mu m), for which laser-excited up-converting phosphors are the invention, are generally Suitable: well-Suited.

Na(Y.Yb, Er)F: X is 0.7 to 0.9, y is 0.09 to 0.29, and Z For example, various phosphor material compositions is 0.05 to 0.01; capable of up-conversion are Suitable for use in the inven tion are shown in Table I.

and Z is 0.0005 to 0.001; and TABLE I

and Z is 0.0005 to 0.001. Phosphor Material Compositions YYb,

- - - 2 X is 0.7 to 0.9, y is 0.05 to 0.12; Z is 0.05 25 Host Material Absorber Ion Emitter Ion Color

(Yoss Yboos Eroos)2O is a relatively efficient Oxysulfides (OS) up-converting phosphor material. YOS Ytterbium Erbium Green For exemplification, but not to limit the invention, Gd2O2S Ytterbium Erbium Red ytterbium(Yb)-erbium(Er)-doped yttrium oxysulfides lumi LaOS Ytterbium Holmium Green neSce in the green after excitation at 950 nm. These are Oxyhalides (OXv) non-linear phosphors, in that the ytterbium acts as an YOF Ytterbium Thulium Blue "antenna” (absorber) for two 950 nm photons and transfers YOCl, Yterbium Terbium Green its energy to erbium which acts as an emitter (activator). The Fluorides (F) critical grain size of the phosphor is given by the quantum 35 YF, Ytterbium Erbium Red yield for green emission and the doping level of both Yb and GdF. Ytterbium Erbium Green Er, which is generally in the range of about 1 to 10 percent, LaF Ytterbium Holmium Green more usually in the range of about 2 to 5 percent. A typical NaYF, Ytterbium Thulium Blue Yb:Er phosphor crystal comprises about 10-30% Yb and BaYF. Ytterbium Thulium Blue BaYF Ytterbium Terbium Green about 1-2%. Er. Thus, a phosphor grain containing Several 40 Gallates (Gakov) thousand formula units ensures the emission of at least one or more photons during a typical laser irradiation time. YGaO, Ytterbium Erbium Red However, the nonlinear relationship between absorption and Y3Ga5O12 Ytterbium Erbium Green emission indicates that intense illumination at the excitation Silicates (SiOy) wavelength(s) may be necessary to obtain Satisfactory signal 45 YSiOs Ytterbium Holmium Green in embodiments employing very Small phosphor particles YSiO, Ytterbium Thulium Blue (i.e., less than about 0.3 mu m). Additionally, it is usually desirable to increase the doping levels of activatoremitter In addition to the materials shown in Table I and variations couples for producing very Small phosphor particles So as to maximize quantum conversion efficiency. 50 thereof, aluminates, phosphates, and Vanadates can be Suit Inorganic microcrystalline phosphors with rare earth acti able phosphor host materials. In general, when Silicates are Vators generally have narrow absorption and line emission used as a host material, the conversion efficiency is rela Spectra. The line emission Spectra are due to f-f transitions tively low. In certain uses, hybrid up-converting phosphor crystals may be made (e.g., combining one or more host within the rare earth ion. These are shielded internal tran

Sitions which result in narrow line emission. 55 material and/or one or more absorber ion and/or one or more In certain applications, Such as where highly Sensitive emitter ion).

detection is required, intense illumination can be provided Exemplary up-converting phosphorS eXcited at about 980 by commercially available Sources, Such as infrared laser nm include, but are not limited to: YosoYbolisEroo)F; Sources (e.g., continuous wave (CW) or pulsed Semiconduc Yo...s 7Ybo.13 Timo.o.o. 1) F3; Yo...so Ybo.1 os Hoo.o.o.2) F3; tor laser diodes). For example, in applications where the 60 Gido...so Ybo.1s Ero.o.) F3; G dos 7Ybo.13 Timo.o.o.) F3; microcrystalline phosphor particle must be very Small and Gido, so Ybo. 1 os Hoooo.2) F3; Yo so Yboos Eroos)2 the quantum conversion efficiency is low, intense laser O2S:Yos, Ybo.1 Tmolool)2O2S: YosoYboios Hoooo.2)2O2S, illumination can increase signal and decrease detection Gdo. so Yboos Eroos)2O2S: G do.s 7Ybo.13 Timo.o.o. 1)2 times. Alternatively, Some applications of the invention may O2S:GdosoyboosHoooo..)2O2S.

require phosphor compositions that have inherently low 65 Exemplary up-converting phosphorS eXcited at about quantum conversion efficiencies (e.g., low doping levels of 1500 nm include, but are not limited to: Yoo. Eroo)2O2S, activator couple), but which have other desirable character Gdoos Eroos)2O2S.

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Preparation of Inorganic Phosphor Labels hydroxy carbonate precursor to the oxySulfide phase by Techniques and methods for manufacture of inorganic including a polysulfide flux for annealing. Using this phosphorS has been described in the art. Up-converting technique, highly efficient OXySulfide particles in the 0.3 to phosphor crystals can be manufactured by those of ordinary 0.4 um diameter range were prepared as a dispersion in skill in the art by various published methods, including but water. Frequently, Sonication can be used to produce a not limited to the following: Yocom et al., (1971) Metallur monodisperse mixture of discrete Spherical particles. After gical Transactions 2: 763; Kano et al., (1972). J. Electro fractionation and coating, these particles can be used as chem. Soc., p. 1561; Wittke et al. (1972).J. Appl. Physics 43: up-converting reporters. Furthermore, this general prepara 595; Van Uitert et al. (1969) Mat. Res. Bull. 4: 381; which tive procedure is Suitable for preparing much Smaller phos are incorporated herein by reference. Other references which phor particles (e.g., 0.1 um diameter or Smaller), which may may be referred to are: be advantageous for various assay formats. Jouart J. P. and Mary G. (1990).J. Luminescence 46: 399; Frequently, Such as with phosphors having an oxySulfide McPherson G. L. and Meyerson S. L. (1991) Chem. Phys. host material, the phosphor particles are preferably dis Lett. (April) p. 325; Oomen et al. (1990).J. Luminescence persed in a polar solvent, such as acetone or DMSO and the 46: 353; NI H and R and SC (1991) Optics Lett. 16 15 like, to generate a Substantially monodisperse emulsion (September); McFarlane R. A. (1991)Optics Lett. 16 (e.g., for a stock Solution). Aliquots of the monodisperse (September); Koch et al. (1990) Appl. Phys. Lett. 56: 1083; Stock Solution may be further diluted into an aqueous Silversmith et al. (1987) Appl. Phys. Lett. 51: 1977; Lenth Solution (e.g., a Solution of avidin in buffered water or W. and McFarlane R. M. (1990) J. Luminescence 45: 346; buffered saline).

Hirao et al. (1991).J. Non-crystalline Solids 135:90; McFar It was found that washing phosphors in acetone or DMSO lane et al. (1988) Appl. Phys. Lett. 52: 1300, incorporated improved Suspendability of inorganic phosphor particles in herein by reference). water. In particular, the phosphor particles prepared with In general, inorganic phosphor particles are milled to a polysulfide flux are preferably resuspended and washed in desired average particle size and distribution by conven hot DMSO and heated for about an hour in a steam bath then tional milling methods known in the art, including milling in 25 allowed to cool to room temperature under continuous a conventional barrel mill with Zirconia and/or alumina balls agitation. The phosphor particles may be pre-washed with for periods of up to about 48 hours or longer. Phosphor acetone (typically heated to boiling) prior to placing the particles used in binding assays are typically about 3.0 to particles in the DMSO. Hot DMSO-treated phosphors were 0.01 um in diameter (or along the long axis if non-spherical), found to be reasonably hydrophilic and form stable Suspen more usually about 2.0 to 0.1 um in size, and more conve sions. A MicrofluidizerTM (Microfluidics Corp.) can be used niently about 1.0 to 0.3 um in size, although phosphor to further improve the dispersion of particles in the mixture. particles larger or Smaller than these dimensions may be DMSO-phosphor Suspensions can be easily mixed with preferred for certain embodiments. Phosphor particle size is water, preferably with Small amounts of Surfactant present. selected by the practitioner on the basis of the desired In general, polysaccharides (e.g., guar gum, Xanthan gum, characteristics and in accordance with the guidelines pro 35 gum arabic, alginate, guaiac gum) can be used to promote Vided herein. Fractions having a particular particle size deaggregation of particles. In a variation, particles are range may be prepared by Sedimentation, generally over an washed in hot DMSO and serially diluted into a 0.1% extended period (i.e., a day or more) with removal or the aqueous gum arabic Solution, which appears to virtually desired size range fraction after the appropriate Sedimenta eliminate water dispersion problems of phosphors. tion time. The Sedimentation proceSS may be monitored, 40 Resuspended phosphors in organic Solvent, Such as such as with a Horiba Particle Analyzer. DMSO, are typically allowed to settle for a suitable period However, milling crystalline materials has Several weak (e.g., about 1-3 days), and the Supernatant which is typically nesses. With milling, the particle morphology is not turbid is used for Subsequent conjugation.

uniform, as milled particles result from random fracture of LudoxTM is a colloidal silica dispersion in water with a larger crystalline particles. Since the Sensitivity of a detec 45 Small amount of organic material (e.g., formaldehyde, tion assay using up-converting inorganic phosphors depends glycols) and a small amount of alkali metal. Ludox" and its on the ability to distinguish between bound and unbound equivalents can be used to coat up-converting phosphor phosphor particles, it is preferable that the particles be of particles which can Subsequently be fired to form a ceramic identical Size and morphology. Size, weight, and morphol Silica coating which cannot be removed from the phosphor ogy of up-converting microcrystalline phosphor particles 50 particles, but which can be readily Silanized with organo can affect the number of potential binding sites per particle functional Silanes (containing thiol, primary amine, and and thus the potential Strength of particle binding to reporter carboxylic acid functionalities) using Standard Silanization and/or analyte. Monodisperse Submicron Spherical particles chemistries (Arkles, B., in: Silicon Compounds: Register of uniform size can be generated by homogeneous precipi and Review; 5th Edition (1991); Anderson, R. G., Larson, G. tation reactions at high dilutions. For example, Small yttrium 55 L., and Smith, C., eds.; p. 59-64, Huls America, Piscataway, hydroxy carbonate particles are formed by the hydrolysis of N.J.).

urea in a dilute yttrium Solution. Similarly, up-converting Phosphor particles can be coated or treated with Surface inorganic phosphors can be prepared by homogeneous pre active agents (e.g., anionic Surfactants such as Aerosol OT) cipitation reactions in dilute conditions. For example, during the milling process or after milling is completed. For (YosYbooseroos)2O3 was prepared as monodisperse 60 example, particles may be coated with a polycarboxylic acid Spherical particles in the Submicron size range by precipi (e.g., Addition XW 330, Hoechst, Frart, Germany or Tamol, tation. See Beverloo et al. (1992) op.cit.) during milling to produce However, after precipitation it is typically necessary to a stable aqueous Suspension of phosphor particles, typically anneal the oxide in air at about 1500 C., which can cause at about pH 6-8. The pH of an aqueous solution of phosphor faceting of the Spherical particles which can generate aggre 65 particles can be adjusted by addition of a suitable buffer and gate formation. Faceting can be Substantially reduced by titration with acid or base to the desired pH range. Depend converting the Small spherical particles of the oxide or ing upon the chemical nature of the coating, Some minor loSS

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in conversion efficiency of the phosphor may occur as a binding reagents, either directly or indirectly, for use in result of coating, however the power available in a laser binding assays to detect and quantitate the presence of excitation Source can compensate for Such reduction in analyte(s) in a Sample. Binding reagents are labeled directly conversion efficiency and ensure adequate phosphor emis by attachment to up-converting reporters (e.g., Surface SO. adsorption, covalent linkage). Binding reagents which can In general, preparation of inorganic phosphor particles be directly labeled include, but are not limited to: primary and linkage to binding reagentS is performed essentially as antibodies (i.e., which bind to a target analyte), Secondary described in Beverloo et al. (1992) op.cit., and Tanke U.S. antibodies (i.e., which bind to a primary antibody or pros Pat. No. 5,043,265 incorporated by reference in their thetic group, Such as biotin or digoxygenin), Staphlococcus entirety herein. Alternatively, a water-insoluble polyfunc aureus Protein A, polynucleotides, Streptavidin, and receptor tional polymer which exhibits glass and melt transition ligands. Binding reagents can also be indirectly labeled; temperatures well above room temperature can be used to thus, a primary antibody (e.g., a rabbit anti-erb-B antibody) coat the up-converting phosphors in a nonaqueous medium.

For example, Such polymer functionalities include: carboxy can be indirectly labeled by noncovalent binding to a lic acids (e.g., 5% acrylic acid/95% methyl acrylate 15 directly labeled Second antibody (e.g., a goat anti-rabbit copolymer), amine (e.g., 5% aminoethyl acrylate/95% antibody linked to an up-converting inorganic phosphor). methyl acrylate copolymer) reducible Sulfonates (e.g., 5% Quantitative detection of the analyte-probe complex may be Sulfonated polystyrene), and aldehydes (e.g., polysaccharide conducted in conjunction with proper calibration of the copolymers). The phosphor particles are coated with water assay for each probe employed. A probe is conveniently insoluble polyfunctional polymers by coacervative encap detected under Saturating excitation conditions using, for Sulation in nonaqueous media, washed, and transferred to a example, a laser Source or focused photodiode Source for Suitable aqueous buffer Solution to conduct the heterobifunc excitation illumination.

tional crosslinking to a protein (e.g., antibody) or polynucle Specific binding assays are commonly divided into homo otide probe molecule. An advantage of using water geneous and heterogeneous assays. In a homogeneous assay, insoluble polymerS is that the polymer microcapsule will not the signal emitted by the bound labeled probe is different migrate from the Surface of the phosphor upon aging the 25 from the signal emitted by the unbound labeled probe, hence encapsulated phosphors in an aqueous Solution (i.e., the two can be distinguished without the need for a physical improved reagent Stability). Another advantage in using Separation Step. In heterogeneous assays, the Signal emitted copolymers in which the encapsulating polymer is only from the bound and unbound labeled probes is identical, partially functionalized is that one can control the degree of hence the two must be physically Separated in order to functionalization, and thus the number of biological probe distinguish between them. The classical heterogeneous spe molecules which can be attached to a phosphor particle, on cific binding assay is the radioimmunoassay (RIA) (Yalow et average. Since the Solubility and coacervative encapsulation al. (1978) Science 200: 1245, which is incorporated herein process will depend on the dominant nonfunctionalized by reference). Other heterogeneous binding assays include component of the copolymer, the functionalized copolymer the radioreceptor assay (Cuatrecasas et al. (1974) Ann. Rev. ratio can be varied over a wide range to generate a range of 35 Biochem. 43: 109), the sandwich radioimmunoassay (U.S. potential crosslinking sites per phosphor, without having to Pat. No. 4,376,110, which is incorporated herein by Substantially change the encapsulation process. reference), and the antibodylectin sandwich assay (EPO 166 A preferred functionalization method employs heterobi 623, which is incorporated herein by reference). Heteroge functional crosslinkers that can be made to link the biologi neous assays are usually preferred, and are generally more cal macromolecule probe to the insoluble phosphor particle 40 Sensitive and reliable than homogeneous assayS. in three steps: (1) bind the crosslinker to the polymer coating Whether a tissue extract is made or a biological fluid on the phosphor, (2) separate the unbound crosslinker from Sample is used, it is often desirable to dilute the Sample in the coated phosphors, and (3) bind the biological macro one or more diluents that do not substantially interfere with molecule to the washed, linked polymer-coated phosphor. Subsequent assay procedures. Generally, Suitable diluents This method prevents undesirable crosslinking interactions 45 are aqueous Solutions containing a buffer System (e.g., 50 between biological macromolecules and So reduces irrevers mM NaH2PO or 5-100 mM Tris, pH 4-pH 10), non ible aggregation as described by Tanke et al. Examples of interfering ionic species (5-500 mM KCl or NaCl, or Suitable heterobifunctional crosslinkers, polymer coating Sucrose), and optionally a nonionic detergent Such as Tween. functionalities, and linkable biological macromolecules When the sample to be analyzed is affixed to a solid support, include, but are not limited to: 50 it is usually desirable to wash the Sample and the Solid Support with diluent prior to contacting with probe. The

Sample, either Straight or diluted, is then analyzed for the diagnostic analyte.

Coating Heterobifunctional Biological In the general method of the invention, an analyte in a Functionality Crosslinker Macromolecule 55 Sample is detected and quantified by contacting the Sample carboxylate N-hydroxysuccimide Proteins (e.g., with a probe-label conjugate that specifically or preferen 1-ethyl-3-(3-dimethylamino Ab, avidin) tially binds to an analyte to form a bound complex, and then propyl)-carbodiimide (EDC) detecting the formation of bound complex, typically by primary amine N-5-azido-2-nitrobenzoyl All having 1° amine measuring the presence of label present in the bound com Oxysuccimide (ANB-NOS)

N-succinimidyl (4-iodoacetyl) 60 plexes. A probe-label conjugate can include a directly aminobenzoate (SIAB) labeled analyte-binding reagent (e.g., a primary antibody thiol (reduced N-succinimidyl (4-iodoacetyl) Proteins linked to an up-converting phosphor) and/or an indirectly sulfonate) aminobenzoate (SIAB) labeled analyte-binding reagent (e.g., a primary antibody that is detected by a labeled second antibody, or a biotiny

Binding ASSayS 65 lated polynucleotide that is detected by labeled streptavidin). Up-converting phosphors and up-converting organic dyes The bound complex(es) are typically isolated from unbound are used as reporters (i.e., detectable markers) to label probe-label conjugate(s) prior to detection of label, usually

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by incorporating at least one washing Step, So as to remove usually performed by first incubating the Sample with a background Signal attributable to label present in unbound blocking or prehybridization solution, followed by incubat probe-label conjugate(s). Hence, it is usually desirable to ing the Sample with probe under binding conditions for a incubate probe-label conjugate(s) with the analyte sample Suitable binding period, followed by Washing or otherwise under binding conditions for a Suitable binding period. removing unbound probe, and finally by detecting the Binding conditions vary, depending upon the nature of the presence, quantity, and/or location of bound probe. The Step probe-label conjugate, target analite, and Specific assay of detecting bound probe can be accomplished by detecting method. Thus, binding conditions will usually differ if the label, if the probe is directly labeled, or by incubating the probe is a polynucleotide used in an in Situ hybridization, in bound complex(es) with a Second binding reagent (e.g., a Northern or Southern blot, or in solution hybridization streptavidin) that is labeled and which binds to the probe, assay. Binding conditions will also be different if the probe thus accomplishing indirect labeling of the probe. is an antibody used in an in Situ histochemical Staining Up-converting labels are attached to probe(s) or Second method or a Western blot (Towbinet al. (1979) Proc. Natl. binding reagents that Specifically or preferentially bind to Acad. Sci. (U.S.A.) 76: 4350, incorporated herein by probe(s) by any of the various methodologies discussed reference). In general, binding conditions are Selected in 15 herein. Additionally, up-converting phosphor particles can accordance with the general binding methods known in the be encapsulated in microSpheres and coated with a probe art. For example, but not for limitation, the following (e.g., a specific antigen or antibody) for use as a labeled binding conditions are provided for general guidance: probe in an immunodiagnostic assay or nucleic acid hybrid For antibody probes: ization assay to detect an analyte in a Sample, Such as the 10–200 mM Tris, pH 6-8; usually 100 mM Tris pH 7.5 presence of an antibody, Virus, or antigen in a bloom Serum 15–250 mM NaCl; usually 150 mM NaCl sample, according to the method of Hari et al. (1990) 0.01-0.5 percent, by volume, Tween 20 Biotechniques 9:342, which is incorporated herein by ref 1 percent bovine Serum albumin erence. Microencapsulation of phosphor can be accom 4–37° C.; usually 4 to 15° C. plished in Several ways known in the art, including coating 25 the phosphor with a monomer Solution and polymerizing the

For polynucleotide probes:

3–10xSSC, pH 6-8; usually 5xSSC, pH 7.5 monomer to generate a polymer shell encasing the phosphor particle. Phosphor particles embedded in a polymer coating, 0-50 percent deionized formamide Such as a gel coating, can be functionalized (e.g., with amino 1-10xDenhardt's Solution groups) for covalent attachment to a binding component. 0-1 percent sodium dodecyl sulfate Similarly, up-converting phosphor particles can be coated 10-200 lug/ml sheared denatured sahnon sperm DNA with probe directly, either by Surface adsorption, by multiple 20°-65 C., usually 37° 45° C. for polynucleotide probes hydrogen bonding, by electroStatic interaction, by Van der longer than 50 bp, usually 55-65 C. for shorter Waals binding, or by covalent linkage to a functional group oligonucleotide probes on a functionalized inorganic phosphor particle (e.g., a Additional examples of binding conditions for antibodies 35 vitroceramic phosphor), for example, by linking an amino and polynucleotides are provided in Several Sources, includ acid Side-chain amine or carboxylate group of a probe ing: Maniatis et al., Molecular Cloning. A Laboratory protein to a carboxylate or amine group, respectively, on a Mantual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and functionalized phosphor particle.

Berger and Kimmel, Methods in Enzoymlogy, Volume 152 In certain embodiments, Such as where Stearic and/or Guide to Molecular Cloning Techniques (1987), Academic 40 charge interference of a bulky up-converting phosphor par Press, Inc., San Diego, Calif.; Young and Davis (1983) Proc. ticle inhibits binding of the linked binding reagent to a Natl. Acad. Sci. (U.S.A.) 80: 1194, which are incorporated target, it is desirable to incorporate a molecular spacer herein by reference. When the probe is a receptor ligand, between the phosphor particle and the binding reagent. For Such as IL-2, 3-interferon, or other polypeptide hormones, example, a derivatized microencapsulated phosphor or Vit cytokines, or lymphokines, Suitable binding conditions gen 45 roceramic phosphor may be conjugated to a heterobifunc erally are those described in the art for performing the tional reagent having a -(CH2)- spacer, where n is respective receptor-ligand binding assay. usually an integer from about 2 to about 50, between Various examples of Suitable binding conditions useful in terminal functional groups. Similarly, phosphors may be immunoassays and immunohistochemistry are discussed, directly derivatized with derivatizing agents (e.g., omega for example, in Harlow and Lane, Antibodies: A Laboratory 50 functionalized silanes) having long intramolecular spacer Manual, Cold Spring Harbor, N.Y. (1988), which is incor chains, wherein a functional group reactive with a desired porated herein by reference. In general, Suitable binding binding reagent is Separated from the Surface of the phos conditions for immunological reactions include an aqueous phor by a Spacer of usually at least about 15 A (i.e., the binding buffer containing a salt (e.g., 5-500 mM NaCl or equivalent of about 10 -CH-Straight-chain groups). In KCl), a buffer (e.g., Tris or phosphate buffer at pH 4-10), 55 Some embodiments, labels are attached by Spacer arms of and optionally a nonionic detergent (e.g., Tween). In Some various lengths to reduce potential Stearic hindrance. Mul embodiments, proteinase inhibitors or Stabilizers may be tiple layers of Spacer arms may also be used (e.g., multiple included. The binding reactions are conducted for a Suitable layers of Streptavidin-biotin linkages). binding period, which, for antibody reactions, are typically Multiple Analyte Detection at least about 1 to 5 minutes, preferably at least about 30 60 Since up-converting phosphors can be differentiated on minutes to Several hours, although typically less than about the basis of the excitation and/or emission wavelength 24 hours, more preferably less than about a few hours or Spectra, up-converting phosphors can be used to detect and less. Binding reactions (including washes) are typically discriminate multiple analyte targets, Such as, for example, carried out a temperature range of about 0°C. to about 45 cell Surface antigens or Soluble macromolecules. C., preferably about 4°C. to about 20°-25° C. 65 For example, Streptavidin, avidin, or another linker mac Binding assays, which include in Situ hybridization, in romolecule (e.g., antidigoxigenin antibody) are attached, Situ binding assays, and immunohistochemical Staining, are respectively, to each of two different phosphors (for

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illustration, designated here as Phosphor #1 and Phosphor different oligopeptides affixed to a Solid Support). One or #2) which differ in their absorption and/or emission spectra more of the Species of first binding component may bind to So as to facilitate discrimination of the two phosphors based a particular analyte (e.g., a muscarinic receptor) in an on absorption and/or emission wavelengths, e.g., one phos analyte Solution that is in contact with the Solid Support. phor may emit in the blue and the other may emit in the Binding of the analyte to one or more of the first binding gree n. For example and not limitation, component Species may then be detected with a Second

Na(YosoYbolisEroo)F emits predominantly in the green, binding component (e.g., an anti-muscarinic receptor antibody) labeled with an up-converting phosphor (either and Na(Yoz-Ybo,TMooo..)F emits predominantly in the directly or through a biotinylated Secondary antibody). blue, and thus these two phosphors may be discriminated on Solid Substrates can be attached to a first binding com the basis of their phosphorescent emissions. Alternatively, ponent which can bind more than one distinct analyte (e.g., two phosphors may produce essentially similar emission may be immunocrossreactive or polyspecific) and/or can be Spectra but may have different excitation wavelengths which attached to multiple first binding component Species which provide a basis for their discrimination in multiple analyte can bind multiple distinct analytes. Similarly, multiple Sec detection. A first binding component (e.g., an antibody) that binds specifically to a first analyte species (e.g., a lympho 15 ond binding component Species with binding Specificities for particular analytes can be employed. When multiple cyte CD4 antigen) and incorporates biotinyl moieties which Second binding component Species are employed, it is typi may be bound by Streptavidin-Phosphor #1 conjugates can be used to quantitatively detect the presence of a first analyte cally

Species desirable to label each Second binding component with a unique up-converting label that can be in a Sample (e.g., a serum sample) by measuring phospho distinguished rescence of Phosphor #1 in analyte-binding component properties. on the basis of its absorption and/or emission complexes. A second binding component (e.g., a probe It is possible to use different absorbers in combination polynucleotide) that binds specifically to a second analyte with

Species (e.g., an HIV-1 sequence) and incorporates digoxy havingvarious Several emitters to produce a collection of phosphors differentiable combinations of excitation and genin moieties (e.g., 11-UTP-digoxygenin) which may be bound by antiligoxigenin-Phosphor #2 conjugates can be 25 emission Spectra. For example but not limitation, Six differ used to quantitatively detect the presence of a Second analyte entiable phosphors may be generated from two absorbers in the Sample by measuring phosphorescence of Phosphor wavelengthemitters.

and three A first absorber, A, has an excitation #2 in analyte-binding component complexes. Thus, by wavelength of 2A, a second of 2A, a first absorber, A has an excitation emitter, E, has an emission line

Simultaneously or contemporaneously detecting the pres at 21, a Second emitter, E2 has an emission ence of multiple phosphor reporters having differentiable a third emitter, Es, has an emission line atline2. at 22, and The Six

Signal characteristics, multiple analytes may be quantita phosphors may be differentiated and the Signal from each tively detected in a Single Sample. individually quantitated by illuminating the Sample with an Sandwich Binding ASSays

Up-converting phosphors labels can be used as reporters excitation wavelength) A and detecting separately the emit for sandwich binding assays (U.S. Pat. No. 4,376,110, which 35 ing the Sampleatwith, 2A),and ted radiation and A, and Separately illuminat is incorporated herein by reference). For example, a mag radiation at 21, 22, and detecting ca. Table Separately the emitted

II shows the Various netic bead, Such as a Superparamagnetic immunobead or absorber:emitter combinations and their excitation and functionalized magnetizable polymer particle (PolySciences, emission wavelengths.

Inc., Warrington, Pa.), can serve as the Solid Substrate which has an immobilized first binding component (e.g., an 40 TABLE II antibody, a polynucleotide, or a lectin) that binds to a first epitope (i.e., a binding locus: an antigenic determinant, Absorber:Emitter Combination Excitation w Emission w Sugar moiety, chemical Substituent, or nucleotide Sequence) A1:E1 WA1 WE1 of an analyte. The analyte binds to the first binding com A1:E2 WA1 AE2 ponent and also to a second binding component (e.g., an 45 A1:E3 WA1 WE3 antibody, a lectin, or a polynucleotide) which binds to a A2:E1 WA2 WE1 Second epitope of the analyte. Thus, the analyte bridges the A2:E2

two binding components to form a Sandwich complex which is immobilized with respect to the Solid substrate. The

Second binding component typically has an attached or 50 Of course, additional absorber:emitter combinations are incorporated label, Such as a biotinyl group which can be possible to provide more than six differentiable phosphor bound to a Streptavidin-coated up-converting phosphor. labels.

Alternatively, the Second binding component can be linked It is also possible to utilize solid substrates of different directly to an up-converting phosphor, Such as through a types which may be distinguished (e.g., by size, color, covalent linkage with a functionalized vitroceramic 55 density, magnetic properties, shape, charge) So that a par up-converting phosphor. ticular type of Solid Substrate is associated with a particular The Sandwich complex comprises the first binding Species of first binding component.

component, an analyte, and the Second binding component, For example and not limitation, the following three brief which is labeled, either directly or indirectly, with an examples are provided to explicate further possible appli up-converting reporter. The Sandwich complex is thus 60 cations of multiple analyte Sandwich assay methods. immobilized on the Solid substrate, although the solid Sub Substrate Differentiation

Strate itself may be mobile (e.g., a Superparamagnetic bead The following example describes the use of distinguish circulating in a sample slurry). The presence and amount of able Substrate types to detect the presence of Specific immu analyte(s) can be quantitatively measured by detecting the noglobulin idiotypes in a sample (e.g., a blood Serum sample presence of up-converting reporter in Sandwich complexes. 65 taken from a patient) which can provide diagnostic infor For example, a Solid Substrate may have a plurality of mation about the immune status of a patient (e.g., is a patient distinct species of first binding component (e.g., an array of Seroreactive with a particular antigen).

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Large Superparamagnetic beads are conjugated to an phosphors provides a measure of the relative abundance of immunogenic Herpesvirus Type II envelope glycoprotein, the APP isoform(s) containing the Y or Z epitopes. medium-sized Superparamagnetic beads are conjugated to Phosphor and Substrate Differentiation

HIV gp120 glycoprotein, and Small Superparamagnetic The following example describes the use of differentiable beads are conjugated to an immunogenic cytomegalovirus up-converting phosphors in conjunction with distinguish envelope glycoprotein. A Serum Sample is taken from a able Substrate types to detect the presence and relative patient and is incubated with a mixture of the Superpara abundance of particular T lymphocyte Subpopulations in a magnetic beads under binding conditions to permit specific blood Sample taken from an individual. Although described binding of immunoglobulins in the Sample with the three here with reference to detecting T cell Subpopulations, immobilized viral glycoprotein Species. The Superparamag analyte multiplexing (i.e., detecting and/or characterizing netic beads are separated from the Sample to remove non multiple analytes in a Sample by using various Solid Sub Specifically bound immunoglobulin and incubated with Strate types and/or up-converting phosphor labels) is up-converting phosphor particles coated with Staphylococ believed to be a generally applicable method. cus aureus Protein A, which binds to IgG, under binding Large Superparamagnetic beads are conjugated to an conditions. Supeiparamagnetic beads having Specifically 15 anti-CD4 antibody, medium-sized Superparamagnetic beads bound IgG are thus labeled with the phosphor-Protein A are conjugated to anti-CD8 antibody, and Small Superpara conjugate. Large, medium, and Small Superparamagnetic magnetic beads are conjugated to an anti-CD28 antibody. An beads are then Separately illuminated with phosphor exci antibody that specifically binds to the CD2 antigen is labeled tation electromagnetic radiation and time-gated emitted with an up-converting phosphor that has an excitation wave phosphorescence is detected. Background attributable to length ), and emits in the red. An antibody that Specifically non-specific binding, if any, is determined and Subtracted binds to the CD45R antigen is labeled with an up-converting using internal standard beads (bovine Serum albumin coated phosphor that has an excitation wavelength ), and emits in Superparamagnetic beads) and positive and negative control the green. An antibody that specifically binds to the CDwó0 Serum Samples. The intensity of phosphorescence associated antigen is labeled with an up-converting phosphor that has with the large, medium, and Small beads provides a measure 25 an excitation wavelength S and emits in the blue. of the amount of antibodies in the Sample which are reactive A blood (or Serum, Sputum, urine, feces, biopsy tissue, with the Herpesvirus Type II envelope glycoprotein, HIV etc.) sample is taken from a patient and is incubated with a gp120 glycoprotein, and cytomegalovirus envelope mixture of the Superparamagnetic beads and phosphor glycoprotein, respectively. This information can be used to labeled antibodies under binding conditions to permit spe determine whether an individual patient has been infected cific binding of cells in the blood sample with the three with the HIV-1, human CMV, and/or Herpes Simplex Type bead-immobilized antibody Species and the three phosphor II viruses. labeled antibody Species. After antigen-antibody binding Phosphor Differentiation occurs, the Superparamagnetic beads are Segregated and The following example describes the use of differentiable examined, either Sequentially or Simultaneously, by illumi up-converting phosphors to detect the presence and relative 35 nation with 2, 2, and ), and quantitative detection of red, abundance of particular isoforms of human APP (amyloid green, and blue emissions, respectively. For example, the precursor protein) in a serum or brain biopsy sample. intensity of -induced red light emission associated with the Various isoforms of APP arise in the brain as a consequence large beads is a rough measure of the amount of cells having of alternative eXon usage and/or alternative proteolytic pro both CD4 and CD2 surface antigens and/or the relative cessing pathways. Thus, although all APP isoforms may 40 abundance of those Surface antigens (e.g., there may be very share a common, hypothetical epitope (X), a particular APP few CD4+ cells that have CD2, but those few cells may have isoform may have a unique epitope (Y), while another APP a large amount of CD2 antigen, and hence a large CD2 isoform has a unique epitope (Z). It is possible that the phosphorescent Signal). Similarly, the intensity of relative abundance of a particular APP isoform in a sample 2-induced green light associated with the large beads is a may be of predictive value or may be pathognomonic for 45 rough measure of the amount of cells having both CD4 and Alzheimer's Disease. CD45R surface antigens and/or the relative abundance of Superparamagnetic beads are conjugated to an antibody those Surface antigens in a Sample.

that binds specifically to a common APP epitope (X) shared In this manner, an analyte Sample, Such as a blood Sample, by all isoforms. A specific antibody reactive with the unique can be "fingerprinted” for the presence and relative Y epitope is labeled with Phosphor #1, which is excited by 50 distribution(s) (e.g., coSegregation and/or correlation) of wavelength), and emits in a wavelength spectrum centered various analyte species. Such an analyte fingerprint may be in the blue. A Specific antibody reactive with the unique Z used for providing diagnostic or therapeutic information, for epitope is labeled with Phosphor #2, which is excited by a example, as to measuring a patient's immune Status or wavelength 2 and emits in a wavelength spectrum centered measuring response to chemotherapy directed against a in the green. A Sample containing APP isoforms is incubated 55 particular blood cell Subset. Similar analyte fingerprints can with the Superparamagnetic beads and labeled Specific anti be used to type pathogenic organisms and viruses, as well as bodies under binding conditions. The Superparamagnetic to order polynucleotide Sequences for gene mapping and/or beads are retrieved from the sample, either individually or in Sequencing. Superparamagnetic beads which can be differ bulk. The beads are illuminated with wavelength) and blue entiated based on size, shape, color, or density can be light emission is detected and measured, and illuminated 60 magnetically trapped individually and Scanned with appro with 2 and green light emission is detected and measured. priate excitation illumination(s) and phosphor emission(s) The intensity of 21-induced blue emission is a measure of characteristic of particular analytes detected. For example, a the APP isoform(s) having the Y epitope, while the intensity unitary detector can Simultaneously or contemporaneously of the 2-induced green emission is a measure of the APP trap the Superparamagnetic bead from a Suspension, deter isoform(s) having the Z epitope. If the emissions from two 65 mine the bead type (size, shape, and/or color), and Scan for phosphors are readily distinguishable, ), and 2 may be presence and abundance of particular phosphors (by illumi identical. The standardized relative intensities of the two nating with excitation wavelength(s) and detecting emitted

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wavelengths). By performing binding assays under dilute a wavelength that produces reactive chemical Species by conditions wherein an average of one analyte or less (e.g., photocatalysis of a compound present in the sample (e.g., a lymphocyte) is bound per microbead, it is possible to type Sample can be doped with buckminsterfullerene). cells individually (e.g., determine the abundance of CD45R Instead of using the emitted radiation directly for photo on each individual CD4" cell) and thus generate more catalytic action on tissue or tumors, an excited form of precise lymphocyte Subpopulation definitions. Biotinylated oxygen, So called Singlet excited oxygen (O2'Ag) can be magnetic beads can also be used to monitor the kinetics of generated by energy transfer from a dye Sensitizer to dis binding Streptavidin to phosphor particles and/or to Segre Solved molecular oxygen. This Scheme makes use of the gate or purify Streptavidin-coated up-converting phosphor tissue penetrating power of near-infrared radiation (red and particles from a reaction. Thus, Streptavidin and ultrared region light, including 970 nm) which reaches the up-converting phosphor particles are mixed in a reaction inorganic up-converting phosphor. Two of the infrared pho vessel under binding conditions for forming Streptavidin tons are converted either into a red, green, or blue photon coated phosphor particles. After a Suitable binding period, depending on the absorption Spectrum of the Sensitizer dye. unbound streptavidin may be removed (e.g., by centrifuga The dye is excited by the up-converted radiation into a triplet tion wherein phosphor particles are collected as the pellet, 15 State which transferS its energy to a dissolved molecular unbound Streptavidin in the Supernatant is decanted, and the oxygen molecule to yield an excited (singlet) oxygren pellet is resuspended), biotinylated magnetic beads are molecule. The cytotoxic activity of Singlet oxygen is well added to the remaining phosphor Suspension in binding documented in photodynamic therapy and other biomedical conditions, and Streptavidin-coated phosphor particles are applications (see, Wagnieres et al. (19–21 Jan. 1990) Future recovered bound to the biotinylated magnetic beads. Directions and Applications of photodynamic Therapy, pp. Photophysical Catalysis by Up-Converting Phosphors 249, SPIE Institutes for Advanced Optical Technologies, Other applications of the invention employ phosphors as Society of Photo-Optical Instrumentation Engineers, Box a photophysical catalyst linked to a probe, where the radia 10, Bellingham, Wash. 98277; Pelegrin et al. (1991) Cancer tion emitted by the phosphor is used, typically in conjunc 67: 2529; Wagnieres et al. (24–25 May 1991) Future Direc tion with a dye molecule, to produce localized intense 25 tions and Applications of Photodynamic Therapy, pp. 219; electromagnetic radiation in an area adjacent to the probe for Folli et al. (Dec. 17, 1991) Fluoresceine Clinique 4; Bra various purposes other than detection (e.g., cytotoxicity, ichotte et al. (May 1991) ENT-Clinic, Lausanne, ionization of chemical species, mutagenesis, etc.). For Switzerland).

example, an antibody that specifically binds to a cell Surface In this application the up-converting phosphor is mixed or antigen, Such as a CD8 antigen on a CD8 lymphocyte, may laced with a Sensitizing dye Such as methylene blue, rose be used as a probe linked to a up-converting phosphor to bengal or phthalocyanine derivatives, Such as localize the phosphor to CD8 lymphocytes. A sample Zn-phthalocyanine. In the first and third case a red-emitting containing CD8 lymphocytes can be incubated with the phosphor is used, whereas for rose bengal a green-emitting anti-CD8 probe-phosphor conjugate and irradiated with an phosphor is best Suited. The phthalocyanine derivatives are excitation wavelength (e.g., from an infrared laser diode), 35 ideally Suited for this purpose because of their total insolu resulting in emission of up-shifted photons (i.e., higher bility in aqueous or biological Solutions. These dyes there frequency electromagnetic radiation) in the vicinity of CD8" fore stay in close proximity to the emitters So that the lymphocytes to which the anti-CD8 probe-phosphor con Specificity of the cell Surface-reporter/probe/dye complex jugate has bound. The emitted radiation may be of a wave becomes the limiting factor. In this case, Specialized com length that is directly mutagenic and/or cytotoxic (e.g., 40 binations of reporter/probe/dye formulations preferably in ultraViolet radiation that can lead to formation of thymine the 0.1 to 0.3-micron Size range must be Synthesized in order dimers, 760–765 nm light is also believed to produce to enable efficient energy transfer: first, up-converted radia chromosomal damage) or may be of a wavelength that can tion is absorbed by the dye as completely as possible; and cause a photolytic decomposition of a chemical present in Second, the dye excited energy (triplet State) is transferred to the environment, leading to local formation of reactive 45 dissolved molecular oxygen. Both processes are very effi Species that may damage adjacent cells (e.g., photodecom cient if the absorption Spectrum of the Sensitizer dye is position of buckminsterfullerene, Co, to Css and C, may matched to the up-converted radiation. This Scheme presents produce free radicals that may cause lipid peroxidation of a step beyond the traditional photodynamic therapy methods cell membranes). in that the red light can be used both for tricking and Since phosphor-emitted radiation is isotropic, it is gener 50 diagnostic as well as for therapeutic purposes after ally desirable to physically separate targets (e.g., CD8 up-converting thus necessitating only one (infrared) light lymphocytes) from non-targets (e.g., CD8 lymphocytes) Source at about 1000 nm. A further advantage is the greater prior to excitation irradiation, So that undesirable damage to range within biological Samples of the infrared radiation non-targets by isotropic emission(s) (i.e., "secondary compared to other known photodynamic therapy excitation damage') is avoided. Physical Separation may be accom 55 schemes (750-850 nm).

plished by various means, including but not limited to: (1) For embodiments employing up-converting phosphors as performing excitation irradiation on a dilute Suspension of photophysical catalysts, it is generally desirable that: (1) the target and non-target cells, wherein the mean distance Sepa wavelength(s) of the excitation radiation do not produce rating individual cells is Sufficient to reduce Secondary Significant photocatalysis of the Substrate compound, (2) the damage to non-targets, and (2) employing hydrodynamic 60 wavelength(s) of the excication radiation are not directly focusing to pass cells (both targets and non-targets) single cytotoxic or mutagenic, and (3) the emitted radiation is file through an illumination Zone (e.g., as in a fluorescence directly cytoloxic and/or is of an appropriate wavelength to activated cell Sorter or the like). Thus, an up-converting produce a biologically effective amount of photodecompo phosphor linked to an anti-DC8" antibody can be used to Sition of a Substrate compound (e.g., buckminsterfullerene, Selectively damage CD8" lymphocytes in a lymphocyte 65 pSoralen, compounds containing azide Substituents or other Sample, where (1) the phosphor emits at a wavelength that photoactivated groups). Alternatively, histidine side chains is either directly cytotoxic and/or (2) the phosphor emits at of polypeptides can be oxidized by light in the presence of

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dye Sensitizers, Such as methylene blue or rose bengal on energy transfer between ions. These processes are (Proteins Structures and Molecular Principies, (1984) described by Auzel (1973) Proc. IEEE 61:758 and Lenth and Creighton (ed.), W. H. Freeman and Company, New York; Macfarlane (March 1992) Optics and Photonic News 3:8. Introduction to Protein Structure, (1991), C. Branden and J. Energy transfer can be efficient in a crystalline host con Tooze, Garland Publishing, New York, N.Y., which are taining many rare earth ions, but not in a Solution where the incorporated herein by reference). Thus, for example, concentration of ions is low and the phonon Structure is leSS up-converting phosphors linked to anti-CD8 antibodies can constrained. In certain cases, these Schemes may not func be used as photophysical catalysts to produce Selective, tion as well for up-conversion in chelates. For example, localized damage to CD8 lymphocytes. In accordance with certain of the Schemes have been demonstrated using crys the invention, essentially any antibody can be linked to an talline host materials at very low temperatures, and may not appropriate up-converting phosphor, either directly or by function as well at room temperature in a chelate. Schemes conjugation to protein A which may then bind the immu that do not involve intermediate relaxation Such as that of noglobulin. Thus, the up-converting photophysical catalysts Smart et al., have advantages in chelates because they can be of the invention may be used to target essentially any desired excited more effectively with pulsed Sources. Higher peak antigen or cell type that can be distinguished by the presence 15 powers can be obtained from diode lasers when they are of an identified antigen. operated in a pulsed mode. The higher peak powers lead to Up-converting Chelates more efficient up-conversion due to the nonlinear depen Certain applications require Small reporters. For example, dence on excitation power.

the transport, ability to Stay in Suspension, the bonding Up-Converting Organic Dyes dynamics, and the tendency toward removal by microphages Similar to the up-converting inorganic phosphor reporters may be improved for smaller reporters. However, the We propose to use "molecular labels whose fluorescence reduced Sensitivity available with Smaller reporters must will be detected by optoelectronic means. Infrared or red also be considered. One type of Small up-converting inor light is exciting the probe-reporter complex bound to a ganic phosphor consists of rare earth ions in chelates. The target, after which light is emitted at Shorter wavelengths use of lanthanide chelates as reporters has been developed 25 with respect to the illuminating Source. This up-converted for biological assays as described above. This prior use of light is free of Scattered light from the Source or autofluo lanthanide chelates involved dowVn-conversion. That is, the rescence by Virtue of its higher energy. Furthermore, autof emission light is at a wavelength which is longer than the luorescence is greatly reduced by Virtue of the excitation in excitation wavelength. Rare earth chelates may be used as the infrared or red Spectral range. The light Source is a pump up-converting reporters through Stepwise excitation Such as laser whose pump pulses are short in order to achieve high shown in FIG.5a, or in FIG.5b (except that all levels would powers and low energy in order to enable non-linear optical be in the same ion). Energy transfer from a Sensitizer ion to processes in the dye. The goal is to excite the Second excited an activator ion cannot be used in the case of a Single rare Singlet state (S) in a dye with a ps pulse from a tunable dye earth ion. Chelates Suitable for use as up-converting phos laser using two red or infrared photons. After pumping the phors include ethylenediaminetetraacetic acid (EDTA), dipi 35 S. State the dye relaxes within a few ps to the fluorescing colinic acid (DPA), diethylenetriaminetetraacetic acid State (S) which can be detected by optoelectronic means. (DTTA), diethylenetriaminepentaacetic acid (DTPA), tet The goal of reaching the S. Slate using two photons enables raazacyclotetradecanetetraacetic acid (TETA), as well as one to take advantage of the increasing two-photon croSS antibiotics, natural chelating proteins, phthalocyanines, and Sections as one approaches the S2 State using two-photon cryptates. Methods for preparation of lanthanide chelates 40 absorption. The non-resonant two-photon absorption croSS and their use in biological assays are described in the sections are on the order of 10' to 10 cm's, whereas the literature (Mukkala et al. (1989) Anal. Biochem. 176: 319, cross Sections corresponding to S absorption are larger by Hemrnila et al. (1984)Anal. Biochem. 137: 335, Soini and two to three orders of magnitude. A few specific examples Kojola (1983) Clin. Chem. 29: 65, Nonisotopic DNA Probe will be mentioned: in general cyanines, Xanthenes, Techniques (1992) Kricka (Ed.) Academic Press, New York, 45 rhodamines, acridines and oxazines are well Suited for this as well as the references on page 6 of this application). purpose. Blue dyes can also be used, but the excitation Up-conversion phosphor reporters can also consist of rare wavelength will be in the red. Rhodamine can be excited at earth ions inside cage compounds Such as fullerene materials 650 to 700 nm using two photons, and fluorescence is following the procedures described by Bethune et al. (1993) expected around 555 nm. Many IR dyes such as IR-140, Nature 366: 123 and references therein. 50 IR-132 and IR-125 can be excited at 1060 nm using two Suitable ions for up-conversion in chelates include photons of the Nd:YAG fundamental, and fluorescence is erbium, neodyrnium, thulium, holnium, and praseodymium. expected in the 850 to 950 nm range. An example of a blue Other candidate ions include the other lanthanide elements, dye is BBQ excited at 480 nm to reach the S. state at 240 the actinide elements, and other metal elements. Stepwise nm, and fluorescence is expected at 390 nm. Many of these excitation Schemes Suitable for up-conversion in lanthanide 55 dyes are only Slightly Soluble in aqueous Solution and are chelates are described in the literature on up-conversion either polar in nature (cyanines) or have polar Subslituents. lasers. Examples include up-conversion in erbium Depending on the nature of the probe, no or only minimal (Silversmith et al. (1986) J. Opt. Soc. Am. A3:128, and attachment chemistry needs to be undertaken because of the Macfarlane et al. (1989) Appl. Phys Lett. 54:2301), neody abundance of functional groups on the dye chromophore. mium (Macfarlane et al. (1988) Appl. Phsy. Lett. 52:1300), 60 Several companies Sell entire lines of dyes: examples are thulium (Nguyen et al. (1989) Appl. Opt. 28:3553 and Allain KODAK, Exciton and Lambda Physik. The scientific foun et al. (1990a) Electron. Lett. 226:166), holmium (Allain et dations of two-photon laser excitation in organic dye mol al. (1990b) Electron. Lett. 26:261), and praseodymium ecules have been treated in a few experimental papers: A. (Smart et al. (1991) Electron. Lett. 27: 1307). Other Penzkofer and W. Leupacher, Optical and Quantum Elecir up-conversion laser Schemes that rely on energy transfer, 65 onics 19 (1987), 327–349; C. H. Chen and M. P. McCann, energy pooling, croSS relaxation, or avalanche absorption are Optics Commun. 63 (1987), 335; J. P. Hermann and J. not appropriate for up-converting chelates because they rely Duculing, Optics Commun. 6 (1972), 101; B. Foucault and J.

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P. Hermann, Optics Commun. 15 (1975), 412; Shichun Li tiple illumination beams are multiplexed (e.g., a pulsed and C. Y. She, Optica Acta 29 (1982), 281–287; D. J. beam is multiplexed with a CW beam), permitting signal Bradley, M. H. R. Hutchinson and H. Koetser, Proc. R. Soc. discrimination between phosphorescence induced by the Lond. A 329 (1972), 105–119. CW Source and phosphorescence induced by the pulsed Resonant Multiphoton Ionization Source, thus allowing the discrimination of multiple phos At very high laser intensities the up-converting organic phor Species having Similar emission Spectra but different dyes are induced to absorb an additional exciting photon in excitation Spectra. For example but not limitation, commer the field of focussed laser radiation. At those high laser cially available gallium arsenide laser diodes can be used as intensities the Fluorescence is Suppressed in favor of absorp an illumination Source for providing near-infrared light. tion of an additional photon. This process usually brings the The ability to use infrared excitation for Stimulating organic dye molecules above the ionization limit in Solution up-converting phosphors provides Several advantages. First, and they stabilize by emitting an electron into the Solvent inexpensive IR and near-IR diode lasers can be used for shell. The result of this three-photon interaction is a molecu Sustained high-intensity excitation illumination, particularly lar ion and an attached or Solvated electron. When this in IR wavelength bands which are not absorbed by water. charge Separation is taking place in an electric field, the 15 This level of high-intensity illumination would not be suit charges drift and generate a Voltage that can be detected in able for use with conventional labels, Such as ordinary an extremely Sensitive manner. This amounts to the mea fluorescent dyes (e.g., FITC), since high-intensity UV or Surement of the transient conductivity in the Solvent System Visible radiation produces extensive photobleaching of the and is usually more Sensitive than light detection. The label and, potentially, damage to the Sample. The ability to disadvantage of this method is that it necessitates electrodes use higher illumination intensities without photobleaching that Sense the moving charges. In that Sense it is not as or Sample damage translates into larger potential Signals, and non-invasive a method as light detection. On the other hand hence more Sensitive assayS.

it bypasses the conversion of light into a photoelectric Signal The compatibility of up-converting labels with the use of which represents an enormous advantage. Every optical diode lasers as illumination Sources provide other distinct System has a restricted Viewing angle that reduces efficiency, 25 advantages over lamp Sources and most other laser Sources. whereas photoionization “senses” always close to 100% of First, diode laser intensity can be modulated directly through the charges generated. Effectively, the non-linear interaction modulation of the drive current. This allows modulation of of the laser field converts every excited organic dye mol the light for time-gated or phase-Sensitive detection ecule into an electric pulse at Sufficiently high field inten techniques, which afford Sensitivity enhancement without Sities that can be routinely achieved using commercial laser the use of an additional modulator. Modulators require Sources. Specific examples are the excitation of Rhodamine high-voltage circuitry and expensive crystals, adding both around 650 to 700 nm, or BBQ excitation around 480 rm. cost and additional size to apparatus. The laser diode or Organic dyes absorbing in the red have to absorb two light-emitting diode may be pulsed through direct current additional photons after being excited into S2 thus making modulation. Second, laser illumination Sources provide illu the whole process a four-photon excitation process, which is 35 mination that is exceptionally monochromatic and can be slower than a three-photon non-linear process. There may, tightly focused on very Small Spot sizes, which provides however, be circumstances where Such a four-photon pro advantages in Signal-to-noise ratio and Sensitivity due to ceSS is desirable. reduced background light outside of the desired excitation Detection Apparatus Spectral region and illuminated Volume. A diode laser affords Detection and quantitation of inorganic up-converting 40 these Significant advantages without the additional expense phosphor(s) is generally accomplished by: (1) illuminating a and size of other conventional or laser Sources. Sample Suspected of containing up-converting phosphors Detection and quantitation of phosphorescent radiation with electromagnetic radiation at an excitation wavelength, from excited up-converting phosphors can be accomplished and (2) detecting phosphorescent radiation at one or more by a variety of means. Various means of detecting phospho emission wavelength band(s). Illumination of the sample is 45 rescent emission(s) can be employed, including but not produced by exposing the Sample to electromagnetic radia limited to: photomultiplier devices, avalanche photodiode, tion produced by at least one excitation Source. Various charge-coupled devices (CCD), CID devices, photographic excitation Sources may be used, including infrared laser film emulsion, photochemical reactions yielding detectable diodes and incandescent filaments, as well as other Suitable products, and Visual observation (e.g., fluorescent light Sources. Optical filters which have high transmissibility in 50 microscopy). If the reporters are organic dyes, resonant the excitation wavelength range(s) and low transmissibility multiphoton ionization can be Sensed using electroStatic in one or more undesirable wavelength band(s) can be position-Sensitive detectors. Detection can employ time employed to filter out undesirable wavelengths from the gated and/or frequency-gated light collection for rejection of Source illumination. Undesirable wavelength ranges gener residual background noise. Time-gated detection is gener ally include those wavelengths that produce detectable 55 ally desirable, as it provides a method for recording long sample autofluoresence and/or are within about 25-100 nm lived emission(s) after termination of illumination; thus, of excitation maxima wavelengths and thus are potential Signal(s) attributable to phosphorescence or delayed fluo Sources of background noise from Scattered excitation illu rescence of up-converting phosphor is recorded, while short mination. Excitation illumination may also be multiplexed lived autofluoresence and Scattered illumination light, if any, and/or collimated; for example, beams of various discrete 60 is rejected. Time-gated detection can be produced either by frequencies from multiple coherent Sources (e.g., lasers) can Specified periodic mechanical blocking by a rotating blade be cellimated and multiplexed using an array of dichroic (i.e., mechanical chopper) or through electronic means mirrors. In this way, Samples containing multiple phosphor wherein prompt signals (i.e., occurring within about 0.1 to Species having different excitation wavelength bands can be 0.3 us of termination of illumination) are rejected (e.g., an illuminated at their excitation frequencies Simultaneously. 65 electronic-controlled, Solid-state optical shutter Such as Illumination may be continuous or pulsed, or may combine Pockel's or Kerr cells). Up-converting phosphors and continuous wave (CW) and pulsed illumination where mul up-converting delayed fluorescent dyes typically have emis

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Sion lifetimes of approximately a few milliseconds (perhaps There may be a plurality of reporters having distinct as much as 10 ms, but typically on the order of 1 ms), emission bands but a common excitation band. In Such a whereas background noise usually decays within about 100 case, the System would include multiple detectors for a nTS. Therefore, when using a pulsed excitation Source, it is Single laser diode. Similarly, there may be a plurality of generally desirable to use time-gated detection to reject reporters having distinct excitation bands but a common prompt signals. emission band. In Such a case, the System would include Since up-converting phosphors are not Subject to multiple laser diodes for a Single detector, and would use photobleaching, very weak emitted phosphor Signals can be time multiplexing techniqueS or the like to Separate the collected and integrated over very long detection times wavelengths.

(continuous illumination or multiple pulsed illumination) to Light from the two Sources is shown as being combined increase Sensitivity of detection. Such time integration can So as to be focused at a Single location by a common be electronic or chemical (e.g., photographic film). When focusing mechanism. This is not necessary, even if it is non-infrared photographic film is used as a means for desired to illuminate the same region of the Sample. detecting weak emitted Signals, up-converting reporters pro Similarly, the collection need not be via a single collection vide the advantage as compared to down-converting phoS 15 mechanism. If it is necessary to preserve all the light, the phors that the excitation Source(s) typically provide illumi combination and Separation elements can include a wave nation in a wavelength range (e.g., infrared and near length division multiplexer and a demultiplexer using dich infrared) that does not produce significant exposure of the roic filters. If loss can be tolerated, 50% beam splitters and film (i.e., is similar to a darkroom safelight). Thus, filters can be used.

up-converting phosphors can be used as convenient ultra The Schematic shows the light passing through the Sample Sensitive labels for immunohistochemical Staining and/or in and being detected in line. As a general matter, the emission Situ hybridization in conjunction with fluorescence microS from the phosphor reporters is generally isotropic, and it copy using an infrared Source (e.g., a infrared laser diode) may be preferred to collect light at an angle from the and photographic film (e.g., Kodak Ektachrome) for signal direction of the incident light to avoid background from the and image detection of visible range luminescence (with or 25 excitation Source. However, Since the excitation and the without an infrared-blocking filter). emission bands are widely separated, Such background is Instrumentation Overview unlikely to be an issue in most cases. Rather, other consid The basic purpose of the instrumentation is to expose the erations may dictate other geometries. For example, it may up-converting phosphor particles of an assay Sample to be desired to detect light traveling back along the path of the near-infrared (NIR) light and to measure the amount of incident radiation So that certain elements in the optical train visible light that is emitted. are shared between the excitation and the detection paths. FIG. 1 is an optical and electronic block diagram illus A typical type of instrument with shared elements is a trating representative apparatus 10 for performing diagnos microscope where the objective is used to focus the excita tics on a Sample 15 according to the present invention., The tion radiation on the Sample and collect the emitted radia invention may be carried out with one or a plurality of 35 tion. A potentially advantageous variation on Such a con reporters. For purposes of illustration, the apparatus shows figuration makes use of the phenomenon of optical trapping. a System wherein two diagnostics are performed on a single In a situation where the reporter is bound to a Small bead, it Sample in which two phosphor reporters are used. The first may be possible to trap the bead in the region near the beam reporter has an excitation band centered at 2 and an focus. The same Source, or a different Source, can be used to emission band centered at 2, while the Second reporter has 40 excite the reporter. The use of an infrared diode laser to trap respective excitation and emission bands centered at 2 and Small particles is described in Sato et al., “Optical trapping 2. Since the reporters of the present invention rely on of Small particles using a 1.3 um compact InGaAsP laser,” multiphoton excitation, wavelengths 2 and 2 are longer Optics Letters, Vol. 16, No. 5 (Mar. 1, 1991), incorporated than wavelengths), and 2. The former are typically in the herein by reference.

near infrared and the latter in the visible. 45 Specific Detection Techniques

A pair of light sources 2001) and 20(2), which may be AS outlined above, multichannel detection uses optical laser diodes or light-emitting diodes (LEDs), provide light at devices Such as filters or dichroic beam. Splitters where the the desired excitation wavelengths, while respective detec emission bands of the phosphor reporters are Sufficiently tors 22(1) and 22(2), which may be photodiodes, detect light Separated. Similarly, it was pointed out that multiple report at the desired emission wavelengths. The emitted radiation 50 erS having a common emission band could be detected using is related to the incident flux by a power law, So efficiency electronic techniques. These electronic techniques will be can be maximized by having the incident beam Sharply described below in conneticon with multiple Sources. focused on the Sample. To this end, light from the two However, the techniques will be first described in the context Sources is combined to a Single path by a Suitable combi of a single channel. The techniques are useful in this context nation element 25, is focused to a Small region by a lens or 55 Since there are Sources of background that are in the same other focusing mechanism 27, and encounters the sample. wavelength range as the Signal Sought to be measured. Light emitted by the phosphor reporters is collected by a lens FIG. 2A shows an apparatus for implementing phase 30, and components in the two emission bands are separated Sensitive detection in the context of a Single channel. Cor by a Suitable Separation element 32 and directed to the responding reference numerals are used for elements corre respective detectors. 60 sponding to those in earlier described figures. In this context, There are a number of possible regimes for driving the control electronics 35 comprises a waveform generator 37 laser diodes and detecting the emitted light in the different and a frequency mixer 40. Waveform generator 37 drives wavelength bands. This is shown generically as a control laser diode 20(1) at a frequency f, and provides a signal at electronics block 35 communicating with the laser diodes f to the frequency mixer. The frequency mixer also receives and detectors. The particular timing and other characteristics 65 the Signal from detector 22(1) and a phase control input of the control electronics will be described below in con Signal. This circuitry provides additional background dis nection with Specific embodiments. crimination because the background has a much shorter

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lifetime than the Signal Sought to be measured (nanoseconds ion (the erbium ion in the example outlined above) without or microSeconds compared to milliseconds). This causes the using energy transfer from another absorbing ion (the ytter Signal and background to have different phases (although bium ion in the example) while taking advantage of resonant they are both modulated at the characteristic frequency of enhancement of intermediate levels. Additionally, the use of the waveform generator). For a discussion of the lifetime different wavelengths for a single reporter can provide dependent phase shift, See Demtroder, Laser SpectroScopy, additional options for excitation-dependent multiplexing Springer-Verlag, New York, 1988, fop. 557-559, incorpo and background discrimination techniques. rated herein by reference). The phase input signal is con Multiple wavelength excitation of a Single phosphor may trolled to maximize the Signal and discriminate against the occur in a number of ways, as shown in FIGS. 5A through 5C. Two laserS may cause Stepwise excitation of a single ion, background. This background discrimination differs from as shown in FIG. 5A. A first laser stimulates excitation from that typical for phase Sensitive detection where the Signal is level 1 to level 2, and a Second laser Stimulates excitation modulated and the background is not. Discrimination from level 2 to level 3, at which level emission occurs. against unmodulated background is also beneficial here, Single ion excitation can also occur using energy transfer as leading to two types of discrimination. shown in FIG. 5B. In this case, a first laser stimulates Because the Signal relies on two-photon excitation, it is 15 excitation from level 1 to level 2, energy transfer occurs possible to use two modulated laser diodes and to detect the from level 2 to level 3, and a second laser stimulates Signal at the Sum or difference of the modulation frequen excitation from level 3 to level 4. In a variation of the latter cies. FIG. 2B ShowS Such an arrangement where first and process, levels 1 and 2 can be in a first ion (i.e., a Sensitizer second laser diodes 2001) and 20(1)" (emitting at the same ion) and levels 3 and 4 in a Second ion (i.e., activator ion) wavelength), or possibly different wavelengths) are modu as shown in FIG. 5C.

lated by signals from waveform generators 37a and 37b In a stepwise excitation Scheme shown in FIG. 5A, energy operating at respective frequencies f and f. The waveform transfer is not required, and thus information on the polar generator output signals are communicated to a first fre ization of the excitation laserS may be preserved and cause quency mixer 42, and a Signal at fit?, is communicated to polarization of the emitted radiation. In this case, depolar a second frequency mixer 45. The signal from detector 22(1) 25 ization of the light may allow for enhanced discrimination and a phase input Signal are also communicated to frequency between Signal and background noise. mixer 45. For the multi-ion multi-laser excitation Scheme shown in FIG. 3 ShowS apparatus for performing gated detection. FIG. 5C, there may be several phosphors that share a Since the background is shorter-lived than the Signal, delay common excitation wavelength. In this case, discrimination ing the detection allows improved discrimination. To this between different phosphors may be performed on the basis end, the laser diode is driven by a pulse generator 50, a of different emission wavelengths and/or through time delayed output of which is used to enable a gated integrator gated, frequency-modulated, and/or phase-Sensitive detec or other gated analyzer 55. tion utilizing modulation of the excitation wavelength(s). FIG. 4 shows an apparatus for performing diagnostics on Specific Instrument Embodiments a Sample using first and Second, reporters having excitation 35 FIG. 6 is a Schematic view Snowing the optical train of a bands centered at 2 and A, and having overlapping emis particular embodiment of apparatus for carrying out the Sion bands near 2. The Sample is irradiated by light from present invention on a Sample using a hand-held probe. This laser diodes 2001) and 20(2) as discussed above in connec embodiment takes the form of a miniaturized instrument tion with FIG. 1. First and second waveform generators comprising a housing 75 (shown in phantom), a hand-held 37(1) and 37(2) drive the laser diodes at respective frequen 40 probe 80, with a fiber optic connecting cable 82. The optical cies f and f, and further provide signals at f and f to and electronics components are located within the housing. respective frequency mixers 60(1) and 60(2). The signal For purposes of illustration, the optical components of a from detector 22(3) is communicated to both frequency 3-channel System are shown. The Sample may contain up to mixers, which also receive respective phase input signals. three reporters having distinct emission bands, for example, Thus, frequency mixer 60(1) provides an output signal 45 in the blue, green, and red portions of the visible Spectrum. corresponding to the amount of emitted light modulated at It is also assumed that the reporters have distinct excitation frequency f, which provides a measure of the presence of bands in the near infrared.

the first reporter in the Sample. Similarly, frequency mixer The output beams from three laser diodes 85a-c are 60(2) provides an output signal corresponding to the amount communicated through graded index (GRIN) lenses 87a-c, of emitted light modulated at frequency f, which provides 50 focused onto the ends of respective fiber segments 88a-c a measure of the presence of the Second reporter in the and coupled into a single fiber 90 by a directional coupler 92 Sample. or other Suitable device. The light emerging from the end of The use of two different wavelengths was discussed above fiber 90 is collimated by a GRIN lens 95, passes through a in the context of two reporters having different excitation dichroic beam splitter 97, and is refocused by a GRIN lens bands. However, the discussion is germane to a single 55 100 onto the end of fiber optic cable 82. The beam splitter reporter situation as well. Since the excitation is a two is assumed to pass the infrared radiation from the laser photon process, there is no requirement that the two photons diodes but reflect visible light.

have the same energy. Rather, it is only necessary that the Hand-held probe 80 includes a handpiece 102, an internal total energy of the two photons fall within the excitation GRIN lens 105, and a frustoconical alignment tip 110. The band. Thus, Since it is relatively Straightforward and inex 60 light emerging from fiber 82 is focused by GRIN lens 105 pensive to provide different wavelengths with laser diodes, at a focus point 115 that is slightly beyond alignment tip 110. there are more possible combinations, i.e., more possible The alignment tip is brought into proximity with the test tube choices of total excitation energy. This allows more latitude holding the Sample So that focus point 115 is in the Sample. in the choice of rare earth ions for up-converters Since the It is assumed that the test tube is transmissive to the laser excitation Steps need not rely on energy transfer coinci 65 radiation.

dences involving a single photon energy. Further, it may be A portion of the light emanating from the region of focus possible to achieve direct Stepwise excitation of the emitting point 115 in the sample is collected by GRIN lens 105,

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focused into fiber 82, collimated by GRIN lens 100, and respective detector elements. This arrangement facilitates reflected at dichroic beam splitter 97. This light may contain the use of filters to the extent that other techniques for wavelengths in up to the three emission bands. rejecting the excitation radiation are not used. Optical filters 120a-c direct the particular components to Optical trapping may be used to transiently immobilize a respective photodetectors 125a–c. Aparticular filter arrange Sample particle for determination of the presence or absence ment is shown where each filter reflects light in a respective of phosphor on the particle. Conveniently, the wavelength emission band, but other arrangements would be used if, for range used to trap Sample particles may be essentially example, one or more of the filters were bandpass filters for identical to an excitation wavelength range for the the emission bands. up-converting phosphor(s) Selected, so that optical trapping The control electronics are not shown, but could incor and excitation illumination is performed with the same porate the time-multiplexed or heterodyne techniques dis Source. FIG. 3 shows a block diagram of an apparatus used cussed above. Such techniques would be necessary, for for Single-beam gradient force trapping of Small particles. example, if the emission bands were not distinct. FIG. 26 is a block diagram of one embodiment of appa FIG. 7A is a schematic of an embodiment of the invention ratus for carrying out the present invention on a Sample in which a charge coupled device (CCD) imaging array 150 15 using a microScope. In this embodiment a Standard micro is used as a detector in combination with a two dimensional Scope is modified to accept infrared Scanning optics and array 152 of peptides or other biologically active Species image processing electronics. A Suitable microScope for deposited on a glass or plastic Substrate. modification is the Zeiss model CLSM-10. The CCD array has a number of individually addressable The microscope is fitted with a HeNe laser A1 for visible photosensitive detector elements 155 with an overlying imaging and an argon laser A2 for both visible and UV passivation layer 157 while the peptide array has a number imaging. Both lasers are mounted internally and are indi of individual binding sites 160. The probe containing the vidually Selectable through a Series of motorized shutters phosphor would be reaction Specific to one or more of the A3. The upconverting phosphors are excited with an exter elements in this peptide array and would therefore become nally mounted IR laser diode. In the preferred embodiment, physically attached to those elements and only those ele 25 two IR laser diodes A4 and A5, operating at two different IR ments. The peptide array is shown as having a one-to-one wavelengths, are coupled to the microScope thereby allow geometric relation to the imaging array in which one pixel ing multiple phosphor reporters to be identified. Laser corresponds to each element in the peptide array. However, diodes A4 and A5 are individually selectable using motor it is also possible to have larger peptide elements that cover ized shutters A6. When an IR beam is selected, it is routed a group of detector elements should Such be necessary. through the microscope's galvanometrically controlled Various of the techniques described above can be used to Scanning mirrorS A7 which Scan the beam in a raster fashion. enable the detector array to distinguish the emissions of the The beam passes through the objective lens (not shown) phosphor from the infrared laser Stimulation. First, it is onto a sample A8 and is reflected back through the objective possible to use a phosphor that responds to IR Stimulation lens to a Set of galvanometrically controlled receiving mir beyond the Sensitivity range of the detector array. An 35 rors A7. Receiving mirrors A7 reflect the light onto pinhole example of Such a phosphor would be Gadolinium oxySul optics A9. If the confocal mode is selected, pinhole A9 limits fide: 10% Erbium. This phosphor is stimulated by 1.5- the detected image to the light collected from the focal micron radiation and emits at 960 nm and 520 nm. The plane. The light is imaged on a photomultiplier tube (PMT) detector array is insensitive to 1.5-micron radiation but is A10. The thickness of the focal plane is proportional to the Sensitive to the up-converted radiation. 40 Size of the pinhole. The Scanning Speed is chosen Such that Further, Since the phosphor emission is relatively slow in sufficient signal intensity is received at the PMT A10. In the rise and fall time it could be time resolved from a pulsed preferred embodiment of this apparatus, a 20 micrometer laser stimulation source by the CCD detector array. The diameter pinhole is used which results in a depth of field of decay time for the up conversion proceSS is a variable about 1 micrometer. If the confocal mode is not Selected, the dependent on the particular emitting transition and the 45 beam is deflected around pinhole optics A9 directly to PMT phosphor host; however, it is normally in the range 500 lis A10.

seconds to 10 ms. This is very slow compared to the laser Once the optical Signal is converted into an electronic one, excitation pulse and the capability of the detector array. it a Standard, composite Video signal can be developed and The techniques for fabricating the CCD array are well displayed as an image on a television monitor A11. The known Since CCD imaging arrays have been commercially 50 image can be manipulated and enhanced through Standard available for many years. A variety of Such devices can be image processing Software. in the preferred embodiment of obtained from David Sarnoff Research Center, Princeton, this apparatus the Software runs on an IBM 486 PC A12. The N.J. Software can be used to perform averaging, filtering, edge The techniques for fabricating the peptide array are detection and overlaying the imageS received from each of described in a paper by Fodor et al., “Light-Directed, 55 the different light Sources.

Spatially Addressable Parallel Chemical Synthesis,” In the confocal mode, it is possible to reconstruct a 3 Science, Vol. 251, pp. 767-773 (Feb. 15, 1991), incorporated dimensional view of sample A8. The reconstruction is herein by reference. The particular array discrete contains formed by Stepping through Sample A8 at Small intervals, 1024 discrete elements in a 1.28 cmx1.28 cm area. making an image of the Sample at each interval. The multiple The embodiment of FIG. 7A shows the peptide array in 60 Sequential images are transferred to an external graphics intimate contact with the CCD array. Indeed it may be machine (not shown) for reconstruction of the sample in 3 possible to deposit the peptides directly on the passivation dimensions. These 3-D images can then be rotated to give layer without a separate Substrate. However, there may be different perspectives of the data Sets, leading to a better Situations where spatially Separated arrays are preferred understanding of the Samples.

FIG. 7B shows an embodiment where the peptide array and 65 FIG. 27 is a block diagram of a microtiter plate reader for the CCD array are separated. An array of lenses 165 collect use with the present invention. Within a light-tight test the light from respective binding sites and focus it on chamber B1 is a near IR laser excitation Source B2, a

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photomultiplier tube (PMT) detector B3, and a sample assay In use, wick D2 wicks up a portion of a sample fluid D8 plate B4. In the preferred embodiment of this apparatus, which is Suspected of containing the target antigens. Target assay plate B4 is a Terasaki HLA plate. This plate is antigens bind to the antibodies present at a capture Surface preferred due to its Small tapered Sample wells which tend D9. Capture surface D9 is positioned at the focal point of to concentrate the Sample material into a relatively Small Source D3. The target antigens can be labeled with target area. The target area in this configuration is still larger phosphor-antibody conjugates either before or after capture. than the diameter of the laser beam. Furthermore, it is In the preferred embodiment wick D2 is formed of glass. In possible that the distribution of the assay material acroSS the this configuration capture Surface D9 is prepared Simply by bottom of the well is not even. Because of these two factors, filling the inside of the capillary with a bubble containing the Simply aiming the laser at the center of the bottom well antibodies of interest. By Silanizing the inner Surface with Surface is unlikely to provide accurate readings. There are organofunctional Silanes, conventional chemistries can be Several approaches that can be used to circumvent this used to covalently link the antibodies or other biological problem. The first approach is to defocus the laser beam

Sufficiently to allow a larger amount of the target area to be macromolecule(s) to the inner tube wall at the site of the interrogated. However, depending upon the output of laser liquid bubble. The Surface energy of the capillary is also B2, defocussing the beam may lower the sensitivity of the 15 easy to modify by Silanization, which will help prevent apparatus to an unacceptable level. Another approach is to nonspecific reagent and antigen adherence to the walls of the raster Scan the laser beam across the bottom of target well. tube.

A third and preferred approach is to Simply automate the In the preferred embodiment of this apparatus, the lower Scanning and data collection System. portion of wick D2 is impregnated with upconverting phos Light from laser B2 passes through a filter B5 and is phors that are conjugated to the target analytes or a croSS focussed by a lens B6 onto an individual sample well of reactive epitope for the capture probe. In use, the phosphor assay plate B4. Plate B4 is mounted on a pair of translators conjugates chromatograph towards capture Surface D9 as B7 which allow positioning in the horizontal and vertical sample fluid D8 is drawn up wick D2. As phosphors directions. In the present configuration translators B7 allow accumulate at capture Surface D9, they will begin to emit approximately 2.5 centimeters of travel; Sufficient to address 25 visible light upon excitation by diode laser D3. The visible 3 sample wells in each direction. Translators B7 are con light emitted by the phosphors is detected by detector D5. trolled by an x-y controller B8. Controller B8 allows for The output of detector D5 is displayed on display D7. The either manual or computerized control. amount of upconverted light reaching the detector is directly A Sample well on plate B4, when containing upconverting proportional to the concentration of labeled target antigen phosphors, will emit visible light which is collected by a lens captured at the capture Surface.

B9, passed through a filter B10, and focussed through a lens The apparatus of FIG. 29 can be designed to simulta B11 and a shutter B12 onto PMT B3. PMT B3 outputs a neously detect more than one target antigen. FIG. 30 illus current which is measured by a picoammeter B13. The PMT trates a three channel configuration using interference filters. Signal is proportional to the phosphor emission intensity. In this configuration capillary wick D2 is placed at the focus Shutter B12, controlled by a shutter driver B14, provides 35 of a small parabolic reflector D10 capable of collecting exposure protection to PMTB3, thereby preventing damage approximately half of the emitted phosphorescence. The which may result from exposure to very intense light beam from diode laser D3 is directed onto capillary wick D2 Sources. Furthermore, overexposure of PMT B3 to light at capture Surface D9 along a direction perpendicular to the causes high dark currents which require Several hours to optical axis of reflector D10. Phosphorescent light from decrease. PMT B3 is cooled for lower dark current and 40 capture surface D9 is collected and collimated by mirror noise. ASSociated with the PMT cooler is a water-cooled D10, directed through a notch filter D11 to reject the pump power supply B15. A power Supply B16 supplies high light, and onto three detectors D5 using three dichroic beam voltage to PMT B3. splitters D12. The reflectance bands of dichroic beamsplit When the apparatus is operated in a computerized mode, ters D12 are matched to the emission bands of the three a computer B17 regulates controller B8 through an interface 45 phosphors used in the detection process. box B18. Picoammeter B13 can also be connected to com In an alternate embodiment of this apparatus, dichroic puter B17, thereby allowing automated data acquisition to beamsplitters D12 could be replaced with three bandpass be performed. The data acquisition procedure moves trans filters used in the transmission mode. By placing the three lator B7 in the X direction to a first position at which location filters on a rotation wheel, a single detector D5 could be a specified number of current readings are taken and the 50 used. Another alternative is to use a diffraction grating and average is calculated. Translator B7 then movres sample B4 a linear detector array to obtain an actual emission Spectrum. a predetermined distance in the X direction to a new location FIG. 31A is an illustration of an embodiment of the where new data is collected. During this process, the data is invention in which a diode laser array F1 and a detector plotted in order to provide the user with an immediate Visual array F2 are combined in a Single device. In the preferred evaluation. After the Scan is completed, the data can be 55 embodiment, arrays F1 and F2 are fabricated on a pair of saved or further data processing can be performed. FIG. 28 Silicon chips F3 with array dimensions of approximately 1 is an illustration of the data for upconverting phosphors in square centimeter. FIG. 31B is a detailed view of a small three test wells. section of the device shown in FIG. 31A. Overlaying FIG. 29 is a schematic view of a second embodiment of detector array F2 is a polymer film F4 of approximately 10 a hand-held probe for carrying out the present invention. 60 to 25 micrometers thickneSS which is used as the capture This embodiment is comprised of a housing Dl and a surface. Arrays F1 and F2 are separated by a spacer F5. capillary wick D2. Within housing Dl is a diode excitation Array F1 is comprised of Fabrey-Perot diode lasers, pref laser D3, a lens assembly D4, a photodiode detector D5, ad erably tuned to 980 nanometers. Lasers of this type are a battery supply D6. A display D7 mounted to one surface easily fabricated in gridded array patterns using conven of housing Dl communicates the results of the test to the 65 tional photolithography techniques. Each individual laser in user. In the preferred embodiment, laser D3 operates in the array F1 has a columnar beam designed to Strike only the 960-980 nanometer range. adjacent portion of capture Surface F4. The required power

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density of the individual laserS is dependent upon the a plurality of Sample collection regions on the basis of the efficiencies of the phosphors being used as well as the Signal(s) detected. A general description of FACS apparatus required detection efficiency. The detectors comprising array and methods in provided in U.S. Pat. Nos. 4,172,227; F2 are chosen to have an extremely low sensitivity in the 4,347,935; 4,661,913; 4,667,830; 5,093,234, 5,094,940; and wavelength region in which laser array F1 operates. If 5,144.224, incorporated herein by reference. It is preferred additional discrimination between the excitation and emis that up-converting phosphors used as labels for FACS meth Sion wavelengths is required, a cutoff filter can be used, ods have excitation range(s) (and preferably also emission preferably incorporated directly into capture Surface F3. range(s)) which do not damage cells or genetic material; Up-converting phosphors F6 are conjugated by any of a generally, radiation in the far red, and infrared ranges are variety of conventional biochemical crosslinking chemis preferred for excitation. It is believed that radiation in the tries to antibody, nucleic acid probes, or other biological range of 200 nm to 400 nm should be avoided, where macromolecules (e.g., carbohydrates, lectins, Streptavidin, possible, and the wavelength range 760 nm to 765 nm may MEC complexes), as well as to biological or chemical be avoided in applications where maintenance of viable cells antigens (F7). Bonded to overlay F3 is a grid array F8 of is desired.

complementary probes or antigens which are bound to 15 Additional Variations capture Surface F3 using the Same crosslinking chemistries. There are Several apparatus design issues relating to the In use, a sample fluid F9 flows between arrays F1 and F2, unique excitation and emission characteristics of upconvert target probes or antigens are captured by grid array F8 and ing phosphors which must be considered when using excited by laser array F1, and the emissions detected by up-converting phosphors with flow cytometry. The first detector array F2. issue is the time required to reach maximum emission Typically, the upconverting phosphors to be used with this intensity. Since upconversion is a two photon process, apparatus are approximately 0.1 to 0.5 micrometers. Since upconverting phosphor emission is time delayed approxi the size of the individual phosphor particles is of the order mately 100 microseconds. The phosphor must remain within of the excitation wavelength, the power of the emission from the excitation beam for this period of time regardless of the the phosphors can be approximated by: 25 flow rate. Therefore given a flow rate between 1 and 10 meters per second with a channel width of 70 to 200 micrometers, the length of the excitation beam must be where f is the phosphorescence efficiency (generally less between 100 and 1000 micrometers. Given that the phos than or equal to 107 cm'W' um"particle), N is the phor emissions Saturate at an excitation intensity of about number of phosphor particles in the light path, D is the 200 watts per Square centimeter, the laser Source typically diameter of the phosphor particles, and I is the power must have a power between 0.01 and 400 milliwatts to density of the excitation Source. achieve phosphor Saturation. This implies that multiple laser Since the emitted power Scales as the Square of the diodes may be required to obtain maximum phosphores excitation intensity, diagnostics using upconverting phos cence at the fastest flow rates.

phors perform better in a microassay format. ASSuming a 35 Another design issue is that associated with the detector. constant power output from the excitation Source, the exci Since there is a considerable Separation between the exci tation power density increases proportionally with the tation and emission wavelengths of the upconverting decrease in detection area, and the number of phosphor phosphors, detection can be performed using a photomulti particles in the light path decreases linearly with a decrease pler tube (PMT), a photodiode, or a CCD array. The phos in the detection area. Since the power of the light emitted 40 phorescence decay time is long, with a decay half life of from the phosphors Scales with the Square of the excitation approximately 300 microseconds. The most sensitive power density, but linearly with the number of phosphors, method of detection is to integrate the Signal measured by P will increase in inverse proportion to the detection area. the PMT. However, 99 percent detection of the available Therefore, a 100x100 array will actually be 100 times more phosphorescent Signal requires that the phosphor remain in sensitive than a 10x10 array. 45 the Sight path of the detector for 5 times the phosphores Fluorescence-activated Cell Sorting cence decay half-life (i.e., 1.5 milliseconds). ASSuming a The up-converting phosphors described herein can be flow rate of 10 meters per second and a channel width of 200 used as phosphorescent labels in fluorescent cell Sorting by micrometers, the PMT must be able to detect over a path flow cytometry. Unlike conventional fluorescent dyes, length of 1.5 centimeters. This path length is also the up-converting phosphors possess the distinct advantage of 50 required Spacing between cells flowing through the not requiring excitation illumination in wavelength ranges cytometer, implying a maximum count rate of 667 cells per (e.g., UV) that damage genetic material and cells. Typically, Second. It is, however, possible to Sacrifice Some detection up-converting phosphor labels are attached to a binding Sensitivity by reducing the detection path length, at least to reagent, Such as an antibody, that binds with high affinity and that required to attain Steady-state emission from the phos Specificity to a cell Surface protein present on a Subset of 55 phors. AS long as a steady-state emission peak is reached by cells in a population of cells in Suspension. The phosphor the phosphor in the excitation window, the peak signal labeled binding component is contacted with the cell SuS received by the PMT should be directly proportional to the pension under binding conditions, So that cells having the concentration of phosphors present. The nonphotobleaching cell Surface protein bind to the labeled binding reagent, property of the phosphors makes this form of detection whereas cells lacking the cell Surface protein do not Sub 60 possible. The loSS in detection Sensitivity corresponding to a Stantially bind to the labeled binding reagent. The Suspended 0.1 centimeter path length (versus a 1.5 centimeter path cells are passed acroSS a Sample detector under conditions length) is approximately a factor of 3. Triggering the emis wherein only about one individual cell is present in a Sample Sion detector can be accomplished by observing the light detection Zone at a time. A Source, typically an IR laser, Scattered by the cell as it passes through the excitation illuminates each cell and a detector, typically a photomul 65 SOCC.

tiplier or photodiode, detects emitted radiation. The detector In environments where absorption of the up-converted controls gating of the cell in the detection Zone into one of phosphor radiation is high, the phosphor microparticles are

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coated with a fluorescent dye or combination of dyes, in the CCD array intensity information by computer analysis Selected proportions, which absorb at the up-converted fre will allow near-real time tracking of the particles in a quency and Subsequently re-radiate at other wavelengths. dynamically evolving or living Systems. Data analysis and Because the Single-photon absorption croSS-Sections for reduction performed by the computer would include a these fluors are typically very high, only a thin layer is convolution of the intrinsic decay of the phosphor emission, required for complete absorption of the phosphor emission. the number of pixels illuminated and their signal level, the This coat particle may then be encapsulated and coated in a orientation of the decaying Signal on the array, and the Suitable antigen or antibody receptor (e.g. microparticle). An intensity contributions from a blur circle from particles example of this layering is depicted schematically in FIG. 9. moving in and out of the focal plane of the array. In an There exists a wide variety of fluorescent dyes with Strong end-on flow detection arrangement, the Size of the blur circle absorption transitions in the visible, and their emission would relate directly to how quickly the particle moves out covers the visible range and extends into the infrared. Most of focus, thereby allowing the velocity of the particle to be have fluorescent efficiencies of 10% or more. In this manner, determined. One possible application would be monitoring the emission wavelengths may be custom-tailored to pass the chemistry and kinetics in a reaction column, through the particle's environment, and optical interference 15 alternatively, the application of this method to flow cytom filters may again used to distinguish between excitation and etry may permit the resolution of cells on the basis of emission wavelengths. If a relatively large wavelength “win hydrodynamic properties (size, shape, density). The method dow' in the test medium exists, then the variety of emission may also be useful for in Vivo diagnostic applications (e.g., wavelengths which may be coated on a Single type of blood perfusion rate).

phosphor is limited only by the number of available dyes and Up-converting phosphor labels may also be used to Sense dye combinations. Discrimination between various reporters the temperature in the region at which the up-converting is then readily carried out using the Spectroscopic and phosphor label is bound. Up-converting phosphor tempera multiplexing techniques described herein. Thus, the number ture measurement methods are described in Berthou H. and of probereporter “fingerprints' which may be devised and Jorgensen C. K. (October, 1990) Optics Lett. 15(19): 1100, used in a heterogenous mixture of multiple targets is virtu 25 incorporated herein by reference. ally unlimited. Matched Label Methods

The principles described above may also be adapted to AS described above, photophysical catalysis, diagnostic driving Species-specific photocatalytic and photochemical assays and other Sampling techniques can take advantage of reactions. In addition to spectroscopic Selection, the long matching the absorption spectrum of a luminescent label emission decay times of the phosphors permit relatively with the emission spectrum of an up-converting label. The Slow reactions or Series of reactions to take place within the matching of an up-converting label with a luminescent label, emission following photoexposure. This is especially useful i.e., a label which emits radiation upon absorption of energy, When the phosphor-catalyst or reactant conjugate enters an represents a step beyond traditional photophysical catalysis environment through which the excitation wavelength can and assay methods.

not penetrate. This Slow release also increases the probabil 35 The term “matched labels' refers to two or more labels ity that more targets will interact with the particle. where at least one label absorbs excitation radiation and The unique decay rates of phosphor particles allow emits energy which in turn excites another label and causes dynamic Studies as well. In a System where continuous it to emit emission radiation. In a matched label pair, an exposure to the excitation Source is not possible, or is excitation label, Such as an up-converting phosphor, absorbs invasive and thereby undesirable, pulsed excitation followed 40 the excitation radiation and emits energy which in turn by delayed fluorescence detection is necessary. After the excites an emission label, Such as a luminescent label. The phosphor reporter has been photoexcited, the Subsequent emission label, in a matched label pair, is excited by the emission from the phosphor or phosphor/dye conjugate energy emitted from the excitation label, and emits emission particle lasts typically about a millisecond. In a dynamic radiation which may be detected in an assay or used in environment, Such as a Static or flowing System with moving 45 photophysical catalysis or photodynamic therapy. targets, the particle will emit a characteristically decaying Additionally, a matched label pair may have one or more intensity of light as it travels relative to the excitation transfer labels in addition to the excitation label and the detection apparatus. Combined with imaging optics appro emission label. Transfer labels are luminescent labels which priate to the Scale of the System and the Velocities within the absorb and relay the excitation energy from the excitation System, a CCD photoelectric Sensor array will be used to 50 label to an emission label, causing the emission label to detect the particle or particles movement acroSS the array's excite and emit radiation.

field of view. The delayed emission of the phosphors, which The matched labels of the invention are convenient and is a well-characterized function of time, makes possible the reliable for photophysical catalysis and for detecting the dynamic tracking of individual particle's positions, direc presence of low concentrations of analytes. By employing tions and Velocities, and optionally calculation of particle 55 matching up-converting and luminescent labels, it is pos size, density, and hydrodynamic conformation. As a particle Sible to detect target analytes with nondestructive visible and moves, it exposes more elements of the array, but with infrared light, thus, producing little or no background inter every-decreasing intensity. The more elements it exposes ference. Furthermore, catalysis, detection and discrimina over a certain fraction of its decay time, the faster it is tion of multiple target analytes may be achieved with moving. Therefore, the integrated intensity pattern of a 60 matched labels.

particle's emission “track’ collected by the array is directly The matched labels may be employed in any traditional related to the velocity of the particle. The particles may be assay or photophysical catalysis method. For example, in refreshed again at any time by the pulsed or chopped CW matched label assays, a Sample Suspected of containing a excitation source. FIG. 10 illustrates this scheme. Although target analyte is contacted with matching up-converting and only a depiction of “side-on' excitation and detection is 65 luminescent labeled probes. The up-converting labeled shown, both side-on and end-on detection and excitation probes and luminescent labeled probes are capable of Spe arrangements, or combinations, are possible. Reduction of cifically binding the target analyte and forming labeled

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probe-target complexes. Having bound the analyte to the exciting the excitation label and observing for emission labeled probes, the Sample is illuminated with illumination radiations from the emission labels, the presence of one or energy to excite the excitation label. The Sample is then both of the two different target analytes may be detected observed for emission radiation from the emission label. The within a single Sample.

detection of an emission from the emission label verifies the (2) In another embodiment, distinct sets of matched presence of the target analyte Since the emission label excitation and emission labels may be employed. For generally only absorbs the energy caused by excitation label example, two different Sets of matched labels each having emissions when the matched label S are attached to the target distinct excitation labels and distinct emission labels may be analyte. The apparatus used to illuminate the Sample with an used in a single Sample. For example, in an assay method, illumination wavelength and to detect an emission wave by exciting the first excitation label and observing emission length from the emission label is the same as described radiation from its matched label, a first analyte may be above. detected. Additionally, in the same Sample, a Second analyte The matched label assays may be carried out as may be detected by exciting the Second excitation label and homogeneous, heterogeneous or competitive assayS. observing emission radiation from its matched label. However, when using matching labeled probes in a homo 15 (3) In another embodiment, a probe having one excitation geneous assay, it is preferred that the matching labels are label may be matched with at least two probes having attached to two probes to form distinct labeled-probe pairs distinct emission labels. Thus, in an assay, different target which bind to the target analyte. The probe may be the same analytes may be detected within a single Sample by exciting or different probe material. For example, an up-converting the excitation label and then observing the different emission label may be attached to a first antigen probe which binds to radiations from the distinct emission labels. one epitope Site on the analyte while the matched lumines (4) Another embodiment of the invention involves using cent label is bound to a different antigen probe which binds matched labels having one or more emission labels which to another epitope Site on the same analyte. emit a detectable emission radiation and at least two distinct Matched label photophysical catalysis can be conducted excitation labels. For example, in an assay on a Single in a manner Similar to the matched label assays described 25 Sample, different target analytes may be detected by Sepa above except that the energy from the emission label may be rately exciting the distinct excitation labels and observing used to produce localized intense electromagnetic radiation the emission radiation from the emission labels. for purposes other than detection. For example, the energy Of course, the various combinations of matched label from the emission label may be used to bring about a pairs discussed above may be employed with different modification in the target analyte or in materials adjacent to probes to detect or, as discussed above for photophysical the target analyte. Another example of photophysical cataly catalysis or photodynamic therapy, effect a multitude of sis involves the use of an up-converting label which excites different target analytes within a Single Sample. a luminescent label causing it to emit its energy to create a Furthermore, these multiplexing embodiments may be car reactive chemical Species. ried out Simultaneously or Sequentially upon a single Both the matched label photophysical catalysis and assays 35 Sample.

of the invention may employ a multitude of different Matched labels may also be used in other assay formats, matched labels and/or different probes within a single (for example a competitive assay), where a first labeled Sample. By varying the labels and probes a wide variety of probe of a matched labeled probe pair binds with a target analytes may undergo photophysical catalysis or be detected analyte to form an initial labeled probe-analyte complex within a single Sample. For example, one Set of matched 40 which may be separated from the original Sample and then labels may contain a luminescent label as the excitation label contacted with the matching labeled probe of the labeled and an up-converting label as its matched emission label probe pair. By contacting the initial probe-analyte with the while another matched Set in the same Sample could contain matching labeled probe, the target analyte may be displaced an up-converting label as the excitation label and a lumi Such that the matching labeled probes may bind together. neScent label as the emission label. 45 Illuminating the resulting Sample excites the excitation label The following four embodiments outline the ability to of the matched label pair. Emission from the emission label conduct multiple assays within a single Sample: then indicates the presence of the target analyte. (1) In a single sample, the matching up-converting and Alternatively, a target analyte may displace a labeled probe luminescent labels may be separately attached to two dis from a bound matched label probe pair. In this situation, tinct probes of the same or different material such that the 50 illumination and the decrease or complete absence of emis matching labels are attached to probes which bind to the Sion radiation indicates the presence of the target analyte. Same analyte or overlapping Sets of analytes. For example, In a preferred embodiment, matched labels involve the a particular up-converting labeled probe may have affinity matching of luminescent labels and up-converting labels. AS for one or Several analytes and the matched luminescent described above, luminescent labels emit radiation, Such as labeled probe may have affinity for a portion of those 55 light, upon absorption of energy. The luminescent labels and analytes as well as other analytes. Thus, use of matched up-converting labels may be phosphorescent, fluorescent, or labeled-probe pairs allows for the detection of analytes chemilumuneScent and generally emit light having a wave which bind to both of the labeled probes or, in other words, length in the infrared, visible or ultraViolet regions. an overlapping Set of analytes for the labeled probes. The up-converting labels of the invention include When employing up-converting and luminescent labels 60 up-converting inorganic phosphors, dyes, chelates and non attached to two distinct probes, the excitation label prefer linear optical compounds. Examples of up-converting dyes ably does not excite an emission label unless both of the and inorganic phosphors Suitable for use in the invention distinct probes are in close proximity, e.g., are bound to the include, but are not limited to, those described above. Same analyte. Thus, different target analytes may be detected Examples of up-converting non-linear optical compounds in an assay. For example, different matched label pairs may 65 include, but are not limited to, 4-(p-nitrolphenyl)-3,4, be attached to two distinct probes which bind to two dihydro-pyrazoc-benzobmorpholine (NDPB), 1-o-tolyl different target analytes. Then, for an assay method, by 3-p-nitrophenyl-2-pyrazoline (TNP), 2-(p-dimethyl

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aminophenyl)ethenyl-phenyl-methylene-propanenitrile general, are predominately spherical in shape. In other (DPP-) and bis-2-(p-dimethylaminophenyl)ethenyl words, the majority of phosphorescent particles exist as methylene-propanenitrile (BDP). Other up-converting individual particles in contrast to clusters of two or more phosphors, dyes and chelates are described in, but are not phosphorescent particles. In a preferred embodiment, the limited to, those described in Copending U.S. application Substantially monodisperse phosphor particles are formed Ser. No. 08/986,196 entitled “Production of Substantially by a fluidized bed production proceSS Such as that described Monodisperse Phosphor Particles, filed on Dec. 5, 1997, in Copending U.S. application Ser. No. 08/986,196 entitled Luminescent Materials, Ullmann's Encyclopedia of Indus “Production of Substantially Monodisperse Phosphor trial Chemistry, 5th Edition, Vol. A15, pgs. 519–557, and in Particles, filed on Dec. 5, 1997. The up-converting labels and luminescent labels may be

Kirk-Othmer Encyclopedia of Chemical Technology, Third attached to their respective probes by conventional methods, edition, Volume 14, pp. 527 ff, the disclosures of which are Such as those described above or other methods known in the incorporated by reference in their entirety herein. art. For example, an up-converting phosphor label may be Luminescent labels of the invention include any material treated with a polymeric material to coat the phosphor to which emits or can be induced to emit a signal. Typically, the form a labeled probe. Another approach involves contacting luminescent labels may be excited with radiation having a 15 an encapsulated luminescent having carboxyl groups on its wavelength of from about 350 to about 1500 nm. A signal surface with Biotin-LC-Hydrazide using 1-ethyl-3-(3- emitted by the luminescent label is generally of a wave dimethylaminopropyl) carbodiimide (EDC) under acidic length in the infrared, visible or ultraViolet region. Also, a conditions to form a stable peptide linkage between the Signal emitted by the luminescent label should be at a large carboxyl groups on the lateX Sphere Surface and the enough down-converting or up-converting (Stokes or anti hydrazide of the Biotin-LC-Hydrazide. Biotin Stokes) shift to enable detection of the signal. Furthermore, LC-Hydrazide and EDC are available from PIERCE located the luminescent emission preferably does not overlap the in Rockford, Ill. Additional methods of attaching labels to emission of its matched up-converting label unless time probes are described in Stavrianopoulos et al. U.S. Pat. No. resolved detection methods are employed. Also, a lumines 4,868,103, and Tanke et al. U.S. Pat. No. 5,043,265, the cent label should not emit a Signal at the same wavelength 25 disclosure of which is incorporated by reference in its as the excitation wavelength of its matched up-converting entirety.

label. With regards to the packaging of the compositions of the Any luminescent labels used in conventional assays or, as invention, the compositions may be in the form of a kit described above, photophysical catalysis methods may be comprising all of the essential ingredients required to con up-converting or down-converting, phosphorescent or fluo For example, the assay duct the desired or photophysical catalysis method.

kit may contain matching up-converting rescent materials. The luminescent labels which are up-converting include, but are not limited to up-converting labeled probes and luminescent labeled probes. The assay kit phosphors, chelates, dyes and non-linear optical compounds, isThepresented kit can be in a convenient, commercially packaged form.

presented as a composition or as an admixture

Such as those described above. Luminescent labels which are down-converting luminescent materials include, but are 35 depending upon the compatibility of the labeled probes. For not limited to, down-converting phosphors, Such as Zinc example an assay kit can be a packaged combination having sulfides activated with silver and those described in Copend one labeled or more containers, devices or the like holding the probes and other materials necessary for particular ing U.S. application Ser. No. 08/986,196 entitled “Produc assay, and usually including written instructions for the tion of Substantially Monodisperse Phosphor Particles,” performing the filed on Dec. 5, 1997, Luminescent Materials, Ullmann's 40 Although theassay. present invention has been described in Encyclopedia of Industrial Chemistry, 5th Edition, Vol. A15, Some detail by way pgs. 519–557, and in Kirk-Othmer Encyclopedia of Chemi understanding, it willofbeillustration for purposes of clarity of apparent that certain changes and cal Technology, Third Edition, Volume 14, pp. 527 ff. the modifications may be practiced within the Scope of the disclosures of which are incorporated by reference in their claims.

entirety. Luminescent label chelates include, but are not 45 The broad scope of this invention is best understood with limited to and those described in Hemmila et al. U.S. Pat.

No. 5,637,509, and Stavrianopoulos et al. U.S. Pat. No. referenceto limit to the following examples, which are not intended the invention in any manner.

4,868,103, the disclosures of which are herein incorporated by reference in their entirety. Luminescent labels which are EXPERIMENTAL EXAMPLES fluorescent dyes include, bit are not limited to, TransFluo 50 Validation of Up-Converting Inorganic Phosphors as Spheres(R (TFS) and those described in Mandel et al. U.S. Reporters

Pat. No. 4,372,745, Brinkley et al. U.S. Pat. No. 5,326,692 Up-converting phosphor particles comprising Sodium and Singer et al. U.S. Pat. No. 5,573,909, the disclosures of yttrium fluoride doped with ytterbium-erbium were milled to which are herein incorporated by reference in their entirety. Submicron size, fractionated by particle size, and coated A preferred luminescent label is a down-converting fluores 55 with polycarboxylic acid. Na(YosoYbos Eroo)F was cho cent dye mixture sold under the tradename TransFluo Sen for its high efficiency upon excitation in the range 940 Spheres(R (TFS) which is commercially available from to 960 nm. A Nd:Yag pumped dye laser/IR dye combination Molecular Probes located in Eugene, Oreg. Generally, pre was used to generate 8-ns to 10-ns duration pulses in the ferred luminescent labels of the invention have a high above frequency range.

quantum efficiency and a high extinction coefficient. 60 The laser pulses were used to illuminate a Suspension of Additionally, while down-converting and up-converting milled phosphor particles in liquid and attached to glass phosphor particle labels of the invention may be prepared by Slides in Situ. The Suspension luminescence observed at right conventional techniques and methods known in the art, it is angles was monitored using a collection lens, a Spatial filter preferred that when particles Such as up-converting phoS in order to filter out Scattered excitation light to the maxi phor particles are used, they should be Substantially mono 65 mum possible extent, and a photomultiplier, Vacuum disperse particles. “Substantially monodisperse particles' photodiode, or simple Solid State photodiode (depending on are particles which are Substantially unagglomerated and, in the light level observed).

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The luminescent Signal level was determined as a function of Solution pH (range: 6-8), grain size, particle loading TABLE III (ug/cm), and the nature of stabilizing anionic Surfactant. Phosphor Phosphor Equivalent Signals were recorded both as a time integral from a boxcar Loading Loading Detection integrator and from a long RC time constant or as a transient 5 Label (ng?well) (particles/well) Sensitivity (M) Signal using a transient digitizer in order to delineate the 100 17OO 90 23,6000,000 + 1,200,000 4 x 102 luminescence lifetime under particular experimental condi 1O-1 17O 9 2,360,000 + 120,000 4 x 1013 tions. In Situ Signals were also measured by laser Scanning 10-2 17 O.9 236,000 + 12,000 4 x 101 microScopy. FIG. 11 is a fluorescence Scan of the phosphor 1O-3 10-4

emission Spectrum incident to excitation with a laser Source 10-5 O.O17 O.OOO9 236 12 4 x 1017 at a wavelength maximum of 977.2 mm; emission maximum 1O-6 O.OO17 O.OOOO9 23.6 1.2 4 x 101 is about 541.0 nm. FIG. 12 is an excitation scan of the phosphor excitation Spectrum, with emission collection win The stock DMSO dispersion had a phosphor density of dow Set at 541.0 nm, excitation maximum for the phosphor 15 1.70+0.09 mg/mL (at 95% confidence limits), determined at the 541.0 nm, emission wavelength is approximately gravimetrically by evaporating 4-1 mL Samples. This trans about 977 nm. FIG. 13 is a time-decay measurement of the lates to 23.6x10° particles/mL (assuming an average particle phosphor luminescence at 541.0 nm after termination of size of 0.3 um and particle density of 5.3 g/mL). The residue excitation illumination; maximal phosphorescence appears after evaporating the Samples over the weekend at at approximately 400 uS with a gradual decay to a lower, 110-120° C. was noticeably yellow, but did phosphoresce stable level of phosphorescence at about 1000 us. FIG. 14 when tested with an IR diode laser.

shows the phosphor emission intensity as a function of Visual green light emanated from all Serial dilutions down excitation illumination intensity; phosphorescence intensity to 10 (i.e., 1.7 tug/mL or 23.6x10° particles/mL) in a 1 mL increases with excitation intensity up to almost about 1000 polypropylenemicrofuge tube using a hand-held diode laser W/cm. 25 in a dark room. The 10' and 10° dilutions were visibly Phosphorescence efficiencies of submicron cloudy. Either 1 ul of each serial dilution, or 0.1 ul of the Na(YosYboEros)F particles were measured. A Ti:sap next higher dilution, were pipetted into a well on the Terisaki phire laser was used as an excitation Source and a spectro 0.1 muIt 1was plate. found that 1 ul fills the bottom of the well and spreads along the edge of the well, but does not photometer and photomultiplier was used as a detection cover the entire Surface. Because of the Statistical and System. Two types of measurement were performed. The pipetting problems associated with Small Volumes with low first was a direct measurement in which the absolute emis particle concentrations, 2 to 4 replicates were prepared of Sion per particle for phosphor Suspensions was measured in each dilution.

emission bands at 540 nm and 660 nm. The calibrated The well of a Terasaki plate holds a 10 ul sample volume. croSS-Sections are shown in FIG. 15, and size-dependence is 35 ASSuming all the phosphor particles contained in this Vol shown graphically in FIG. 16. This corresponded to a ume adhere to the bottom of the Sample well, we can phosphorescence cross-section of approximately 1x10' estimate an equivalent detection sensitivity (Table III). It cm for 0.3 um particles with excitation light at 975 nm and should be noted that 10 to 10M is the normal range of an intensity of approximately 20 W/cm. The emission enzyme-linked Surface assayS.

efficiency of dry phosphor powder of about 25 um was also 40 Control Sample Results measured. On the basis of known values for the absorption The control Samples were Scanned using a prototype cross-section of Yb" in crystalline hosts (Lacovara et al. up-conversion fluorimeter device (David Samnoff Research (1991) Op. Lett. 16:1089, incorporated herein by reference) Center). The Samples were Scanned by moving the plate in and the measured dependence of the phosphorescence emis 50 um increments, using a motorized X-Y positioning Stage, Sion on particle Size, a phosphorescence cross-section of 45 relative to the focal point of an infrared diode laser. approximately 1x10 cm was found. The difference The IR diode laser was operated at 63 mW (100 mA). The between these two measurements may be due to a difference beam bottom was focused to 2.4x10 cm at the focal point. As the of the sample well is about 1.4x10 cm (1365 um in phosphorescence efficiency between dry phosphor and aqueous Suspensions, or due to absorption of multiply Scat 50 diameter), the beam covers less than 17% of the well bottom tered photons in the dry phosphor. On the basis of either of Surface at any individual position. The well also has sloping side walls which widen from bottom to top of the sample these croSS-Section estimates, the cross-section is Sufficiently well large to allow detection of Single Submicron phosphor par laser and are also interrogated by a progressively divergent beam. Neglecting losses in the optics, the IR light ticles at moderate laser intensities. At laser intensities of roughly 10 W/cm, the phosphorescence scales as the laser intensity at the focal point (bottom of the sample well) was 55 approximately 26–27 W/cm at 980 nm wavelength. A intensity to the 1.5 power.

Phosphor Particle Performance: Sensitivity of Detection photomultipler tube (PMT) was used for detection of the visible (upconverted) light emitted from the sample. Since

A Series of Terasaki plates containing Serial dilutions of the laser beam width was Smaller than the Surface area at the monodisperse 0.3 um up-converting phosphor particles con bottom of the Sample well, the plate was aligned by visual Sisting of (Yose Yboos Eroos). OS were tested for 60 inspection against the focal point of the diode laser So that up-conversion fluorescence under IR diode laser illumina the laser was centered in the middle well (C6 when reading tion in a prototype instrument. wells C5, C6 and C7, and D6 when reading wells D5, D6, The phosphor particles were prepared by Settling in and D7).

DMSO and were serially diluted into a 0.1% acqueous gum The PMT signal (amps) was recorded at each plate arabic Solution. This appeared to completely eliminate any 65 position and numerically integrated over the width of the water dispersion problems. The Serial dilutions used are Sample well (approximately 4000 um). Several Scans were listed in Table III. made at different positions in the 10° to 10° dilution sample

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S1 52 wells to determine the uniformity of the particle distribution. as Small, presumably monodisperse particles, and 35% being The background Signal was determined by integrating the Significantly larger, presumably aggregates. Only 60% of the average dark field current of the PMT over a 4000 um Smaller Subpopulation appeared to have significant quanti distance, which yields an integrated background Signal of ties of active Ab (determined by FITC fluorescence). Of the 1x10 ua-m. The integration products of the samples wells purported aggregates, about 90% appeared to contain active were Scaled to this background Signal, and are shown in FIG. Ab (by FITC fluorescence). This suggests that less than 40% 19. of the phosphor-Abconjugates were of an appropriate size Immimodiagnostic Sample Detection (nominal 0.3 um) and exhibited anti-mouse IgG activity. A A Series of IgG/anti-IgG Samples for demonstrating the Similar fraction of phosphor-Ab conjugates (31%) were capabilities of the up-converting phosphor reporters in a active but carried a significantly larger phosphor reporter. immunosorbant assay format was prepared. These Samples The PMT signal (amps) was recorded at each plate consisted of six individual wells (positive samples) coated position and numerically integrated over the width of the with antigen (mouse IgG) and bovine Serum albumin (BSA), Sample well approximately 4000 um). The average signals and six wells coated with BSA alone (negative controls). (with 95% confidence limits) are:

Nominal 0.3 um (Yose Yboos Broo)2O2S phosphor particles 15 Average of Positive Samples=1.30x10"+1.25x10" ua-m coated with goat anti-mouse IgG antibody (anti-IgG) were Average of Negative Controls=4.20x10+6.82x10 then used as the reporter-antibody conjugate. Six wells (C5, Ala-m

C6, C7, D5, D6, and D7) of a clear polystyrene Terasaki The positive Samples and negative controls are Statisti plate were coated with mouse IgG by incubating at 37 C., cally different at the 99.9% confidence level. The positive against 5 till of a 100 ug/ull mouse IgG Solution in phosphatesamples emit on average 30.0+29.7 times more light than the buffered saline (PBS). After 1 h, this solution was aspirated negative controls.

off and each sample well was washed with 10 ul of 3% BSA Linkage of Phosphors to Biological Macromolecules in PBS. This was immediately aspirated off and replaced In order to delineate further the parameters for with 20 uL of 3% BSA in PBS. Each sample well was up-converting phosphors as biochemical reporters, biologi post-coated with BSA by incubating against the 20 u, of 25 cal linkers were attached to phosphor particles. Sodium BSA/PBS solution for 1 h at 37° C. The post-coat solution yttrium fluoride-ytterbium/erbium phosphor particles were was aspirated off and the plates Stored at 4 C. overnight. coated with Streptavidin. The excitation and emission Spec These wells were considered in positive Samples. The same tral properties of the phosphor alone and the phosphor Six wells in a Second Terasaki plate were prepared in an coated with streptavidin were measured (FIGS. 17A, 17B, identical fashion, except they were not coated with mouse 18A, and 18B) and both the uncoated and streptavidin IgG. This Second Set of Sample wells were considered coated phosphors were almost identical in their absorption negative controls. and emission properties, indicating that the attachment of Phosphor-Antibody Conjugate macromolecular linkers (e.g., proteins) have little if any A Solution of (YosYboosEroos)O2S phosphor particles effect on the phosphorescent properties of the up-converting was prepared by Suspending the dry phosphors into DMSO. 35 phosphor. The Streptavidin-coated phosphors were then spe The initial particle density was approximately 107 particles/ cifically bound to biotinylated magnetic beads, demonstrat mL as determined by counting the number of particles ing the applicability of linker-conjugated inorganic phos contained in the field of an optical microScope. It should be phors as reporters in biochemical assays, Such as noted that the 0.3 um fundamental particle size was below immunoassays, immunohistochemistry, nucleic acid the resolution limits of the microScope. This Solution was 40 hybridizations, and other assayS. Magnetic bead technology allowed to settle undisturbed for 3 days. The Supernatant, allows for the easy Separation of biotin-bound Streptavidin which was turbid and presumably contained mostly mono coated phosphor from a Solution, and is particularly well disperse Smaller particles was used for Subsequent conjuga Suited for Sandwich assays wherein the magnetic bead is the tion. Solid Substrate.

Goat anti-mouse IgG antibody (Ab) was conjugated (by 45 Advantageously, Streptavidin-biotin chemistry is widely adsorption) onto the DMSO fractionated phosphor particles. used in a variety of biological assays, for which This was done by mixing 200ull of the Absolution (in 0.1M up-converting phosphor reporters are Suited. FIG. 20 shows Tris-HCl, pH 7.2) with 100 uL of the phosphor suspension Schematically, for example and not limitation, one embodi in DMSO. Several different Ab concentrations were tried in ment of an immunoassay for detecting an analyte in a the range of 0.025 to 1 lug?u L. A concentration of 0.25 ug?u L 50 Solution by binding the analyte (e.g., an antigen target) to a appeared to result in the most efficient coating (i.e., maxi biotinylated antibody, wherein the analyte forms a Sandwich mum Ab utilization with a minimum of clumping of the complex immobilized on a Solid Substrate (e.g., a magnetic phosphor particles). The phosphors were equilibrated over bead) by linking a first binding component bound directly to night at room temperature with the Ab in this DMSO/Tris the Solid Substrate to a second binding component (e.g., the Solution with gentle agitation. The resulting phosphor-Ab 55 biotinylated antibody); a Streptavidin-coated up-converting conjugates were centrifuged from this Solution and resus phosphor then binds specifically to the biotinylated antibody pended in a 3 tug/ull BSA solution in PBS for post-coating. in the Sandwich and Serves to report formation of the The resulting BSA/PSA resuspension was used directly for Sandwich complex on the Solid Substrate (which is a measure the assay. of the analyte concmagnetic bead., it is Solid Substrate is a The degree of Ab adsorption to the phosphors, and 60 magnetic bead, it is readily removed from the Sample residual Ab activity, was determined by titrating the Solution by magnetic Separation and the amount of phosphor phosphor-bound Ab with a fluorescein isothiocyanate attached to the bead(s) in Sandwich complex(es) are deter (FITC) conjugated-mouse IgG. The resulting FITC-labeled mined by measuring Specific up-converting phosphores phosphors were passed through a Cyteron Absolute flow cence. Thus, Sandwich complex phosphorescence provides a cytometer, which was also capable of measuring the relative 65 quantitative measure of analyte concentration. Size of the particles. Two distinct size Subpopulations were Biotinylated polynucleotides are also conveniently used observed with about 65% of the counted particles appearing as hybridization probes, which can be bound by

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S3 S4

Streptavidin-coated up-converting phosphors to report Monodisperse upconverting phosphor particles were hybrid formation. silanized with thiopropyltriethoxysilane (Huls) following Background Phosphorescence in Biological Samples the procedure detailed by Arkles (in: Silicone Compounds: Background Signals were determined in two biological Register and Review, Hills America, pgs. 59-75, 1991). Samples for determination of potential background in immu This consisted of adding thiopropyltriethoxysilane (2 g) noassayS. Sputum and urine were used as Samples in the and 95% aq. ethanol (100 mL) to a 500 mL Erlenmeyer flask Same apparatus as used for the phosphorescence Sensitivity and stirred for 2 minutes. Approximately 8 mL of the 65 measurements (Supra). No background levels were found mg/mL phosphor Suspension in DMSO was then added to above the system noise levels set by the photomultiplier dark the mixture. This Suspension was stirred for an additional 2 current. This noise level allows detection of Signals from on 1O minutes, then transferred to centrifuge tubes and centrifuged the order of a few hundred particles cm.

This is close to a single particle in the detection volume twice with the to Separate

phosphor particles. The pellets were washed aq. ethanol centrifuging each time. The of the System.

A photomultiplier is a preferred choice for a detector for resulting particles were collected and dried overnight under high Sensitivity measurements of up-converting phosphors vacuum at approximately 30° C.A quantity (127 mg) of dry Since photomultipliers can be selected to produce high 15 silanized phosphors were resuspended in 1.5 mL of DMSO quantum efficiency at the up-converted (i.e., emitted) wave (phosphor Stock).

lengths and virtually no response in the range of the longer A solution containing 1.19 mg of avidin (Pierce) in 1.0 excitation wavelengths. mL of borate buffer (954 mg sodium borate decahydrate and Detection of Cell Antigens with Phosphor-Labeled Antibod 17.7 mL of 0.1N NC1 in 50 mL of deionized water, pH 8.3) ies was prepared (Avidin Stock). Another Solution containing Streptavidin is attached to the up-converting phosphor 1.7 mg of N-Succinimide(4-iodoacetyl) aminobenzoate particles as described, Supra. The mouse lymphoma cell line, (Pierce Chemical) in 1.2 mL of DMSO was prepared (SIAB EL-4, is probed with a hamster anti-CD3 antibody which stock). A quantity (10 mu L) of the SIAB stock was added specifically bind, to the 30 kD cell Surface EL-4 CD3 to the 1.0 mL of Avidin stock and stirred at room temperature Tlymphocyte differentiation antigen. The primary hamster 25 30 minto allow the N-hydroxysuccimide ester of the SLAB antibody is then specifically bound by a biotinylated goat to react with primary amines on the avidin (Avidin-SIAB antihamster Secondary antibody. The biotinylated Secondary Stock).

antibody is then detected with the streptavidin-phosphor A 20 mL Scintillation vial was prepared containing 10 mL conjugate. This type of multiple antibody attachment and of borate buffer (pH 8.3). The following additions were then labeling is termed antibody layering. made to this vial: 21.6 till of the avidin-SIAB stock solution Addition of multiple layers (e.g., binding the primary followed by 1.5 mL of the phosphor stock. This reaction hamster Ab with a goat-antihamster Ab, followed by binding mixture was stirred at room temperature in the dark over with a biotinylated rabbit-antigoat Ab) are used to increase night to allow the SIAB activated avidin to react with the the distance Separating the phosphor from the target. The thiol groups present on the Silanized phosphor Surface and layering effect on Signal intensity and target detection Speci 35 resulting in the covalent linkage of avidin to the phosphor ficity is calibrated and optimized for the individual applica particles.

tion by performing layer antibody layering from one layer After the overnight incubation 1.0 mL of the reaction (primary antibody is biotinylated) to at least five layers and mixture was centrifuged (1 min at 10,000 g) and the Super ascertaining the optimal number of layers for detecting CD3 natant removed. The pellet was resuspended in 1.0 mL of on EL-4 cells. 40 phosphate buffered saline (pH 7.2, Pierce) and centrifuged FIG. 21 Schematically portrays simultaneous detection of again to wash any uncongugated protein from the phos two EL-4 cell Surface antigens using phosphors which can phors. This washing process was repeated. The washed be distinguished on the basis of excitation and/or emission pellet was resuspended in 1.0 mL of phosphate buffered Spectra. Detection of both antigens in the Scheme shown in Saline and used directly in diagnostic assays as described FIG. 21 uses a biotinylated terminal antibody which is 45 below.

conjugated to Streptavidin-coated phosphor (#1 or #2) prior Measurement Apparatus to incubation with the Ab-layered sample. Thus, the A modified SLM Aminco 48000 Fluorimeter was used to phosphor-antibody Specificity is retained through the unusu measure the fluorescence spectrum from the phosphor ally strong (K, approx. 1x10"M") non-covalent bond Samples. The modifications to this device consisted of between streptavidin and biotin which is pre-formed before 50 adding a laser diode (David Sarnoff CD-299R-FA #13) incubation with the primary antibody-bound Sample. Quan which was input to the fluorimeter through port 3. The laser titation of each antigen is accomplished by detecting the diode emits at ) =985.1 nm. Spectral data provided by the distinct signal(s) attributable to each individual phosphor David Sarnoff Research Center also shows a small peak at Species. Phosphorescent Signals can be distinguished on the 980.2 nm. This peak has 15% the intensity of the peak at 985 basis of excitation spectrum, emission spectrum, fluores 55 nm. A 5.08 cm focal length lens was used to collimate the cence decay time, or a combination of these or other diode laser beam. The power of the IR laser light was properties. FIG. 22 shows a Schematic of an apparatus for measured as 6.1 mW at the cuvette location with a drive phase-Sensitive detection, which affords additional back current of 75 mA. The beam was not focused at the center ground discrimination. The pulse or frequency mixer is Set of the cuvette. This is true for the standard visible light from to pass the Signal and discriminate against the background 60 the fluorimeter excitation monochromator as well. The laser following frequency calibration for maximum background diode beam is diverging as it enters the cuvette holder and rejection. is approximately 4 mm (H)x2 mm (V) by the time it reaches Covalent Conjugation of Upconverting Phosphor Label to the center of the cell, neglecting the changes in refractive Avidin index of the cell wall and the liquid. An up converting y trium -ytterbium-erbium 65 Light emitted is Scanned with a monochromator and

(Yose Ybooseroo) oxysulfide (OS) phosphor was linked to detected by a photomultiplying tube (PMT) 90 from the avidin by the following procedure: direction of the excitation light. The detection limits for the

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modified SLM Aminco 48000 were determined by serial dilution to be 4x10'M (240,000 phosphor particles per TABLE IV mL) in PBS. Phosphor emission peaks in the spectrum were Cell Cell seen at wavelengths of 406+2 nm, 434+2 nm, 522+2 nm, and Surface Surface 548+2 nm. The largest peak was at 548 nm. The intensity of Type of goat Avidin Phosphor FITC the 548 nm peak was used to discriminate Samples. Tube anti-mouse IgG Conjugate Signal Signal 1. biotinylated Avidin-Phosphor --

Linkage of Avidin-Phosphor Conjugate to Cell Surface 2 biotinylated Phosphor Marker 3 biotinylated Avidin 1O 4 biotinylated Avidin-FITC --

A lymphoblastoid cell line (Human Genetic Mutant Cell 5 FITC labelled Avidin-Phosphor -- Repository #GM07092) was cultured in RPMI 1640 media 6 FITC labelled Phosphor -- containing 15% heat inactivated fetal calf Serum. A Suspen sion of cells (107 cells) was centrifuged and resuspended in The remainder of the Samples were used to resuspended an equal volume of phosphate buffered salin (PBS) pH 7.4. 15 paramagnetic, polystyrene beads bound with Sheep anti Cells were washed two times in PBS and resuspended to a mouse IgG. For each of the six samples, 3x107 beads were final concentration of 5x10 cells/ml. These cells were then prewashed with blocking buffer for 1 hour at room tempera incubated with a mouse IgG1 monoclonal antibody to ture in Eppendorf tubes. The buffer was removed by aspi human B microglobulin, a Class I histocompatibility anti ration while the tubes were in a magnetic rack. The magnetic gen in polystyrene centrifuge tubes. The cells were immu beads with anti-mouse IgG were allowed to bind to the antibody labelled cells for 1 hour at room temperature with noprecipitated for 30 minutes at 4 C. with an antibody intermittent resuspension. The magnetic beads were then concentration of 10 ug/ml. The cells were harvested by collected on a magnetic rack, washed four times in blocking centrifugation, washed twice in PBS, resuspended in PBS buffer, resuspended in 100 till blocking buffer, transferred to and then aliquoted (250 uD) into six fresh centrifuge tubes. 25 a fresh tube, and up-converting phosphorescence was mea Four of these Samples received biotinylated goat anti-mouse Sured on the fluorimeter.

IgG, while the remaining two received FITC.-labelled goat To Scan for phosphor emission, the emission monochro anti-mouse IgG. These immunoprecipitations were per mator bandwidth was Set to 8 nm and the spectra were formed at 4 C. for 30 minutes in volume of 400 till with a scanned from 500 to 700 nm with a step size of 2 nm. final second antibody concentration of 20 mu g/ml. The cells Samples were also measured for FITC signal by exciting the samples with 37 uM at ) =490 nm with a 2 nm bandwidth.

were harvested and washed in PBS as above but were resuspended in 50 uL of blocking buffer (0.2% purified Since the excitation wavelength (490 nm) and the emission wavelength (514 nm) are very close for FITC, higher casein in PBS, Tropix, Bedford, Mass.). The cell-antibody resolution was required to get Separable Signals than with complexes were blocked in this solution for 30 minutes at 35 phosphor labelling. The intensity of the 490 nm signal was room temperature and then transferred to fresh tubes. 240 uW/cm at the center of the well. FITC emission spectra were Scanned at 0.5 nm increments from 450 nm to 750 nm.

A pre-blocked Suspension (40 uD) of either avidin with a 2 nm bandwidth on the emission monochromator. Phosphor conjugate, avidin-FITC, avidin, or unconjugated Sample 1 is the positive control and clearly yielded the Phosphor was added to four of the cell Samples conjugated 40 highest emission signal. Sample 2 indicates that any non with the biotinylated anti-mouse IgG (H&L). In addition, an Specific adsorption of the phosphors to the Sample is limited equal amount of pre-blocked avidin-Phosphorer unconju and is readily discriminated from Signal attributable to gated Phosphor was added to the remaining two cell Samples avidin-conjugated phosphor and showing that avidin linked immunoprecipitated with the non-biotinylated FITC phosphors can specifically bind only when they are conju labelled anti-mouse IgG (H&L). The avidin reporter conju 45 gated with the probe, in this example through the biotin gates or negative controls were pre-blocked as follows. avidin linkage. Sample 3 is the negative control which Avidin-Phosphor and Phosphor alone was diluted in block contains no phosphors, only avidin. Sample 4 shows FITC ing buffer by adding 10 till of a 6.7 mg/ml Suspension to a conjugated avidin. Although FITC signals were observed on final volume of 100 u. Avidin-FITC and the avidin alone 50 the the cell Surface by laser microScopy, the Signals were below level of detection on the fluorimeter for measurement of controls were also diluted in blocking buffer by adding 27 till FITC, and since there was no phosphor in the sample there of 2.5 mg/ml Solution to a final volume of 100 lull. These was no significant phosphor Signal. Samples 5 and show that reagents were blocked at room temperature for 3 hours with FITC-conjugated primary antibodies can be detected and intermittent resuspension and then added to 50 till of cells that the presence of phosphor or avidin-phosphor does not labelled with biotinylated or non-biotinylated second anti 55 Significantly disrupt binding of the primary antibody to its body. The avidin-biotin reactions were performed at room target antigen.

temperature for 30 minutes with occasional resuspension. Linkage of Avidin-Phosphor Conjugate to DNA The reactions were Stopped by harvesting the cells by Plasmid DNA (25 ug) was nick translated in the presence centrifugation and washing twice in blocking buffer. The 60 of 20 mM dGTP, 20 mM dCTP, 20 mM biotin-14 DATP, 13 mM dTTP, and 7 mM digoxigenin-11 dUTP and purified by samples were resuspended in 100 lull of blocking buffer and ethanol precipitation. The average size of the biotinylated, allowed to settle for 4-5 minutes. Slides for imaging were digoxygenin labelled fragments was estimated to be between prepared by pipetting 5 till of Settled cells from the bottom 200-300 nucleotides as estimated by gel electrophoresis. of the tube. Cells were imaged by confocal laser microscopy Approximately 20 ug DNA was immunoprecipitated for 1 under appropriate conditions to observe cell surface FITC 65 hour at 22 C. with 10 ug/ml mouse monoclonal anti and upconverting phosphor Signals. The observations are digoxigenin IgG1 solution (PBS) in a 200 uL volume. An Summarized in Table IV. equivalent reaction containing no DNA was also prepared.

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Each of the two samples were then aliquoted (50 uL) into that require no Sample washing StepS. Such diagnostic three fresh Eppendorf tubes. assays that do not require the removal of unbound phosphor The avidin-conjugates were blocked for 1 hour at room labels from the Sample are herein termed homogeneous temperature by diluting 500 lug of an avidin-phosphor assays, and can also be termed pseudohomogeneous assayS. Suspension, unconjugated phosphor Suspension, or avidin Homogeneous ASSay Example 1 solution in 300 till of blocking buffer. For each of the One embodiment of a homogeneous assay consists of the samples (summarized below in Table V) 50 lull of the use of an upconverting phosphor label linked to an appro anti-digoxigenin conjugates was added to 150 u of pre priate probe (e.g., an antibody or DNA). The phosphor blocked avidin-conjugates or avidin and were incubated for labeled probe specifically binds to a target (e.g., antigen or 30 minutes at room temperature. nucleic acid) that is linked to a capturing Surface. A Suitable Unbound avidin-conjugates were removed by resus capture Surface can be the tip of a light carrying optical fiber pended 3x107 paramagnetic beads linked with sheep anti (FIG. 23) or the bottom surface of a sample container (FIG. mouse IgG (pre-blocked in blocking buffer). After incuba 24). Upon incubation of the target-labelled capture Surface tion for 30 minutes at room temperature with intermittent with the phosphor-labelled probe, phosphor particles will resuspension, the beads were Separated on a magnetic rack and washed 4 to 6 times in PBS. The antibody-DNA bound 15 accumulate at the capture Surface as a function of the amount beads were then measured on the fluorimeter. of target present on the capturing Surface. The target may be The samples were scanned from 500 to 700 nm with a linked directly to the capturing Surface or may be immobi bandwidth of 8 nm and step size of 2 nm. Each PMT value lized by interaction with a binding agent (e.g., specific reported (Table V) represents an average over 5 scans. antibody reactive with target, polynucleotide that binds Sample 1 is expected to provide the highest PMT signal target) that is itself linked to the capturing Surface (Such as since biotinylated DNA is present and can bind to the in a Sandwich immunoassay, for example).

avidin-linked phosphors. Sample 2 indicates the level of Detection of the phosphor bound to the capture Surface is nonspecific adsorption of the phosphors to the Sample which effected using an excitation light that is focused from a low is found to be insignificant since the PMT signal is observed intensity beam of large cross-section to a high intensity to be the same as that of the negative control (sample 4) 25 beam of small cross-section with the focal point of the beam which contains no phosphors. Sample 3 is another control being an or very near the capture Surface. Focusing of the and shows that the avidin-linked phosphors do not bind to excitation light is accomplished by transmission through the paramagnetic beads in the absence of DNA. Samples 5 optical elements that have a very Small focal length, Such and 6 show results of FITC-labeled avidin used to validate asSay. that the beam diverges and becomes leSS intense, within a Short distance of the capture Surface.

TABLE V

Since the intensity of the light emitted from the upcon

Verting phosphor labels is proportional to the excitation light

Upconverting Phosphor Nucleic Acid Diagnostic ASSay Results intensity raised to a power of two or greater, phosphors near the focal point of the excitation Source will emit significantly

Sam- PMTSignal PMTSignal 35 more light than those remaining in Suspension in the Sample ple DNA Reporter (V (a 546 nm) (V (a 514 nm) away from the capture Surface. Therefore, binding of upcon 1 DNA labeled with Avidin 6.1297 2.4788 Verting phosphor linked probes to the capture Surface will digoxigenin and linked yield an increase in emitted light intensity measured from biotin Phosphor 2 DNA labeled with Silanized 1.0528 4.4022 the Sample as a whole or as measured from a control Sample digoxigenin and Phosphor 40 in which phosphors do not bind to the capture Surface. biotin Emitted light intensity may be plotted as a function of target

linked 1.63O2 3.5779 concentration using for Standardization (calibration) a Series

Phosphor of Samples containing predetermined concentrations of tar 4 DNA labelled Avidin 0.8505 2.8067 get. The emitted light intensity from a test sample (unknown with digoxigenie 45 concentration of target) can be compared to the standard

and biotin

curve thus generated to determine the concentration of with digoxigenin target.

6 No DNA FITC-Avidin 1.0899 3.5779 Examples of Suitable homogeneous assay formats include, hut are not limited to, immunodiagnostic Sandwich 50 assays and antigen and/or antibody Surface competition

Phosphor Downconversion Evaluation asSayS.

A Sample of the (Yose Yboos.Eroo).OS phosphors were Homogeneous ASSay Example 2

Scanned for the presence of a downconverted Signal. This Another embodiment allows for the accumulation of was accomplished by exciting a Sample of the monodisperse phosohors described above (4x10'’M in DMSO) with 1.3 55 upconverting phosphor linked probes at the detection Sur mW of monochromatic light at 350 nm with a 6 mm face by the application of centrifugal or gravitational Set tling. In this embodiment an upconverting phosphor is bandwidth for the excitation Source. Detection was accom plished by scanning this sample from 350 to 800 nm with a linked to multiple probes. All the probes must bind to the monochomator bandwidth of 8 nm. Scanning was performed Same target, although Said binding can be accomplished at in 2 nm increments. No downconversion was observed. different locations (e.g., as antibody probes may target Moreover, no downconversion was seen at the excitation 60 different epitopes on a single antigen). The multiprobe wavelengths cited by Tanke et al. (U.S. Pat. No. 5,043,265) phosphor can then be used to effect the aggregation of Thus, the upconverting phosphors tested are unlike those targets in Solution or Suspension in the Sample. This aggre reported in Tanke et al. gation will result in the formation of a large insoluble phosphor-probe-target complex that precipitates from Solu

HOMOGENEOUS ASSAYS 65 tion or Suspension (FIG. 25). The aggregated complex The multiphoton activation process characteristic of containing phosphors accumulates at a detection Surface upconverting phosphors can be exploited to produce assays while nonaggregated material remains in Solution or SuS

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pension. Detection is accomplished as described in the microns and a carboxylate modified surface (free -COOH above example using a sharply converging excitation beam. groups exposed).

Evaluation of Up-converting Chelate The TFS particle was reacted with EZ-link Biotin-LC Up-conversion has been performed in rare earth chelate Hydrazide using 1-ethyl-3-(3-dimethylaminopropyl) carbo and rare earth Salt Solutions. Chelate of erbium and neody diimide (EDC) suspended at 0.2% solids in phosphate mium have been prepared with ethylenediaminetetraacetic buffered Saline. An avidin horseradish peroxidase conjugate acid (EDNA) and dipicolinic acid (DPA). The erbium che (Avidin-HRP) was reconstituted in water to 1 mg/ml. To late were pumped using light near 793.5 nm from a Ti:sap fixed volumes of the TFS-biotin various concentrations of phire laser (the excitation scheme of Macfarlane (1989) Avidin-HRP, 0.1 to 100 micrograms/ml were added. The Appl. Phys. Lett. 54:2301). This approach produced upcon TFS-biotin/Avidin-HRP mixture was then incubated at room version but not satisfactorily, which we attribute to weak temperature for 30 minutes after which the mixture was absorption for the first step due to the increase in linewidth centrifuged and aspirated to form a dry pellet. The dry pellet in the chelate over the low temperature crystal used for the was suspended in 100 microliters of Biotin-LC-Hydrazide up-conversion laser. and incubated for 30 minutes in the dark. The neodymium chelate were excited with light near 580 15 The mixture was observed for changes in color as an nm from a Nd:YAG-pumped dye laser (following the exci indication of HRP activity due to coupling of the Avidin HRP to TFS-Biotin. An intense blue color was observed at tation scheme of Macfarlane et al. (1988) Appl. Phys. Lett. all concentrations of Avidin-HRP 52:1300). An emission spectrum for the emitted Matching Label Example 3 up-converted light at 380 nm is shown in FIG. 32. We The conjugation of an up-converting label to a probe was estimate the up-conversion cross section to be 102.7 cm for Studied.

this experiment. One milliliter of a 0.1 mg/ml up-converting phosphor We have also observed up-conversion in thulium acetate label Solution was centrifuged for ten minutes. The Super hexahydrate and holmium chloride hexahydrate in Solution natant was then removed and 3 ml of Hepes buffer (pH 6.3) following the excitation schemes of Allain et al. (1990) was added. The up-converting phosphor particles were then Electron. Lett. 26:166, and Allain et al. (1990) Electron. Lett. 25 Sonicated until the particles dispersed. Another 7 ml of 26:261, respectively. The salts were dissolved in heavy Hepes buffer was added to the dispersion. The dispersion water, and excitation was performed using a krypton laser. was then centrifuged for ten minutes. The Supernatant was Although the up-conversion was weak, the up-conversion then removed. Three milliliters of a 0.11 mg/ml Solution of should be improved if chelated compounds are used instead Neutravidin in Hepes buffer, available from PIERCE of of dissolved salts. Rockford, Ill., was then added to the up-converting phos MATCHING LABEL, EXAMPLES phor particles. This mixture was then stirred for two hours Matching Label Example 1 in the dark at room temperature and then centrifuged. The A study was made of the excitation of an up-converting Supernatant was removed and 10 ml of Hepes buffer was label where its Subsequent energy emission excites a lumi added without dispersing the particles. The particles were neScent label causing the luminescent label to emit radiation 35 then centrifuged. The Step of removing the Supernatant, at a wavelength which can be detected. Specifically, the adding Hepes buffer without dispersing the particles and Secondary fluorescent emission of an infrared excited centrifuging the particles was then repeated an additional up-converting phosphor label coupled with a luminescent two times. This was followed by twice adding 3 ml of Hepes label was studied. buffer to the particles, Sonicating the particles until they The up-converting label, a phosphor, (Y.Yb, Tm).O.S 40 dispersed, adding an additional 7 ml Hepes buffer, centri phosphor having an emission radiation of 488 nm, was fuging the particles and then removing the Supernatant. Then dispersed in water by Sonification and then diluted to 0.1 3 ml of a Hepes buffer having a pH of 8.5 and containing mg/ml in water. The luminescent label, TransFluoSpheres (R) 0.1% bovine serum albumin was added to the particles. The (TFS) having an excitation wavelength of 488 nm and a particles were then Sonicated until dispersed and an addi particle diameter of 0.35 microns, was dispersed in water by 45 tional 7 ml of the Hepes pH 8.5 buffer was added. The Sonification and diluted in water to 0.2% Solids. Ten micro particles were then centrifuged and the Supernatant was liters of the phosphor and ten microliters of TFS were then removed. Then the particles were avidinylated in 2 ml of mixed and dried on a glass slide at 60° C. and then mounted Hepes buffer having a pH of 8.5 and containing 0.1% bovine on a coverSlip. The glass slide was then Subjected to Serum albumin.

excitation radiation having a wavelength of 488 nm which 50 Although the present invention has been described in produced a very bright TFS fluorescence against a mottled Some detail by way of illustration for purposes of clarity of green background. The glass slide was also Subjected to an understanding, it will be apparent that certain changes and excitation radiation having a wavelength of 900 to 1000 nm. modifications may be practiced within the Scope of the A faint bluewhite luminescence characteristic of the phoS claims.

phor was detected by Visual observation as well as areas of 55 The claimed invention is:

dim yellow/green fluorescence. The yellow/green fluores 1. A composition for detecting an analyte in a Sample cence represented the presence of TFS:phosphor aggregates using matched labels, comprising:

in which the TFS was excited by the phosphor emission a pair of matched up-converting and luminescent labeled radiation. probes, wherein the up-converting labeled probe is an Matching of Example 2 60 inorganic phosphor particle comprising at least one rare The attachment of a luminescent label to a probe was earth element and a phosphor host material, and the Studied. Specifically, the biotinylation of a Secondary lumi luminescent label is matched to the up-converting neScent latex particle with a linker arm containing a biotin label.

complex was Studied. The Secondary luminescent Studied 2. The composition according to claim 1, wherein the was a TransFluoSpheres (E) (TFS) latex particle available 65 probes are antibodies, polynucleotides, polypeptide from Molecular Probes of Eugene, Oregon, having an exci hormones, receptor ligands, Streptavidins, StaphylococcuS tation radiation of 488 nm, a particle diameter of 0.35 aureus Protein A, lectins, antigens or mixtures thereof.

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3. The composition according to claim 1, wherein the 12. A method according to claim 6, wherein the Sample is target analyte is a polynucleotide, a polypeptide, a virus, a contacted with two or more matched labeled probe pairs, the microorganism, a hapten, a mammalian cell, a hormone, a matched labeled probe pairs having matched labels with glycoprotein, a blood clotting factor, a lipoprotein, a drug, a different excitation label wavelengths and different emission polysaccharide, a metabolite, a pollutant, a pesticide or label wavelengths, and wherein the matched probe pairs mixtures thereof.

4. The composition according to claim 1, wherein the bind to different target analytes. composition is in the form of an assay kit. 13. A method according to claim 6, wherein the Sample is 5. The composition according to claim 1, wherein the contacted with two or more matched labeled probe pairs, the up-converting label is an up-converting inorganic phosphor matched labeled probe pairs having matched labels with and comprises at least one rare earth element and the probes Similar excitation label wavelengths and different emission are antibodies, polynucleotides, polypeptide hormones, label wavelengths, and wherein the matched probe pairs receptor ligands, Streptavidins, StaphylococcuS aureuS Pro bind to different target analytes.

tein A, lectins, antigens or mixtures thereof. 14. A method according to claim 6, wherein the Sample is 6. A method for detecting an analyte in a Sample with the 15 contacted with two or more matched labeled probe pairs, the composition of claim 1 using matched labels, comprising the matched labeled probe pairs having matched labels with Steps of: different excitation label wavelengths and Similar emission contacting a Sample Suspected of containing a target label wavelengths, and wherein the matched probe pairs analyte with matched up-converting and luminescent bind to different target analytes.

labeled probe pairs, wherein the up-converting labeled 15. A method according to claim 6, wherein the Sample is probe and the luminescent labeled probes are capable contacted with two or more matched labeled probe pairs, the of binding the target analyte to form a labeled probe matched labeled probe pairs having matched labels with target complex; Similar excitation and emission label wavelengths, and illuminating the Sample to excite a label of the labeled 25 wherein the matched labels are attached to probes which probe-target complex, and bind affinitively to different target analytes. observing the Sample for emission radiation from the 16. A method according to claim 6, wherein the target matched label of the labeled probe-target complex, analyte is a polynucleotide, a polypeptide, a virus, a thereby detecting the presence or absence of the ana microorganism, a hapten, a mammalian cell, a hormone, a lyte. glycoprotein, a blood clotting factor, a lipoprotein, a drug, a 7. A method according to claim 6, wherein the polysaccharide, a metabolite, a pollutant, a pesticide and up-converting label is an excitation label and the lumines mixtures thereof.

cent label is an emission label.

8. A method according to claim 7, wherein the illumina antibodies,17. A method according to claim 6, wherein the probes are polynucleotides, polypeptide hormones, receptor tion Step excites the excitation label with radiation having a 35 ligands, Streptavidins, wavelength ranging from about 700 to about 1500 nm. StaphylococcuS aureus Protein A, 9. A method according to claim 6, wherein the lumines lectins, antigens and mixtures thereof.

cent label is an excitation label and the up-converting label 18. A method according to claim 6, wherein one of the is an emission label. labeled probes is bound to a Solid Support. 10. A method according to claim 9, wherein the illumi 40 19. A method according to claim 6, further comprising nation Step excites the excitation label with radiation having before the illumination Step, the Step of Separating the a wavelength ranging from about 350 to about 1500 nm. labeled-probe target complex from any unbound labeled 11. A method according to claim 10, wherein the illumi probes.

nation Step excites the excitation label with radiation having a wavelength ranging from about 350 to about 700 nm.