Up-converting reporters for biological and other assays

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Marks in Cell Separation and Analysis,” Cell Separation 5,102,786 4/1992 Cohen et al. .. ... 435/7.9 Science and Technology (1991) 3:41-58. 5,132,242 7/1992 Cheung ............. ... 436/5O1 Lenth and Macfarlane, Lasers, Optics & Photoincs News, 5,141,740 8/1992 Rajagopalan et al. 424/9.364 (1992) 3:8–15.

5,166,948 11/1992 Gavrilovic et al. .................... 372/70 Louge et al., “Optical Fiber Measurements of Particle Veloc 5,185,265 2/1993 Steen et al. ............................ 436/63 ity Using Laser-induced Phosphorescence, Applied Optics 5,188,942 2/1993 Reddington et al. .................... 435/5 (1991) 30:1976-1981.

5,196,709 3/1993 Berndt et al. ...... 250/458.1 Lovgren et al., “Detection of Lanthanide Chelates by Tim 5,208,651 5/1993 Buican ....... ... 356/346 e-resolved Fluorescence, in Nonisotopic Dna Probe Tech 5,247.339 9/1993 Ogino ..................................... 356/73 5,324,633 6/1994 Fodor et al. ............................. 435/6 niques” Academic Press (1992) 227–261. 5,326.692 7/1994 Brinkley et al. ......................... 435/6 Manashirov et al., “Effect of the Purity of Initial Substances 5,399,315 3/1995 Paz-Pujalt et al. .................... 422/56 of Luminescence Intensity of Erbium in Anti-Stroke Lumi 5,512,493 4/1996 Mathis et al. ...... ... 436/537 nophores,” Chemical Abstracts, (1989) 110:457 Abstract 5,573,909 11/1996 Singer et al. ............................ 435/6 No. 3075OB.

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OTHER PUBLICATIONS Alternative Reagents for Labeling of Antibodies with Metal B.J. Tromberg et al. “Proc. SPIE-INT" Soc. Opt. Eng., Ions,” Anal. Bio. (1989) 176:319–325. Nguyen et al., “Blue-green (450-nm) Upconversion Tm":

(1991) 1427:101–108. Ylf Laser." Applied Optics (1989) 28:3553–3555. Camus et al., “Two-photon absorption spectroscopy in P.A. Santa Cruz, et al., “Quim, Nova” (1983) 6:149-151. ytterbium”, J. Phys. B. Atom. Molec. Phys. (1978) Rich and Pinow, Exploring the Ultimate Efficiency in Infra 11:L395-L397. red-to-Visible Converting Phosphors Activated with Er and Campiglia, A.D. et al., “Utilization of an Inorganic Phosphor Sensitized with Yb, J. Appl. Phys. (1972) 43:2357-2365. as a Reference Signal in Solid-Surface Room Temperature Schindele and Renzoni, “Ultra Fluors: New Fluorophores Phosphorimetry”, Anal. Chem. (1988) vol. for Immunlogical Applications”, J. Clin. Immun. (1990) 60(g):2165-2167. 13:182-186.

D.C. Yeh et al., “J. Appl. Phys”., (1988) 63:4644–4650. Seveus et al., “Time-Resolved Fluorescence Imaging of D.C. Yeh et al., “Phys. Rev. B", (1989) 39:80–90. Europium chelate Label in Immunohistochemistry and in Diamandis and Christopoulos, Detection of Lanthanide Che Situ Hybridization,” Cytometry (1992) 13:329-338. lates and Multiple Labeling Strategies Based on Time-re Silversmith et al., “Green Infrared-Pumped Erbium Upcon solved Fluorescence, in Nonisotopic DNA Probe Tech version Laser,” J. Opt. Soc. Am. (1982) 3:128-12. niques, Academic Press (1992) 263-274. Smart et al., “Cw Room Temperature Upconversion Lasing D.R. Tallant et al., J. Chem. Phys., (1975) 63:2074–2085. at Blue, Green and Red Wavelengths in Infrared-Pumped Eichstein et al., “Laser-excited Time-resolved Solid-phase Pr-doped Fluoride Fibre.” Electronics Letters (1991)

Fluoroimmunoassays with the New Europium Chelate 4, Soini and Kojola, “Time-resolved Fluorometer for Lan 7-bis (chlorosulfophenyl)-1, 10-phenanthroline-2, 9-dicarboxylic Acid as Label.” Anal. Chem. (1988) thanide Cachelates-a New Generation of Nonisotopic 60:1069-1074. Immunoassays,” Clin Chem. (1983) 29/1:65–68. Evangelista et al., “Enzyme-amplified Lanthanide Lumines Soules and Hoffman, “Luminescent Materials (Phosphors).” cence for Enzyme Detection in Bioanalytical ASSayS, Encyclopedia of Chemical Technology, (1981) Third Edi

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

Xu and Hemmila, “Co-fluorescence Enhancement System

Hemmila et al., “Europium as a Label in Time-resolved Based on Pivaloyltrifluoroacetone and Yttrium for the Immunofluorometric Assays,” Anal. Biol. (1984) Simultaneous Detection of Europium, Terbium, Samarium 137:335-343. and Dysprosium,” Anal. Chimica Acta. (1992) 256:9-16. Johnson et al., “Infrared-to-visible Conversion by Rare-e- Wojciechowski, et al., “Infrared-to-Blue Up-converting arth Ions in Crystals,” J. Appl. Phys. (1972) 43:3. Phosphor.” Electron Technology (1978) 11:3:31-47. Johnston and Wright, “Trace Analysis of Nonfluorescent Moser, K., et al., “Infrared Spectral Distribution of Photo Ions by Associate Clustering with a Fluorescent Probe.” conductivity and Up-conversion in GaP Light Emitting Anal. Chem. (1979) 51:1774–1780. Diodes,” J. Appl Phys, (1985) 57:12:5438–5444. Kano et al., NaLnF:YB", (Ln:Y.Gd, La): Efficient Green Tanabe, S., et al., “Up-conversion Fluorescences of Te0 emitting Infrared-excited Phosphors, J. Electrochem. Soc. and Ga-O-Based Oxide Glasses Containing Er” Journal (1972) 119:1561–1564. of Non-Crystalline Solids, (1990) 122:79-82.

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Franz, K. A., et al., “Luminescent Materials.” Ullmann's Wollenberger, L.V. et al. “Detection of DNA Using Upcon Encyclopedia of Industrial Chemistry, 5th Edition, verting Phosphor Reporter Probes,” SRI International. A15:519-558. Riris, H. et al., “A Compact Upconverting Phosphor Detec “TransFluoSpheres Fluorescent Microspheres-A Break tion System for Wick Assays, SRI International. through in Microsphere Technology,” Molecular Probes, Mufti, N.A., et al., “Design and Manufacture of Capillary 114-115. Wicks for Ultrasensitive Detection of Antigenic and Nucleic Wright, W.H., et al. “High-Sensitivity Immunoassay Using Acid Analytes,” SRI International. A Novel Upconverting Phosphor Reporter, SRI Interna tional. * cited by examiner

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Visible Light Out = Negative Somple

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Coated n 4. Phosphor E. Optic

Target linked

Capture Surface

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

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ADO ANTIGEN

COATED

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EMITTED LIGHT

FOCAL (HIGH

INTENSITY

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EXCITATION LIGHT

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COMPETITIVE HOMOGENOUS

ANTIGEN CAPTURE ASSAY

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

Homogeneous Immunoprecipitation Assay

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FIG. 31 A FS 10 x 10 Gridded Array of 980 nm Diode Lasers

Aqueous

Sample Flow

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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 Radioisotopic labels possess Several advantages, Such as: BIOLOGICAL AND OTHER ASSAYS very high Sensitivity of detection, very low background

CROSS REFERENCE TO RELATED

Signal, and accurate measurement with precision radiometric

APPLICATION

instruments (Scintillation and gamma counters) or with inexpensive and Sensitive autoradiographic techniques.

This application is a divisional application of application However, radioisotopic labels also have Several Ser. No. 09/016,402 filed Jan. 30, 1998, U.S. Pat. No. disadvantages, Such as: potential health hazards, difficulty in 6,159,686, which is a continuation-in-part of application Ser. disposal, Special licensing requirements, and instability No. 08/962,673 filed Nov. 3, 1997, which is a continuation (radioactive decay and radiolysis). Further, the fact that in-part of application Ser. No. 08/482,203 filed Jun. 7, 1995, radioisotopic labels typically do not produce a strong (i.e., now U.S. Pat. No. 5,698,397 which is a division of appli non-Cerenkov) signal in the ultraViolet, infrared, or visible cation Ser. No. 08/416,023 filed Mar. 30, 1995, now U.S. portions of the electromagnetic spectrum makes radioiso Pat. No. 5,674,698, which is a continuation-in-part of appli topes generally unsuitable as labels for applications, Such as cation Ser. No. 08/381,006 (now abandoned), filed Jan. 30, microScopy, image spectroscopy, and flow cytometry, that 1995, which is a continuation of Ser. No. 07/946,068 filed 15 employ optical methods for detection. Sep. 14, 1992 (now abandoned). This application also claims For these and other reasons, the fields of clinical priority benefits to provisional application Ser. No. 60/037, chemistry, water and air monitoring, and biomedical 392 filed Feb. 2, 1997. Each of these applications is spe research have Sought alternative detectable labels that do not cifically 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 25 (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 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 calo 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 35 Signal amplification, Since a single enzyme molecule typi medicine. Examples of Such detection methods include: cally has a persistent capacity to catalyze the transformation immunoassays, immunochemical Staining for microscopy, 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 40 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 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 45 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 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 50 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 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 examples radioimmunoassays (RIA) have been 55 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 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, 60 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 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 65 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. an appropriate excitation frequency and the resultant Spec

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tral emissions can be detected by electro-optical Sensors or as a noise Source. More importantly perhaps, phycobilipro light microScopy. teins and cyanine dyes possess Several distinct disadvan A wide variety of fluorescent dyes are available and offer tages: (1) emission in the red, far red, and near infrared a Selection of excitation and emission spectra. It is possible region is not well-Suited for detection by the human eye, to Select fluorophores having emission spectra that are hampering the use of phycobiliprotein and cyanine labels in Sufficiently different So as to permit multitarget detection and optical fluorescence microscopy, (2) cyanines, discrimination with multiple probes, wherein each probe phycobiliproteins, and the coupled accessory molecules Species is linked to a different fluorophore. Because the (e.g., Azure A) are organic molecules Susceptible to pho Spectra of fluorophores can be discriminated on the basis of tobleaching and undergoing undesirable chemical interac both narrow band excitation and selective detection of tions with other reagents, and (3) emitted radiation is down emission Spectra, two or more distinct target Species can be converted, i.e., of longer wavelength(s) than the absorbed detected and resolved (Titus et al. (1982) J. Immunol. excitation radiation. For example, AZure A absorbs at 632 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). 15 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 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 25 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 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: 35 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 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. 40 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 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 45 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 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 50 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 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 55 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 fluorescence, excitation light Scattering and refraction, etc.) tral overlap between excitation frequencies and emission that must be rejected (e.g., by filters or time-gated signal frequencies. An alternative approach has been to use cyanine 60 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). 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 65 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

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S 6 organic dye, Hoechst 33258, using a stream of Strongly case of a single reporter, the apparatus includes a laser diode focused laser pulses (Denk et al. (1990) Science 248: 73). capable of emitting light at one or more wavelengths in the The organic fluorphore used by Denk et al. was significantly reporter's excitation band and a detector that is Sensitive to 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 different emission bands. Where the excitation bands differ, having a wavelength shorter than the pump light (i.e., multiple up-converted emission). 15 laser diodes emitting at respective appropriate Thus, there exists a significant need in the art for labels wavelengths are combined using a wavelength division and detection methods that permit Sensitive optical and/or multiplexer or other Suitable techniques, Such as frequency spectroscopic detection of specific label signal(s) with labeling, frequency modulation, and lock-in detector device. essentially total rejection of nonspecific background noise, If the emission bands are different (whether or not the and which are compatible with intact viable cells and bands excitation bands are different), light in the different emission aqueous or airborne environments. is separated and Sent to multiple detectors. If the 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 25 diodes can be modulated at different characteristic frequen by virtue of prior invention. cies and lock-in detection performed. SUMMARY OF THE INVENTION 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 35 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 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 40 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 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 45 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 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 50 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 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 55 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 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 60 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 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 65 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

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ceSSes involving either direct absorption or energy transfer, biochemical, or immunological reaction, including binding Such spatially-controlled deposition of energy can be used to reactions. For example, the invention may be used to moni produce localized damage and/or to probe the chemical tor the progreSS of ligand-binding reactions, polynucleotide 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 15 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 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 25 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 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 heterobifunctional 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 35 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/ 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 40 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 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. 45 FIGS. 5A, 5B, 5C show schematically energy state tran The invention also provides biochemical assay methods Sitions 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 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 50 array magnetically and/or optically trapping particles that com bindingused to detect emissions from a large plurality of Sites, prise the analyte and the labeled probe. In one embodiment, a Sandwich assay is performed, wherein an immobilized lensFIG. 7B shows the CCD array used in conjunction with a array;

probe, immobilized on a particle, binds to a predetermined analyte, producing an immobilization of the bound analyte 55 FIG. 8 shows an embodiment using optical trapping, on the particle; a Second probe, labeled with an FIG. 9 ShowS Schematically dye coating and encapsula up-converting label can then bind to the bound analyte to tion of an up-converting phosphor particle; produce a bound Sandwich complex containing an 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 60 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 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 65 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,

Page 39

FIG. 13 is a time-decay measurement of the phosphor FIG. 31B is a detailed view of a small section of the luminescence at 541.0 nm after termination of excitation device shown in FIG. 31A.

illumination for sodium yttrium fluoride-ytterbium/erbium; FIG. 32 is an emission Spectrum for up-conversion from FIG. 14 shows the phosphor emission intensity as a neodymium chelated in EDTA.

function of excitation illumination intensity for a Sodium 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(YosYboEroos) F. Unless defined otherwise, all technical and Scientific following excitation with 200 W/cm 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(YosYbo.12 Eroos)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 15 of the present invention, the following terms are defined

FIG. 17B shows a fluorescence spectrum scan of an up-converting phosphor reporter coated with Streptavidin in below.

Hepes-buffered saline induced by excitation with a 970-nm 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 up-converting phosphor reporter in Hepes-buffered Saline excited label is typically electromagnetic radiation in the 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 up-converting phosphor reporter coated with Streptavidin in labels, which means that the chemical Substituent typically absorbs at least two photons at an excitation frequency and

Hepes-buffered saline with monochromatic detection of 25 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 (YosYboosEros)2O2S showing the relationship 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 35 phosphor particle. The label can alternatively comprise a FIG. 21 shows schematically the detection and discrimi lanthanide ion in a chelate or cage compound. nation of two cell Surface antigens with Specific antibodies AS used herein, a "probe' refers to a binding component labeled with two phosphors with distinct phosphorescence which binds preferentially to one or more targets (e.g., characteristics. antigenic epitopes, polynucleotide Sequences, macromo FIG. 22 shows a Schematic of an apparatus for phase 40 lecular receptors) with an affinity Sufficient to permit dis Sensitive detection. crimination of labeled probe bound to target from nonspe FIG. 23 show a Schematic of a competitive homogeneous cifically bound labeled probe (i.e., background). Generally, assay using phosphors as labels and fiber optic illumination the probe-target binding is a non-covalent interaction with a at a capture Surface. binding affinity (KD) of at least about 1x10''', prefer FIG. 24 show a Schematic of a competitive homogeneous 45 ably with at least about 1x107M, and more preferably with antigen capture assay using phosphors as labels and a an affinity of at least about 1x10M or greater. Antibodies convergent illumination beam focused on the capture Sur typically have a binding affinity for cognate antigen of about face. 1x10"M" or more. For example but not limitation, probes FIG. 25 shows a Schematic of a homogeneous immuno 50 of the invention include: antibodies, polypeptide hormones, precipitation assay using phosphors as labels and a conver tein A, receptorStreptavidin, polynucleotides, StaphlyOCOccus aureuS pro gent illumination beam focused on the capture Surface hormones), leucineligands (e.g., Steroid or polypeptide Zipper polypeptides, lectins, antigens wherein the capture Surface collects immunoprecipitates. (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 55 AS used herein, a “probe-label conjugate' and a "labeled using a microscope.

FIG. 27 is a block diagram of a microtiter plate reader for probe' a probe.

refer to a combination comprising a label attached to

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 phosphors in three test wells. 60 a label (e.g., multiple antibody molecules may be attached to FIG. 29 is a schematic view of a second embodiment of a Submicron-size inorganic up-converting phosphor bead). a hand-held probe for carrying out the present invention. Various attachment chemistries can be employed to link a FIG. 30 illustrates a three channel configuration using label to a probe, including, but not limited to, the formation interference filters. of covalent bonds, hydrogen bonds, ionic bonds, electro FIG. 31A is an illustration of an embodiment of the 65 Static interactions, and Surface tension (phase boundary) invention in which a diode laser array F1 and a detector interactions. Attachment of label-can also involve incorpo array F2 are combined in a single device. ration of the label into or onto microSpheres, microparticles,

Page 40

immunobeads, and Superparamagnetic magnetic beads sponding to a particular mRNA sequence, a portion of a (PolySciences, Inc., Warrington, Pa.; Bangs Laboratories, genomic clone, a Synthetic oligonucleotide having Sufficient Inc.,979 Keystone Way, Carmel, Ind. 46032). For example, Sequence homology to a known target Sequence (e.g., a 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 polyacrolein) 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 15 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 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 25 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). 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 imnmunoglobulin 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 35 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 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 40 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 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 45 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 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 50 with, illumination of the up-converting label with one or antibodies, immunoglobulins 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., 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 55 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 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 60 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). 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 65 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

Page 41

marker) of the location of the probe(s). The up-converting from available Sources (e.g., handbooks and published labels can be attached to various probes, Such as antibodies, references) or may be obtained by generating a standard Streptavidin, protein A, polypeptide ligands of cellular ization curve measuring quantum conversion efficiency as a 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 host material such as barium-yttrium-fluoride. The ytter

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

quantum conversion efficiency to produce a detectable Sig The optimum ratio of absorber (e.g., ytterbium) to the nal; however, for any particular application, the size of the emitting center (e.g., erbium, thulium, or holmium) varies, phosphor particle(s) to be used should be selected at the depending upon the Specific absorber/emitter couple. For discretion of the practitioner. For instance, Some applica 55 example, the absorber:emitter ratio for Yb:Er couples is tions (e.g., detection of a non-abundant cell Surface antigen) typically in the range of about 20:1 to about 100:1, whereas may require a highly Sensitive phosphor label that need not the absorber:emitter ratio for Yb:Tm and Yb:Ho couples is be Small but must have high conversion efficiency and/or typically in the range of about 500:1 to about 2000:1. These absorption cross-section, while other applications (e.g., different ratios are attributable to the different matching detection of an abundant nuclear antigen in a permeablized 60 energy levels of the Er, Tm, or Ho with respect to the Yb cell) may require a very Small phosphor particle that can level in the crystal. For most applications, up-converting readily diffuse and penetrate Subcellular structures, but phosphors may conveniently comprise about 10-30% Yb which need not have high conversion efficiency. Therefore, and either: about 1-2% Er, about 0.1-0.05% Ho, or about the optimal size of inorganic phosphor particle is application 0.1-0.05% Tm, although other formulations may be dependent and is Selected by the practitioner on the basis of 65 employed.

quantum efficiency data for the various phosphors of the Some embodiments of the invention employ inorganic invention. Such conversion efficiency data may be obtained phosphors that are optimally excited by infrared radiation of

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about 950 to 1000 nm, preferably about 960 to 980 nm. For quantum conversion efficiencies (e.g., low doping levels of example but not limitation, a microcrystalline inorganic activator couple), but which have other desirable character 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 5 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 for extremely Sensitive signal detection, particularly when maxima in the red or green portions of the visible Spectrum, intense depending upon the phosphor host, ytterbium-holmium radiation.laser illumination is used as the Source of excitation Thus, the unique property of up-conversion of couples generally emit maximally in the green portion, photon energy by up-converting phosphors makes possible ytterbium-thulium typically have an emission maximum in the detection of very Small particles of microcrystalline the blue range, and ytterbium/erbium usually emit maxi inorganic phosphors. For practical implementation of phos mally in the green range. For example, Yoo YbooErooF phors as ultrasensitive reporters, particularly as intracellular emits maximally in the green portion of the Spectrum. 15 reporters, it is essential that the grain size of the phosphor be Although up-converting inorganic phosphor crystals of as Small as practicable (typically less than about 0.3 to 0.1 various formulae are Suitable for use in the invention, the mu m), for which laser-excited up-converting phosphors are following formulae, provided for example and not to limit well-Suited.

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

Na(Y.Yb, Ho.)F: X is 0.7 to 0.9, y is 0.0995 to 0.2995, TABLE I

Phosphor Material Compositions

Host Material Absorber Ion Emitter Ion Color

YYb,

to 0.12. Oxysulfides (OS)

(Yoss Yboos Eroos)2O is a relatively efficient YOS Ytterbium Erbium Green up-converting phosphor material. Gd2O2S Ytterbium Erbium Red For exemplification, but not to limit the invention, LaOS Ytterbium Holmium Green ytterbium(Yb)-erbium(Er)-doped yttrium oxysulfides lumi Oxyhalides (OXv) nesce in the green after excitation at 950 nm. These are YOF Ytterbium Thulium Blue non-linear phosphors, in that the ytterbium acts as an YOCl, Yterbium Terbium Green “antenna' (absorber) for two 950 nm photons and transfers 35 Fluorides (F) its energy to erbium which acts as an emitter (activator). The YF, Ytterbium Erbium Red critical grain size of the phosphor is given by the quantum GdF. Ytterbium Erbium Green yield for green emission and the doping level of both Yb and LaF Ytterbium Holmium Green Er, which is generally in the range of about 1 to 10 percent, NaYF, Ytterbium Thulium Blue more usually in the range of about 2 to 5 percent. A typical 40 BaYF, Ytterbium Thulium Blue

Yb:Er phosphor crystal comprises about 10-30% Yb and Gallates (G.Q.)

about 1-2%. Er. Thus, a phosphor grain containing Several thousand formula units ensures the emission of at least one YGaO. Ytterbium Erbium Red or more photons during a typical laser irradiation time. YGSO12 Ytterbium Erbium Green However, the nonlinear relationship between absorption and 45 Silicates (SiO) emission indicates that intense illumination at the excitation YSiOs Ytterbium Holmium Green wavelength(s) may be necessary to obtain Satisfactory signal YSiO, Ytterbium Thulium Blue in embodiments employing very Small phosphor particles (i.e., less than about 0.3 mu m). Additionally, it is usually In addition to the materials shown in Table I and variations desirable to increase the doping levels of activator/emitter 50 thereof, aluminates, phosphates, and Vanadates can be Suit couples for producing very Small phosphor particles So as to able phosphor host materials. In general, when Silicates are maximize quantum conversion efficiency. used as a host material, the conversion efficiency is rela Inorganic microcrystalline phosphors with rare earth acti tively low. In certain uses, hybrid up-converting phosphor Vators generally have narrow absorption and line emission crystals may be made (e.g., combining one or more host Spectra. The line emission spectra are due to f-f transitions 55 material and/or one or more absorber ion and/or one or more within the rare earth ion. These are shielded internal tran emitter ion).

Sitions which result in narrow line emission. Exemplary up-converting phosphorS eXcited at about 980 In certain applications, Such as where highly Sensitive nm include, but are not limited to: YosoYbolisEroo)Fs, detection is required, intense illumination can be provided Yo...s 7Ybo.13 Timo.o.o. 1) F3; Yo...so Ybo.1 os Hoo.o.o.2) F3; by commercially available Sources, Such as infrared laser 60 Gido. so Ybo.1s Ero.o.2) F3; G do. 87Ybo.13 Timo.o.o 1) F3; Sources (e.g., continuous wave (CW) or pulsed Semiconduc Gdoso Ybo.1os Hoooo...)Fa; Yoss Yboos Eroos)2O2S, tor laser diodes). For example, in applications where the Yo.87Ybo.13 Tmolool)2O2S: YosoYbo.1osHoooo.2)2O2S, microcrystalline phosphor particle must be very Small and Gdo so Yboos Eroos)2O2S: Gdo.87Ybo.13Tmo.o.o.)2O2S, the quantum conversion efficiency is low, intense laser Gdosoyboioshoooo2)2O2S.

illumination can increase signal and decrease detection 65 Exemplary up-converting phosphorS eXcited at about times. Alternatively, Some applications of the invention may 1500 nm include, but are not limited to: Yoo. Eroo)2O2S, require phosphor compositions that have inherently low Gdoos Eroos)2O2S.

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Preparation of Inorganic Phosphor Labels including a polysulfide flux for annealing. Using this Techniques and methods for manufacture of inorganic technique, highly efficient OXySulfide particles in the 0.3 to phosphorS has been described in the art. Up-converting 0.4 um diameter range were prepared as a dispersion in phosphor crystals can be manufactured by those of ordinary water. Frequently, Sonication can be used to produce a skill in the art by various published methods, including but monodisperse mixture of discrete Spherical particles. After not limited to the following: Yocom et al., (1971) Metallur fractionation and coating, these particles can be used as gical Transactions 2: 763; Kano et al., (1972). J. Electro up-converting reporters. Furthermore, this general prepara chem. Soc., p. 1561; Wittke et al. (1972).J. Appl. Physics 43: tive procedure is Suitable for preparing much Smaller phos 595; Van Uitert et al. (1969) Mat. Res. Bull. 4: 381; which phor particles (e.g., 0.1 um diameter or Smaller), which may are incorporated herein by reference. Other references which be advantageous for various assay formats. may be referred to are: Jouart J. P. and Mary G. (1990).J. Frequently, Such as with phosphors having an oxySulfide Luminescence 46: 39; McPherson G. L. and Meyerson S. L. host material, the phosphor particles are preferably dis (1991) Chem. Phys. Lett. (April) p. 325; Oomen et al: (1990) persed in a polar solvent, such as acetone or DMSO and the J. Luminescence 46: 353; NIH and R and SC (1991) Optics like, to generate a Substantially monodisperse emulsion Lett. 16 (September); McFarlane R. A. (1991) Optics Lett. 15 (e.g., for a stock Solution). Aliquots of the monodisperse 16 (September); Koch et al. (1990) Appl. Phys. Lett. 56: Stock Solution may be further diluted into an aqueous 1083; Silversmith et al. (1987) Appl. Phys. Lett. 51: 1977; Solution (e.g., a Solution of avidin in buffered water or Lenth W. and McFarlane R. M. (1990).J. Luminescence 45: buffered saline).

346; Hirao et al. (1991) J. Non-crystalline Solids 135:90; It was found that washing phosphors in acetone or DMSO McFarlane et al. (1988) Appl. Phys. Lett. 52: 1300, incor improved Suspendability of inorganic phosphor particles in porated 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 allowed to cool to room temperature under continuous a conventional barrel mill with Zirconia and/or alumina balls 25 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 gum arabic, alginate, guaiac gum) can be used to promote Vided herein. Fractions having a particular particle size 35 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, Resuspended phosphors in organic Solvent, Such as such as with a Horiba Particle Analyzer. 40 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 Small amount of organic material (e.g., formaldehyde, tion assay using up-converting inorganic phosphors depends 45 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 particles, but which can be readily Silanized with organo can affect the number of potential binding sites per particle 50 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 L., and Smith, C., eds.; p. 59-64, Huls America, Piscataway, hydroxy carbonate particles are formed by the hydrolysis of 55 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 example, particles may be coated with a polycarboxylic acid Spherical particles in the Submicron size range by precipi 60 (e.g., Addition XW 330, Hoechst, Frankfurt, Germany or tation. Tamol, see Beverloo et al. (1992) op.cit.) during milling to However, after precipitation it is typically necessary to produce a stable aqueous Suspension of phosphor particles, anneal the oxide in air at about 1500 C., which can cause typically at about pH 6-8. The pH of an aqueous solution of faceting of the Spherical particles which can generate aggre phosphor particles can be adjusted by addition of a Suitable gate formation. Faceting can be Substantially reduced by 65 buffer and titration with acid or base to the desired pH range. converting the Small spherical particles of the oxide or Depending upon the chemical nature of the coating, Some hydroxy carbonate precursor to the oxySulfide phase by minor loSS in conversion efficiency of the phosphor may

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occur as a result of coating, however the power available in binding assays to detect and quantitate the presence of a laser excitation Source can compensate for Such reduction analyte(s) in a Sample. Binding reagents are labeled directly in conversion efficiency and ensure adequate phosphor emis by attachment to up-converting reporters (e.g., Surface Sion. 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), Staph lococcuS 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. can be indirectly labeled by noncovalent binding to a For example, Such polymer functionalities include: carboxy lic acids (e.g., 5% acrylic acid/95% methyl acrylate directlyantibody labeled Second antibody (e.g., a goat anti-rabbit linked to an up-converting inorganic phosphor).

copolymer), amine (e.g., 5% aminoethyl acrylate/95% 15 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 from the signal emitted by the unbound labeled probe, hence encapsulated phosphors in an aqueous Solution (i.e., 25 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 Biochem. 43: 109), the sandwich radioimmunoassay (U.S. potential crosslinking sites per phosphor, without having to 35 Pat. No. 4,376,110, which is incorporated herein by Substantially change the encapsulation process. reference), and the antibody/lectin sandwich assay (EP0166 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 Sensitive and reliable than homogeneous assayS. in three steps: (1) bind the crosslinker to the polymer coating 40 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 are aqueous Solutions containing a buffer System (e.g., 50 between biological macromolecules and So reduces irrevers 45 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: it is usually desirable to wash the Sample and the Solid 50 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 Sample is detected and quantified by contacting the Sample carboxylate N-hydroxysuccimide Proteins (e.g., 55 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

Oxysuccimide (ANB-NOS) measuring the presence of label present in the bound com N-succinimidyl (4-iodoacetyl) plexes. A probe-label conjugate can include a directly aminobenzoate (SIAB) 60 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 lated polynucleotide that is detected by labeled streptavidin). Up-converting phosphors and up-converting organic dyes 65 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 binding reagents, either directly or indirectly, for use in by incorporating at least one washing Step, So as to remove

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background Signal attributable to label present in unbound usually performed by first incubating the Sample with a probe-label conjugate(s). Hence, it is usually desirable to blocking or prehybridization solution, followed by incubat incubate probe-label conjugate(s) with the analyte sample ing the Sample with probe under binding conditions for a under binding conditions for a Suitable binding period. Suitable binding period, followed by Washing or otherwise Binding conditions vary, depending upon the nature of the removing unbound probe, and finally by detecting the probe-label conjugate, target analyte, and Specific assay presence, quantity, and/or location of bound probe. The Step method. Thus, binding conditions will usually differ if the of detecting bound probe can be accomplished by detecting probe is a polynucleotide used in an in Situ hybridization, in label, if the probe is directly labeled, or by incubating the a Northern or Southern blot, or in solution hybridization bound complex(es) with a Second binding reagent (e.g., assay. Binding conditions will also be different if the probe streptavidin) that is labeled and which binds to the probe, is an antibody used in an in Situ histochemical Staining thus accomplishing indirect labeling of the probe. method or a Western blot (Towbinet al. (1979) Proc. Natl. Up-converting labels are attached to probe(s) or Second Acad. Sci. (U.S.A.) 76: 4350, incorporated herein by binding reagents that Specifically or preferentially bind to reference). In general, binding conditions are Selected in probe(s) by any of the various methodologies discussed accordance with the general binding methods known in the 15 herein. Additionally, up-converting phosphor particles can art. For example, but not for limitation, the following be encapsulated in microSpheres and coated with a probe binding conditions are provided for general guidance: (e.g., a specific antigen or antibody) for use as a labeled For antibody probes: probe in an immunodiagnostic assay or nucleic acid hybrid 10–200 mM Tris, pH 6-8; usually 100 mM Tris pH 7.5 ization assay to detect an analyte in a Sample, Such as the 15–250 mM NaCl; usually 150 mM NaCl presence of an antibody, Virus, or antigen in a bloom Serum 0.01-0.5 percent, by volume, Tween 20 sample, according to the method of Hari et al. (1990) 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 For polynucleotide probes: 25 the phosphor with a monomer Solution and polymerizing the 3–10xSSC, pH 6-8; usually 5xSSC, pH 7.5 monomer to generate a polymer shell encasing the phosphor 0-50 percent deionized formamide particle. Phosphor particles embedded in a polymer coating, 1-10xDenhardt's Solution Such as a gel coating, can be functionalized (e.g., with amino 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 salmon 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 Manual (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 Phosphorit1 and Phosphorit2) different oligopeptides affixed to a Solid Support). One or which differ in their absorption and/or emission spectra So as more of the Species of first binding component may bind to to facilitate discrimination of the two phosphors based on a particular analyte (e.g., a muscarinic receptor) in an absorption and/or emission wavelengths, e.g., one phosphor analyte Solution that is in contact with the Solid Support. may emit in the blue and the other may emit in the green. For Binding of the analyte to one or more of the first binding example and not limitation, Na(YosoYbolisEroo)F emits component Species may then be detected with a Second predominantly in the green, and Na(Yoz-Ybo, Tmooo...)F binding component (e.g., an anti-muscarinic receptor emits predominantly in the blue, and thus these two phos antibody) labeled with an up-converting phosphor (either phors may be discriminated on the basis of their phospho directly or through a biotinylated Secondary antibody). rescent emissions. Alternatively, two phosphors may pro Solid Substrates can be attached to a first binding com duce essentially similar emission Spectra but may have ponent which can bind more than one distinct analyte (e.g., different excitation wavelengths which provide a basis for may be immunocrossreactive or polyspecific) and/or can be their discrimination in multiple analyte detection. A first attached to multiple first binding component Species which binding component (e.g., an antibody) that binds specifically can bind multiple distinct analytes. Similarly, multiple Sec to a first analyte species (e.g., a lymphocyte CD4 antigen) 15 ond binding component Species with binding Specificities and incorporates biotinyl moieties which may be bound by for particular analytes can be employed. When multiple Streptavidin-Phosphorif1 conjugates can be used to quanti Second binding component Species are employed, it is typi tatively detect the presence of a first analyte in a Sample cally desirable to label each Second binding component (e.g., a serum Sample) by measuring phosphorescence of Species with a unique up-converting label that can be Phosphorif1 in analyte-binding component complexes. A distinguished on the basis of its absorption and/or emission Second binding component (e.g., a probe polynucleotide) properties.

that binds specifically to a second analyte species (e.g., an It is possible to use different absorbers in combination HIV-1 sequence) and incorporates digoxygenin moieties with various emitters to produce a collection of phosphors (e.g., 11-UTP-digoxygenin) which may be bound by having Several differentiable combinations of excitation and antidigoxigenin-Phosphorif2 conjugates can be used to 25 emission Spectra. For example but not limitation, Six differ quantitatively detect the presence of a Second analyte in the entiable phosphors may be generated from two absorbers Sample by measuring phosphorescence of Phosphorit2 in and three emitters. A first absorber, A, has an excitation analyte-binding component complexes. Thus, by Simulta wavelength of 2A, a Second absorber, A has an excitation neously or contemporaneously detecting the presence of wavelength of 2A, a first emitter, E, has an emission line multiple phosphor reporters having differentiable Signal at 21, a Second emitter, E2 has an emission line at 22, and characteristics, multiple analytes may be quantitatively a third emitter, Es, has an emission line at 2. The Six detected in a Single Sample. phosphors may be differentiated and the Signal from each Sandwich Binding ASSays individually quantitated by illuminating the sample with an Up-converting phosphors labels can be used as reporters excitation wavelength ) and detecting separately the emit for sandwich binding assays (U.S. Pat. No. 4,376,110, which 35 ted radiation at 21, 22, and 23, and Separately illuminat is incorporated herein by reference). For example, a mag ing the Sample with 2A and detecting Separately the emitted netic bead, Such as a Superparamagnetic immunobead or radiation at 2, 22, and 2. Table II shows the Various functionalized magnetizable polymer particle (PolySciences, absorber:emitter combinations and their excitation and Inc., Warrington, Pa.), can serve as the Solid Substrate which emission wavelengths.

has an immobilized first binding component (e.g., an 40 antibody, a polynucleotide, or a lectin) that binds to a first TABLE II epitope (i.e., a binding locus: an antigenic determinant, Absorber:

Sugar moiety, chemical Substituent, or nucleotide Sequence) Emitter of an analyte. The analyte binds to the first binding com Combination Excitation w Emission w ponent and also to a second binding component (e.g., an 45 antibody, a lectin, or a polynucleotide) which binds to a

Second epitope of the analyte. Thus, the analyte bridges the 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 incorporated label, Such as a biotinyl group which can be bound to a Streptavidin-coated up-converting phosphor. Of course, additional absorber:emitter combinations are Alternatively, the Second binding component can be linked possible to provide more than six differentiable phosphor directly to an up-converting phosphor, Such as through a labels.

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

up-converting reporter. The Sandwich complex is thus 60 For example and not limitation, the following three brief immobilized on the Solid substrate, although the solid Sub examples are provided to explicate further possible appli Strate itself may be mobile (e.g., a Superparamagnetic bead cations of multiple analyte Sandwich assay methods. circulating in a sample slurry). The presence and amount of Substrate Differentiation analyte(s) can be quantitatively measured by detecting the The following example describes the use of distinguish presence of up-converting reporter in Sandwich complexes. 65 able Substrate types to detect the presence of Specific immu For example, a Solid Substrate may have a plurality of noglobulin idiotypes in a sample (e.g., a blood Serum sample distinct species of first binding component (e.g., an array of taken from a patient) which can provide diagnostic infor

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mation about the immune Status of a patient to (e.g., is a phosphors are readily distinguishable, ), and 2 may be patient Seroreactive with a particular antigen). identical. The standardized relative intensities of the two 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 15 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. Superparamagnetic beads having Specifically 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 25 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 an excitation wavelength ), 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 35 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 nation with 21, 2, and 2, 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 21-induced red light emission associated with Various isoforms of APP arise in the brain as a consequence 40 the large beads is a rough measure of the amount of cells of alternative eXon usage and/or alternative proteolytic pro having both CD4 and CD2 surface antigens and/or the cessing pathways. Thus, although all APP isoforms may relative abundance of those Surface antigens (e.g., there may share a common, hypothetical epitope (X), a particular APP be very few CD4+ cells that have CD2, but those few cells isoform may have a unique epitope (Y), while another APP may have a large amount of CD2 antigen, and hence a large isoform has a unique epitope (Z). It is possible that the 45 CD2 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 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 50 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 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 55 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 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 60 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 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 65 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 mine the bead type (size, shape, and/or color), and Scan for

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presence and abundance of particular phosphors (by illumi Sample, where (1) the phosphor emits at a wavelength that nating with excitation wavelength(s) and detecting emitted is either directly cytotoxic and/or (2) the phosphor emits at 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, 15 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, 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) oxygen mol pellet is resuspended), biotinylated magnetic beads are ecule. 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. (Jan. 19-21, 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 25 10, Bellingham, Wash. 98277; Pelegrin et al. (1991) Cancer tion emitted by the phosphor is used, typically in conjunc 67: 2529; Wagnieres et al. (May 24–25, 1991) Future tion with a dye molecule, to produce localized intense Directions and Applications of Photodynamic Therapy, pp. electromagnetic radiation in an area adjacent to the probe for 219; Folli et al. (Dec. 17, 1991) Fluoresceine Clinique 4; various purposes other than detection (e.g., cytotoxicity, Braichotte 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 be ngal or phthalocyanine de rivative S, 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 35 phosphor is used, whereas for rose bengal agreen-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), 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 40 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., 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 45 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 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 50 a step beyond the traditional photodynamic therapy methods cell membranes). in that the red light can be used both for tracking and Since phosphor-emitted radiation is isotropic, it is gener 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 55 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 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 60 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 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 cytotoxic and/or is of an appropriate wavelength to activated cell Sorter or the like). Thus, an up-converting 65 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 pSoralen, compounds containing azide Substituents or other

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photoactivated groups). Alternatively, histidine side chains energy pooling, croSS relaxation, or avalanche absorption are of polypeptides can be oxidized by light in the presence of not appropriate for up-converting chelates because they rely dye Sensitizers, Such as methylene blue or rose bengal on energy transfer between ions. These processes are (Proteins Structures and Molecular Principles, (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 15 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 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 25 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 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 down-conversion. That is, the rescence by virtue of its higher energy. Furthermore, auto emission light is at a wavelength which is longer than the fluorescence 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 35 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 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. State using two photons enables raazacyclotetradecanetetraacetic acid (TETA), as well as 40 one to take advantage of the increasing two-photon croSS antibiotics, natural chelating proteins, phthalocyanines, and Sections as one approaches the S. State using two-photon cryptates. Methods for preparation of lanthanide chelates 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 Hemmila et al. (1984) Anal. Biochem. 137: 335, Soini and 45 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 (E&) Academic Press, New York, 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 50 650 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. 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, neodymium, thulium, holmium, and praseodymium. expected in the 850 to 950 nm range. An example of a blue Other candidate ions include the other lanthanide elements, 55 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 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 Substituents. 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 60 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 Phys. Lett. 52:1300), 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 65 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 Elec up-conversion laser Schemes that rely on energy transfer, tronics 19 (1987), 327–349; C. H. Chen and M. P. McCann,

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Optics Commun. 63 (1987), 335; J. P. Hermann and J. Illumination may be continuous or pulsed, or may combine Duculing, Optics Commun. 6 (1972), 101; B. Foucault and J. continuous wave (CW) and pulsed illumination where mul 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 A329 (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 15 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 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 25 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, 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 run. cost and additional size to apparatus. The laser diode or Organic dyes absorbing in the red have to absorb two 35 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 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. 40 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 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, 45 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 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 50 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 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 55 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 ally desirable, as it provides a method for recording long sample autofluoresence and/or are within about 25-100 um 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 60 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 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 collimated and multiplexed using an array of dichroic (i.e., mechanical chopper) or through electronic means mirrors. In this way, Samples containing multiple phosphor 65 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. electronic-controlled, Solid-state optical shutter Such as

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Pockel's or Kerr cells). Up-converting phosphors and of the control electronics will be described below in con up-converting delayed fluorescent dyes typically have emis nection with Specific embodiments. 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 ns. 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 15 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 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) 25 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 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 35 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 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 40 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 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 45 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. 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 50 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 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 connection with multiple Sources. focused on the Sample. To this end, light from the two 55 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 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 60 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. 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 65 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 the Signal from detector 22(1) and a phase control input

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Signal. This circuitry provides additional background dis dences involving a single photon energy. Further, it may be crimination because the background has a much shorter possible to achieve direct Stepwise excitation of the emitting 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, pp. 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, 15 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 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 21, or possibly different wavelengths) are as shown in FIG. 5C.

modulated by Signals from waveform generators 37a and In a stepwise excitation Scheme shown in FIG. 5A, energy 37b operating at respective frequencies f and f. The transfer is not required, and thus information on the polar waveform generator output signals are communicated to a 25 ization of the excitation laserS may be preserved and cause first frequency mixer 42, and a signal at fit?, is communi polarization of the emitted radiation. In this case, depolar cated to a Second frequency mixer 45. The Signal from ization of the light may allow for enhanced discrimination detector 22(1) and a phase input signal are also communi between Signal and background noise.

cated to frequency 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. 35 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 FIG. 6 is a Schematic view Snowing the optical train of a bands centered at 2 and 22, 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 40 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 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 45 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 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 50 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 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 55 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 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 60 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 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 65 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 radiation.

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A portion of the light emanating from the region of focus the CCD array are separated. An array of lenses 165 collect point 115 in the sample is collected by GRIN lens 105, the light from respective binding sites and focus it on 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. Optical filters rejecting the excitation radiation are not used. 120a–C direct the particular components to respective pho Optical trapping may be used to transiently immobilize a todetectors 125a–c. A particular filter arrangement is shown Sample particle for determination of the presence or absence where each filter reflects light in a respective emission band, of phosphor on the particle. Conveniently, the wavelength but other arrangements would be used if, for example, one range used to trap Sample particles may be essentially or more of the filters were bandpass filters for the emission identical to an excitation wavelength range for the 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 for Single-beam gradient force trapping of Small particles.

cussed above. Such techniques would be necessary, for FIG. 26 is a block diagram of one embodiment of appa example, if the emission bands were not distinct. 15 ratus for carrying out the present invention on a Sample FIG. 7A is a schematic of an embodiment of the invention using a microScope. In this embodiment a Standard micro in which a charge coupled device (CCD) imaging array 150 Scope is modified to accept infrared Scanning optics and is used as a detector in combination with a two dimensional image processing electronics. A Suitable microScope for array 152 of peptides or other biologically active Species modification is the Zeiss model CLSM-10. deposited on a glass or plastic Substrate. The CCD array has The microscope is fitted with a HeNe laser A1 for visible a number of individually addressable photosensitive detec imaging and an argon laser A2 for both visible and UV tor elements 155 with an overlying passivation layer 157 imaging. Both lasers are mounted internally and are indi while the peptide array has a number of individual Binding vidually Selectable through a Series of motorized shutters sites 160. The probe containing the phosphor would be A3. The upconverting phosphors are excited with an exter reaction Specific to one or more of the elements in this 25 nally mounted IR laser diode. In the preferred embodiment, peptide array and would therefore become physically two IR laser diodes A4 and A5, operating at two different IR attached to those elements and only those elements. The wavelengths, are coupled to the microScope thereby allow peptide array is shown as having a one-to-one geometric ing multiple phosphor reporters to be identified. Laser relation to the imaging array in which one pixel corresponds diodes A4 and A5 are individually selectable using motor to each element in the peptide array. However, it is also ized shutters A6. When an IR beam is selected, it is routed possible to have larger peptide elements that cover a group through the microscope's galvanometrically controlled of detector elements should Such be necessary. Scanning mirrorS A7 which Scan the beam in a raster fashion. Various of the techniques described above can be used to The beam passes through the objective lens (not shown) enable the detector array to distinguish the emissions of the onto a Sample A8 and is reflected back through the objective phosphor from the infrared laser Stimulation. First, it is 35 lens to a Set of galvanometrically controlled receiving mir possible to use a phosphor that responds to IR Stimulation rors A7. Receiving mirrors A7 reflect the light onto pinhole beyond the Sensitivity range of the detector array. An optics A9. If the confocal mode is selected, pinhole A9 limits example of Such a phosphor would be Gadolinium oxySul the detected image to the light collected from the focal fide: 10% Erbium. This phosphor is stimulated by 1.5- plane. The light is imaged on a photomultiplier tube (PMT) micron radiation and emits at 960 nm and 520 nm. The 40 A10. The thickness of the focal plane is proportional to the detector array is insensitive to 1.5-micron radiation but is Size of the pinhole. The Scanning Speed is chosen Such that Sensitive to the up-converted radiation. sufficient signal intensity is received at the PMT A10. In the Further, Since the phosphor emission is relatively slow in preferred embodiment of this apparatus, a 20 micrometer rise and fall time it could be time resolved from a pulsed diameter pinhole is used which results in a depth of field of laser stimulation source by the CCD detector array. The 45 about 1 micrometer. If the confocal mode is not Selected, the decay time for the upconversion proceSS is a variable beam is deflected around pinhole optics A9 directly to PMT dependent on the particular emitting transition and the A10.

phosphor host; however, it is normally in the range 500 uS Once the optical Signal is converted into an electronic one, seconds to 10 ms. This is very slow compared to the laser a Standard, composite Video signal can be developed and excitation pulse and the capability of the detector array. 50 displayed as an image on a television monitor A11. The The techniques for fabricating the CCD array are well image can be manipulated and enhanced through Standard known Since CCD imaging arrays have been commercially image processing Software, in the preferred embodiment of available for many years. A variety of Such devices can be this apparatus the Software runs on an IBM 486 PC A12. The obtained from David Sarnoff Research Center, Princeton, Software can be used to perform averaging, filtering, edge N.J. 55 detection and overlaying the imageS received from each of The techniques for fabricating the peptide array are the different light Sources.

described in a paper by Fodor et al., “Light-Directed, In the confocal mode, it is possible to reconstruct a 3 Spatially Addressable Parallel Chemical Synthesis,” dimensional view of sample A8. The reconstruction is Science, Vol. 251, pp. 767-773 (Feb. 15, 1991), incorporated formed by Stepping through Sample A8 at Small intervals, herein by reference. The particular array discrete contains 60 making an image of the Sample at each interval. The multiple 1024 discrete elements in a 1.28 cmx1.28 cm area. Sequential images are transferred to an external graphics The embodiment of FIG. 7A shows the peptide array in machine (not shown) for reconstruction of the sample in 3 intimate contact with the CCD array. Indeed it may be dimensions. These 3-D images can then be rotated to give possible to deposit the peptides directly on the passivation different perspectives of the data Sets, leading to a better layer without a separate Substrate. However, there may be 65 understanding of the Samples.

Situations where spatially Separated arrays are preferred FIG. 27 is a block diagram of a microtiter plate reader for FIG. 7B shows an embodiment where the peptide array and use with the present invention. Within a light-tight test

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chamber B1 is a near IR laser excitation Source B2, a user. In the preferred embodiment, laser D3 operates in the photomultiplier tube (PMT) detector B3, and a sample assay 960-980 nanometer range.

plate B4. In the preferred embodiment of this apparatus, In use, wick D2 wicks up a portion of a sample fluid D8 assay plate B4 is a Terasaki HLA plate. This plate is which is Suspected of containing the target antigens. Target preferred due to its Small tapered Sample wells which tend antigens bind to the antibodies present at a capture Surface to concentrate the Sample material into a relatively Small D9. Capture surface D9 is positioned at the focal point of target area. The target area in this configuration is still larger Source D3. The target antigens can be labeled with than the diameter of the laser beam. Furthermore, it is phosphor-antibody conjugates either before or after capture. possible that the distribution of the assay material acroSS the In the preferred embodiment wick D2 is formed of glass. In bottom of the well is not even. Because of these two factors, this configuration capture Surface D9 is prepared Simply by Simply aiming the laser at the center of the bottom well filling the inside of the capillary with a bubble containing the Surface is unlikely to provide accurate readings. There are antibodies of interest. By Silanizing the inner Surface with Several approaches that can be used to circumvent this organofunctional Silanes, conventional chemistries can be problem. The first approach is to defocus the laser beam used to covalently link the antibodies or other biological Sufficiently to allow a larger amount of the target area to be 15 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 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 25 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 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 35 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 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 40 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 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. 45 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 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 50 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 used. Another alternative is to use a diffraction grating and average is calculated. Translator B7 then moves 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 55 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 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. 60 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. to 25 micrometers thickneSS which is used as the capture This embodiment is comprised of a housing D1 and a surface. Arrays F1 and F2 are separated by a spacer F5. capillary wick D2. Within housing D1 is a diode excitation Array F1 is comprised of Fabrey-Perot diode lasers, pref laser D3, a lens assembly D4, a photodiode detector D5, and 65 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 D1 communicates the results of the test to the tional photolithography techniques. Each individual laser in

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array F1 has a columnar beam designed to Strike only the tiplier or photodiode, detects emitted radiation. The detector adjacent portion of capture Surface F4. The required power controls gating of the cell in the detection Zone into one of 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 rum may MHC complexes), as well as to biological or chemical 15 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 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 25 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: flow rate. Therefore given a flow rate between 1 and 10 P=fND-L 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 Wumparticle), 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 35 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 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 40 phosphors, detection can be performed using a photomulti particles in the light path decreases linearly with a decrease plier tube (PMT), a photodiode, or a CCD array. The in the detection area. Since the power of the light emitted phosphorescence decay time is long, with a decay half life from the phosphors Scales with the Square of the excitation of 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. 45 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. 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 50 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 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 55 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 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 60 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 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 65 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 SOCC.

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In environments where absorption of the up-converted shown, both side-on and end-on detection and excitation phosphor radiation is high, the phosphor microparticles are arrangements, or combinations, are possible. Reduction of 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, 15 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 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 25 ture measurement methods are described in Berthou H. and of probe/reporter “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 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 35 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 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 40 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 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 45 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 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 50 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 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 55 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 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 60 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 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 65 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 luminescent labeled probes. The up-converting labeled

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probes and luminescent labeled probes are capable of Spe in an assay. For example, different matched label pairs may cifically binding the target analyte and forming labeled be attached to two distinct probes which bind to two probe-target complexes. Having bound the analyte to the different target analytes. Then, for an assay method, by labeled probes, the Sample is illuminated with illumination exciting the excitation label and observing for emission energy to excite the excitation label. The Sample is then radiations from the emission labels, the presence of one or observed for emission radiation from the emission label. The both of the two different target analytes may be detected detection of an emission from the emission label verifies the within a single Sample.

presence of the target analyte Since the emission label (2) In another embodiment, distinct sets of matched generally only absorbs the energy caused by excitation label excitation and emission labels may be employed. For emissions when the matched labels are attached to the target example, two different Sets of matched labels each analyte. The apparatus used to illuminate the Sample with an having distinct excitation labels and distinct emission illumination wavelength and to detect an emission wave labels may be used in a single Sample. For example, in length from the emission label is the same as described an assay method, by exciting the first excitation label above. and observing emission radiation from its matched The matched label assays may be carried out as 15 label, a first analyte may be detected. Additionally, in homogeneous, heterogeneous or competitive assayS. the same Sample, a Second analyte may be detected by However, when using matching labeled probes in a homo exciting the Second excitation label and observing geneous assay, it is preferred that the matching labels are emission radiation from its matched label. attached to two probes to form distinct labeled-probe pairs (3) In another embodiment, a probe having one excitation which bind to the target analyte. The probe may be the same label may be matched with at least two probes having or different probe material. For example, an up-converting distinct emission labels. Thus, in an assay, different label may be attached to a first antigen probe which binds to target analytes may be detected within a single Sample one epitope Site on the analyte while the matched lumines by exciting the excitation label and then observing the cent label is bound to a different antigen probe which binds different emission radiations from the distinct emission to another epitope Site on the same analyte. 25 labels.

Matched label photophysical catalysis can be conducted (4) Another embodiment of the invention involves using in a manner Similar to the matched label assays described matched labels having one or more emission labels above except that the energy from the emission label may be which emit a detectable emission radiation and at least used to produce localized intense electromagnetic radiation two distinct excitation labels. For example, in an assay for purposes other than detection. For example, the energy on a Single Sample, different target analytes may be from the emission label may be used to bring about a detected by Separately exciting the distinct excitation modification in the target analyte or in materials adjacent to labels and observing the emission radiation from the the target analyte. Another example of photophysical cataly emission labels.

sis involves the use of an up-converting label which excites Of course, the various combinations of matched label a luminescent label causing it to emit its energy to create a 35 pairs discussed above may be employed with different reactive chemical Species. probes to detect or, as discussed above for photophysical Both the matched label photophysical catalysis and assays catalysis or photodynamic therapy, effect a multitude of of the invention may employ a multitude of different different target analytes within a Single Sample. matched labels and/or different probes within a single Furthermore, these multiplexing embodiments may be car Sample. By varying the labels and probes a wide variety of 40 ried out Simultaneously or Sequentially upon a single analytes may undergo photophysical catalysis or be detected Sample.

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

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and inorganic phosphors Suitable for use in the invention conventional techniques and methods known in the art, it is include, but are not limited to, those described above. preferred that when particles Such as up-converting phos Examples of up-converting non-linear optical compounds phor particles are used, they should be Substantially mono include, but are not limited to, 4-(p-nitrophenyl)-3,4, disperse particles. "Substantially monodisperse particles' dihydro-pyrazoc-benzobmorpholine (NDPB), 1-o-tolyl are particles which are Substantially unagglomerated and, in 3-p-nitrophenyl-2-pyrazoline (TNP), 2-(p-dimethyl general, are predominately spherical in shape. In other aminophenyl)ethenyl-phenyl-methylene-propanedinitrile words, the majority of phosphorescent particles exist as (DPP) and bis-2-(p-dimethylaminophenyl)ethenyl individual particles in contrast to clusters of two or more methylene-propanedinitrile (BDP). Other up-converting phosphorescent particles. In a preferred embodiment, the phosphors, dyes and chelates are described in, but are not Substantially monodisperse phosphor particles are formed limited to, those described in Copending U.S. application by a fluidized bed production proceSS Such as that described Ser. No. 08/986,196 entitled “Production of Substantially in Copending U.S. application Ser. No. 08/986,196 entitled Monodisperse Phosphor Particles, filed on Dec. 5, 1997, “Production of Substantially Monodisperse Phosphor Luminescent Materials, Ullmann's Encyclopedia of Indus Particles, filed on Dec. 5, 1997.

trial Chemistry, 5th Edition, Vol. A15, pgs. 519–557, and in 15 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 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 enough down-converting or up-converting (Stokes or anti 25 carboxyl groups on the lateX Sphere Surface and the Stokes) shift to enable detection of the signal. Furthermore, hydrazide of the Biotin-LC-Hydrazide. Biotin 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 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, described above, photophysical catalysis methods may be the compositions may be in the form of a kit up-converting or down-converting, phosphorescent or fluo 35 comprising duct the all of the essential ingredients required to con desired assay or photophysical catalysis method.

rescent materials. The luminescent labels which are

For example, the kit may contain matching up-converting up-converting include, but are not limited to up-converting labeled phosphors, chelates, dyes and non-linear optical compounds, is presented probes and luminescent labeled probes. The assay kit Such as those described above. Luminescent labels which in a convenient, commercially packaged form. are down-converting luminescent materials include, but are 40 The kit can be presented as a composition or as an admixture not limited to, down converting phosphors, Such as Zinc depending upon the compatibility of the labeled probes. For sulfides activated with silver and those described in Copend example an assay kit can be a packaged combination having ing U.S. application Ser. No. 08/986,196 entitled “Produc one or more containers, devices or the like holding the tion of Substantially Monodisperse Phosphor Particles,” labeled probes and other materials necessary for particular assay, and usually including written instructions for the filed on Dec. 5, 1997, Luminescent Materials, Ullmann's 45 performing the assay.

Encyclopedia of Industrial Chemistry, 5th Edition, Vol. A15, pgs. 519–557, and in Kirk-Othmer Encyclopedia of Chemi Some detail bytheway

Although present invention has been described in cal Technology, Third Edition, Volume 14, pp. 527 ff. the understanding, it willofbeillustration for purposes of clarity of apparent that certain changes and disclosures of which are incorporated by reference in their modifications may be practiced within the Scope of the entirety. Luminescent label chelates include, but are not 50 claims.

limited to and those described in Hemmila et al. U.S. Pat.

No. 5,637,509, and Stavrianopoulos et al. U.S. Pat. No. reference The broad scope of this invention is best understood with 4,868,103, the disclosures of which are herein incorporated to limit theto invention the following examples, which are not intended in any manner.

by reference in their entirety. Luminescent labels which are fluorescent dyes include, but are not limited to, TransFluo 55 EXPERIMENTAL EXAMPLES Spheres(R (TFS) and those described in Mandel et al. U.S. Validation of Up-Converting Inorganic Phosphors as Pat. No. 4,372,745, Brinkley et al. U.S. Pat. No. 5,326,692 Reporters and Singer et al. U.S. Pat. No. 5,573,909, the disclosures of Up-converting phosphor particles comprising Sodium which are herein incorporated by reference in their entirety. yttrium fluoride doped with ytterbium-erbium were milled to A preferred luminescent label is a down-converting fluores 60 Submicron size, fractionated by particle size, and coated cent dye mixture sold under the tradename TransFluo with polycarboxylic acid. Na(YosYbos Eroo)F was cho Spheres(R (TFS) which is commercially available from Sen for its high efficiency upon excitation in the range 940 Molecular Probes located in Eugene, Oreg. Generally, pre to 960 nm. A Nd:Yag pumped dye laser/IR dye combination ferred luminescent labels of the invention have a high was used to generate 8-ns to 10-ns duration pulses in the quantum efficiency and a high extinction coefficient. 65 above frequency range.

Additionally, while down-converting and up-converting The laser pulses were used to illuminate a Suspension of phosphor particle labels of the invention may be prepared by milled phosphor particles in liquid and attached to glass

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49 SO

Slides in situ. The Suspension luminescence observed at right arabic Solution. This appeared to completely eliminate any angles was monitored using a collection lens, a Spatial filter water dispersion problems. The Serial dilutions used are in order to filter out Scattered excitation light to the maxi listed in Table III.

mum possible extent, and a photomultiplier, Vacuum photodiode, or simple Solid State photodiode (depending on TABLE III the light level observed). Phosphor Phosphor Equivalent The luminescent Signal level was determined as a function Loading Loading Detection of Solution pH (range: 6-8), grain size, particle loading Label (ng?well) (particles/well) Sensitivity (M) (ug/cm), and the nature of stabilizing anionic Surfactant. 100 17OO 90 23,600,000 + 1,200,000 4 x 1012 Signals were recorded both as a time integral from a boxcar 1O-1 17O 9 2,360,000 + 120,000 4 x 10 integrator and from a long RC time constant or as a transient 1O-2 17 O.9 236,000 + 12,000 4 x 101 Signal using a transient digitizer in order to delineate the 1O-3 17 O.09 23,600 + 1,200 4, 10-15 luminescence lifetime under particular experimental condi 10-4

tions. In Situ Signals were also measured by laser Scanning 1O O.OO17 O.OOOO9 23.6 1.2 4 x 101 microScopy. FIG. 11 is a fluorescence Scan of the phosphor 15 emission Spectrum incident to excitation with a laser Source at a wavelength maximum of 977.2 mm; emission maximum The stock DMSO dispersion had a phosphor density of is about 541.0 nm. FIG. 12 is an excitation scan of the 1.70+0.09 mg/mL (at 95% confidence limits), determined phosphor excitation Spectrum, with emission collection win gravimetrically by evaporating 4-1 mL Samples. This trans dow Set at 541.0 nm, excitation maximum for the phosphor lates to 23.6x10° particles/mL (assuming an average particle at the 541.0 nm, emission wavelength is approximately size of 0.3 um and particle density of 5.3 g/mL). The residue about 977 nm. FIG. 13 is a time-decay measurement of the after evaporating the Samples over the weekend at 110-120° C. was noticeably yellow, but did phosphoresce phosphor luminescence at 541.0 nm after termination of when tested with an IR diode laser. excitation illumination; maximal phosphorescence appears Visual green light emanated from all Serial dilutions down at approximately 400 uS with a gradual decay to a lower, 25 to 10 (i.e., 1.7 tug/mL or 23.6x10° particles/mL) in a 1 mL stable level of phosphorescence at about 1000 us. FIG. 14 polypropylenemicrofuge tube using a hand-held diode laser shows the phosphor emission intensity as a function of in a dark room. The 10' and 10° dilutions were visibly excitation illumination intensity; phosphorescence intensity cloudy. Either 1 ul of each serial dilution, or 0.1 ul of the increases with excitation intensity up to almost about 1000 next higher dilution, were pipetted into a well on the Terisaki W/cm. plate. It was found that 1 ul fills the bottom of the well and Phosphorescence efficiencies of submicron 0.1 mu 1 spreads along the edge of the well, but does not Na(YosYboEroos)F particles were measured. A Ti:sap cover the entire Surface. Because of the Statistical and phire laser was used as an excitation Source and a spectro pipetting problems associated with Small volumes with low photometer and photomultiplier was used as a detection particle concentrations, 2 to 4 replicates were prepared of System. Two types of measurement were performed. The 35 each dilution.

first was a direct measurement in which the absolute emis The well of a Terasaki plate holds a 10 ul sample volume. Sion per particle for phosphor Suspensions was measured in ASSuming all the phosphor particles contained in this Vol emission bands at 540 nm and 660 nm. The calibrated ume adhere to the bottom of the Sample well, we can croSS-Sections are shown in FIG. 15, and size-dependence is estimate an equivalent detection sensitivity (Table III). It shown graphically in FIG. 16. This corresponded to a 40 should be noted that 10 to 10M is the normal range of phosphorescence cross-section of approximately 1x10' enzyme-linked Surface assayS.

cm for 0.3 um particles with excitation light at 975 nm and Control Sample Results an intensity of approximately 20 W/cm. The emission The control Samples were Scanned using a prototype efficiency of dry phosphor powder of about 25 um was also up-conversion fluorimeter device (David Sarnoff Research measured. On the basis of known values for the absorption 45 Center). The Samples were Scanned by moving the plate in cross-section of Yb" in crystalline hosts (Lacovara et al. 50 um increments, using a motorized X-Y positioning Stage, (1991) Op. Lett. 16:1089, incorporated herein by reference) relative to the focal point of an infrared diode laser. and the measured dependence of the phosphorescence emis The IR diode laser was operated at 63 mW (100 mA). The Sion on particle Size, a phosphorescence cross-section of beam was focused to 2.4x10 cm at the focal point. As the approximately 1x10 cm was found. The difference 50 bottom of the sample well is about 1.4x10 cm (1365 um between these two measurements may be due to a difference diameter), the beam covers less than 17% of the well bottom in phosphorescence efficiency between dry phosphor and Surface at any individual position. The well also has sloping aqueous Suspensions, or due to absorption of multiply Scat side walls which widen from bottom to top of the sample tered photons in the dry phosphor. On the basis of either of well and are also interrogated by a progressively divergent these croSS-Section estimates, the cross-section is Sufficiently 55 laser beam. Neglecting losses in the optics, the IR light large to allow detection of Single Submicron phosphor par intensity at the focal point (bottom of the sample well) was ticles at moderate laser intensities. At laser intensities of approximately 26–27 W/cm at 980 nm wavelength. A roughly 10 W/cm, the phosphorescence scales as the laser photomultiplier tube (PMT) was used for detection of the intensity to the 1.5 power. visible (upconverted) light emitted from the sample. Since Phosphor Particle Performance: Sensitivity of Detection 60 the laser beam width was Smaller than the Surface area at the A Series of Terasaki plates containing Serial dilutons of bottom of the Sample well, the plate was aligned by visual monodisperse 0.3 um up-converting phosphor particles con inspection against the focal point of the diode laser So that Sisting of (Yose Yboos.Eroo)2O2S were tested for if the laser was centered in the middle well (C6 when reading up-conversion fluorescence under IR diode laser illumina wells C5, C6 and C7, and D6 when reading wells D5, D6, tion in a prototype instrument. 65 and D7).

The phosphor particles were prepared by Settling in The PMT signal (amps) was recorded at each plate DMSO and were serially diluted into a 0.1% acqueous gum position and numerically integrated over the width of the

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Sample well (approximately 4000 um). Several Scans were Size of the particles. Two distinct size Subpopulations were made at different positions in the 10° to 10' dilution sample observed with about 65% of the counted particles appearing 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 Immunodiagnostic 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), 15 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 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 phosphate samples 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 25 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 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 35 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. 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 40 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 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 45 Suited for Sandwich assays wherein the magnetic bead is the tion. Solid Substrate.

Goat anti-mouse IgG antibody (Ab) was conjugated (by 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 50 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 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 55 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 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 60 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 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 65 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 quantitative measure of analyte concentration.

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Biotinylated polynucleotides are also conveniently used Covalent Conjugation of Upconverting Phosphor Label to as hybridization probes, which can be bound by Avidin

Streptavidin-coated up-converting phosphors to report An up converting y trium -ytterbium-erbium hybrid formation. (Yose Yboos Broo) oxysulfide (OS) phosphor was linked to Background Phosphorescence in Biological Samples avidin by the following procedure:

Monodisperse upconverting phosphor particles were

Background Signals were determined in two biological silanized with thiopropyltriethoxysilane (Huls) following Samples for determination of potential background in immu the procedure detailed by Arkles (in: Silicone Compounds: noassayS. Sputum and urine were used as Samples in the Register and Review, Hills America, pgs. 59-75, 1991). Same apparatus as used for the phosphorescence Sensitivity 1O This consisted of adding thiopropyltriethoxysilane (2 g) measurements (Supra). No background levels were found and 95% aq. ethanol (100 mL) to a 500 mL Erlenmeyer flask above the system noise levels set by the photomultiplier dark and stirred for 2 minutes. Approximately 8 mL of the 65 current. This noise level allows detection of Signals from on mg/mL phosphor Suspension in DMSO was then added to the order of a few hundred particles/cm. the mixture. This Suspension was stirred for an additional 2 This is close to a single particle in the detection volume 15 minutes, then transferred to centrifuge tubes and centrifuged of the System. to Separate the phosphor particles. The pellets were washed A photomultiplier is a preferred choice for a detector for twice with 95% aq. ethanol centrifuging each time. The high Sensitivity measurements of up-converting phosphors resulting particles were collected and dried overnight under Since photomultipliers can be selected to produce high vacuum at approximately 30° C. A quantity (127 mg) of dry quantum efficiency at the up-converted (i.e., emitted) wave silanized phosphors were resuspended in 1.5 mL of DMSO lengths and virtually no response in the range of the longer (phosphor Stock).

excitation wavelengths. A solution containing 1.19 mg of avidin (Pierce) in 1.0 Detection of Cell Antigens with Phosphor-Labeled Antibod mL of borate buffer (954 mg sodium borate decahydrate and ies 17.7 mL of 0.1N NC1 in 50 mL of deionized water, pH 8.3) Streptavidin is attached to the up-converting phosphor 25 was prepared (Avidin Stock). Another Solution containing particles as described, Supra. The mouse lymphoma cell line, 1.7 mg of N-Succinimidyl(4-iodoacetyl) aminobenzoate EL-4, is probed with a hamster anti-CD3 antibody which (Pierce Chemical) in 1.2 mL of DMSO was prepared (SIAB stock). A quantity (10 mu L) of the SIAB stock was added specifically binds 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 30 minto allow the N-hydroxysuccimide ester of the SIAB 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 conjugate. This type of multiple antibody attachment and ofAborate 20 mL Scintillation vial was prepared containing 10 mL buffer (pH 8.3). The following additions were then labeling is termed antibody layering. 35 made to this vial: 21.6 till of the avidin-SLAB 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 40 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. 45 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 50 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 55 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 60 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 65 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

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the center of the cell, neglecting the changes in refractive index of the cell wall and the liquid. TABLE IV Light emitted is Scanned with a monochromator and Cell Cell detected by a photomultiplying tube (PMT) 90° from the Surface Surface direction of the excitation light. The detection limits for the Type of goat Avidin Phosphor FITC modified SLM Aminco 48000 were determined by serial Tube anti-mouse IgG Conjugate Signal Signal dilution to be 4x10'M (240,000 phosphor particles per 1. biotinylated Avidin-Phosphor -- mL) in PBS. Phosphor emission peaks in the spectrum were 2 biotinylated Phosphor 3 biotinylated Avidin seen at wavelengths of 406+2 nm, 434+2 nm, 522+2 nm, and 1O 4 biotinylated Avidin-FITC -- 548+2 nm. The largest peak was at 548 nm. The intensity of 5 FITC labelled Avidin-Phosphor -- the 548 nm peak was used to discriminate Samples. 6 FITC labelled Phosphor -- Linkage of Avidin-Phosphor Conjugate to Cell Surface

Marker The remainder of the Samples were used to resuspend A lymphoblastoid cell line (Human Genetic Mutant Cell 15 paramagnetic, polystyrene beads bound with Sheep anti mouse IgG. For each of the six samples, 3x107 beads were

Repository #GM07092) was cultured in RPMI 1640 media prewashed containing 15% heat inactivated fetal calf Serum. A Suspen with blocking buffer for 1 hour at room tempera sion of cells (107 cells) was centrifuged and resuspended in ture in Eppendorf tubes. The buffer was removed by aspi ration while the tubes were in a magnetic rack. The magnetic an equal volume of phosphate buffered saline (PBS) pH 7.4. beads

Cells were washed two times in PBS and resuspended to a antibody with anti-mouse IgG were allowed to bind to the final concentration of 5x10 cells/ml. These cells were then labelled cells for 1 hour at room temperature with intermittent resuspension. The magnetic beads were then incubated with a mouse IgG1 monoclonal antibody to collected on a magnetic rack, washed four times in blocking human fB microglobulin, a Class I histocompatibility anti buffer, resuspended in 100 till blocking buffer, transferred to gen in polystyrene centrifuge tubes. The cells were immu 25 a fresh tube, and up-converting phosphorescence was mea noprecipitated for 30 minutes at 4 C. with an antibody Sured on the fluorimeter.

concentration of 10 ug/ml. The cells were harvested by To Scan for phosphor emission, the emission monochro centrifugation, washed twice in PBS, resuspended in PBS mator bandwidth was Set to 8 nm and the spectra were and then aliquoted (250 uD) into six fresh centrifuge tubes. scanned from 500 to 700 nm with a step size of 2 nm. Four of these Samples received biotinylated goat anti-mouse Samples were also measured for FITC signal by exciting the IgG, while the remaining two received FITC-labelled goat samples with 37 uM at ) =490 nm with a 2 nm bandwidth. anti-mouse IgG. These immunoprecipitations were per Since the excitation wavelength (490 nm) and the emission formed at 4 C. for 30 minutes in volume of 400 uL with a wavelength (514 nm) are very close for FITC, higher final second antibody concentration of 20 mu g/ml. The cells resolution was required to get Separable Signals than with were harvested and washed in PBS as above but were 35 phosphor labelling. The intensity of the 490 nm signal was resuspended in 50 uL of blocking buffer (0.2% purified 240 uW/cm at the center of the well. FITC emission spectra casein in PBS, Tropix, Bedford, Mass.). The cell-antibody were Scanned at 0.5 nm increments from 450 nm to 750 nm. complexes were blocked in this solution for 30 minutes at with a 2 nm bandwidth on the emission monochromator. room temperature and then transferred to fresh tubes. Sample 1 is the positive control and clearly yielded the A pre-blocked Suspension (40 uD) of either avidin 40 highest emission signal. Sample 2 indicates that any non Phosphor conjugate, avidin-FITC, avidin, or unconjugated Specific adsorption of the phosphors to the Sample is limited Phosphor was added to four of the cell Samples conjugated and is readily discriminated from Signal attributable to with the biotinylated anti-mouse IgG (H&L). In addition, an avidin-conjugated phosphor and showing that avidin linked equal amount of pre-blocked avidin-Phosphor er unconju phosphors can specifically bind only when they are conju gated Phosphor was added to the remaining two cell Samples 45 gated with the probe, in this example through the biotin immunoprecipitated with the non-biotinylated FITC avidin linkage. Sample 3 is the negative control which labelled anti-mouse IgG (H&L). The avidin reporter conju contains no phosphors, only avidin. Sample 4 shows FITC gates or negative controls were pre-blocked as follows. conjugated avidin. Although FITC signals were observed on Avidin-Phosphor and Phosphor alone was diluted in block the cell Surface by laser microScopy, the Signals were below ing buffer by adding 10 till of a 6.7 mg/ml Suspension to a 50 the level of detection on the fluorimeter for measurement of final volume of 100 u. Avidin-FITC and the avidin alone FITC, and since there was no phosphor in the sample there controls were also diluted in blocking buffer by adding 27 till was no significant phosphor Signal. Samples 5 and show that of 2.5 mg/ml Solution to a final volume of 100 lull. These FITC-conjugated primary antibodies can be detected and reagents were blocked at room temperature for 3 hours with that the presence of phosphor or avidin-phosphor does not intermittent resuspension and then added to 50 till of cells 55 Significantly disrupt binding of the primary antibody to its labelled with biotinylated or non-biotinylated second anti target antigen.

body. The avidin-biotin reactions were performed at room Linkage of Avidin-Phosphor Conjugate to DNA temperature for 30 minutes with occasional resuspension. Plasmid DNA (25 ug) was nick translated in the presence The reactions were Stopped by harvesting the cells by of 20 mM dGTP, 20 mM dCTP, 20 mM biotin-14 DATP, 13 centrifugation and washing twice in blocking buffer. The 60 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 220 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 blocked avidin-conjugates or avidin and were incubated for priate probe (e.g., an antibody or DNA). The phosphor 30 minutes at room temperature. labeled probe specifically binds to a target (e.g., antigen or Unbound avidin-conjugates were removed by resuspend nucleic acid) that is linked to a capturing Surface. A Suitable ing 3x10" paramagnetic beads linked with sheep anti-mouse capture Surface can be the tip of a light carrying optical fiber IgG (pre-blocked in blocking buffer). After incubation for 30 (FIG. 23) or the bottom surface of a sample container (FIG. minutes at room temperature with intermittent resuspension, 24). Upon incubation of the target-labelled capture Surface the beads were Separated on a magnetic rack and washed 4 with the phosphor-labelled probe, phosphor particles will to 6 times in PBS. The antibody-DNA bound beads were 15 accumulate at the capture Surface as a function of the amount 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 is found to be insignificant since the PMT signal is observed effected intensity using an excitation light that is focused from a low beam of large cross-section to a high intensity to be the same as that of the negative control (sample 4) which contains no phosphors. Sample 3 is another control 25 beam of small cross-section with the focal point of the beam and shows that the avidin-linked phosphors do not bind to being an or very near the capture Surface. Focusing of the the paramagnetic beads in the absence of DNA. Samples 5 excitation light is accomplished by transmission through and 6 show results of FITC-labeled avidin used to validate optical elements that have a very Small focal length, Such 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 PMT PMT the focal point of the excitation Source will emit significantly

Signal Signal 35 more light than those remaining in Suspension in the Sample

(V (a) (V (a away from the capture Surface. Therefore, binding of upcon

Sample DNA Reporter 546 mm) 514 nm) Verting phosphor linked probes to the capture Surface will 1. DNA labeled with Avidin linked 6.1297 2.4788 yield an increase in emitted light intensity measured from digOxigenin and Phosphor the Sample as a whole or as measured from a control Sample biotin 40 in which phosphors do not bind to the capture Surface. 2 DNA labeled with Silanized Phosphor 1.0528 4.4022 Emitted light intensity may be plotted as a function of target digOxigenin and biotin concentration using for Standardization (calibration) a Series 3 No DNA Avidin linked 1.63O2 3.5779 of Samples containing predetermined concentrations of tar Phosphor get. The emitted light intensity from a test sample (unknown 4 DNA labelled with Avidin O.85OS 2.8067 45 concentration of target) can be compared to the standard digOxigenie and biotin curve thus generated to determine the concentration of 5 DNA labelled with FITC - Avidin 1.0484 8.4394 target.

digOxigenin Examples of Suitable homogeneous assay formats 6 No DNA FITC - Avidin 1.0899 3.5779 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 Ybooser0.06)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 upconverting phosphor linked probes at the detection Sur phosphors described above (4x10'’M in DMSO) with 1.3 55 face by the application of centrifugal or gravitational Set mW of monochromatic light at 350 nm with a 16 nm tling. In this embodiment an upconverting phosphor is bandwidth for the excitation Source. Detection was accom linked to multiple probes. All the probes must bind to the plished by scanning this sample from 350 to 800 nm with a Same target, although Said binding can be accomplished at monochomator bandwidth of 8 nm. Scanning was performed different locations (e.g., as antibody probes may target in 2 nm increments. No downconversion was observed. 60

Moreover, no downconversion was seen at the excitation 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 tation scheme of Macfarlane et al. (1988) Appl. Phys. Lett. HRP to TFS-Biotin. An intense blue color was observed at 52:1300). An emission spectrum for the emitted all concentrations of Avidin-HRP up-converted light at 380 nm is shown in FIG. 32. We Matching Label Example 3 estimate the up-conversion cross section to be 107 cm for The conjugation of an up-converting label to a probe was this experiment. Studied.

We have also observed up-conversion in thulium acetate One milliliter of a 0.1 mg/ml up-converting phosphor hexahydrate and holmium chloride hexahydrate in Solution label Solution was centrifuged for ten minutes. The Super following the excitation schemes of Allain et al. (1990) natant was then removed and 3 ml of Hepes buffer (pH 6.3) Electron. Lett. 26:166, and Allain et al. (1990) Electron. Let. 25 was added. The up-converting phosphor particles were then 26:261, respectively. The salts were dissolved in heavy Sonicated until the particles dispersed. Another 7 ml of water, and excitation was performed using a krypton laser. Hepes buffer was added to the dispersion. The dispersion Although the up-conversion was weak, the up-conversion was then centrifuged for ten minutes. The Supernatant was should be improved if chelated compounds are used instead then removed. Three milliliters of a 0.11 mg/ml solution of of dissolved salts.

Neutravidin in Hepes buffer, available from PIERCE of

Matching Label Examples Rockford, Ill., was then added to the up-converting phos Matching Label Example 1 phor particles. This mixture was then stirred for two hours A study was made of the excitation of an up-converting in the dark at room temperature and then centrifuged. The label where its Subsequent energy emission excites a lumi Supernatant was removed and 10 ml of Hepes buffer was neScent label causing the luminescent label to emit radiation 35 added without dispersing the particles. The particles were at a wavelength which can be detected. Specifically, the then centrifuged. The Step of removing the Supernatant, Secondary fluorescent emission of an infrared excited adding Hepes buffer without dispersing the particles and up-converting phosphor label coupled with a luminescent centrifuging the particles was then repeated an additional label was studied. two times. This was followed by twice adding 3 ml of Hepes The up-converting label, a phosphor, (Y.Yb, Tm).O.S 40 buffer to the particles, Sonicating the particles until they phosphor having an emission radiation of 488 nm, was dispersed, adding an additional 7 ml Hepes buffer, centri dispersed in water by Sonification and then diluted to 0.1 fuging the particles and then removing the Supernatant. Then mg/ml in water. The luminescent label, TransFluoSpheres(R) 3 ml of a Hepes buffer having a pH of 8.5 and containing (TFS) having an excitation wavelength of 488 nm and a 0.1% bovine serum albumin was added to the particles. The particle diameter of 0.35 microns, was dispersed in water by 45 particles were then Sonicated until dispersed and an addi Sonification and diluted in water to 0.2% Solids. Ten micro tional 7 ml of the Hepes pH 8.5 buffer was added. The liters of the phosphor and ten microliters of TFS were then particles were then centrifuged and the Supernatant was mixed and dried on a glass slide at 60° C. and then mounted removed. Then the particles were avidinylated in 2 ml of on a coverSlip. The glass slide was then Subjected to Hepes buffer having a pH of 8.5 and containing 0.1% bovine excitation radiation having a wavelength of 488 nm which 50 Serum albumin.

produced a very bright TFS fluorescence against a mottled Although the present invention has been described in green background. The glass slide was also Subjected to an Some detail by way of illustration for purposes of clarity of excitation radiation having a wavelength of 900 to 1000 nm. understanding, it will be apparent that certain changes and A faint blue/white luminescence characteristic of the phoS modifications may be practiced within the Scope of the phor was detected by Visual observation as well as areas of 55 claims.

dim yellow/green fluorescence. The yellow/green fluores What is claimed is:

cence represented the presence of TFS:phosphor aggregates 1. A photophysical catalysis method which induces in which the TFS was excited by the phosphor emission mutagenesis or cytotoxicity in a cell or causes photolytic radiation. decomposition of a chemical present in the cell Matching of Example 2 60 environment, comprising the Steps of: The attachment of a luminescent label to a probe was (a) contacting a cell with a pair of matched up-converting Studied. Specifically, the biotinylation of a Secondary lumi and luminescent labeled probes under conditions Suf neScent latex particle with a linker arm containing a biotin ficient to bind the up-converting labeled probe and the complex was Studied. The Secondary luminescent Studied luminescent labeled probe to the cell to form a labeled was a TransFluoSpheres(R (TFS) latex particle available 65 probe-cell complex, wherein the up-converting label is from Molecular Probes of Eugene, Oreg., having an exci an inorganic phosphor particle comprising at least one tation radiation of 488 nm, a particle diameter of 0.35 rare earth element and a phosphor host material; and

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wherein the up-converting label or the luminescent Sensitizer Such that the dye Sensitizer is excited to a label is an excitation label and the other is an emission triplet State to induce cytotoxic activity in the cell. label, and, 3. A method according to claim 2, wherein the excited (b) illuminating the labeled probe-cell complex to excite Sensitizer dye transferS its energy to a dissolved molecular the excitation label which in turn excites the emission 5 oxygen molecule to yield an excited oxygen molecule. label Such that the emission label emits radiation at a 4. A photophysical catalysis method which induces mutagenesis wavelength which is mutagenic or cytotoxic to the cell decomposition or cytotoxicity in a cell or causes photolytic or is of a wavelength that causes photolytic decompo environment, comprising of a chemical present in the cell the Steps of:

Sition of a chemical present in the cell environment. (a) contacting a cell with an up-converting probe under 2. A photophysical catalysis method which induces cyto- 10 conditions Sufficient to bind the up-converting labeled toxicity in a cell, comprising the Steps of: probe to the cell to form a labeled probe-cell complex, (a) contacting a cell with a pair of matched up-converting wherein the up-converting label is an inorganic phos and dye Sensitizer labeled probes under conditions phor particle comprising at least one rare earth element Sufficient to bind the up-converting labeled probe and and a phosphor host material; and the dye sensitizer labeled probe to the cell to form a (b) illuminating the labeled probe-cell complex Such that labeled probe-cell complex, wherein the up-converting the label emits radiation at a wavelength which is label is an inorganic phosphor particle comprising at mutagenic or cytotoxic to the cell or is of a wavelength least one rare earth element and a phosphor host that causes photolytic decomposition of a chemical material; and, present in the cell environment. (b) illuminating the labeled probe-cell complex to excite 2O the up-converting label which in turn excites the dye k . . . .