Up-converting reporters for biological and other assays using laser excitation techniques

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OTHER PUBLICATIONS Lenth and Macfarlane, Lasers, Optics & Photonics News, Moser, K., et al., “Infrared Spectral Distribution of Photo (1992) 3:8–15.

conductivity and Up-conversion in GaP Light Emitting Louge et al., “Optical Fiber Measurements of Particle Veloc Diodes”, J. Appl Phys, (1985) 57:12:5438–5442. ity Using Laser-induced Phosphorescence, Applied Optics Tanabe, S., et al., “Up-conversion Fluorescences of Te0 (1991) 30:1976-1981.

and Ga-O-Based Oxide Glasses Containing Er'”, Journal Manashirov et al., “Effect of the Purity of Initial Substances of Non-Crystalline Solids, (1990) 122:79-82. on Luminescence Intensity of Erbium in Anti-Stroke Lumi Allain et al., “Room Temperature CW Tunable Green nophores,” Chemical Abstracts, (1989) 110:457 Abstract

Upconversion Holmium Fibre Laser,” Electronics Letters (1990) 26:261-263. McFarlane, “Dual Wavelength Visible Upconversion Laser,” Allain et al., “Blue Upconversion Fluorozirconate Fibre Appl. Phys. Letts. (1989) 54:2301-2302. Laser”, Electronic Letters, (1990) 26:166-168. McFarlane, “Violet Cw Neodymium Upconversion Laser,” Auzel, “Materials and Devices Using Double-pumped Appl. Phys. Letts. (1988). 54: 1300–1302. Phosphors and Energy Transfer”, Proceedings of the IEEE Nguyen et al., “Blue-green (450-nm) Upconversion (1973) 61:758-786. Tm:Ylf Laser.” Applied Optics (1989) 28:3553-3555. Berthou and Jorgensen "Optical-fibre temperature Sensor Rich and Pinow, Exploring the Ultimate Efficiency in Infra based on upconversion-excited fluorescence', Optic Letters red-to-Visible Coverting Phosphors Activated with Er and (1990) 15:1100–1102. Sensitized with Yb, J. Appl. Phys. (1972) 43:2357-2365. Bethune et al., “Atoms in carbon cages: the Structure and Schindele and Renzoni, “Ultra Fluors: New Fluorophores properties of endohedral fullerences”, Nature (1993) for Immunological Applications”, J. Clin. Immun. (1990) 366:123-128. 13:182-186.

Beverloo et al., “Inorganic phosphors as new luminscent Seveus et al., “Time-Resolved Fluorescence Imaging of labels for immunocytochemistry and time-resolved microS Europium chelate Label in Immunochistochemistry and in copy", Cytometry (1990) 11:784-792. Situ Hybridization,” Cytometry (1992) 13:329–338. Camus et al., “Two-photon absorption spectroscopy in Silversmith et al., “Green Infrared-Pumped Erbium Upcon ytterbium”, J. Phys. B. Atom. Molec. Phys. (1978) version Laser,” J. Opt. Soc. Am. (1982) 3:128-12.

Campiglia, A.D. et al., “Utilization of an Inorganic Phosphor Soini and Kojola,“Time-resolved Fluorometer for Lan as a Reference Signal in Solid-Surface Room Temperature thanide Cachelates-a New Generation of Nonisotopic Phosphorimetry”, Anal. Chem. (1988) vol. Immunoassays,” Clin Chem. (1983) 29/1:65–68. 60(g):2165-2167. Soules and Hoffman, “Luminescent Materials (Phosphors).” Diamandis and Christopoulos, Detection of Lanthanide Che Encylclopedia of Chemical Technology, (1981) Third Edi lates and Multiple Labeling Strategies Based on Time-re tion, 14:527–545.

solved Fluorescence, in Nonisotopic DNA Probe Tech Tiffany, “Fluorometry, Nephelometry, and Turbidimetry,” niques, Academic Press (1992) 263-274. Textbook of Clinical Chemistry, (1986) 78-90. Eichstein et al., “Laser-excited Time-resolved Solid-phase Xu and Hemmila, “Co-fluorescence Enhancement System Fluoroimmunoassays with the New Europium Chelate 4, Based on Pivaloyltrifluoroacetone and Yttriuim for the 7-bis (chlorosulfophenyl)-1, 10-phenanthroline-2, Simultaneous Detection of Europium, Terbium, Samarium 9-dicarboxylic Acid as Label.” Anal. Chem. (1988) and Dysprosium.” Anal. Chimica Acta. (1992) 256:9-16. 60:1069-1074. Lovgren, J. et al., Jul. 12, 1994, J. Immunol. Methods, vol. Gudgin Templeton et al., “Time Resolved Fluorescence 173(1), pp. 119–125.

Detection of Enzyme-amplified Lanthanide Luminescence Beverloo et al, Cytometry, 1992, vol. 13(6), pp. 561-570. for Nucleic Acid Hybridization Assays.” Clin. Chem. (1991) Lovgren, T et al., Oct. 1997, vol. 43(10) pp. 1937–1943, 37/9:1509-1512. Clinical Chemistry.

Johnson et al., “Infrared-to-visible Conversion by Rare-e- Heinonen, Petal, Clin. Chem., 1977, 43(7), pp. 1142-1150. arth Ions in Crystals,” J. Appl Phys. (1972) 43:3. Evangelista, R.A. et al, Analytical Biochem., vol. 197(1), Johnston and Wright, “Trace Analysis of Nonfluorescent 1991, pp. 213–224.

Ions by Associate Clustering with a Fluorescent Probe.” Hemmilä, I. et al., 1984, Analytical Biochem., vol. 137(2), Anal. Chem. (1979) 51:1774–1780. pp. 335-343.

Kano et al., NaLnF:YB",(Ln:Y.Gd,La): Efficient Xu et al, Analyst, Nov. 1991, vol. 116(11), pp. 1155-1158. Green-emitting Infrared-excited Phosphors, J. Electro chem. Soc. (1972) 119:1561–1564. Mukkala, V.M. et al., Analytical Biochem., vol. 176 #2, (Feb. Leif and Vallarino, “Rare-earth Chelates as Fluorescent 1, 1989), pp. 319-325.

Marks in Cell Separation and Analysis,” Cell Separation Beverloo, HB et al, Anal. Biochem., Jun. 1992, vol. 203(2), Science and Technology (1991) 3:41-58. pp. 326-334.

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THERMAL

CONTROLLER

MONITOR

THERMISTOR

RELAY

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BEAM

EXPANDER

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LENS

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FIG. 8

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Visible Light Out = Negative Sample

Infrared

Coated n Fiber Opti Phosphor Fo pTIC Phosphor

Target linked

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

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Coated Phosphor Phosphor

Emitted Light NO CAPTURE is of Low AT SURFACE Intensity

Focal point (high intensity excitation)

Excitation Light of low intensity

CAPTURE AT

SURFACE

Emitted Light is of High

Intensity

Focal point (high intensity excitation)

Excitation Light of low intensity Competitive Homogeneous Antigen Capture Assay

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FIG 51A

10 x 10 Gridded Array of 980 nm Diode Lasers

Aqueous

Sample Flow

MMTMN

10 GriddedDetectors

Array

Individual

Diode Laser

DXXX XXXXXXX XXXXXX XXXXX-XXXX,

Aqueous

Silicon Chip Sample Flow F9 Support Matrix N C O

PvP PPPre-Acco-F4wrksvarves

10-25 um Polymer film

Overlay Used as

Individual. Photodiode Capture Surface Detector in Array

Coniugated Antigen Capture Probe Bonded to

PhoSchor

FIG 51B

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UP-CONVERTING REPORTERS FOR disposal, Special licensing requirements, and instability BIOLOGICAL AND OTHER ASSAYS USING (radioactive decay and radiolysis). Further, the fact that LASER EXCITATION TECHNIQUES radioisotopic labels typically do not produce a strong (i.e.,

CONTINUING DATA

non-Cerenkov) signal in the ultraViolet, infrared, or visible portions of the electromagnetic spectrum makes radioiso

This application is a continuation of application Ser. No. topes generally unsuitable as labels for applications, Such as 08/416,023, filed Mar. 30, 1995, now U.S. Pat. No. 5,674, microScopy, image spectroscopy, and flow cytometry, that 698 which is a continuation-in-part of application Ser. No. employ optical methods for detection. 08/381,006, filed Jan. 30, 1995, now abandoned, which is a For these and other reasons, the fields of clinical continuation of application Ser. No. 07/946,068, filed Sep. chemistry, water and air monitoring, and biomedical 14, 1992, now abandoned. research have Sought alternative detectable labels that do not require radioisotopes. Examples of Such non-radioactive

BACKGROUND OF THE INVENTION labels include: (1) enzymes that catalyze conversion of a The invention relates generally to detectable labels and 15 chromogenic Substrate to an insoluble, colored product (e.g., compositions useful in assay methods for detecting Soluble, alkaline phosphatase, B-galactosidase, horse radish Suspended, or particulate Substances or analytes Such as peroxidase) or catalyze a reaction that yields a fluorescent or proteins, carbohydrates, nucleic acids, bacteria, Viruses, and luminescent product (e.g., luciferase) (Beck and Koster eukaryotic cells and more Specifically relates to composi (1990) Anal. Chem, 62: 2258; Durrant, I. (1990) Nature 346: tions and methods that include luminescent (phosphorescent 297; Analytical Applications of Bioluminescence and or fluorescent) labels. Chemiluminescence (1984) Kricka et al. (Eds.) Academic Methods for detecting specific macromolecular species, Press, London), and (2) direct fluorescent labels (e.g., fluo Such as proteins, drugs, and polynucleotides, have proven to rescein isothiocyanate, rhodamine, Cascade blue), which absorb electromagnetic energy in a particular absorption be very valuable analytical techniques in biology and wavelength medicine, particularly for characterizing the molecular com 25 one or morespectrum longer and Subsequently emit visible light at (i.e., less energetic) wavelengths.

position of normal and abnormal tissue samples and genetic material. Many different types of Such detection methods are Using enzymes and phosphorescent/fluorescent or calo widely used in biomedical research and clinical laboratory rimetric detectable labels offers the Significant advantage of medicine. Examples of Such detection methods include: Signal amplification, Since a single enzyme molecule typi immunoassays, immunochemical Staining for microscopy, cally has a persistent capacity to catalyze the transformation fluorescence-activated cell Sorting (FACS), nucleic acid of a chromogenic substrate into detectable product. With hybridization, water Sampling, air Sampling, and others. appropriate reaction conditions and incubation time, a Single Typically, a detection method employs at least one ana enzyme molecule can produce a large amount of product, lytical reagent that binds to a Specific target macromolecular and hence yield considerable signal amplification. However, Species and produces a detectable Signal. These analytical 35 detection methods that employ enzymes as labels disadvan reagents typically have two components: (1) a probe tageously require additional procedures and reagents in macro molecule, for example, an antibody or order to provide a proper concentration of Substrate under oligonucleotide, that can bind a target macromolecule with conditions Suitable for the production and detection of the a high degree of specificity and affinity, and (2) a detectable colored product. Further, detection methods that rely on label, Such as a radioisotope or covalently-linked fluorescent 40 enzyme labels typically require prolonged time intervals for dye molecule. In general, the binding properties of the probe generating detectable quantities of product, and also gener macromolecule define the Specificity of the detection molecule. ate an insoluble product that is not attached to the probe method, and the detectability of the associated label deter mines the sensitivity of the detection method. The sensitivity An additional disadvantage of enzyme labels is the dif of detection is in turn related to both the type of label 45 ficulty of detecting multiple target Species with enzyme employed and the quality and type of equipment available to labeled probes. It is problematic to optimize reaction con detect it. ditions and development time(s) for two or more discrete For example, radioimmunoassays (RIA) have been enzyme label Species and, moreover, there is often consid among the most Sensitive and Specific analytical methods erable spectral overlap in the chromophore end products used for detecting and quantitating biological macromol 50 which makes discrimination of the reaction products diffi ecules. Radioimmunoassay techniques have been used to cult.

detect and measure minute quantities of Specific analytes, Fluorescent labels do not offer the Signal amplification Such as polypeptides, drugs, Steroid hormones, advantage of enzyme labels, nonetheless, fluorescent labels polynucleotides, metabolites, and tumor markers, in biologi possess significant advantages which have resulted in their cal Samples. Radioimmunoassay methods employ immuno 55 widespread adoption in immunocytochemistry. Fluorescent globulins labeled with one or more radioisotopes as the labels typically are Small organic dye molecules, Such as analytical reagent. Radiation (C, f, or y) produced by decay fluorescein, Texas Red, or rhodamine, which can be readily of the attached radioisotope label Serves as the Signal which conjugated to probe molecules, Such as immunoglobulins or can be detected and quantitated by various radiometric Staph. aureus Protein A. The fluorescent molecules methods. 60 (fluorophores) can be detected by illumination with light of Radioisotopic labels possess Several advantages, Such as: an appropriate excitation frequency and the resultant Spec very high Sensitivity of detection, Very low background tral emissions can be detected by electro-optical Sensors or Signal, and accurate measurement with precision radiometric light microScopy.

instruments (Scintillation and gamma counters) or with A wide variety of fluorescent dyes are available and offer inexpensive and Sensitive autoradiographic techniques. 65 a Selection of excitation and emission spectra. It is possible However, radioisotopic labels also have Several to Select fluorophores having emission spectra that are disadvantages, Such as: potential health hazards, difficulty in Sufficiently different So as to permit multitarget detection and

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discrimination with multiple probes, wherein each probe (e.g., Azure A) are organic molecules Susceptible to pho Species is linked to a different fluorophore. Because the tobleaching and undergoing undesirable chemical interac Spectra of fluorophores can be discriminated on the basis of tions with other reagents, and (3) emitted radiation is down both narrow band excitation and selective detection of converted, i.e., of longer wavelength(s) than the absorbed emission Spectra, two or more distinct target Species can be excitation radiation. For example, AZure A absorbs at 632 detected and resolved (Titus et al. (1982) J. Immunol. nm and emits at 645 nm, and allophycocyanin absorbs at 645 Methods 50: 193; Nederlof et al. (1989) Cytometry 10:20; nm and emits at 655 nm, and therefore autofluorescence and Ploem, J. S. (1971) Ann. NY Acad. Sci. 177: 414). background noise from Scattered excitation light is not Unfortunately, detection methods which employ fluores eliminated.

cent labels are of limited Sensitivity for a variety of reasons. Another alternative class of fluorophore that has been First, with conventional fluorophores it is difficult to dis 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 15 al. (1992) Cytometry 13: 329). Down-converting lanthanide 50-100 nm to a longer wavelength than the excitation (i.e., chelates are inorganic phosphors which possess a large absorption) wavelength. Typically, both the absorption and downward Stokes shift (i.e., emission maxima is typically at emission bands are located in the UV/visible portion of the least 100 nm greater than absorption maxima) which aids in Spectrum. Further, the lifetime of the fluorescence emission the discrimination of Signal from Scattered excitation light. is usually short, on the order of 1 to 100 ns. Unfortunately, Lanthanide phosphors possess emission lifetimes that are these general characteristics of organic dye fluorescence are Sufficiently long (i.e., greater than 1 us) 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. 25 phosphors possess narrow-band emission, which facilitates 27: 36). Autofluorescence of optical lenses and reflected wavelength discrimination against background noise and excitation light are additional Sources of background noise Scattered excitation light, particularly when a laser excita in the visible spectrum (Beverloo et al. (1991) Cytometry 11: tion source is utilized (Reichstein et al. (1988) Anal. Chem. 784; Beverloo et al. (1992) Cytometry 13: 561). Therefore, 60: 1069). Recently, enzyme-amplified lanthanide lumines the limit of detection of Specific fluorescent signal from cence using down-converting lanthanide chelates has been typical fluorophores is limited by the Significant background proposed as a fluorescent labeling technique (Evangelista et noise contributed by nonspecific fluorescence and reflected al. (1991) Anal. Biochem. 197: 213; Gudgin-Templeton et al. excitation light. (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 35 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 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. 40 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 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 45 reduce autofluorescence. Tanke et al. (U.S. Pat. No. 5,043, emits in the far red or near infrared region of the Spectrum 265) report down-converting phosphor particles as labels for where nonspecific fluorescent noise is reduced. Phycobilip immunoglobulins and polynucleotides.

roteins are used in conjunction with accessory molecules However, the down-converting lanthanide phosphor of that effect a large Stokes shift via energy transfer mecha Beverloo et al. and the europium chelate of Seveus et al. nisms (U.S. Pat. No. 4,666,862; Oi et al. (1982).J. Cell. Biol. 50 require excitation wavelength maxima that are in the ultra 93: 891). Phycobiliprotein labels reduce the degree of spec Violet range, and thus produce Significant Sample autofluo tral overlap between excitation frequencies and emission rescence and background noise (e.g., Serum and/or fixative frequencies. An alternative approach has been to use cyanine fluorescence, excitation light Scattering and refraction, etc.) dyes which absorb in the yellow or red region and emit in the that must be rejected (e.g., by filters or time-gated signal red or far red where autofluorescence is reduced (Mujumbar 55 rejection). Further, excitation with ultraviolet irradiation et al. (1989) Cytometry 10: 11). damageS nucleic acids and other biological macromolecules, However, with both the phycobiliproteins and the cyanine posing Serious problems for immunocytochemical applica dyes the emission frequencies are red-shifted (i.e., frequency tions where it is desirable to preserve the viability of living downshifted) and emission lifetimes are short, therefore cells and retain cellular structures (e.g., FACS, cyto background autofluorescence is not completely eliminated 60 architectural microscopy).

as a noise Source. More importantly perhaps, phycobilipro Laser Scanning fluorescence microScopy has been used teins and cyanine dyes possess Several distinct disadvan for two-photon excitation of a UV-excitable fluorescent tages: (1) emission in the red, far red, and near infrared organic dye, Hoechst 33258, using a stream of Strongly region is not well-Suited for detection by the human eye, focused laser pulses (Denk et al. (1990) Science 248: 73). hampering the use of phycobiliprotein and cyanine labels in 65 The organic fluorphore used by Denk et al. was significantly optical fluorescence microscopy, (2) cyanines, photobleached by the intense, highly focused laser light phycobiliproteins, and the coupled accessory molecules during the course of imaging. Motsenbocker et al. (EP 476

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S 6 35 556) describes a method to increase luminol chemilumi and directed to the detector. An electrical Signal representing neScence by adding a dye catalyst that absorbS long wave the intensity of light in the emission band provides a length radiation (deep red light) and Subsequently reacts measure of the amount of reporter present. Depending on the with molecular oxygen to generate an oxidant which can detector's Spectral response, it may be necessary to provide itself react with luminol and produce oxidized luminol a filter to block the excitation light. which emits blue light. Gavrilovic (U.S. Pat. No. 5,166,948) Simultaneous detection of multiple reporters is possible, discloses a method and apparatus for optical pumping of at least where the reporters have different excitation bands or infrared pump light to a visible or ultraViolet emission light different emission bands. Where the excitation bands differ, having a wavelength shorter than the pump light (i.e., multiple laser diodes emitting at respective appropriate up-converted emission). wavelengths are combined using a wavelength division Thus, there exists a significant need in the art for labels multiplexer or other Suitable techniques, Such as frequency and detection methods that permit Sensitive optical and/or labeling, frequency modulation, and lock-in detector device. spectroscopic detection of specific label signal(s) with If the emission bands are different (whether or not the essentially total rejection of nonspecific background noise, bands excitation bands are different), light in the different emission and which are compatible with intact viable cells and emission 15 is separated and Sent to multiple detectors. If the aqueous or airborne environments. bands overlap, a single detector may be used, but other detection techniques are used. One example is to use

The references discussed herein are provided solely for time multiplexing techniques So that only one reporter is their disclosure prior to the filing date of the present appli emitting at a given time. Alternatively, the different laser cation. Nothing herein is to be construed as an admission diodes can be modulated at different characteristic frequen that the inventors are not entitled to antedate Such disclosure cies and lock-in detection performed. by virtue of prior invention. Detection methods and detection apparatus of the present SUMMARY OF THE INVENTION invention enable the ultrasensitive detection of up-converting phosphors and up-converting organic dyes by

The present invention provides labels, detection methods, 25 exploiting what is essentially the total absence of back and detection apparatus which permit ultrasensitive detec ground noise (e.g., autofluorescence, Serum/fixative tion of cells, biological macromolecules, and other analytes, fluorescence, excitation light Scatter) that are advantageous which can be used for multiple target detection and target characteristics of up-converting labels. Some embodiments discrimination. The up-converting labels of the invention of the invention utilize time-gated detection and/or permit essentially total rejection of non-specific background wavelength-gated detection for optimizing detection autofluorescence and are characterized by excitation and Sensitivity, discriminating multiple samples, and/or detect emitted wavelengths that are typically in the infrared or ing multiple probes on a Single Sample. Phase-Sensitive Visible portions of the Spectrum, respectively, and thus avoid detection can also be used to provide discrimination between the potentially damaging effects of ultraViolet radiation. The Signal(s) attributable to an up-converting phosphor and up-converting labels of the invention convert long 35 background noise (e.g. autofluorescence) which has a dif wavelength excitation radiation (e.g., near-IR) to emitted ferent phase shift.

radiation at about one-half to one-third the wavelength of the Up-converting organic dyes, Such as red-absorbing dyes, excitation wavelength. Since background fluorescence in the also can be used in an alternate embodiment that converts Visible range is negligible if near-IR excitation wavelengths the photons absorbed by the dye into a transient Voltage that are used, the use of up-converting labels provides essentially 40 can be measured using electrodes and conventional elec background-free detection of 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 whose presence can be detected and quantified by measuring invention, up-converting phosphors (i.e., which absorb mul 45 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 Furthermore, essentially all photoions formed in the irradi other probe molecule. In an another embodiment, 50 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 tronic Signal that is readily measured with relatively simple many other applications. 55 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 certain variations of the invention, laser irradiation of an diodes or light-emitting diodes (LEDs), and the detector 60 up-converting label can modify the immediate molecular may be any convenient detector, Such as a photodiode. In the environment through laser-induced photochemical pro case of a single reporter, the apparatus includes a laser diode ceSSes involving either direct absorption or energy transfer; capable of emitting light at one or more wavelengths in the Such spatially-controlled deposition of energy can be used to reporter's excitation band and a detector that is Sensitive to produce localized damage and/or to probe the chemical at least Some wavelengths in the reporter's emission band. 65 environment of a defined location. In Such embodiments, the The laser light is preferably focused to a Small region in the up-converting label can preferably act as a photophysical Sample, and light emanating from that region is collected catalyst.

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The invention provides methods for producing targeted performing fluorescence-activated cell Sorting (FACS) by damage (e.g., catalysis) in chemical or biological materials, flow cytometry using excitation radiation that is in the wherein a probe is employed to localize a linked infrared portion of the Spectrum and does not significantly up-converting label to a position near a targeted biological damage cells. This provides a significant advantage over structure that is bound by the probe. The localized present FACS methods which rely on excitation illumination up-converting label is excited by one or more excitation in the ultraViolet portion of the Spectrum, including wave wavelengths and emits at a shorter wavelength which may lengths which are known to produce DNA lesions and be directly cytotoxic or genotoxic (e.g., by producing free damage cells.

radicals Such as Superoxide, and/or by generating thymine The invention also provides compositions comprising at thymine dimers), or which may induce a local photolytic least one fluorescent organic dye molecule attached to an chemical reaction to produce reactive chemical Species in inorganic up-converting phosphor. The fluorescent organic the immediate vicinity of the label, and hence in the vicinity 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 15 porphyrins, and phthalocyanines, and may optionally be targeted damage to biological Structures, Such as cells, complexed with a heavy metal. The fluorescent organic dye tissues, neoplasms, vasculature, or other anatomical or his may be adsorbed to the inorganic up-converting phosphor tological Structures. crystal and/or may be covalently attached to a coated Embodiments of the present invention also include inorganic up-converting phosphor, a derivatized vitrocer up-converting phosphors which can also be excited by an amic up-converting phosphor, or a microencapsulated inor electron beam or other beam of energetic radiation of ganic up-converting phosphor. Frequently, covalent conju Sufficient energy and are cathodoluminescent. Such 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 phor with at least two electrons is employed to generate a 25 BRIEF DESCRIPTION OF THE DRAWINGS Visible-light or band emission. FIG. 1 is an optical and electronic block diagram illus The invention also provides for the Simultaneous detec trating representative apparatus for performing diagnostics tion of multiple target Species by exploiting the multiphoton on a Sample according to the present invention; excitation and Subsequent background-free fluorescence detection of Several up-converting phosphors or tiveFIG. 2A shows apparatus for implementing phase Sensi detection in the context of a single channel;

up-converting dyes. In one embodiment, Several phosphorS/ dyes are Selected which have overlapping absorption bands FIG. 2B shows apparatus where first and second laser which allow simultaneous excitation at one wavelength (or diodes are modulated by Signals from waveform generators, in a narrow bandwidth), but which vary in emission char FIG. 3 shows apparatus for performing gated detection; acteristics Such that each probe-label Species is endowed 35 FIG. 4 shows an apparatus for performing diagnostics on with a distinguishable fluorescent "fingerprint.” By using a Sample using first and Second reporters excitation bands various methods and devices, the presence and concentra centered at 2 and 22, respectively, and having overlapping tion of each of the phosphors or dyes can be determined. emission bands near 2,

The invention also provides biochemical assay methods FIG. 5A, 5B, 5C show schematically energy state transi for determining the presence and concentration of one or 40 tions in multi-photon excitation Schemes. more analytes, typically in Solution. The assay methods FIG. 6 shows a miniaturized instrument using a hand-held employ compositions of probes labeled with up-converting probe, phosphors and/or up-converting dyes and apparatus for FIG. 7A shows the use of a charge-coupled device (CCD) magnetically and/or optically trapping particles that com array used to detect emissions from a large plurality of prise the analyte and the labeled probe. In one embodiment, 45 binding Sites, a Sandwich assay is performed, wherein an immobilized probe, immobilized on a particle, binds to a predetermined lensFIG. 7B shows the CCD array used in conjunction with a array;

analyte, producing an immobilization of the bound analyte FIG. 8 shows an embodiment using optical trapping, on the particle; a Second probe, labeled with an up-converting label can then bind to the bound analyte to 50 tionFIG. of 9 ShowS Schematically dye coating and encapsula an up-converting phosphor particle;

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

sizes, colors, and/or shapes with distinct immobilized probe

(S), and/or various excitation wavelengths, it is possible to 55 FIG. 11 is a phosphor emission spectrum of Sodium perform multiple assays essentially simultaneously or con yttrium fluoride-ytterbium/erbium up-converting phosphor temporaneously. This multipleX advantage affords detection with an excitation laser Source at a wavelength maximum of and quantitation of multiple analyte species in a single 977.2 mm; emission maximum is about 541.0 nm, Sample. The assay methods are also useful for monitoring FIG. 12 is an excitation scan of the sodium yttrium the progreSS of a reaction, Such as a physical, chemical, 60 fluoride-ytterbium/erbium phosphor excitation Spectrum, biochemical, or immunological reaction, including binding with emission collection window set at 541.0 nm, reactions. For example, the invention may be used to moni FIG. 13 is a time-decay measurement of the phosphor tor the progreSS of ligand-binding reactions, polynucleotide luminescence at 541 nm after termination of excitation hybridization reactions, including hybridization kinetics and illumination for sodium yttrium fluoride-ytterbium/erbium; thermodynamic stability of hybridized polynucleotides. 65 FIG. 14 shows the phosphor emission intensity as a The invention also provides methods, up-converting function of excitation illumination intensity for a Sodium labels, and compositions of labeled binding reagents for yttrium fluoride-ytterbium/erbium phosphor;

Page 39

FIG. 15 shows effective single-photon phosphorescence DESCRIPTION OF SPECIFIC EMBODIMENTS cross-section for 0.3 um particles of Na(YosYboEros) F.

following excitation with 200 W/cm at 970 nm. Definitions FIG. 16 shows size-dependence of phosphorescence Unless defined otherwise, all technical and Scientific cross-section for Na(YosYboEroos)F particles. terms used herein have the same meaning as commonly FIG. 17A shows a fluorescence Scan of an up-converting understood by one of ordinary skill in the art to which this phosphor reporter in Hepes-buffered Saline induced by exci invention belongs. Although any methods and materials tation with a 970-nm laser Source; Similar or equivalent to those described herein can be used FIG. 17B shows a fluorescence spectrum scan of an in the practice or testing of the present invention, the up-converting phosphor reporter coated with Streptavidin in preferred methods and materials are described. For purposes Hepes-buffered saline induced by excitation with a 970-nm of the present invention, the following terms are defined laser Source; below.

FIG. 18A shows an excitation spectrum scan of an AS used herein, “label” refers to a chemical Substituent up-converting phosphor reporter in Hepes-buffered Saline 15 that produces, under appropriate excitation conditions, a with monochromatic detection of emission at 541 nm, detectable optical Signal. The optical Signal produced by an FIG. 18B shows an excitation spectrum scan of an excited label is typically electromagnetic radiation in the up-converting phosphor reporter coated with Streptavidin in near-infrared, visible, or ultraViolet portions of the Spectrum. Hepes-buffered saline with monochromatic detection of The labels of the invention are up-converting labels, which emission at 541 nm, means that the chemical Substituent absorbs at least two FIG. 19 shows the integrated signal obtained from photons at an excitation frequency and Subsequently emits Samples of (YosYboosEroos).O2S showing the relation electromagnetic energy at an emission frequency higher than ship between phosphor concentration and up-converted Sig the excitation frequency. Thus, there is generally a signifi nal; cant Stokes shift between the original excitation frequency FIG. 20 shows schematically one embodiment of an and the final emission frequency. A label is generally

Sandwich immunoassay for detecting an analyte in a Solution attached to a probe to Serve as a reporter that indicates the by binding the analyte (e.g., an antigen target) to a biotiny presence and/or location of probe. The invention encom lated antibody and to an immobilized antibody, wherein the passes organic and inorganic up-converting labels, but pref analyte forms a Sandwich complex immobilized on a Solid erably phors employs up-converting inorganic lanthanide phos as labels. Thus, a typical label of the invention is a

Substrate Superparamagnetic microbead; and Submicron-size up-converting lanthanide phosphor particle. FIG.21 shows schematically detection and discrimination The label can alternatively of two cell Surface antigens with Specific antibodies labeled chelate or cage compound. comprise a lanthanide ion in a with two phosphors with distinct phosphorescence charac teristics. AS used herein, a "probe' refers to a binding component 35 which binds preferentially to one or more targets (e.g.,

FIG. 22 shows a Schematic of an apparatus for phase antigenic epitopes, polynucleotide Sequences, macromo Sensitive detection.

FIG. 23 show a Schematic of a competitive homogeneous lecular receptors) with an affinity Sufficient to permit dis crimination of labeled probe bound to target from nonspe assay using phosphors as labels and fiber optic illumination cifically bound labeled probe (i.e., background). Generally, at a capture Surface. 40 the probe-target binding is a non-covalent interaction with a FIG.24A & FIG.24B show a schematic of a competitive binding affinity (K) of at least about 1x10'M', preferably homogeneous antigen capture assay using phosphors as with at least about 1x107M, and more preferably with an labels and a convergent illumination beam focused on the affinity of at least about 1x10 M' or greater. Antibodies capture Surface. typically have a binding affinity for cognate antigen of about FIG. 25 shows a Schematic of a homogeneous immuno 45 1x10" M' or more. For example but not limitation, probes precipitation assay using phosphors as labels and a conver of the invention include: antibodies, polypeptide hormones, gent illumination beam focused on the capture Surface polynucleotides, Streptavidin, StaphlyOCOccus aureuS pro wherein the capture Surface collects immunoprecipitates. tein A, receptor ligands (e.g., Steroid or polypeptide FIG. 26 shows a block diagram of one embodiment of hormones), leucine Zipper polypeptides, lectins, antigens apparatus for carrying out the present invention on a Sample 50 (polypeptide, carbohydrate, nucleic acid, and hapten using a microscope. epitopes), and others.

FIG. 27 is a block diagram of a microtiter plate reader for AS used herein, a “probe-label conjugate' and a "labeled use with the present invention. probe' refer to a combination comprising a label attached to FIG. 28 is an illustration of the data for upconverting 55 a probe. In certain embodiments, more than one label phosphors in three test wells. Substituent may be attached to a probe. Alternatively, in FIG. 29 is a schematic view of a second embodiment of Some embodiments more than one probe may be attached to a hand-held probe for carrying out the present invention. a label (e.g., multiple antibody molecules may be attached to FIG. 30 illustrates a three channel configuration using Variousa Submicron-size inorganic up-converting phosphor bead). interference filters. attachment chemistries can be employed to link a 60 label to a probe, including, but not limited to, the formation

FIG. 31A is an illustration of an embodiment of the of covalent bonds, hydrogen bonds, ionic bonds, electro invention in which a diode laser array F1 and a detector Static interactions, and Surface tension (phase boundary) array F2 are combined in a single device. interactions. Attachment of label can also involve incorpo FIG. 31B is a detailed view of a small section of the ration of the label into or onto microSpheres, microparticles, device shown in FIG. 31A. 65 immunobeads, and Superparamagnetic magnetic beads FIG. 32 is an emission spectrum for up-conversion from (PolySciences, Inc., Warrington, Pa.; Bangs Laboratories, neodymium chelated in EDTA. Inc. 979 Keystone way, Carmel, Ind. 46032). For example,

Page 40

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 Sequences.

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

encoded by a variable region gene (about 116 amino acids) As used herein, the term “label excitation wavelength” and one of the other aforementioned constant region genes, refers to an electromagnetic radiation wavelength that, when e.g., gamma (encoding about 330 amino acids). One form of 40 absorbed immunoglobulin constitutes the basic Structural unit of an fluorescentbyemissionan up-converting label, produces a detectable from the up-converting label, wherein antibody. This form is a tetramer and consists of two the fluorescent emission identical pairs of immunoglobulin chains, each pair having higher frequency radiation)is that of a shorter wavelength (i.e., one light and one heavy chain. In each pair, the light and length. AS used herein, the termthe"label label excitation wave emission wave heavy chain variable regions are together responsible for 45 length” refers to a wavelength that is emitted from an binding to an antigen, and the constant regions are respon up-converting label Subsequent to, or contemporaneously sible for the antibody effector functions. In addition to with, illumination of the up-converting label with one or antibodies, immunoglobulins may exist in a variety of other more excitation wavelengths, label emission wavelengths of forms including, for example, Fv, Fab, and F(ab'), as well up-converting as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., 50 radiation) than labels the are shorter (i.e., higher frequency corresponding excitation wavelengths.

Eur: J. Immunol. 17, 105 (1987)) and in single chains (e.g., Both label excitation wavelengths and label emission wave

(1988) and Bird et al., Science, 242, 423–426 (1988)). (See, Species, and are readily determined by up-converting lengths are characteristic to individual label performing simple generally, Hood et al., “Immunology’, Benjamin, N.Y., 2nd excitation and emission Scans.

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

Page 41

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 bium and erbium act as an activator couple in a phosphor physically adsorbed to the Surface of the phosphor particle host material

(Beverloo et al. (1991) op.cit., which is incorporated herein bium such as barium-yttrium-fluoride. The ytter ions act by reference). Alternatively, various inorganic phosphor radiatively to excite as the absorber, and transfer energy non the erbium ions. The emission is thus coating techniques can be employed including, but not 15 characteristic of the erbium limited to: Spray drying, plasma deposition, and derivatiza ion’s energy levels. tion with functional groups (e.g., -COOH, -NH2, Up-Converting Microcrystalline Phosphors -CONH2) attached by a silane coupling agent to -SiOH Although the invention can be practiced with a variety of moieties coated on the phosphor particle or incorporated into up-converting inorganic phosphors, it is believed that the a vitroceramic phosphor particle comprising Silicon oxide(s) preferred embodiment(s) employ one or more phosphors and up-converting phosphor compositions. Vitroceramic derived from one of several different phosphor host phosphor particles can be aminated with, for example, materials, each doped with at least one activator couple. aminopropyltriethoxysilane for the purpose of attaching Suitable phosphor host materials include: Sodium yttrium amino groups to the vitroceramic Surface on linker fluoride (NaYF), lanthanum fluoride (LaF), lanthanum molecules, however other omega-functionalized Silanes can 25 oxysulfide, yttrium oxysulfide, yttrium fluoride (YF), be Substituted to attach alternative functional groups. yttrium gallate, yttrium aluminum garnet, gadolinium fluo Probes, Such as proteins or polynucleotides may then be ride (GdF), barium yttrium fluoride (BaYFs, BaYF), and directly attached to the vitroceramic phosphor by covalent gadolinium oxysulfide. Suitable activator couples are linkage, for example through Siloxane bonds or through selected from: ytterbium/erbium, ytterbium/thulium, and carbon-carbon bonds to linker molecules (e.g., organo ytterbium/holmium. Other activator couples suitable for functional Sillylating agents) that are covalently bonded to or up-conversion may also be used. By combination of these adsorbed to the Surface of a phosphor particle. Covalent host materials with the activator couples, at least three conjunction between the up-converting inorganic phosphor phosphors with at least three different emission spectra (red, particles and proteins e.g., avidin, immunogobulin) can be green, and blue Visible light) are provided. Generally, the accomplished with homobifunctional, or preferably 35 absorber is ytterbium and the emitting center can be Selected heterobifunctional, crosslinkers. For example, Surface Slian from: erbium, holmium, terbium, and thulium; however, ization of the phosphors with tri(ethoxy)thiopropyl Silane other up-converting phosphors of the invention may contain leaves a phosphor Surface with a thiol functionality to which other absorbers and/or emitters. The molar ratio of absorber: a protein (e.g., antibody) or any compound containing a emitting center is typically at least about 1:1, more usually primary amine can be grafted using conventional 40 at least about 3:1 to 5:1, preferably at least about 8:1 to 10:1, N-succinimidyl(4-iodoacetyl)amino-benzoate (SIAB) more preferably at least about 11:1 to 20:1, and typically less chemistry (Weltman et al. (1983). Other silanization and than about 250:1, usually less than about 100:1, and more croSS-linking methods compatible with the inorganic phos usually less than about 50:1 to 25:1, although various ratios phors may be used at the discretion of the practitioner. may be Selected by the practitioner on the basis of desired Microcrystalline up-converting phosphor particles are 45 characteristics (e.g., chemical properties, manufacturing typically Smaller than about 7 microns in diameter, prefer efficiency, absorption cross-section, excitation and emission ably less than about 1 micron in diameter (i.e., Submicron), wavelengths, quantum efficiency, or other considerations). and more preferably are 0.1 to 0.3 microns or less in The ratio(s) chosen will generally also depend upon the diameter. It is generally most preferred that the phosphor particular absorber-emitter couple(s) selected, and can be particles are as Small as possible while retaining Sufficient 50 calculated from reference values in accordance with the quantum conversion efficiency to produce a detectable Sig desired characteristics.

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

invention. Such conversion efficiency data may be obtained Some embodiments of the invention employ inorganic from available Sources e.g., handbooks and published phosphors that are optimally excited by infrared radiation of

Page 42

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-terbium 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 pum), 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 tion capable of up-conversion are Suitable for use in the inven is 0.05 to 0.01; 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

Oxysulfides (OS)

(Yose Yboos Eroos) O 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 (OX) 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 critical grain size of the phosphor is given by the quantum YF,

GdF.

Ytterbium

Ytterbium

Erbium

Erbium

Red

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 BaYF Ytterbium Terbium Green about 1-2%. Er. Thus, a phosphor grain containing Several Gallates (Gao) thousand formula units ensures the emission of at least one YGaO. Ytterbium Erbium Red or more photons during a typical laser irradiation time. Y3Ga5O12 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 um). 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 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.

Page 43

Preparation of Inorganic Phosphor Labels hydroxy carbonate precursor to the oxySulfide phase by Techniques and methods for manufacture of inorganic including a polysulfide flux for annealing. Using this phosphorS has been described in the art. Up-converting technique, highly efficient OXySulfide particles in the 0.3 to phosphor crystals can be manufactured by those of ordinary 0.4 um diameter range were prepared as a dispersion in skill in the art by various published methods, including but water. Frequently, Sonication can be used to produce a not limited to the following: Yocom et al. (1971) Metallur monodisperse mixture of discrete Spherical particles. After gical Transactions 2: 763; Kano et al. (1972) J. Electro fractionation and coating, these particles can be used as chem. Soc., p. 1561; Wittke et al. (1972).J. Appl. Physics 43: up-converting reporters. Furthermore, this general prepara 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 may be referred to are: Jouart J. P. and Mary G (1990).J. be Frequently, advantageous for various assay formats.

Such as with phosphors having an oxySulfide

Luminescence 46: 39; McPherson G. L. and Meyerson S. L.

(1991) Chem. Phys. Lett. (April) p. 325; Oomen et al. (1990) host material, the phosphor particles are preferably dis J. Luminescence 46:353; NIH and Rand S.C. (1991) Optics 15 persed in a polar solvent, such as acetone or DMSO and the Lett. 16 (September); McFarlane R. A. (1991) Optics Lett. like, to generate a Substantially monodisperse emulsion 16 (September); Koch et al. (1990) Appl. Phys. Lett. 56: (e.g., for a stock Solution). Aliquots of the monodisperse 1083; Silversmith et al. (1987) Appl. Phys. Lett. 51: 1977; Stock Solution may be further diluted into an aqueous Lenth W and McFarlane R. M. (1990).J. Luminescence 45: Solution (e.g., a Solution of avidin in buffered water or 346; Hirao et al. (1991) J. Non-crystalline Solids 135:90; buffered saline).

McFarlane et al. (1988) Appl. Phys. Lett. 52: 1300, incor It was found that washing phosphors in acetone or DMSO porated herein by reference). improved Suspendability of inorganic phosphor particles in In general, inorganic phosphor particles are milled to a water. In particular, the phosphor particles prepared with polysulfide flux are preferably resuspended and washed in desired average particle size and distribution by conven hot DMSO and heated for about an hour in a steam bath then tional milling methods known in the art, including milling in 25 a conventional barrel mill with Zirconia and/or alumina balls allowed to cool to room temperature under continuous for periods of up to about 48 hours or longer. Phosphor agitation. The phosphor particles may be pre-washed with particles used in binding assays are typically about 3.0 to acetone (typically heated to boiling) prior to placing the 0.01 um in diameter (or along the long axis if non-spherical), particles in the DMSO. Hot DMSO-treated phosphors were more usually about 2.0 to 0.1 um in size, and more conve found to be reasonably hydrophilic and form stable Suspen niently about 1.0 to 0.3 um in size, although phosphor sions. A MicrofluidizerTM (Microfluidics Corp.) can be used particles larger or Smaller than these dimensions may be to further improve the dispersion of particles in the mixture. preferred for certain embodiments. Phosphor particle size is DMSO-phosphor Suspensions can be easily mixed with selected by the practitioner on the basis of the desired water, preferably with mall amounts of Surfactant present. In general, olysaccharides (e.g., guar gum, Xanthan gum, gum characteristics and in accordance with the guidelines pro 35

Vided herein. Fractions having a particular particle size arabic, lginate, guaiac gum) can be used to promote deaggre range may be prepared by Sedimentation, generally over an gation of particles. In a variation, particles are washed in hot DMSO and serially diluted into a 0.1% aqueous gum arabic extended period (i.e., a day or more) with removal or the Solution, desired size range fraction after the appropriate Sedimenta Sion problems which appears to virtually eliminate water disper tion time. The Sedimentation proceSS may be monitored, 40 of phosphors.

such as with a Horiba Particle Analyzer. Resuspended phosphors in organic Solvent, Such as However, milling crystalline materials has Several weak DMSO, are typically allowed to settle for a suitable period nesses. With milling, the particle morphology is not (e.g., about 1-3 days), and the Supernatant which is typically uniform, as milled particles result from random fracture of turbid is used for Subsequent conjugation. larger crystalline particles. Since the Sensitivity of a detec 45 LudoxTM is a colloidal silica dispersion in water with a tion assay using up-converting inorganic phosphors depends Small amount of organic material (e.g., formaldehyde, on the ability to distinguish between bound and unbound glycols) and a small amount of alkali metal. Ludox" and its phosphor particles, it is preferable that the particles be of equivalents can be used to coat up-converting phosphor identical Size and morphology. Size, weight, and morphol particles which can Subsequently be fired to form a ceramic ogy of up-converting microcrystalline phosphor particles 50 Silica coating which cannot be removed from the phosphor can affect the number of potential binding sites per particle particles, but which can be readily Silanized with organo and thus the potential Strength of particle binding to reporter functional Silanes (containing thiol, primary amine, and and/or analyte. Monodisperse Submicron Spherical particles carboxylic acid functionalities) using Standard Silanization of uniform size can be generated by homogeneous precipi chemistries (Arkles, B, in: Silicon Compounds: Register and tation reactions at high dilutions. For example, Small yttrium 55 Review; 5th Edition (1991); Anderson, R. G., Larson, G. L., hydroxy carbonate particles are formed by the hydrolysis of and Smith, C, eds.; p. 59-64, Huls America, Piscataway, urea in a dilute yttrium Solution. Similarly, up-converting N.J.).

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

Page 44

Depending upon the chemical nature of the coating, Some binding reagents, either directly or indirectly, for use in minor loSS in conversion efficiency of the phosphor may binding assays to detect and quantitate the presence of occur as a result of coating, however the power available in analyte(s) in a Sample. Binding reagents are labeled directly a laser excitation Source can compensate for Such reduction by attachment to up-converting reporters (e.g., Surface in conversion efficiency and ensure adequate phosphor emis adsorption, covalent linkage). Binding reagents which can SO. be directly labeled include, but are not limited to: primary In general, preparation of inorganic phosphor particles antibodies (i.e., which bind to a target analyte), Secondary and linkage to binding reagentS is performed essentially as antibodies (i.e., which bind to a primary antibody or pros described in Beverloo et al. (1992) op.cit., and Tanke U.S. thetic group, Such as biotin or digoxygenin), Staph lococcuS Pat. No. 5,043,265. Alternatively, a water-insoluble poly aureuS Protein A, polynucleotides, Streptavidin, and receptor functional 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 directly labeled Second antibody (e.g., a goat anti-rabbit lic acids (e.g., 5% acrylic acid/95% methyl acrylate 15 antibody linked to an up-converting inorganic phosphor). copolymer), amine (e.g., 5% aminoethyl acrylate/95% Quantitative detection of the analyte-probe complex may be methyl acrylate copolymer) reducible Sulfonates (e.g., 5% conducted in conjunction with proper calibration of the Sulfonated polystyrene), and aldehydes (e.g., polysaccharide assay for each probe employed. A probe is conveniently copolymers). The phosphor particles are coated with water detected under Saturating excitation conditions using, for insoluble polyfunctional polymers by coacervative encap example, a laser Source or focused photodiode Source for Sulation in nonaqueous media, washed, and transferred to a excitation illumination.

Suitable aqueous buffer Solution to conduct the heterobifunc Specific binding assays are commonly divided into homo tional crosslinking to a protein (e.g., antibody) or polynucle otide probe molecule. An advantage of using water the signalandemittedgeneous heterogeneous assays. In a homogeneous assay, by the bound labeled probe is different insoluble polymerS is that the polymer microcapsule will not from the signal emitted by the unbound labeled probe, hence migrate from the Surface of the phosphor upon aging the 25 encapsulated phosphors in an aqueous Solution (i.e., the two can be distinguished without the need for a physical improved reagent Stability). Another advantage in using Separation Step. In heterogeneous assays, the Signal emitted copolymers in which the encapsulating polymer is only from the bound and unbound labeled probes is identical, partially functionalized is that one can control the degree of hence the two must be physically Separated in order to functionalization, and thus the number of biological probe cific bindingbetween distinguish assay is them. The classical heterogeneous spe the radioimmunoassay (RIA) (Yalow et molecules which can be attached to a phosphor particle, on al. (1978) Science 200: 1245, which is incorporated herein average. Since the Solubility and coacervative encapsulation by reference). Other heterogeneous process will depend on the dominant nonfunctionalized the radioreceptor assay (Cuatrecasasbinding assays include

component of the copolymer, the functionalized copolymer ratio can be varied over a wide range to generate a range of 35 Biochem. 43: 109), the sandwich radioimmunoassay (U.S. potential crosslinking sites per phosphor, without having to Pat. No. 4,376,110, which is incorporated herein by Substantially change the encapsulation process. reference), and the antibody/lectin sandwich assay (EP0166 A preferred functionalization method employs heterobi neous 623, which is incorporated herein by reference). Heteroge assays are usually preferred, and are generally more functional crosslinkers that can be made to link the biologi 40 Sensitive and reliable than homogeneous assayS. cal macromolecule probe to the insoluble phosphor particle Whether a tissue extract is made or a biological fluid in three steps: (1) bind the crosslinker to the polymer coating Sample is used, it is often desirable to dilute the Sample in on the phosphor, (2) separate the unbound crosslinker from one or more diluents that do not substantially interfere with the coated phosphors, and (3) bind the biological macro Subsequent assay procedures. Generally, Suitable diluents molecule to the washed, linked polymer-coated phosphor. 45 are aqueous Solutions containing a buffer System (e.g., 50 This method prevents undesirable crosslinking interactions mM NaH2PO or 5-100 mM Tris, pH4-pH10), non between biological macromolecules and So reduces irrevers interfering ionic species (5-500 mM KCl or NaCl, or ible aggregation as described by Tanke et al. Examples of Sucrose), and optionally a nonionic detergent Suitable heterobifunctional crosslinkers, polymer coating When the sample to be analyzed is affixed to aSuch as Tween. solid support, functionalities, and linkable biological macromolecules 50 it is usually desirable to wash the Sample and the Solid include, but are not limited to: Support with diluent prior to contacting with probe. The Sample, either Straight or diluted, is then analyzed for the

Coating Heterobifunctional Biological diagnostic analyte.

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

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

Binding ASSayS that is detected by a labeled second antibody, or a biotiny 65 lated polynucleotide that is detected by labeled streptavidin).

Up-converting phosphors and up-converting organic dyes The bound complex(es) are typically isolated from unbound are used as reporters (i.e., detectable markers) to label probe-label conjugate(s) prior to detection of label, usually

Page 45

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

For polynucleotide probes:

3–10x SSC, pH 6-8; usually 5x SSC, pH 7.5 monomer to generate a polymer shell encasing the phosphor article. Phosphor particles embedded in a polymer coating, 0-50 percent deionized formamide Such as a gel coating, can be functionalized (e.g., with amino 1-10x Denhardt's Solution groups) for covalent attachment to a binding component. 0-1 percent sodium dodecyl sulfate Similarly, up-converting phosphor particles can be coated 10-200 lug/ml sheared denatured 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 Enzymology, 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 minutes to Several hours, although typically less than about Since up-converting phosphors can be differentiated on 24 hours, more preferably less than about a few hours or the basis of the excitation and/or emission wavelength less. Binding reactions (including washes) are typically Spectra, up-converting phosphors can be used to detect and carried out a temperature range of about 0°C. to about 45 discriminate multiple analyte targets, Such as, for example, C., preferably about 4°C. to about 20°-25° C. 65 cell Surface antigens or Soluble macromolecules. Binding assays, which include in Situ hybridization, in For example, Streptavidin, avidin, or another linker mac Situ binding assays, and immunohistochemical Staining, are romolecule (e.g., antidigoxigenin antibody) are attached,

Page 46

respectively, to each of two different phosphors (for For example, a Solid Substrate may have a plurality of illustration, designated here as Phosphorit1 and Phosphorit2) distinct species of first binding component (e.g., an array of which differ in their absorption and/or emission spectra So as different oligopeptides affixed to a Solid Support). One or to facilitate discrimination of the two phosphors based on more of the Species of first binding component may bind to absorption and/or emission wavelengths, e.g., one phosphor a particular analyte (e.g., a muscarinic receptor) in an may emit in the blue and the other may emit in the green. For analyte Solution that is in contact with the Solid Support. example and not limitation, Na(YosoYbolisEroo)F emits Binding of the analyte to one or more of the first binding predominantly in the green, and Na(YozaYbo27Tmooo..)F. component Species may then be detected with a Second emits predominantly in the blue, and thus these two phos binding component (e.g., an anti-muscarinic receptor phors may be discriminated on the basis of their phospho antibody) labeled with an up-converting phosphor (either rescent emissions. Alternatively, two phosphors may pro directly or through a biotinylated Secondary antibody). duce essentially similar emission Spectra but may have Solid Substrates can be attached to a first binding com different excitation wavelengths which provide a basis for ponent which can bind more than one distinct analyte (e.g., their discrimination in multiple analyte detection. A first may be immunocrossreactive or polyspecific) and/or can be binding component (e.g., an antibody) that binds specifically 15 attached to multiple first binding component Species which to a first analyte species (e.g., a lymphocyte CD4 antigen) can bind multiple distinct analytes. Similarly, multiple Sec and incorporates biotinyl moieties which may be bound by ond binding component Species with binding Specificities Streptavidin-Phosphorif1 conjugates can be used to quanti for particular analytes can be employed. When multiple tatively detect the presence of a first analyte in a Sample Second binding component Species are employed, it is typi (e.g., a serum Sample) by measuring phosphorescence of cally desirable to label each Second binding component Phosphorif1 in analyte-binding component complexes. A Species with a unique up-converting label that can be Second binding component (e.g., a probe polynucleotide) distinguished on the basis of its absorption and/or emission that binds specifically to a second analyte species (e.g., an properties.

HIV-1 sequence) and incorporates digoxygenin moieties It is possible to use different absorbers in combination (e.g., 11-UTP-digoxygenin) which may be bound by 25 with antidigoxigenin-Phosphorif2 conjugates can be used to havingvarious Several emitters to produce a collection of phosphors differentiable combinations of excitation and quantitatively detect the presence of a Second analyte in the emission Spectra.

Sample by measuring phosphorescence of Phosphorit2 in entiable phosphorsFormay example but not limitation, Six differ be generated from two absorbers analyte-binding component complexes. Thus, by Simulta and three emitters. A first absorber, A, has an excitation neously or contemporaneously detecting the presence of wavelength of 2A, a Second absorber, A has an excitation multiple phosphor reporters having differentiable Signal wavelength of 2A, a first emitter, E, has an emission line characteristics, multiple analytes may be quantitatively at 2, a Second emitter, E2 has an emission line at 22, and detected in a single Sample. a third emitter, E, has an emission line at 2Es. The six Sandwich Binding ASSayS phosphors may be differentiated and the Signal from each 35 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 ted radiation at 21, 22, and 23, and Separately illuminat is incorporated herein by reference). For example, a mag ing the Sample with 2A2 and detecting Separately the netic bead, Such as a Superparamagnetic immunobead or emitted radiation at 21, 2, and 2. Table II shows the functionalized magnetizable polymer particle (PolySciences, 40 various absorber:emitter combinations and their excitation Inc., Warrington, Pa.), can serve as the Solid Substrate which and emission wavelengths.

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

Sugar moiety, chemical Substituent, or nucleotide Sequence) 45 Absorber:Emitter Combination Excitation w Emission w of an analyte. The analyte binds to the first binding com A1:E1 WA1 WE1 ponent and also to a second binding component (e.g., an A1:E2 WA1 AE2 antibody, a lectin, or a polynucleotide) which binds to a A1:E3 WA1 WE3 Second epitope of the analyte. Thus, the analyte bridges the A2:E1 WA2 WE1 two binding components to form a Sandwich complex which 50 A2:E2 WA2 AE2

is immobilized with respect to the Solid substrate. The

Second binding component typically has an attached or incorporated label, Such as a biotinyl group which can be Of course, additional absorber:emitter combinations are bound to a Streptavidin-coated up-converting phosphor. possible to provide more than six differentiable phosphor Alternatively, the Second binding component can be linked 55 labels.

directly to an up-converting phosphor, Such as through a It is also possible to utilize solid substrates of different covalent linkage with a functionalized vitroceramic types which may be distinguished (e.g., by size, color, up-converting phosphor. density, magnetic properties, shape, charge) So that a par The Sandwich complex comprises the first binding ticular type of Solid Substrate is associated with a particular component, an analyte, and the Second binding component, 60 Species of first binding component.

which is labeled, either directly or indirectly, with an For example and not limitation, the following three brief up-converting reporter. The Sandwich complex is thus examples are provided to explicate further possible appli immobilized on the Solid substrate, although the solid Sub cations of multiple analyte Sandwich assay methods. Strate itself may be mobile (e.g., a Superparamagnetic bead Substrate Differentiation circulating in a sample slurry). The presence and amount of 65 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. able Substrate types to detect the presence of Specific immu

Page 47

noglobulin idiotypes in a sample (e.g., a blood Serum sample the APP isoform(s) having the Y epitope, while the intensity taken from a patient) which can provide diagnostic infor of the 2-induced green emission is a measure of the APP mation about the immune status of a patient (e.g., is a patient isoform(s) having the Z epitope. If the emissions from two Seroreactive with a particular antigen). phosphors are readily distinguishable, 2 and 2 may be Large Superparamagnetic beads are conjugated to an identical. The standardized relative intensities of the two immunogenic Herpesvirus Type II envelope glycoprotein, phosphors provides a measure of the relative abundance of medium-sized Superparamagnetic beads are conjugated to the APP isoform(s) containing the Y or Z epitopes. HIV gp120 glycoprotein, and Small Superparamagnetic Phosphor and Substrate Differentiation beads are conjugated to an immunogenic cytomegalovirus The following example describes the use of differentiable envelope glycoprotein. A Serum Sample is taken from a up-converting phosphors in conjunction with distinguish patient and is incubated with a mixture of the Superpara able Substrate types to detect the presence and relative magnetic beads under binding conditions to permit specific abundance of particular T lymphocyte Subpopulations in a binding of immunoglobulins in the Sample with the three blood Sample taken from an individual. Although described immobilized viral glycoprotein Species. The Superparamag here with reference to detecting T cell Subpopulations, netic beads are separated from the Sample to remove non 15 analyte multiplexing (i.e., detecting and/or characterizing Specifically bound immunoglobulin and incubated with multiple analytes in a Sample by using various Solid Sub up-converting phosphor particles coated with Staphylococ Strate types and/or up-converting phosphor labels) is cus aureuS Protein A, which binds to IgG, under binding believed to be a generally applicable method. conditions. Superparamagnetic beads having Specifically Large Superparamagnetic beads are conjugated to an bound IgG are thus labeled with the phosphor-Protein A anti-CD4 antibody, medium-sized Superparamagnetic beads conjugate. Large, medium, and Small Superparamagnetic are conjugated beads are then Separately illuminated with phosphor exci magnetic beads to are anti-CD8 antibody, and Small Superpara conjugated to an anti-CD28 antibody. An tation electromagnetic radiation and time-gated emitted antibody that specifically binds to the CD2 antigen is labeled phosphorescence is detected. Background attributable to with an up-converting phosphor non-specific binding, if any, is determined and Subtracted length ), and emits in the red. Anthatantibody 25 has an excitation wave using internal standard beads (bovine Serum albumin coated binds to the CD45R antigen is labeled with anthat Specifically

Superparamagnetic beads) and positive and negative control phosphor that has an excitation wavelength ), and emits in Serum Samples. The intensity of phosphorescence associated with the large, medium, and Small beads provides a measure the green. An antibody that specifically binds to the CDwó0 antigen is labeled with an up-converting phosphor that has of the amount of antibodies in the Sample which are reactive with the Herpesvirus Type II envelope glycoprotein, HIV an excitation wavelength ), and emits in the blue. gp120 glycoprotein, and cytomegalovirus envelope A blood (or Serum, Sputum, urine, feces, biopsy tissue, etc.)

glycoprotein, respectively. This information can be used to mixture sample is taken from a patient and is incubated with a of the Superparamagnetic beads and phosphor determine whether an individual patient has been infected labeled antibodies with the HIV-1, human CMV, and/or Herpes Simplex Type cific binding of cells 35 under binding conditions to permit spe

II viruses. in the blood sample with the three bead-immobilized antibody Species and the three phosphor

Phosphor Differentiation labeled antibody Species. After antigen-antibody binding The following example describes the use of differentiable occurs, the Superparamagnetic beads are Segregated and up-converting phosphors to detect the presence and relative 40 examined, either Sequentially or Simultaneously, by illumi abundance of particular isoforms of human APP (amyloid nation with 2, 2, and ), and quantitative detection of red, precursor protein) in a serum or brain biopsy sample. green, and blue emissions, respectively. For example, the Various isoforms of APP arise in the brain as a consequence intensity of 21-induced red light emission associated with of alternative eXon usage and/or alternative proteolytic pro the large beads is a rough measure of the amount of cells cessing pathways. Thus, although all APP isoforms may 45 having both CD4 and CD2 surface antigens and/or the share a common, hypothetical epitope (X), a particular APP relative abundance of those Surface antigens (e.g., there may isoform may have a unique epitope (Y), while another APP be very few CD4 cells that have CD2, but those few cells isoform has a unique epitope (Z). it is possible that the may have a large amount of CD2 antigen, and hence a large relative abundance of a particular APP isoform in a sample CD2 phosphorescent signal).

may be of predictive value or may be pathognomonic for 50 Similarly, the intensity of 2.2-induced green light associ Alzheimer's Disease. ated with the large beads is a rough measure of the amount Superparamagnetic beads are conjugated to an antibody of cells having both CD4 and CD45R surface antigens that binds specifically to a common APP epitope (X) shared and/or the relative abundance of those Surface antigens in a by all isoforms. A specific antibody reactive with the unique Sample.

Y epitope is labeled with Phosphor #1, which is excited by 55 In this manner, an analyte Sample, Such as a blood Sample, wavelength ), and emits in a wavelength spectrum centered can be "fingerprinted” for the presence and relative in the blue. A Specific antibody reactive with the unique Z distribution(s) (e.g., coSegregation and/or correlation) of epitope is labeled with Phosphor #2, which is excited by a various analyte species. Such an analyte fingerprint may be wavelength), and emits in a wavelength spectrum centered used for providing diagnostic or therapeutic information, for in the green. A Sample containing APP isoforms is incubated 60 example, as to measuring a patient's immune Status or with the Superparamagnetic beads and labeled Specific anti measuring response to chemotherapy directed against a bodies under binding conditions. The Superparamagnetic particular blood cell Subset. Similar analyte fingerprints can beads are retrieved from the sample, either individually or in be used to type pathogenic organisms and viruses, as well as bulk. The beads are illuminated with wavelength) and blue to order polynucleotide Sequences for gene mapping and/or light emission is detected and measured, and illuminated 65 Sequencing.

with 2 and green light emission is detected and measured. Superparamagnetic beads which can be differentiated The intensity of 21-induced blue emission is a measure of based on size, shape, color, or density can be magnetically

Page 48

trapped individually and Scanned with appropriate excitation damage') is avoided. Physical Separation may be accom illumination(s) and phosphor emission(s) characteristic of plished by various means, including but not limited to: (1) particular analytes detected. For example, a unitary detector performing excitation irradiation on a dilute Suspension of can simultaneously or contemporaneously trap the Super target and non-target cells, wherein the mean distance Sepa paramagnetic bead from a Suspension, determine the bead rating individual cells is Sufficient to reduce Secondary type (size, shape, and/or color), and Scan for presence and damage to non-targets, and (2) employing hydrodynamic abundance of particular phosphors (by illuminating with focusing to pass cells (both targets and non-targets) single excitation wavelength(s) and detecting emitted file through an illumination Zone (e.g., as in a fluorescence wavelengths). activated cell Sorter or the like). Thus, an up-converting phosphor linked to an anti-CD8" antibody can be used to

By performing binding assays under dilute conditions Selectively damage CD8" lymphocytes in a lymphocyte wherein an average of one analyte or less (e.g., lymphocyte) Sample, where (1) the phosphor emits at a wavelength that is bound per microbead, it is possible to type cells individu is either directly cytotoxic and/or (2) the phosphor emits at ally (e.g., determine the abundance of CD45R on each a wavelength that produces reactive chemical Species by individual CD4+ cell) and thus generate more precise lym 15 photocatalysis of a compound present in the sample (e.g., a phocyte Subpopulation definitions. Sample can be doped with buckminsterfullerene). Biotinylated magnetic beads can also be used to monitor Instead of using the emitted radiation directly for photo the kinetics of binding Streptavidin to phosphor particles catalytic action on tissue or tumors, an excited form of and/or to Segregate or purify Streptavidin-coated oxygen, So called singlet excited oxygen (O'Ag) can be up-converting phosphor particles from a reaction. Thus, generated by energy transfer from a dye Sensitizer to dis Streptavidin and up-converting phosphor particles are mixed Solved molecular oxygen. This Scheme makes use of the in a reaction vessel under binding conditions for forming tissue penetrating power of near-infrared radiation (red and Streptavidin-coated phosphor particles. After a Suitable bind ultrared region light, including 970 nm) which reaches the ing period, unbound Streptavidin may be removed (e.g., by inorganic up-converting phosphor. Two of the infrared pho centrifugation wherein phosphor particles are collected as 25 tons are converted either into a red, green, or blue photon the pellet, unbound Streptavidin in the Supernatant is depending

The dye is on the absorption Spectrum of the Sensitizer dye.

excited by the up-converted radiation into a triplet decanted, and the pellet is resuspended), biotinylated mag State which transferS its energy to a dissolved molecular netic beads are added to the remaining phosphor Suspension oxygen molecule to yield an excited (singlet) oxygen mol in binding conditions, and Streptavidin-coated phosphor ecule. The cytotoxic activity of Singlet oxygen is well particles are recovered bound to the biotinylated magnetic documented in photodynamic therapy and other biomedical beads.

applications (see, Wagnieres et al. (19–21 Jan. 1990) Future

Photophysical Catalysis by Up-Converting Directions and Applications of Photodynamic Therapy, pp. Phosphors 249, SPIE Institutes for Advanced Optical Technologies,

Society of Photo-Optical Instrumentation Engineers, Box

Other applications of the invention employ phosphors as 35 10, Bellingham, Wash. 98277; Pelegrin et al. (1991) Cancer a photophysical catalyst linked to a probe, where the radia 67: 2529; Wagnieres et al. (24–25 May 1991) Future Direc tion emitted by the phosphor is used, typically in conjunc tions and Applications of Photodynamic Therapy, pp. 219; tion with a dye molecule, to produce localized intense Folli et al. (17 Dec. 1991) Fluoresceine Clinique 4; Bra electromagnetic radiation in an area adjacent to the probe for ichotte et al. (May 1991) ENT-Clinic, Lausanne, various purposes other than detection (e.g., cytotoxicity, 40 Switzerland).

ionization of chemical species, mutagenesis, etc.). For In this application the up-converting phosphor is mixed or example, an antibody that specifically binds to a cell Surface laced with a Sensitizing dye Such as methylene blue, rose antigen, Such as a CD8 antigen on a CD8 lymphocyte, may bengal or phthalocyanine derivatives, Such as be used as a probe linked to a up-converting phosphor to Zn-phthalocyanine. In the first and third case a red-emitting localize the phosphor to CD8 lymphocytes. A sample 45 phosphor is used, whereas for rose bengal agreen-emitting containing CD8 lymphocytes can be incubated with the phosphor is best Suited. The phthalocyanine derivatives are anti-CD8 probe-phosphor conjugate and irradiated with an ideally Suited for this purpose because of their total insolu excitation wavelength (e.g., from an infrared laser diode), bility in aqueous or biological Solutions. These dyes there resulting in emission of up-shifted photons (i.e., higher fore stay in close proximity to the emitters So that the frequency electromagnetic radiation) in the vicinity of CD8" 50 Specificity of the cell Surface-reporter/probe/dye complex lymphocytes to which the anti-CD8" probe-phosphor con becomes the limiting factor. In this case, Specialized com jugate has bound. The emitted radiation may be of a wave binations of reporter/probe/dye formulations preferably in length that is directly mutagenic and/or cytotoxic (e.g., the 0.1 to 0.3-micron Size range must be Synthesized in order ultraViolet radiation that can lead to formation of thymine to enable efficient energy transfer: first, up-converted radia dimers, 760–765 nm light is also believed to produce 55 tion is absorbed by the dye as completely as possible; and chromosomal damage) or may be of a wavelength that can Second, the dye excited energy (triplet State) is transferred to cause a photolytic decomposition of a chemical present in dissolved molecular oxygen. Both processes are very effi the environment, leading to local formation of reactive cient if the absorption Spectrum of the Sensitizer dye is Species that may damage adjacent cells (e.g., photodecom matched to the up-converted radiation.

position of buckminsterfullerene, Co, to Css and C, may 60 This Scheme presents a step beyond the traditional pho produce free radicals that may cause lipid peroxidation of todynamic therapy methods in that the red light can be used cell membranes). both for tracking and diagnostic as well as for therapeutic Since phosphor-emitted radiation is isotropic, it is gener purposes after up-converting thus necessitating only one ally desirable to physically separate targets (e.g., CD8 (infrared) light source at about 1000 nm. A further advantage lymphocytes) from non-targets (e.g., CD8 lymphocytes) 65 is the greater range within biological Samples of the infrared prior to excitation irradiation, So that undesirable damage to radiation compared to other known photodynamic therapy non-targets by isotropic emission(s) (i.e., "secondary excitation schemes (750-850 nm).

Page 49

For embodiments employing up-converting phosphors as Other candidate ions include the other lanthanide elements, photophysical catalysts, it is generally desirable that: (1) the the actinide elements, and other metal elements. Stepwise wavelength(s) of the excitation radiation do not produce excitation Schemes Suitable for up-conversion in lanthanide Significant photocatalysis of the Substrate compound, (2) the chelates are described in the literature on up-conversion wavelength(s) of the excitation radiation are not directly lasers. Examples include up-conversion in erbium cytotoxic or mutagenic, and (3) the emitted radiation is (Silversmith et al. (1986) J. Opt. Soc. Am. A3: 128, and directly cytotoxic and/or is of an appropriate wavelength to Macfarlane et al. (1989) Appl. Phys. Lett. 54: 2301), neody produce a biologically effective amount of photodecompo mium (Macfarlane et al. (1988) Appl. Phys. Lett. 52: 1300), Sition of a Substrate compound (e.g., buckminsterfullerene, thulium (Nguyen et al. (1989) Appl. Opt. 28: 3553 and pSoralen, compounds containing azide Substituents or other Allain et al. (1990a) Electron. Lett. 226: 166), holmium photoactivated groups). Alternatively, histidine side chains (Allain et al. (1990b) Electron. Lett. 26:261), and praseody of polypeptides can be oxidized by light in the presence of mium (Smart et al. (1991) Electron. Lett. 27: 1307). Other dye Sensitizers, Such as methylene blue or rose bengal up-conversion laser Schemes that rely on energy transfer, (Proteins, Structures and Molecular Principles, (1984) energy pooling, croSS relaxation, or avalanche absorption are Creighton (ed.), W.H. Freeman and Company, New York; 15 not appropriate for up-converting chelates because they rely Introduction to Protein Structure, (1991), C. Branden and J. on energy transfer between ions. These processes are Tooze, Garland Publishing, New York, N.Y., which are described by Auzel (1973) Proc. IEEE 61: 758 and Lenth incorporated herein by reference). Thus, for example, and Macfarlane (March 1992) Optics and Photonic News 3: up-converting phosphors linked to anti-CD8 antibodies can 8. Energy transfer can be efficient in a crystalline host be used as photophysical catalysts to produce Selective, containing many rare earth ions, but not in a Solution where localized damage to CD8 lymphocytes. In accordance with the concentration of ions is low and the phonon Structure is the invention, essentially any antibody can be linked to an leSS constrained.

appropriate up-converting phosphor, either directly or by In certain cases, these Schemes may not function as well conjugation to protein A which may then bind the immu for up-conversion in chelates. For example, certain of the noglobulin. Thus, the up-converting photophysical catalysts 25 Schemes have been demonstrated using crystalline host of the invention may be used to target essentially any desired materials at very low temperatures, and may not function as antigen or cell type that can be distinguished by the presence well at room temperature in a chelate. Schemes that do not of an identified antigen. involve intermediate relaxation Such as that of Smart et al. Up-converting Chelates have advantages in chelates because they can be excited Certain applications require Small reporters. For example, more effectively with pulsed Sources. Higher peak powers the transport, ability to Stay in Suspension, the bonding can be obtained from diode lasers when they are operated in a pulsed mode. The higher peak powers lead to more dynamics, and the tendency toward removal by microphages efficient up-conversion due to the nonlinear dependence on may be improved for smaller reporters. However, the reduced Sensitivity available with Smaller reporters must 35 excitation power.

also be considered. Up-Converting Organic Dyes One type of Small up-converting inorganic phosphor Similar to the up-converting inorganic phosphor reporters consists of rare earth ions in chelates. The use of lanthanide chelates as reporters has been developed for biological We propose to use "molecular labels whose fluorescence will be detected by optoelectronic means. Infrared or red assays as described on pages 6 and 7 of this application. This 40 light is exciting the probe-reporter complex bound to a prior use of lanthanide chelates involved down-conversion. target, after which light is emitted at Shorter wavelengths That is, the emission light is at a wavelength which is longer with respect to the illuminating Source. This up-converted than the excitation wavelength. light is free of Scattered light from the Source or autofluo Rare earth chelates may be used as up-converting report rescence by Virtue of its higher energy. Furthermore, autof erS through Stepwise excitation Such as shown in FIG. 5a, or 45 luorescence is greatly reduced by Virtue of the excitation in in FIG. 5b (except that all levels would be in the same ion). the infrared or red Spectral range. The light Source is a pump Energy transfer from a Sensitizer ion to an activator ion laser whose pump pulses are short in order to achieve high cannot be used in the case of a single rare earth ion. powers and low energy in order to enable non-linear optical Chelates Suitable for use as up-converting phosphors processes in the dye. The goal is to excite the Second excited include ethylenediaminetetraacetic acid (EDTA), dipicolinic 50 Singlet State (S) in a dye with a ps pulse from a tunable dye acid (DPA), diethylenetriaminetetraacetic acid (DTTA), laser using two red or infrared photons. After pumping the diethylenetriaminepentaacetic acid (DTPA), tetraazacy S. State the dye relaxes within a few pS to the fluorescing clotetradecanetetraacetic acid (TETA), as well as antibiotics, State (S) which can be detected by optoelectronic means. natural chelating proteins, phthalocyanines, and cryptates. The goal of reaching the S. State using two photons enables Methods for preparation of lanthanide chelates and their use 55 one to take advantage of the increasing two-photon croSS in biological assays are described in the literature (Mukkala Sections as one approaches the S. State using two-photon et al. (1989) Anal. Biochem. 176:319, Hemmila et al. (1984) absorption. The non-resonant two-photon absorption croSS Anal. Biochem. 137: 335, Soini and Kojola (1983) Clin. sections are on the order of 10' to 10 cm's, whereas the Chem. 29: 65, Nonisotopic DNA Probe Techniques (1992) croSS Sections corresponding to S absorption are larger by Kricka (Ed.) Academic Press, New York, as well as the 60 two to three orders of magnitude. A few specific examples references on page 6 of this application). Up-conversion will be mentioned: in general cyanines, Xanthenes, phosphor reporters can also consist of rare earth ions inside rhodamines, acridines and oxazines are well Suited for this cage compounds Such as fullerene materials following the purpose. Blue dyes can also be used, but the excitation procedures described by Bethune et al. (1993) Nature 366: wavelength will be in the red. Rhodamine can be excited at 123 and references therein. 65 650 to 700 nm using two photons, and fluorescence is Suitable ions for up-conversion in chelates include expected around 555 nm. Many IR dyes such as IR-140, erbium, neodymium, thulium, holmium, and praseodymium. IR-132 and IR-125 can be excited at 1060 nm using two

Page 50

photons of the Nd:YAG fundamental, and fluorescence is excitation Source. Various excitation Sources may be used, expected in the 850 to 950 nm range. An example of a blue including infrared laser diodes and incandescent filaments, dye is BBQ excited at 480 nm to reach the S. state at 240 as well as other suitable sources. Optical filters which have nm, and fluorescence is expected at 390 nm. Many of these high transmissibility in the excitation wavelength range(s) dyes are only slightly Soluble in aqueous Solution and are and low transmissibility in one or more undesirable wave either polar in nature (cyanines) or have polar Substituents. length band(s) can be employed to filter out undesirable Depending on the nature of the probe, no or only minimal wavelengths from the Source illumination. Undesirable attachment chemistry needs to be undertaken because of the wavelength ranges generally include those wavelengths that abundance of functional groups on the dye chromophore. produce detectable Sample autofluoresence and/or are within Several companies Sell entire lines of dyes: examples are about 25-100 nm of excitation maxima wavelengths and KODAK, Exciton and Lambda Physik. The scientific foun thus are potential Sources of background noise from Scat dations of two-photon laser excitation in organic dye mol tered excitation illumination. Excitation illumination may ecules have been treated in a few experimental papers: A. also be multiplexed and/or collimated; for example, beams Penzkofer and W. Leupacher, Optical and Quantum Elec of various discrete frequencies from multiple coherent Sources (e.g., lasers) can be collimated and multiplexed tronics 19 (1987), 327–349; C. H. Chen and M. P. McCann, 15 using

Optics Commun. 63 (1987), 335; J. P. Hermann and J. containing an array of dichroic mirrors. In this way, Samples Duculing, Optics Commun. 6 (1972), 101; B. Foucault and J. multiple phosphor Species having different exci P. Hermann, Optics Commun. 15 (1975), 412; Shichun Li tation wavelength bands can be illuminated at their excita and C. Y. She, Optica Acta 29 (1982), 281–287; D. J. tion frequencies simultaneously. Illumination may be con Bradley, M. H. R. Hutchinson and H. Koetser, Proc. R. Soc. tinuous or pulsed, or may combine continuous wave (CW) Lond. A 329 (1972), 105–119. and pulsed illumination where multiple illumination beams are multiplexed (e.g., a pulsed beam is multiplexed with a

Resonant Multiphoton Ionization CW beam), permitting signal discrimination between phos phorescence induced by the CW Source and phosphores

At very high laser intensities the up-converting organic 25 cence induced by the pulsed Source, thus allowing the dyes are induced to absorb an additional exciting photon in discrimination of multiple phosphor Species having similar the field of focussed laser radiation. At those high laser emission spectra but different excitation spectra. For Intensities the fluorescence is Suppressed in favor of absorp example but not limitation, commercially available gallium tion of an additional photon. This process usually brings the arsenide laser diodes can be used as an illumination Source organic dye molecules above the ionization limit in Solution for providing near-infrared light.

and they stabilize by emitting an electron into the Solvent The ability to use infrared excitation for Stimulating shell. The result of this three-photon interaction is a molecu up-converting phosphors provides Several advantages. First, lar ion and an attached or Solvated electron. When this charge separation is taking place in an electric field, the inexpensive IR and near-IR diode lasers can be used for Sustained high-intensity excitation illumination, particularly charges drift and generate a Voltage that can be detected in 35 in IR wavelength bands which are not absorbed by water. an extremely Sensitive manner. This amounts to the mea This level of high-intensity illumination would not be suit Surement of the transient conductivity in the Solvent System able for use with conventional labels, Such as ordinary and is usually more Sensitive than light detection. The fluorescent dyes (e.g., FITC), since high-intensity UV or disadvantage of this method is that it necessitates electrodes Visible radiation produces extensive photobleaching of the that Sense the moving charges. In that Sense it is not as 40 label and, potentially, damage to the Sample. The ability to non-invasive a method as light detection. On the other hand use higher illumination intensities without photobleaching it bypasses the conversion of light into a photoelectric Signal or Sample damage translates into larger potential Signals, and which represents an enormous advantage. Every optical hence more Sensitive assayS.

System has a restricted Viewing angle that reduces efficiency, The compatibility of up-converting labels with the use of whereas photoionization “senses” always close to 100% of 45 diode lasers as illumination Sources provide other distinct the charges generated. Effectively, the non-linear interaction advantages over lamp Sources and most other laser Sources. of the laser field converts every excited organic dye mol First, diode laser intensity can be modulated directly through ecule into an electric pulse at Sufficiently high field inten modulation of the drive current. This allows modulation of Sities that can be routinely achieved using commercial laser the light for time-gated or phase-Sensitive detection Sources. Specific examples are the excitation of Rhodamine 50 techniques, which afford Sensitivity enhancement without around 650 to 700 nm, or BBQ excitation around 480 nm.

Organic dyes absorbing in the red have to absorb two the use of an additional modulator. Modulators require high-voltage circuitry and expensive crystals, adding both additional photons after being excited into S thus making the whole process a four-photon excitation process, which is cost and additional size to apparatus. The laser diode or light-emitting diode may be pulsed through direct current slower than a three-photon non-linear process. There may, 55 modulation. Second, laser illumination Sources provide illu however, be circumstances where Such a four-photon pro mination that is exceptionally monochromatic and can be ceSS is desirable.

tightly focused on very Small Spot sizes, which provides

Detection Apparatus advantages in Signal-to-noise ratio and Sensitivity due to reduced background light outside of the desired excitation

Detection and quantitation of inorganic up-converting 60 Spectral region and illuminated Volume. A diode laser affords phosphor(s) is generally accomplished by: (1) illuminating a these Significant advantages without the additional expense Sample Suspected of containing up-converting phosphors and size of other conventional or laser Sources. with electromagnetic radiation at an excitation wavelength, Detection and quantitation of phosphorescent radiation and (2) detecting phosphorescent radiation at one or more from excited up-converting phosphors can be accomplished emission wavelength band(s). 65 by a variety of means. Various means of detecting phospho Illumination of the Sample is produced by exposing the rescent emission(s) can be employed, including but not Sample to electromagnetic radiation produced by at least one limited to: photomultiplier devices, avalanche photodiode,

Page 51

charge-coupled devices (CCD), CID devices, photographic A pair of light sources 20(1) and 20(2), which may be film emulsion, photochemical reactions yielding detectable laser diodes or light-emitting diodes (LEDs), provide light at products, and Visual observation (e.g., fluorescent light the desired excitation wavelengths, while respective detec microscopy). If the reporters are organic dyes, resonant tors 22(1) and 22(2), which may be photodiodes, detect light multiphoton ionization can be Sensed using electroStatic at the desired emission wavelengths. The emitted radiation position-Sensitive detectors. Detection can employ time is related to the incident flux by a power law, So efficiency gated and/or frequency-gated light collection for rejection of can be maximized by having the incident beam Sharply residual background noise. Time-gated detection is gener focused on the Sample. To this end, light from the two ally desirable, as it provides a method for recording long Sources is combined to a Single path by a Suitable combi lived emission(s) after termination of illumination; thus, nation element 25, is focused to a Small region by a lens or Signal(s) attributable to phosphorescence or delayed fluo other focusing mechanism 27, and encounters the Sample. rescence of up-converting phosphor is recorded, while short Light emitted by the phosphor reporters is collected by a lens lived autofluoresence and Scattered illumination light, if any, 30, and components in the two emission bands are Separated is rejected. Time-gated detection can be produced either by by a Suitable Separation element 32 and directed to the Specified periodic mechanical blocking by a rotating blade 15 respective detectors.

(i.e., mechanical chopper) or through electronic means There are a number of possible regimes for driving the wherein prompt signals (i.e., occurring within about 0.1 to laser diodes and detecting the emitted light in the different 0.3 us of termination of illumination) are rejected (e.g., an wavelength bands. This is shown generically as a control electronic-controlled, Solid-state optical shutter Such as electronics block 35 communicating with the laser diodes Pockel's or Kerr cells). Up-converting phosphors and and detectors. The particular timing and other characteristics up-converting delayed fluorescent dyes typically have emis of the control electronics will be described below in con Sion lifetimes of approximately a few milliseconds (perhaps nection with Specific embodiments.

as much as 10 ms, but typically on the order of 1 ms), There may be a plurality of reporters having distinct whereas background noise usually decays within about 100 emission bands but a common excitation band. In Such a ns. Therefore, when using a pulsed excitation Source, it is 25 case, the System would include multiple detectors for a generally desirable to use time-gated detection to reject Single laser diode. Similarly, there may be a plurality of prompt signals. reporters having distinct excitation bands but a common Since up-converting phosphors are not Subject to emission band. In Such a case, the System would include photobleaching, very weak emitted phosphor Signals can be multiple laser diodes for a Single detector, and would use collected and integrated over very long detection times time multiplexing techniqueS or the like to Separate the (continuous illumination or multiple pulsed illumination) to wavelengths.

increase Sensitivity of detection. Such time integration can be electronic or chemical (e.g., photographic film). When So Light as to from the two Sources is shown as being combined be focused at a Single location by a common non-infrared photographic film is used as a means for focusing mechanism.

detecting weak emitted Signals, up-converting reporters pro desired to illuminate This 35 is not necessary, even if it is vide the advantage as compared to down-converting phoS Similarly, the collection needsame the not region of the Sample.

be via a single collection phors that the excitation Source(s) typically provide illumi mechanism. If it is necessary to preserve nation in a wavelength range (e.g., infrared and near combination and Separation elements can all the light, the include a wave infrared) that does not produce significant exposure of the length division multiplexer and a demultiplexer using film (i.e., is similar to a darkroom safelight). Thus, 40 roic filters. If loss can be tolerated, 50% beam splittersdich and up-converting phosphors can be used as convenient ultra filters can be used.

Sensitive labels for immunohistochemical Staining and/or in The Schematic shows the light passing through the Sample Situ hybridization in conjunction with fluorescence microS copy using an infrared Source (e.g., a infrared laser diode) from and being detected in line. As a general matter, the emission and photographic film (e.g., Kodak Ektachrome) for signal 45 may be the phosphor reporters is generally isotropic, and it and image detection of visible range luminescence (with or direction preferred of the to collect light at an angle from the incident light to avoid background from the without an infrared-blocking filter).

excitation Source. However, Since the excitation and the

Instrumentation Overview emission bands are widely separated, Such background is The basic purpose of the instrumentation is to expose the 50 unlikely to be an issue in most cases. Rather, other consid up-converting phosphor particles of an assay Sample to erations may dictate other geometries. For example, it may near-infrared (NIR) light and to measure the amount of be desired to detect light traveling back along the path of the visible light that is emitted. incident radiation So that certain elements in the optical train FIG. 1 is an optical and electronic block diagram illus are shared between the excitation and the detection paths. trating representative apparatus 10 for performing diagnos 55 A typical type of instrument with shared elements is a tics on a Sample 15 according to the present invention. The microScope where the objective is used to focus the excita invention may be carried out with one or a plurality of tion radiation on the Sample and collect the emitted radia reporters. For purposes of illustration, the apparatus shows tion. A potentially advantageous variation on Such a con a System wherein two diagnostics are performed on a single figuration makes use of the phenomenon of optical trapping. Sample in which two phosphor reporters are used. The first 60 In a situation where the reporter is bound to a Small bead, it reporter has an excitation band centered at 2 and an may be possible to trap the bead in the region near the beam emission band centered at 2," while the Second reporter has focus. The same Source, or a different Source, can be used to respective excitation and emission bands centered at , and excite the reporter. The use of an infrared diode laser to trap 2. Since the reporters of the present invention rely on Small particles is described in Sato et al., “Optical trapping multiphoton excitation, wavelengths 2 and 2 are longer 65 of Small particles using a 1.3 um compact InGaAsP laser,” than wavelengths), and 2'. The former are typically in the Optics Letters, Vol. 16, No. 5 (Mar. 1, 1991), incorporated near infrared and the latter in the visible. herein by reference.

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Specific Detection Techniques mixers, which also receive respective phase input Signals. Thus,

AS outlined above, multichannel detection uses optical corresponding frequency mixer 60(1) provides an output signal devices Such as filters or dichroic beam Splitters where the frequency f, which to the amount of emitted light modulated at emission bands of the phosphor reporters are Sufficiently the first reporter in the provides a measure of the presence of Separated. Similarly, it was pointed out that multiple report Sample. Similarly, frequency mixer 60(2) provides an erS having a common emission band could be detected using of emitted light modulated output signal corresponding to the amount electronic techniques. These electronic techniques will be a measure of the presenceatoffrequency f, which provides the Second reporter in the described below in connection with multiple Sources.

However, the techniques will be first described in the context Sample.

of a single channel. The techniques are useful in this context The use of two different wavelengths was discussed above Since there are Sources of background that are in the same in the context of two reporters having different excitation wavelength range as the Signal Sought to be measured. bands. However, the discussion is germane to a single FIG. 2A shows an apparatus for implementing phase reporter photon situation as well. Since the excitation is a two process, there is no requirement that the two photons

Sensitive detection in the context of a single channel. Cor 15 have the same energy.

responding reference numerals are used for elements corre total energy of the twoRather, it is only necessary that the photons fall within the excitation sponding to those in earlier described figures. In this context, band. Thus, Since it is relatively Straightforward and inex control electronics 35 comprises a waveform generator 37 pensive to provide different wavelengths with laser diodes, and a frequency mixer 40. Waveform generator 37 drives there are more possible combinations, i.e., laser diode 20(1) at a frequency f, and provides a signal at choices of total excitation energy. This allowsmore possible more latitude f to the frequency mixer. The frequency mixer also receives the Signal from detector 22(1) and a phase control input in the choice of rare earth ions for up-converters Since the Signal. This circuitry provides additional background dis excitation Steps need not rely on energy transfer coinci crimination because the background has a much shorter possible to achievea direct dences involving single photon energy. Further, it may be

Stepwise excitation of the emitting lifetime than the Signal Sought to be measured (nanoseconds 25 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 dependent phase shift, see Demtröder, Laser Spectroscopyy different additional wavelengths for a single reporter can provide 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 background. This background discrimination differs from occur in a number of ways, as shown in FIGS. 5A through that typical for phase Sensitive detection where the Signal is 35 5C. Two laserS may cause Stepwise excitation of a single ion, modulated and the background is not. Discrimination as shown in FIG. 5A. A first laser stimulates excitation from against unmodulated background is also beneficial here, level 1 to level 2, and a Second laser Stimulates excitation leading to two types of discrimination. from level 2 to level 3, at which level emission occurs. Because the Signal relies on two-photon excitation, it is shown ion

Single

excitation can also occur using energy transfer as

FIG. 5B. In this case, a first laser stimulates possible to use two modulated laser diodes and to detect the 40 excitation from

Signal at the Sum or difference of the modulation frequen from level 2 tolevel 1 to level 2, energy transfer occurs level 3, and a second laser stimulates cies. FIG. 2B ShowS Such an arrangement where first and excitation from level 3 to level 4. In a variation of the latter second laser diodes 2001) and 20(1)" (emitting at the same process, levels 1 and 2 can be in a first ion (i.e., a Sensitizer wavelength), or possibly different wavelengths) are modu lated by signals from waveform generators 37a and 37b 45 ion) and levels 3 and 4 in a Second ion (i.e., activator ion) operating at respective frequencies f and f. The waveform as shown in FIG. 5C.

generator output signals are communicated to a first fre In a stepwise excitation Scheme shown in FIG. 5A, energy quency mixer 42, and a Signal at fit?, is communicated to transfer is not required, and thus information on the polar a Second frequency mixer 45. The signal from detector 22(1) ization of the excitation laserS may be preserved and cause and a phase input Signal are also communicated to frequency 50 polarization of the emitted radiation. In this case, depolar mixer 45. ization of the light may allow for enhanced discrimination FIG. 3 ShowS apparatus for performing gated detection. between Signal and background noise.

Since the background is shorter-lived than the Signal, delay For the multi-ion multi-laser excitation Scheme shown in ing the detection allows improved discrimination. To this FIG. 5C, there may be several phosphors that share a end, the laser diode is driven by a pulse generator 50, a 55 common excitation wavelength. In this case, discrimination delayed output of which is used to enable a gated integrator between different phosphors may be performed on the basis or other gated analyzer 55. of different emission wavelengths and/or through time FIG. 4 shows an apparatus for performing diagnostics on gated, frequency-modulated, and/or phase-Sensitive detec a Sample using first and Second, reporters having excitation tion utilizing modulation of the excitation wavelength(s). bands centered at 2 and 22, and having overlapping emis 60 Specific Instrument Embodiments Sion bands near 2. The Sample is irradiated by light from laser diodes 2001) and 20(2) as discussed above in connec FIG. 5 is a schematic view showing the optical train of a tion with FIG. 1. First and second waveform generators particular embodiment of apparatus for carrying out the 37(1) and 37(2) drive the laser diodes at respective frequen present invention on a Sample using a hand-held probe. This cies f and f, and further provide signals at f and f to 65 embodiment takes the form of a miniaturized instrument respective frequency mixers 60(1) and 60(2). The signal comprising a housing 75 (shown in phantom), a hand-held from detector 22(3) is communicated to both frequency probe 80, with a fiber optic connecting cable 82. The optical

Page 53

and electronics components are located within the housing. Further, Since the phosphor emission is relatively slow in For purposes of illustration, the optical components of a rise and fall time it could be time resolved from a pulsed 3-channel System are shown. The Sample may contain up to laser stimulation source by the CCD detector array. The three reporters having distinct emission bands, for example, decay time for the upconversion proceSS is a variable in the blue, green, and red portions of the visible Spectrum. dependent on the particular emitting transition and the It is also assumed that the reporters have distinct excitation phosphor host; however, it is normally in the range 500 uS bands in the near infrared. seconds to 10 ms. This is very slow compared to the laser The output beams from three laser diodes 85a-c are excitation pulse and the capability of the detector array. communicated through graded index (GRIN) lenses 87a-c, The techniques for fabricating the CCD array are well focused onto the ends of respective fiber segments 38a–c known Since CCD imaging arrays have been commercially and coupled into a single fiber 90 by a directional coupler 92 or other Suitable device. The light emerging from the end of available for many years. A variety of Such devices can be fiber 90 is collimated by a GRIN lens 95, passes through a N.J. obtained from David Sarnoff Research Center, Princeton, dichroic beam splitter 97, and is refocused by a GRIN lens 100 onto the end of fiber optic cable 82. The beam splitter The techniques for fabricating the peptide array are is assumed to pass the infrared radiation from the laser 15 described in a paper by Fodor et al., “Light-Directed, diodes but reflect visible light. Spatially Addressable Parallel Chemical Synthesis,” Hand-held probe 80 includes a handpiece 102, an internal Science, Vol. 251, pp. 767-773 (Feb. 15, 1991), incorporated GRIN lens 105, and a frustoconical alignment tip 110. The herein by reference. The particular array described contains light emerging from fiber 82 is focused by GRIN lens 105 1024 discrete elements in a 1.28 cmx1.28 cm area. at a focus point 115 that is slightly beyond alignment tip 110. The embodiment of FIG. 7A shows the peptide array in The alignment tip is brought into proximity with the test tube intimate contact with the CCD array. Indeed it may be holding the Sample So that focus point 115 is in the Sample. possible to deposit the peptides directly on the passivation It is assumed that the test tube is transmissive to the laser layer without a separate Substrate. However, there may be radiation.

Situations where spatially Separated arrays are preferred

A portion of the light emanating from the region of focus FIG. 7B shows an embodiment where the peptide array and point 115 in the sample is collected by GRIN lens 105, the CCD array are separated. An array of lenses 165 collect focused into fiber 82, collimated by GRIN lens 100, and the light from respective binding sites and focus it on reflected at dichroic beam splitter 97. This light may contain respective detector elements. This arrangement facilitates wavelengths in up to the three emission bands. Optical filters the use of filters to the extent that other techniques for 120a–C direct the particular components to respective pho rejecting the excitation radiation are not used. todetectors 125a–c. A particular filter arrangement is shown Optical trapping may be used to transiently immobilize a where each filter reflects light in a respective emission band, Sample particle for determination of the presence or absence but other arrangements would be used if, for example, one of phosphor on the particle. Conveniently, the wavelength or more of the filters were bandpass filters for the emission 35 range used to trap Sample particles may be essentially bands. identical to an excitation wavelength range for the The control electronics are not shown, but could incor up-converting phosphor(s) Selected, so that optical trapping porate the time-multiplexed or heterodyne techniques dis and excitation illumination is performed with the same cussed above. Such techniques would be necessary, for Source. FIG. 8 shows a block diagram of an apparatus used example, if the emission bands were not distinct. 40 for Single-beam gradient force trapping of Small particles. FIG. 7A is a schematic of an embodiment of the invention FIG. 26 is a block diagram of one embodiment of appa in which a charge coupled device (CCD) imaging array 150 ratus for carrying out the present invention on a Sample is used as a detector in combination with a two dimensional using a microScope. In this embodiment a Standard micro array 152 of peptides or other biologically active Species Scope is modified to accept infrared Scanning optics and deposited on a glass or plastic Substrate. The CCD array has 45 image processing electronics. A Suitable microScope for a number of individually addressable photosensitive detec modification is the Zeiss model CLSM-10. tor elements 155 with an overlying passivation layer 157 The microscope is fitted with a HeNe laser A1 for visible while the peptide array has a number of individual binding imaging and an argon laser A2 for both visible and UV sites 160. The probe containing the phosphor would be imaging. Both lasers are mounted internally and are indi reaction Specific to one or more of the elements in this 50 vidually Selectable through a Series of motorized shutters peptide array and would therefore become physically A3. The upconverting phosphors are excited with an exter attached to those elements and only those elements. The nally mounted IR laser diode. In the preferred embodiment, peptide array is shown as having a one-to-one geometric two IR laser diodes A4 and A5, operating at two different IR relation to the imaging array in which one pixel corresponds wavelengths, are coupled to the microScope thereby allow to each element in the peptide array. However, it is also 55 ing multiple phosphor reporters to be identified. Laser possible to have larger peptide elements that cover a group diodes A4 and A5 are individually selectable using motor of detector elements should Such be necessary. ized shutters A6. When an IR beam is selected, it is routed Various of the techniques described above can be used to through the microscope's qalvanometrically controlled enable the detector array to distinguish the emissions of the Scanning mirrorS A7 which Scan the beam in a raster fashion. phosphor from the infrared laser Stimulation. 60 The beam passes through the objective lens (not shown) First, it is possible to use a phosphor that responds to IR onto a Sample A8 and is reflected back through the objective Stimulation beyond the Sensitivity range of the detector lens to a Set of galvanometrically controlled receiving mir array. An example of Such a phosphor would be Gadolinium rors A7. Receiving mirrors A7 reflect the light onto pinhole oxysulfide: 10% Erbium. This phosphor is stimulated by optics A9. If the confocal mode is selected, pinhole A9 limits 1.5-micron radiation and emits at 960 nm and 520 nm. The 65 the detected image to the light collected from the focal detector array is insensitive to 1.5-micron radiation but is plane. The light is imaged on a photomultiplier tube (PMT) Sensitive to the up-converted radiation. A10. The thickness of the focal plane is proportional to the

Page 54

Size of the pinhole. The Scanning Speed is chosen Such that Sources. Furthermore, overexposure of PMT B3 to light sufficient signal intensity is received at the PMT A10. In the causes high dark currents which require Several hours to preferred embodiment of this apparatus, a 20 micrometer decrease. PMT B3 is cooled for lower dark current and diameter pinhole is used which results in a depth of field of noise. ASSociated with the PMT cooler is a water-cooled about 1 micrometer. If the confocal mode is not Selected, the power supply B15. A power Supply B16 supplies high beam is deflected around pinhole optics A9 directly to PMT voltage to PMT B3.

A10. When the apparatus is operated in a computerized mode, Once the optical Signal is converted into an electronic one, a computer B17 regulates controller B8 through an interface a Standard, composite Video signal can be developed and box B18. Picoammeter B13 can also be connected to com displayed as an image on a television monitor A11. The puter B17, thereby allowing automated data acquisition to image can be manipulated and enhanced through Standard be performed. The data acquisition procedure moves trans image processing Software. In the preferred embodiment of lator B7 in the X direction to a first position at which location this apparatus the software runs on an IBM 486 PC A12. The a specified number of current readings are taken and the Software can be used to perform averaging, filtering, edge average is calculated. Translator B7 then moves sample B4 detection and overlaying the images received from each of 15 a predetermined distance in the X direction to a new location the different light Sources. where new data is collected. During this process, the data is In the confocal mode, it is possible to reconstruct a 3 plotted in order to provide the user with an immediate visual dimensional view of Sample A3. The reconstruction is evaluation. After the Scan is completed, the data can be formed by Stepping through Sample A8 at Small intervals, saved or further data processing can be performed. FIG. 28 making an image of the Sample at each interval. The multiple is an illustration of the data for upconverting phosphors in Sequential images are transferred to an external graphics three test wells.

machine (not shown) for reconstruction of the sample in 3 FIG. 29 is a schematic view of a second embodiment of dimensions. These 3-D images can then be rotated to give a hand-held probe for carrying out the present invention. different perspectives of the data sets, leading to a better 25 This embodiment is comprised of a housing D1 and a understanding of the Samples. capillary wick D2. Within housing D1 is a diode excitation FIG. 27 is a block diagram of a microtiter plate reader for laser D3, a lens assembly D4, a photodiode detector D5, and use with the present invention. Within a light-tight test a battery supply D6. A display D7 mounted to one surface chamber B1 is a near IR laser excitation Source B2, a of housing D1 communicates the results of the test to the photomultiplier tube (PMT) detector B3, and a sample assay user. In the preferred embodiment, laser D3 operates in the plate B4. In the preferred embodiment of this apparatus, 960-980 nanometer range.

assay plate B4 is a Terasaki HLA plate. This plate is In use, wick D2 wicks up a portion of a sample fluid D8 preferred due to its Small tapered Sample wells which tend which is Suspected of containing the target antigens. Target to concentrate the Sample material into a relatively Small antigens bind to the antibodies present at a capture Surface target area. The target area in this configuration is still larger 35 D9. Capture surface D9 is positioned at the focal point of than the diameter of the laser beam. Furthermore, it is Source D3. The target antigens can be labeled with possible that the distribution of the assay material acroSS the phosphor-antibody conjugates either before or after capture. bottom of the well is not even. Because of these two factors, In the preferred embodiment wick D2 is formed of glass. In Simply aiming the laser at the center of the bottom well this configuration capture Surface D9 is prepared Simply by Surface is unlikely to provide accurate readings. There are 40 filling the inside of the capillary with a bubble containing the Several approaches that can be used to circumvent this antibodies of interest. By Silanizing the inner Surface with problem. The first approach is to defocus the laser beam organofunctional Silanes, conventional chemistries can be Sufficiently to allow a larger amount of the target area to be used to covalently link the antibodies or other biological interrogated. However, depending upon the output of laser macromolecule(s) to the inner tube wall at the site of the B2, defocussing the beam may lower the sensitivity of the 45 liquid bubble. The Surface energy of the capillary is also apparatus to an unacceptable level. Another approach is to easy to modify by Silanization, which will help prevent raster Scan the laser beam across the bottom of target well. nonspecific reagent and antigen adherence to the walls of the A third and preferred approach is to Simply automate the tube.

Scanning and data collection System. In the preferred embodiment of this apparatus, the lower Light from laser B2 passes through a filter B5 and is 50 portion of wick D2 is impregnated with upconverting phos focussed by a lens B6 onto an individual sample well of phors that are conjugated to the target analytes or a croSS assay plate B4. Plate B4 is mounted on a pair of translators reactive epitope for the capture probe. In use, the phosphor B7 which allow positioning in the horizontal and vertical conjugates chromatograph towards capture Surface D9 as directions. In the present configuration translators B7 allow sample fluid D8 is drawn up wick D2. As phosphors approximately 2.5 centimeters of travel; Sufficient to address 55 accumulate at capture Surface D9, they will begin to emit 3 sample wells in each direction. Translators B7 are con visible light upon excitation by diode laser D3. The visible trolled by an x-y controller B8. Controller B8 allows for light emitted by the phosphors is detected by detector D5. either manual or computerized control. The output of detector D5 is displayed on display D7. The A Sample well on plate B4, when containing upconverting amount of upconverted light reaching the detector is directly phosphors, will emit visible light which is collected by a lens 60 proportional to the concentration of labeled target antigen B9, passed through a filter B10, and focussed through a lens captured at the capture Surface.

B11 and a shutter B12 onto PMT B3. PMT B3 outputs a The apparatus of FIG. 29 can be designed to simulta current which is measured by a picoammeter B13. The PMT neously detect more than one target antigen. FIG. 30 illus Signal is proportional to the phosphor emission intensity. trates a three channel configuration using interference filters. Shutter B12, controlled by a shutter driver B14, provides 65 In this configuration capillary wick D2 is placed at the focus exposure protection to PMTB3, thereby preventing damage of a small parabolic reflector D10 capable of collecting which may result from exposure to very intense light approximately half of the emitted phosphorescence. The

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beam from diode laser D3 is directed onto capillary wick D2 phors perform better in a microassay format. ASSuming a at capture Surface D9 along a direction perpendicular to the constant power output from the excitation Source, the exci optical axis of reflector D10. Phosphorescent light from tation power density increases proportionally with the capture surface D9 is collected and collimated by mirror decrease in detection area, and the number of phosphor D10, directed through a notch filter D11 to reject the pump particles in the light path decreases linearly with a decrease light, and onto three detectors D5 using three dichroic beam in the detection area. Since the power of the light emitted splitters D12. The reflectance bands of dichroic beamsplit from the phosphorS Scales with the Square of the excitation ters D12 are matched to the emission bands of the three power density, but linearly with the number of phosphors, phosphors used in the detection process. P will increase in inverse proportion to the detection area. In an alternate embodiment of this apparatus, dichroic Therefore, a 100x100 array will actually be 100 times more beamsplitters D12 could be replaced with three bandpass sensitive than a 10x10 array.

filters used in the transmission mode. By placing the three filters on a rotation wheel, a single detector D5 could be Fluorescence-Activated Cell Sorting used. Another alternative is to use a diffraction grating and The up-converting phosphors described herein can be a linear detector array to obtain an actual emission Spectrum. 15 used as phosphorescent labels in fluorescent cell Sorting by FIG. 31A is an illustration of an embodiment of the flow cytometry. Unlike conventional fluorescent dyes, invention in which a diode laser array F1 and a detector up-converting phosphors possess the distinct advantage of array F2 are combined in a single device. In the preferred not requiring excitation illumination in wavelength ranges embodiment, arrays F1 and F2 are fabricated on a pair of (e.g., UV) that damage genetic material and cells. Typically, Silicon chips F3 with array dimensions of approximately 1 up-converting phosphor labels are attached to a binding square centimeter. FIG. 31B is a detailed view of a small reagent, Such as an antibody, that binds with high affinity and section of the device shown in FIG. 31A. overlaying detec Specificity to a cell Surface protein present on a Subset of tor array F2 is a polymer film F4 of approximately 10 to 25 cells in a population of cells in Suspension. The phosphor micrometers thickness which is used as the capture Surface. 25 labeled binding component is contacted with the cell Sus Arrays F1 and F2 are separated by a spacer F5. Array F1 is pension under binding conditions, So that cells having the comprised of Fabrey-Perot diode lasers, Preferably tuned to cell Surface protein bind to the labeled binding reagent, 980 nanometers. Lasers of this type are easily fabricated in whereas cells lacking the cell Surface protein do not Sub gridded array patterns using conventional photolithography Stantially bind to the labeled binding reagent. The Suspended techniques. Each individual laser in array F1 has a columnar cells are passed acroSS a Sample detector under conditions beam designed to Strike only the adjacent portion of capture wherein only about one individual cell is present in a Sample surface F4. The required power density of the individual detection Zone at a time. A Source, typically an IR laser, laserS is dependent upon the efficiencies of the phosphors illuminates each cell and a detector, typically a photomul being used as well as the required detection efficiency. The tiplier or photodiode, detects emitted radiation. The detector detectors comprising array F2 are chosen to have an 35 controls gating of the cell in the detection Zone into one of extremely low Sensitivity in the wavelength region in which a plurality of Sample collection regions on the basis of the laser array F1 operates. If additional discrimination between Signal(s) detected. A general description of FACS apparatus the excitation and emission Wavelengths is required, a cutoff and methods in provided in U.S. Pat. Nos. 4,172,227; filter can be used, Preferably incorporated directly into 4,347,935; 4,661,913; 4,667,830; 5,093,234, 5,094,940; and capture Surface F3. 40 5,144.224, incorporated herein by reference. It is preferred

Upconverting phosphors F6 are conjugated by any of a that up-converting phosphors used as labels for FACS meth variety of conventional biochemical crosslinking chemis ods have excitation range(s) (and preferably also emission tries to antibody, nucleic acid probes, or other biological range(s)) which do not damage cells or genetic material; macromolecules (e.g., carbohydrates, lectins, Streptavidin, generally, radiation in the far red, and infrared ranges are MHC complexes), as well as to biological or chemical 45 preferred for excitation. It is believed that radiation in the antigens (F7). Bonded to overlay F3 is a grid array F8 of range of 200 nm to 400 nm should be avoided, where complementary probes or antigens which are bound to possible, and the wavelength range 760 nm to 765 nm may capture Surface F3 using the Same crosslinking chemistries. be avoided in applications where maintenance of viable cells In use, a sample fluid F9 flows between arrays F1 and F2, is desired.

target probes or antigens are captured by grid array F8 and 50 excited by laser array F1, and the emissions detected by Additional Variations detector array F2. There are Several apparatus design issues relating to the Typically, the upconverting phosphors to be used with this unique excitation and emission characteristics of upconvert apparatus are approximately 0.1 to 0.5 micrometers. Since ing phosphors which must be considered when using the size of the individual phosphor particles is of the order 55 up-converting phosphors with flow cytometry. The first of the excitation wavelength, the power of the emission from issue is the time required to reach maximum emission the phosphors can be approximated by: intensity. Since upconversion is a two photon process, upconverting phosphor emission is time delayed approxi

P=fNDI mately 100 microseconds. The phosphor must remain within 60 the excitation beam for this period of time regardless of the where f is the phosphorescence efficiency (generally less flow rate. Therefore given a flow rate between 1 and 10 than or equal to 107 cm'W' um' particle'), N is the meters per second with a channel width of 70 to 200 number of phosphor particles in the light path, D is the micrometers, the length of the excitation beam must be diameter of the phosphor particles, and I is the power between 100 and 1000 micrometers. Given that the phos density of the excitation Source. 65 phor emissions Saturate at an excitation intensity of about Since the emitted power Scales as the Square of the 200 watts per Square centimeter, the laser Source typically excitation intensity, diagnostics using upconverting phos must have a power between 0.01 and 400 milliwatts to

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achieve phosphor Saturation. This implies that multiple laser emission following photoexposure. This is especially useful diodes may be required to obtain maximum phosphores when the phosphor-catalyst or reactant conjugate enters an cence at the fastest flow rates. environment through which the excitation wavelength can Another design issue is that associated with the detector. not penetrate. This Slow release also increases the probabil Since there is a considerable Separation between the exci ity that more targets will interact with the particle. tation and emission wavelengths of the up-converting The unique decay rates of phosphor particles allow phosphors, detection can be performed using a photomulti dynamic Studies as well. In a System where continuous pler tube (PMT), a photodiode, or a CCD array. The phos exposure to the excitation Source is not possible, or is phorescence decay time is long, with a decay half life of invasive and thereby undesirable, pulsed excitation followed approximately 300 microseconds. The most sensitive by delayed fluorescence detection is necessary. After the method of detection is to integrate the Signal measured by phosphor reporter has been photoexcited, the Subsequent the PMT. However, 99 percent detection of the available emission from the phosphor or phosphor/dye conjugate phosphorescent Signal requires that the phosphor remain in particle lasts typically about a millisecond. In a dynamic the Sight path of the detector for 5 times the phosphores environment, Such as a Static or flowing System with moving cence decay half-life (i.e., 1.5 milliseconds). ASSuming a 15 targets, the particle will emit a characteristically decaying flow rate of 10 meters per second and a channel width of 200 intensity detection of light as it travels relative to the excitation/ apparatus. Combined with imaging optics appro micrometers, the PMT must be able to detect over a path length of 1.5 centimeters. This path length is also the priate to the Scale of the System and the Velocities within the required Spacing between cells flowing through the System, a CCD photoelectric Sensor array will be used to detect the particle or particles movement acroSS the array's cytometer, implying a maximum count rate of 667 cells per field of view. The delayed emission of the phosphors, which Second. It is, however, possible to Sacrifice Some detection is a well-characterized function of time, makes possible the Sensitivity by reducing the detection path length, at least to dynamic tracking of individual particle's positions, direc that required to attain Steady-state emission from the phos tions and Velocities, and optionally calculation of particle phors. AS long as a steady-state emission peak is reached by size, density, and hydrodynamic conformation. As a particle the phosphor in the excitation window, the peak signal 25 moves, it exposes more elements of the array, but with received by the PMT should be directly proportional to the every-decreasing intensity. The more elements it exposes concentration of phosphors present. The nonphotobleaching over a certain fraction of its decay time, the faster it is property of the phosphors makes this form of detection moving. Therefore, the integrated intensity pattern of a possible. The loSS in detection Sensitivity corresponding to a particle's emission “track’ collected by the array is directly 0.1 centimeter path length (versus a 1.5 centimeter path related to the velocity of the particle. The particles may be length) is approximately a factor of 3. Triggering the emis refreshed again at any time by the pulsed or chopped CW Sion detector can be accomplished by observing the light excitation source. FIG. 10 illustrates this scheme. Although Scattered by the cell as it passes through the excitation shown, only a depiction of “side-on' excitation and detection is SOCC. both side-on and end-on detection and excitation In environments where absorption of the up-converted 35 the CCD arrayorintensity arrangements, combinations, are possible. Reduction of phosphor radiation is high, the phosphor microparticles are will allow near-real timeinformation by computer analysis tracking of the particles in a coated with a fluorescent dye or combination of dyes, in dynamically evolving or living Systems. Data analysis and Selected proportions, which absorb at the up-converted fre reduction performed by the computer would include a quency and Subsequently re-radiate at other wavelengths. convolution of the intrinsic decay of the phosphor emission, Because the Single-photon absorption croSS-Sections for 40 the number of pixels illuminated and their signal level, the these fluors are typically very high, only a thin layer is orientation of the decaying Signal on the array, and the required for complete absorption of the phosphor emission. intensity contributions from a blur circle from particles This coat particle may then be encapsulated and coated in a moving in and out of the focal plane of the array. In an endon Suitable antigen or antibody receptor (e.g. microparticle). An flow detection arrangement, the Size of the blur circle would example of this layering is depicted schematically in FIG. 9. 45 relate directly to how quickly the particle moves out of There exists a wide variety of fluorescent dyes with Strong focus, thereby allowing the velocity of the particle to be absorption transitions in the visible, and their emission determined. One possible application would be monitoring covers the visible range and extends into the infrared. Most the chemistry and kinetics in a reaction column, have fluorescent efficiencies of 10% or more. In this manner, alternatively, the application of this method to flow cytom the emission wavelengths may be custom-tailored to pass 50 etry may permit the resolution of cells on the basis of through the particle's environment, and optical interference hydrodynamic properties (size, shape, density). The method filters may again used to distinguish between excitation and may also be useful for in Vivo diagnostic applications (e.g., emission wavelengths. If a relatively large wavelength “win blood perfusion rate).

dow' in the test medium exists, then the variety of emission Up-converting phosphor labels may also be used to Sense wavelengths which may be coated on a Single type of 55 the temperature in the region at which the up-converting phosphor is limited only by the number of available dyes and phosphor label is bound. Up-converting phosphor tempera dye combinations. Discrimination between various reporters ture measurement methods are described in Berthou H and is then readily carried out using the Spectroscopic and Jorgensen C. K. (October, 1990) Optics Lett. 15(19): 1100, multiplexing techniques described herein. Thus, the number incorporated herein by reference.

of probe/reporter “fingerprints” which may be devised and 60 Although the present invention has been described in used in a heterogenous mixture of multiple targets is virtu Some detail by way of illustration for purposes of clarity of ally unlimited. understanding, it will be apparent that certain changes and The principles described above may also be adapted to modifications may be practiced within the Scope of the driving Species-specific photocatalytic and photochemical claims.

reactions. In addition to spectroscopic Selection, the long 65 The broad scope of this invention is best understood with emission decay times of the phosphors permit relatively reference to the following examples, which are not intended Slow reactions or Series of reactions to take place within the to limit the invention in any manner.

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EXPERIMENTAL EXAMPLES aqueous Suspensions, or due to absorption of multiply Scat tered photons in the dry phosphor. On the basis of either of

Validation of UP-Converting Inorganic Phosphors these croSS-Section estimates, the cross-section is Sufficiently as Reporters large to allow detection of Single Submicron phosphor par ticles at moderate laser intensities. At laser intensities of

Up-converting phosphor particles comprising Sodium roughly 10 W/cm, the phosphorescence Scales as the laser yttrium fluoride doped with ytterbium-erbium were milled to intensity to the 1.5 power.

Submicron size, fractionated by particle size, and coated with polycarboxylic acid. Na(YosoYbos.Eroo)F was cho Phosphor Particle Performance: Sensitivity of Sen for its high efficiency upon excitation in the range 940 Detection to 960 nm. A Nd:Yag pumped dye laser/IR dye combination A Series of Terasaki plates containing Serial dilutions of was used to generate 8-ns to 10-nS duration pulses in the monodisperse 0.3 um up-converting phosphor particles con above frequency range. Sisting of (Yoss Yboos Eroos)2O2S were tested for The laser pulses were used to illuminate a Suspension of up-conversion fluorescence under IR diode laser illumina milled phosphor particles in liquid and attached to glass 15 tion in a prototype instrument.

Slides in situ. The Suspension luminescence observed at right The phosphor particles were prepared by Settling in angles was monitored using a collection lens, a Spatial filter DMSO and were serially diluted into a 0.1% acqueous gum in order to filter out Scattered excitation light to the maxi arabic Solution. This appeared to completely eliminate any mum possible extent, and a photomultiplier, Vacuum photodiode, or simple Solid State photodiode (depending on water dispersion problems. The Serial dilutions used are listed in Table III.

the light level observed).

The luminescent Signal level was determined as a function TABLE III of Solution pH (range: 6-8), grain size, particle loading (ug/cm), and the nature of stabilizing anionic Surfactant. Phosphor Phosphor Equivalent

Detection

Signals were recorded both as a time integral from a boxcar 25 Loading Loading Sensitivity integrator and from a long RC time constant or as a transient Label (ng?well) (particles/well) (M) Signal using a transient digitizer in order to delineate the luminescence lifetime under particular experimental condi 100 17OO 90 23,600,000 + 1,200,000 4 x 102 tions. In Situ Signals were also measured by laser Scanning 1O-1

microScopy. FIG. 11 is a fluorescence Scan of the phosphor 1O-3 17 O.O9 23,600 + 1,200 4 x 10 emission Spectrum incident to excitation with a laser Source 10-4

at a wavelength maximum of 977.2 mm; emission maximum 1O-6 OOO17 O.OOOO9 23.6 1.2 4 x 101 is about 541.0 nm. FIG. 12 is an excitation scan of the phosphor excitation Spectrum, with emission collection win dow Set at 541.0 nm, excitation maximum for the phosphor 35 The stock DMSO dispersion had a phosphor density of at the 541.0 nm, emission wavelength is approximately 1.70+0.09 mg/mL (at 95% confidence limits), determined about 977 nm. FIG. 13 is a time-decay measurement of the gravimetrically by evaporating 4-1 mL Samples. This trans phosphor luminescence at 541.0 nm after termination of lates to 23.6x10° particles/mL (assuming an average particle excitation illumination; maximal phosphorescence appears size of 0.3 um and particle density of 5.3 g/mL). The residue at approximately 400 uS with a gradual decay to a lower, 40 after evaporating the Samples over the weekend at stable level of phosphorescence at about 1000 us. FIG. 14 110-120° C. was noticeably yellow, but did phosphoresce shows the phosphor emission intensity as a function of when tested with an IR diode laser.

excitation illumination intensity; phosphorescence intensity Visual green light emanated from all Serial dilutions down increases with excitation intensity up to almost about 1000 to 10 (i.e., 1.7 ug/mL or 23.6x10° particles/mL) in a 1 mL W/cm. 45 polypropylene microfuge tube using a hand-held diode laser Phosphorescence efficiencies of submicron in a dark room. The 10' and 10° dilutions were visibly Na(YosYboEros)F particles were measured. A Ti:sap cloudy. Either 1 ul of each serial dilution, or 0.1 ul of the phire laser was used as an excitation Source and a spectro next higher dilution, were pipetted into a well on the Terisaki photometer and photomultiplier was used as a detection plate. It was found that 1 ul fills the bottom of the well and System. Two types of measurement were performed. The 50 0.1 ul spreads along the edge of the well, but does not cover first was a direct measurement in which the absolute emis the entire Surface. Because of the Statistical and pipetting Sion per particle for phosphor Suspensions was measured in problems associated with Small Volumes with low particle emission bands at 540 nm and 660 nm. The calibrated concentrations, 2 to 4 replicates were prepared of each croSS-Sections are shown in FIG. 15, and size-dependence is dilution.

shown graphically in FIG. 16. This corresponded to a 55 The well of a Terasaki plate holds a 10 ul sample volume. phosphorescence cross-section of approximately 1x10' ASSuming all the phosphor particles contained in this Vol cm for 0.3 um particles with excitation light at 975 nm and ume adhere to the bottom of the Sample well, we can an intensity of approximately 20 W/cm. The emission estimate an equivalent detection sensitivity (Table III). It efficiency of dry phosphor powder of about 25 um was also should be noted that 10 to 10M is the normal range of measured. On the basis of known values for the absorption 60 enzyme-linked Surface assayS.

cross-section of Yb" in crystalline hosts (Lacovara et al. Control Sample Results (1991) Op. Lett. 16: 1089, incorporated herein by reference) and the measured dependence of the phosphorescence emis The control Samples were Scanned using a prototype Sion on particle Size, a phosphorescence cross-section of up-conversion fluorimeter device (David Sarnoff Research approximately 1x10 cm was found. The difference 65 Center). The Samples were Scanned by moving the plate in between these two measurements may be due to a difference 50 um increments, using a motorized X-Y positioning Stage, in phosphorescence efficiency between dry phosphor and relative to the focal point of an infrared diode laser.

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The IR diode laser was operated at 63 mW (100 mA). The Goat anti-mouse IgG antibody (Ab) was conjugated (by beam was focused to 2.4x10 cm at the focal point. As the adsorption) onto the DMSO fractionated phosphor particles. bottom of the sample well is about 1.4x10 cm (1365 um This was done by mixing 200 uLof the Absolution (in 0.1M diameter), the beam covers less than 17% of the well bottom Tris-HCl, pH 7.2) with 100 uL of the phosphor Suspension Surface at any individual position. The well also has sloping in DMSO. Several different Ab concentrations were tried in side walls which widen from bottom to top of the sample the range of 0.025 to 1 lug?u L. A concentration of 0.25 ug?u L well and are also interrogated by a progressively divergent appeared to result in the most efficient coating (i.e., maxi laser beam. Neglecting losses in the optics, the IR light mum Ab utilization with a minimum of clumping of the intensity at the focal point (bottom of the sample well) was phosphor particles). The phosphors were equilibrated over approximately 26–27 W/cm at 980 nm wavelength. A 1O night at room temperature with the Ab in this DMSO/Tris photomultipler tube (PMT) was used for detection of the Solution visible (upconverted) light emitted from the sample. Since conjugateswithwere gentle agitation. The resulting phosphor-Ab centrifuged from this Solution and resus the laser beam width was Smaller than the Surface area at the bottom of the Sample well, the plate was aligned by Visual pended in a 3 tug/mL BSA solution in PBS for post-coating. inspection against the focal point of the diode laser So that The resulting BSA/PSA resuspension was used directly for the laser was centered in the middle well (C6 when reading 15 the assay.

wells C5, C6 and C7, and D6 when reading wells D5, D6, The degree of Ab adsorption to the phosphors, and and D7). residual Ab activity, was determined by titrating the The PMT signal (amps) was recorded at each plate phosphor-bound Ab with a fluorescein isothiocyanate position and numerically integrated over the width of the (FITC) conjugated-mouse IgG. The resulting FITC-labeled Sample well (approximately 4000 um). Several Scans were phosphors were passed through a Cyteron Absolute flow made at different positions in the 10° to 10” dilution sample cytometer, which was also capable of measuring the relative wells to determine the uniformity of the particle distribution. Size of the particles. Two distinct size Subpopulations were The background Signal was determined by integrating the observed with about 65% of the counted particles appearing average dark field current of the PMT over a 4000 um as Small, presumably monodisperse particles, and 35% being distance, which yields an integrated background Signal of 25 Significantly larger, presumably aggregates. Only 60% of the 1x10" ua-m. The integration products of the samples wells Smaller Subpopulation appeared to have significant quanti were Scaled to this background Signal, and are shown in FIG. ties of active Ab (determined by FITC fluorescence). Of the 19. purported aggregates, about 90% appeared to contain active Immunodiagnostic Sample Detection Ab (by FITC fluorescence). This suggests that less than 40% of the phosphor-Ab conjugates were of an appropriate size

A Series of IgG/anti-IgG Samples for demonstrating the (nominal 0.3 um) and exhibited anti-mouse IgG activity. A capabilities of the up-converting phosphor reporters in a Similar fraction of phosphor-Ab conjugates (31%) were immunosorbant assay format was prepared. These samples active but carried a Significantly larger phosphor reporter. consisted of six individual wells (positive samples) coated The PMT signal (amps) was recorded at each plate with antigen (mouse IgG) and bovine Serum albumin (BSA), 35 position and numerically integrated over the width of the and six wells coated with BSA alone (negative controls). Sample well (approximately 4000 um). The average signals Nominal 0.3 um (YosYbos.Eroo)2O2S phosphor particles (with 95% confidence limits) are:

coated with goat anti-mouse IgG antibody (anti-IgG) were then used as the reporter-antibody conjugate.

Six wells (C5, C6, C7, D5, D6, and D7) of a clear 40 Average of Positive Samples=1.30x10'+1.25x10" ua-m polystyrene Terasaki plate were coated with mouse IgG by Average of Negative Controls=4.20x10-6.82x10 ua-m incubating at 37 C. against 5 till of a 100 ug/ull mouse IgG solution in phosphate buffered saline (PBS). After 1 h, this The positive Samples and negative controls are Statistically Solution was aspirated off and each Sample well was washed 45 different at the 99.9% confidence level. The positive samples with 10 ul of 3% BSA in PBS. This was immediately emit on average 30.0+29.7 times more light than the nega aspirated off and replaced with 20 uL of 3% BSA in PBS. tive controls.

Each sample well was post-coated with BSA by incubating against the 20 lull of BSA/PBS solution for 1 h at 37° C. The Linkage of Phosphors to Biological post-coat Solution was aspirated off and the plates Stored at Macromolecules 4 C. overnight. These wells were considered in positive 50

Samples. The same Six wells in a Second Terasaki plate were In order to delineate further the parameters for prepared in an identical fashion, except they were not coated up-converting phosphors as biochemical reporters, biologi with mouse IgG. This second set of sample wells were cal linkers were attached to phosphor particles. Sodium yttrium fluoride-ytterbium/erbium phosphor particles were considered negative controls. 55 coated with Streptavidin. The excitation and emission Spec Phosphor-Antibody Conjugate tral properties of the phosphor alone and the phosphor A Solution of (YosYboosEroos)O2S phosphor particles coated with streptavidin were measured (FIGS. 17A, 17B, was prepared by Suspending the dry phosphors into DMSO. 18A, and 18B) and both the uncoated and streptavidin The initial particle density was approximately 107 particles/ coated phosphors were almost identical in their absorption mL as determined by counting the number of particles 60 and emission properties, indicating that the attachment of contained in the field of an optical microScope. It should be macromolecular linkers (e.g., proteins) have little if any noted that the 0.3 um fundamental particle size was below effect on the phosphorescent properties of the up-converting the resolution limits of the microScope. This Solution was phosphor. The Streptavidin-coated phosphors were then spe allowed to settle undisturbed for 3 days. The Supernatant, cifically bound to biotinylated magnetic beads, demonstrat which was turbid and presumably contained mostly mono 65 ing the applicability of linker-conjugated inorganic phos disperse Smaller particles was used for Subsequent conjuga phors as reporters in biochemical assays, Such as tion. immunoassays, immunohistochemistry, nucleic acid

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49 SO hybridizations, and other assayS. Magnetic bead technology ficity is calibrated and optimized for the individual applica allows for the easy Separation of biotin-bound Streptavidin tion by performing layer antibody layering from one layer coated phosphor from a Solution, and is particularly well (primary antibody is biotinylated) to at least five layers and Suited for Sandwich assays wherein the magnetic bead is the ascertaining the optimal number of layers for detecting CD3 Solid Substrate. on EL-4 cells.

Advantageously, Streptavidin-biotin chemistry is widely FIG. 21 Schematically portrays Simultaneous detection of used in a variety of biological assays, for which two EL-4 cell Surface antigens using phosphors which can up-converting phosphor reporters are Suited. FIG. 20 shows be distinguished on the basis of excitation and/or emission Schematically, for example and not limitation, one embodi Spectra. Detection of both antigens in the Scheme shown in ment of an immunoassay for detecting an analyte in a FIG. 21 uses a biotinylated terminal antibody which is Solution by binding the analyte (e.g., an antigen target) to a conjugated to Streptavidin-coated phosphor (#1 or #2) prior biotinylated antibody, wherein the analyte forms a Sandwich to incubation with the Ab-layered sample. Thus, the complex immobilized on a Solid Substrate (e.g., a magnetic phosphor-antibody Specificity is retained through the unusu bead) by linking a first binding component bound directly to ally strong (KD approx. 1x10"M") non-covalent bond the Solid Substrate to a second binding component (e.g., the 15 between streptavidin and biotin which is pre-formed before biotinylated antibody); a Streptavidin-coated up-converting incubation with the primary antibody-bound Sample. Quan phosphor then binds specifically to the biotinylated antibody titation of each antigen is accomplished by detecting the in the Sandwich and Serves to report formation of the distinct signal(s) attributable to each individual phosphor Sandwich complex on the Solid Substrate (which is a measure Species. Phosphorescent Signals can be distinguished on the of the analyte concentration). When the solid substrate is abasis of excitation spectrum, emission spectrum, fluores magnetic bead, it is readily removed from the Sample cence decay time, or a combination of these or other Solution by magnetic Separation and the amount of phosphor properties.

attached to the bead(s) in Sandwich complex(es) are deter FIG. 22 shows a Schematic of an apparatus for phase mined by measuring Specific up-converting phosphores Sensitive detection, which affords additional background cence. Thus, Sandwich complex phosphorescence provides a 25 discrimination. The pulse or frequency mixer is set to pass quantitative measure of analyte concentration. the Signal and discriminate against the background follow Biotinylated polynucleotides are also conveniently used ing frequency calibration for maximum background rejec as hybridization probes, which can be bound by tion.

Streptavidin-coated up-converting phosphors to report hybrid formation. Covalent Conjugation of Upconverting Phosphor Label to Avidin

Background Phosphorescence in Biological An up converting yttrium -ytterbium-erbium

Samples (YoseYboos Eroo) oxysulfide (OS) phosphor was linked to

Background Signals were determined in two biological 35 avidin by the following procedure:

Samples for determination of potential background in immu Monodisperse upconverting phosphor particles were noassayS. Sputum and urine were used as Samples in the silanized with thiopropyltriethoxysilane (Huls) following Same apparatus as used for the phosphorescence Sensitivity the procedure detailed by Arkles (in: Silicone Compounds: measurements (Supra). No background levels were found Register and Review, Hills America, pgs. 59–75, 1991). This above the system noise levels set by the photomultiplier dark 40 consisted of adding thiopropyltriethoxysilane (2 g) and 95% current. This noise level allows detection of Signals from on aq. ethanol (100 mL) to a 500 mL Erlenmeyer flask and the order of a few hundred particles/cm. This is close to a stirred for 2 minutes. Approximately 8 mL of the 65 mg/mL Single particle in the detection volume of the System. phosphor Suspension in DMSO was then added to the A photomultiplier is a preferred choice for a detector for mixture. This Suspension was stirred for an additional 2 high Sensitivity measurements of up-converting phosphors 45 minutes, then transferred to centrifuge tubes and centrifuged Since photomultipliers can be selected to produce high to Separate the phosphor particles. The pellets were washed quantum efficiency at the up-converted (i.e., emitted) wave twice with 95% aq. ethanol centrifuging each time. The lengths and virtually no response in the range of the longer resulting particles were collected and dried overnight under excitation wavelengths. vacuum at approximately 30° C. A quantity (127 mg) of dry 50 silanized phosphors were resuspended in 1.5 mL of DMSO

Detection of Cell Antigens with Phosphor-Labeled (phosphor Stock).

Antibodies

A solution containing 1.19 mg of avidin (Pierce) in 1.0

Streptavidin is attached to the up-converting phosphor mL of borate buffer (954 mg sodium borate decahydrate and particles as described, Supra. The mouse lymphoma cell line, 17.7 mL of 0.1N NC1 in 50 mL of deionized water, pH 8.3) EL-4, is probed with a hamster anti-CD3 antibody which 55 was prepared (Avidin Stock). Another Solution containing specifically binds to the 30 kD cell Surface EL-4 CD3 T 1.7 mg of N-Succinimidyl(4-iodoacetyl) aminobenzoate lymphocyte differentiation antigen. The primary hamster (Pierce Chemical) in 1.2 mL of DMSO was prepared (SIAB antibody is then specifically bound by a biotinylated goat stock). A quantity (10 ul) of the SIAB stock was added to antihamster Secondary antibody. The biotinylated Secondary the 1.0 mL of Avidin stock and stirred at room temperature antibody is then detected with the streptavidin-phosphor 60 30 minto allow the N-hydroxysuccimide ester of the SIAB conjugate. This type of multiple antibody attachment and to react with primary amines on the avidin (Avidin-SIAB labeling is termed antibody layering. Stock).

Addition of multiple layers (e.g., binding the primary A 20 mL Scintillation vial was prepared containing 10 mL hamster Ab with a goat-antihamster Ab, followed by binding of borate buffer (pH 8.3). The following additions were then with a biotinylated rabbit-antigoat Ab) are used to increase 65 made to this vial: 21.6 till of the avidin-SIAB stock solution the distance Separating the phosphor from the target. The followed by 1.5 mL of the phosphor stock. This reaction layering effect on Signal intensity and target detection Speci mixture was stirred at room temperature in the dark over

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S1 52 night to allow the SIAB activated avidin to react with the casein in PBS, Tropix, Bedford, Mass.). The cell-antibody thiol groups present on the Silanized phosphor Surface and complexes were blocked in this solution for 30 minutes at resulting in the covalent linkage of avidin to the phosphor room temperature and then transferred to fresh tubes. particles. A pre-blocked Suspension (40 uD) of either avidin After the overnight incubation 1.0 mL of the reaction Phosphor conjugate, avidin-FITC, avidin, or unconjugated mixture was centrifuged (1 min at 10,000 g) and the Super Phosphor was added to four of the cell Samples conjugated natant removed. The pellet was resuspended in 1.0 mL of with the biotinylated anti-mouse IgG (H&L). In addition, an phosphate buffered saline (pH 7.2, Pierce) and centrifuged equal amount of pre-blocked avidin-Phosphor or unconju again to wash any unconjugated protein from the phosphors. gated Phosphor was added to the remaining two cell Samples This washing process was repeated. The washed pellet was immunoprecipitated with the non-biotinylated FITC resuspended in 1.0 mL of phosphate buffered Saline and used labelled anti-mouse IgG (H&L). The avidin reporter conju directly in diagnostic assays as described below. gates or negative controls were pre-blocked as follows. Avidin-Phosphor and Phosphor alone was diluted in block

Measurement Apparatus ing buffer by adding 10 till of a 6.7 mg/ml Suspension to a 15 final volume of 100 ul. Avidin-FITC and the avidin alone

A modified SLM Aminco 48000 Fluorimeter was used to controls were also diluted in blocking buffer by adding 27 till measure the fluorescence spectrum from the phosphor of 2.5 mg/ml Solution to a final volume of 100 L. These Samples. The modifications to this device consisted of reagents were blocked at room temperature for 3 hours with adding a laser diode (David Sarnoff CD-299R-FA #13) intermittent resuspension and then added to 50 till of cells which was input to the fluorimeter through port 3. The laser labelled with biotinylated or non-biotinylated second anti diode emits at ) =985.1 nm. Spectral data provided by the body. The avidin-biotin reactions were performed at room David Sarnoff Research Center also shows a small peak at temperature for 30 minutes with occasional resuspension. 980.2 nm. This peak has 15% the intensity of the peak at 985 The reactions were Stopped by harvesting the cells by

centrifugation and washing twice in blocking buffer. The

A 5.08 cm focal length lens was used to collimate the 25 samples were resuspended in 100 lull of blocking buffer and diode laser beam. The power of the IR laser light was allowed to settle for 4-5 minutes. Slides for imaging were measured as 6.1 mW at the cuvette location with a drive prepared by pipetting 5 till of Settled cells from the bottom current of 75 mA. The beam was not focused at the center of the tube. Cells were imaged by confocal laser microScopy of the cuvette. This is true for the standard visible light from under appropriate conditions to observe cell surface FITC the fluorimeter excitation monochromator as well. The laser and up-converting phosphor Signals. The observations are diode beam is diverging as it enters the cuvette holder and Summarized in Table IV.

is approximately 4 mm (H)x2 mm (V) by the time it reaches the center of the cell, neglecting the changes in refractive TABLE IV index of the cell wall and the liquid.

Cell Cell

Light emitted is Scanned with a monochromator and 35 Surface Surface detected by a photomultiplying tube (PMT) 90° from the Type of goat Avidin Phosphor FITC direction of the excitation light. The detection limits for the Tube anti-mouse IgG Conjugate Signal Signal modified SLM Aminco 48000 were determined by serial 1. biotinylated Avidin-Phosphor -- dilution to be 4x10M (240,000 phosphor particles per 2 biotinylated Phosphor mL) in PBS. Phosphor emission peaks in the spectrum were 40 3 biotinylated Avidin seen at wavelengths of 406+2 nm, 434+2 nm, 522+2 nm, and 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.

Linkage of Avidin-Phosphor Conjugate to Cell 45 The remainder of the Samples were used to resuspend Surface Marker paramagnetic, polystyrene beads bound with Sheep anti A lymphoblastoid cell line (Human Genetic Mutant Cell mouse IgG. For each of the six samples, 3x107 beads were Repository #GM07092) was cultured in RPMI 1640 media pre-washed with blocking buffer for 1 hour at room tem containing 15% heat inactivated fetal calf Serum. A Suspen perature in Eppendorf tubes. The buffer was removed by sion of cells (10 cells) was centrifuged and resuspended in 50 aspiration while the tubes were in a magnetic rack. The an equal volume of phosphate buffered saline (PBS) pH 7.4. magnetic beads with anti-mouse IgG were allowed to bind Cells were washed two times in PBS and resuspended to a to the antibody labelled cells for 1 hour at room temperature final concentration of 5x10° cells/ml. These cells were then with intermittent resuspension. The magnetic beads were incubated with a mouse IgG1 monoclonal antibody to then collected on a magnetic rack, washed four times in human B-microglobulin, a Class I histocompatibility anti 55 blocking buffer, resuspended in 100 u, blocking buffer, gen in polystyrene centrifuge tubes. The cells were immu transferred to a fresh tube, and up-converting phosphores noprecipitated for 30 minutes at 4 C. with an antibody cence was measured 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. 60 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 till with a wavelength (514 nm) are very close for FITC, higher final Second antibody concentration of 20 tug/ml. The cells 65 resolution was required to get Separable Signals than with were harvested and washed in PBS as above but were 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

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S3 S4 were Scanned at 0.5 nm increments from 450 nm to 750 nm.

with a 2 nm bandwidth on the emission monochromator. TABLE V Sample 1 is the positive control and clearly yielded the Upconverting Phosphor Nucleic Acid Diagnostic Assay Results highest emission signal. Sample 2 indicates that any non

Specific adsorption of the phosphors to the Sample is limited PMTSignal PMTSignal and is readily discriminated from Signal attributable to Sample DNA Reporter (V (G. 546 mm) (VG) 514 nm) avidin-conjugated phosphor and showing that avidin linked 1. DNA labeled Avidin 6.1297 2.4788 phosphors can specifically bind only when they are conju with linked gated with the probe, in this example through the biotin digOxigenin Phosphor and biotin avidin linkage. Sample 3 is the negative control which 2 DNA labeled Silanized 1.0528 4.4022 contains no phosphors, only avidin. Sample 4 shows FITC with Phosphor conjugated avidin. Although FITC signals were observed on digOxigenin and biotin the cell Surface by laser microScopy, the Signals were below 3 No DNA Avidin 1.63O2 3.5779 the level of detection on the fluorimeter for measurement of 15 linked FITC, and since there was no phosphor in the sample there Phosphor

was no significant phosphor Signal. Samples 5 and 6 show with that FITC-conjugated primary antibodies can be detected digOxigenin and that the presence of phosphor or avidin-phosphor does and biotin

not significantly disrupt binding of the primary antibody to with Avidin its target antigen. digOxigenin and biotin

Linkage of Avidin-Phosphor Conjugate to DNA Avidin

Plasmid DNA (25ug) was nick translated in the presence 25 of 20 mM dGTP, 20 mM dCTP, 20 mM biotin-14 DATP, 13 mM dTTP, and 7 mM digoxigenin-11 dUTP and purified by Phosphor Downconversion Evaluation ethanol precipitation. The average size of the biotinylated, A Sample of the (YosYboosEroos).O2S phosphors were digoxygenin labelled fragments was estimated to be between Scanned for the presence of a downconverted Signal. This 200-300 nucleotides as estimated by gel electrophoresis. was accomplished by exciting a Sample of the monodisperse Approximately 20 ug DNA was immunoprecipitated for 1 phosphors described above (4x10'’M in DMSO) with 1.3 hour at 22 C. with 10 ug/ml mouse monoclonal anti mW of monochromatic light at 350 nm with a 16 nm digoxigenin IgG1 solution (PBS) in a 200 uL volume. An bandwidth for the excitation Source. Detection was accom equivalent reaction containing no DNA was also prepared. 35 plished by scanning this sample from 350 to 800 nm with a Each of the two samples were then aliquoted (50 uL) into monochomator bandwidth of 8 nm. Scanning was performed three fresh Eppendorf tubes. in 2 nm increments. No downconversion was observed. Moreover, no downconversion was seen at the excitation

The avidin-conjugates were blocked for 1 hour at room wavelengths cited by Tanke et al. (U.S. Pat. No. 5,043,265). temperature by diluting 500 lug of an avidin-phosphor Thus, the upconverting phosphors tested are unlike those Suspension, unconjugated phosphor Suspension, or avidin 40 reported in Tanke et al.

solution in 300 till of blocking buffer. For each of the samples (summarized below in Table V) 50 lull of the HOMOGENEOUS ASSAYS anti-digoxigenin conjugates was added to 150 u of pre

The multiphoton activation process characteristic of blocked avidin-conjugates or avidin and were incubated for upconverting 30 minutes at room temperature. 45 phosphors can be exploited to produce assays that require no Sample washing StepS. Such diagnostic

Unbound avidin-conjugates were removed by resuspend assays that do not require the removal of unbound phosphor ing 3x10" paramagnetic beads linked with sheep anti-mouse labels from the Sample are herein termed homogeneous IgG (pre-blocked in blocking buffer). After incubation for 30 assays, and can also be termed pseudohomogeneous assayS. minutes at room temperature with intermittent resuspension, 50 the beads were Separated on a magnetic rack and washed 4 Homogeneous ASSay Example 1 to 6 times in PBS. The antibody-DNA bound beads were One embodiment of a homogeneous assay consists of the then measured on the fluorimeter.

use of an upconverting phosphor label linked to an appro

The samples were scanned from 500 to 700 nm with a priate probe (e.g., an antibody or DNA). The phosphor bandwidth of 8 nm and step size of 2 nm. Each PMT value 55 labeled probe specifically binds to a target (e.g., antigen or reported (Table V) represents an average over 5 scans. nucleic acid) that is linked to a capturing Surface. A Suitable Sample 1 is expected to provide the highest PMT signal capture Surface can be the tip of a light carrying optical fiber since biotinylated DNA is present and can bind to the (FIG. 23) or the bottom surface of a sample container (FIG. avidin-linked phosphors. Sample 2 indicates the level of 24A and 24B). Upon incubation of the target-labelled cap nonspecific adsorption of the phosphors to the Sample which 60 ture Surface with the phosphor-labelled probe, phosphor is found to be insignificant since the PMT signal is observed particles will accumulate at the capture Surface as a function to be the same as that of the negative control (sample 4) of the amount of target present on the capturing Surface. The which contains no phosphors. Sample 3 is another control target may be linked directly to the capturing Surface or may and shows that the avidin-linked phosphors do not bind to be immobilized by interaction with a binding agent (e.g., the paramagnetic beads in the absence of DNA. Samples 5 65 Specific antibody reactive with target, polynucleotide that and 6 show results of FITC-labeled avidin used to validate binds target) that is itself linked to the capturing Surface asSay. (Such as in a Sandwich immunoassay, for example).

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SS S6

Detection of the phosphor bound to the capture Surface is up-converted light at 380 nm is shown in FIG. 32. We effected using an excitation light that is focused from a low estimate the up-conversion cross Section to be 107-27 >> intensity beam of large croSS-Section to a high intensity cms.o.2>> for this experiment.

beam of small cross-section with the focal point of the beam We have also observed up-conversion in thulium acetate being at or very near the capture Surface. Focusing of the hexahydrate and holmium chloride hexahydrate in Solution excitation light is accomplished by transmission through following the excitation schemes of Allain et al. (1990) optical elements that have a very Small focal length, Such Electron. Lett. 26: 166, and Allain et al. (1990) Electron. that the beam diverges and becomes leSS intense, within a Lett. 26:261, respectively. The salts were dissolved in heavy Short distance of the capture Surface. water, and excitation was performed using a krypton laser. Since the intensity of the light emitted from the upcon Although the up-conversion was weak, the up-conversion Verting phosphor labels is proportional to the excitation light should be improved if chelated compounds are used instead intensity raised to a power of two or greater, phosphors near of dissolved salts.

the focal point of the excitation Source will emit significantly Although the present invention has been described in more light than those remaining in Suspension in the Sample Some detail by way of illustration for purposes of clarity of away from the capture Surface. Therefore, binding of upcon 15 understanding, it will be apparent that certain changes and Verting phosphor linked probes to the capture Surface will modifications may be practiced within the Scope of the yield an increase in emitted light intensity measured from claims.

the Sample as a whole or as measured from a control Sample We claim:

in which phosphors do not bind to the capture Surface. 1. A method for detecting an analyte in a Sample, com Emitted light intensity may be plotted as a function of target prising the Steps of concentration using for Standardization (calibration) a Series contacting a Sample containing a target analyte with a of Samples containing predetermined concentrations of tar labeled probe to Specifically bind the target analyte and get. The emitted light intensity from a test sample (unknown form a labeled probe-target complex, wherein the concentration of target) can be compared to the Standard labeled probe comprises a probe attached to an curve thus generated to determine the concentration of 25 target. up-converting chelate complex comprising a rare earth ion and a ligand and being capable of converting

Examples of Suitable homogeneous assay formats excitation radiation to emission radiation of a shorter include, but are not limited to, immunodiagnostic Sandwich wavelength;

assays and antigen and/or antibody Surface competition Separating any bound labeled probe from the labeled asSayS. probe-target complex;

Homogeneous ASSay Example 2 illuminating the labeled probe-target complex with exci Another embodiment allows for the accumulation of tation radiation, and upconverting phosphor linked probes at the detection Sur 35 detecting emission radiation of at least one label emission face by the application of centrifugal or gravitational Set wavelength, wherein the emission radiation has a tling. In this embodiment an upconverting phosphor is shorter wavelength than the excitation radiation. linked to multiple probes. All the probes must bind to the contacting 2. A method of claim 1, further comprising, before the Same target, although said binding can be accomplished at chelate complex Step, the Step of attaching the up-converting different locations (e.g., as antibody probes may target 40 3. A method according to the probe to form the labeled probe. different epitopes on a single antigen). The multiprobe ion is Selected from the group to claim 1, wherein the rare earth phosphor can then be used to effect the aggregation of ion, thulium ion, a holmium ion of an erbium ion, neodymium targets in Solution or Suspension in the Sample. This aggre and a praseodymium ion. 4. A method according to claim gation will result in the formation of a large insoluble Selected from the group ethylenediaminetetraacetic 3, wherein the ligand is phosphor-probe-target complex that precipitates from Solu 45 dipicolinic acid, diethylenetriaminetetraacetic acid, diethyl acid, tion or Suspension (FIG. 25). The aggregated complex enetriaminepentaacetic acid, tetraazacyclotetradecanetet containing phosphors accumulates at a detection Surface while nonaggregated material remains in Solution or SuS raacetic acid, an antibiotic, a protein, a phthalocyanine and pension. Detection is accomplished as described in the a cryptate. 5. A method for detecting an analyte in a Sample, com above example using a sharply converging excitation beam. 50 prising the Steps of

Evaluation of Up-Converting Chelates contacting a Sample containing a target analyte with a Up-conversion has been performed in rare earth chelates probe to specifically bind the target analyte and form a probe-target complex;

and rare earth Salt Solutions. Chelates of erbium and neody contacting the probe-target complex with a label to form mium have been prepared with ethylenediaminetetraacetic 55 acid (EDTA) and dipicolinic acid (DPA). The erbium che a labeled probe-target complex, wherein the label is an lates were pumped using light near 793.5 nm from a up-converting chelate complex comprising a rare earth Ti:sapphire laser (the excitation Scheme of Macfarlane ion and a ligand and being capable of converting (1989) Appl. Phys. Lett 54: 2301). This approach produced excitation radiation to emission radiation of a shorter upconversion but not Satisfactorily, which we attribute to 60 wavelength;

weak absorption for the first Step due to the increase in Separating any unbound label from the labeled probe linewidth in the chelate over the low temperature crystal target complex;

used for the up-conversion laser. illuminating the labeled probe-target complex with exci The neodymium chelates were excited with light near 580 tation radiation; and nm from a Nd:YAG-pumped dye laser (following the exci 65 detecting emission radiation of at least one label emission tation scheme of Macfarlane et al. (1988) Appl. Phys. Lett wavelength, wherein the emission radiation has a 52: 1300). An emission spectrum for the emitted shorter wavelength than the excitation radiation.

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6. A method for detecting an analyte in a Sample, com differentiating the labeled probe-target complex from any prising the Steps of unbound labeled probe in the Sample, contacting a Sample containing a target analyte with a illuminating the labeled probe-target complex with exci labeled probe to Specifically bind the target analyte and tation radiation; and form a labeled probe-target complex, wherein the 5 detecting emission radiation of at least one label emission labeled probe comprises a probe attached to an wavelength from the labeled probe-target complex, up-converting chelate complex comprising a rare earth wherein the emission radiation has a shorter wave ion and a ligand and wherein the up-converting chelate length than the excitation radiation. complex is capable of converting excitation radiation to emission radiation of a shorter wavelength; k k k k k