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

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OTHER PUBLICATIONS Seveus et al. (1992), Time-Resolved Fluorescence Imaging Diamandis and Christopoulos (1992), Detection of Lan of Europium Chelate Label in Immunohistochemistry and in thanide Chelates and Multiple Labeling Strategies Based on Situ Hybridization. Cytometry 13:329-338. Time-Resolved Fluorescence, in Nonisotopic DNA Probe Silversmith et al. 1986, Green Infrared-Pumped Erbium Techniques, pp. 263-274, L.J. Kricka, Ed., Academic Press. Upconversion Laser, J. Opt. Soc. Am. A3, P128, PDP12. Evangelista et al. (1991), Enzyme-Amplified Lanthanide Smart et al. (1991), CW Room Temperature Upconversion Luminescence for Enzyme Detection in Bioanalytical Lasing at Blue, Green and Red Wavelengths in Assays, Anal. Biol. 197:213-224. Infrared-Pumped Pr'-Doped Fluoride Fibre, Electronics Gudgin Templeton et al. (1991), Time Resolved Fluores Letters 27:1307-1309.

cence Detection of Enzyme-Amplified Lanthanide Lumi Soini and Kojola (1983), Time-Resolved Fluorometer for nescence for Nucleic Acid Hybridization Assays, Clin. Lanthanide Chelates-A New Generation of Nonisotopic Chem, 37/9:1509-1512. Immunoassays, Clin. Chem. 29/1:65-68. Hemmila et al. (1984), Europium as a Label in Time-Re solved Immunofluorometric Assays, Anal. Bio. Allain et al. (1990), Room Temperature CW Tunable Green 137:335-343. Upconversion Holmium Fibre Laser, Electroncis Letters Johnson et al. (1972), Infrared-To-Visible Conversion By 26:261-263.

Rare-Earth Ions in Crystals, J. Appl. Phys. vol.43, No. 3. Allain et al. (1990), Blue Upconversion Fluorozirconate Johnston and Wright (1979), Trace Analysis of Nonfluores Fibre Laser, Electronics Letters 26:166-168. cent Ions By Associative Clustering with a Fluorescent Auzel (1973), Materials and Devices Using Dou Probe, Anal. Chem. 51:1774-1780. ble-Pumped Phosphors and Energy Transfer, Proceedings of Kano et al. (1972), NaLnF:Yb"Er", (Ln:YGd La): Effi the IEEE 61:758-786.

cient Green-Emitting Infrared-Excited Phosphors, J. Elec Berthou and Jorgensen (1990), Optical-Fiber Temperature

Nguyen et al. (1989), Blue-Green (450-NM) Upconversion Sensor Based on Upconversion-Excited Fluorescence, Tm:YLF Laser, Applied Optics 28:3553-3555. Optics Letters 15:1100-1102.

Reichstein et al. (1988), Laser-Excited Time-Resolved Beverloo et al. (1992), Preparation and Microscopic Visu Solid-Phase Fluoroimmunoassays with the New Europium alization of Multicolor Luminescent Immunophosphors, Chelate 4.7-Bis (Chlorosulfophenyl)-1,10-Phenanthro Cytometry 13:561-570.

line-2.9-Dicarboxylic Acid as Label, Anal. Chem. Beverloo et al. (1990), Inorganic Phosphors as New Lumi 60:1069-1074. nescent Labels for Immunocytochemistry and Time-Re Rich and Pinnow (1972), Exploring The Ultimate Efficiency solved Microscopy, Cytometry 11:784-792. in Infrared-To-Visible Converting Phosphors Activated

With Er and Sensitized With Yb, J. Appl. Phys. Bethune etal. (1993), Atoms in Carbon Cages: The Structure 43:2357-2365. and Properties of Endohedral Fullerences, Nature Schindele and Renzoni (1990), Ultra Fluors: New Fluoro 366:123-128.

phores for Immunological Applications, J. Clin. Immun. Campiglia, A D et al, Anal. Chem. vol. 60(19), pp. 13:182-186. 2165-2167, 1988.

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Visible Light Out = Negative Sample

Infrared

Coated th Phosphor E. Optic Phosphor

Target linked

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

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Add Antigen

Coated Phosphor Phosphor Anitgen Emitted Light NO CAPTURE is of LOW AT SURFACE Intensity

Focal point (high intensity excitation)

Excitation Light of low intensity

CAPTURE AT

Emitted Light SURFACE is of High

Intensity

Focal point (high intensity excitation)

FIG. 24B Excitation Light of low intensity Competitive Homogeneous Antigen Capture Assay

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

Homogeneous immunoprecipitation Assay

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FIG 51A F3 10 x 10 Gridded Array of 980 nm Diode Lasers

- Aqueous

Sample Flow

MMT 44-444444 10 x 10 Gridded Array

of Photodiode Detectors

individual

Diode Loser in Array

Sample Flow F9 east sees

Raay' 0-25 um Polymer film individual Photodiode Overlay Capture Used as

Surface

Detector in Array

Antibody Immund Or Nudeic Acid Conjugated Antigen Capture Probe Bonded to

PhoSchor

FIG. 31B

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UP-CONVERTNG REPORTERS FOR radioisotopic labels typically do not produce a strong (i.e., BOLOGICAL AND OTHER ASSAYS USNG non-Cerenkov) signal in the ultraviolet, infrared, or visible LASER EXCITATION TECHNIQUES portions of the electromagnetic spectrum makes radioiso topes generally unsuitable as labels for applications, such as

CONTINUING DATA microscopy, image spectroscopy, and flow cytometry, that This application is a continuation-in-part of application employ optical methods for detection. Ser. No. 08/381,006, filed Jan. 30, 1995, now abandoned, For these and other reasons, the fields of clinical which is a continuation of application Ser. No. 071946,068, chemistry, water and air monitoring, and biomedical filed Sep. 14, 1992, now abandoned. research have sought alternative detectable labels that do not 10 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 chromogenic substrate to an insoluble, colored product (e.g., compositions useful in assay methods for detecting soluble. alkaline phosphatase, B-galactosidase, horseradish 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 15 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 rescein isothiocyanate, rhodamine, Cascade blue), which such as proteins, drugs, and polynucleotides, have proven to absorb be very valuable analytical techniques in biology and electromagnetic energy in a particular absorption medicine, particularly for characterizing the molecular com wavelength spectrum and subsequently emit visible light at position of normal and abnormal tissue samples and genetic one or more longer (i.e., less energetic) wavelengths. material. Many different types of such detection methods are 25 Using enzymes and phosphorescent/fluorescent or colo 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. 30 appropriate reaction conditions and incubation time, a single Typically, a detection method employs at least one ana and enzyme molecule can produce a large amount of product, lytical reagent that binds to a specific target macromolecular hence yield considerable signal amplification. However, species and produces a detectable signal. These analytical detection tageously methods that employ enzymes as labels disadvan require additional procedures and reagents in reagents typically have two components: (1) a probe macromolecule, for example, an antibody or 35 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 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 ate an insoluble product that is not attached to the probe method, and the detectability of the associated label deter molecule.

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 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. 45 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 macronol which makes discrimination of the reaction products diffi ecules. Radioimmunoassay techniques have been used to cult, detect and measure minute quantities of specific analytes, 50 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 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 (o, B, or y) produced by decay 55 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. (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 (scintilation and gamma counters) or with A wide variety of fluorescent dyes are available and offer inexpensive and sensitive autoradiographic techniques. 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 65 sufficiently different so as to permit multitarget detection and disposal, special licensing requirements, and instability discrimination with multiple probes, wherein each probe (radioactive decay and radiolysis). Further, the fact that species is linked to a different fluorophore. Because the

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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. N.Y. 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 10 chelates (Soini and Lovgren (1987) CRC Crit. Rev. Anal. ground signals. Most common fluorophores are aromatic Chen. 18: 105; Leif et al. (1977) Clin. Chem. 23: 1492; organic molecules which have broad absorption and emis Soini and Hemmila (1979) Clin. Chem. 25: 353; Seveus et sion spectra, with the emission maximum red-shifted al. (1992) Cytometry 13:329). Down-converting lanthanide 50-100 nm to a longer wavelength than the excitation (i.e., chelates are inorganic phosphors which possess a large absorption) wavelength. Typically, both the absorption and 15 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 20 in time-gated detection methods which can reduce, but not by other reagents (e.g., fixative or serum), or autofluores totally eliminate, noise caused by shorter-lived autofluores cence or the sample itself (Jongkind et al. (1982) Exp. Cell cence and scattered excitation light. Further, lanthanide Res. 138: 409; Aubin, J. E. (1979).J. Histochem, Cytochem. phosphors possess narrow-band emission, which facilitates 27: 36). Autofluorescence of optical lenses and reflected wavelength discrimination against background noise and excitation light are additional sources of background noise 25 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 30 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 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 35 render them unsuitable for cytochemical staining. Beverloo integrate a weak fluorescent signal over a long detection et al. (op.cit.) have described a particular down-converting time, since the dye molecules decompose as a function of lanthanide phosphor (yttrium oxysulfide activated with incident irradiation in the UV and near-UV bands. europium) that produces a signal in aqueous solutions which However, because fluorescent labels are attractive for can be detected by time-resolved methods. Seveus et al. various applications, several alternative fluorophores having (op.cit.) have used down-converting europium chelates in advantageous properties for sensitive detection have been conjunction with time-resolved fluorescence microscopy to proposed. One approach has been to employ organic dyes reject the signal from prompt fluorescence and thereby comprising a phycobiliprotein acceptor molecule dye that reduce autofluorescence. 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 45 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. 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 50 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 redregion 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 rejection). Further, excitation with ultraviolet irradiation et al. (1989) Cytometry 19:11). damages nucleic acids and other biological macromolecules, However, with both the phycobiliproteins and the cyanine 55 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 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 The organic fluorphore used by Denket al. was significantly optical fluorescence microscopy, (2) cyanines, photobleached by the intense, highly focused laser light phycobiliproteins, and the coupled accessory molecules 65 during the course of imaging. Motsenbocker et al. (EP 476 (e.g., Azure A) are organic molecules susceptible to pho 556) describes a method to increase luminol chemilumines tobleaching and undergoing undesirable chemical interac cence by adding a dye catalyst that absorbs long wavelength

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S 6 radiation (deep red light) and subsequently reacts with measure of the amount of reporter present. Depending on the molecular oxygen to generate an oxidant which can itself detector's spectral response, it may be necessary to provide react with luminol and produce oxidized luminol which a filter to block the excitation light. emits blue light. Gavrilovic (U.S. Pat. No. 5,166,948) dis Simultaneous detection of multiple reporters is possible, closes 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 multiplexer or other suitable techniques, such as frequency

Thus, there exists a significant need in the art for labels labeling, frequency modulation, and lock-in detector device. and detection methods that permit sensitive optical and/or 10 If the emission bands are different (whether or not the spectroscopic detection of specific label signal(s) with excitation bands are different), light in the different emission essentially total rejection of nonspecific background noise, bands is separated and sent to multiple detectors. If the and which are compatible with intact viable cells and emission bands overlap, a single detector may be used, but aqueous or airborne environments. 15 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 INVENTON invention enable the ultrasensitive detection of up-converting phosphors and up-converting organic dyes by

The present invention provides labels, detection methods, 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, 25 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 background noise (e.g. autofluorescence) which has a dif wavelength excitation radiation (e.g., near-ER) to emitted ferent phase shift.

radiation at about one-half to one-third the wavelength of the 35 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 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 the transient photoconductivity following the excitationirra 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 45 tion is used to provide a quantitative measurement of the one or more probes, such as an immunoglobulin, number and/or concentration of dye molecules in a sample. polynucleotide, streptavidin, Protein A, receptor ligand, or Furthermore, essentially all photoions formed in the irradi other probe molecule. In an another embodiment, ated sample contribute to the signal, whereas photons are up-converting organic dyes serve as the label. The organic emitted isotopically and only a fraction can be collected dye labels and phosphor labels of the invention are highly 50 using optics. Measurement of the transient photocurrent compatible with automated diagnostic testing, microscopic effectively transfers the conversion of photons into an elec imaging applications, and coded particle detection, among tronic signal that is readily measured with relatively simple many other applications. and inexpensive sensors such as electrodes. The nature of the invention provides considerable flex In some embodiments, the present invention utilizes one ibility in the apparatus for carrying out the methods. As a 55 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 inradiation of an diodes or light-emitting diodes (LEDs), and the detector up-converting label can modify the immediate molecular may be any convenient detector, such as a photodiode. In the environment through laser-induced photochemical pro case of a single reporter, the apparatus includes a laser diode 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. 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 65 catalyst.

and directed to the detector. An electrical signal representing The invention provides methods for producing targeted the intensity of light in the emission band provides a damage (e.g., catalysis) in chemical or biological materials,

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wherein a probe is employed to localize a linked flow cytometry using excitation radiation that is in the up-converting label to a position near a targeted biological infrared portion of the spectrum and does not significantly structure that is bound by the probe. The localized damage cells. This provides a significant advantage over up-converting label is excited by one or more excitation present FACS methods which rely on excitation illumination wavelengths and emits at a shorter wavelength which may in the ultraviolet portion of the spectrum, including wave be directly cytotoxic or genotoxic (e.g., by producing free radicals such as Superoxide, and/or by generating thymine lengths which are known to produce DNA lesions and damage cells.

thymine dimers), or which may induce a local photolytic The invention also provides compositions comprising at chemical reaction to produce reactive chemical species in least one fluorescent organic dye molecule attached to an the immediate vicinity of the label, and hence in the vicinity O inorganic up-converting phosphor. The fluorescent organic of the targeted biological material. Thus, targeting probes labeled with one or more up-converting labels (e.g., an dye molecule is selected from the group consisting of: up-converting inorganic phosphor) may be used to produce rhodamines, cyanines, Xanthenes, acridines, oxazines, targeted damage to biological structures, such as cells, porphyrins, with and phthalocyanines, and may optionally be tissues, neoplasms, vasculature, or other anatomical or his 15 complexed may be adsorbed a heavy metal. The fluorescent organic dye to the inorganic up-converting phosphor tological structures.

crystal

Embodiments of the present invention also include inorganic up-convertingand/or may be covalently attached to a coated up-converting phosphors which can also be excited by an amic up-converting phosphor, phosphor, a derivatized vitrocer electron beam or other beam of energetic radiation of ganic up-converting phosphor.orFrequently, a microencapsulated inor covalent conju sufficient energy and are cathodoluminescent. Such 20 gation between the up-converting inorganic phosphor electron-stimulated labels afford novel advantages in elimi particles and proteins (e.g., avidin, immunoglobulin) nating background in ultrasensitive biomolecule detection accomplished with heterobifunctional crosslinkers. can be methods. Typically, stimulation of the up-converting phos phor with at least two electrons is employed to generate a BRIEF DESCRIPTION OF THE DRAWINGS visible-light or UV band emission. 25

The invention also provides for the simultaneous detec FIG. 1 is an optical and electronic block diagram illus tion of multiple target species by exploiting the multiphoton trating representative apparatus for performing diagnostics excitation and subsequent background-free fluorescence on a sample according to the present invention; detection of several up-converting phosphors or FIG. 2A shows apparatus for implementing phase sensi up-converting dyes. In one embodiment, several phosphors/ 30 tive detection in the context of a single channel; 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 FIG. 4 shows ah apparatus for performing diagnostics on with a distinguishable fluorescent "fingerprint.” By using 35 a sample various methods and devices, the presence and concentra centered atusing 1 first and second reporters excitation bands and , respectively, and having overlapping tion of each of the phosphors or dyes can be determined. emission bands near A:

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 more analytes, typically in solution. The assay methods tions in multi-photon excitation schemes. employ compositions of probes labeled with up-converting FIG. 6 shows a miniaturized instrument using a hand-held phosphors and/or up-converting dyes and apparatus for probe;

magnetically and/or optically trapping particles that com FIG. 7A shows the use of a charge-coupled device (CCD) prise the analyte and the labeled probe. In one embodiment, array used to detect emissions from a large plurality of a sandwich assay is performed, wherein an immobilized 45 binding sites;

probe, immobilized on a particle, binds to a predetermined FIG. 7B shows the CCD array used in conjunction with a analyte, producing an immobilization of the bound analyte lens array;

on the particle; a second probe, labeled with an FIG. 8 shows an embodiment using optical trapping; up-converting label can then bind to the bound analyte to FIG. 9 shows schematically dye coating and encapsula produce a bound sandwich complex containing an 50 tion of an up-converting phosphor particle;.

up-converting label bound to a particle. By combining different probe-label combinations, particles of various ingFIG. 10 shows schematically an apparatus for determin sizes, colors, and/or shapes with distinct immobilized probe properties of velocity particle a target;

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

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FIG. 15 shows effective single-photon phosphorescence DESCRIPTION OF SPECIFIC EMBODIMENTS cross-section for 0.3 um particles of Na(YoYboEros)F. Definitions following excitation with 200W/cm at 970 nm. Unless defined otherwise, all technical and scientific FIG. 16 shows size-dependence of phosphorescence terms used herein have the same meaning as commonly cross-section for Na(YosYboBros)f particles. 5 understood by one of ordinary skill in the art to which this

FIG. 17A shows a fluorescence scan of an up-converting phosphor reporter in Hepes-buffered saline induced by exci invention similar or belongs. Although any methods and materials equivalent to those described herein can be used tation with a 970-nm laser source; in the practice or testing of the present invention, the FIG. 17B shows a fluorescence spectrum scan of an preferred methods and materials are described. For purposes up-converting phosphor reporter coated with streptavidin in 10 of the present invention, the following terms are defined Hepes-buffered saline induced by excitation with a 970-nm below, laser Source; As used herein, "label” refers to a chemical substituent FIG. 18A shows an excitation spectrum scan of an that produces, under appropriate excitation conditions, a up-converting phosphor reporter in Hepes-buffered saline 15 detectable optical signal. The optical signal produced by an with monochromatic detection of emission at 541 mm; excited label is typically electromagnetic radiation in the FIG. 18B shows an excitation spectrum scan of an near-infrared, visible, or ultravioletportions of the spectrum. up-converting phosphor reporter coated with streptavidin in The labels of the invention are up-converting labels, which Hepes-buffered saline with monochromatic detection of means that the chemical substituent absorbs at least two emission at 541 nm, photons at an excitation frequency and subsequently emits FIG. 19 shows the integrated signal obtained from electromagnetic energy at an emission frequency higher than samples of (YosYboosroos)2O2S showing the relation the excitation frequency. Thus, there is generally a signifi ship between phosphor concentration and up-converted sig cant Stokes shift between the original excitation frequency nal; and the final emission frequency. A label is generally FIG. 20 shows schematically one embodiment of an 25 attached to a probe to serve as a reporter that indicates the sandwich immunoassay for detecting an analyte in a solution presence and/or location of probe. The invention encom by binding the analyte (e.g., an antigen target) to a biotiny passes organic and inorganic up-converting labels, but pref lated antibody and to an immobilized antibody, wherein the erably employs up-converting inorganic lanthanide phos analyte forms a sandwich complex immobilized on a solid phors 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 comprise a lanthanide ion in a chelate or cage compound.

of two cell surface antigens with specific antibodies labeled with two phosphors with distinct phosphorescence charac As used herein, a "probe' refers to a binding component teristics. which binds preferentially to one or more targets (e.g., 35 antigenic epitopes, polynucleotide sequences, macromo

FIG. 22 shows a schematic of an apparatus for phase lecular receptors) with an affinity sufficient to permit dis sensitive detection.

FIG. 23 show a schematic of a competitive homogeneous crimination of labeled probe bound to target from nonspe cifically bound labeled probe (i.e., background). Generally, assay using phosphors as labels and fiber optic illumination the probe-target binding is a non-covalent interaction with a at a capture surface. binding affinity (K) of at least about 1x10'M', preferably FIG. 24A and 24B show a schematic of a competitive homogeneous antigen capture assay using phosphors as with at least about 1x10'M', and more preferably with an affinity of at least about 1x10M or greater. Antibodies labels and a convergent illumination beam focused on the typically have a binding affinity for cognate antigen of about capture surface. 1x10'M' or more. For example but not limitation, probes FIG. 25 shows a schematic of a homogeneous immuno 45 of the invention include: antibodies, polypeptide hormones, precipitation assay using phosphors as labels and a conver polynucleotides, streptavidin, Staphlyococcus aureus pro gent illumination beam focused on the capture surface tein A, receptor ligands (e.g., steroid or polypeptide wherein the capture surface collects immunoprecipitates. hormones), leucine Zipper polypeptides, lectins, antigens FIG. 26 shows a block diagram of one embodiment of (polypeptide, carbohydrate, nucleic acid, and hapten apparatus for carrying out the present invention on a sample 50 epitopes), and others.

using a microscope. As used herein, a "probe-label conjugate” and a "labeled FIG. 27 is a block diagram of a microtiter plate reader for probe" refer to a combination comprising a label attached to use with the present invention. a probe. In certain embodiments, more than one label FIG. 28 is an illustration of the data for upconverting 55 substituent may be attached to a probe. Alternatively, in phosphors in three test wells. some embodiments more than one probe may be attached to FIG. 29 is a schematic view of a second embodiment of a label (e.g., multiple antibody molecules may be attached to a hand-held probe for carrying out the present invention. a submicron-size inorganic up-converting phosphor bead). FIG. 30 illustrates a three channel configuration using Various attachment chemistries can be employed to link a interference filters. 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 static interactions, and surface tension (phase boundary) invention in which a diode laser array F and a detector interactions. Attachment of label can also involve incorpo array F2 are combined in a single device. ration of the label into or onto microspheres, microparticles, FIG. 31B is a detailed view of a small section of the immunobeads, and superparamagnetic magnetic beads device shown in FIG. 31A. 65 (Polysciences, Inc., Warrington, Pa.; Bangs Laboratories, FIG. 32 is an emission spectrum for up-conversion from Inc. 979 Keystone Way, Carmel, Ind. 46032). For example, neodymium chelated in EDTA. inorganic up-converting phosphor particles can be encapsu

Page 39

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” 10 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., hCH, insulin, albumin), glyco of "Specific hybrids hybridization" is defined herein as the formation between a probe polynucleotide and a target proteins (e.g., immunoglobulins, thrombomodulin, polynucleotide, wherein the probe polynucleotide preferen Y-glutamyltranspeptidase; Goodspeed et al. (1989) Gene 76: 15 tially hybridizes to the target 1), lipoproteins, viruses, microorganisms (e.g., pathogenic least one discrete band can beDNA such that, for example, at 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 20 somal location characteristic of a unique or repetitive biological fluid), and pharmaceuticals (i.e., prescribed or sequence. In some instances, a target sequence may be over-the-counter drugs listed in the Physicians Drug Refer present in more than one target polynucleotide species (e.g., ence and/or Merck Manual, or illegal substances such as a particular target sequence may occur in multiple members intoxicants or anabolic steroids). of a gene family or in a known repetitive sequence). It is As used herein, the term "antibody" refers to a protein 25 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

(IgG, IgG, IgG, IgG), delta, epsilon and mu constant mental method selected by the practitioner. Various guide 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 (1987), Academic Press, Inc., San Diego, Calif., Dunn et al. COOH-terminus. Full-length immunoglobulin “heavy 35 (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 absorbed immunoglobulin constitutes the basic structural unit of an fluorescentbyemission an up-converting label, produces a detectable from the up-converting label, wherein antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having the fluorescent emission is of a shorter wavelength (i.e., one light and one heavy chain. In each pair, the light and higher frequency radiation) that the label excitation wave heavy chain variable regions are together responsible for length. As used herein, the term "label emission wave length” refers to a wavelength that is emitted from an binding to an antigen, and the constant regions are respon 45 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 forms including, for example, Fv, Fab, and F(ab'), as well more excitation wavelengths; label emission wavelengths of as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., up-converting radiation) than labels are shorter (i.e., higher frequency the corresponding excitation wavelengths.

Eur, J. Immunol. 17, 105 (1987)) and in single chains (e.g., 50 Both label excitation wavelengths and label emission wave Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 lengths are characteristic to individual up-converting label (1988) and Bird et al., Science, 242,423-426 (1988)). (See, species, and are readily determined by performing simple generally, Hood et al., “Immunology", Benjamin, N.Y., 2nd excitation and emission scans.

ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 Invention Overview (1986)). Thus, not all immunoglobulins are antibodies. (See, 55

U.S. Ser. No. 07/634.278, which is incorporated herein by The subject invention encompasses fluorescent labels that reference, and Co et al. (1991) Proc. Natl. Acad. Sci. are excited by an excitation wavelength and subsequently (U.S.A.) 88: 2869, which is incorporated herein by emit electromagnetic radiation at up-shifted frequencies reference). (i.e., at higher frequencies than the excitation radiation). As used herein, "probe polynucleotide' refers to a poly In accordance with the present invention, labels compris nucleotide that specifically hybridizes to a predetermined ing up-converting inorganic phosphors and/or up-converting target polynucleotide. For example but not limitation, a organic dyes are provided for various applications. The probe polynucleotide may be a portion of a cDNA corre up-converting labels of the invention may be attached to one sponding to a particular mRNA sequence, a portion of a or more probe(s) to serve as a reporter (i.e., a detectable genomic clone, a synthetic oligonucleotide having sufficient 65 marker) of the location of the probe(s). The up-converting sequence homology to a known target sequence (e.g., a labels can be attached to various probes, such as antibodies, telomere repeat TTAGGG or an Alu repetitive sequence) for streptavidin, protein A, polypeptide ligands of cellular

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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 up-converting lanthanide phosphor particles may be coated willAlthough

the properties of the up-converting phosphors described in detail in a later section, it is useful to with a polycarboxylic acid (e.g., AdditionXW 330, Hoechst, outline the basic mechanisms

Frankfurt, Germany) during milling and various proteins been found to occur in certaininvolved. Up-conversion has (e.g., immunoglobulin, streptavidin or protein A) can be earth ions in certain crystal materials. Forcontaining materials example, rare ytter physically adsorbed to the surface of the phosphor particle 10 bium and erbium act as an activator couple in a phosphor (Beverloo et al. (1991) op.cit., which is incorporated herein host material such as barium-yttrium-fluoride. The ytter by reference). Alternatively, various inorganic phosphor bium ions act as the absorber, and transfer energy non coating techniques can be employed including, but not radiatively limited to: spray drying, plasma deposition, and derivatiza characteristicto ofexcite the erbium ions. The emission is thus tion with functional groups (e.g., -COOH, -NH2, 15 Up-ConvertingtheMicrocrystalline erbium ion's energy levels. Phosphors -CONH2) attached by a silane coupling agent to -SiOH moieties coated on the phosphorparticle or incorporated into Although the invention can be practiced with a variety of a vitroceramic phosphor particle comprising silicon oxide(s) up-converting inorganic phosphors, it is believed that the and up-converting phosphor compositions. Vitroceramic preferred embodiment(s) employ one or more phosphors phosphor particles can be aminated with, for example, derived from one of several different phosphor host aminopropyltriethoxysilane for the purpose of attaching materials, each doped with at least one activator couple. amino groups to the vitroceramic surface on linker Suitable phosphor host materials include: sodium yttrium molecules, however other omega-functionalized silanes can fluoride (NaYF), lanthanum fluoride (LaF), lanthanum be substituted to attach alternative functional groups. oxysulfide, yttrium oxysulfide, yttrium fluoride (YF), Probes, such as proteins or polynucleotides may then be 25 yttrium gallate, yttrium aluminum garnet, gadolinium fluo directly attached to the vitroceramic phosphor by covalent ride (Gdf), barium yttrium fluoride (BaYF BaYF), and linkage, for example through siloxane bonds or through gadolinium oxysulfide. Suitable activator couples are carbon-carbon bonds to linker molecules (e.g., organofunc selected from: ytterbium/erbium, ytterbium/thulium, and tional silylating agents) that are covalently bonded to or ytterbium?holmium. Other activator couples suitable for adsorbed to the surface of a phosphor particle. Covalent up-conversion may also be used. By combination of these conjugation between the up-converting inorganic phosphor host materials with the activator couples, at least three particles and proteins (e.g., avidin, immunoglobulin) can be phosphors with at least three different emission spectra (red, accomplished with homobifunctional, or preferably green, and blue visible light) are provided. Generally, the heterobifunctional, crosslinkers. For example, surface slian absorber is ytterbium and the emitting center can be selected ization of the phosphors with tri(ethoxy)thiopropyl silane 35 from: erbium, holmium, terbium, and thulium; however, leaves a phosphor surface with a thiol functionality to which other up-converting phosphors of the invention may contain a protein (e.g., antibody) or any compound containing a other absorbers and/or emitters. The molar ratio of absorber: primary amine can be grafted using conventional emitting center is typically at least about 1:1, more usually N-succinimidyl(4-iodoacetyl)amino-benzoate (SLAB) at least about 3:1 to 5:1, preferably at least about 8:1 to 10:1. chemistry (Weltman et al. (1983). Other silanization and more preferably at least about 11:1 to 20:1, and typically less cross-linking methods compatible with the inorganic phos than about 250:1, usually less than about 100:1, and more phors may be used at the discretion of the practitioner. usually less than about 50:1 to 25:1, although various ratios Microcrystalline up-converting phosphor particles are may be selected by the practitioner on the basis of desired typically smaller than about 3 microns in diameter, prefer characteristics (e.g., chemical properties, manufacturing ably less than about 1 micron in diameter (i.e., submicron), 45 efficiency, absorption cross-section, excitation and emission and more preferably are 0.1 to 0.3 microns or less in wavelengths, quantum efficiency, or other considerations). diameter. It is generally most preferred that the phosphor The ratio(s) chosen will generally also depend upon the particles are as small as possible while retaining sufficient particular absorber-emitter couple(s) selected, and can be quantum conversion efficiency to produce a detectable sig calculated from reference values in accordance with the nal; however, for any particular application, the size of the 50 desired characteristics.

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

from available sources (e.g., handbooks and published 65 Some embodiments of the invention employ inorganic references) or may be obtained by generating a standard phosphors that are optimally excited by infrared radiation of ization curve measuring quantum conversion efficiency as a about 950 to 1000 nm, preferably about 960 to 980 nm. For

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example but not limitation, a microcrystalline inorganic activator couple), but which have other desirable character phosphor of the formula YF:YbooEro exhibits a lumi istics (e.g., manufacturing efficiency, ease of derivatization, nescence intensity maximum at an excitation wavelength of etc.); such low efficiency up-converting phosphors are pref about 980 nm. Inorganic phosphors of the invention typi erably excited with laser illumination at a frequency at or cally have emission maxima that are in the visible range. For near (i.e., within about 25 to 75 nm) an absorption maximum example, specific activator couples have characteristic emis of the material. The fact that no other light is generated in the system other than from the up-converting phosphor allows sion spectra: ytterbium-erbium couples have emission for extremely sensitive signal detection, particularly when maxima in the red or green portions of the visible spectrum, depending upon the phosphor host; ytterbium-holmium intense laser illumination is used as the source of excitation radiation. Thus, the unique property of up-conversion of couples generally emit maximally in the green portion, 10 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, YosoYboEroof phors as ultrasensitive reporters, particularly as intracellular emits maximally in the green portion of the spectrum.

Although up-converting inorganic phosphor crystals of 15 as small as practicable that reporters, it is essential the grain size of the phosphor be (typically less than about 0.3 to 0.1 various formulae are suitable for use in the invention, the um), 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(YYb.Er.)F: x is 0.7 to 09, y is 0.09 to 0.29, and Z capable of up-conversion are suitable for use in the inven is 0.05 to 0.01; tion are shown in Table I.

and Z is 0.0005 to 0.001; and TABLE I Na(YYb,Tm)F: x is 0.7 to 09, y is 0.0995 to 0.2995, Phosphor Material Compositions and Z is 0.0005 to 0.001. 25

Host Material Absorber on Emitter Ion Color

(Yoss Yboos Eroos)2O3 is a relatively efficient Oxysulfides (OS) up-converting phosphor material. YOS Ytterbium Erbium Green For exemplification, but not to limit the invention, 30 Gd2OS Ytterbium Erbium Red ytterbium(Yb)-erbium(Er)-doped yttrium oxysulfides lumi LaOS

Oxyhalides (OX)

Ytterbium Holmium Green nesce in the green after excitation at 950 nm. These are non-linear phosphors, in that the ytterbium acts as an YOF Ytterbium Thulium Blue "antenna" (absorber) for two 950 nm photons and transfers YOCl, Yterbium Terbium Green its energy to erbium which acts as an emitter (activator). The 35 Fluorides (F) critical grain size of the phosphor is given by the quantum YF, Ytterbium Erbium Red yield for green emission and the doping level of both Yb and GdF Ytterbium Erbium Green Er, which is generally in the range of about 1 to 10 percent, LaF

NaYF,

Ytterbium

Ytterbium

Holmium

Thulium

Green

Blue more usually in the range of about 2 to 5 percent. A typical 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 (Ga.0) thousand formula units ensures the emission of at least one or more photons during a typical laser irradiation time. YGaO Ytterbium Erbium Red

However, the nonlinear relationship between absorption and Silicates (SiOy) emission indicates that intense illumination at the excitation 45 wavelength(s) may be necessary to obtain satisfactory signal YSiO Ytterbium Holmium Green in embodiments employing very small phosphor particles YSiO, Ytterbium Thulium Blue (i.e., less than about 0.3 pm). Additionally, it is usually desirable to increase the doping levels of activator?emitter In addition to the materials shown in Table I and variations couples for producing very small phosphor particles so as to thereof, aluminates, phosphates, and vanadates can be suit maximize quantum conversion efficiency. able phosphor host materials. In general, when silicates are Inorganic microcrystalline phosphors with rare earth acti used as a host material, the conversion efficiency is rela vators generally have narrow absorption and line emission tively low. In certain uses, hybrid up-converting phosphor spectra. The line emission spectra are due to f-f transitions crystals may be made (e.g., combining one or more host within the rare earth ion. These are shielded internal tran 55 material and/or one or more absorberion and/or one or more sitions which result in narrow line emission. emitter ion).

In certain applications, such as where highly sensitive Exemplary up-converting phosphors excited at about 980 detection is required, intense illumination can be provided nm include, but are not limited to: YosoYbosBroo)P3; by commercially available sources, such as infrared laser Yo.87Ybo. 13Tmo.o.o.1)P3; Yo so Ybo.19s. Hoo-oo:2)Fa; sources (e.g., continuous wave (CW) or pulsed semiconduc Gido.soYbo.1s Ero.o.2)Fa; Gido.87Ybo.13 Tino.o.o.1)Fa; tor laser diodes). For example, in applications where the Gido soybo.19s Hoo.oo2)F3; Yoss Yboos Eroos)2O2S: microcrystalline phosphor particle must be very small and Yos7Ybo.13Tmooo1)2O2S: YosoYbo.19s Hoo-oo2)2O2S: the quantum conversion efficiency is low, intense laser Gdossyboos Broos)2O2S: Gido.87Ybo.13Tino.oo1)2O2S: illumination can increase signal and decrease detection Gdosoybo.19shoooo2)2O2S:

times. Alternatively, some applications of the invention may 65 Exemplary up-converting phosphors excited at about require phosphor compositions that have inherently low 1500 nm include, but are not limited to: YooBroos)OS; quantum conversion efficiencies (e.g., low doping levels of

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Preparation of Inorganic Phosphor Labels including a polysulfide flux for annealing. Using this Techniques and methods for manufacture of inorganic technique, highly efficient oxysulfide particles in the 0.3 to phosphors has been described in the art. Up-converting 0.4 m diameter range were prepared as a dispersion in phosphor crystals can be manufactured by those of ordinary water, Frequently, sonication can be used to produce a skill in the art by various published methods, including but monodisperse mixture of discrete spherical particles. After not limited to the following: Yocom et al. (1971) Metalur fractionation and coating, these particles can be used as gical Transactions 2: 763; Kano et al. (1972) J. Electro up-converting reporters. Furthermore, this general prepara chem. Soc., p. 1561;Wittke et al. (1972).J. Appl. Physics 43: tive procedure is suitable for preparing much smaller phos 595; Van Uitert et al. (1969) Mat. Res. Bull. 4: 381; which phor particles (e.g., 0.1 m diameter or Smaller), which may are incorporated herein by reference. Other references which 10 be advantageous for various assay formats. may be referred to are: Jouart JP and Mary G (1990).J. Frequently, such as with phosphors having an oxysulfide Luminescence 46: 39; McPherson GL and Meyerson SL host material, the phosphor particles are preferably dis (1991) Chem. Phys. Lett. (April) p.325; Oomen et al. (1990) persed in a polar solvent, such as acetone or DMSO and the J. Luminescence 46:353; NIH and Rand SC (1991) Optics like, to generate a substantially monodisperse emulsion Lett. 16 (Sept.); McFarlane RA (1991) Optics Lett, 16 15 (e.g., for a stock solution). Aliquots of the monodisperse (Sept.); Koch et al. (1990) Appl. Phys. Lett. 56: 1083; stock solution may be further diluted into an aqueous Silversmith et al. (1987) Appl. Phys. Lett. 51: 1977; Lenth W solution (e.g., a solution of avidin in buffered water or and McFarlane RM (1990).J. Luminescence 45: 346; Hirao buffered saline).

et al. (1991).J. Non-crystalline Solids 135:90; McFarlane et It was found that washing phosphors in acetone or DMSO al. (1988) Appl. Phys. Lett. 52: 1300, incorporated herein by 20 improved suspendability of inorganic phosphor particles in reference). water. In particular, the phosphor particles prepared with In general, inorganic phosphor particles are milled to a polysulfide flux are preferably resuspended and washed in desired average particle size and distribution by conven hot DMSO and heated for about an hour in a steam bath then tional milling methods known in the art, including milling in allowed to cool to room temperature under continuous a conventional barrel mill with zirconia and/or alumina balls 25 agitation. The phosphor particles may be pre-washed with for periods of up to about 48 hours or longer. Phosphor acetone (typically heated to boiling) prior to placing the particles used in binding assays are typically about 3.0 to particles in the DMSO. Hot DMSO-treated phosphors were 0.01 m in diameter (or along the long axis if nonspherical), found to be reasonably hydrophilic and form stable suspen more usually about 2.0 to 0.1 um in size, and more conve sions. A MicrofluidizerTM (Microfluidics Corp.) can be used niently about 1.0 to 0.3 pm in size, although phosphor to further improve the dispersion of particles in the mixture. particles larger or smaller than these dimensions may be DMSO-phosphor suspensions can be easily mixed with preferred for certain embodiments. Phosphor particle size is water, preferably with small amounts of surfactant present. selected by the practitioner on the basis of the desired In general, polysaccharides (e.g., guar gum, Xanthan gum, characteristics and in accordance with the guidelines pro gum arabic, alginate, guaiac gum) can be used to promote vided herein. Fractions having a particular particle size 35 deaggregation of particles. In a variation, particles are range may be prepared by sedimentation, generally over an washed in hot DMSO and serially diluted into a 0.1% extended period (i.e., a day or more) with removal or the aqueous gum arabic solution, which appears to virtually desired size range fraction after the appropriate sedimenta eliminate water dispersion problems of phosphors. tion time. The sedimentation process may be monitored, Resuspended phosphors in organic solvent, such as such as with a Horiba Particle Analyzer. DMSO, are typically allowed to settle for a suitable period However, milling crystalline materials has several weak (e.g., about 1-3 days), and the supernatant which is typically nesses. With milling, the particle morphology is not turbid is used for subsequent conjugation. uniform, as milled particles result from random fracture of LudoxTM is a colloidal silica dispersion in water with a larger crystalline particles. Since the sensitivity of a detec small amount of organic material (e.g., formaldehyde, tion assay using up-converting inorganic phosphors depends 45 glycols) and a small amount of alkalimetal. LudoxTM and its on the ability to distinguish between bound and unbound equivalents can be used to coat up-converting phosphor phosphor particles, it is preferable that the particles be of particles which can subsequently be fired to form a ceramic identical size and morphology. Size, weight, and morphol silica coating which cannot be removed from the phosphor ogy of up-converting microcrystalline phosphor particles particles, but which can be readily silanized with organo can affect the number of potential binding sites per particle 50 functional silanes (containing thiol, primary amine, and and thus the potential strength of particle binding to reporter carboxylic acid functionalities) using standard silanization and/or analyte. Monodisperse submicron spherical particles chemistries (Arkles, B, in: Silicon Compounds: Register and of uniform size can be generated by homogeneous precipi Review; 5th Edition (1991); Anderson, RG, Larson, GL, and tation reactions at high dilutions. For example, smallyttrium Smith, C, eds.; p.59-64, Huls America, Piscataway, N.J.). hydroxy carbonate particles are formed by the hydrolysis of 55 Phosphor particles can be coated or treated with surface urea in a dilute yttrium solution. Similarly, up-converting active agents (e.g., anionic surfactants such as Aerosol OT) inorganic phosphors can be prepared by homogeneous pre during the milling process or after milling is completed. For cipitation reactions in dilute conditions. For example, example, particles may be coated with a polycarboxylic acid (YossyboosEroos.O. was prepared as monodisperse (e.g., Addition XW 330, Hoechst, Frankfurt, Germany or spherical particles in the submicron size range by precipi Tamol, see Beverloo et al. (1992) op.cit.) during milling to tation. produce a stable aqueous suspension of phosphor particles, However, after precipitation it is typically necessary to typically at about pH 6-8. The pH of an aqueous solution of anneal the oxide in air at about 1500 C., which can cause phosphor particles can be adjusted by addition of a suitable faceting of the spherical particles which can generate aggre buffer and titration with acid or base to the desired pH range. gate formation. Faceting can be substantially reduced by 65 Depending upon the chemical nature of the coating, some converting the small spherical particles of the oxide or minor loss in conversion efficiency of the phosphor may hydroxy carbonate precursor to the oxysulfide phase by occur as a result of coating, however the power available in

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a laser excitation source can compensate for such reduction adsorption, covalent linkage). Binding reagents which can in conversion efficiency and ensure adequate phosphor emis be directly labeled include, but are not limited to: primary S1O. antibodies (i.e., which bind to a target analyte), secondary In general, preparation of inorganic phosphor particles antibodies (i.e., which bind to a primary antibody or pros and linkage to binding reagents is performed essentially as thetic group. Such as biotin or digoxygenin), Staphlococcus described in Beverloo et al. (1992) op.cit., and Tanke U.S. aureus Protein A, polynucleotides, streptavidin, and receptor Pat. No. 5,043,265. Alternatively, a water-insoluble poly ligands. Binding reagents can also be indirectly labeled; functional polymer which exhibits glass and melt transition thus, a primary antibody (e.g., a rabbit anti-erb-B antibody) temperatures well above room temperature can be used to can be indirectly labeled by noncovalent binding to a coat the up-converting phosphors in a nonaqueous medium. 10 directly labeled second antibody (e.g., a goat anti-rabbit For example, such polymer functionalities include: carboxy antibody linked to an up-converting inorganic phosphor). lic acids (e.g., 5% acrylic acid/95% methyl acrylate Quantitative detection of the analyte-probe complex may be copolymer), amine (e.g., 5% aminoethyl acrylate/95% conducted in conjunction with proper calibration of the methyl acrylate copolymer) reducible sulfonates (e.g., 5% assay for each probe employed. A probe is conveniently sulfonated polystyrene), and aldehydes (e.g., polysaccharide detected under saturating excitation conditions using, for copolymers). The phosphor particles are coated with water 15 example, a laser source or focused photodiode source for insoluble polyfunctional polymers by coacervative encap excitation illumination.

sulation in nonaqueous media, washed, and transferred to a Specific binding assays are commonly divided into homo suitable aqueous buffer solution to conduct the heterobifunc geneous and heterogeneous assays. In a homogeneous assay, tional crosslinking to a protein (e.g., antibody) or polynucle the signal emitted by the bound labeled probe is different otide probe molecule. An advantage of using water from the signal emitted by the unbound labeled probe, hence insoluble polymers is that the polymer microcapsule will not the two can be distinguished without the need for a physical migrate from the surface of the phosphor upon aging the separation step. In heterogeneous assays, the signal emitted encapsulated phosphors in an aqueous solution (i.e., from the bound and unbound labeled probes is identical, improved reagent stability). Another advantage in using hence the two must be physically separated in order to copolymers in which the encapsulating polymer is only distinguish between them. The classical heterogeneous spe partially functionalized is that one can control the degree of cific binding assay is the radioimmunoassay (RIA) (Yalow et functionalization, and thus the number of biological probe al. (1978) Science 200: 1245, which is incorporated herein molecules which can be attached to a phosphor particle, on by reference). Other heterogeneous binding assays include average. Since the solubility and coacervative encapsulation the radioreceptor assay (Cuatrecasas et al. (1974) Ann. Rey. process will depend on the dominant nonfunctionalized 30 Biochem, 43: 109), the sandwich radioimmunoassay (U.S. component of the copolymer, the functionalized copolymer Pat. No. 4.376,110, which is incorporated herein by ratio can be varied over a wide range to generate a range of reference), and the antibody/lectin sandwich assay (EPO 166 potential crosslinking sites per phosphor, without having to 623, which is incorporated herein by reference). Heteroge substantially change the encapsulation process. neous assays are usually preferred, and are generally more A preferred functionalization method employs heterobi 35 sensitive and reliable than homogeneous assays. functional crosslinkers that can be made to link the biologi Whether a tissue extract is made or a biological fluid cal macromolecule probe to the insoluble phosphor particle sample is used, it is often desirable to dilute the sample in in three steps: (1) bind the crosslinker to the polymer coating one or more diluents that do not substantially interfere with on the phosphor, (2) separate the unbound crosslinker from Subsequent assay procedures. Generally, suitable diluents the coated phosphors, and (3) bind the biological macro are aqueous solutions containing a buffer system (e.g., 50 molecule to the washed, linked polymer-coated phosphor. mM NaH2PO or 5-100 mM Tris, pH4-pH10), non This method prevents undesirable crosslinking interactions interfering ionic species (5-500 mM KCl or NaCl, or between biological macromolecules and so reduces irrevers sucrose), and optionally a nonionic detergent such as Tween. ible aggregation as described by Tanke et al. Examples of When the sample to be analyzed is affixed to a solid support, suitable heterobifunctional crosslinkers, polymer coating 45 it is usually desirable to wash the sample and the solid functionalities, and linkable biological macromolecules support with diluent prior to contacting with probe. The include, but are not limited to: sample, either straight or diluted, is then analyzed for the diagnostic analyte.

In the general method of the invention, an analyte in a

Coating Heterobifunctional Biological 50 sample is detected and quantified by contacting the sample Functionality Crosslinker Macromolecule with a probe-label conjugate that specifically or preferen carboxylate N-hydroxysuccimide Proteins (e.g., tially binds to an analyte to form a bound complex, and then 1-ethyl-3-(3-dimethylamino Ab, avidin) detecting the formation of bound complex, typically by propyl)-carbodiimide (EDC) measuring the presence of label present in the bound com primary amine N-5-azido-2-nitrobenzoyl All having 1° amine 55 plexes. A probe-label conjugate can include a directly oxysuccimide (ANB-NOS)

N-succinimidyl(4-iodoacetyl) labeled analyte-binding reagent (e.g., a primary antibody aminobenzoate (SIAB) linked to an up-converting phosphor) and/or an indirectly thiol (reduced N-succinimidyl(4-iodoacetyl) Proteins labeled analyte-binding reagent (e.g., a primary antibody sulfonate) aminobenzoate (SLAB) that is detected by a labeled second antibody, or a biotiny lated polynucleotide that is detected by labeled streptavidin).

Binding Assays The bound complex(es) are typically isolated from unbound Up-converting phosphors and up-converting organic dyes probe-label conjugate(s) prior to detection of label, usually are used as reporters (i.e., detectable markers) to label by incorporating at least one washing step, so as to remove binding reagents, either directly or indirectly, for use in background signal attributable to label present in unbound binding assays to detect and quantitate the presence of 65 probe-label conjugate(s). Hence, it is usually desirable to analyte(s) in a sample. Binding reagents are labeled directly incubate probe-label conjugate(s) with the analyte sample by attachment to up-converting reporters (e.g., surface under binding conditions for a suitable binding period.

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Binding conditions vary, depending upon the nature of the removing unbound probe, and finally by detecting the probe-label conjugate, target analyte, and specific assay presence, quantity, and/or location of bound probe. The step method. Thus, binding conditions will usually differ if the of detecting bound probe can be accomplished by detecting probe is a polynucleotide used in an in situ hybridization, in label, if the probe is directly labeled, or by incubating the a Northern or Southern blot, or in solution hybridization bound complex(es) with a second binding reagent (e.g., assay. Binding conditions will also be different if the probe streptavidin) that is labeled and which binds to the probe, is an antibody used in an in situ histochemical staining thus accomplishing indirect labeling of the probe. method or a Western blot (Towbin et al. (1979) Proc. Natl. Up-converting labels are attached to probe(s) or second Acad. Sci. (U.S.A.) 76: 4350, incorporated herein by binding reagents that specifically or preferentially bind to reference). In general, binding conditions are selected in probe(s) by any of the various methodologies discussed accordance with the general binding methods known in the herein. Additionally, up-converting phosphor particles can art. For example, but not for limitation, the following be encapsulated in microspheres and coated with a probe binding conditions are provided for general guidance: (e.g., a specific antigen or antibody) for use as a labeled For antibody probes: probe in an immunodiagnostic assay or nucleic acid hybrid 10-200 mM Tris, pH 6-8; usually 100 mM Tris pH 7.5 15 ization assay to detect an analyte in a sample, such as the 15-250 mM NaCl; usually 150 mM. NaCl presence of an antibody, virus, or antigen in a blood serum 0.01-0.5 percent, by volume, Tween 20 sample, according to the method of Hari et al. (1990) Biotechniques 9: 342, which is incorporated herein by 1 percent bovine serum albumin reference. Microencapsulation of phosphor can be accom 49–37° C.; usually 4° to 15° C. 20 plished in several ways known in the art, including coating For polynucleotide probes: the phosphor with a monomer solution and polymerizing the 3-10x SSC, pH 6-8; usually 5x SSC, pH 7.5 monomer to generate a polymer shell encasing the phosphor 0-50 percent deionized formamide particle. Phosphor particles embedded in a polymer coating, 1-10x Denhardt's solution such as a gel coating, can be functionalized (e.g., with amino 25 groups) for covalent attachment to a binding component.

0-1 percent sodium dodecyl sulfate Similarly, up-converting phosphor particles can be coated 10–200 pg/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 30 on a functionalized inorganic phosphor particle (e.g., a Additional examples of binding conditions for antibodies 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, 35 In certain embodiments, such as where stearic and/or Guide to Molecular Cloning Techniques (1987), Academic 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, B-interferon, or other polypeptide hormones, example, a derivatized microencapsulated phosphor or vit cytokines, or lymphokines, suitable binding conditions gen 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, 45 directly derivatized with derivatizing agents (e.g., omega for example, in Harlow and Lane, Antibodies: A Laboratory functionalized silanes) having long intramolecular spacer Manual, Cold Spring Harbor, New York (1988), which is chains, wherein a functional group reactive with a desired incorporated herein by reference. In general, suitable bind binding reagent is separated from the surface of the phos ing conditions for immunological reactions include an aque phor by a spacer of usually at least about 15 A (i.e., the ous binding buffer containing a salt (e.g., 5-500 mM NaCl 50 equivalent of about 10 -CH2-straight-chain groups). In or KCl), a buffer (e.g., Tris or phosphate buffer at pH 4-10), 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 55 Multiple Analyte Detection at least about 1 to 5 minutes, preferably at least about 30 Since up-converting phosphors can be differentiated on minutes to several hours, although typically less than about the basis of the excitation and/or emission wavelength 24 hours, more preferably less than about a few hours or spectra, up-converting phosphors can be used to detect and less. Binding reactions (including washes) are typically discriminate multiple analyte targets, such as, for example, carried out a temperature range of about 0°C. to about 45° cell surface antigens or soluble macromolecules. C., preferably about 4° C. to about 20°-25° C. For example, streptavidin, avidin, or another linker mac Binding assays, which include in situ hybridization, in romolecule (e.g., antidigoxigenin antibody) are attached, situ binding assays, and immunohistochemical staining, are respectively, to each of two different phosphors (for usually performed by first incubating the sample with a illustration, designated here as Phosphorf1 and Phosphoriz) blocking or prehybridization solution, followed by incubat 65 which differ in their absorption and/or emission spectra so as ing the sample with probe under binding conditions for a to facilitate discrimination of the two phosphors based on suitable binding period, followed by washing or otherwise absorption and/or emission wavelengths; e.g., one phosphor

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may emit in the blue and the other may emit in the green. For Binding of the analyte to one or more of the first binding example and not limitation, Na(Yoo Ybo. Eroo) emits component species may then be detected with a second predominantly in the green, and Na(YozaYbo2Tmooo..)F binding component (e.g., an anti-muscarinic receptor emits predominantly in the blue, and thus these two phos antibody) labeled with an up-converting phosphor (either phors may be discriminated on the basis of their phospho directly or through a biotinylated secondary antibody). rescent emissions. Alternatively, two phosphors may pro Solid substrates can be attached to a first binding com duce essentially similar emission spectra but may have ponent which can bind more than one distinct analyte (e.g., different excitation wavelengths which provide a basis for may be immunocrossreactive or polyspecific) and/or can be their discrimination in multiple analyte detection. A first attached to multiple first binding component species which binding component (e.g., an antibody) that binds specifically 1O can bind multiple distinct analytes. Similarly, multiple sec to a first analyte species (e.g., a lymphocyte CD4 antigen) ond binding component species with binding specificities and incorporates biotinyl moieties which may be bound by for particular analytes can be employed. When multiple streptavidin-Phosphorf1 conjugates can be used to quanti second binding component species are employed, it is typi tatively detect the presence of a first analyte in a sample cally desirable to label each second binding component (e.g., a serum sample) by measuring phosphorescence of 15 species with a unique up-converting label that can be Phosphort 1 in analyte-binding component complexes. A distinguished on the basis of its absorption and/or emission second binding component (e.g., a probe polynucleotide) properties.

that binds specifically to a second analyte species (e.g., an It is possible to use different absorbers in combination HIV-1 sequence) and incorporates digoxygenin moieties with various emitters to produce a collection of phosphors (e.g., 11-UTP-digoxygenin) which may be bound by having several differentiable combinations of excitation and antidigoxigenin-Phosphorf2 conjugates can be used to emission spectra. For example but not limitation, six differ quantitatively detect the presence of a second analyte in the entiable phosphors may be generated from two absorbers sample by measuring phosphorescence of Phosphor-2 in and three emitters. A first absorber, A, has an excitation analyte-binding component complexes. Thus, by simulta wavelength of , a second absorber, A had an excitation neously or contemporaneously detecting the presence of 25 wavelength of Wa, a first emitter, E, has an emission line multiple phosphor reporters having differentiable signal at Wei, a second emitter, E has an emission line at A2, and characteristics, multiple analytes may be quantitatively a third emitter, E, has an emission line at W, The six detected in a single sample. phosphors may be differentiated and the signal from each Sandwich Binding Assays individually quantitated by illuminating the sample with an Up-converting phosphors labels can be used as reporters 30 excitation wavelength A1 and detecting separately the for sandwich binding assays (U.S. Pat. No. 4,376,110, which emitted radiation at A1, A2, and Aca, and separately illu is incorporated herein by reference). For example, a mag minating the sample with WA2 and detecting separately the netic bead, such as a superparamagnetic immunobead or emitted radiation at W, A, and W. Table II shows the functionalized magnetizable polymer particle (Polysciences, various absorber:emitter combinations and their excitation Inc., Warrington, Pa.), can serve as the solid substrate which 35 and emission wavelengths.

has an immobilized first binding component (e.g., an antibody, a polynucleotide, or a lectin) that binds to a first TABLE epitope (i.e., a binding locus: an antigenic determinant, Absorber: Emitter Combination Excitation. Emission A. sugar moiety, chemical substituent, or nucleotide sequence) of an analyte. The analyte binds to the first binding com A1E1 AA1 E1 ponent and also to a second binding component (e.g., an A1E2 A1 E2 antibody, a lectin, or a polynucleotide) which binds to a A1E3 A1 E3

second epitope of the analyte. Thus, the analyte bridges the A2E2 AA2 E2 two binding components to form a sandwich complex which A2E3 A2 E3 is immobilized with respect to the solid substrate. The 45 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 labels.

directly to an up-converting phosphor, such as through a 50 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, density, magnetic properties, shape, charge) so that a par up-converting phosphor.

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, species of first binding component, which is labeled, either directly or indirectly, with an 55 examples For example and not limitation, the following three brief up-converting reporter. The sandwich complex is thus 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 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 For example, a solid substrate may have a plurality of noglobulin idiotypes in a sample (e.g., a blood serum sample distinct species of first binding component (e.g., an array of taken from a patient) which can provide diagnostic infor different oligopeptides affixed to a solid support). One or mation about the immune status of a patient (e.g., is a patient more of the species of first binding component may bind to 65 seroreactive with a particular antigen). a particular analyte (e.g., a muscarinic receptor) in an Large superparamagnetic beads are conjugated to an analyte solution that is in contact with the solid support. immunogenic Herpesvirus Type II envelope glycoprotein,

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medium-sized superparamagnetic beads are conjugated to Phosphor and Substrate Differentiation HIV gp120 glycoprotein, and small superparamagnetic The following example describes the use of differentiable beads are conjugated to an immunogenic cytomegalovirus envelope glycoprotein. A serum sample is taken from a up-converting phosphors in conjunction with distinguish able substrate types to detect the presence and relative patient and is incubated with a mixture of the superpara abundance of particular T lymphocyte subpopulations in a magnetic beads under binding conditions to permit specific blood sample taken from an individual. Although described binding of immunoglobulins in the sample with the three here with reference immobilized viral glycoprotein species. The superparamag analyte multiplexing to detecting T cell subpopulations, (i.e., detecting and/or characterizing netic beads are separated from the sample to remove non specifically bound immunoglobulin and incubated with 10 multiple analytes in a sample by using various solid sub up-converting phosphor particles coated with Staphylococ believed to beand/or strate types up-converting phosphor labels) is a generally applicable method.

cus aureus Protein A, which binds to IgG, under binding Large Superparamagnetic beads are conjugated to an conditions. Superparamagnetic beads having specifically anti-CD4 antibody, medium-sized superparamagnetic beads bound IgG are thus labeled with the phosphor-Protein A are conjugated to anti-CD8 antibody, and small superpara conjugate. Large, medium, and Small superparamagnetic beads are then separately illuminated with phosphor exci 15 magnetic beads are conjugated to an anti-CD28 antibody. An tation electromagnetic radiation and time-gated emitted antibody that specifically binds to the CD2 antigenis labeled phosphorescence is detected. Background attributable to with an up-converting phosphor that has an excitation wave length and emits in the red. An antibody that specifically non-specific binding, if any, is determined and subtracted binds using internal standard beads (bovine serum albumin coated phosphorto the CD45R antigenis labeled with an up-converting Superparamagnetic beads) and positive and negative control that has an excitation wavelength and emits in serum samples. The intensity of phosphorescence associated the green. An antibody that specifically binds to the CDw80 with the large, medium, and small beads provides a measure antigen is labeled with an up-converting phosphor that has of the amount of antibodies in the sample which are reactive an excitation wavelength A and emits in the blue. with the Herpesvirus Type II envelope glycoprotein, HIV A blood (or serum, sputum, urine, feces, biopsy tissue, gp120 glycoprotein, and cytomegalovirus envelope 25 etc.) sample is taken from a patient and is incubated with a glycoprotein, respectively. This information can be used to mixture of the superparamagnetic beads and phosphor determine whether an individual patient has been infected labeled antibodies under binding conditions to permit spe with the HIV-1, human CMV, and/or Herpes SimplexType cific binding of cells in the blood sample with the three II viruses. bead-immobilized antibody species and the three phosphor 30 labeled antibody species. After antigen-antibody binding

Phosphor Differentiation occurs, the Superparamagnetic beads are segregated and The following example describes the use of differentiable examined, either sequentially or simultaneously, by illumi up-converting phosphors to detect the presence and relative nation with 1, 2, and Aa, and quantitative detection of red, abundance of particular isoforms of human APP (amyloid green, and blue emissions, respectively. For example, the precursor protein) in a serum or brain biopsy sample. 35 intensity of A-induced red light emission associated with Various isoforms of APP arise in the brain as a consequence the large beads is a rough measure of the amount of cells of alternative exon usage and/or alternative proteolytic pro having both CD4 and CD2 surface antigens and/or the cessing pathways. Thus, although all APP isoforms may relative abundance of those surface antigens (e.g., there may share a common, hypothetical epitope (X), a particular APP be very few CD4 cells that have CD2, but those few cells isoform may have a unique epitope (Y), while another APP may have a large amount of CD2 antigen, and hence a large isoform has a unique epitope (Z). It is possible that the CD2 phosphorescent signal). Similarly, the intensity of relative abundance of a particular APP isoform in a sample A2-induced green light associated with the large beads is a may be of predictive value or may be pathognomonic for rough measure of the amount of cells having both CD4 and Alzheimer's Disease. CD45R surface antigens and/or the relative abundance of Superparamagnetic beads are conjugated to an antibody 45 those surface antigens in a sample.

that binds specifically to a common APP epitope (X) shared In this manner, an analyte sample, such as a blood sample, by all isoforms. A specific antibody reactive with the unique can be "fingerprinted” for the presence and relative Y epitope is labeled with Phosphor #1, which is excited by distribution(s) (e.g., cosegregation and/or correlation) of wavelength and emits in a wavelength spectrum centered various analyte species. Such an analyte fingerprint may be in the blue. A specific antibody reactive with the unique Z. 50 used for providing diagnostic or therapeutic information, for epitope is labeled with Phosphor #2, which is excited by a example, as to measuring a patient's immune status or wavelength and emits in a wavelength spectrum centered measuring response to chemotherapy directed against a in the green. A sample containing APP isoforms is incubated particular blood cell subset. Similar analyte fingerprints can with the Superparamagnetic beads and labeled specific anti be used to type pathogenic organisms and viruses, as well as bodies under binding conditions. The superparamagnetic 55 to order polynucleotide sequences for gene mapping and/or beads are retrieved from the sample, either individually or in Sequencing.

bulk. The beads are illuminated with wavelength and blue Superparamagnetic beads which can be differentiated light emission is detected and measured, and illuminated based on size, shape, color, or density can be magnetically with A and green light emission is detected and measured. trapped individually and scanned with appropriate excitation The intensity of A-induced blue emission is a measure of illumination(s) and phosphor emission(s) characteristic of the APP isoform(s) having the Yepitope, while the intensity particular analytes detected. For example, a unitary detector of the A-induced green emission is a measure of the APP can simultaneously or contemporaneously trap the super isoform(s) having the Z epitope. If the emissions from two paramagnetic bead from a suspension, determine the bead phosphors are readily distinguishable, A and W may be type (size, shape, and/or color), and scan for presence and identical. The standardized relative intensities of the two 65 abundance of particular phosphors (by illuminating with phosphors provides a measure of the relative abundance of excitation wavelength(s) and detecting emitted the APP isoform(s) containing the Y or Z epitopes. wavelengths).

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By performing binding assays under dilute conditions is either directly cytotoxic and/or (2) the phosphor emits at wherein an average of one analyte or less (e.g., lymphocyte) a wavelength that produces reactive chemical species by is bound per microbead, it is possible to type cells individu photocatalysis of a compound present in the sample (e.g., a ally (e.g., determine the abundance of CD45R on each sample can be doped with buckminsterfullerene). individual CD4'cell) and thus generate more precise lym Instead of using the emitted radiation directly for photo phocyte subpopulation definitions. catalytic action on tissue or tumors, an excited form of Biotinylated magnetic beads can also be used to monitor oxygen, so called singlet excited oxygen (OAg) can be the kinetics of binding streptavidin to phosphor particles generated by energy transfer from a dye sensitizer to dis and/or to segregate or purify streptavidin-coated solved molecular oxygen. This scheme makes use of the up-converting phosphor particles from a reaction. Thus, 10 tissue penetrating power of near-infrared radiation (red and streptavidin and up-converting phosphor particles are mixed ultrared region light, including 970 nm) which reaches the in a reaction vessel under binding conditions for forming inorganic up-converting phosphor. Two of the infrared pho streptavidin-coated phosphorparticles. After a suitable bind tons are converted either into a red, green, or blue photon ing period, unbound streptavidin may be removed (e.g., by depending on the absorption spectrum of the sensitizer dye. centrifugation wherein phosphor particles are collected as 15 The dye is excited by the up-converted radiation into a triplet the pellet, unbound streptavidin in the supernatant is state which transfers its energy to a dissolved molecular decanted, and the pellet is resuspended), biotinylated mag oxygen molecule to yield an excited (singlet) oxygen mol netic beads are added to the remaining phosphor suspension ecule. The cytotoxic activity of singlet oxygen is well in binding conditions, and streptavidin-coated phosphor documented in photodynamic therapy and other biomedical particles are recovered bound to the biotinylated magnetic applications (see, Wagnieres et al. (19-21 Jan. 1990) Future beads. Directions and Applications of Photodynamic Therapy, pp. Photophysical Catalysis by Up-Converting Phosphors 249, SPIE Institutes for Advanced Optical Technologies, Other applications of the invention employ phosphors as Society of Photo-Optical Instrumentation Engineers, Box a photophysical catalyst linked to a probe, where the radia 10, Bellingham, Wash, 98277; Pelegrin et al. (1991) Cancer tion emitted by the phosphor is used, typically in conjunc 25 67:2529; Wagnieres et al. (24–25 May 1991) Future Direc tion with a dye molecule, to produce localized intense tions and Applications of Photodynamic Therapy, pp. 219; electromagnetic radiation in an area adjacent to the probe for Folli et al. (17 Dec. 1991) Fluoresceine Clinique 4; Bra various purposes other than detection (e.g., cytotoxicity, ichotte et al. (May 1991) ENT-Clinic, Lausanne, ionization of chemical species, mutagenesis, etc.). For Switzerland).

example, an antibody that specifically binds to a cell surface 30 In this application the up-converting phosphoris mixed or antigen, such as a CD8 antigen on a CD8"lymphocyte, may laced with a sensitizing dye such as methylene blue, rose be used as a probe linked to a up-converting phosphor to bengal or phthalocyanine derivatives, such as localize the phosphor to CD8 lymphocytes. A sample Zn-phthalocyanine. In the first and third case a red-emitting containing CD8 lymphocytes can be incubated with the phosphor is used, whereas for rose bengal a green-emitting anti-CD8 probe-phosphor conjugate and irradiated with an 35 phosphor is best suited. The phthalocyanine derivatives are excitation wavelength (e.g., from an infrared laser diode), ideally suited for this purpose because of their total insolu resulting in emission of upshifted photons (i.e., higher bility in aqueous or biological solutions. These dyes there frequency electromagnetic radiation) in the vicinity of CD8 fore stay in close proximity to the emitters so that the lymphocytes to which the anti-CD8 probe-phosphor con specificity of the cell surface-reporter/probe?olye complex jugate has bound. The emitted radiation may be of a wave becomes the limiting factor. In this case, specialized com length that is directly mutagenic and/or cytotoxic (e.g., binations of reporter/probe/dye formulations preferably in ultraviolet radiation that can lead to formation of thymine the 0.1 to 0.3-micron size range must be synthesized in order dimers, 760–765 nm light is also believed to produce to enable efficient energy transfer: first, up-converted radia chromosomal damage) or may be of a wavelength that can tion is absorbed by the dye as completely as possible; and cause a photolytic decomposition of a chemical present in 45 second, the dye excited energy (triplet state) is transferred to the environment, leading to local formation of reactive dissolved molecular oxygen. Both processes are very effi species that may damage adjacent cells (e.g., photodecom cient if the absorption spectrum of the sensitizer dye is position of buckminsterfullerene, Co, to Css and C2, may matched to the up-converted radiation. produce free radicals that may cause lipid peroxidation of This scheme presents a step beyond the traditional pho cell membranes). 50 todynamic therapy methods in that the red light can be used Since phosphor-emitted radiation is isotropic, it is gener both for tracking and diagnostic as well as for therapeutic ally desirable to physically separate targets (e.g., CD8" purposes after up-converting thus necessitating only one lymphocytes) from non-targets (e.g., CD8 lymphocytes) (infrared) light source at about 1000 nm. Afurther advantage prior to excitation inradiation, so that undesirable damage to is the greaterrange within biological samples of the infrared non-targets by isotropic emission(s) (i.e., "secondary 55 radiation compared to other known photodynamic therapy damage") is avoided. Physical separation may be accom excitation schemes (750-850 nm).

plished by various means, including but not limited to: (1) For embodiments employing up-converting phosphors as performing excitation irradiation on a dilute suspension of photophysical catalysts, it is generally desirable that: (1) the target and non-target cells, wherein the mean distance sepa wavelength(s) of the excitation radiation do not produce rating individual cells is sufficient to reduce secondary significant photocatalysis of the substrate compound, (2) the damage to non-targets, and (2) employing hydrodynamic wavelength(s) of the excitation radiation are not directly focusing to pass cells (both targets and non-targets) single cytotoxic or mutagenic, and (3) the emitted radiation is file through an illumination zone (e.g., as in a fluorescence directly cytotoxic and/or is of an appropriate wavelength to activated cell sorter or the like). Thus, an up-converting produce a biologically effective amount of photodecompo phosphor linked to an anti-CD8' antibody can be used to 65 sition of a substrate compound (e.g., buckminsterfullerene, selectively damage CD8 lymphocytes in a lymphocyte psoralen, compounds containing azide substituents or other sample, where (1) the phosphor emits at a wavelength that photoactivated groups). Alternatively, histidine side chains

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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; 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 schemes have been demonstrated using crystalline host of the invention may be used to targetessentially any desired 15 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 more effectively with pulsed sources. Higher peak powers

Certain applications require small reporters. For example, 20 can the transport, ability to stay in suspension, the bonding a pulsed be obtained from diode lasers when they are operated in mode. The higher peak powers lead to more dynamics, and the tendency toward removal by microphages efficient up-conversion may be improved for smaller reporters. However, the excitation power. due to the nonlinear dependence on reduced sensitivity available with smaller reporters must Up-Converting Organic Dyes also be considered.

One type of small up-converting inorganic phosphor 25 Similar to the up-converting inorganic phosphor reporters consists of rare earth ions in chelates. The use of lanthanide we propose to use "molecular" labels whose fluorescence chelates as reporters has been developed for biological will be detected by optoelectronic means. Infrared or red light is exciting the probe-reporter complex bound to a assays as described on pages 6 and 7 of this application. This target, after which light is emitted at shorter wavelengths prior use of lanthanide chelates involved down-conversion.

That is, the emission light is at a wavelength which is longer 30 with respect to the illuminating source. This up-converted light is free of scattered light from the source or autofluo than the excitation wavelength.

Rare earth chelates may be used as up-converting report rescence by virtue of its higher energy. Furthermore, autof luorescence is greatly reduced by virtue of the excitation in ers through stepwise excitation such as shown in FIG. 5a, or the infrared or red spectral range. The light source is a pump in FIG.5b (except that all levels would be in the same ion). 35 laser whose pump pulses are short in order to achieve high Energy transfer from a sensitizer ion to an activator ion powers and low energy in order to enable nonlinear optical cannot be used in the case of a single rare earth ion. processes in the dye. The goal is to excite the second excited Chelates suitable for use as up-converting phosphors singlet state (S2) in a dye with aps pulse from a tunable dye include ethylenediaminetetraacetic acid (EDTA), dipicolinic laser using two red or infrared photons. After pumping the acid (DPA), diethylenetriaminetetraacetic acid (DTTA), S. state the dye relaxes within a few ps to the fluorescing diethylenetriaminepentaacetic acid (DTPA), tetraazacy state (S) which can be detected by optoelectronic means. clotetradecanetetraacetic acid (TETA), as well as antibiotics, The goal of reaching the S. state using two photons enables natural chelating proteins, phthalocyanines, and cryptates. one to take advantage of the increasing two-photon cross Methods for preparation of lanthanide chelates and their use sections as one approaches the S. state using two-photon in biological assays are described in the literature (Mukkala 45 absorption. The non-resonant two-photon absorption cross et al. (1989) Anal. Biochem. 176: 319. Hemmila et al. (1984) sections are on the order of 10 to 10 cm's, whereas the Anal. Biochen. 137: 335, Soini and Kojola (1983) Clin, cross sections corresponding to S absorption are larger by Chem. 29: 65. Nonisotopic DNA Probe Techniques (1992) two to three orders of magnitude. A few specific examples Kricka (Ed.) Academic Press, New York, as well as the will be mentioned: in general cyanines, xanthenes, references on page 6 of this application). Up-conversion 50 rhodamines, acridines and oxazines are well suited for this phosphor reporters can also consist of rare earth ions inside purpose. Blue dyes can also be used, but the excitation cage compounds such as fullerene materials following the wavelength will be in the red. Rhodamine can be excited at procedures described by Bethune et al. (1993) Nature 366: 650 to 700 nm using two photons, and fluorescence is 123 and references therein. expected around 555 nm. Many IR dyes such as IR-140, Suitable ions for up-conversion in chelates include 55 IR-132 and IR-125 can be excited at 1060 nm using two erbium, neodymium, thulium, holmium, and praseodymium. photons of the Nd:YAG fundamental, and fluorescence is Other candidate ions include the other lanthanide elements, expected in the 850 to 950 nm range. An example of a blue the actinide elements, and other metal elements. Stepwise dye is BBQ excited at 480 nm to reach the S. state at 240 excitation schemes suitable for up-conversion in lanthanide nm, and fluorescence is expected at 390 nm. Many of these chelates are described in the literature on up-conversion dyes are only slightly soluble in aqueous solution and are lasers. Examples include up-conversion in erbium either polar in nature (cyanines) or have polar substituents. (Silversmith et al. (1986) J. Opt. Soc. Am. A3: 128, and Depending on the nature of the probe, no or only minimal Macfarlane et al. (1989) Appl. Phys. Lett. 54: 2301), neody attachment chemistry needs to be undertaken because of the mium (Macfarlane et al. (1988) Appl. Phys. Lett. 52: 1300), abundance of functional groups on the dye chromophore. thulium (Nguyen et al. (1989) Appl. Opt. 28: 3553 and 65 Several companies sell entire lines of dyes: examples are Allain et al. (1990a) Electron. Lett. 226: 166), holmium KODAK, Exciton and Lambda Physik. The scientific foun (Allain et al. (1990b) Electron. Lett. 26:261), and praseody dations of two-photon laser excitation in organic dye mol

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ecules have been treated in a few experimental papers: A. sources (e.g., lasers) can be collimated and multiplexed Penzkofer and W. Leupacher, Optical and Quantum Elec using an array of dichroic mirrors. In this way, samples tronics 19 (1987), 327-349; C. H. Chen and M. P. McCann, containing multiple phosphor species having different exci Optics Commun. 63 (1987), 335; J. P. Hermann and J. tation wavelength bands can be illuminated at their excita Duculing. Optics Commun. 6 (1972), 101; B. Foucault and J. tion frequencies simultaneously. Illumination may be con P. Hermann, Optics Commun. 15 (1975), 412; Shichun Li tinuous or pulsed, or may combine continuous wave (CW) and C. Y. She, Optica Acta 29 (1982), 281-287; D. J. and pulsed illumination where multiple illumination beams Bradley, M. H. R. Hutchinson and H. Koetser, Proc. R. Soc. are multiplexed (e.g., a pulsed beam is multiplexed with a Lond. A 329 (1972), 105-119. CW beam), permitting signal discrimination between phos Resonant Multiphoton Ionization 10 phorescence induced by the CW source and phosphores cence induced by the pulsed source, thus allowing the

At very high laser intensities the up-converting organic discrimination of multiple phosphor species having similar dyes are induced to absorb an additional exciting photon in emission spectra but different excitation spectra. For the field of focussed laser radiation. At those high laser example but not limitation, commercially available gallium intensities the fluorescence is suppressed in favor of absorp 15 arsenide laser diodes can be used as an illumination source tion of an additional photon. This process usually brings the for providing near-infrared light.

organic dye molecules above the ionization limit in solution The ability to use infrared excitation for stimulating and they stabilize by emitting an electron into the solvent up-converting phosphors provides several advantages. First, shell. The result of this three-photon interaction is a molecu inexpensive IR and near-IR diode lasers can be used for lar ion and an attached or solvated electron. When this sustained high-intensity excitation illumination, particularly charge separation is taking place in an electric field, the in charges drift and generate a voltage that can be detected in ThisIRlevel wavelength bands which are not absorbed by water. of high-intensity illumination would not be suit an extremely sensitive manner. This amounts to the mea able for use with conventional labels, such as ordinary surement of the transient conductivity in the solvent system fluorescent dyes (e.g., FITC), since high-intensity UV or and is usually more sensitive than light detection. The 25 visible radiation produces extensive photobleaching of the disadvantage of this method is that it necessitates electrodes label and, potentially, damage to the sample. The ability to that sense the moving charges. In that sense it is not as use higher illumination intensities without photobleaching non-invasive a method as light detection. On the other hand or sample damage translates into larger potential signals, and it bypasses the conversion of light into a photoelectric signal hence more sensitive assays.

which represents an enormous advantage. Every optical The compatibility of up-converting labels with the use of system has a restricted viewing angle that reduces efficiency, diode lasers as illumination sources provide other distinct whereas photoionization “senses" always close to 100% of advantages over lamp sources and most other laser sources. the charges generated. Effectively, the non-linear interaction First, diode laser of the laser field converts every excited organic dye mol modulation of theintensity can be modulated directly through drive current. This allows modulation of ecule into an electric pulse at sufficiently high field inten 35 the light for time-gated or phase-sensitive detection sities that can be routinely achieved using commercial laser sources. Specific examples are the excitation of Rhodamine techniques, the use of which afford sensitivity enhancement without an additional modulator. Modulators require around 650 to 700 nm, or BBQ excitation around 480 nm,

Organic dyes absorbing in the red have to absorb two high-voltage circuitry and expensive crystals, adding both additional photons after being excited into S, thus making light-emitting diode size cost and additional may to apparatus. The laser diode or be pulsed through direct current the whole process a four-photon excitation process, which is modulation. Second, laser illumination sources provide illu slower than a three-photon non-linear process. There may, mination that is exceptionally monochromatic and can be however, be circumstances where such a four-photon pro tightly focused on very small spot sizes, which provides cess is desirable. advantages in signal-to-noise ratio and sensitivity due to Detection Apparatus 45 reduced background light outside of the desired excitation Detection and quantitation of inorganic up-converting 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 50 from excited up-converting phosphors can be accomplished emission wavelength band(s). 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, excitation source. Various excitation sources may be used, charge-coupled devices (CCD), CID devices, photographic including infrared laser diodes and incandescent filaments, 55 film emulsion, photochemical reactions yielding detectable as well as other suitable sources. Optical filters which have products, and visual observation (e.g., fluorescent light high transmissibility in the excitation wavelength range(s) microscopy). If the reporters are organic dyes, resonant and low transmissibility in one or more undesirable wave multiphoton ionization can be sensed using electrostatic length band(s) can be employed to filter out undesirable position-sensitive detectors. Detection can employ time wavelengths from the source illumination. Undesirable gated and/or frequency-gated light collection for rejection of wavelength ranges generally include those wavelengths that residual background noise. Time-gated detection is gener produce detectable sample autofluoresence and/or are within ally desirable, as it provides a method for recording long about 25-100 nm of excitation maxima wavelengths and lived emission(s) after termination of illumination; thus, thus are potential sources of background noise from scat signal(s) attributable to phosphorescence or delayed fluo tered excitation illumination. Excitation illumination may 65 rescence of up-converting phosphoris recorded, while short also be multiplexed and/or collimated; for example, beams lived autofluoresence and scattered illumination light, if any, of various discrete frequencies from multiple coherent is rejected. Time-gated detection can be produced either by

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

sion lifetimes of approximately a few milliseconds (perhaps There may be a plurality of reporters having distinct as much as 10 ms, but typically on the order of 1 ms), emission bands but a common excitation band. In such a whereas background noise usually decays within about 100 10 ns. Therefore, when using a pulsed excitation source, it is 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 multiple laser diodes for a single detector, and would use photobleaching, very weak emitted phosphor signals can be 15 time collected and integrated over very long detection times wavelengths. multiplexing techniques or the like to separate the (continuous illumination or multiple pulsed illumination) to increase sensitivity of detection. Such time integration can Light from the two sources is shown as being combined be electronic or chemical (e.g., photographic film). When So as to be focused at a single location by a common non-infrared photographic film is used as a means for focusing mechanism. This is not necessary, even if it is detecting weak emitted signals, up-converting reporters pro 20 desired to illuminate the same region of the sample. vide the advantage as compared to down-converting phos Similarly, the collection need not be via a single collection phors that the excitation source(s) typically provide illumi mechanism. If it is necessary to preserve all the light, the nation in a wavelength range (e.g., infrared and near combination and separation elements can include a wave infrared) that does not produce significant exposure of the 25 length division multiplexer and a demultiplexer using dich film (i.e., is similar to a darkroom safelight). Thus, roic filters. If loss can be tolerated, 50% beam splitters 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 and being detected in line. As a general matter, the emission copy using an infrared source (e.g., a infrared laser diode) 30 from the phosphor reporters is generally isotropic, and it and photographic film (e.g., Kodak Ektachrome) for signal may be preferred to collect light at an angle from the and image detection of visible range luminescence (with or direction of 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 35 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. incidentradiation 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 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 45 In a situation where the reporter is bound to a small bead, it reporter has an excitation band centered at and an may be possible to trap the bead in the region near the beam emission band centered at ' while the second reporter has focus. The same source, or a different source, can be used to respective excitation and emission bands centered at A and excite the reporter. The use of an infrared diode laser to trap A. Since the reporters of the present invention rely on small particles is described in Sato et al., "Optical trapping multiphoton excitation, wavelengths and are longer 50 of small particles using a 1.3 um compact InCaAsP laser.” than wavelengths and A'. 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.

A pair of light sources 2001) and 2002), which may be Specific Detection Techniques laser diodes or light-emitting diodes (LEDs), provide light at As outlined above, multichannel detection uses optical the desired excitation wavelengths, while respective detec 55 devices such as filters or dichroic beam splitters where the tors 22(1) and 22(2), which may be photodiodes, detect light emission bands of the phosphor reporters are sufficiently at the desired emission wavelengths. The emitted radiation separated. Similarly, it was pointed out that multiple report is related to the incident flux by a power law, so efficiency ers having a common emission band could be detected using can be maximized by having the incident beam sharply electronic techniques. These electronic techniques will be focused on the sample. To this end, light from the two described below in connection with multiple sources. sources is combined to a single path by a suitable combi However, the techniques will be first described in the context nation element 25, is focused to a small region by a lens or of a single channel. The techniques are useful in this context other focusing mechanism 27, and encounters the sample. since there are sources of background that are in the same Light emitted by the phosphor reporters is collected by a lens wavelength range as the signal sought to be measured. 30, and components in the two emission bands are separated 65 FIG. 2A shows an apparatus for implementing phase by a suitable separation element 32 and directed to the sensitive detection in the context of a single channel. Cor respective detectors. responding reference numerals are used for elements corre

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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., more possible laser diode 20(1) at a frequency f. and provides a signal at choices of total excitation energy. This allows more latitude f to the frequency mixer. The frequency mixer also receives in the choice of rare earth ions for up-converters since the the signal from detector 22(1) and a phase control input excitation steps need not rely on energy transfer coinci signal. This circuitry provides additional background dis dences involving a single photon energy. Further, it may be crimination because the background has a much shorter possible to achieve direct stepwise excitation of the emitting lifetime than the signal sought to be measured (nanoseconds ion (the erbium ion in the example outlined above) without or microseconds compared to milliseconds). This causes the O using energy transfer from another absorbing ion (the ytter signal and background to have different phases (although bium ion in the example) while taking advantage of resonant they are both modulated at the characteristic frequency of enhancement of intermediate levels. Additionally, the use of the waveform generator). For a discussion of the lifetime different wavelengths dependent phase shift, see Demtröder, Laser Spectroscopy, additional options for forexcitation-dependent a single reporter can provide multiplexing

Springer-Verlag, New York, 1988, pp. 557-559, incorpo 15 and background discrimination techniques. rated herein by reference). The phase input signal is con Multiple wavelength excitation of a single phosphor may trolled to maximize the signal and discriminate against the occur in a number of ways, as shown in FIGS. 5A through background. This background discrimination differs from 5C. Two that typical for phase sensitive detection where the signal is as shownlasers may cause stepwise excitation of a single ion, in FIG. 5A. A first laser stimulates excitation from modulated and the background is not. Discrimination 20 level 1 to level 2, and a second laser stimulates excitation against unmodulated background is also beneficial here, from level 2 to level 3, at which level emission occurs. leading to two types of discrimination.

Because the signal relies on two-photon excitation, it is Singleshown ion excitation can also occur using energy transfer as in FIG. 5B. In this case, a first laser stimulates possible to use two modulated laser diodes and to detect the excitation signal at the sum or difference of the modulation frequen 25 from levelfrom level 1 to level 2, energy transfer occurs 2 to 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 2001)" (emitting at the same wavelength, or possibly different wavelengths) are modu process, levels 1 and 2 can be in a first ion (i.e., a sensitizer lated by signals from waveform generators 37a and 37b as shownlevels ion) and 3 and 4 in a second ion (i.e., activator ion) in FIG. 5C.

operating at respective frequencies f andf. The waveform 30 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 f-f 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 polarization of the emitted radiation. In this case, depolar mixer 45. 35 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 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 W and A2, and having overlapping emis Specific instrument Embodiments sion bands near . The sample is irradiated by light from 45 FIG. 6 is a schematic view showing the optical train of a laser diodes 2001) and 2002) as discussed above in connec particular embodiment of apparatus for carrying out the tion with FIG. 1. First and second waveform generators present invention on a sample using a hand-held probe. This 37(1) and 37(2) drive the laser diodes at respective frequen embodiment takes the form of a miniaturized instrument cies f and f, and further provide signals at f and f to comprising a housing 75 (shown in phantom), a hand-held respective frequency mixers 60(1) and 60(2). The signal 50 probe 80, with a fiber optic connecting cable 82. The optical from detector 22(3) is communicated to both frequency and electronics components are located within the housing. mixers, which also receive respective phase input signals. For purposes of illustration, the optical components of a Thus, frequency mixer 60(1) provides an output signal 3-channel system are shown. The sample may contain up to corresponding to the amount of emitted light modulated at three reporters having distinct emission bands, for example, frequency f, which provides a measure of the presence of 55 in the blue, green, and red portions of the visible spectrum. the first reporter in the sample. Similarly, frequency mixer It is also assumed that the reporters have distinct excitation 60(2) provides an output signal corresponding to the amount bands in the near infrared.

of emitted light modulated at frequency f, which provides The output beams from three laser diodes 85a-c are a measure of the presence of the second reporter in the communicated through graded index (GRN) lenses 87a-c, sample. focused onto the ends of respective fiber segments 88a-c The use of two different wavelengths was discussed above and coupled into a single fiber 90 by a directional coupler 92 in the context of two reporters having different excitation or other suitable device. The light emerging from the end of bands. However, the discussion is germane to a single fiber 90 is collimated by a GRIN lens 95, passes through a reporter situation as well. Since the excitation is a two dichroic beam splitter 97, and is refocused by a GRIN lens photon process, there is no requirement that the two photons 65 100 onto the end of fiber optic cable 82. The beam splitter have the same energy. Rather, it is only necessary that the is assumed to pass the infrared radiation from the laser total energy of the two photons fall within the excitation diodes but reflect visible light.

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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 115that is slightly beyond alignment tip 110. The embodiment of FIG. 7A shows the peptide array in The alignment tipis 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, 10 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. Aparticular filter arrangement is shown 15 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 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 20 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. 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 25 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 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 30 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 vidually selectable through a series of motorized shutters peptide array and would therefore become physically 35 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 ing multiple phosphor reporters to be identified. Laser possible to have larger peptide elements that cover a group diodes A4 and AS 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 galvanometrically controlled enable the detector array to distinguish the emissions of the scanning mirrors A7 which scan the beaminaraster fashion. phosphor from the infrared laser stimulation. The beam passes through the objective lens (not shown) First, it is possible to use a phosphor that responds to IR 45 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 the detected image to the light collected from the focal detector array is insensitive to 1.5-micron radiation but is 50 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 Further, since the phosphor emission is relatively slow in size of the pinhole. The scanning speed is chosen such that rise and fall time it could be time resolved from a pulsed sufficient signal intensity is received at the PMTA10. In the laser stimulation source by the CCD detector array. The preferred embodiment of this apparatus, a 20 micrometer decay time for the upconversion process is a variable 55 diameter pinhole is used which results in a depth of field of dependent on the particular emitting transition and the about 1 micrometer. If the confocal mode is not selected, the phosphor host; however, it is normally in the range 500 us beam is deflected around pinhole optics A9 directly to PMT seconds to 10 ms. This is very slow compared to the laser A10, excitation pulse and the capability of the detector array. Once the optical signalis converted into an electronic one, The techniques for fabricating the CCD array are well a standard, composite video signal can be developed and known since CCD imaging arrays have been commercially displayed as an image on a television monitor A11. The available for many years. A variety of such devices can be image can be manipulated and enhanced through standard obtained from David Sarnoff Research Center, Princeton, image processing software. In the preferred embodiment of N.J. - this apparatus the software runs on an IBM 486 PCA12. The The techniques for fabricating the peptide array are 65 software can be used to perform averaging, filtering, edge described in a paper by Fodor et al., "Light-Directed, detection and overlaying the images received from each of Spatially Addressable Parallel Chemical Synthesis,” the different light sources.

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In the confocal mode, it is possible to reconstruct a 3 evaluation. After the scan is completed, the data can be dimensional view of sample A8. The reconstruction is saved or further data processing can be performed. FIG. 28 formed by stepping through sample A8 at small intervals, is an illustration of the data for upconverting phosphors in making an image of the sample at each interval. The multiple three test wells.

sequential images are transferred to an external graphics FIG. 29 is a schematic view of a second embodiment of machine (not shown) for reconstruction of the sample in 3 a hand-held probe for carrying out the present invention. dimensions. These 3-D images can then be rotated to give different perspectives of the data sets, leading to a better This embodiment is comprised of a housing D1 and a capillary wick D2. Within housing D1 is a diode excitation understanding of the samples.

FIG. 27 is a block diagram of a microtiter plate reader for 10 laser D3, asupply lens assembly D4, a photodiode detector D5, and use with the present invention. Within a light-tight test aofbattery housing D1

D6. A display D7 mounted to one surface communicates the results of the test to the chamber B1 is a near IR laser excitation source B2, a 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 15 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 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 20 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 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 bean 25 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 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 nonspecific reagent and antigen adherence to the walls of the raster scan the laser beam across the bottom of target well. 30 tube.

A third and preferred approach is to simply automate the 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 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 35 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 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 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 45 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 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 sources. Furthermore, overexposure of PMT B3 to light beam from diode laser D3 is directed onto capillary wick D2 causes high dark currents which require several hours to at capture surface D9 along a direction perpendicular to the decrease. PMT B3 is cooled for lower dark current and optical axis of reflector D10. Phosphorescent light from noise. Associated with the PMT cooler is a water-cooled capture surface D9 is collected and collimated by mirror power supply B15. A power supply B16 supplies high 55 D10, directed through a notch filter D11 to reject the pump voltage to PMTB3. light, and onto three detectors D5 using three dichroic beam When the apparatus is operated in a computerized mode, splitters D12. The reflectance bands of dichroic beamsplit a computer B17 regulates controller B8 through an interface ters D12 are matched to the emission bands of the three box B18. Picoammeter B13 can also be connected to com phosphors used in the detection process. puter B17, thereby allowing automated data acquisition to In an alternate embodiment of this apparatus, dichroic be performed. The data acquisition procedure moves trans beamsplitters D12 could be replaced with three bandpass lator B7 in the x direction to a first position at which location filters used in the transmission mode. By placing the three a specified number of current readings are taken and the filters on a rotation wheel, a single detector D5 could be average is calculated. Translator B7 then moves sample B4 used. Another alternative is to use a diffraction grating and a predetermined distance in the x direction to a new location 65 a linear detector array to obtain an actual emission spectrum. where new data is collected. During this process, the data is FIG. 31A is an illustration of an embodiment of the plotted in order to provide the user with an immediate visual invention in which-a diode laser array F1 and a detector

Page 54

array F2 are combined in a single device. In the preferred upconverting phosphor labels are attached to a binding embodiment, arrays F1 and F2 are fabricated on a pair of reagent, such as an antibody, that binds with high affinity and silicon chips F3 with array dimensions of approximately 1 specificity to a cell surface protein present on a subset of square centimeter. FIG. 31B is a detailed view of a small cells in a population of cells in suspension. The phosphor section of the device shown in FIG. 31A. Overlaying labeled binding component is contacted with the cell sus detector array F2 is a polymer film F4 of approximately 10 pension under binding conditions, so that cells having the to 25 micrometers thickness which is used as the capture cell surface protein bind to the labeled binding reagent, surface. Arrays F1 and F2 are separated by a spacer F5. whereas cells lacking the cell surface protein do not sub Array F1 is comprised of Fabrey-Perot diode lasers, pref stantially bind to the labeled binding reagent. The suspended erably tuned to 980 nanometers. Lasers of this type are O cells are passed across a sample detector under conditions easily fabricated in gridded array patterns using conven wherein only about one individual cellis present in a sample tional photolithography techniques. Each individual laser in detection zone at a time. A source, typically an IR laser, array F1 has a columnar beam designed to strike only the illuminates each cell and a detector, typically a photomul adjacent portion of capture surface F4. The required power tiplier or photodiode, detects emitted radiation. The detector density of the individual lasers is dependent upon the 15 controls gating of the cell in the detection zone into one of efficiencies of the phosphors being used as well as the a plurality of sample collection regions on the basis of the required detection efficiency. The detectors comprising array signal(s) detected. A general description of FACS apparatus F2 are chosen to have an extremely low sensitivity in the and methods in provided in U.S. Pat. Nos. 4,172,227; wavelength region in which laser array F1 operates. If 4,347,935; 4,661,913; 4,667,830; 5,093.234; 5,094.940; and additional discrimination between the excitation and emis 20 5.144.224, incorporated herein by reference. It is preferred sion wavelengths is required, a cutoff filter can be used, that up-converting phosphors used as labels for FACS meth preferably incorporated directly into capture surface F3. ods have excitation range(s) (and preferably also emission Upconverting phosphors F6 are conjugated by any of a range(s)) which do not damage cells or genetic material; variety of conventional biochemical crosslinking chemis generally, radiation in the far red, and infrared ranges are tries to antibody, nucleic acid probes, or other biological 25 preferred for excitation. It is believed that radiation in the macromolecules (e.g., carbohydrates, lectins, streptavidin, range of 200 nm to 400 nm should be avoided, where MHC complexes), as well as to biological or chemical possible, and the wavelength range 760 nm to 765 nm may antigens (F7). Bonded to overlay F3 is a grid array F8 of be avoided in applications where maintenance of viable cells complementary probes or antigens which are bound to is desired.

capture surface F3 using the same crosslinking chemistries. 30 Additional Variations

In use, a sample fluid F9 flows between arrays F1 and F2, There are several apparatus design issues relating to the target probes or antigens are captured by grid array F8 and unique excitation and emission characteristics of upconvert excited by laser array F1, and the emissions detected by ing phosphors which must be considered when using detector array F2. up-converting phosphors with flow cytometry. The first 35 issue is the time required to reach maximum emission

Typically, the upconverting phosphors to be used with this apparatus are approximately 0.1 to 0.5 micrometers. Since intensity. Since upconversion is a two photon process, upconverting phosphor emission is time delayed approxi the size of the individual phosphor particles is of the order of the excitation wavelength, the power of the emission from mately 100 microseconds. The phosphor must remain within the phosphors can be approximated by: the excitation beam for this period of time regardless of the flow rate. Therefore given a flow rate between 1 and 10

P=Fnd. meters per second with a channel width of 70 to 200 micrometers, the length of the excitation beam must be where f is the phosphorescence efficiency (generally less between 100 and 1000 micrometers. Given that the phos than or equal to 107 cm'Wumparticle'), N is the phor emissions saturate at an excitation intensity of about number of phosphor particles in the light path, D is the 45 200 watts per square centimeter, the laser source typically diameter of the phosphor particles, and I is the power must have a power between 0.01 and 400 milliwatts to density of the excitation source. achieve phosphor saturation. This implies that multiple laser Since the emitted power scales as the square of the diodes may be required to obtain maximum phosphores excitation intensity, diagnostics using upconverting phos cence at the fastest flow rates.

phors perform better in a microassay format. Assuming a Another design issue is that associated with the detector. constant power output from the excitation source, the exci Since there is a considerable separation between the exci tation power density increases proportionally with the tation and emission wavelengths of the upconverting decrease in detection area, and the number of phosphor phosphors, detection can be performed using a photomulti particles in the light path decreases linearly with a decrease pler tube (PMT), a photodiode, or a CCD array. The phos in the detection area. Since the power of the light emitted 55 phorescence decay time is long, with a decay half life of from the phosphors scales with the square of the excitation approximately 300 microseconds. The most sensitive power density, but linearly with the number of phosphors, method of detection is to integrate the signal measured by P will increase in inverse proportion to the detection area. the PMT. However, 99 percent detection of the available Therefore, a 100x100 array will actually be 100 times more phosphorescent signal requires that the phosphor remain in sensitive than a 10x10 array. the sight path of the detector for 5 times the phosphores Fluorescence-activated Cell Sorting cence decay half-life (i.e., 1.5 milliseconds). Assuming a The up-converting phosphors described herein can be flow rate of 10 meters per second and a channel width of 200 used as phosphorescent labels in fluorescent cell sorting by micrometers, the PMT must be able to detect over a path flow cytometry. Unlike conventional fluorescent dyes, length of 1.5 centimeters. This path length is also the up-converting phosphors possess the distinct advantage of 65 required spacing between cells flowing through the not requiring excitation illumination in wavelength ranges cytometer, implying a maximum count rate of 667 cells per (e.g., UV) that damage genetic material and cells. Typically, second. It is, however, possible to sacrifice some detection

Page 55

sensitivity by reducing the detection path length, at least to is a well-characterized function of time, makes possible the that required to attain steady-state emission from the phos dynamic tracking of individual particle's positions, direc phors. As long as a steady-state emission peak is reached by tions and velocities, and optionally calculation of particle the phosphor in the excitation window, the peak signal size, density, and hydrodynamic conformation. As a particle received by the PMT should be directly proportional to the moves, it exposes more elements of the array, but with concentration of phosphors present. The nonphotobleaching every-decreasing intensity. The more elements it exposes property of the phosphors makes this form of detection over a certain fraction of its decay time, the faster it is moving. Therefore, the integrated intensity pattern of a possible. The loss in detection sensitivity corresponding to a particle's 0.1 centimeter path length (versus a 1.5 centimeter path related to emission “track" collected by the array is directly the velocity of the particle. The particles may be length) is approximately a factor of 3. Triggering the emis 10 refreshed again sion detector can be accomplished by observing the light excitation source.at FIG. any time by the pulsed or chopped CW 10 illustrates this scheme. Although scattered by the cell as it passes through the excitation only a depiction of “side-on" excitation and detection is

SOCC

shown, both side-on and end-on detection and excitation

In environments where absorption of the up-converted arrangements, or combinations, are possible. Reduction of phosphor radiation is high, the phosphor microparticles are 15 the CCD array intensity information by computer analysis coated with a fluorescent dye or combination of dyes, in will allow near-real time tracking of the particles in a selected proportions, which absorb at the up-converted fre dynamically evolving or living systems. Data analysis and quency and subsequently re-radiate at other wavelengths. reduction performed by the computer would include a Because the single-photon absorption cross-sections for convolution of the intrinsic decay of the phosphor emission, these fluors are typically very high, only a thin layer is 20 the number of pixels illuminated and their signal level, the required for complete absorption of the phosphor emission. orientation of the decaying signal on the array, and the This coat particle may then be encapsulated and coated in a intensity contributions from a blur circle from particles suitable antigen or antibody receptor (e.g. microparticle). An moving in and out of the focal plane of the array. In an example of this layering is depicted schematically in FIG. 9. end-on flow detection arrangement, the size of the blur circle There exists a wide variety of fluorescent dyes with strong 25 would relate directly to how quickly the particle moves out absorption transitions in the visible, and their emission of focus, thereby allowing the velocity of the particle to be covers the visible range and extends into the infrared. Most determined. One possible application would be monitoring have fluorescent efficiencies of 10% or more. In this manner, the chemistry and kinetics in a reaction column, the emission wavelengths may be custom-tailored to pass alternatively, the application of this method to flow cytom through the particle's environment, and optical interference 30 etry may permit the resolution of cells on the basis of filters may again used to distinguish between excitation and hydrodynamic properties (size, shape, density). The method emission wavelengths. If a relatively large wavelength “win may also be useful for in vivo diagnostic applications (e.g., dow” in the test medium exists, then the variety of emission blood perfusion rate), wavelengths which may be coated on a single type of Up-converting phosphor labels may also be used to sense phosphoris limited only by the number of available dyes and 35 the temperature in the region at which the up-converting dye combinations. Discrimination between various reporters phosphor label is bound. Up-converting phosphor tempera is then readily carried out using the spectroscopic and ture measurement methods are described in Berthou H and multiplexing techniques described herein. Thus, the number Jorgensen CK (October, 1990) Optics Lett. 15(19): 1100, of probe/reporter "fingerprints" which may be devised and incorporated herein by reference.

used in a heterogenous mixture of multiple targets is virtu Although the present invention has been described in ally unlimited. some detail by way of illustration for purposes of clarity of The principles described above may also be adapted to understanding, it will be apparent that certain changes and driving species-specific photocatalytic and photochemical modifications may be practiced within the scope of the reactions. In addition to spectroscopic selection, the long claims.

emission decay times of the phosphors permit relatively 45 The broad scope of this invention is best understood with slow reactions or series of reactions to take place within the reference to the following examples, which are not intended emission following photoexposure. This is especially useful to limit the invention in any manner. W when the phosphor-catalyst or reactant conjugate enters an EXPERMENTALEXAMPLES environment through which the excitation wavelength can not penetrate. This slow release also increases the probabil 50 Reporters of Up-Converting Inorganic Phosphors as Validation ity that more targets will interact with the particle.

The unique decay rates of phosphor particles allow Up-converting phosphor particles comprising sodium dynamic studies as well. In a system where continuous yttrium fluoride doped with ytterbium-erbium were milled to exposure to the excitation source is not possible, or is submicron size, fractionated by particle size, and coated invasive and thereby undesirable, pulsed excitation followed 55 with polycarboxylic acid. Na(YooYbosBroo).P. was cho by delayed fluorescence detection is necessary. After the sen for its high efficiency upon excitation in the range 940 phosphor reporter has been photoexcited, the subsequent to 960 nm. ANd:Yagpumped dye laser/IR dye combination emission from the phosphor or phosphor/dye conjugate was used to generate 8-ns to 10–ns duration pulses in the particle lasts typically about a millisecond. In a dynamic above frequency range.

environment, such as a static or flowing system with moving The laser pulses were used to illuminate a suspension of targets, the particle will emit a characteristically decaying milled phosphor particles in liquid and attached to glass intensity of light as it travels relative to the excitation/ slides in situ. The suspension luminescence observed at right detection apparatus. Combined with imaging optics appro angles was monitored using a collection lens, a spatial filter priate to the scale of the system and the velocities within the in order to filter out scattered excitation light to the maxi system, a CCD photoelectric sensor array will be used to 65 mum possible extent, and a photomultiplier, vacuum detect the particle or particles movement across the array's photodiode, or simple solid state photodiode (depending on field of view. The delayed emission of the phosphors, which the light level observed).

Page 56

The luminescent signal level was determined as a function of solution pH (range: 6-8), grain size, particle loading TABLE I (g/cm), and the nature of stabilizing anionic surfactant. Equivalent Signals were recorded both as a time integral from a boxcar Phosphor Phosphor Detection integrator and from a long RC time constant or as a transient Loading Loading Sensitivity signal using a transient digitizer in order to delineate the Label (ng?well) (particles well) (M) luminescence lifetime under particular experimental condi 100 1700 90 23,600,000 it 1,200,000 4 x 10? tions. In situ signals were also measured by laser scanning 10-1 1709 2,360,000 it 120,000 4 x 10-13 microscopy. FIG. 11 is a fluorescence scan of the phosphor 10 10-2 170.9 236,000 + 12,000 4 x 10

emission spectrum incident to excitation with a laser source 10. 17 - 0.09 23,600 + 1,200 4,10-15

at a wavelength maximum of 977.2 nmi; emission maximum 10 OO17 OOOO9 236 t 12 4 x 107 is about 541.0 nm. FIG. 12 is an excitation scan of the 10 00017 + 0.00009 23.6 t 1.2 4 x 101 phosphor excitation spectrum, with emission collection win dow set at 541.0 nmi; excitation maximum for the phosphor 5 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 m and particle density of 5.3 g/mL). The residue at approximately 400 us with a gradual decay to a lower, 20 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 increases with excitation intensity up to almost about 1000 to Visual

green light emanated from all serial dilutions down

W/cm. 25 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(YosYboEroos)F particles were measured. A Ti:sap cloudy. Either 1 pil of each serial dilution, or 0.1 pil 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 30 plate. It was found that 1 pil fills the bottom of the well and system. Two types of measurement were performed. The 0.1 pil 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 35 dilution.

shown graphically in FIG. 16. This corresponded to a 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 25um was also should be noted that 10' to 10 M is the normal range measured. On the basis of known values for the absorption of 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) The control samples were scanned using a prototype and the measured dependence of the phosphorescence emis up-conversion fluorimeter device (David Sarnoff Research sion on particle size, a phosphorescence cross-section of 45 Center). The samples were scanned by moving the plate in approximately 1x10 cm was found. The difference 50 pum increments, using a motorized X-Y positioning stage, between these two measurements may be due to a difference relative to the focal point of an infrared diode laser. in phosphorescence efficiency between dry phosphor and The IR diode laser was operated at 63 mW (100 mA). The aqueous suspensions, or due to absorption of multiply scat tered photons in the dry phosphor. On the basis of either of 50 beam bottom was focused to 24x10 cm at the focal point. As the of the sample well is about 1.4x10 cm (1365 m these cross-section estimates, the cross-section is sufficiently diameter), the beam covers less than 1.7% of the well bottom large to allow detection of single submicron phosphor par surface at any individual position. The well also has sloping ticles at moderate laser intensities. At laser intensities of roughly 10 Wlcm, the phosphorescence scales as the laser well side walls which widen from bottom to top of the sample intensity to the 1.5 power. and are also interrogated by a progressively divergent 55 laser beam. Neglecting losses in the optics, the IR light

Phosphor Particle Performance: Sensitivity of Detection intensity at the focal point (bottom of the sample well) was A series of Terasaki plates containing serial dilutions of approximately 26–27 W/cm at 980 nm wavelength. A monodisperse 0.3 um up-converting phosphor particles con photomultipler tube (PMT) was used for detection of the sisting of (YosYboosBroos)2O2S were tested for visible (upconverted) light emitted from the sample. Since the laser beam width was smaller than the surface area at the up-conversion fluorescence under IR diode laser illumina bottom of the sample well, the plate was aligned by visual tion in a prototype instrument. inspection against the focal point of the diode laser so that The phosphor particles were prepared by settling in the laser was centered in the middle well (C6 when reading DMSO and were serially diluted into a 0.1% aqueous gun wells C5, C6 and C7, and D6 when reading wells D5, D6, arabic solution. This appeared to completely eliminate any 65 and n7).

water dispersion problems. The serial dilutions used are The PMT signal (amps) was recorded at each plate listed in Table I. position and numerically integrated over the width of the

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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 m as Small, presumably monodisperse particles, and 35% being distance, which yields an integrated background signal of 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 Ab (by FITC fluorescence). This suggests that less than 40%

Immunodiagnostic Sample Detection 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 15 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), position and numerically integrated over the width of the and six wells coated with BSA alone (negative controls). sample well (approximately 4000 pm). The average signals Nominal 0.3 um (Yoss YboosBroos)2O2S phosphor particles (with 95% confidence limits) are:

coated with goat anti-mouse IgG antibody (anti-IgG) were 20 Average of Positive Samples=130x10 +1.25x10'a-m then used as the reporter-antibody conjugate. Average of Negative Controls=4.20x10-6.82x10 Six wells (C5, C6, C7, D5, D6, and D7) of a clear a polystyrene Terasaki plate were coated with mouse IgG by The positive samples and negative controls are statisti incubating at 37° C. against 5 L of a 100 g/L mouse IgG cally different at the 99.9% confidence level. The positive solution in phosphate buffered saline (PBS). After 1 h, this samples emit on average 30.0+29.7 times more light than the solution was aspirated off and each sample well was washed 25 negative controls.

with 10 L of 3% BSA in PBS. This was immediately Linkage of Phosphors to Biological Macromolecules aspirated off and replaced with 20 uL of 3% BSA in PBS. In order to delineate further the parameters for Each sample well was post-coated with BSA by incubating up-converting phosphors as biochemical reporters, biologi against the 20L of BSA/PBS solution for 1 h at 37° C. The 30 cal linkers were attached to phosphor particles. Sodium post-coat solution was aspirated off and the plates stored at yttrium fluoride-ytterbium/erbium phosphor particles were 4° C. overnight. These wells were considered in positive coated with streptavidin. The excitation and emission spec samples. The same six wells in a second Terasaki plate were prepared in an identical fashion, except they were not coated tral properties of the phosphor alone and the phosphor coated with streptavidin were measured (FIGS. 17A, 17B, with mouse IgG. This second set of sample wells were 35 18A, and 18B) and both the uncoated and streptavidin considered negative controls. coated phosphors were almost identical in their absorption Phosphor-Antibody Conjugate and emission properties, indicating that the attachment of A solution of (Yoss YboosBroos)OS phosphor particles macromolecular linkers (e.g., proteins) have little if any was prepared by suspending the dry phosphors into DMSO. effect on the phosphorescent properties of the up-converting The initial particle density was approximately 10' particles/ phosphor, The streptavidin-coated phosphors were then spe mL as determined by counting the number of particles cifically bound to biotinylated magnetic beads, demonstrat contained in the field of an optical microscope. It should be ing the applicability of linker-conjugated inorganic phos noted that the 0.3 pm fundamental particle size was below phors as reporters in biochemical assays, such as the resolution limits of the microscope. This solution was immunoassays, immunohistochemistry, nucleic acid allowed to settle undisturbed for 3 days. The supernatant, hybridizations, and other assays. Magnetic bead technology which was turbid and presumably contained mostly mono allows for the easy separation of biotin-bound streptavidin disperse smaller particles was used for subsequent conjuga coated phosphor from a solution, and is particularly well tion. suited for sandwich assays wherein the magnetic bead is the Goat anti-mouse IgG antibody (Ab) was conjugated (by solid substrate.

adsorption) onto the DMSO fractionated phosphor particles. 50 Advantageously, streptavidin-biotin chemistry is widely This was done by mixing 200L of the Absolution (in 0.1M used in a variety of biological assays, for which Tris-HCl, pH 7.2) with 100 L of the phosphor suspension up-converting phosphor reporters are suited. FIG. 20 shows in DMSO. Several different Ab concentrations were tried in schematically, for example and not limitation, one embodi the range of 0.025 to 1 g/L. A concentration of 0.25 g/ul ment of an immunoassay for detecting an analyte in a appeared to result in the most efficient coating (i.e., maxi 55 solution by binding the analyte (e.g., an antigen target) to a mum Ab utilization with a minimum of clumping of the biotinylated antibody, wherein the analyte forms a sandwich phosphor particles). The phosphors were equilibrated over complex immobilized on a solid substrate (e.g., a magnetic night at room temperature with the Ab in this DMSO/Tris bead) by linking a first binding component bound directly to solution with gentle agitation. The resulting phosphor-Ab the solid substrate to a second binding component (e.g., the conjugates were centrifuged from this solution and resus biotinylated antibody); a streptavidin-coated up-converting pended in a 3 ug/mL BSA solution in PBS for post-coating. phosphor then binds specifically to the biotinylated antibody The resulting BSA/PSA resuspension was used directly for in the sandwich and serves to report formation of the the assay. sandwich complex on the solid substrate (which is a measure The degree of Ab adsorption to the phosphors, and of the analyte concentration). When the solid substrate is a residual Ab activity, was determined by titrating the 65 magnetic bead, it is readily removed from the sample phosphor-bound Ab with a fluorescein isothiocyanate solution by magnetic separation and the amount of phosphor (FITC) conjugated-mouse IgG. The resulting FITC-labeled attached to the bead(s) in sandwich complex(es) are deter

Page 58

mined by measuring specific up-converting phosphores Covalent Conjugation of Upconverting Phosphor Label to cence. Thus, sandwich complex phosphorescence provides a Avidin quantitative measure of analyte concentration. An upconverting ytrium-ytterbium-erbium Biotinylated polynucleotides are also conveniently used (Yossybooseroos) oxysulfide (OS) phosphor was linked to as hybridization probes, which can be bound by avidin by the following procedure:

streptavidin-coated up-converting phosphors to report Monodisperse upconverting phosphor particles were hybrid formation.

Background Phosphorescence in Biological Samples silanized with thiopropyltriethoxysilane (Huls) following the procedure

Background signals were determined in two biological Register and Review, detailed by Arkles (in: Silicone Compounds: samples for determination of potential background in immu 10 Hills America, pgs. 59-75, 1991). This noassays. Sputum and urine were used as samples in the consisted of adding thiopropyltriethoxysilane (2 g) and 95% same apparatus as used for the phosphorescence sensitivity aq. ethanol (100 mL) to a 500 mL Erlenmeyer flask and measurements (supra). No background levels were found stirred for 2 minutes. Approximately 8 mL of the 65 mg/mL above the system noise levels set by the photomultiplier dark phosphor suspension in DMSO was then added to the current. This noise level allows detection of signals from on 15 mixture. This suspension was stirred for an additional 2 the order of a few hundred particles/cm. This is close to a minutes, then transferred to centrifuge tubes and centrifuged single particle in the detection volume of the system. to separate the phosphor particles. The pellets were washed A photomultiplier is a preferred choice for a detector for twice with 95% aq. ethanol centrifuging each time. The high sensitivity measurements of up-converting phosphors resulting particles were collected and dried overnight under since photomultipliers can be selected to produce high vacuum at approximately 30°C. A quantity (127mg) of dry quantum efficiency at the up-converted (i.e., emitted) wave 20 silanized phosphors were resuspended in 1.5 mL of DMSO lengths and virtually no response in the range of the longer (phosphor stock).

excitation wavelengths. A solution containing 1.19 mg of avidin (Pierce) in 1.0 Detection of Cell Antigens with Phosphor-Labeled Anti mL of borate buffer (954 mg sodium borate decahydrate and bodies 17.7 mL of 0.1N NC1 in 50 mL of deionized water, pH 8.3) Streptavidin is attached to the up-converting phosphor 25 was prepared (Avidin stock). Another solution containing particles as described, supra. The mouse lymphoma cell line, 1.7 mg of N-succinimidyl(4-iodoacetyl) aminobenzoate EL-4, is probed with a hamster anti-CD3 antibody which (Pierce Chemical) in 1.2 mL of DMSO was prepared (SIAB specifically binds to the 30 kD cell surface EL-4 CD3 T stock). A quantity (10 uI) of the SLAB stock was added to lymphocyte differentiation antigen. The primary hamster the 1.0 mL of Avidin stock and stirred at room temperature antibody is then specifically bound by a biotinylated goat 30 antihamster secondary antibody. The biotinylated secondary 30 minto allow the N-hydroxysuccimide ester of the SIAB antibody is then detected with the streptavidin-phosphor to react with primary amines on the avidin (Avidin-SIAB stock).

conjugate. This type of multiple antibody attachment and labeling is termed antibody layering. A20 mL scintillation vial was prepared containing 10 mL Addition of multiple layers (e.g., binding the primary 35 of borate buffer (pH 8.3). The following additions were then hamster Ab with a goat-antihamster Ab, followed by binding made to this vial: 21.6L of the avidin-SIAB stock solution with a biotinylated rabbit-antigoat Ab) are used to increase followed by 1.5 mL of the phosphor stock. This reaction the distance separating the phosphor from the target. The mixture was stirred at room temperature in the dark over layering effect on signal intensity and target detection speci night to allow the SLAB activated avidin to react with the ficity is calibrated and optimized for the individual applica thiol groups present on the silanized phosphor surface and tion by performing layer antibody layering from one layer resulting in the covalent linkage of avidin to the phosphor (primary antibody is biotinylated) to at least five layers and particles.

ascertaining the optimal number of layers for detecting CD3 After the overnight incubation 1.0 mL of the reaction on EL-4 cells. mixture was centrifuged (1 min at 10,000 g) and the super FIG. 21 schematically portrays simultaneous detection of 45 natant removed. The pellet was resuspended in 1.0 mL of two EL-4 cell surface antigens using phosphors which can phosphate buffered saline (pH 7.2, Pierce) and centrifuged be distinguished on the basis of excitation and/or emission again to wash any unconjugated protein from the phosphors. spectra. Detection of both antigens in the scheme shown in This washing process was repeated. The washed pellet was FIG. 21 uses a biotinylated terminal antibody which is resuspended in 1.0 mL of phosphate buffered saline and used conjugated to streptavidin-coated phosphor (#1 or #2) prior 50 directly in diagnostic assays as described below. to incubation with the Ab-layered sample. Thus, the Measurement Apparatus phosphor-antibody specificity is retained through the unusu A modified SLMAminco 48000 Fluorimeter was used to ally strong (K approx. 1x10" M') non-covalent bond measure the fluorescence spectrum from the phosphor between streptavidin and biotin which is pre-formed before samples. The modifications to this device consisted of incubation with the primary antibody-bound sample. Quan 55 adding a laser diode (David Sarnoff CD-299R-FA #13) titation of each antigen is accomplished by detecting the which was input to the fluorimeter through port 3. The laser distinct signal(s) attributable to each individual phosphor diode emits at A=985.1 nm. Spectral data provided by the species. Phosphorescent signals can be distinguished on the David Sarnoff Research Center also shows a small peak at basis of excitation spectrum, emission spectrum, fluores 980.2 mm. This peak has 15% the intensity of the peak at 985 cence decay time, or a combination of these or other properties. A 5.08 cm focal length lens was used to collimate the FIG. 22 shows a schematic of an apparatus for phase diode laser beam. The power of the IR laser light was sensitive detection, which affords additional background measured as 6.1 mW at the cuvette location with a drive discrimination. The pulse or frequency mixer is set to pass current of 75 mA. The beam was not focused at the center the signal and discriminate against the background follow 65 of the cuvette. This is true for the standard visible light from ing frequency calibration for maximum background the fluorimeter excitation monochromator as well. The laser rejection. diode beam is diverging as it enters the cuvette holder and

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is approximately 4 mm (H)X2 mm (V) by the time it reaches the center of the cell, neglecting the changes in refractive TABLE TV index of the cell wall and the liquid. Cell Cell Light emitted is scanned with a monochromator and 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 4x10'M (240,000 phosphor particles per 2 biotinylated Phosphor - --- mL) in PBS. Phosphor emission peaks in the spectrum were 10 3

biotinylated biotinylated

Avidin

seen at wavelengths of 4062 nm, 434+2 nm, 522+2 nm, and 5 FTTC labelled Avidin-Phosphor - -- 5482 nm. The largest peak was at 548 nm. The intensity of 6 FEC labeled Phosphor - -- the 548 nm peak was used to discriminate samples.

Linkage of Avidin-Phosphor Conjugate to Cell Surface The remainder of the samples were used to resuspend Marker paramagnetic, polystyrene beads bound with sheep anti

A lymphoblastoid cell line (Human Genetic Mutant Cell mouse IgG. For each of the six samples, 3x10' 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 aspiration while the tubes were in a magnetic rack. The an equal volume of phosphate buffered saline (PBS) pH 7.4. 20 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/mi. These cells were then with intermittent resuspension. The magnetic beads were incubated with amouse IgG1 monoclonal antibody to human then collected on a magnetic rack, washed four times in 3-microglobulin, a Class I histocompatibility antigen in blocking buffer, resuspended in 100 L blocking buffer, polystyrene centrifuge tubes. The cells were immunopre 25 transferred to a fresh tube, and up-converting phosphores cipitated for 30 minutes at 4°C. with an antibody concen cence was measured on the fluorimeter. tration of 10 g/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 L) into six fresh centrifuge tubes. scanned from 500 to 700 nm with a step size of 2 nm. Four of these samples received biotinylated goat antimouse 30 Samples were also measured for FTTC signal by exciting the IgG, while the remaining two received FITC-labelled goat samples with 37 IM at A-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 L with a wavelength (514 nm) are very close for FITC, higher final second antibody concentration of 20 pg/ml. The cells resolution was required to get separable signals than with were harvested and washed in PBS as above but were 35 phosphor labelling. The intensity of the 490 nm signal was resuspended in 50 L of blocking buffer (0.2% purified 240W/cm at the center of the well. FITC emission spectra casein in PBS, Tropix, Bedford, Mass.). The cell-antibody were scanned at 0.5 nm increments from 450 nm to 750 nm. complexes were blocked in this solution for 30 minutes at with a 2 nm bandwidth on the emission monochromator. room temperature and then transferred to fresh tubes. Sample 1 is the positive control and clearly yielded the A pre-blocked suspension (40 L) of either avidin highest emission signal. Sample 2 indicates that any non Phosphor conjugate, avidin-FITC, avidin, or unconjugated specific adsorption of the phosphors to the sample is limited Phosphor was added to four of the cell samples conjugated and is readily discriminated from signal attributable to with the biotinylated anti-mouse IgG (H&L). In addition, an avidin-conjugated phosphor and showing that avidin linked equal amount of pre-blocked avidin-Phosphor or unconju phosphors can specifically bind only when they are conju gated Phosphor was added to the remaining two cell samples 45 gated with the probe, in this example through the biotin immunoprecipitated with the non-biotinylated FITC avidin linkage. Sample 3 is the negative control which labelled anti-mouse IgG (H&L). The avidin reporter conju contains no phosphors, only avidin. Sample 4 shows FTTC gates or negative controls were preblocked as follows. conjugated avidin. Although FTTC signals were observed on Avidin-Phosphor and Phosphor alone was diluted in block the cell surface by laser microscopy, the signals were below ing buffer by adding 10 pull of a 6.7 mg/ml suspension to a 50 the level of detection on the fluorimeter for measurement of final volume of 100 L. Avidin-FITC and the avidin alone was FTTC, and since there was no phosphor in the sample there controls were also diluted in blocking buffer by adding 27 L no significant phosphor signal. Samples 5 and 6 show of 2.5 mg/ml solution to a final volume of 100 pull. These that FITC-conjugated primary antibodies can be detected reagents were blocked at room temperature for 3 hours with and that the presence of phosphor or avidin-phosphor does not significantly disrupt binding of the primary antibody to intermittent resuspension and then added to 50 uL of cells 55 its labelled with biotinylated or non-biotinylated second anti target antigen.

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

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Each of the two samples were then aliquoted (50 uL) into Moreover, no downconversion was seen at the excitation three fresh Eppendorf tubes. wavelengths cited by Tanke et al. (U.S. Pat. No. 5,043,265). The avidin-conjugates were blocked for 1 hour at room Thus, the upconverting phosphors tested are unlike those temperature by diluting 500 g of an avidin-phosphor reported in Tanke et al.

suspension, unconjugated phosphor suspension, or avidin HOMOGENEOUS ASSAYS solution in 300 L of blocking buffer. For each of the samples (summarized below in Table V) 50 L of the The multiphoton activation process characteristic of anti-digoxigenin conjugates was added to 150 L of pre upconverting phosphors can be exploited to produce assays blocked avidin-conjugates or avidin and were incubated for that require no sample washing steps. Such diagnostic 30 minutes at room temperature. O assays that do not require the removal of unbound phosphor Unbound avidin-conjugates were removed by resuspend labels from the sample are herein termed homogeneous ing 3x10' paramagnetic beads linked with sheep anti-mouse assays, and can also be termed pseudohomogeneous assays. IgG (pre-blocked in blocking buffer). After incubation for 30 Homogeneous Assay Example 1 minutes at room temperature with intermittent resuspension, 15 One embodiment of a homogeneous assay consists of the the beads were separated on a magnetic rack and washed 4 use of an upconverting phosphor label linked to an appro to 6 times in PBS. The antibody-DNA bound beads were priate probe (e.g., an antibody or DNA). The phosphor then measured on the fluorimeter. labeled probe specifically binds to a target (e.g., antigen or The samples were scanned from 500 to 700 nm with a nucleic acid) that is linked to a capturing surface. A suitable bandwidth of 8 nm and step size of 2 nm. Each PMT value 20 capture surface can be the tip of a light carrying optical fiber reported (Table V) represents an average over 5 scans. (FIG. 23) or the bottom surface of a sample container (FIG. Sample 1 is expected to provide the highest PMT signal 24A and 24B). upon incubation of the target-labelled capture since biotinylated DNA is present and can bind to the surface with the phosphor-labelled probe, phosphor particles avidin-linked phosphors. Sample 2 indicates the level of will accumulate at the capture surface as a function of the nonspecific adsorption of the phosphors to the sample which 25 amount of target present on the capturing surface. The target is found to be insignificant since the PMT signal is observed may be linked directly to the capturing surface or may be to be the same as that of the negative control (sample 4) immobilized by interaction with a binding agent (e.g., spe which contains no phosphors. Sample 3 is another control cific antibody reactive with target, polynucleotide that binds and shows that the avidin-linked phosphors do not bind to target) that is itself linked to the capturing surface (such as the paramagnetic beads in the absence of DNA. Samples 5 30 in a sandwich immunoassay, for example). and 6 show results of FTTC-labeled avidin used to validate Detection of the phosphor bound to the capture surface is assay. effected using an excitation light that is focused from a low intensity beam of large cross-section to a high intensity

TABLE V beam of small cross-section with the focal point of the beam 35 being at or very near the capture surface. Focusing of the

Upconverting Phosphor Nucleic Acid Diagnostic Assay Results excitation light is accomplished by transmission through PMTSignal PMTSignal optical elements that have a very small focal length, such

Sample DNA Reporter (VG 546 m) (V (a 514 nm) that the beam diverges and becomes less intense, within a

short distance of the capture surface.

with linked Since the intensity of the light emitted from the upcon digoxigenin Phosphor verting phosphor labels is proportional to the excitation light

and biotin

intensity raised to a power of two or greater, phosphors near with Phosphor the focal point of the excitation source will emit significantly digoxigenin more light than those remaining in suspension in the sample and biotin 45 away from the capture surface. Therefore, binding of upcon

linked 16302 3.5779 verting phosphor linked probes to the capture surface will

Phosphor yield an increase in emitted light intensity measured from 4 DNA labelled Avidin 0.8505 2.8067 the sample as a whole or as measured from a control sample with in which phosphors do not bind to the capture surface. and biotin 50 Emitted light intensity may be plotted as a function of target 5 DNA labelled FTC- 1.0484 8.4394 concentration using for standardization (calibration) a series with Avidin of samples containing predetermined concentrations of tar digoxigenin get. The emitted light intensity from a test sample (unknown and biotin concentration of target) can be compared to the standard

Avidin 55 curve thus generated to determine the concentration of target.

Examples of suitable homogeneous assay formats

Phosphor Downconversion Evaluation include, but are not limited to, immunodiagnostic sandwich A sample of the (Yoss Ybooseroos)2O2S phosphors were assays and antigen and/or antibody surface competition scanned for the presence of a downconverted signal. This assays.

was accomplished by exciting a sample of the monodisperse Homogeneous Assay Example 2 phosphors described above (4x10M in DMSO) with 1.3 Another embodiment allows for the accumulation of mW of monochromatic light at 350 nm with a 16 mm upconverting phosphor linked probes at the detection sur bandwidth for the excitation source. Detection was accom face by the application of centrifugal or gravitational set plished by scanning this sample from 350 to 800 nm with a 65 tling. In this embodiment an upconverting phosphor is monochomator bandwidth of 8 nm. Scanning was performed linked to multiple probes. All the probes must bind to the in 2 nm increments. No downconversion was observed. same target, although said binding can be accomplished at

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different locations (e.g., as antibody probes may target 3. A method according to claim 2, wherein the different epitopes on a single antigen). The multiprobe up-converting inorganic phosphor comprises sodium phosphor can then be used to effect the aggregation of yttrium fluoride ytterbium erbium or yttrium ytterbium targets in solution or Suspension in the sample. This aggre erbium oxysulfide.

gation will result in the formation of a large insoluble 4. A method of claim 1, further comprising, before the phosphor-probe-target complex that precipitates from solu contacting step, the step of attaching the up-converting tion or suspension (FIG. 25). The aggregated complex inorganic phosphor particle to the binding component to containing phosphors accumulates at a detection surface form the labeled binding component.

while nonaggregated material remains in solution or sus 5. A method according to claim 4, wherein the binding pension. Detection is accomplished as described in the O component is attached to the label by covalent or noncova above example using a sharply converging excitation beam. lent binding.

Evaluation of Up-converting Chelates 6. A method according to claim 5, wherein the binding Up-conversion has been performed in rare earth chelates component is streptavidin or avidin and the target analyte is and rare earth salt solutions. Chelates of erbium and neody 15 7. A methodtarget a biotinylated analyte.

mium have been prepared with ethylenediaminetetraacetic component-target complex 1,is wherein of claim the labeled binding separated from the unbound acid (EDTA) and dipicolinic acid (DPA). The erbium che labeled probe by immobilization on a solid support. lates were pumped using light near 793.5 nm from a

Ti:sapphire laser (the excitation scheme of Macfarlane separating 8. A method according to claim 7, wherein the step of (1989) Appl. Phys. Lett54: 2301). This approach produced labeled binding unbound labeled binding component from the upconversion but not satisfactorily, which we attribute to performed by washing component-target complex in the sample is weak absorption for the first step due to the increase in to remove suspendible the sample with an aqueous solution or soluble unbound labeled binding linewidth in the chelate over the low temperature crystal component.

used for the up-conversion laser.

9. A method of claim 7, wherein the labeled binding

The neodymium chelates were excited with light near 580 25 component nm from a Nd:YAG-pumped dye laser (following the exci target complex is bound to a first binding com tation scheme of Macfarlane et al. (1988) Appl. Phys. Lett ponent on the solid support to form a sandwich complex. 10. A method according to claim 1, wherein the target 52: 1300). An emission spectrum for the emitted up-converted light at 380 nm is shown in FIG. 32. We analyte is selected from the group consisting of: polynucleotides, polypeptides, viruses, microorganisms, estimate the up- conversion cross section to be 30 haptens, mammalian cells, steroid hormones, glycoproteins, 10-27)>cm2>> for this experiment. lipoproteins, biotinylated magnetic beads, prescribed or We have also observed up-conversion in thulium acetate hexahydrate and holmium chloride hexahydrate in solution over-the-counter drugs of abuse.

drugs, illegal substances, intoxicants and following the excitation schemes of Allain et al. (1990) 11. A method according to claim 1, wherein the step of Electron. Lett. 26: 166, and Allain et al. (1990) Electron. 35 illuminating with a label excitation wavelength is performed Lett. 26:261, respectively. The salts were dissolved in heavy with an infrared laser diode or light-emitting diode. water, and excitation was performed using a krypton laser.

Although the up-conversion was weak, the up-conversion laser diode or light-emittingtodiode 12. A method according claim 1, wherein the infrared emits pulsed illumination.

should be improved if chelated compounds are used instead 13. A method according to claim 12, wherein the infrared of dissolved salts.

laser diode or light-emitting diode is pulsed through direct

Although the present invention has been described in current modulation.

some detail by way of illustration for purposes of clarity of 14. A method according to claim 12, wherein the step of understanding, it will be apparent that certain changes and detecting light emission of at least one label emission modifications may be practiced within the scope of the wavelength is performed by time-gated or lock-in detection. claims. 45 15. A method according to claim 11, wherein the step of We claim: detecting light emission is performed with phase-sensitive 1. A method for detecting an analyte in a sample, com detection.

prising the steps of: 16. A method according to claim 11, wherein the laser contacting a sample containing a target analyte with a diode or light-emitting diode has peak emissions in the range labeled binding component to specifically bind the 50 of 960-980 nm and at approximately 1500 nm. target analyte and form a labeled binding component 17. A method according to claim 1, wherein said step of target complex, wherein the labeled binding component detecting light emissionis performed with a photomultiplier, comprises a binding component attached to an photodiode, a charge coupled device, a charge injection up-converting inorganic phosphor particle comprising device, or photographic film emulsion.

at least one rare earth element and a phosphor host 55 18. A method According to claim 1, wherein the target material and being capable of converting excitation analyte is immobilized in a histological tissue section or a radiation to emission radiation of a shorter wavelength; solid support.

separating any unbound labeled binding component from 19. A method according to claim 1, wherein the binding the labeled binding component-target complex; component is selected from the group consisting of: illuminating the labeled binding component-target com antibodies, polynucleotides, polypeptide hormones, plex with excitation radiation; and streptavidin. Staphylococcus aureus Protein A, lectins, and detecting emission radiation of at least one label emission antigens.

wavelength, wherein the emission radiation has a 20. A method for detecting an analyte in a sample, shorter wavelength than the excitation radiation. comprising the steps of:

2. A method according to claim 1, wherein said 65 contacting a sample containing a target analyte with a up-converting inorganic phosphor comprises ytterbium and binding component to specifically bind the target ana erbium in a phosphor host material. lyte and form a binding component-target complex;

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contacting the binding component-target complex with a target analyte and form a labeled binding component label to form a labeled binding component-target target complex, wherein the labeled binding component complex, the label comprising an up-converting inor comprises a binding component attached to an ganic phosphor particle comprising at least one rare up-converting inorganic phosphor particle comprising earth element and a phosphor host material and being at least one rare earth element and a phosphor host capable of converting excitation radiation to emission material and being capable of converting excitation radiation of a shorter wavelength; radiation to emission radiation of a shorter wavelength; separating any unbound label from the labeled binding differentiating the labeled binding component-target com component-target complex; plex from any unbound labeled binding component in illuminating the labeled binding component-target com 10 the sample;

plex with excitation radiation; and illuminating the labeled binding component-target com detecting emission radiation of at least one label emission plex with excitation radiation; and wavelength, wherein the emission radiation has a shorter wavelength than the excitation radiation. 15 detecting emission radiation of at least one label emission 21. A method according to claim 20, wherein the binding wavelength from the labeled binding component-target component is a primary antibody and the up-converting complex, wherein the emission radiation has a shorter inorganic phosphor is bound to the binding component wavelength than the excitation radiation. through a secondary antibody. 30. The method of claim 29, wherein the labeled binding 22. A method according to claim 21, wherein said sec component-target complex is illuminated with a confocal ondary antibody is biotinylated and said up-converting inor 20 beam having a focal point at the contact surface and being ganic phosphor is bound to streptavidin. divergent at points other than the contact surface. 23. A method according to claim 20, wherein the target 31. A method of claim 29, further comprising, before the analyte is a polynucleotide and the binding component is a contacting step, the step of attaching the up-converting biotinylated polynucleotide which hybridizes to the target 25 inorganic phosphor particle to the binding component to polynucleotide under binding conditions. form the labeled binding component. 24. A method according to claim 23, wherein the 32. A method of claim 29, wherein the differentiating step up-converting inorganic phosphor comprises an comprises contacting the labeled binding component-target up-converting phosphor particle and streptavidin. complex with a contact surface wherein the labeled binding 25. A method of claim 20 further comprising, before the component-target complex is localized at the contact surface contacting step, the step of attaching the up-converting as compared to the unbound labeled binding component; and inorganic phosphor particle to the binding component to the illuminating step comprises illuminating the labeled form the labeled binding component. binding component-target complex at the contact surface 26. A method for detecting a biotinylated analyte in a with excitation radiation.

sample, comprising the steps of: 35 33. The method of claim 32, wherein binding component contacting a sample containing a biotinylated analyte with target complexes are localized to the contact surface by a a labelled binding component to specifically bind the method selected from the group consisting of: biotinylated analyte and form a labeled binding magnetic localization of magnetic beads to said contact component-target complex, wherein the labeled bind surface, wherein said binding component-target com ing component comprises a streptavidin-coated plexes are localized on the magnetic beads relative to up-converting inorganic phosphor particle, the unbound labeled binding component; up-converting inorganic phosphor particle comprising gravitational sedimentation of binding component-target at least one rare earth element and a phosphor host complexes from unbound labelled binding component, material and being capable of converting excitation wherein said sedimented binding component-target radiation to emission radiation of a shorter wavelength; 45 complexes are localized on the contact surface relative separating any unbound labeled binding component from to unbound labeled binding component; the labeled binding component-target complex; filtration over a contact surface wherein said binding illuminating the labeled binding component-target com component-target complexes are localized on the con plex with excitation radiation; and tact surface relative to unbound labeled binding com detecting emission radiation of at least one label emission 50 ponent;

wavelength, wherein the emission radiation has a antibody capture;

shorter wavelength than the excitation radiation. affinity adsorption; and 27. A method according to claim 26, wherein the target is nucleic acid hybridization.

a biotinylated magnetic bead. 34. A method of claim 29, wherein the differentiating and 28. A method of claim 26 further comprising, before the 55 illuminating steps are accomplishied by confocal excitation. contacting step, the step of attaching the up-converting 35. A method of claim 29 wherein the differentiating, inorganic phosphor particle to the binding component to illuminating, and detecting steps are accomplished by con form the labeled binding component. focal excitation and confocal detection. 29. A method for detecting an analyte in a sample, 36. A method of claim29, wherein the differentiating step comprising the steps of: is accomplished by size discrimination. contacting a sample containing a target analyte with a labeled binding component to specifically bind the ; : :: *k sk