Simultaneous Use of Time-Resolved Fluorescence and Anti-Stokes

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide ch...
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Anal. Chem. 2005, 77, 2826-2834

Simultaneous Use of Time-Resolved Fluorescence and Anti-Stokes Photoluminescence in a Bioaffinity Assay Katri Kuningas,* Terhi Rantanen, Ulla Karhunen, Timo Lo 1 vgren, and Tero Soukka

Department of Biotechnology, University of Turku, Tykisto¨katu 6A, FIN-20520 Turku, Finland

A bioaffinity assay is described where anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates are measured from a single microtitration well without any disturbance from these label technologies to each other. Up-converting phosphor (UPC-phosphor) bioconjugate was produced by grinding the commercial, micrometersized UPC-phosphors to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. Experiments were carried out in standard 96-well microtitration plates to determine detection limits, linearity, and cross-talk of UPC-phosphor and europium chelate. In numbers of molecules the lower limits of detection for UPC-phosphor were roughly 3 × 103 particles in solution and 1 × 104 particles in solid phase, and for europium label same values were 9 × 106 and 9 × 107 molecules. Linearity of detection was for UPCphosphor 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase and for europium label over 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase. The cross-talk between the two labels was practically nonexistent. In this study we show that up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with long-lifetime lanthanide chelates. Time-resolved fluorescence of lanthanide chelates is a label technology where the unique fluorescence properties of lanthanides are utilized to measure the specific signal of the label after the background fluorescence has already decayed. The label is excited with ultraviolet light, and emission occurs at visible wavelengths. Time-resolved fluorometry was first introduced to the research world in the early 1980s,1,2 after which there has been constant research and development going on concerning improved lanthanide labels that could be used in different kinds of immunoassay systems.3-5 Currently, time-resolved fluorometry * Corresponding author. Telephone: +358-2-333-8056. Fax: +358-2-333-8050. E-mail: [email protected]. (1) Siitari, H.; Hemmila¨, I.; Soini, E.; Lo¨vgren, T.; Koistinen, V. Nature 1983, 301, 258-260. (2) Hemmila¨, I.; Dakubu, S.; Mukkala, V. M.; Siitari, H.; Lo¨vgren, T. Anal. Biochem. 1984, 137, 335-343. (3) Soini, E.; Lo ¨vgren, T. CRC Crit. Rev. Anal. Chem. 1987, 18, 105-154.

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and lanthanide labels have established their use in several bioaffinity assay applications.6-9 In anti-Stokes photoluminescence, the label is excited with infrared light and emission occurs at shorter, visible wavelengths.10,11 Thus, in contrast to long-lifetime fluorescence of lanthanide chelates where the wavelength increases and energy decreases, in this up-conversion process the wavelength decreases and the light of lower energy is converted to the light of higher energy. The up-convertive anti-Stokes photoluminescence is based on unique features of anti-Stokes phosphors or up-converting phosphors (UPC-phosphors); to our knowledge no other material possesses this up-conversion property at conventional excitation light intensities. UPC-phosphors, which consist of certain lanthanides embedded in a crystalline host lattice, convert infrared to visible light via the sequential absorption of two low-energy photons.12,13 In contrast to two-photon excitation,14 the photons do not need to be absorbed simultaneously, but in a microsecond time scale, enabling the excitation of up-converting phosphors with relatively low laser power. The phenomenon of up-conversion was first reported already in 1966,15,16 but it was not employed in biomedical research until in the early 1990s.17 Many advantages of UPC-phosphors have made these particulates very attractive to be used as labels. First, no autofluorescence is produced with infrared excitation; in principle, the only sources of background signal are stray light (4) Lo¨vgren, T.; Merio¨, L.; Mitrunen, K.; Ma¨kinen, M. L.; Ma¨kela¨, M.; Blomberg, K.; Palenius, T.; Pettersson, K. Clin. Chem. 1996, 42, 1196-1201. (5) Ha¨rma¨, H.; Soukka, T.; Lo ¨vgren, T. Clin. Chem. 2001, 47, 561-568. (6) Ha¨rma¨, H.; Soukka, T.; Lo ¨nnberg, S.; Paukkunen, J.; Tarkkinen, P.; Lo¨vgren, T. Luminescence 2000, 15, 351-355. (7) Nurmi, J.; Lilja, H.; Ylikoski, A. Luminescence 2000, 15, 381-388. (8) Pettersson, K.; Katajama¨ki, T.; Irjala, K.; Leppanen, V.; Majamaa-Voltti, K.; Laitinen, P. Luminescence 2000, 15, 399-407. (9) Karvinen, J.; Laitala, V.; Ma¨kinen, M. L.; Mulari, O.; Tamminen, J.; Hermonen, J.; Hurskainen, P.; Hemmila¨, I. Anal. Chem. 2004, 76, 14291436. (10) Guggenheim, H. J.; Johnson, L. F. Appl. Phys. Lett. 1969, 15, 51-52. (11) Johnson, L. F.; Guggenheim, H. J.; Rich, T. C.; Ostermayer, F. W. J. Appl. Phys. 1972, 43, 1125-1137. (12) Niedbala, R. S.; Feindt, H.; Kardos, K.; Vail, T.; Burton, J.; Bielska, B.; Li, S.; Milunic, D.; Bourdelle, P.; Vallejo, R. Anal. Biochem. 2001, 293, 2230. (13) Auzel, F. Proc. SPIE 2002, 4766, 179-190. (14) Koskinen, J. O.; Vaarno, J.; Meltola, N. J.; Soini, J. T.; Ha¨nninen, P. E.; Luotola, J.; Waris, M. E.; Soini, A. E. Anal. Biochem. 2004, 328, 210-218. (15) Auzel, F. C. R. Seances Acad. Sci., Ser. B 1966, 262, 1016-1019. (16) Auzel, F. C. R. Seances Acad. Sci., Ser. B 1966, 263, 819-821. (17) Zarling, D. A.; Rossi, M. J.; Peppers, N. A.; Kane, J.; Faris, G. W.; Dyer, M. J.; Ng, S. Y.; Schneider, L. V. U.S. Pat. 5,674,698, 1997. 10.1021/ac048186y CCC: $30.25

© 2005 American Chemical Society Published on Web 03/24/2005

and detector noise.18 Although time-resolved measurements are possible because of the long lifetime of UPC-phosphors,19 temporal resolution is not needed to avoid background.18 Further, the antiStokes photoluminescence signal of UPC-phosphors can be measured by direct excitation both from solution and from solid phase without any additional development steps. Also, because the up-conversion process occurs within the host lattice, the optical properties of phosphors are almost completely unaffected by their environment. Finally, UPC-phosphors are not susceptible to photobleaching; the same sample can be measured several times and even after a long period without any change in the signal measured. The main disadvantage in using UPC-phosphors as labels in bioaffinity assays is probably the nonspesific binding of these particles when they are conjugated to biomolecules.20,21 This unwanted property of phosphor conjugates can originate from multiple sources, but particle size, shape, and the surface layer of phosphors may have a quite large effect. Colloidal, submicrometer-sized phosphor particles suitable for bioaffinity assays, can be produced by grinding the originally larger phosphor material smaller.22-24 Also, there are protocols for preparing nanocrystalline particles directly.25-31 Preparation of these nanopowders can be more complicated than producing submicrometersized particles with grinding, but the advantage is the resulting more homogeneous particle suspension. In grinding of the phosphor material, the outcome is usually a mixture of phosphor particles with different sizes and the purification of monosized particles is practically impossible. However, methods for producing submicrometer-sized inorganic phosphor particles with grinding and for conjugation of these particles to biomolecules have been reported earlier,22,32 and these methods can be employed for UPCphosphors also. Bioconjugates of UPC-phosphors have previously been used in immunochromatographic and DNA hybridization assays.12,18,21,33-36 (18) Hampl, J.; Hall, M.; Mufti, N. A.; Yao, Y. M.; MacQueen, D. B.; Wright, W. H.; Cooper, D. E. Anal. Biochem. 2001, 288, 176-187. (19) Soukka, T.; Kuningas, K.; Rantanen, T.; Haaslahti, V.; Lo¨vgren, T. Submitted for publication in J. Fluoresc. (20) Wright, W. H.; Mufti, N. A.; Tagg, N. T.; Webb, R. R.; Schneider, L. V. Proc. SPIE 1997, 2985, 248-255. (21) Zijlmans, H. J. M. A. A.; Bonnet, J.; Burton, J.; Kardos, K.; Vail, T.; Niedbala, R. S.; Tanke, H. J. Anal. Biochem. 1999, 267, 30-36. (22) Beverloo, H. B.; van Schadewijk, A.; Bonnet, J.; van der Geest, R.; Runia, R.; Verwoerd, N. P.; Vrolijk, J.; Ploem, J. S.; Tanke, H. J. Cytometry 1992, 13, 561-570. (23) Mende, S.; Stenger, F.; Peukert, W.; Schwedes, J. Powder Technol. 2003, 132, 64-73. (24) Stenger, F.; Peukert, W. Chem. Eng. Technol. 2003, 26, 177-183. (25) Tessari, G.; Bettinelli, M.; Speghini, A.; Ajo`, D.; Pozza, G.; Depero, L. E.; Allieri, B.; Sangaletti, L. Appl. Surf. Sci. 1999, 144-145, 686-689. (26) Igarashi, T.; Ihara, M.; Kusunoki, T.; Ohno, K.; Isobe, T.; Senna, M. Appl. Phys. Lett. 2000, 76, 1549-1551. (27) Martinez-Rubio, M. I.; Ireland, T. G.; Fern, G. R.; Silver, J.; Snowden, M. J. Langmuir 2001, 17, 7145-7149. (28) Schmechel, R.; Kennedy, M.; von Seggern, H.; Winkler, H.; Kolbe, M.; Fischer, R. A.; Li, X. M.; Benker, A.; Winterer, M.; Hahn, H. J. Appl. Phys. 2001, 89, 1679-1686. (29) Hirai, T.; Orikoshi, T.; Komasawa, I. Chem. Mater. 2002, 14, 3576-3583. (30) Patra, A.; Friend, C. S.; Kapoor, R.; Prasad, P. N. J. Phys. Chem. B 2002, 106, 1909-1912. (31) Yi, G.; Sun, B.; Yang, F.; Chen, D.; Zhou, Y.; Cheng, J. Chem. Mater. 2002, 14, 2910-2914. (32) Beverloo, H. B.; van Schadewijk, A.; Zijlmans, H. J. M. A. A.; Tanke, H. J. Anal. Biochem. 1992, 203, 326-334. (33) Niedbala, R. S.; Vail, T. L.; Feindt, H.; Li, S.; Burton, J. L. Proc. SPIE 2000, 3913, 193-203.

The purpose of this work was to present, for the first time, the simultaneous measurement of anti-Stokes photoluminescence of inorganic lanthanide phosphors and time-resolved fluorescence of lanthanide chelates from a single microtitration well without any disturbance from these label technologies to each other. We show that neither anti-Stokes photoluminescence signals nor timeresolved fluorescence signals have en effect on the signals in the measurement window of the other label. In addition, we demonstrate the utilization of UPC-phosphors as labels in bioaffinity assays in a common, 96-well platform. The phosphor label used in this work was produced by grinding the commercial, micrometersized UPC-phosphor material to colloidal, submicrometer-sized phosphor particles and by attaching these phosphors to biomolecules. EXPERIMENTAL SECTION Reagents. Up-converting anti-Stokes phosphor PTIR550/F was purchased from Phosphor Technology Ltd. (Stevenage, England). Polycarboxylic acid Additol XW330 was obtained from Surface Specialties Austria GmbH (Werndorf, Austria), N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDAC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) were from Fluka (Buchs, Switzerland), streptavidin was from Spa-BioSpa (Milan, Italy), Tween 20 and Tween 85 were from E. Merck (Darmstadt, Germany), bovine serum albumin fraction V (BSA) was from Bioreba (Nyon, Switzerland), and MaxiSorp microtitration wells were purchased from Nunc A/S (Roskilde, Denmark). 2-(((N-(Biotinoyl)amino)hexanoyl)amino)ethylamine (bio-NH2) was obtained from Molecular Probes, Inc. (Eugene, OR), and biotinamidohexanoic acid N-hydroxysuccinimide ester (NHS-LC-biotin) was from Pierce Biotechnology (Rockford, IL). Terbium(III) chelate of N1-(4-isothiocyanatobenzyl)diethylenetriamineN1,N2,N3,N4-tetrakis(acetic acid) (Tb(III)-N1-ITC) as well as Delfia enhancement solution (DES) and enhancer solution (DE) were purchased from Perkin Elmer Life and Analytical Sciences (Wallac Oy, Turku, Finland). Streptavidin-coated normal-capacity microtitration wells, assay buffer (50 mM Tris-HCl, pH 7.8, containing 9 g/L NaCl, 0.5 g/L NaN3, 5 g/L BSA, 0.1 g/L Tween 40, 0.5 g/L bovine γ-globulin, and 20 µM DTPA), and wash solution (5 mM Tris-HCl, pH 7.8, containing 9 g/L NaCl, 0.05 g/L Tween 20, and 1 g/L Germall II) were obtained from Innotrac Diagnostics Oy (Turku, Finland). Biotinylation of BSA. BSA was biotinylated with a 35-fold molar excess of NHS-LC-biotin in 50 mM carbonate buffer, pH 9.3. Freshly prepared solution of NHS-LC-biotin in dimethylformamide (DMF) was added to BSA solution, and the reaction mixture with a total volume of 200 µL, containing 0.1 g/mL BSA and 15% (v/v) DMF, was incubated for 3 h at room temperature. Finally, biotinylated BSA (bio-BSA) was purified with NAP-5 and NAP-10 columns (Amersham Biosciences AB, Uppsala, Sweden) using aqueous solution of NaCl (9 g/L) and stored at -20 °C. The protein concentration of bio-BSA was measured with Protein Assay kit (Bio-Rad, Hercules, CA) and the biotinylation of BSA was (34) van de Rijke, F.; Zijlmans, H.; Li, S.; Vail, T.; Raap, A. K.; Niedbala, R. S.; Tanke, H. J. Nat. Biotechnol. 2001, 19, 273-276. (35) Corstjens, P. L. A. M.; Zuiderwijk, M.; Nilsson, M.; Feindt, H.; Niedbala, R. S.; Tanke, H. J. Anal. Biochem. 2003, 312, 191-200. (36) Zuiderwijk, M.; Tanke, H. J.; Niedbala, R. S.; Corstjens, P. L. A. M. Clin. Biochem. 2003, 36, 401-403.

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confirmed using an assay based on europium-labeled streptavidin (SA-Eu) (Perkin Elmer Life and Analytical Sciences). In the assay, three replicates of appropriate dilutions of bio-BSA in 50 µL of assay buffer were first incubated in streptavidin wells for 30 min at room temperature with slow shaking (Delfia Plateshake, Perkin Elmer Life and Analytical Sciences). The wells were washed once with wash solution (Delfia Platewash, Perkin Elmer Life and Analytical Sciences), and 75 µL of SA-Eu was added so that there was excess of streptavidin compared to BSA in every well. Wells were incubated for 30 min with slow shaking, washed three times, and developed with DES, and finally, europium fluorescence was measured with Victor 1420 Multilabel Counter (Perkin Elmer Life and Analytical Sciences) at 615 nm using the normal europium measurement protocol. Labeling of Aminobiotin with Intrinsically Fluorescent Nonadentate Europium Chelate. Intrinsically fluorescent nonadentate europium chelate, {2,2′,2′′,2′′′-{[2-(4-isothiocyanatophenyl)ethylimino]bis(methylene)bis{4-{[4-(R-galactopyranoxy)phenyl]ethynyl}pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III)37 was coupled to bio-NH2 using a 25-fold molar excess of the biotin. Bio-NH2 was first dissolved in DMF and 100 mM carbonate buffer, pH 9.5, so that the proportion of DMF was less than 20% (v/v). Europium chelate dissolved in Milli-Q water was added, and the reaction with the total volume of 90 µL was left to incubate overnight in the dark at room temperature. Europium-labeled biotin (bio-Eu) was purified from the reaction mixture with reverse-phase HPLC using a 150 × 10 mm, 3 µm Hypersil-Keystone C-18 column (Thermo Electron Corp., West Palm Beach, FL) and a linear acetonitrile gradient in aqueous 50 mM triethylammonium acetate buffer. The bioconjugate was stored at 4 °C. Labeling of Aminobiotin with Terbium Chelate. Bio-NH2 was labeled with Tb(III)-N1-ITC according to the instructions of the Delfia terbium labeling kit (Perkin Elmer Life and Analytical Sciences) using a 10-fold molar excess of the chelate. Bio-NH2, dissolved just prior labeling in DMF, and terbium chelate, dissolved in Milli-Q water, were incubated overnight in 50 mM carbonate buffer, pH 9.8, in the dark at room temperature. The total reaction volume was 300 µL of which 10% (v/v) was DMF. The labeling mixture was purified with reverse-phase HPLC as the reaction mixture of bio-Eu, and terbium-labeled biotin (bioTb) was stored at 4 °C. Coating of Microtitration Wells. Special, yellow, lowfluorescence MaxiSorp microtitration wells were blocked with BSA as described earlier38 and also coated with streptavidin by physical adsorption to prepare low-capacity, streptavidin-coated microtitration wells. In the coating, the wells were first incubated overnight at 35 °C with 20 ng of streptavidin in 60 µL of 100 mM Na2HPO4, 50 mM citric acid, and 9 g/L NaCl. The coated wells were washed twice and saturated overnight at room temperature with 250 µL of 50 mM Tris-HCl buffer, pH 7.0, containing 9 g/L NaCl, 0.5 g/L NaN3, 6 g/L D-sorbitol and 2 g/L BSA. The wells were aspirated, dried in a laminar hood for 2 h, and stored at 4 °C in a sealed package with desiccant. (37) von Lode, P.; Rosenberg, J.; Pettersson, K.; Takalo, H. Anal. Chem. 2003, 75, 3193-3201. (38) Kokko, L.; Sandberg, K.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2004, 503, 155-162.

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Preparation and Characterization of Sub-micrometerSized UPC-Phosphors. Sub-micrometer-sized UPC-phosphors were prepared as described earlier by Soukka et al.19 Briefly, micrometer-sized UPC-phosphor particles were first ground in a small bead-mill, and thereafter phosphor particles with certain size and density were purified among the grinding suspension. To characterize the submicrometer-sized UPC-phosphors, particle size, concentration, and anti-Stokes photoluminescence of the phosphors were determined. First, the size distribution profile of the phosphors was measured in measurement buffer (10 mM borate buffer, pH 8.5, containing 1 g/L Tween 20) using Coulter N4 plus submicrometer particle size analyzer (Beckman Coulter, Fullerton, CA). Three replicate measurements of each sample were performed according to the manufacturer’s instructions. Thereafter, the concentration of phosphor solution was determined by measuring the mass of the phosphors present in a certain volume of purified phosphor slurry. For mass measurements, VectaSpin Micro centrifuge tube filters with Anopore 0.02 µm membrane (Whatman, Maidstone, England) were first dried at 40 °C and weighed. An aliquot of 80 µL of phosphor suspension was then centrifuged at 7000 rpm (Sorvall MC 12V, F-12/ M18, Sorvall Products, L. P., Newtown, CT) in dried tube filters. Resulting phosphor pellets on the filters were washed with 350 µL of ethanol using centrifugation, dried at 40 °C, and finally, tube filters with phosphor pellets were weighed again. To determine whether the anti-Stokes photoluminescence properties of the phosphors had been affected by grinding, antiStokes photoluminescence of the submicrometer-sized UPCphosphors and the original phosphor material was measured with Plate Chameleon (Hidex Oy, Turku, Finland) equipped with infrared laser excitation.19 Six replicates of the dilutions of both submicrometer-sized phosphors and the original phosphor material in 150 µL of the measurement buffer with the phosphor concentration of 0.08 mg/mL, were first incubated in noncoated microtitration wells for 5 min at room temperature with slow shaking. Anti-Stokes photoluminescence of the UPC-phosphors was then measured at 535 nm, using continuous laser excitation at 980 nm, emission filter of 535/50 nm (center wavelength ) 535 nm, half-width ) 50 nm, peak transmittance g 60%; Bk Interferenzoptik Elektronic, Nabburg, Germany), and a measurement time of 2 s. Conjugation of Sub-micrometer-Sized UPC-Phosphors to Streptavidin. Sub-micrometer-sized UPC-phosphors were conjugated to streptavidin according to modified procedures from Kubitschko et al.39 and Beverloo et al.22 After careful bath sonication (Finnsonic m03, Finnsonic Oy, Lahti, Finland), phosphors were suspended in aqueous solution of Additol XW330, pH 9, to a concentration of 10 mg/mL. The total volume of this suspension was 750 µL, and the final concentration of Additol XW330 was 2.5% (v/v). Phosphor suspension was slowly agitated overnight at room temperature in Rotamix RK at 17 rpm (HetoHolten A/S, Allerød, Denmark) to let Additol XW330, an ammonium salt of poly(acrylic acid), to develop carboxylic acid groups onto the surface of phosphor particles. The next day, to remove excess of poly(acrylic acid) from the phosphor solution, (39) Kubitschko, S.; Spinke, J.; Bruckner, T.; Pohl, S.; Oranth, N. Anal. Biochem. 1997, 253, 112-122.

the carboxylated phosphors were washed four times with Milli-Q water and once with 20 mM MES, pH 6.1, using centrifugation at 12 000 rpm and bath sonication. Finally, the phosphors were resuspended in 750 µL of 20 mM MES, pH 6.1, for the activation step of carboxyl groups on phosphor particles. Among the phosphors, 15 µL of such, freshly prepared solutions of both sulfoNHS and EDAC in 20 mM MES, pH 6.1, were added that the concentrations of sulfo-NHS and EDAC were 30 mM in the final reaction mixture. Phosphors were incubated for 1 h in rotation, washed once with 20 mM MES, pH 6.1, and resuspended with bath sonication in 500 µL of the same buffer. After that, 250 µL of streptavidin diluted in 20 mM MES, pH 6.1, was added, and the conjugation reaction, in which the streptavidin concentration was 0.5 mg/mL and the phosphor concentration around 10 mg/mL, was left to rotate slowly for 2.5 h. Reaction was stopped by adding 2 M glycine, pH 11, to the final concentration of 50 mM in the reaction and continuing incubation for 30 min. Phosphors were washed three times with measurement buffer and finally resuspended using bath sonication into 750 µL of storage buffer (5 mM borate buffer, pH 8.5, containing 2 g/L Tween 85, 5 g/L BSA, and 0.5 g/L NaN3). Streptavidin-coated phosphors (SA-phosphors) were stored at room temperature in slow rotation. Characterization of UPC-Phosphors Conjugated to Streptavidin. The size of the streptavidin-coated phosphor particles, the concentration of the phosphor solution, and the amount of streptavidin attached onto the phosphors were determined to characterize the SA-phosphors. First, the size distribution profile of SA-phosphors was measured as the size distribution profile of uncoated, submicrometer-sized phosphors. Second, the concentration of the phosphor solution was determined by comparing the anti-Stokes photoluminescence signals of SA-phosphors to the signals of uncoated UPC-phosphors with known concentration. Both uncoated and streptavidin-coated phosphors were diluted similarly in measurement buffer, 100 µL of the dilutions in two replicates were incubated in noncoated microtitration wells for 5 min at room temperature with slow shaking, and anti-Stokes photoluminescence was measured with Plate Chameleon at 535 nm. The amount of streptavidin attached onto phosphors and the amount of streptavidin free in the phosphor solution were measured using an assay based on pure streptavidin standards and terbium-labeled biotin (bio-Tb). In the assay, free, unconjugated streptavidin present in SA-phosphor solution was first separated from the streptavidin-conjugated phosphors by centrifuging an aliquot of SA-phosphor solution in a centrifuge tube filter (VectaSpin Micro with Anopore 0.02 µm membrane) at 10 000 rpm for 10 min. The principle of the separation is that, during centrifugation, free streptavidins in the phosphor solution with very small diameter pass through the filter pores to eluate, but larger, streptavidin-conjugated phosphor particles stop onto the filter. After separation, original SA-phosphor solution, eluate, pure streptavidin, and bio-Tb were diluted in assay buffer. Thereafter, dilutions of bio-Tb and either eluate, phosphor solution, or streptavidin standards, respectively, were mixed in small eppendorf tubes to a total volume of 150 µL. Reactions were incubated for 15 min at room temperature in slow rotation to allow bio-Tb to bind to any streptavidin-containing molecules present in the reactions. Next, 50 µL of each of the reactions were incubated in

two replicates in normal-capacity streptavidin wells for 30 min with slow shaking. Finally, the wells were washed twice and developed with both DES and DE, and terbium fluorescence was measured with Victor at 545 nm using normal terbium measurement protocol. The principle in the assay is that the more there has been free streptavidin or other streptavidin-containing molecules in the reactions, the less terbium fluorescence is measured from the wells. This is because bio-Tb bound to streptavidin present in solution cannot anymore bind to streptavidin immobilized in the wells due to the slow dissociation of biotin and streptavidin from each other. Dilution Series of SA-Phosphor and Bio-Eu. Dilution series of SA-phosphor and bio-Eu were used to determine the detection limits, linearity, and cross-talk of these label conjugates from liquid and solid phases. Concentration ranges from 0.004 to 400 µg/mL for SA-phosphor and from 4 × 10-4 to 400 nM for bio-Eu in assay buffer were analyzed. In liquid-phase measurements, 75 µL of the dilutions of SA-phosphor and bio-Eu in four replicates were incubated in BSA-blocked microtitration wells for 10 min at room temperature with slow shaking. After incubation, europium fluorescence at 615 nm was measured with Victor and phosphorassociated anti-Stokes photoluminescence at 535 nm was measured with Plate Chameleon. Both of these signals were measured from both bio-Eu and SA-phosphor to assess also the amount of cross-talk between the two labels, europium chelate and UPCphosphor. Special low-fluorescence, streptavidin-coated, normal-capacity microtitration wells were used in solid-phase measurements, because these wells provide lower background for europium label. Just prior to use, the wells were prewashed once to remove any loosely bound streptavidin. In bio-Eu measurements, 75 µL of bioEu dilutions in four replicates were incubated for 1 h in streptavidin-wells, unbound bio-Eu was washed away four times, and the wells were left to dry at room temperature. Finally, signals from bio-Eu were measured with Victor at 615 nm and with Plate Chameleon at 535 nm. In Victor, europium fluorescence was measured from the surface of the wells using the normal europium emission filter D615 and europium measurement protocol (delay time, 400 µs; window time, 400 µs; cycle time, 1000 µs with 1000 repeats) with maximum Xe-flash excitation energy. In SA-phosphor measurements, prewashed streptavidin wells were first saturated with bio-BSA by incubating 10 µg/mL bio-BSA in 50 µL of assay buffer in the wells for 30 min. Excess of bio-BSA was washed away once, after which 75 µL of SA-phosphor dilutions in four replicates was incubated for 1 h in the wells. The wells were washed four times and left to dry at room temperature, and, finally, signals from the phosphors were measured with Plate Chameleon at 535 nm and with Victor at 615 nm. In Plate Chameleon, a measuring protocol was used, where nine 2 s readings in a 3 × 3 raster were measured from the bottom of the microtitration well and an average of signals was calculated. The measurement raster was centered at the middle of the well, and the distance between the measuring points was 1 mm. Measurement of Time-Resolved Fluorescence and AntiStokes Photoluminescence from a Single Microtitration Well. SA-phosphor and bio-Eu were used in the same bioaffinity assays to show that both time-resolved fluorescence and anti-Stokes photoluminescence can be measured from a single microtitration Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

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well. Two types of assays, assay I and assay II with similar principles, were carried out in low-fluorescence, low-capacity streptavidin wells. In assay I, bio-BSA was first diluted in assay buffer in the concentration range from 0.001 to 2 µg/ mL, after which 50 µL of these dilutions in four replicates was incubated in prewashed streptavidin wells for 30 min at room temperature with slow shaking. Bio-Eu diluted to 50 nM with assay buffer was then added to the wells in 25 µL and incubated for 15 min. The wells were washed once, and, subsequently, 75 µL of SA-phosphor in 0.04 mg/mL concentration was added to the wells. Phosphor solution was incubated for 15 min, and the wells were washed four times and left to dry at room temperature. Europium fluorescence at 615 nm was measured with Victor, and anti-Stokes photoluminescence at 535 nm was measured with Plate Chameleon from the surface of the wells. In assay II, bio-Eu was first diluted in assay buffer from 0.01 to 100 nM and the dilutions were then incubated in prewashed streptavidin wells for 30 min. BioBSA diluted to 4 µg/mL with assay buffer was then added to the wells in 25 µL and incubated for 15 min. As in the assay I, the wells were washed, SA-phosphors were added and incubated, and, after washing and drying, the signals from the surface of the wells were measured. RESULTS AND DISCUSSION Characteristics of Bio-BSA. Protein yield in the biotinylation of BSA was about 80%. Using europium-labeled streptavidin, it was deduced that about 40% of BSA molecules had at least two biotins attached to their surfaces. Only BSA molecules with two or more biotins were detected in the assay, since one biotin is needed for the attachment of bio-BSA to streptavidin well and another one for binding bio-BSA to SA-Eu. Preparation and Characterization of Sub-micrometerSized UPC-Phosphor Particles. According to the manufacturer, the particle size of the original PTIR550/F anti-Stokes phosphor was between 2.3 and 6.1 µm (50% of particle volume),19 so this phosphor was too large to be used in the bioaffinity assays as such. Particulates with micrometer size do not stay in solution; rather, they tend to sediment in the bottom of the container. Consequently, their conjugation to biomolecules is difficult, and even though bioconjugates could be produced, their binding is poor and nonspecific due to steric hindrances and sedimentation. Thus, a colloidal suspension of up-converting phosphor particles, suitable for use in the assays, was produced, by grinding the original phosphor material. According to the size distribution profile generated with Coulter N4 plus, most of the PTIR550 phosphors in the resulting phosphor suspension after grinding and purifications were in the diameter range of 200-400 nm (Figure 1a), and average particle size in the phosphor suspension was about 280 nm. The storage concentration of the phosphor suspension after grinding and purifications was measured to be 97 mg/mL. To assess the quality of the phosphor solution and its photoluminescence properties, anti-Stokes photoluminescence and the mass concentration of the phosphors were used to calculate the parameter photoluminescence per mass for both the original phosphor material and for submicrometer-sized phosphors. This parameter was 430 000 ( SD 61 000 counts/µg for the original phosphor material and 63 000 ( SD 3 300 counts/µg for the submicrometer-sized phosphors. The determination of the photolu2830 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

Figure 1. Size distribution profiles of submicrometer-sized UPCphosphors after grinding and purification (a) and after streptavidin coating (b). Weight distribution profiles of the phosphors were measured with Coulter N4 plus submicrometer particle size analyzer. Most of the particles in both analyses were in the diameter range of 200-400 nm, and the average particle size was determined to be about 280 nm.

minescence per mass value for the original phosphor material could have been susceptible to errors, because of the tendency of large particles to sediment in the bottom of microtitration wells, rendering the photoluminescence measurements difficult to perform accurately. Still, photoluminescence per mass parameter was clearly smaller for the submicrometer-sized phosphors than for the original phosphor material. As has been reported earlier,17 this can be due to problems related to grinding. First, grinding beads tend to ground also in the grinding process, and they contaminate the phosphor suspension. Because of the similar size and density with UPC-phosphors, ground beads behave like the phosphors in the purification process and are left in some amount in the purified phosphor solution. Thus, the smaller photoluminescence per mass value measured for the submicrometer-sized UPC-phosphors may be due to the presence of ground beads among the phosphor particles, which increases the measured mass for the phosphors and, consequently, affects the photoluminescence signals negatively. Second, the smaller size itself may have an effect on the intensity of the photoluminescence signals of submicrometer-sized phosphors. Supposedly, grinding changes

the structure of the phosphors so that it is not anymore as optimal as it was for the up-conversion process to happen. Also, quenching processes, which are said to have no effect on UPC-phosphors generally,12 may diminish the photoluminescence emission from the submicrometer-sized phosphors with larger surface area per mass. These quenching effects may explain the multiple fluorescence decay times described for UPC-phosphors in solvent and only a single-exponential decay observed from solid phosphor material.19 It should be possible to improve the photoluminescence intensity of ground phosphors by coating the particles with a thin, protective surface layer to eliminate the quenching effects of the environment. In general, the specific signal of the phosphors can also be improved considerably by increasing the laser power, with no effect on the background fluorescence. Preparation and Characterization of UPC-Phosphors Conjugated to Streptavidin. According to the anti-Stokes photoluminescence signal measurements, the phosphor yield in the conjugation of phosphor particles to streptavidin was 80% and the storage concentration of SA-phosphor solution was determined to be about 8 mg/mL. The size distribution profile of the SAphosphors was similar to the size distribution profile of unconjugated UPC-phosphors (Figure 1b). Thus, the conjugation protocol used does not change the particle size. The concentration of streptavidin in the phosphor solution was determined to be 22 ng of streptavidin/(1 µg of phosphors). Roughly 4% of this whole streptavidin amount was shown to be unconjugated, free streptavidin in solution, when measured 1 day after the conjugation reaction, but, after 2 weeks in storage, the amount of free streptavidin was determined to be 8%. SA-phosphors were stored in rotation to prevent the slow aggregation and sedimentation observable with the submicrometer-sized particles when held stationary. However, due to the rotary movement, phosphors may shear against each other, facilitating the dissociation of streptavidin and polyacryclic acid from particle surfaces. Although slight detachment of polyacryclic acid and streptavidin from the phosphors was expected due to the passive adsorption of poly(acrylic acid)s on the phosphor particles, the storage in rotation probably enhanced this dissociation. Nonreversible dissociation of streptavidins from the phosphor particles decreases gradually the reactivity of the phosphor label, but additional data are needed to evaluate the effect of the instability of the phosphor bioconjugates on routine use of these particles in bioaffinity assays. It should be possible to improve further the coating procedure of the SAphosphors by cross-linking the poly(acrylic acid)s on the surface of the phosphor particles before conjugation of the particles to streptavidins and produce stabilized phosphor bioconjugates more suitable for longer-term storage. Dilution Series of SA-Phosphor and Bio-Eu. Detection limits, linearity, and cross-talk of SA-phosphor and bio-Eu were determined using dilution series measurements to compare the performances of the two different types of labels, UPC-phosphor and europium chelate. The lower limits of detection, calculated as the mean of background + 3 SD, were for bio-Eu, 2 × 10-4 nM in solution and 0.002 nM in solid-phase measurements (Figure 2), and for SA-phosphor, 0.002 µg/mL in solution and 0.01 µg/ mL in solid phase measurements (Figure 3). To compare the performance of the two labels with each other, we converted these concentration values to numbers of molecules. For SA-phosphor,

Figure 2. Dilution curves and cross-talk signals of bio-Eu. Europium fluorescence of bio-Eu from solution (filled triangles) and from surface (filled circles) were measured with Victor 1420 multilabel counter, and anti-Stokes photoluminescence or cross-talk signals from solution (open triangles) and from surface (open circles) were measured with Plate Chameleon equipped with infrared laser excitation. The lower limits of detection (dotted lines) for bio-Eu, calculated as the mean of background + 3 SD, were 2 × 10-4 nM in solution and 0.002 nM in solid-phase measurements. cts, counts.

Figure 3. Dilution curves and cross-talk signals of SA-phosphor. Anti-Stokes photoluminescence signals of SA-phosphor from solution (filled triangles) and from surface (filled circles) were measured with Plate Chameleon equipped with infrared laser excitation, and europium fluorescence or cross-talk signals from solution (open triangles) and from surface (open circles) were measured with Victor 1420 multilabel counter. The lower limits of detection (dotted lines) for SA-phosphor, calculated as the mean of background + 3 SD, were 0.002 µg/mL in solution and 0.01 µg/mL in solid-phase measurements.

values were calculated by assuming that all SA-phosphors were spherical, with a diameter of 280 nm, and had the same densities. With these assumptions we first deduced the volume and mass of one phosphor molecule and then the detection limits in numbers of molecules using the concentration values of lower limits of detection, the volume of the label solution in the microtitration well, and the mass of one phosphor particle. For bio-Eu, the lower limits of detection in numbers of molecules were roughly 9 × 106 molecules in solution and 9 × 107 molecules in solid phase and for SA-phosphor, 3 × 103 particles in solution and Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

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1 × 104 particles in solid phase. However, the actual numbers of SA-phosphors and bio-Eu detected were even smaller. The diameter of the bottom of the microtitration well used in the measurements is 6.5 mm according to the manufacturer, so the bottom area of the well is roughly 33 mm2. The area excited in the wells in both plate readers is smaller than the whole well area; in Victor, according to the specifications received from the manufacturer, the excited area with Xe-flash lamp is at least 3 mm2 (1 × 3 mm), which is 9% of the bottom area, and in Plate Chameleon the area excited with laser is measured to be at most 0.24 mm2 (around 0.3 × 0.8 mm),19 being only 0.7% of the bottom area of the well. Thus, the actual amounts detected in the measurements, based on our assumptions, would be for europium label around 8 × 105 molecules in solution and 8 × 106 molecules in solid-phase measurements and for UPC-phosphor around 20 particles in solution and less than 100 particles in solid phase. For UPC-phosphor, similar values have been reported also before by Niedbala et al.12 Although these calculated values are rough estimations, they indicate that less UPC-phosphors than europium chelates can be detected and demonstrate the potential sensitivity of the phosphors as labels. The linearity of detection of bio-Eu was over 5 orders of magnitude in solution and over 4 orders of magnitude in solidphase measurements (Figure 2). For the SA-phosphor the response was linear for 5 orders of magnitude in solution and over 4 orders of magnitude in solid phase (Figure 3). These results show that the excellent dynamic range achieved with lanthanide labels and time-resolved fluorescence is achieved also with UPCphosphors and anti-Stokes photoluminescence. The cross-talk, as can be seen in Figures 2 and 3, was practically nonexistent between the two bioconjugates. No anti-Stokes photoluminescence signal was detected from bio-Eu at visible wavelengths when excited with infrared light (Figure 2). All cross-talk signals of bioEu (Figure 2) were below or very close to the lower limits of detection of SA-phosphor (Figure 3), so they would not have any effect on the signals of SA-phosphor if measured from the same microtitration well. The variation observed in the cross-talk signals of bio-Eu is due to fluctuation in background signals. In the SAphosphor measurements, a very small europium fluorescence signal was detected from the strongest SA-phosphor dilution in solution, but not in solid phase measurement (Figure 3). Most likely, the cross-talk signal resulted from small europium contamination in the UPC-phosphor material. However, as the europium fluorescence measured from SA-phosphor (Figure 3) was close to the lower limit of detection of bio-Eu (Figure 2), and since the very strong phosphor concentrations are impractical for use in bioaffinity assays, the minor cross-talk can be easily avoided. Measurement of Time-Resolved Fluorescence and AntiStokes Photoluminescence from a Single Microtitration Well. As was already demonstrated with the dilution series measurements, both time-resolved fluorescence and anti-Stokes photoluminescence can be measured from the same microtitration well without disturbance from these label technologies to each other. This was further confirmed with two bioaffinity assays, assay I and assay II, with similar principles, using bio-BSA, SA-phosphor, and bio-Eu (Figure 4). In assay I, variable concentrations of bioBSA were first allowed to bind onto immobilized streptavidin in the microtitration wells, after which a constant amount of bio-Eu 2832 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005

Figure 4. Principle of two bioaffinity assays (assay I and assay II) for measuring time-resolved fluorescence (bio-Eu) and anti-Stokes photoluminescence (SA-phosphor) from a single microtitration well. In assay I, bio-BSA is first incubated in streptavidin wells and then bio-Eu is added. In assay II, the situation is vice versa. After washings, in both assay types, SA-phosphor is used to detect bio-BSA molecules bound on solid phase. Either the concentration of bio-BSA or bio-Eu is changed while the amounts of other assay components are kept constant.

Figure 5. Binding curves from assay I with bio-BSA as a variable component. Anti-Stokes photoluminescence of SA-phosphors bound to bio-BSA (filled circles) was measured with Plate Chameleon equipped with infrared laser excitation, and time-resolved fluorescence of bio-Eu (filled triangles) was measured with Victor 1420 multilabel counter.

was added to occupy the remaining streptavidin binding sites. In assay II, variable concentrations of bio-Eu were first allowed to bind onto streptavidins, and thereafter, constant amounts of bioBSA were added. After washing, in both assays, a constant amount of SA-phosphor was used to bind bio-BSA-molecules attached to streptavidins. Resulting binding curves from assay I and assay II were just as expected. In assay I (Figure 5), where the amount of bio-BSA is raised in the low-capacity streptavidin wells, less bio-Eu can bind to solid phase when there is more bio-BSA. SA-phosphor can bind only bio-BSA molecules with two or more biotins and not bio-Eu, whose only biotin is already attached to streptavidin immobilized in the wells. Thus, the phosphor signal rises with the amount of bio-BSA. However, because bio-Eu is such a small molecule compared to bio-BSA, bio-BSA can never totally exclude

Figure 6. Binding curves from assay II with bio-Eu as a variable component. Time-resolved fluorescence of bio-Eu (filled triangles) was measured with Victor 1420 multilabel counter, and anti-Stokes photoluminescence of SA-phosphors bound to bio-BSA (filled circles) was measured with Plate Chameleon equipped with infrared laser excitation.

bio-Eu from the binding sites in the streptavidin well. So, although the maximum anti-Stokes signal is already reached, the europium signal is still higher than the background signal. In assay II (Figure 6), less bio-BSA can bind to streptavidin wells when the amount of bio-Eu increases and, thus, anti-Stokes photoluminescence decreases. However, anti-Stokes photoluminescence stays elevated also with low bio-BSA concentrations, due to the nonspecific binding of the UPC-phosphor bioconjugates onto the solid phase. Anti-Stokes photoluminescence signals in the bioaffinity assays were highly dependent on the quality of SA-phosphor. For improved binding of the phosphor bioconjugate, the amount of streptavidin on the surface of phosphor particles should be increased, on the basis of the work of Soukka et al. who have shown that when there are more biomolecules attached on the surface of phosphor particles, the affinity of the bioconjugates is higher.40 In addition, the nonreversible dissociation of streptavidins from phosphor bioconjugates should be diminished, since free streptavidin, if present in the assay, can block the bio-BSA binding sites from SA-phosphors. Further, the binding of the SA-phosphor to solid phase is also dependent on the quality of the bio-BSA used. Only 40% of bio-BSA molecules in this study were determined to contain two or more biotins, which limits the attachment of SA-phosphors to solid phase. Sub-micrometer-sized, irregularly shaped particles are still kinetically rather slow and susceptible to steric hindrances. Thus, SA-phosphors may not be able to find all the possible binding sites in the bio-BSA-saturated streptavidin wells, lowering anti-Stokes photoluminescence signals measured in the assays. On the other hand, higher anti-Stokes photoluminescence signals would be detected in the assays, if signals at all emission wavelengths of the UPC-phosphors would be measured.19 Only part of the emission signal from the phosphor was now measured at 535 nm; the red emission of the PTIR550/F phosphor at 665 nm was not (40) Soukka, T.; Ha¨rma¨, H.; Paukkunen, J.; Lo¨vgren, T. Anal. Chem. 2001, 73, 2254-2260.

detected at all. For comparison, in europium labels, emission is largely measured in a single peak. Finally, as discussed earlier, because of the small, focused laser beam in Plate Chameleon, only a fraction of phosphor particles bound on the solid phase are excited. The capability of using time-resolved fluorescence labels together with anti-Stokes phosphors enables the development of multiplexed assays. Besides europium, also the other lanthanide labels, terbium, samarium, and dysprosium, could be used together with anti-Stokes phosphors. Further, there are different kinds of UPC-phosphors emitting light at either red, green, or blue wavelengths.21 UPC-phosphors could, thus, complement the lanthanide labels, and multianalyte assays could be performed from a single sample in a common microtitration well platform. Although the bioaffinity assays described above are only model assays, the assay configuration should be applicable also for real samples. Europium and other lanthanide chelate-based labels have already established their use in various assays for determining the analytes from biological sample matrixes,41-43 and also UPCphosphors have been utilized for assaying different specimens.21,33 Therefore, the combination of these two detection technologies is anticipated to work also with real samples. An assay utilizing an up-converting phosphor label and europium label has several advantages over dual-label assays accomplished with two different time-resolved fluorescent lanthanide chelates. First, dual-label assays for determining various analyte pairs have been developed, utilizing usually either the combinations of Eu3+ and Tb3+ or Eu3+ and Sm3+ chelate bioconjugates as labels.44-47 However, for now, all these assays have been based on the principle of dissociative fluorescence enhancement, requiring the development of the chelate with a special enhancement solution, or even two different enhancement solutions in the case of Tb3+, before the fluorescence can be measured. For comparison, no additional enhancement steps are needed in the measurement of anti-Stokes photoluminescence of UPC-phosphor and time-resolved fluorescence of intrinsically fluorescent europium chelate used in this study. Another disadvantage in dual-label assays frequently encountered is the weaker detectability of the other lanthanides compared to Eu3+, affecting the sensitivities of the assays.44,48 Due to minimal background of anti-Stokes photoluminescence measurements, originating only from the photomultiplier tube, and the intense photoluminescence signals obtained from UPC-phosphors, the sensitivity problem can be easily avoided in the dual-label assay utilizing UPC-phosphor and europium chelate. Finally, the major advantage of simultaneous use of the two different measurement technologies, antiStokes photoluminescence of UPC-phosphors and time-resolved (41) Nurmikko, P.; Pettersson, K.; Piironen, T.; Hugosson, J.; Lilja, H. Clin. Chem. 2001, 47, 1415-1423. (42) von Lode, P.; Rainaho, J.; Pettersson, K. Clin. Chem. 2004, 50, 1026-1035. (43) Hagren, V.; Crooks, S. R. H.; Elliott, C. T.; Lo¨vgren, T.; Tuomola, M. J. Agric. Food Chem. 2004, 52, 2429-2433. (44) Hemmila¨, I.; Holttinen, S.; Pettersson, K.; Lo ¨vgren, T. Clin. Chem. 1987, 33, 2281-2283. (45) Qin, Q. P.; Christiansen, M.; Lo¨vgren, T.; Norgaard-Pedersen, B.; Pettersson, K. J. Immunol. Methods 1997, 205, 169-175. (46) Eriksson, S.; Vehnia¨inen, M.; Janse´n, T.; Meretoja, V.; Saviranta, P.; Pettersson, K.; Lo ¨vgren, T. Clin. Chem. 2000, 46, 658-666. (47) Zhu, L.; Leinonen, J.; Zhang, W. M.; Finne, P.; Stenman, U. H. Clin. Chem. 2003, 49, 97-103. (48) Xu, Y. Y.; Pettersson, K.; Blomberg, K.; Hemmila¨, I.; Mikola, H.; Lo ¨vgren, T. Clin. Chem. 1992, 38, 2038-2043.

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fluorometry of lanthanide-based chelates, is the absence of crosstalk and the effect of photobleaching. Often in double-label assays utilizing the two lanthanide chelates, mathematical corrections have to be employed after fluorescence measurements to subtract the unwanted spillover signals originating from slight overlapping of the measurement windows of the labels.48,49 According to the results obtained in this study, no correction coefficients are required when UPC-phosphor and europium chelate bioconjugates are used in the same assay. Also, for example in the assays utilizing Eu3+ and Tb3+, ultraviolet excitation for measuring europium fluorescence may partially bleach the fluorescence of Tb3+ due to very similar excitation wavelengths of these lanthanides. By utilizing an anti-Stokes label along with europium label, this problem is avoided, since neither the ultraviolet excitation has an effect on the signals of UPC-phosphors nor the infrared excitation has an effect on the signals of europium chelate, irrespective of the excitation and measurement order of these two labels. Besides utilizing multiple labels, also microarray-type assays can be used for multianalyte assays. In principle, the microtitration well can be scanned with the small laser excitation beam to enable the measurement of different analytes in multiple spots on the microtitration well surface50 to be performed also with UPCphosphors. The possibility to measure anti-Stokes photoluminescence of UPC-phosphors and time-resolved fluorescence of lanthanide chelates simultaneously from the single microtitration well and the potential for utilizing the phosphor bioconjugates in an array-in-well approach shows the significant versatility of the up-converting phosphors as labels in bioaffinity assays. (49) Xu, Y. Y.; Hemmila¨, I. A. Anal. Chim. Acta 1992, 256, 9-16. (50) Ekins, R.; Chu, F.; Biggart, E. Clin. Chim. Acta 1990, 194, 91-114.

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CONCLUSIONS In this work, we have shown that it is possible to use UPCphosphors and more conventional europium labels together in a bioaffinity assay. Anti-Stokes photoluminescence signals of UPCphosphors and time-resolved fluorescence signals of europium label are measured from the same microtitration well without any effect from these different signals to each other. No additional enhancement steps are needed in the measurements and spatial resolution is retained. In principle, the simultaneous use of these two label technologies, anti-Stokes photoluminescence of UPCphosphors and time-resolved fluorescence based on lanthanide chelates, would enable the development of multiparameter assays using different labels for different analytes to be measured from the same sample well. Employment of UPC-phosphors in bioaffinity assays is, however, still in its infancy. Grinding, purification, conjugation to biomolecules, and utilization of these particles in bioaffinity assays need still a lot of optimization. Nevertheless, in the future up-converting anti-Stokes photoluminescent phosphors could be employed in bioaffinity assays as very potential labels with significant advantages either alone or together with longlifetime lanthanide chelates. ACKNOWLEDGMENT Tekes, the National Technology Agency of Finland, and the Academy of Finland (Grant No. 209417) are gratefully acknowledged for supporting this research.

Received for review December 9, 2004. Accepted February 5, 2005. AC048186Y