Homogeneous Assay Technology Based on Upconverting Phosphors

Anal. Chem. 2005, 77, 7348-7355

Homogeneous Assay Technology Based on Upconverting Phosphors Katri Kuningas,* Terhi Rantanen, Telle Ukonaho, Timo Lo 1 vgren, and Tero Soukka

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

Upconversion photoluminescence can eliminate problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. We have demonstrated a luminescence resonance energy-transfer-based assay utilizing inorganic upconverting (UPC) lanthanide phosphor as a donor and fluorescent protein as an acceptor. UPC phosphors are excited at near-infrared and they have narrow-banded anti-Stokes emission at visible wavelengths enabling measurement of the proximity-dependent sensitized emission with minimal background. The acceptor alone does not generate any direct emission at shorter wavelengths under near-infrared excitation. A competitive model assay for biotin was constructed using streptavidin-conjugated Er3+,Yb3+-doped UPC phosphor as a donor and biotinylated phycobiliprotein as an acceptor. UPC phosphor was excited at near-infrared (980 nm) and sensitized acceptor emission was measured at red wavelength (600 nm) by using a microtitration plate fluorometer equipped with an infrared laser diode and suitable excitation and emission filters. Lower limit of detection was in the subnanomolar concentration range. Compared to time-resolved fluorometry, the developed assay technology enabled simplified instrumentation. Excitation at near-infrared and emission at red wavelengths render the technology also suitable to analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in high-throughput screening.

Various approaches have been introduced in the past decade to overcome the problems encountered in homogeneous assays. New label technologies based on photoluminescence have gained much interest, since several types of physical and chemical interactions can be used to modulate the emission of a photoluminescent label within specific immunological complexes.1,2 Potentially very sensitive assays have been constructed by utilizing proximity-based techniques, e.g., luminescent oxygen channeling immunoassay (LOCI)4,5 and time-resolved luminescence resonance energy-transfer (LRET) assays based on lanthanide cryptates,6 lanthanide chelates,7 and lanthanide-chelate dyed nanoparticles.8 Moreover, inorganic lanthanide-doped core/shell nanoparticles suitable for LRET assays and possessing very high energy-transfer efficiency have been recently reported.9 However, there are still some major disadvantages inherent to these assay methods when employed in clinical applications. For example, due to the ultraviolet excitation of the donor molecule, time-resolved luminescence resonance energy-transfer assays are unsuitable for analysis of complex biological samples having strong absorption of light at the ultraviolet region, such as whole blood. Moreover, the time-resolved ultraviolet-excited fluorometer required for these assays is rather expensive for use in routine diagnostic applications. Relatively new types of photoluminescent label molecules, introduced in biomedical research in the late 1990s,10,11 are upconverting (UPC) rare earth phosphors with unique photoluminescence features.12 UPC phosphor particles, consisting of certain lanthanide dopants embedded in a crystalline host lattice, can convert infrared excitation light into emission at visible

In the development of simple, rapid and inexpensive, yet sensitive, reliable, and quantitative ligand binding assays, homogeneous assay methods have received much attention. In these assays, the signal of the labeled component is modulated by binding, eliminating the need to separate the bound and the free label.1 This shortens the total assay time and significantly simplifies the construction of an instrument required to perform an assay automatically. However, the nonseparation nature of these assays also sets some major limitations to their performance; homogeneous assay technologies can be very prone to interference from biological sample material, the universal employment of the technologies to different analytes and assay formats can be difficult, and sensitivity of the assays is often inadequate.2,3

(2) Mathis, G. Clin. Chem. 1993, 39, 1953-1959. (3) Ullman, E. F. J. Chem. Educ. 1999, 76, 781-788. (4) Ullman, E. F.; Kirakossian, H.; Singh, S.; Wu, Z. P.; Irvin, B. R.; Pease, J. S.; Switchenko, A. C.; Irvine, J. D.; Dafforn, A.; Skold, C. N.; Wagner, D. B. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5426-5430. (5) Ullman, E. F.; Kirakossian, H.; Switchenko, A. C.; Ishkanian, J.; Ericson, M.; Wartchow, C. A.; Pirio, M.; Pease, J.; Irvin, B. R.; Singh, S.; Singh, R.; Patel, R.; Dafforn, A.; Davalian, D.; Skold, C.; Kurn, N.; Wagner, D. B. Clin. Chem. 1996, 42, 1518-1526. (6) Mathis, G. Clin. Chem. 1995, 41, 1391-1397. (7) Hemmila¨, I. J. Biomol. Screening 1999, 4, 303-308. (8) Kokko, L.; Sandberg, K.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2004, 503, 155-162. (9) Meyer, C.; Haase, M.; Hoheisel, W.; Bohmann, K., Patent application WO 2004/096944, 2004. (10) Wright, W. H.; Mufti, N. A.; Tagg, N. T.; Webb, R. R.; Schneider, L. V. Proc. SPIE 1997, 2985, 248-255. (11) 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. Patent 5,674,698, 1997. (12) Soukka, T.; Kuningas, K.; Rantanen, T.; Haaslahti, V.; Lo¨vgren, T. J. Fluoresc. 2005, 15, 513-528.

* Corresponding author: (telephone) +358-2-333-8056; (fax) +358-2-333-8050; (e-mail) [email protected]. (1) Hemmila¨, I. Clin. Chem. 1985, 31, 359-370.

7348 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

10.1021/ac0510944 CCC: $30.25

© 2005 American Chemical Society Published on Web 10/06/2005

wavelengths via the sequential absorption of two or more lowenergy photons.13 No other material possesses this capability for upconvertive anti-Stokes photoluminescence at conventional excitation light intensities, and the phosphor emission can be measured entirely free of autofluorescence and scattered excitation light, enabling immunoassays with low background fluorescence signals.14 Many advantages of UPC phosphors have already rendered these particulates very attractive for use as labels in various bioaffinity assays.15-19 However, at least to our knowledge, no reports of the use of upconverting phosphor particles in homogeneous, LRET assays have been published. We have developed a novel homogeneous upconversion luminescence resonance energy-transfer assay technology utilizing a particulate upconverting rare earth phosphor as a donor and a fluorescent phycobiliprotein as an acceptor. A model assay for biotin is described, in which the streptavidin-conjugated donor is excited at near-infrared and the sensitized emission from the biotinylated acceptor is measured at visible wavelength. Because of the extremely narrow and sharp emission bands of the UPC phosphors characteristic to luminescent lanthanide ions, practically no donor emission is detectable at the measurement wavelength of the sensitized emission from the acceptor. Due to the unique nature of upconversion, no direct acceptor emission, autofluorescence, or scattered excitation light is generated at visible wavelengths under near-infrared excitation, enabling measurements without temporal resolution. EXPERIMENTAL SECTION Reagents. Infrared to visible UPC anti-Stokes phosphors FCD546-1 and FCD-546-2 with structural compositions of La 2O2S:Yb3+,Er3+ and Y2O2S:Yb3+,Er3+, respectively, were obtained from Luminophor SPF (Stavropol, Russia). The photoluminescence and chemical characteristics of these phosphors have been described previously.12 Fluorescent phycobiliprotein, B-phycoerythrin (BPE), was purchased from Cyanotech Corp. (Kailua-Kona, HI), supplied in 100 mM sodium phosphate buffer, pH 7.0, containing 60% (w/ v) ammonium sulfate and 0.02% (w/v) sodium azide, with protein concentration of 10 mg/mL. Additol XW330,20 an aqueous solution of an ammonium salt of a poly(acrylic acid) (MW 30 000-50 000), was obtained from Surface Specialties Austria GmbH (Werndorf, Austria). Streptavidin was purchased from Spa-BioSpa (Milan, Italy), biotinamidohexanoic acid N-hydroxysuccinimide ester (NHS-LC-biotin) was from Pierce Biotechnology (Rockford, IL), N-(3-dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDAC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) (13) Auzel, F. Proc. SPIE 2002, 4766, 179-190. (14) Corstjens, P. L. A. M.; Li, S.; Zuiderwijk, M.; Kardos, K.; Abrams, W. R.; Niedbala, R. S.; Tanke, H. J. IEE Proc.-Nanobiotechnol. 2005, 152, 64-72. (15) 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. (16) 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. (17) 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. (18) Zuiderwijk, M.; Tanke, H. J.; Niedbala, R. S.; Corstjens, P. L. A. M. Clin. Biochem. 2003, 36, 401-403. (19) Kuningas, K.; Rantanen, T.; Karhunen, U.; Lo ¨vgren, T.; Soukka, T. Anal. Chem. 2005, 77, 2826-2834. (20) 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.

were from Fluka (Buchs, Switzerland), Tween 20 and Tween 85 were from E. Merck (Darmstadt, Germany), d-biotin was obtained from Sigma-Aldrich (St. Louis, MO), and bovine serum albumin fraction V (BSA) was from Bioreba (Nyon, Switzerland). Black Costar Half Area microtitration wells were purchased from Corning Inc. (Corning, NY). 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), and Delfia enhancement solution (DES) and enhancer solution (DE) were purchased from PerkinElmer Life and Analytical Sciences (Wallac Oy, Turku, Finland). Spectral Characterizations. Anti-Stokes photoluminescence emission spectra of the UPC phosphors, suspended in dimethyl sulfoxide in appropriate dilutions, were measured using Varian Cary Eclipse fluorescence spectrophotometer (Varian Scientific Instruments, Mulgrave, Australia) equipped with an infrared laser diode module C2021-F1 (Roithner Lasertechnik, Vienna, Austria).12 For spectral characterizations of the fluorescent protein (BPE), 0.5 mg of the protein was first dialyzed against 150 mM NaCl using cellulose tubular membrane (CelluSep T2, flat width 10 mm, MWCO 6000-8000, Membrane Filtration Products, Inc., Seguin, TX) to remove ammonium sulfate and azide. The dialyzation was performed overnight at 4 °C, protected from light, according to the instructions of the phycobiliprotein and dialyzation membrane manufacturers. Protein concentration after dialyzation was determined by absorbance measurements at 546 nm, using molar absorption coefficient 546 nm ) 2 400 000 M-1 cm-1.21 Fluorescence excitation and emission spectra of the BPE was then measured with a Varian Cary Eclipse fluorescence spectrophotometer from the protein solution diluted in Tris-saline-azide buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.5 g/L NaN3) in poly(methyl methacrylate) cuvettes (3.5-mL capacity and 10 mm × 10 mm light path) with four clear sides (Kartell, Noviglio, Italy). The emission spectrum from BPE was measured from 565 to 750 nm by 0.75nm resolution with 5-nm emission slit, utilizing the fluorescence mode of the fluorescence spectrophotometer and an excitation wavelength of 555 nm with excitation slit of 10 nm. The excitation spectrum from BPE was measured from 250 to 575 nm by 0.75nm resolution with 5-nm excitation slit, utilizing the fluorescence mode of the fluorescence spectrophotometer and an emission wavelength of 585 nm with emission slit of 10 nm. Biotinylation of the Fluorescent Protein. The dialyzed BPE was biotinylated with a 25-fold molar excess of NHS-LC-biotin. Freshly prepared solution of NHS-LC-biotin in dimethylformamide (DMF) was added into protein solution containing 8.5 mg/mL phycobiliprotein in 50 mM carbonate buffer, pH 9.3. The reaction mixture with a total volume of 500 µL, containing ∼2% (v/v) DMF, was incubated in rotation at 17 rpm (Rotamix RK, Heto-Holten A/S, Allerød, Denmark) protected from light for 3 h at room temperature. Finally, biotinylated B-phycoerythrin (bio-BPE) was purified with NAP-5 and NAP-10 columns (Amersham Biosciences AB, Uppsala, Sweden) using aqueous solution of 150 mM NaCl. Protein concentration was determined by absorbance measurement, and the biotinylation of the protein was confirmed by a (21) Kronick, M. N. J. Immunol. Methods 1986, 92, 1-13.

Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

7349

competitive, heterogeneous binding assay for biotin. In the assay, d-biotin in a concentration range from 0 to 500 nM or appropriate dilutions of biotinylated protein were first added in a 50-µL volume of assay buffer into microtitration wells coated with 20 ng of streptavidin.19 The wells were incubated for 20 min at room temperature with slow shaking (Delfia Plateshake, PerkinElmer Life and Analytical Sciences) and thereafter, 1.2 nM biotin conjugate of Tb-chelate19 was added to the wells in 25 µL of assay buffer to compete for the streptavidin-binding sites. Incubation was continued for 30 min, and the wells were washed twice with wash solution (Delfia Platewash, PerkinElmer Life and Analytical Sciences). For terbium fluorescence signal development, DES and DE were then used according to manufacturer’s instructions. Finally, terbium fluorescence was measured with Victor 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences) at 545 nm using the standard terbium measurement protocol. The biotinylated phycobiliprotein preparation was stabilized by addition of 0.1% (w/v) BSA and 0.05% (w/v) NaN3 and stored at 4 °C protected from light. Streptavidin Coating of the UPC Phosphors. Commercial, micrometer-sized UPC phosphor particles were bead-milled and purified according to the procedure by Soukka et al.12 to produce colloidal suspension of submicrometer-sized particles. Concentration of phosphor suspension was obtained by weighting dried phosphors,19 and size distribution profile was measured in measurement buffer (10 mM borate buffer, pH 8.5, containing 1 g/L Tween 20) using Coulter N4plus submicrometer particle size analyzer (Beckman Coulter, Fullerton, CA). Thereafter, the submicrometer-sized phosphors were conjugated to streptavidin.19 Briefly, 7.5 mg of phosphors was first suspended into 750 µL of 2.5% (v/v) Additol XW330, pH 9, in Milli-Q water, to a concentration of 10 mg/mL using a bath sonicator (Finnsonic m03; Finnsonic Oy, Lahti, Finland). The suspension was slowly agitated overnight at room temperature in Rotamix RK at 17 rpm (HetoHolten A/S). Thereafter, the poly(acrylic acid)-coated phosphors were washed four times with Milli-Q water and once with 20 mM MES, pH 6.1, using centrifugation (12 000 rpm; Sorvall MC 12V, F-12/M18, Sorvall Products, Newtown, CT) and bath sonication. Finally, phosphors were resuspended into 780 µL of 20 mM MES, pH 6.1, containing 20 mM EDAC and 30 mM sulfo-NHS, and the suspension was incubated for 1 h in rotation. The activated phosphors were washed once with 20 mM MES, pH 6.1, resuspended into 750 µL of 20 mM MES, pH 6.1, containing 0.5 mg/mL streptavidin, and left to rotate slowly for 2.5 h before stopping the reaction by addition of 2 M glycine, pH 11, to a final concentration of 50 mM. The incubation was continued for 30 min, the coated phosphors were washed three times with measurement buffer and finally suspended into 750 µL of 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 were stored at room temperature in slow rotation. Concentration of the coated phosphors was determined by measuring anti-Stokes photoluminescence at 535 nm in comparison with a known concentration of uncoated phosphors. Size distribution profile was measured similarly to uncoated phosphors. The amount of streptavidin attached onto phosphors and concentration of free, unbound streptavidin present in phosphor suspension were measured using a heterogeneous, competitive binding 7350


assay for streptavidin.19 First, unconjugated streptavidin was separated from a known volume of phosphor suspension by using a centrifuge tube filter (VectaSpin Micro with Anopore 0.02-µm membrane; Whatman, Maidstone, England) and collecting the filtrate. In the assay, 0-50 ng of streptavidin in 75 µL of assay buffer, or appropriate dilutions of phosphor suspension and filtrate, were mixed with 75 µL of 4 nM biotin conjugate of Tb-chelate and the reactions were incubated for 15 min at room temperature in slow rotation. Thereafter, 50-µL volume of each of the reactions was transferred to normal-capacity streptavidin-coated wells, and the wells were incubated for 30 min at room temperature with slow shaking to capture all unbound biotin conjugates of Tbchelate. Finally, the wells were washed twice and the signal was developed and measured similarly to the heterogeneous biotinbinding assay. Titration of the Biotinylated Fluorescent Protein. Optimal acceptor concentration in the homogeneous upconversion LRET assay was first determined by the titration of bio-BPE. Total reaction volume in the titration assays was 50 µL, containing the following reagent concentrations. Into black, half-area microtitration wells, 20 µL of assay buffer without d-biotin or with 10 µM d-biotin and 15 µL of 0.05 mg/mL streptavidin-conjugated UPC phosphor in assay buffer were first added. The wells were incubated for 15 min at room temperature in slow shaking, and thereafter, 15 µL of bio-BPE in assay buffer in a concentration range from 0 nM to 16 nM was added into the wells. Four replicates of each acceptor concentration were utilized. The wells were protected from light with aluminum foil, and the incubation was continued for 15 min. The anti-Stokes photoluminescence from the phosphors at 550 nm and upconversion LRET emission from the phycobiliproteins at 600 nm were then measured with Plate Chameleon (Hidex Oy, Turku, Finland) equipped with a 200mW infrared laser module (Roithner Lasertechnik) and RG-850 long-pass excitation filter (Andover Corp., Salem, NH).12 The photomultiplier tube in the plate fluorometer was replaced with Hamamatsu R4632 (Hamamatsu Photonics K.K.). Emission light at both measurement wavelengths was collected for 2000 ms under continuous laser excitation at 980 nm. At 550 nm, anti-Stokes photoluminescence emission was measured employing a bandpass emission filter of 535/50 nm (center wavelength, 535 nm; half-width, 50 nm; peak transmittance, g60%; Bk Interferenzoptik Elektronic, Nabburg, Germany), combined with an absorptive neutral density filter (optical density 2.0; i.e., average transmittance 1%; Thorlabs, Newton, NJ). At 600 nm, sensitized emission from the energy transfer was measured employing a band-pass emission filter of 600/40 nm (peak T g 80%; Chroma Technology Corp.), combined with a short-pass filter glass KG-1 (Andover Corp.). In addition to the measurement after 15 min of incubation, two similar measurements were also carried out in the assay at time points 45 min and 2 h. Titration of the Streptavidin-Conjugated UPC Phosphors. The concentration of streptavidin-conjugated UPC phosphor donor was optimized by utilizing a similar assay setup than was employed in the titration of bio-BPE. In the titration of FCD-546-1 streptavidin-phosphor, a concentration range from 0 to 0.1 mg/mL phosphor in the assay buffer was analyzed, using acceptor concentrations of 1 and 2 nM. Four replicates of each phosphor concentration were studied. In the titration of streptavidin-

Figure 1. Emission spectrum of the Er3+,Yb3+-doped UPC phosphor (FDC-546-1) and emission and excitation spectra of BPE. Emission spectrum of FCD-546-1 phosphor (solid line) overlaps with the excitation spectrum of BPE (wide, dashed line). Emission spectrum of BPE (narrow, dashed line) is separated from the phosphor emission, and the sensitized emission of BPE can be measured at 600 nm using a band-pass emission filter and continuous laser excitation at 980 nm. au, arbitrary unit.

conjugated FCD-546-2 phosphor, a concentration range from 0 to 0.2 mg/mL phosphor was analyzed, utilizing bio-BPE concentrations of 0.5 and 1.5 nM. D-Biotin, streptavidin-phosphor, and bioBPE were added to black, half-area microtitration wells, and incubations and measurements were performed as in the titration of the biotinylated phycobiliprotein. Competitive Ligand-Binding Assay for Biotin. The assay setup in a competitive, homogeneous assay for biotin was similar to that in the titrations of the biotinylated fluorescent protein and streptavidin-conjugated phosphors. A concentration range from 0 to 102.4 nM d-biotin in assay buffer was analyzed, each in four replicates, and the concentration of 10 µM d-biotin was used to verify the background level. When streptavidin-conjugated FCD546-1 phosphor was the donor, the assays were carried out utilizing 0.01 or 0.05 mg/mL phosphor and 1 nM bio-BPE in assay buffer. With streptavidin-conjugated FCD-546-2 phosphor, either 0.01 mg/mL phosphor and 0.5 nM bio-BPE or 0.05 mg/mL phosphor and 1.5 nM bio-BPE were employed. RESULTS Spectral Principle of the Homogeneous Upconversion LRET Assay. According to spectral characterizations, the fluorescent protein BPE has an excitation maximum at ∼550 nm. This rather wide excitation band overlaps well with the emission spectra of the upconverting phosphors (Figure 1), enabling the luminescence resonance energy-transfer between streptavidin-conjugated UPC phosphor donor and biotinylated fluorescent protein acceptor (Figure 2). Furthermore, due to the extremely narrow and sharp emission bands of the phosphors, it is possible to measure the sensitized emission from the phycobiliprotein acceptor completely free of donor emission at only tens of nanometers aside from the emission bands of the phosphors by using a suitable band-pass emission filter (Figure 1). The large anti-Stokes shift characteristic to upconverting rare earth phosphors enables total elimination of the scattered excitation laser light from the measurements by an inexpensive combination of long-pass and short-pass filters, blocking excitation at wavelengths below 850 nm, and preventing infrared excitation light from entering to the photomultiplier tube.12 Because of the Er3+ and Yb3+ ions in their crystal structure, FCD-546-1 and FCD-546-2 UPC phosphors have emission maxi-

Figure 2. Principle of the competitive homogeneous upconversion LRET assay using biotin as a model analyte. Er3+,Yb3+-doped UPC phosphor (donor) produces emission at 550 nm upon excitation at 980 nm. Binding sites of streptavidin-conjugated donor are occupied either by biotin or bio-BPE. Bound bio-BPE (acceptor) is excited via LRET, and the sensitized acceptor emission can be measured at 580-600 nm.

mums around 550 and 660 nm (Figure 1). In this study, emission maximum at 550 nm and the emission minimum next to this band at range 580-620 nm were employed. It is noted that, by using acceptors with suitable absorption and emission maximums, the second emission band of the phosphors at 660 nm and the emission minimum above 700 nm could also be utilized, enabling measurement of the sensitized acceptor emission at far-red (700740 nm) wavelengths. Characterization of the Biotinylated Phycobiliprotein and Streptavidin-Coated Phosphors. Bio-BPE was characterized for its ability to tightly bind immobilized streptavidin; over 90% of the protein expressed at least a single reactive biotin residue, ensuring the functionality of bio-BPE in the assays. The size distribution obtained for the streptavidin-coated UPC phosphors indicated only ∼10-nm increase to original distributions of uncoated phosphors; original size distributions were 240-350 nm (average 310 nm) for FCD-546-1 preparation and 270-350 nm (320 nm) for FCD546-2. Obtained phosphor and streptavidin concentrations in the stock solutions of the streptavidin-coated phosphors were 12.1 mg/mL and 206 µg/mL for FCD-546-1 preparation and 9.2 mg/ mL and 185 µg/mL for FCD-546-2. The degree of free, unbound streptavidin was 10% for FCD-546-1 preparation and 1.4% for FCDAnalytical Chemistry, Vol. 77, No. 22, November 15, 2005

7351

546-2. Based on obtained average size and density information12 of the phosphor material the average number of bound streptavidins per phosphor particle were 15 000 and 20 000 for FCD546-1 and FCD-546-2 preparations, respectively. However, due to the wide size distributions of the phosphor particles, these values for streptavidin-binding sites are only indicative. Titration of the Phycobiliprotein and UPC Phosphor Bioconjugates. The homogeneous competitive assay for biotin (Figure 2) was used as a model assay to study the upconversion LRET principle. The assay was performed with two different streptavidin-conjugated UPC phosphors (FCD-546-1, FCD-546-2) to demonstrate the general applicability of the principle. Optimal acceptor and donor amounts for the biotin assays were determined by utilizing only two biotin concentrations, either zero or excess of d-biotin. The aim was to enable measurements with high specific, proximity-based resonance energy-transfer signals and minimal background fluorescence signals, i.e., to maximize the signal-to-background ratios. Furthermore, optimization was important to avoid the possible situation where fluorescent proteins on the surface of UPC phosphor particles would nonradiatively quench the fluorescence of neighboring proteins and produce considerably reduced signals. It is noted that the final upconversion LRET signals for the assays were calculated using eq 1. The equation was employed to correct the sensitized acceptor emission signals measured at 600 nm (I600) for variation in laser intensity, because a change in laser power would have caused a second power change in the emission intensity of UPC phosphors,22 resulting in a respective change in upconversion LRET signals. Before correcting the values, the instrumental background originating from the photomultiplier dark current was subtracted from all signals. The correction was based on the normalization of the phosphor emission signals measured at 550 nm (I550) by an average of reference signals measured from the phosphors alone, i.e., without acceptor in the same reaction well (I*550). To obtain signal values consisting only of energy-transfer signals, background signals measured from the phosphors at 600 nm (I*600) were subtracted from the corrected values. The correction based on donor emission was possible due to the submicrometer size of UPC phosphors used in this study (diameter ∼300 nm); in these particles only emissive ions located near the particle surfaces are within the distance requirement of luminescence resonance energy transfer9,23 and thus, no significant decrease in the donor emission can be observed.

upconversion LRET ) [I600(I*550/I550)] - I*600

(1)

Maximum proximity-based resonance energy-transfer signals for every acceptor or donor concentration analyzed were obtained from the reactions performed without d-biotin, in which streptavidin-conjugated phosphors and biotinylated phycobiliproteins can freely bind to each other. In contrast, in the reactions carried out with excess of d-biotin, no specific binding can occur, because the streptavidin binding sites of the donor particles are completely (22) Heer, S.; Ko ¨mpe, K.; Gu ¨ del, H. U.; Haase, M. Adv. Mater. 2004, 16, 21022105. (23) Bazin, H.; Pre´audat, M.; Trinquet, E.; Mathis, G. Spectrochim. Acta, Part A 2001, 57, 2197-2211.

7352


blocked by biotin. Signals detected in these reactions were the background signals, originating mainly from the reabsorption of the UPC phosphor emission by the unbound fluorescent proteins, due to the rather large size of the phosphors used. Since only emissive ions located near the particle surfaces can participate in the LRET,9,23 considerably larger fraction of emissive ions in the core of the particles produce signals only through nonproximitybased reabsorptive energy-transfer,24 increasing linearly with the acceptor or donor concentrations. By analyzing various phycobiliprotein and UPC phosphor concentrations, it was possible to determine the concentrations giving the largest difference between the nonradiative resonance energy-transfer signals and reabsorptive energy-transfer signals, i.e., the largest signal-to-background ratio. To eliminate the potential quenching effects from the measurements, the optimal concentrations of phycobiliprotein and UPC phosphor bioconjugates for the biotin assays were considered to be those in which the signal-to-background ratios were high but resonance energy-transfer signals still showed no saturation after 2 h of incubation. In the acceptor titration, best signal-to-background ratios close to 10 were obtained when lower phycobiliprotein concentrations were utilized. For example, when FCD-546-1 streptavidinphosphor and bio-BPE were used, high ratios were detected with the bio-BPE concentrations from 0.125 to 2 nM, maximum signalto-background ratio being observed with 1 nM bio-BPE after 2 h of incubation (Figure 3a). In all the assays, 2 nM concentration was the point after which the proximity-based energy-transfer signals no longer grew linearly, i.e., started to saturate. The signal increase observed after this saturation point in the higher acceptor concentrations was most likely due to the reabsorptive energy transfer and nonspecific binding. In the donor titrations, the highest signal-to-background ratios up to 10 were detected for FCD-546-1 bioconjugate when 0.025 and 0.05 mg/mL phosphor concentrations and 1 nM bio-BPE were used (Figure 3b). As in the acceptor titrations, even higher signalto-background ratios (up to ∼20) were obtained with FCD-546-2 bioconjugate but energy-transfer signals were lower compared to FCD-546-1 (data not shown). With both phosphors, the use of higher acceptor concentration increased the energy-transfer signals but decreased the signal-to-background ratios, due to the increase also in background signals. Depending on the acceptor concentration used, assay signals started to saturate with phosphor concentrations higher than 0.05 mg/mL. Thus, donor concentrations of ∼0.05 mg/mL were considered to give the highest possible resonance energy-transfer signals and good signal-tobackground ratio in the biotin assay. According to both acceptor and donor titrations, reaction kinetics with both phosphors were rather fast, since the upconversion LRET signals were almost the same after 15 min and 2 h of incubation. In the titrations of phycobiliprotein and UPC phosphor concentrations, high specific proximity-based energy-transfer signals and signal-to-background ratios were detected, comparable to those observed previously in time-resolved fluorescence resonance energy-transfer-based assays using particulate lanthanide-chelate dyed nanoparticle as a donor.8 However, it is anticipated that even (24) Valanne, A.; Lindroos, H.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2005, 539, 251-256.

Figure 3. Optimization of acceptor and donor concentrations for the biotin assay using streptavidin-conjugated FCD-546-1 phosphor as a donor and bio-BPE as an acceptor. (a) Acceptor titration using 0.05 mg/mL phosphor donor and (b) donor titration using 1 nM bioBPE as the acceptor. Maximal upconversion LRET signals (squares), background signals (circles), and maximal signal to background (Sg/ Bg) ratios (triangles) after 15 min (solid lines, filled symbols) and 2 h (dashed lines, open symbols) of incubation.

greater signal-to-background ratios can be obtained, if smaller UPC phosphor particles capable for efficient upconversion emission22,25 are employed as donors. By using smaller phosphor particles, a larger proportion of the emissive ions in the particles would participate in the nonradiative resonance energy-transfer because of the shortened distances,9 and less nonproximity-based reabsorptive energy-transfer would be observed. This would lead to lower background signals and, thus, higher signal-to-background ratios. Due to the upconversion process, potentially very low background fluorescence could be obtained, since no antiStokes emission is produced by assay components other than the UPC phosphor donor. For example in this study, in the absence of UPC phosphors, background signals measured at 600 nm were equivalent to the dark current of the photomultiplier tube (below (25) Suyver, J. F.; Aebischer, A.; Biner, D.; Gerner, P.; Grimm, J.; Heer, S.; Kra¨mer, K. W.; Reinhard, C.; Gu ¨ del, H. U. Opt. Mater. 2005, 27, 11111130.

50 counts/s), irrespective of the acceptor concentration analyzed or the presence of d-biotin in the assay buffer (data not shown). Furthermore, signals measured from the phosphors alone at 600 nm were also very low due to the narrow emission bands of the phosphors.12 In addition to lower background and higher signalto-background ratios, smaller phosphor particles could provide even faster kinetics in the measurements, enabling very rapid, yet sensitive and quantitative homogeneous immunoassays to be performed. Competitive Ligand-Binding Assay for Biotin. In a typical competitive assay, an analyte to be determined and a labeled analyte derivative (tracer) are added simultaneously in a reaction well to compete for the limited amount of binding sites. In this study, a variation of a competitive assay was used; a back-titration assay where the analyte (biotin) and the tracer (biotinylated acceptor) are added to the reaction well at different time points was performed to facilitate the manual assay procedure and to allow better sensitivity in the assays.26,27 Acceptor and donor concentrations determined to be optimal by the titration analyses were used in the biotin assays. In addition, assays were also carried out using 5-fold smaller donor concentrations to improve the sensitivity of the upconversion luminescence resonance energy-transfer -based assays. By using 0.01 mg/mL streptavidin-conjugated FCD-546-1 phosphor and 1 nM bio-BPE, a sensitive assay with IC50 values (concentrations that inhibited 50% of the maximum signal) of ∼0.7 nM, after both 15 min and 2 h of incubation, and dynamic range close to 2 orders of magnitude could be constructed (Figure 4a). The bulky size of the biotinylated phycobiliproteins, in addition to the low concentration employed, however, rendered the assay relatively slow. Similar results were also obtained with the FCD-546-2 phosphor bioconjugate and bio-BPE when 0.0075 mg/mL phosphor and 0.5 nM bio-BPE were used (Figure 4b). Signal levels were slightly lower compared to those obtained with FCD-546-1 phosphor, but they could be increased by using a larger phosphor amount (Figure 4c). However, according to the nature of competitive assays, the use of higher phosphor concentrations shifted the working range of the assays to higher biotin concentrations. In addition, when more phosphor was used, there was a very sudden signal decrease observed in the biotin titration curves with all donor-acceptor pairs, rendering the dynamic range of the assays rather narrow. This decrease could be explained by the different sizes of the fluorescent proteins, streptavidin, and biotin and by the several biotin-binding sites in the streptavidin molecule. First, because of the very small size of biotin, more than one biotin can bind to one streptavidin. However, no more than one biotinylated fluorescent protein can fit in one streptavidin conjugated to UPC phosphor, due to the larger size of the phycobiliprotein (240 kDa) compared to streptavidin (60 kDa). Moreover, when bound, the fluorescent protein can also block the binding of other proteins to adjacent streptavidins, although the binding of biotin is still possible. In the assay, when biotin is absent or its concentration is low, there are free binding sites available in UPC phosphor bioconjugates for the biotinylated acceptor to bind, also enabling energy transfer to happen. But when the amount of biotin (26) Rodbard, D.; Ruder, H. J.; Vaitukaitis, J.; Jacobs, H. S. J. Clin. Endocrinol. Metab. 1971, 33, 343-355. (27) Piran, U.; Silbert-Shostek, D.; Barlow, E. H. Clin. Chem. 1993, 39, 879883.


7353

increases, at a certain point all the streptavidin binding sites are suddenly fully saturated with biotin, preventing protein acceptors to bind and leading to a decrease in the signals.

Figure 4. Standard curves of upconversion LRET assays for biotin. Standard curves after incubation time of 15 min (solid lines, filled squares) and 2 h (dashed lines, open squares) using (a) 0.01 mg/ mL streptavidin-conjugated FCD-546-1 phosphor and 1 nM bio-BPE; (b) 0.0075 mg/mL FCD-546-2 streptavidin conjugate and 0.5 nM bioBPE; and (c) 0.0375 mg/mL FCD-546-2 bioconjugate and 1.5 nM bio-BPE. IC50 values for the assays were from 0.7 (a) to 9 nM (c). 7354 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

DISCUSSION Elevated autofluorescence and variable absorption properties of sample material have been major problems when using homogeneous photoluminescence-based assay techniques in biological samples. These problems were partially solved by timeresolved luminescence resonance energy-transfer assays using long-lifetime lanthanide chelates and cryptates as donors.2 Temporal resolution provided a means of avoiding the short-lifetime autofluorescence and direct acceptor emission from the measurements, enabling highly sensitive assays to be performed. However, the ultraviolet excitation of the lanthanide donors has hindered the use of these assays with whole blood, which has efficient absorption of light at the ultraviolet region. Moreover, the timeresolved detection mode renders the instrumentation required rather complicated and expensive. The upconversion-based homogeneous assay technique introduced in this study eliminates the need for both time-resolved detection and ultraviolet excitation. Inorganic upconverting lanthanide phosphors used as donors can be excited with infrared laser light and their emission occurs at visible wavelengths. This unique nature of the upconversion process enables the measurement of the sensitized acceptor emission completely free of autofluorescence and direct acceptor emission. An inexpensive infrared laser diode used as the excitation light source further reduces the cost of the instrumentation for the assay technique. Moreover, infrared excitation of the donor together with emission measurement at red (580-620 nm) or at far-red (700-740 nm) above the major absorption of most of the compounds present in biological samples can also enable measurements in strongly colored samples such as whole blood. The requirement for the assay would be the careful selection of a suitable small molecular acceptor dye, not affected by the fluorescence quenching effects of whole blood.28,29 A competitive assay for biotin was used as a model to demonstrate the principle of the homogeneous, upconversion LRET-based assay. In addition to biotin, competitive assays for other small molecules can be performed with similar assay design. Moreover, it is anticipated that the technology will also be applicable to homogeneous noncompetitive sandwich-type immunoassays when recombinant antibody fragments, for example, Fab or single-chain scFv fragments, are utilized to provide the donoracceptor distances more favorable for resonance energy-transfer assays.8 Besides B-phycoerythrin used in this study, a wellperforming assay was also constructed by utilizing R-phycoerythrin as an acceptor. Furthermore, preliminary results show that in addition to fluorescent proteins, conventional small molecular fluorescent acceptors can also be used in the assay, introducing even wider applicability of the principle. The assay strategy based on a particulate label and a protein or a small molecular dye is one of the most significant advantages of the current upconversion proximity-based assay compared to the luminescent oxygen channeling immunoassay,5 where two large particles are em(28) Abugo, O. O.; Nair, R.; Lakowicz, J. R. Anal. Biochem. 2000, 279, 142150. (29) Abugo, O. O.; Herman, P.; Lakowicz, J. R. J. Biomed. Opt. 2001, 6, 359365.

ployed. Although delayed chemiluminescence emission measurable at shorter wavelengths than irradiation ensures very sensitive assays to be performed with the LOCI, a particle pair formation can be unfavorable in some applications due to steric and kinetic issues. The lower limit of detection in the current assay was in the subnanomolar concentration range. Thus, the sensitivity was comparable to that of the previously reported homogeneous proximity-based immunoassays where europium(III) chelate-dyed nanoparticles were used.8,24 In the homogeneous immunoassay for estradiol described by Kokko et al.,8 the IC50 values were from 0.5 to 4 nM estradiol, depending on the amount of detection antibody in the reaction, while in the present assay system the IC50 values were from 0.7 to 9 nM depending on the phosphor concentration used. It is anticipated that the sensitivity and reproducibility of the upconversion LRET assay could still be improved by, first, optimizing further the assay conditions. Second, more studies are required to improve the stability of the phosphor bioconjugates. As reported previously, the coating method of the phosphors has been associated with the slow dissociation of streptavidin from the surface of phosphor particles.19 As a consequence, free streptavidin in the phosphor preparation can compete for the biotin-binding sites in the assay, thus decreasing the assay sensitivity. Another issue affecting the capacity of the described assay is the still rather large size of the phosphor particles used as donors, increasing the signal variation and decreasing the proximity-based energy-transfer efficiency in the assay system. More efficient energy transfer could be obtained with particles preferably less than 40 nm in diameter, in which larger proportion of the emissive ions could participate in the nonradiative energy-transfer due to shortened distances.9 Although particles of this size are very difficult to produce by grinding, there

are reports showing that nanometer-sized UPC phosphors capable for efficient upconversion emission can be newly synthesized.22,25 We believe that the upconversion LRET technology will provide an attractive assay system platform to be used in clinical laboratory and in high-throughput screening assays, where colorful and fluorescent samples are of concern. CONCLUSIONS We have developed a novel homogeneous assay technology based on upconversion luminescence resonance energy-transfer. The use of inorganic upconverting lanthanide phosphors as donors provides significant and unique advantages. Sensitized acceptor emission can be measured at visible wavelengths completely free of autofluorescence and direct acceptor emission without need for temporal resolution in the detection. The continuous photoluminescence counting and the use of laser diode excitation simplify the detection of upconversion LRET compared to timeresolved fluorometry. Furthermore, the employed wavelength range is above the major absorption in biological samples with potential advantages in clinical applications. ACKNOWLEDGMENT This study was supported by the National Technology Agency of Finland (TEKES) and the Academy of Finland (Grant 209417), and in part by the grant from the Instrumentarium Science Foundation denoted to K.K. The authors acknowledge technological support from Hidex Oy in anti-Stokes photoluminescence measurement. Received for review June 21, 2005. Accepted September 6, 2005. AC0510944


7355

Homogeneous Assay Technology Based on Upconverting Phosphors

Recommend Documents