Upconversion Fluorescence Resonance Energy Transfer in a

and purified using SoftLink Soft Release Avidin Resin-based affinity chromatography (Promega Corp., Madison, WI). Terbium(III) chelate of ...
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Anal. Chem. 2006, 78, 4690-4696

Upconversion Fluorescence Resonance Energy Transfer in a Homogeneous Immunoassay for Estradiol Katri Kuningas,* Telle Ukonaho, Henna Pa 1 kkila 1 , Terhi Rantanen, Jaana Rosenberg, Timo Lo 1 vgren, and Tero Soukka

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

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17β-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches. Assays used in clinical diagnostics ought to be sensitive yet also rapid and automatable to enable the analysis of hundreds or thousands of samples in central laboratories or single samples in point-of-care situations, reliably and cost-efficiently. The same requirements are met in high-throughput screening assays where the number of compounds to be studied is constantly increasing due to the advantages in the fields of genomics and proteomics. High sensitivity and wide dynamic ranges can be achieved by using heterogeneous assays based on time-resolved fluorescence and long-lifetime lanthanide chelates.1-3 These assays are also quantitative and can be performed very rapidly.4 However, * Corresponding author: (telephone) +358-2-333-8085; (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) Soini, E.; Lo ¨vgren, T. CRC Crit. Rev. Anal. Chem. 1987, 18, 105-154. (3) Soukka, T.; Paukkunen, J.; Ha¨rma¨, H.; Lo¨nnberg, S.; Lindroos, H.; Lo¨vgren, T. Clin. Chem. 2001, 47, 1269-1278. (4) von Lode, P.; Rainaho, J.; Pettersson, K. Clin. Chem. 2004, 50, 1026-1035.

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heterogeneous assays always include at least one separation step, which complicates the construction of an automated instrument.5 To eliminate the cumbersome separation steps, numerous homogeneous assays have been constructed. In these assays the signal of the labeled component is modulated by binding, eliminating the need to separate the bound and the free label.6,7 Among the most sensitive luminescence-based homogeneous assays are time-resolved fluorescence resonance energy-transfer (TR-FRET) assays, utilizing the unique fluorescent properties of lanthanide chelates,8 lanthanide cryptates,7 or lanthanide chelate-dyed nanoparticles.9 In FRET, energy is transferred from a fluorescent donor molecule via a nonradiative dipole-dipole interaction to an acceptor molecule.10 The inverse association of the energy transfer to the sixth power of the distance between the donor and the acceptor requires that the two molecules are in close proximity. In addition, the fluorescence spectrum of the donor has to overlap, at least partially, the excitation spectrum of the acceptor. In TRFRET assays, the long-lived emission from the lanthanide donor extends the observed lifetime of the sensitized acceptor emission, allowing energy-transfer signals to be measured in a time-resolved manner without background interference from the assay matrix or disturbance from the direct short-lived acceptor emission.11 Moreover, by using suitable emission filters, the sensitized acceptor emission can be separated from the donor emission. Temporal and spectral resolution provide TR-FRET assays potential sensitivity down to picomolar range, and the assays can also be robust and have wide energy-transfer distances (up to 10 nm).12,13 However, the sensitivity of the assays may still be compromised by the background from the long-lived donor emission at the emission wavelength of the acceptor.5 Furthermore, the absorption of ultraviolet excitation light by the constituents of sample matrixes can attenuate the excitation of the lanthanide donor.14 (5) Blomberg, K.; Hurskainen, P.; Hemmila¨, I. Clin. Chem. 1999, 45, 855861. (6) Soini, E.; Hemmila¨, I. Clin. Chem. 1979, 25, 353-361. (7) Mathis, G. Clin. Chem. 1993, 39, 1953-1959. (8) Hemmila¨, I.; Malminen, O.; Mikola, H.; Lo¨vgren, T. Clin. Chem. 1988, 34, 2320-2322. (9) Kokko, L.; Sandberg, K.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2004, 503, 155-162. (10) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819-846. (11) Morrison, L. E. Anal. Biochem. 1988, 174, 101-120. (12) Selvin, P. R.; Hearst, J. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1002410028. (13) Hemmila¨, I. J. Biomol. Screening 1999, 4, 303-308. 10.1021/ac0603983 CCC: $33.50

© 2006 American Chemical Society Published on Web 05/17/2006

A novel homogeneous FRET-based assay utilizing an upconverting phosphor (UCP) as a donor was recently described by constructing a model assay for biotin.15 Inorganic UCPs, consisting of certain lanthanide dopants (e.g., Yb3+ and Er3+) embedded in a crystalline host lattice, can convert infrared excitation light into emission at visible wavelengths via the sequential absorption of two or more low-energy photons.16 No biological material possesses this capability for upconversion and produces anti-Stokes photoluminescence at conventional excitation light intensities. Thus, phosphor emission can be measured entirely free of autofluorescence and scattered excitation light, enabling immunoassays with low background fluorescence signals.17 The novel upconversion FRET (UC-FRET) principle, where an UCP donor is excited at near-infrared and sensitized acceptor emission is measured at visible wavelength, has significant advantages over the previous luminescence-based methods and can potentially alleviate the limitations of TR-FRET assays. First, there is no absorption of infrared excitation light by the biological samples that could attenuate the excitation of the UCP donor. Second, practically no donor emission is detectable at the measurement wavelength of the sensitized acceptor emission due to the extremely narrow and sharp emission bands of the UCPs characteristic to luminescent lanthanide ions. Third, because of 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. Considering the assay automation, the homogeneous assay format where continuous photon counting and an inexpensive laser diode excitation light source are utilized can reduce the costs and simplify the construction of the detection instrumentation compared to the timeresolved fluorometer with pulsed ultraviolet-light excitation.15 Previously, the principle of UC-FRET was demonstrated by a model assay for biotin where a streptavidin-conjugated UCP was used as a donor and a biotinylated fluorescent phycobiliprotein was used as an acceptor.15 In the current study, a competitive homogeneous immunoassay for 17β-estradiol (E2) was constructed, using an UCP coated with an E2-specific recombinant antibody Fab fragment (Fab S16) as a donor and an E2-conjugated small molecular dye, Oyster-556, as an acceptor. The assay was tested both in buffer and in male serum. EXPERIMENTAL SECTION Reagents. Infrared to visible anti-Stokes phosphor FCD-546-1 with a structural composition of La2O2S:Yb3+,Er3+ was obtained from Luminophor SPF (Stavropol, Russia). The photoluminescence and chemical properties of the phosphor have been characterized before.18 The small-molecular acceptor dye Oyster556, a structural analogue to Cy3 dye, was purchased from Denovo Biolabels GmbH (Muenster, Germany). 17β-Estradiol [1,3,5(10)estratriene-3,17β-diol] (E2) was from Sigma Chemical Co. (St. (14) Hemmila¨, I.; Webb, S. Drug Discovery Today 1997, 2, 373-381. (15) Kuningas, K.; Rantanen, T.; Ukonaho, T.; Lo ¨vgren, T.; Soukka, T. Anal. Chem. 2005, 77, 7348-7355. (16) 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. (17) Wright, W. H.; Mufti, N. A.; Tagg, N. T.; Webb, R. R.; Schneider, L. V. Proc. SPIE 1997, 2985, 248-255. (18) Soukka, T.; Kuningas, K.; Rantanen, T.; Haaslahti, V.; Lo¨vgren, T. J. Fluoresc. 2005, 15, 513-528.

Louis, MO). E2-specific antibody fragment (Fab S16)9,19 and biotinylated Fab S16 were kindly provided by Leena Kokko (Department of Biotechnology, University of Turku, Turku, Finland). Biotinylation of the Fab fragment was performed as described earlier,20 with 3-(N-maleimidopropionyl)biocytin (Sigma Chemical Co.) and purified using SoftLink Soft Release Avidin Resin-based affinity chromatography (Promega Corp., Madison, WI). Terbium(III) chelate of N1-(4-isothiocyanatobenzyl)diethylenetriamine-N1,N2,N3,N4-tetrakis(acetic acid) (Tb(III)-N1-ITC), 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), Delfia enhancement solution (DES), and Delfia enhancer solution (DE) were purchased from PerkinElmer Life and Analytical Sciences (Wallac, Turku, Finland). Streptavidin-coated normal-capacity microtitration wells and assay buffer (50 mM Tris-HCl, pH 7.8, containing 9 g/L NaCl, 0.5 g/L NaN3, 5 g/L bovine serum albumin fraction V (BSA), 0.1 g/L Tween 40, 0.5 g/L bovine γ-globulin, and 20 µM DTPA) were obtained from Innotrac Diagnostics (Turku, Finland). Tween 20 was from E. Merck (Darmstadt, Germany), and BSA was from Bioreba (Nyon, Switzerland). Spectral Characterizations. Anti-Stokes photoluminescence emission spectrum of the FCD-546-1 UCP was measured as described earlier.15,18 Fluorescence excitation and emission spectra of Oyster-556 were measured using a Varian Cary Eclipse fluorescence spectrophotometer (Varian Scientific Instruments, Mulgrave, Australia) from a dye solution diluted in tris-salineazide buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.5 g/L NaN3) in poly(methyl methacrylate) cuvettes with four clear sides (3.5-mL capacity and 10 mm × 10 mm light path; Kartell, Noviglio, Italy). The emission spectrum was measured from 560 to 850 nm by 0.75-nm resolution with 5-nm emission slit, utilizing the fluorescence mode of the fluorescence spectrophotometer and an excitation wavelength of 550 nm with excitation slit of 10 nm. The excitation spectrum was measured from 400 to 568 nm by 0.75nm resolution with 5-nm excitation slit, utilizing the fluorescence mode of the fluorescence spectrophotometer and an emission wavelength of 578 nm with emission slit of 10 nm. Terbium(III) Chelate-Labeled E2. Amino derivative of E2 (6-oxoestradiol 6[O-(6-aminohexyl)oxime)]) (E2-NH2), synthesized according to a published method,21 was labeled with an 8-fold molar excess of the Tb(III)-N1-ITC in 50 mM carbonate buffer, pH 9.8, containing 15% (v/v) dimethylformamide (DMF) and 5% (v/v) absolute ethanol to dissolve the E2-NH2. The labeling reaction with a total volume of 67 µL, containing 1.3 g/L E2-NH2, was incubated overnight at room temperature, protected from light, and stopped by addition of 1 mol/L Tris-HCl, pH 7.2, to a final concentration of 100 mM. Terbium(III) chelate-labeled E2 (E2-Tb) was purified using reversed-phase high-performance liquid chromatography (HPLC) with a 150 × 4.6 mm, 3-µm HypersilKeystone C18 column (Thermo Electron, Waltham, MA) and a gradient from 95% A and 5% B to 50% B in 15 min and to 100% B in 30 min, and a flow rate of 1.0 mL/min (A, aqueous 20 mM triethylammonium acetate (TEAA); B, 20 mM TEAA/50% acetonitrile). The HPLC fractions producing terbium fluorescence were (19) Lamminma¨ki, U.; Westerlund-Karlsson, A.; Toivola, M.; Saviranta, P. Protein Sci. 2003, 12, 2549-2558. (20) Korpima¨ki, T.; Hagren, V.; Brockmann, E. C.; Tuomola, M. Anal. Chem. 2004, 76, 3091-3098. (21) Mikola, H.; Ha¨nninen, E. Bioconjugate Chem. 1992, 3, 182-186.

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analyzed by a competitive heterogeneous immunoassay for E2, modified from the previously published procedure,19 to identify the fraction containing the E2-Tb. Briefly, 40 ng of biotinylated Fab S16 in 80 µL of assay buffer was incubated in normal-capacity, streptavidin-coated microtitration wells for 1 h, at room temperature, in slow shaking on a Delfia plate shaker (PerkinElmer Life and Analytical Sciences). The wells were washed once with wash solution using Delfia plate washer (PerkinElmer Life and Analytical Sciences), and a series of E2 standards (0-5 nM) and appropriate dilutions of the HPLC fractions in 100 µL of assay buffer were added. After 1 h of incubation, the wells were washed twice and 120 µL of 5 nM europium(III) N1-chelate labeled E2NH222 in assay buffer was added. The incubation was continued for 30 min, the wells were washed four times, and the signal was developed using DES according to the manufacturer’s instructions. Europium fluorescence was measured with a Victor 1420 Multilabel Counter (PerkinElmer Life and Analytical Sciences) at 615 nm using the standard europium measurement protocol. E2-Tb was stored at +4 °C. Fab-Coated FCD-546-1 UCP. Commercial, micrometersized UCP material was bead-milled and purified according to the procedure by Soukka et al.18 to produce a colloidal suspension of submicrometer-sized particles. The concentration of the phosphor suspension was obtained by weighting dried phosphors,23 and the 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 phosphors were covalently coated with Fab S16 using a published method.23 In the coating, 0.125 mg/mL Fab was used. The coated phosphors were stored in a 5 mM borate buffer, pH 8.5, containing 0.5 g/L Tween 20, 5 g/L BSA, and 0.5 g/L NaN3, at room temperature and in rotation in a self-made rack turning the container around its vertical axis at a speed of 5 rpm. Concentration of the Fab S16-coated UCP was determined by measuring its anti-Stokes photoluminescence at 535 nm in comparison with a known concentration of uncoated phosphors.23 The size distribution profile of the coated phosphors was measured similarly to uncoated phosphors. The amount of Fab attached onto phosphor particles and the concentration of free, unbound Fab present in phosphor suspension were measured using a heterogeneous, competitive immunoassay for Fab S16. First, unconjugated Fab S16 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, 0-160 ng of Fab S16 in 75 µL of assay buffer, or appropriate dilutions of the phosphor suspension and filtrate, were mixed with 75 µL of 5 nM E2-Tb in assay buffer, and the reactions were incubated for 30 min at room temperature in rotation at 17 rpm (Rotamix RK, HetoHolten A/S, Allerød, Denmark). Simultaneously, Fab S16-coated microtitration wells were prepared by incubating 30 ng of biotinylated Fab S16 in 40 µL of assay buffer in the streptavidin-coated wells for 30 min at room temperature in slow shaking. The wells were washed once, and thereafter, a 50-µL volume of each of the reactions was transferred to Fab-coated wells and incubated for (22) Mikola, H.; Sundell, A. C.; Ha¨nninen, E. Steroids 1993, 58, 330-334. (23) Kuningas, K.; Rantanen, T.; Karhunen, U.; Lo ¨vgren, T.; Soukka, T. Anal. Chem. 2005, 77, 2826-2834.

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30 min to capture all unbound E2-Tb. Finally, the wells were washed twice and the signal was developed using DES and DE according to manufacturer’s instructions. Terbium fluorescence was measured with a Victor 1420 Multilabel Counter at 545 nm using the standard terbium measurement protocol. E2 Amino Derivative with a Long Spacer Arm. Reagents for the synthesis of a long-chain amino derivative of E2 ((6aminohexyl)-{[(1,3,5(10)-estratriene-3,17β-diol-6-ylidene)amino]oxy}hexanamide) (E2-LC-NH2) were purchased either from Acros Organics (Geel, Belgium) or Sigma-Aldrich (St. Louis, MO). The solvents were p.a. grade either from Riedel-de Hae¨n (Seelze, Germany) or Fluka (Buchs, Switzerland). Briefly, 6-aminocaproic acid was first protected with tert-butyl carbamate group (BOC), and thereafter, it was activated with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The succinimidyl ester of the BOCprotected 6-aminocaproic acid was coupled to 6-oxoestradiol 6[O(6-aminohexyl)oxime)], prepared as described earlier,21 in the mixture of pyridine, water, and triethylamine (9/1.5/0.1 v/v/v). The reaction was stirred overnight at room temperature and evaporated to dryness, and the product was purified by flash chromatography using first 1% methanol in dichloromethane and finally 3% methanol in dichloromethane. The BOC protection was removed with stirring in trifluoroacetic acid for 90 min at room temperature, and the reaction mixture was evaporated to dryness. The final product, E2-LC-NH2, was used without further purification. E2 Conjugate of Oyster-556. Succinimidyl ester of the Oyster-556 was coupled with a 2.5-fold molar excess of the E2LC-NH2 to create the E2 conjugate of Oyster-556 (E2-Oyster556). The dye was dissolved in dry DMF and the E2-LC-NH2 in absolute ethanol. The coupling reaction with a total volume of 84 µL, containing 1.2 mg/mL Oyster-556 in 71 mM carbonate buffer, pH 9.3, with 14% (v/v) DMF and 9.5% (v/v) absolute ethanol, was incubated overnight at +35 °C, protected from light. E2-Oyster556 was purified from the reaction using the reversed-phase HPLC as the E2-Tb described earlier. Colored HPLC fractions were analyzed using the competitive, heterogeneous immunoassay for E2 described above to identify the fraction containing the E2Oyster-556. The concentration of the dye in the conjugate was determined by absorbance measurement at 556 nm. The E2Oyster-556 was stored at +4 °C, protected from light. Competitive Homogeneous Immunoassay for E2. The assay procedure used in the competitive homogeneous immunoassay for E2 was similar to that in the competitive homogeneous biotin assay described earlier.15 First, E2 dilutions (0-19.5 and 10 000 nM; end concentrations in the total reaction volume of 80 µL) in 32 µL of assay buffer in eight replicates and 24 µL of Fab S16-coated UCP (0.015 mg/mL) in assay buffer were added into black, half-area microtitration wells (Costar Corning, Corning, NY) and incubated for 15 min at room temperature with slow shaking. Thereafter, 24 µL of the E2-Oyster-556 (5 nM) in assay buffer was added, and the incubation was continued for 45 min, protected from light with aluminum foil. Donor emission from the UCP and sensitized acceptor emission from the Oyster-556 were measured at 535 nm and at 600 nm, respectively, with Plate Chameleon (Hidex Oy, Turku, Finland) equipped with a 200-mW infrared laser module (Roithner Lasertechnik, Vienna, Austria), RG-850 longpass excitation filter (Andover Corp., Salem, NH), and Hamamatsu

R4632 photomultiplier tube (Hamamatsu Photonics, Shizuoka, Japan).18 A band-pass 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), and an emission filter of 600/40 nm (peak T g 80%; Chroma Technology Corp., Rockingham, VT), combined with a short-pass filter glass KG-1 (Andover Corp.) were employed in the measurement. Emission light was collected for 2000 ms under continuous laser excitation at 980 nm. Energy-transfer signals were corrected for variation in laser intensity by the normalized donor emission signals as described previously.15 The signals were converted to molar concentrations of E2, and a sigmoidal standard curve was fitted to the data by using the program Origin 6.0 (OriginLab Corp., Northampton, MA) and the Logistic function y ) (A1 - A2)/[1 + (x/x0)p)] + A2, where A1 and A2 correspond to the maximum and the minimum values of the response, respectively, p is the slope, and x0 is the IC50 value. E2 Assay in Male Serum. Serum samples were collected from three male volunteers at the Department of Biotechnology, University of Turku. Samples were pooled and stored frozen at -20 °C before use in the assay. The assay protocol in serum was otherwise the same as in buffer, but 32 µL of serum spiked with E2 (0-58.5 nM, 10 000 nM) was first added into the microtitration wells and incubations were carried out in a temperature-controlled iEMS Incubator/Shaker (Thermo Labsystems, Helsinki, Finland) at +36 °C and 900 rpm. Assay Kinetics. Kinetics of the homogeneous E2 assay was studied in buffer using three different E2 concentrations, 0, 1, and 10 000 nM E2, in three replicates. After 15 min of incubation of the E2 dilutions and the Fab S16-coated UCP, photoluminescence signals were measured at 535 nm and at 600 nm to give the signal levels at time point 0 min. E2-Oyster-556 was added and donor emission from the UCP and sensitized acceptor emission from the Oyster-556 were measured after 5-90 min of continuous incubation. RESULTS Characteristics of the Donor and Acceptor Bioconjugates. Coating of the FCD-546-1 phosphor with Fab S16 did not change significantly the size distribution profile of the bead-milled phosphor particles. Before coating, the size distribution was 210310 nm (average diameter, 271 nm) and after coating 210-350 nm (283 nm); thus, there was only a ∼10 nm increase observed in the average diameter. The phosphor concentration in the stock solution of Fab-coated UCP was determined to be 8.5 mg/mL and Fab S16 concentration 91.5 µg/mL of which the degree of free, unbound Fab was 4.9%. Based on the density information of the phosphor material18 and obtained average size, the average number of bound Fab fragments per phosphor was 9400. However, the value for Fab S16 binding sites is only indicative because of the wide size distribution of the phosphor particles. The coated phosphors were stored in rotation to prevent the slow sedimentation observable with the submicrometer-sized particles when held stationary. Oyster-556 was coupled with a long-chain E2 amino derivative (E2-LC-NH2) to minimize the possible steric hindrances between E2 and acceptor dye, which could impair the functionality of the

Figure 1. Principle of the UC-FRET-based homogeneous immunoassay for E2. UCP conjugated to anti-E2-Fab (donor) can bind E2 (analyte) and E2-Oyster-556 (acceptor). Upon excitation at 980 nm, the Er3+,Yb3+-doped UCP donor produces anti-Stokes emission at 520-550 nm and can excite the attached acceptors by FRET. The intensity of the sensitized emission from the acceptor measured at 600 nm is inversely proportional to the analyte concentration in the reaction.

homogeneous E2 immunoassay. The HPLC fraction containing both E2 and Oyster-556 could be identified by analyzing the colored fractions (containing the dye) with the competitive heterogeneous E2 assay. However, due to the standardization based on free E2, this assay could not be used to determine the accurate E2 concentration of the E2-Oyster-556 conjugate. Dye concentration of the conjugate was determined to be 17.5 µM by absorbance measurement at 556 nm. The functionality of the conjugate (i.e., whether E2 and Oyster-556 were coupled to each other) was verified by its performance in the homogeneous E2 immunoassay. Spectral Principle of the Homogeneous Assay. Homogeneous immunoassay for E2 was based on UC-FRET15 between the Fab S16-coated UCP donor and the E2-conjugated small molecular acceptor dye, Oyster-556 (Figure 1). Upon excitation at 980 nm the Er3+,Yb3+-doped phosphor produces anti-Stokes photoluminescence emission at 535 nm and at 665 nm (Figure 2). The 535nm emission band overlaps with the excitation spectrum of the Oyster-556, enabling resonance energy transfer from the UCP to the E2-Oyster-556 attached to Fab S16 binding sites on the phosphor surface. Although, the actual emission maximum of Oyster-556 is at 570 nm, the broad emission band of the dye enables the measurement of the sensitized acceptor emission at 600 nm using suitable band-pass emission filters. At the 580620-nm region, the phosphor donor has an emission minimum with a signal level corresponding to the background noise and being at least 2000 times smaller than at the emission maximum of the phosphor (Figure 2). Thus, the resonance energy-transfer signals can be measured without any significant background from the donor emission. Moreover, due to the large anti-Stokes shift characteristic of upconverting rare earth phosphors, scattered excitation laser light can be totally eliminated from the measurement by a 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.18 Assay Performance. In the homogeneous E2 assay, E2 dilutions (analyte), either in buffer or in serum, were first incubated together with the phosphor donor. According to a principle of a back-titration assay,24 the labeled analyte (E2Oyster-556) was added to the reaction later to allow better Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

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Figure 2. Anti-Stokes photoluminescence emission spectrum of the FCD-546-1 UCP (wide solid line) and fluorescence excitation (dashed line) and emission (narrow solid line) spectra of the Oyster-556. Emission spectrum of the FCD-546-1 phosphor overlaps with the excitation spectrum of the Oyster-556. UCP donor has a true emission minimum at 580-620 nm where the sensitized acceptor emission from the energy transfer can be measured. The spectra have been normalized to the same intensity levels. au, arbitrary unit.

sensitivity25 and to facilitate the manual pipetting. By using 0.015 mg/mL UCP donor and 5 nM E2-Oyster-556 in the assay reactions, standard curves with IC50 values (concentration that inhibited 50% of the maximum signal) of 1 nM (0.3 ng/mL) in buffer and 3 nM (0.8 ng/mL) in serum were obtained (Figure 3). Lower limits of detection using the defined donor and acceptor concentrations were 0.4 (0.1 ng/mL) and 0.9 nM (0.2 ng/mL), respectively, calculated as the concentrations corresponding to a signal 3SD below the mean of the zero calibrators. The highest measurable calibrator concentrations were 3.1 nM (0.8 ng/mL) in buffer and 9.3 nM (2.5 ng/mL) in serum, if the cutoff was defined as the concentration where the within-run CV based on calculated concentrations was below 20%. Maximum resonance energy-transfer signals in the assay were obtained from the reactions without E2, where the acceptor and the donor can freely bind to each other, and minimum signals from the reactions with 10 000 nM E2, where no Fab S16 binding sites are left on the phosphor surface for the acceptor to bind. The ratio between maximum and minimum signals, or the signal-to-background ratio, was 34 in buffer and 15 in serum. Assay Kinetics. Energy-transfer signals for the E2 standard curves were measured after 45 min of incubation. At this point, maximum signal level could already be measured, as determined by the kinetic experiments (Figure 4). Assay kinetics was studied using only three E2 concentrations (0, 1, and 10 000 nM) for the measurements to be fast and simple. The idea was to keep the incubation as similar as in the actual E2 immunoassay, causing minimal interference through repeated measurements. When there was no E2, maximum signals could be measured, reaching equilibrium after 30 min of incubation. With 1 nM E2, energy(24) Rodbard, D.; Ruder, H. J.; Vaitukaitis, J.; Jacobs, H. S. J. Clin. Endocrinol. Metab. 1971, 33, 343-355. (25) Piran, U.; Silbert-Shostek, D.; Barlow, E. H. Clin. Chem. 1993, 39, 879883.

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Figure 3. Standard curves (filled squares) and within-assay precision profiles (CV, open squares) of the homogeneous E2 immunoassay in buffer (A) and in male serum (B) after 45 min of incubation. The standard curves and the precision profiles are based on means and CVs of eight replicates. IC50 values of the assay were 1 nM in buffer and 3 nM in serum. cts, counts.

transfer signals were ∼50% of the maximum through all measurement points, as expected by the IC50 value of the E2 standard curve. A slight increase in the signals until 90 min of incubation could be explained by the slow replacement of the E2 with the E2-Oyster-556. With 10 000 mol/L E2, minimum assay signals were measured, unchangeable through the assay. DISCUSSION The homogeneous assay principle based on UC-FRET was described earlier using a streptavidin-coated UCP as a donor and a biotinylated fluorescent protein as an acceptor.15 In the current study, UCP particles were coated with a recombinant antibody Fab fragment to demonstrate the feasibility of the UC-FRET in an immunoassay in serum. Moreover, a small molecular dye was introduced as an acceptor to expand further the applications of the assay method. The E2-specific antibody Fab fragment used in this study has been produced in our laboratory and subjected to several protein engineering studies to improve its binding properties toward E2.19

Figure 4. Kinetics of the UC-FRET-based homogeneous E2 immunoassay in buffer. Sensitized acceptor emission with 0 (circles), 1 (squares), or 10 000 nM (triangles) E2 after 5-90 min of incubation. The values presented are the mean of three replicates. The error bars indicate the SD of the value. cts, counts.

Fab S16 has been used before in a competitive, heterogeneous, time-resolved, fluorescence-based E2 immunoassay19 as well as in a competitive, homogeneous, FRET-based assay using Fab S16coated europium(III)-chelate dyed nanoparticles as a donor.9 Heterogeneous assays are generally considered to be more sensitive than their homogeneous counterparts,5 and accordingly, slightly lower IC50 concentration of ∼0.3 nM was achieved in the heterogeneous E2 assay.19 In the homogeneous nanoparticle-based assay, very comparable IC50 values with the UC-FRET assay were obtained, IC50 values varying between 0.5 and 3.5 nM depending on the Fab S16 amount on the particle surface and the donor concentration in the assay.9 However, the detection range in the UC-FRET-based immunoassay was still limited compared to the previous studies due to the rather large variation. The variation can originate from the wide size distribution and irregular shape of the phosphor particles and fluctuation in the laser intensity during the measurement,15 as well as the excitation of only a minor proportion of the whole reaction volume by the narrow laser beam.23 In serum, a few differences were observed in the standard curve compared to buffer. This was expected due to the nonspecific binding interactions and the presence of endogenous steroidbinding proteins in serum,26-28 increasing the binder concentration in the assay reactions. Increased nonspecific binding between the acceptor and the donor elevated the minimum signals in serum while the binding of the E2-Oyster-556 and E2 to the serum proteins both decreased the maximum level of resonance energytransfer signals, shifted the IC50 value to the higher E2 concentrations, and widened the dynamic range of the assay. However, the purpose of this study was only to construct a model for the utilization of UC-FRET in an immunoassay in biological matrix. For the E2 assay to be employed in the analysis of clinical samples, in addition to the elevated incubation temperature, the effect of (26) Brock, P.; Eldred, E. W.; Woiszwillo, J. E.; Doran, M.; Schoemaker, H. J. Clin. Chem. 1978, 24, 1595-1598. (27) Slaats, E. H.; Kennedy, J. C.; Kruijswijk, H. Clin. Chem. 1987, 33, 300302. (28) Masters, A. M.; Ha¨hnel, R. Clin. Chem. 1989, 35, 979-984.

the steroid-binding proteins could be eliminated or minimized by the adjustment of reaction pH or addition of blockers such as 8-anilino-1-naphthalenesulfonic acid, salicylic acid, or danazol to assay buffer.26,29 Because of the unique nature of upconversion, no autofluorescence or scattered excitation light is produced from any biological material at visible wavelengths under infrared excitation.16 In principle, the anti-Stokes photoluminescence background in the UCP-based assay could be equivalent to that achieved in luminescence counting where the dark counts of the photomultiplier set the limit of detection. However, the assay background (the minimum signals measured from the reactions with 10 000 nM E2) in the competitive UC-FRET-based E2 immunoassay was somewhat elevated due to the issues related to nonspecific binding interactions but also to the submicrometer size of the phosphor donor used. In the submicrometer-sized particles, only the emissive ions located near the particle surface are within the distance requirement of FRET.10,30 A considerably larger fraction of the ions in the core of the particles produces only background signals through nonproximity-based reabsorptive energy transfer,31 increasing linearly with acceptor or donor concentrations. However, the background caused by the reabsorptive energy transfer could be diminished in the UC-FRET assay by using smaller UCP particles. In these nanophosphors, preferably less than 50 nm in diameter, a larger proportion of the emissive ions would participate in the nonradiative resonance energy transfer because of the shortened distances, and less nonproximity-based reabsorptive energy transfer would be observed.30 Noteworthy is that, despite the smaller particle size, the specific activity of the UCP should not be diminished. Rather, stronger anti-Stokes photoluminescence emission efficiency from the phosphor donor should be obtained to increase the FRET signals and to be able to exploit the significant advantages of the UC-FRET principle in the construction of homogeneous assays with superior sensitivity. According to the second-order dependence of the photoluminescence emission on the excitation power, if two photons are involved in the upconversion process,32 instant increase in the signal levels can be obtained by raising the laser excitation power. Nevertheless, it is envisioned that if smaller monosized particles could be used in the UC-FRET assay, the variation of the assay would also be improved, increasing further the assay sensitivity. These particles are also expected to have less undesired steric interactions and effects on biochemical binding reactions30 and, thus, could accelerate the assay kinetics considerably. Moreover, smaller particles would have less tendency for sedimentation and shaking could be omitted, having no effect on the reaction velocity in a homogeneous solution and being advantageous for automation.5 Compared to the fluorescent phycobiliprotein used in a model assay for UC-FRET,15 the small-molecular acceptor dye in this study enabled higher signal-to-background ratios to be measured. In addition to Oyster-556, other dyes such as Alexa Fluor 555, Atto 565, and Cy 3B were applicable in the assay (data not shown). (29) Pandey, P. K.; Shrivastav, T. G.; Kumari, G. L.; Rao, P. N.; Grover, P. K.; Murthy, H. G. K. Clin. Chim. Acta 1990, 190, 175-184. (30) Meyer, C.; Haase, M.; Hoheisel, W.; Bohmann, K., 2004. (31) Valanne, A.; Lindroos, H.; Lo ¨vgren, T.; Soukka, T. Anal. Chim. Acta 2005, 539, 251-256. (32) Heer, S.; Ko ¨mpe, K.; Gu ¨ del, H. U.; Haase, M. Adv. Mater. 2004, 16, 21022105.

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The possibility of using both fluorescent proteins and smallmolecular dyes as acceptors enhances the potential applications of the assay technology, as has been already demonstrated in TRFRET-based assays.33,34 Fluorescent phycobiliproteins can have exceptionally high extinction coefficients and high quantum efficiencies, conferring potentially very good sensitivities for fluorescent assays.35 However, the larger size can be considered as a disadvantage that could favor the use of dyes over fluorescent proteins in some applications. The size of the proteins might cause steric and kinetic problems, and the inherent nonspecific binding of the protein molecules could compromise the assay sensitivity. The conjugation of the proteins to analytes or antibodies can also be difficult, and the number of analyte molecules coupled to the protein may become numerous, which in particle-based assays could lead to cross-linking and aggregation of the donor particles. In the structure of the small-molecular dyes, there is usually only one functional group where biomolecules can be conjugated and a monoreactive acceptor can be obtained. Finally, due to the structure of the proteins held together through noncovalent bonds, there is also a potential stability problem with the phycobiliproteins; in very dilute solutions, the protein might fall apart into subunits, losing its excellent fluorescent properties.35 CONCLUSIONS In this study, a competitive homogeneous immunoassay for E2 in serum based on the use of a recombinant antibody fragment (33) Bazin, H.; Pre´audat, M.; Trinquet, E.; Mathis, G. Spectrochim. Acta, Part A 2001, 57, 2197-2211. (34) Hemmila¨, I.; Laitala, V. J. Fluoresc. 2005, 15, 529-542. (35) Kronick, M. N. J. Immunol. Methods 1986, 92, 1-13.

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and UC-FRET between an UCP donor and a small-molecular acceptor dye was demonstrated. The UC-FRET principle has significant and unique advantages that eliminate the problems associated with autofluorescence and scattered excitation light in homogeneous luminescence-based assays without need for temporal resolution. Moreover, the excitation of the UCP donor is not attenuated because there is no absorption of infrared light by the biological samples. The particulate label still sets some limitations to the assay, and more experience on the use of inorganic UCPs as reporters in immunoassays is required. By further optimization, UC-FRET can extend the applications of FRET and enable the construction of rapid and very sensitive assays to be utilized in diagnostic applications and also in highthroughput screening approaches. ACKNOWLEDGMENT This study was supported by the Finnish Funding Agency for Technology and Innovation (Tekes), the Academy of Finland (Grant 209417) and the Graduate School of In Vitro Diagnostics in Finland. A grant from the Instrumentarium Science Foundation denoted to K.K. is also gratefully acknowledged. The authors are thankful for technological support from Hidex Oy in anti-Stokes photoluminescence measurement.

Received for review March 3, 2006. Accepted April 11, 2006. AC0603983