Development of a Visible-Light-Sensitized Europium Complex for

Feb 12, 2010 - All bright-field imaging, normal luminescence imaging, and time-resolved ..... This work was supported by the National Natural Science ...
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Anal. Chem. 2010, 82, 2529–2535

Development of a Visible-Light-Sensitized Europium Complex for Time-Resolved Fluorometric Application Lina Jiang,† Jing Wu,‡ Guilan Wang,† Zhiqiang Ye,*,† Wenzhu Zhang,† Dayong Jin,§ Jingli Yuan,*,† and James Piper§ State Key Laboratory of Fine Chemicals, Department of Chemistry, Dalian University of Technology, Dalian 116012, China, School of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, China, and MQ Photonics Centre, Faculty of Science, Macquarie University, NSW 2109, Sydney, Australia The time-resolved luminescence bioassay technique using luminescent lanthanide complexes as labels is a highly sensitive and widely used bioassay method for clinical diagnostics and biotechnology. A major drawback of the current technique is that the luminescent lanthanide labels require UV excitation (typically less than 360 nm), which can damage living biological systems and is holding back further development of time-resolved luminescence instruments. Herein we describe two approaches for preparing a visible-light-sensitized Eu3+ complex in aqueous media for time-resolved fluorometric applications: a dissociation enhancement aqueous solution that can be excited by visible light for ethylenediaminetetraacetate (EDTA)-Eu3+ detection and a visible-lightsensitized water-soluble Eu3+ complex conjugated bovine serum albumin (BSA) for biolabeling and timeresolved luminescence bioimaging. In the first approach, a weakly acidic aqueous solution consisting of 4,4′bis(1′′,1′′,1′′,2′′,2′′,3′′,3′′-heptafluoro-4′′,6′′-hexanedion6′′-yl)-o-terphenyl (BHHT), 2-(N,N-diethylanilin-4-yl)4,6-bis(3,5-dimethylpyrazol-1-yl)-1,3,5-triazine (DPBT), and Triton X-100 was prepared. This solution shows a strong luminescence enhancement effect for EDTA-Eu3+ with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm) and a long luminescence lifetime (520 µs), to provide a novel dissociation enhancement solution for time-resolved luminescence detection of EDTA-Eu3+. In the second approach, a ternary Eu3+ complex, 4,4′-bis(1′′,1′′,1′′,2′′,2′′,3′′,3′′-heptafluoro-4′′,6′′-hexanedion-6′′-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu3+-DPBT, was covalently bound to BSA to form a water-soluble BSA-BHHCTEu3+-DPBT conjugate. This biocompatible conjugate is of the visible-light excitable feature in aqueous media with a wide excitation wavelength range from UV to visible light (a maximum at 387 nm), a long luminescence lifetime (460 µs), and a higher quantum yield * To whom correspondence should be addressed. Phone and Fax: +86-41184706293. E-mail: [email protected] (Z.Y.); [email protected] (J.Y.). † Dalian University of Technology. ‡ Liaoning Normal University. § Macquarie University. 10.1021/ac100021m  2010 American Chemical Society Published on Web 02/12/2010

(27%). The conjugate was successfully used for streptavidin (SA) labeling and time-resolved luminescence imaging detection of three environmental pathogens, Giardia lamblia, Cryptosporidium muris, and Cryptosporidium parvum, in water samples. Our strategy gives a general idea for designing a visible-light-sensitized Eu3+ complex for time-resolved luminescence bioassay applications. The time-resolved (or time-gated) luminescence bioassay technique using luminescent lanthanide (mainly Eu3+ and Tb3+) complexes as labels has attracted much attention since the first application was reported in 1983,1 and several commercially available assay systems, such as the DELFIA (dissociation enhanced lanthanide fluoroimmunoassay) system,2-4 CyberFluor (or FIAgen) system,5,6 enzyme-amplified time-resolved fluorometric system,7-9 and TRACE (time-resolved amplified cryptate emission) system,10-14 have been successfully developed and widely used in the areas of clinical diagnostics and biotechnology. In contrast to the conventional organic fluorescence dyes, luminescent lanthanide complexes display several unique spectral and temporal properties, including long luminescence lifetime, large Stokes shift, and sharp emission profile, which enable them to be suitable as biolabels for microsecond time-resolved luminescence measurement to effectively eliminate the short-lived background noise from the raw biological samples and scattering (1) Siitari, H.; Hemmila¨, I.; Soini, E.; Lo ¨vgren, T.; Koistinen, V. Nature 1983, 301, 258–260. (2) Hemmila¨, I. Clin. Chem. 1985, 31, 359–370. (3) Soini, E.; Lo ¨vgren, T. CRC Crit. Rev. Anal. Chem. 1987, 18, 105–154. (4) Hemmila¨, I. Scand. J. Clin. Lab. Invest. 1988, 48, 389–400. (5) Diamandis, E. P.; Christopoulos, T. K. Anal. Chem. 1990, 62, 1149A–1157A. (6) Dickson, E. F. G.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1995, 66, 207–235. (7) Evangelista, R. A.; Pollak, A.; Templeton, E. F. G. Anal. Biochem. 1991, 197, 213–224. (8) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1992, 64, 342–346. (9) Veiopoulou, C. J.; Lianidou, E. S.; Ioannou, P. C.; Efstathiou, C. E. Anal. Chim. Acta 1996, 335, 177–184. (10) Mathis, G. Clin. Chem. 1993, 39, 1953–1959. (11) Mathis, G. Clin. Chem. 1995, 41, 1391–1397. (12) Mathis, G.; Socquet, F.; Viguier, M.; Darbouret, B. Anticancer Res. 1997, 17, 3011–3014. (13) Mathis, G. J. Clin. Ligand Assay 1997, 20, 141–147. (14) Cummings, R. T.; McGovern, H. M.; Zheng, S.; Park, Y. W.; Hermes, J. D. Anal. Biochem. 1999, 269, 79–93.

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from nearby optics, to provide highly sensitive detections for the analytes in complicated samples.15-17 Until now, the most widely used time-resolved luminescence bioassay system for clinical diagnostics is the DELFIA system. A key technique for this system is the use of a weakly acidic (pH 2.5-3.2) dissociation enhancement solution consisting of a β-diketone (2-naphthoyltrifluoroacetone, 2-thenoyltrifluoroacetone, or pivaloyltrifluoroacetone), tri-n-octylphosphine oxide (TOPO), and Triton X-100 for transforming the Eu3+ ions from the nonluminescent Eu3+ complex [isothiocyanatophenyl-ethylenediamine tetraacetate-Eu3+ or N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N4-tetraacetate-Eu3+] labeled immunocomplexes into a highly luminescent Eu3+-β-diketonate-TOPO complex before the measurement.2-4 In such a system, one of the major drawbacks is its lower power UV excitation source (xenon flash lamp) in the most commercially available instruments (such as the Perkin-Elmer DELFIA 1234 or Victor 1420 timeresolved luminescence counter) because the luminescent Eu3+-βdiketonate-TOPO complex formed in the enhancement solution requires a shorter wavelength excitation (420 nm), V-2A filters (excitation filter, 380-420 nm; dichroic mirror, 430 nm; emission filter, >450 nm), and a cooled color CCD camera system (RET-2000R-F-CLR-12-C, Qimaging Ltd.), was used for the normal luminescence imaging measurement with an exposure time of 6 s. The microscope, equipped with a 30 W xenon flashlamp, UV-2A and V-2A filters, and a timeresolved digital black-and-white CCD camera system (ImagexTGi, Photonic Research Systems Ltd.), was used for the timeresolved luminescence imaging measurement with the following conditions: delay time, 100 µs; gate time, 1 ms; lamp pulse width, 6 µs; exposure time, 120 s. Preparation of the Dissociation Enhancement Solution for EDTA-Eu3+. After Triton X-100 (0.025%, 0.05%, 0.1%, and 0.2%, respectively) was added to a 0.1 M potassium acid phthalate solution, the pH of the solution was adjusted to 3.2 with concentrated HCl. To the solution were added quantitative amounts of DPBT and BHHT (dissolved in 150 and 50 µL of acetone, respectively. The concentration of DPBT was fixed to 60 µM, and that of BHHT was changed from 10 to 80 µM) with stirring and ultrasonication. After the solution was incubated at room temperature for 3 days, it was filtered through a 0.22 µm (39) Yuan, J. L.; Matsumoto, K.; Kimura, H. Anal. Chem. 1998, 70, 596–601. (40) Ye, Z. Q.; Tan, M. Q.; Wang, G. L.; Yuan, J. L. Anal. Chem. 2004, 76, 513–518.

filter to remove trace amount of undissolved materials. The solution was stored at room temperature before use. Preparation of the BSA-BHHCT-Eu3+-DPBT Conjugate. To 1.3 mL of 0.05 M carbonate buffer of pH 9.3 containing 7.53 mg of BSA (∼1.1 × 10-4 mmol) was added dropwise 200 µL of ethanol containing 6.07 mg of BHHCT (7.54 × 10-3 mmol) with stirring. After stirring for 1 h at room temperature, the BHHCT-labeled BSA was separated by gel filtration chromatography on a Sephadex G-50 column using 0.05 M NaHCO3 of pH 8.5 as the eluent. The fractions of BHHCT-labeled BSA were collected, and the labeling ratio (BHHCT to BSA) was determined by using the previous method39 to be ∼30. To the BSA-BHHCT solution was added NaN3 (0.1%), and then the pH was adjusted to 6.2 with 1 M HCl. To 2.0 mL of the above BSA-BHHCT solution (containing ∼1.5 × 10-3 mmol of BHHCT) were added 1.8 mL of dimethyl sulfoxide containing 1.52 mg of DPBT (3.65 × 10-3 mmol) and 0.53 mg of EuCl3 · 6H2O (1.46 × 10-3 mmol) with stirring. After incubating at 52 °C for 2 h, the solution was filtrated through a 0.45 µm filter to remove trace amounts of undissolved materials. The solution was further separated by gel filtration chromatography on a Sephadex G-50 column to remove the unreacted DPBT using 0.1 M phosphate buffer of pH 7.0 as the eluent. The fractions containing the BSA-BHHCTEu3+-DPBT conjugate were collected and stored at 4 °C before use. Preparation of the BSA-BHHCT-Eu3+-DPBT ConjugateLabeled SA. To 0.54 mL of the above BSA-BHHCT-Eu3+DPBT conjugate solution (containing ∼0.2 mg of BSA) was added 0.2 mg of SA and 20 µL of 1% glutaraldehyde. After stirring at 4 °C for 23 h, 1.0 mg of NaBH4 was added, and the solution was incubated for 1 h at room temperature. To the solution was added 0.6 mg of NaN3 and 1.3 mg of BSA, and then the solution was stored at 4 °C before use. Luminescence Imaging Detection of the Environmental Pathogen. After three environmental pathogens, G. lamblia, C. muris, and C. parvum, were immunostained with their monoclonal antibody, biotinylated secondary antibody, and the Eu3+ complexlabeled SA, respectively, the normal luminescence and timeresolved luminescence imaging detections were carried out. The details of the experiments are described as follows. G. lamblia. An amount of 5 µL of G. lamblia solution (2 × 106 cysts/mL) was mixed with 16 µL of mouse anti-Giardia antibody (∼50 µg/mL), 20 µL of biotinylated rabbit antimouse IgG antibody (∼45 µg/mL), and 10 µL of the BSA-BHHCTEu3+-DPBT conjugate-labeled SA in a tube. After incubation for 24 h at room temperature, the cysts were separated by centrifugation at 500 rpm, washed several times with distilled water to remove the unreacted materials, and then spotted on a glass slide for luminescence microscopy imaging detection. To confirm the nonspecific binding of the Eu3+ complex on Giardia cysts, a control experiment in the absence of antiGiardia antibody was also carried out with the same method. C. muris. An amount of 10 µL of C. muris solution (2.5 × 105 cysts/mL) was mixed with 8 µL of anti-C. muris antibody (∼50 µg/mL), 8 µL of biotinylated rabbit antimouse IgM antibody (∼45 µg/mL), and 8 µL of the BSA-BHHCT-Eu3+DPBT conjugate-labeled SA in a tube. After incubation for 10 h Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Figure 2. Time-resolved excitation (300-500 nm) and emission (500-710 nm) spectra of EDTA-Eu3+ complex (1.0 µM) in four dissociation enhancement solutions containing 60 µM DPBT and BHHT and different concentrations of Triton X-100 (a, 0.025%; b, 0.05%; c, 0.1%; d, 0.2%). The spectra were measured with the following conditions: excitation wavelength, 387 nm; emission wavelength, 615 nm; delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm; emission slit, 5 nm.

at room temperature, the cysts were treated and imaged with the same method as described in the imaging of Giardia. A control experiment in the absence of anti-C. muris antibody was also carried out with the same method. C. parvum. An amount of 10 µL of C. parvum solution (1.25 × 106 cysts/mL) was mixed with 16 µL of anti-C. parvum antibody (∼50 µg/mL), 20 µL of biotinylated rabbit antimouse IgM antibody (∼45 µg/mL), and 8 µL of the BSA-BHHCTEu3+-DPBT conjugate-labeled SA in a tube. After incubation for 10 h at room temperature, the cysts were treated and imaged with the same method as described in the imaging of Giardia. A control experiment in the absence of anti-C. parvum antibody was also carried out with the same method. RESULTS AND DISCUSSION Characterization and Application of the Dissociation Enhancement Solution. BHHT-Eu3+ is a strongly luminescent Eu3+ complex. Similar to almost luminescent β-diketonateEu3+ complexes, this complex is not water-soluble and requires a shorter wavelength excitation (a maximum excitation peak at ∼330 nm).39 However, when DPBT was added to the organic solution (e.g., chloroform and dichloromethane) of BHHT-Eu3+, the maximum excitation peak of the complex could be red-shifted to ∼406 nm. This phenomenon can be attributed to the formation of the ternary complex BHHT-Eu3+-DPBT, which can alter the sensitization process of the Eu3+ complex from the triplet pathway mechanism to the singlet pathway mechanism.37 In this work, an aqueous micellar solution at pH 3.2 containing BHHT, DPBT, and Triton X-100 was prepared as a visible-light-sensitized dissociation enhancement solution for the time-resolved luminescence detection of EDTA-Eu3+. First, by mixing EDTA-Eu3+ solution with the dissociation enhancement solutions prepared with different conditions (different concentrations of Triton X-100 and BHHT), the effects of the concentrations of Triton X-100 and BHHT on the luminescence properties of the Eu3+ complex were investigated. Figure 2 shows time-resolved excitation and emission spectra of four dissociation enhancement solutions containing 1.0 µM 2532

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EDTA-Eu3+, 60 µM DPBT, and BHHT, and different concentrations of Triton X-100 (0.025%, 0.05%, 0.1%, and 0.2%). All the solutions showed a broad excitation spectrum between 300 and 450 nm with a peak at ∼387 nm, and a strong and sharp emission peak at 615 nm with several side emission peaks centered at 585, 594, 647, and 692 nm, respectively. With the increase of Triton X-100 concentration from 0.025% to 0.05%, the luminescence intensity at 615 nm was 1.5-fold increased, but further increase of Triton X-100 concentration caused the decrease of luminescence intensity (9.1% and 36.8% decreased with the increase of Triton X-100 concentration from 0.05% to 0.1% and 0.05% to 0.2%, respectively). Because higher Triton X-100 concentration is beneficial for the dissolution of DPBT and BHHT in aqueous solution, and the luminescence intensity is only a little decreased in the solution of 0.1% Triton X-100, the concentration of 0.1% for Triton X-100 is considered to be an optimal concentration for the dissociation enhancement solution. Instead of Triton X-100, the utility of other surfactants including Tween 20 and cetyltrimethyl ammonium bromide (CTAB) for preparing the dissociation enhancement solution was also determined. Unfortunately, in the solutions prepared with these surfactants, the effective 387 nm excited luminescence enhancement was not observed when they were mixed with EDTA-Eu3+, the luminescence intensity of EDTA-Eu3+ in the Tween 20 or CTAB solution was ∼10-fold or ∼150-fold weaker than that in the Triton X-100 solution. The effect of BHHT concentration (10, 30, 60, and 80 µM, respectively) in the dissociation enhancement solution containing 0.1% Triton X-100 and 60 µM DPBT on the luminescence intensity of EDTA-Eu3+ (2.5 µM) was investigated. Because the concentrations of BHHT and DPBT in four solutions were high enough to displace EDTA from the micromolar concentration level of EDTA-Eu3+ to form BHHT-Eu3+-DPBT complex in the micellar solutions, the luminescence intensities of four solutions, 588 ± 11 (average ± SD), did not show remarkable difference. Thus, 60 µM BHHT in the dissociation enhancement solution was selected as an optimal concentration for the dissociation enhancement solution, because that could provide a wider dynamic range (a higher up limit) than other lower concentration solutions. The luminescence lifetime of the Eu3+ complex formed in this solution was determined to be 520 µs, indicating that the luminescence of the Eu3+ complex formed in the dissociation solution has a long luminescence lifetime for 100 µs level time-resolved luminescence detection. The kinetics of the ligand displacing reaction for the formation of luminescent Eu3+ complex from EDTA-Eu3+ complex in the dissociation enhancement solution was also determined. Figure 3 shows the reaction kinetic curves of the dissociation enhancement solution with different concentrations of EDTA-Eu3+. Upon addition of EDTA-Eu3+, the luminescence intensity of the solution was rapidly increased and reached the maximum value within ∼10 min. This result demonstrates that the ligand displacing reaction between the dissociation enhancement solution and EDTA-Eu3+ is rapid enough for the detection of a EDTA-Eu3+-type biolabel if the solution is used for the dissociation enhancement-type time-resolved luminescence bioassay.

Figure 3. Kinetic curves of the BHHT-Eu3+-DPBT formation in the dissociation enhancement solution upon additions of different concentrations of EDTA-Eu3+ (a, 2.5 µM; b, 1.0 µM; c, 0.25 µM). The measurements were carried out with the following conditions: delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation wavelength, 387 nm; emission wavelength, 615 nm; excitation slit, 10 nm; emission slit, 5 nm; data interval, 0.1 s.

Figure 4. Time-resolved excitation (300-500 nm) and emission (500-710 nm) spectra of the dissociation enhancement solution in the presence of different concentrations of EDTA-Eu3+. The spectra were measured with the following conditions: excitation wavelength, 387 nm; emission wavelength, 615 nm; delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm; and emission slit, 5 nm.

To investigate the performance of the new dissociation enhancement solution for quantitative time-resolved luminescence detection of EDTA-Eu3+, the time-resolved excitation and emission spectra of the solution in the presence of different concentrations of EDTA-Eu3+ were determined. As shown in Figure 4, the excitation peak at 387 nm has no change with the concentration change of EDTA-Eu3+, and the spectra show a good dose-dependent luminescence response to the EDTAEu3+ addition, indicating that the visible-light-excitable property of the enhancement solution for EDTA-Eu3+ is stable even at low Eu3+ concentration levels. The solutions in a 96-well microtiter plate were further measured on a more sensitive time-resolved luminescence counter, the Perkin-Elmer Victor 1420 multilabel counter, with a 340 nm xenon flash lamp as an excitation source. The result is shown in Figure 5A. In this case, the dose-dependent luminescence enhancement shows a good linearity that can be expressed as log(signal) ) 0.979 log[EDTA-Eu3+] + 13.800 (r ) 0.999) in the concentration

Figure 5. Calibration curves for time-resolved luminescence detection of EDTA-Eu3+ using two dissociation enhancement solutions prepared with BHHT-DPBT-Triton X-100 (A) and TTA-TOPO-Triton X-100 (B), respectively. The measurement was carried out on a Perkin-Elmer Victor 1420 multilabel counter with the following conditions: excitation wavelength, 340 nm; emission wavelength, 615 nm; delay time, 0.2 ms; window time (counting time), 0.4 ms; cycling time, 1.0 ms.

range of 0.1 nM to 0.1 µM with a detection limit of ∼0.1 nM. The detection limit of the present result is ∼3 orders of magnitude higher than that of the previously reported DELFIA system due to the strong effect of a very high background level for our dissociation enhancement solution. A reliable explanation about this phenomenon is considered by the fact that our dissociation enhancement solution was prepared in an open chemistry laboratory with general distilled water and the solution could be contaminated by the Eu(III)-containing house dust (this is also a main drawback of the DELFIA system because trace amounts of Eu3+ could cause the remarkable increase of the background for the dissociation enhancement solution5,6); therefore, the background level of the solution was very high. To evaluate the performances of the present system and the DELFIA system under the same conditions, an aqueous micellar solution of 0.1 M phthalate-HCl at pH 3.2 containing 60 µM 2-thenoyltrifluoroacetone (TTA), 60 µM TOPO, and 0.1% Triton X-100 was prepared and used for the time-resolved luminescence detection of EDTA-Eu3+ in a 96-well microtiter plate. As shown in Figure 5B, the dose-dependent luminescence enhancement of EDTA-Eu3+ in this solution also shows a good linearity that can be expressed as log(signal) ) 0.861 log[EDTA-Eu3+] + 11.383 (r ) 0.998) in the concentration range of 0.1 nM to 1.0 µM with a detection limit of ∼0.3 nM. In comparison with the BHHT-DPBT system, both the slope and luminescence intensity of the calibration curve using the TTA-TOPO system are smaller, which suggests that the sensitivity using the BHHT-DPBT system should be higher than that using the TTA-TOPO system. In addition, when a TTA-DPBT-Triton X-100 solution was used for the detection of EDTA-Eu3+, it was found that the luminescence of EDTA-Eu3+ in this solution was remarkably weak compared with that in the TTA-TOPO-Triton X-100 solution. This result indicates that the bidentate β-diketone-DPBT systems are not available for the preparation of water-based visible-light-sensitized luminescence enhancement solution for EDTA-Eu3+. Because of the lack of a commercially available LED (with a maximum output Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Scheme 1. Preparation Principle of the BSA-BHHCT-Eu3+-DPBT Conjugate

over 380 nm) excited instrument and the limitation of our laboratory conditions, the measurements on an LED-excited system and further improvement for the preparation of dissociation enhancement solution were not carried out. Preparation and Characterization of the BSA-BHHCTEu3+-DPBT Conjugate. The ternary Eu3+ complex, BHHCTEu3+-DPBT, is a highly luminescent complex having a maximum excitation peak at 408 nm in chloroform. However, due to its water-insoluble property, this complex is difficult to use directly as a biolabel for time-resolved luminescence bioassays. In this work, by covalent binding BHHCT-Eu3+DPBT to BSA, a visible-light-sensitized and water-soluble conjugate, BSA-BHHCT-Eu3+-DPBT, was prepared. The principle of the conjugate preparation is shown in Scheme 1. After BSA was conjugated with BHHCT by the formation of an amide conjugation (-SO2-NH-)39 to form a water-soluble BSA-BHHCT conjugate, the BSA-BHHCT-Eu3+-DPBT conjugate was easily prepared by reacting the BSA-BHHCT conjugate with DPBT and Eu3+ in the aqueous media. Time-resolved excitation (300-500 nm) and emission (500-710 nm) spectra of BHHCT-Eu3+-DPBT in chloroform and the BSA-BHHCT-Eu3+-DPBT conjugate in 0.05 M NaHCO3 buffer of pH 8.5 were measured, respectively. As shown in Figure 6, the chloroform solution of BHHCT-Eu3+-DPBT complex has two excitation peaks at 408 and 336 nm and a strong emission peak at 612 nm. It is noteworthy that the aqueous solution of the BSA-BHHCT-Eu3+-DPBT conjugate shows two excitation peaks at 387 and 338 nm, and a strong emission peak at 610 nm, indicating that the visible-lightexcitable property of the BHHCT-Eu3+-DPBT complex is almost retained after forming the BSA-BHHCT-Eu3+-DPBT conjugate in an aqueous buffer except that the relative peak intensities in excitation spectrum of 450-300 nm are distorted due to the effects of BSA binding and water and buffer molecules. The luminescence lifetime and quantum yield26 of the BSA-BHHCT-Eu3+-DPBT conjugate in 0.05 M NaHCO3 buffer of pH 8.5 were measured to be 460 µs and 27%, respectively. This quantum yield is higher than those of almost the water-soluble Eu3+ complexes with aromatic amine derivative ligands,41 indicating that the BSA-BHHCT-Eu3+-DPBT conjugate in aqueous media is highly luminescent with a long luminescence lifetime for time-resolved measurement application. 2534

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Figure 6. Time-resolved excitation (300-500 nm) and emission (500-710 nm) spectra of BHHCT-Eu3+-DPBT in chloroform (black lines) and the BSA-BHHCT-Eu3+-DPBT conjugate in 0.05 M NaHCO3 buffer of pH 8.5 (red lines). The spectra were measured with the following conditions: excitation wavelength, 387 nm; emission wavelength, 612 nm; delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation slit, 10 nm; emission slit, 5 nm.

Preparation and Bioimaging Application of the BSABHHCT-Eu3+-DPBT Conjugate-Labeled SA. In this work, the BSA-BHHCT-Eu3+-DPBT conjugate-labeled SA was prepared by cross-linking the primary amino groups of BSA and SA with glutaraldehyde.42 Although some primary amino groups of BSA have been bound to the BHHCT-Eu3+-DPBT complex (∼30 primary amino groups per BSA molecule were conjugated to the Eu3+ complex), it was found that the unreacted primary amino groups (a BSA molecule has 59 primary amino groups43) in the BSA-BHHCT-Eu3+-DPBT conjugate were still reactive for the cross-linking reaction. To evaluate the utility of the BSA-BHHCT-Eu3+-DPBT conjugate-labeled SA for luminescence bioimaging, the labeled SA was used for the time-resolved luminescence imaging detections of three environmental pathogens, G. lamblia, C. muris, and C. parvum, in the water samples with both UV and visible light excitations. Figure 7 shows the bright-field (Figure 7a), luminescence (Figure 7, parts b and c), and time-resolved luminescence (Figure 7, parts d and e) images of Giardia cysts, C. muris oocysts, and C. parvum oocysts that have been incubated with antibody, biotinylated secondary antibody, and the BSA-BHHCT-Eu3+-DPBT conjugate-labeled SA. In contrast to the conventional luminescence images, since the autofluorescence from the cells was completely suppressed with the time-resolved mode, highly sensitive and specific time-resolved luminescence images for three pathogens were obtained. These results demonstrate that time-resolved luminescence bioimaging technique using the as-prepared conjugate as a label can substantially eliminate the interference of short-lived background luminescence, to provide a highly specific and sensitive detection method for environmental microorganisms. In addition, the strong luminescence signals shown in the images excited by visible light, which were comparable to the images excited by UV light, further demonstrated the efficacy of the (41) Latva, M.; Takalo, H.; Mukkala, V. M.; Matachescu, C.; Rodrı´guez-Ubis, J. C.; Kankare, J. J. Lumin. 1997, 75, 149–169. (42) Yuan, J. L.; Wang, G. L.; Kimura, H.; Matsumoto, K. Anal. Biochem. 1997, 254, 283–287. (43) Evangelista, R. A.; Pollak, A.; Allore, B.; Templeton, E. F.; Morton, R. C.; Diamandis, E. P. Clin. Biochem. 1988, 21, 173–178.

Figure 7. Bright-field (a), luminescence (b and c, excited with 330-380 and 380-420 nm, respectively), and time-resolved luminescence (d and e, excited with 330-380 and 380-420 nm, respectively) images of G. lamblia (A), C. muris (B), and C. parvum (C) immunostained by the BSA-BHHCT-Eu3+-DPBT conjugate-labeled SA in the water samples. Scale bars, 10 µm. The time-resolved luminescence images are shown in pseudocolor treated by a SimplePCI software (ref 23).

BSA-BHHCT-Eu3+-DPBT conjugate as a biolabel for timeresolved luminescence bioassays with visible light excitation. The control experiments in the absence of the specific antibodies were conducted to evaluate the nonspecific binding of the BSA-BHHCT-Eu3+-DPBT conjugate-labeled SA on the pathogens. The results showed that no specific Eu3+ luminescence could be observed from the cysts, indicating that the nonspecific binding of the labeled SA on the pathogens is negligible. CONCLUSIONS In the present work, two Eu3+ complex aqueous systems that can be excited by visible light have been successfully developed for dissociation enhancement-based time-resolved luminescence detection and biolabeling as well as time-resolved luminescence bioimaging detection, respectively. The two systems, which represent two main application fields of the time-resolved luminescence technique, suggest that a practical strategy to solve the UV-excitation problem in the current timeresolved luminescence technique has been developed. The visible-light-excitable dissociation enhancement solution for EDTA-Eu3+ provides an essential reagent for developing the

next generation of time-resolved luminescence bioassay instruments, in which solid-state LEDs or laser diodes with low cost, small size, high excitation power, and maximum outputs over 380 nm can be used to replace the xenon flash lamp as an excitation source. The visible-light-excitable and water-soluble Eu3+ biolabel can be expected for using in a variety of timeresolved luminescence bioimaging detections of living microorganisms, cells, and tissues, to facilitate investigations of their biological functions. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 20835001, 20975017), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 200801410003), and Macquarie University Research Fellowship Scheme.

Received for review January 5, 2010. Accepted January 28, 2010. AC100021M

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