New Class of Tetradentate β-Diketonate-Europium Complexes That

May 31, 2012 - Luminescent lanthanide complexes that can be covalently bound to proteins have shown great utility as biolabels for highly sensitive ...
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New Class of Tetradentate β-Diketonate-Europium Complexes That Can Be Covalently Bound to Proteins for Time-Gated Fluorometric Application Lin Zhang,† Yanjiao Wang,† Zhiqiang Ye,*,† Dayong Jin,‡ and Jingli Yuan*,† †

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, P. R. China MQ Photonics Centre, Faculty of Science, Macquarie University, NSW 2109, Sydney, Australia

‡

S Supporting Information *

ABSTRACT: Luminescent lanthanide complexes that can be covalently bound to proteins have shown great utility as biolabels for highly sensitive time-gated luminescence bioassays in clinical diagnostics and biotechnology discoveries. In this work, three new tetradentate β-diketonate−europium complexes that can be covalently bound to proteins to display strong and longlived Eu3+ luminescence, 1,2-bis[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-4-chlorosulfobenzene-Eu3+ (BHHBCB-Eu3+), 1,2-bis[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]-4-chlorosulfobenzene-Eu3+ (BPPBCB-Eu3+), and 1,2-bis[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-4-chlorosulfobenzene-Eu3+ (BTBBCB-Eu3+), have been designed and synthesized as biolabels for time-gated luminescence bioassay applications. The luminescence spectroscopy characterizations of the aqueous solutions of three complex-bound bovine serum albumin reveal that BHHBCB-Eu3+ has the strongest luminescence with the largest quantum yield (40%) and longest luminescence lifetime (0.52 ms) among the complexes, which is superior to the other currently available europium biolabels. The BHHBCB-Eu3+-labeled streptavidin was prepared and used for both the time-gated luminescence immunoassay of human prostate specific antigen and the time-gated luminescence microscopy imaging of a pathogenic microorganism Cryptosporidium muris. The results demonstrated the practical utility of the new Eu3+ complex-based biolabel for time-gated luminescence bioassay applications.

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assay system (DELFIA system),1−3,16 this kind of complex is unsuitable for direct use as a biolabel due to the relatively low stability in aqueous solution (dissociation of the complexes at lower concentration will quench their luminescence) and the lack of a reactive group in the complexes for covalent binding to biomolecules. For this reason, several chlorosulfonylated tetradentate β-diketone ligands that can form stable and highly luminescent complexes with Eu3+ ions in aqueous solutions for labeling biomolecules, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl (BHHCT),17 4,4′-bis(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)chlorosulfo-o-terphenyl (BPPCT),18 4,4′-bis(1″,1″,1″-trifluoro2″,4″-butanedion-4″-yl)-chlorosulfo-o-terphenyl (BTBCT),19 1,10-bis(4″-chlorosulfo-1′,1″-diphenyl-4′-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone (BCDOT),20 1,10-bis(8′-chlorosulfo-dibenzothiophene-2′-yl)-4,4,5,5,6,6,7,7-octafluorodecane- 1,3,8,10-tetraone (BCOT),21 and 1,10-bis(5′-chlorosulfothiophene-2′-yl)-4,4,5,5,6,6,7,7- octafluorodecane-1,3,8,10tetraone (BCTOT),22 have been developed for time-gated luminescence bioassay applications in recent years. It is worth noting that, among the above β-diketones, BPPCT and BTBCT are the simple analogues of BHHCT

INTRODUCTION As one of the most highly sensitive bioassay methods, timegated (or time-resolved) luminescence bioassay technique using lanthanide complexes as labels has been comprehensively studied and widely used for clinical diagnostics and biotechnology discoveries over the past two decades.1−9 The most important advantage of this technique is that the strong autofluorescence from complicated biological samples and scattering light from nearby optics can be easily eliminated by the time-gated detection mode, since the specific luminescence signal from the lanthanide complex label is super long-lived compared to the background fluorescence. On the basis of this feature, several powerful time-gated luminescence bioassay methods, such as time-gated luminescence immunoassay (TRFIA), DNA hybridization assay, microscopy imaging assay, and high-speed cytometry assay, have been successfully developed for the highly sensitive detection of various analytes in complicated biological and environmental samples.10−13 The main luminescent lanthanide complex-based biolabels used in the current time-gated luminescence bioassay technique are the Eu3+ and Tb3+ complexes with various β-diketone and aromatic amine derivative ligands.1−7,14,15 Although several classic bidentate β-diketonate-Eu3+ complexes, such as 2naphthoyltrifluoroacetonate-Eu3+ (β-NTA-Eu3+) and 2-thenoyltrifluoroacetonate-Eu3+ (TTA-Eu3+), have been successfully used in the dissociation enhanced lanthanide fluoroimmuno© 2012 American Chemical Society

Received: February 14, 2012 Revised: May 5, 2012 Published: May 31, 2012 1244

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without significant improvement, while BCDOT, BCOT, and BCTOT have the problem of protein−protein cross-linking owing to their two chlorosulfonyl groups; only the Eu3+ complex of BHHCT has shown good utility for a variety of time-gated luminescence bioassays, such as TR-FIAs of various antibodies and antigens,5,7 hybridization assays of DNAs,23,24 and microscopy imaging assays of environmental pathogens.25−27 This complex was also found to be useful for preparing highly luminescent europium nanobiolabels,28−31 and biosensors.32,33 As a biolabel, BHHCT-Eu3+ has advantages including being easy to conjugate to proteins through the formation of sulfonamide (protein-NH-SO2-label) without the protein cross-linking and strong luminescence with a larger quantum yield (∼27%) and longer luminescence lifetime (∼380 μs) in an aqueous buffer (bound to BSA).4,17 However, because BHHCT is a 1,2-diphenylbenzene derivative, the angle between the three benzene rings is hard to change, which makes the two bidentate β-diketone groups in BHHCT short of flexibility to simultaneously coordinate to a Eu3+ ion, to reduce the emission efficiency of its Eu3+ complex. To further improve the luminescence properties of βdiketonate-Eu3+ biolabels, especially those of BHHCT-Eu3+ analogues, in this work, three new chlorosulfonylated tetradentate β-diketonate-Eu3+ complexes that can display strong and long-lived Eu 3+ luminescence, 1,2-bis[4′(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]4-chlorosulfobenzene-Eu 3+ (BHHBCB-Eu 3+ ), 1,2-bis[4′(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]-4chlorosulfobenzene-Eu3+ (BPPBCB-Eu3+), and 1,2-bis[4′(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-4-chlorosulfobenzene-Eu3+ (BTBBCB-Eu3+), were designed and synthesized as biolabels for the time-gated luminescence bioassay application. Since there are two freely rotatable methylene groups introduced into the three benzene rings to enable the two bidentate β-diketone groups to simultaneously coordinate a Eu3+ ion easier, three such Eu3+ complexes can be expected to have higher emission efficiency and longer luminescence lifetime than those of BHHCT-Eu3+ analogues. To examine the practical utility of the new Eu3+ complexes as biolabels for time-gated luminescence bioassays, the BHHBCB-Eu3+-labeled streptavidin (SA) was prepared and used for the TR-FIA of human prostate specific antigen (PSA) and time-gated luminescence microscopy imaging of an environmental pathogen Cryptosporidium muris. Figure 1 shows the structures of three BHHCT analogues and the newly synthesized βdiketones.

Article

RESULTS AND DISCUSSION

Design, Synthesis, and Characterization of the New βDiketonate-Eu3+ Complexes. It has been known for a while that some β-diketones can form strongly luminescent complexes with lanthanide ions (mainly Eu3+, Sm3+, Tb3+, and Dy3+). The early application of these complexes to bioassays was pioneered by the successful establishment of highly sensitive TR-FIA technique in 1980s,1,3,34 in which isothiocyanatophenyl-ethylenediamine tetraacetate-Eu3+ or N1(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N4-tetraacetate-Eu3+ was employed for labeling antibodies or antigens. After the immune reaction, the immunocomplexes were reacted with a luminescence enhancement solution consisting of Triton X-100, β-diketone (2-naphthoyltrifluoroacetone, 2-thenoyltrifluoroacetone, or pivaloyltrifluoroacetone), and tri-n-octylphosphine oxide (TOPO) at pH 3.2 to transform Eu3+ from the nonluminescent Eu3+ label into a highly luminescent β-diketonate-Eu3+-TOPO ternary complex, so that the time-gated luminescence measurement could be carried out. Because this method suffers from the problem that the luminescence enhancement solution could be easily contaminated by the Eu(III)-containing experimental materials and environments (e.g., solvents, reagents, house dust, etc.), many efforts have been initiated to develop highly luminescent lanthanide complexes that can be directly used for biolabeling to avoid the use of luminescence enhancement solutions. However, the current status is still that there are only a few of luminescent lanthanide complexes, such as BHHCT-Eu3+ analogues and some lanthanide complexes with aromatic amine derivatives,35−47 suitable for direct biolabeling due to the strict requirements of strong luminescence and high stability in aqueous buffers and an appropriate biomoleculecoupling group for a lanthanide complex biolabel. To develop a better luminescent Eu3+ complex biolabel, we identify a new strategy of increasing the flexibility in the structure of BHHCT-type β-diketone, so that the two bidentate β-diketone groups can simultaneously coordinate to a Eu3+ ion easier, to improve the emission efficiency of its Eu3+ complex. In this work, by introducing two freely rotatable methylene groups into the three benzene rings of the 1,2-diphenylbenzene skeleton, a new class of chlorosulfonylated tetradentate βdiketones that can form strongly luminescent complexes with Eu3+ ions in aqueous solutions, BHHBCB, BPPBCB, and BTBBCB, were synthesized for improving the luminescence properties of BHHCT-Eu3+ analogues. The molecular modeling results of BHHCT-Eu3+ and BHHBCB-Eu3+ calculated using a Gaussian 09 software reveal that the four oxygen atoms in BHHBCB are more equally coordinated to the central Eu3+ ion than those in BHHCT (Figure 2), and the atomic population charges of Eu3+ in BHHCT-Eu3+ and BHHBCB-Eu3+ are 2.790 and 2.924, respectively. Three new chlorosulfonylated tetradentate β-diketones were synthesized according to the procedure shown in Scheme 1. After 1,2-bis(4′-acetyl-benzyl)-benzene was synthesized by using the Suzuki cross-coupling reaction between 1,2dibromomethyl-benzene and 4-acetyl-phenylboronic acid,48 three tetradentate β-diketones, 1,2-bis[4′-(1″,1″,1″,2″,2″,3″,3″heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-benzene, 1,2-bis[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]benzene, and 1,2-bis[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″yl)-benzyl]-benzene were synthesized by the Claisen condensation reaction between 1,2-bis(4′-acetyl-benzyl)-benzene

Figure 1. Structures of BHHCT analogues and new β-diketones BHHBCB, BPPBCB, and BTBBCB. 1245

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Figure 2. Molecular modeling results of BHHCT-Eu3+ and BHHBCB-Eu3+ calculated using Gaussian 09 software.

7.8. Three Eu3+ complexes displayed similar excitation and emission patterns with the maximum excitations at 327, 328, and 324 nm, and a sharp main emission peak at 608 nm (5D0 → 7F2 transition of Eu3+) for BHHBCB-Eu3+, BPPBCB-Eu3+, and BTBBCB-Eu3+, respectively. The luminescence properties of three Eu3+ complexes in 0.05 M borate buffer of pH 9.1 were measured, and the results were summarized in Table 1. It can

Scheme 1. Synthesis Procedure of Three New Chlorosulfonylated Tetradentate β-Diketones

Table 1. Luminescence Properties of BHHBCB-Eu3+, BPPBCB-Eu3+, and BTBBCB-Eu3+ (bound to BSA) in 0.05 M Borate Buffer of pH 9.1 complex (bound to BSA) 3+

BHHBCB-Eu BPPBCB-Eu3+ BTBBCB-Eu3+

and CnF2n+1CO2Et (n = 1−3) in dry diethyl ether in the presence of NaOCH3.49 Finally, the chlorosulfonylated βdiketones were obtained with high yields by reacting the βdiketones with HSO3Cl.17 The three compounds were wellcharacterized by the NMR, MS, and elementary analyses. The water-soluble conjugates of three Eu3+ complex-labeled bovine serum albumins (BSA), BSA-BHHBCB-Eu3+, BSABPPBCB-Eu3+, and BSA-BTBBCB-Eu3+, were prepared for characterizing the luminescence properties of the Eu 3+ complexes in aqueous buffers. Figure 3 shows the time-gated excitation and emission spectra of the conjugates of Eu3+ complexes (1.0 × 10−6 M) in 0.05 M Tris-HCl buffer of pH

λex,max (nm)

λem,max (nm)

ϕ (%)

τ (ms)

327 328 324

608 608 608

40 36 34

0.52 0.49 0.48

be observed that, among three Eu3+ complexes, BHHBCB-Eu3+ has both the highest quantum yield and the longest luminescence lifetime than those of BPPBCB-Eu3+ and BTBBCB-Eu3+, which indicates that BHHBCB-Eu3+ is the most suitable biolabel for highly sensitive time-gated luminescence bioassays. Furthermore, compared to BHHCTEu3+, the emissions of all three new Eu3+ complexes showed higher efficiency and longer lifetimes, which demonstrated the validity of our molecular structure design in this work. The new Eu3+ complexes also exhibited excellent stabilities in the biological buffer. As shown in Figure S1, the luminescence intensities of BHHBCB-Eu3+ at low concentrations (1−10 nM) are stable in 0.05 M Tris-HCl buffer of pH 7.8. To further compare the stability of BHHBCB-Eu3+ with that of BHHCTEu3+, the luminescence intensity changes of the BSA conjugated BHHBCB-Eu3+ (1.0 × 10−6 M) and BHHCT-Eu3+ (1.0 × 10−6 M) in the presence of different concentrations of EDTA in 0.05 M Tris-HCl buffer of pH 7.8 were investigated. As shown in Figure S2, the luminescence intensity of BHHCT-Eu3+ is decreased in the presence of 1.0 × 10−4 M of EDTA, whereas that of BHHBCB-Eu3+ remains stable even in the presence of 1.0 × 10−2 M of EDTA. These results clearly indicate that the stability of BHHBCB-Eu3+ is much higher than that of BHHCT-Eu3+ in aqueous buffers. The time-gated luminescence detection sensitivities of three Eu3+ complexes (bound to BSA) in 0.05 M Tris-HCl buffer of pH 7.8 were further measured on a more sensitive time-gated luminescence counter, Perkin-Elmer Victor 1420 multilabel counter, using 96-well microtiter plates as the cuvettes. As shown in Figure 4, the luminescence intensities displayed good linear relationships to the Eu3+ complex concentrations with

Figure 3. Time-gated excitation and emission spectra of the BSA conjugates of three Eu3+ complexes (1.0 × 10−6 M; red, BHHBCBEu3+; blue, BPPBCB-Eu3+; black, BTBBCB-Eu3+) in 0.05 M Tris-HCl buffer of pH 7.8. 1246

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Figure 4. Time-gated luminescence measurements of different concentrations of BHHBCB-Eu3+ (●), BPPBCB-Eu3+ (○), and BTBBCB-Eu3+ (□) in 0.05 M Tris-HCl buffer of pH 7.8 (the BSA bound complexes were used for the measurements).

Figure 5. Calibration curve of TR-FIA for human PSA using the BHHBCB-Eu3+-labeled SA (bg: background). The inset shows the plot of background-subtracted signal against human PSA concentration.

wide dynamic ranges. The detection limits, calculated as the concentrations corresponding to three standard deviations of the background signals, are 2.5 × 10−13 M, 2.8 × 10−13 M, and 4.6 × 10−13 M for BHHBCB-Eu3+, BPPBCB-Eu3+, and BTBBCB-Eu3+, respectively. These results indicate that the time-gated luminescence measurements using the new Eu3+ complexes as labels are more sensitive than that using BHHCTEu3+ as a label (the detection limit of BHHCT-Eu3+ under the same condition is 8.4 × 10−13 M).4,17 TR-FIA of Human PSA Using BHHBCB-Eu3+ as a Label. As an important tumor marker, the human PSA measurement with various immunoassay methods, i.e., enzyme immunoassay,50 radioimmunoassay,51 chemiluminescence immunoassay,52 and TR-FIA,53,54 has been widely used for the clinical diagnoses of malignant tumors, such as prostatic cancer55 and breast cancer.56 To evaluate the utility of the new Eu3+ complexes as labels for quantitative TR-FIA, the BHHBCBEu3+-labeled SA was prepared and used for the solid-phase TRFIA of human PSA. The BHHBCB-Eu3+-labeled SA was prepared by using a previously reported indirect method established for conjugating BHHCT-Eu3+ to SA.57 In the method, SA was first conjugated to BSA by cross-linking SA and BSA with glutaraldehyde, and then, the SA-BSA conjugate was labeled with BHHBCB-Eu3+. Because this method enables much more BHHBCB-Eu3+ molecules to be labeled to a SA molecule, as a result, an average of ∼47 BHHBCB-Eu3+ molecules were conjugated to each SA-BSA conjugate to yield the Eu3+ complex-labeled SA with an approximate composition of SA-BSA(BHHBCBEu3+)47. The TR-FIA of human PSA was carried out using a 96-well microtiter plate as the solid-phase carrier. After the wells were coated with antihuman PSA monoclonal antibody, the standard human PSA solutions with different concentrations and biotinylated antihuman PSA polyclonal antibody were successively added into the wells for reacting with the PSA antibody to form the sandwich-type immunocomplex, (antiPSA antibody)-PSA-(biotinylated anti-PSA antibody), on the surface of the wells. Finally, the BHHBCB-Eu3+-labeled SA was added into each well of the plate to react with the biotinylated antibody. After washing, the plate was directly used for the solid-phase time-gated luminescence measurement on the Perkin-Elmer Victor 1420 multilabel counter. Figure 5 shows the calibration curve of TR-FIA for human PSA using the

BHHBCB-Eu3+-labeled SA. When background-subtracted signal vs antigen concentration was plotted (the inset in Figure 5), the calibration curve showed a good linearity that could be expressed as log (signal) = 0.858 log[PSA] + 3.508 (r = 0.999). The detection limit, calculated as the concentration corresponding to three standard deviations of the background signal, is 57 pg/mL, which is low enough for the detection of PSA in human sera,50−54 and indicates that BHHBCB-Eu3+ can be used as a label for highly sensitive TR-FIA applications. Time-Gated Luminescence Microscopy Imaging of Cryptosporidium muris Using BHHBCB-Eu3+ as a Label. The waterborne pathogen cryptosporidium is an intestinal parasite in humans and animals that can cause a number of diarrheal disease outbreaks worldwide.58 In general, its detection can be carried out by using the immunofluorescence microscopy imaging technique.59,60 However, the food or environmental water samples of cryptosporidium are usually complicated, containing large amounts of strongly autofluorescent algae, organic debris, and mineral particles, which would obscure the immunofluorescence microscopy imaging result. It is noteworthy that the recent advances in time-gated luminescence microscopy imaging have demonstrated the background-suppression power for detecting pathogens in complicated environmental samples.25−27,29,30 To further confirm the utility of the new Eu3+ complexes as labels for time-gated luminescence bioassays, the BHHBCB-Eu3+-labeled SA was used for the immunoluminescence microscopy imaging of Cryptosporidium muris in both the steady-state and timegated modes. Figure 6 shows the bright-field, steady-state luminescence and time-gated luminescence images of Cryptosporidium muris oocysts that have been immunostained by anti-cryptosporidium antibody, biotinylated secondary antibody, and the BHHBCBEu3+-labeled SA in water and fruit juice samples. It should be mentioned that the strong red luminescence signals could be clearly observed from the BHHBCB-Eu3+-stained pathogen oocysts in a water sample in both steady-state and time-gated modes, but for the autofluorescence-rich juice sample, the steady-state luminescence imaging suffered the effect of autofluorescence from the coexisting substances within the sample, while the time-gated luminescence imaging provided highly specific background-free images of the oocysts. These results demonstrated that BHHBCB-Eu3+ is a favorably useful 1247

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distillation and purification. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification. 1 H NMR spectra were measured on a Bruker Avance spectrometer (400 MHz). Mass spectra were measured on a HP1100LC/MSD electrospray ionization mass spectrometry (ESI-MS). Elemental analysis was carried out on a Vario-EL analyzer. Absorption spectra were measured on a Perkin-Elmer Lambda 35 UV−vis spectrometer. Time-gated luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer with the conditions of delay time, 0.2 ms; gate time, 1.0 ms; cycle time, 20 ms; excitation slit, 10 nm; and emission slit, 5 nm. The luminescence quantum yields (ϕ1) of the Eu3+ complexes were measured with a previous method.62 The TR-FIA of human PSA was carried out with 96-well microtiter plate (PerkinElmer Life Sciences) as the solid-phase carrier and measured on a Perkin-Elmer Victor 1420 multilabel counter. All bright-field, steady-state, and time-gated luminescence imaging measurements were carried out on a laboratoryuse luminescence microscope.62 Synthesis of 1,2-Bis(4′-acetyl-benzyl)-benzene. In an ice−water bath, 2.46 g (10 mmol) of 1,2-bis(bromomethyl)benzene, 5.91 g (36 mmol) of 4-acetyl-phenylboronic acid and 6.90 g (50 mmol) of K2CO3 were dissolved in the mixture of acetone (60 mL) and water (20 mL) in a flask. After 70.9 mg (0.4 mmol) of PdCl2 was added, the solution was heated to 50 °C with stirring, and then the reaction was allowed to continue for 12 h under an argon atmosphere. The solvents were evaporated, and the residue was extracted with 2 × 100 mL chloroform, and then the organic phase was dried with Na2SO4. After evaporation of the solvent, the crude product was purified by silica gel column chromatography with petroleum ether/ ethyl acetate (2/1, v/v) as eluent to afford the target compound 1,2-bis(4′-acetyl-benzyl)-benzene as a white powder (1.58 g, 49% yield). 1H NMR (400 MHz, CDCl3): δ = 7.83 (d, J = 8.0 Hz,4H), 7.24−7.27 (m, 2H), 7.11−7.15 (m, 6H), 3.96 (s, 4H), 2.57 (s, 6H). Syntheses of Three 1,2-Bis[4′-(fluoroalkyl-β-diketonyl)-benzyl]-benzenes. To 30 mL of dry diethyl ether solution containing 2.92 mmol (1.0 g) of 1,2-bis(4′-acetyl-benzyl)benzene and 8.75 mmol of CnF2n+1CO2Et (2.04 g C3F7CO2Et, 1.68 g C2F5CO2Et, or 1.25 g CF3CO2Et) was added 8.75 mmol (0.47 g) of NaOCH3 with stirring. After the solution was stirred for 42 h at room temperature, 20 mL of 15% H2SO4 was added, and the mixture was further stirred for 20 min. Diethyl ether was evaporated, and the precipitate was collected and washed with distilled water. The crude product was recrystallized from ethanol to afford the target compound as yellow crystals (50− 60% yields for the three compounds). 1H NMR (400 MHz, CDCl3) for 1,2-bis[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-benzene: δ = 8.00 (d, J = 8.0 Hz, 4H), 7.29 (d, J = 5.6 Hz, 4H), 7.27 (d, J = 8.4 Hz, 4H), 6.92 (s, 2H), 4.14 (s, 4H); for 1,2-bis[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″pentanedion-5″-yl)-benzyl]-benzene: δ = 8.00 (d, J = 8.0 Hz, 4H), 7.25−7.30 (m, 8H), 6.92 (s, 2H), 4.14 (s, 4H); for 1,2bis[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-benzene: δ = 7.96 (d, J = 8.0 Hz, 4H), 7. 23−7.30 (m, 8H), 6.84 (s, 2H), 4.14 (s, 4H). Syntheses of BHHBCB, BPPBCB, and BTBBCB. To 4.0 mL of HSO3Cl was gradually added 1.0 g of the above 1,2bis[4′-(fluoroalkyl-β-diketonyl)-benzyl]-benzene with stirring. After stirring for 5 h at room temperature, the solution was added dropwise to 200 mL of ice−water with stirring. The

Figure 6. Bright-field (left), steady-state (middle), and time-gated (right) luminescence images of Cryptosporidium muris oocysts immunostained by the BHHBCB-Eu3+-labeled SA in water (top) and fruit juice (bottom) samples. Scale bar: 10 μm.

biolabel for time-gated immuno-luminescence microscopy imaging to effectively eliminate the interference from shortlived background fluorescence.

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CONCLUSIONS In summary, a new class of chlorosulfonylated tetradentate βdiketonate-Eu3+ complexes, BHHBCB-Eu3+, BPPBCB-Eu3+, and BTBBCB-Eu3+, has been successfully designed, synthesized, and characterized as biolabels for time-gated luminescence bioassay applications. Our demonstrations of TR-FIA for human PSA and luminescence microscopy imaging for pathogen Cryptosporidium muris using the BHHBCB-Eu3+labeled SA approved the practical utility of the label for timegated luminescence bioassays. Compared to the Eu3+ complex biolabel BHHCT-Eu3+, the new biolabels show the further improved luminescence properties with higher emission efficiency and longer luminescence lifetimes, which reveals a new underpinning theory in the structure design of tetradentate β-dikatonate-Eu3+ complex biolabels. Our new class of chlorosulfonylated tetradentate β-diketonate-Eu3+ complexes should enhance various time-gated luminescence bioassay applications piloted by BHHCT-Eu3+ or the other lanthanide complex biolabels. Furthermore, the successful development of the new Eu3+ complex biolabels suggests that the lanthanide complex-based biolabels could be improved through the rational design of ligands, which would be a useful strategy for developing new lanthanide biolabels with better luminescence properties.

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EXPERIMENTAL SECTION Materials and Physical Measurements. Mouse monoclonal and goat polyclonal antihuman PSA antibodies were purchased from OEM Concepts Co. Biotinylated goat antihuman PSA antibody was prepared and used according to a previous method.61 The standard solutions of human PSA were prepared by diluting human PSA antigen (Biogenesis Ltd.) with 0.05 M Tris-HCl buffer of pH 7.8 containing 5% BSA, 0.9% NaCl, and 0.1% NaN3. BSA and SA were purchased from Sigma. Cryptosporidium muris and its monoclonal antibody (IgM) were purchased from Anapure Bioscientific Co. Ltd. Rabbit antimouse IgM antibody was purchased from Beijing Biosynthesis Biotechnology Co. Ltd. Biotinylated rabbit antimouse IgM antibody was prepared and used according to a previous method.61 Diethyl ether was used after appropriate 1248

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precipitate was filtered, washed with cold water, and then dried under vacuum for more than 48 h to afford the target compound as a yellow powder (80−90% yields for the three compounds). 1H NMR (400 MHz, CDCl3) for BHHBCB: δ = 8.02−8.06 (m, 6H), 7.66 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 8.8 Hz, 2H), 6.92 (s, 2H), 4.41 (s, 2H), 4.36 (s, 2H); for BPPBCB: δ = 7.92−7.96 (m, 6H), 7.57 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.8 Hz, 4H), 6.83 (s, 2H), 4.32 (s, 2H), 4.26 (s, 2H); for BTBBCB: δ = 8.01−8.06 (m, 6H), 7.67 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 8.8 Hz, 4H), 6.85 (s, 2H), 4.41 (s, 2H), 4.35 (s, 2H). 13C NMR (100 MHz, acetone-d6) for BHHBCB: δ = 39.27, 39.51, 93.28, 126.56, 129.16, 129.55, 129.67, 130.44, 130.49, 130.56, 130.62, 131.90, 133.48, 142.08, 143.60, 146.90, 147.14, 148.50, 176.72, 177.08; for BPPBCB: δ = 38.35, 38.59, 93.81, 125.65, 128.34, 128.36, 128.37, 129.59, 129.73, 130.77, 132.53, 141.17, 142.71, 146.17, 146.39, 147.60, 185.00; for BTBBCB: δ = 39.26, 39.51, 94.56, 126.56, 129.27, 129.64, 130.48, 130.61, 131.79, 133.44, 142.07, 143.61, 147.06, 147.29, 148.50. Elemental analysis results: calcd. (%) for BHHBCB (C32H19ClF14O6S·2.5H2O), C 43.78, H 2.16; found (%), C 43.53, H 2.38; calcd. (%) for BPPBCB (C30H19ClF10O6S·H2O), C 47.98, H 2.80; found (%), C 47.54, H 2.43; calcd. (%) for BTBBCB (C28H19ClF6O6S·H2O), C 51.67, H 3.23; found (%), C 51.46, H 2.90. API-ES-MS (negative mode) results: for BHHBCB, m/z 833.0 (45%), [M-H]−; for BPPBCB, m/z 733.0 (100%), [M-H]−; for BTBBCB, m/z 633.0 (100%), [M-H]−. Preparation of the Eu3+ Complex-Labeled BSA. To the solution of 10.0 mg BSA dissolved in 2.0 mL of 0.05 M carbonate buffer at pH 9.3 was added dropwise 9.0 μmol of the chlorosulfonylated tetradentate β-diketone (7.9, 6.8, and 5.9 mg for BHHBCB, BTBBCB, and BTBBCB, respectively) dissolved in 400 μL of ethanol with stirring. After 2 h of stirring at room temperature, the β-diketone-labeled BSA was separated from the unreacted β-diketone (the hydrolyzed product) by Sephadex G-50 column chromatography with 0.05 M NH4HCO3 of pH 8.0 as eluent. To estimate the labeling ratio (the bound β-diketone numbers per BSA molecule), the molar extinction coefficient of the β-diketone at its maximum absorption wavelength (328, 326, and 324 nm for BHHBCB, BTBBCB, and BTBBCB, respectively) was measured by using the unchromatographed solution appropriately diluted with 0.05 M NH4HCO3 of pH 8.0 (3.08 × 104, 2.65 × 104, and 1.60 × 104 cm−1 M−1 for BHHBCB, BTBBCB, and BTBBCB, respectively). Assuming that the molar extinction coefficient of the β-diketone did not change before and after the BSA binding, the labeling ratio was calculated (∼34, ∼18, and ∼17 for BHHBCB-BSA, BTBBCB-BSA, and BTBBCB-BSA, respectively). After EuCl3 (1.5-fold concentrations of the β-diketone) and NaN3 (0.1%) were added into the labeled BSA solution, the Eu3+ complex-labeled BSA solution was obtained, and then stored at 4 °C before use. Preparation of the BHHBCB-Eu3+-Labeled SA. To 0.6 mL of 0.1 M phosphate buffer at pH 7.1 containing 0.4 mg SA and 0.4 mg BSA was added 0.1 mL of 1% glutaraldehyde. After the solution was stirred for 24 h at 4 °C, 0.4 mg of NaBH4 was added, and then the solution was further incubated for 2 h at room temperature. The solution was dialyzed twice each for 24 h against 3 L of 0.9% NaCl solution at 4 °C. After 10 mg NaHCO3 was added, the solution’s pH was adjusted to 9.1 with 1.0 M of NaOH. To the solution was added dropwise 1.0 mg of BHHBCB dissolved in 40 μL of dimethyl sulfoxide (DMSO), and then the solution was stirred for 1 h at room temperature.

The solution was dialyzed twice each for 24 h against 3 L of 0.9% NaCl solution at 4 °C to delete the unreacted BHHBCB. After 0.63 mg of EuCl3·6H2O was added, the solution was 50fold diluted with 0.05 M Tris-HCl buffer of pH 7.8 containing 0.2% BSA, 0.9% NaCl, and 0.1% NaN3, and then stored at −20 °C before use. Because the actual species of the SA−BSA conjugate cross-linked by glutaraldehyde were rather complicated, the composition of the BHHBCB-Eu3+-labeled SA−BSA conjugate prepared by the above method was approximately determined to be SA-BSA(BHHBCB-Eu3+)47. TR-FIA of Human PSA. After antihuman PSA monoclonal antibody (diluted to 10 μg/mL with 0.1 M carbonate buffer of pH 9.6) was coated on the wells (45 μL per well) of a 96-well microtiter plate by physical adsorption,63 45 μL of human PSA standard solutions with different concentrations were added to the wells. The plate was incubated at 37 °C for 1 h and washed twice with 0.05 M Tris-HCl buffer of pH 7.8 containing 0.05% Tween 20 and once with 0.05 M Tris-HCl buffer of pH 7.8. Then, 45 μL aliquots of the biotinylated antibody (∼1.1 μg/ mL) were added to each well, and the plate was incubated at 37 °C for 1 h. After washing, 45 μL aliquots of the BHHBCBEu3+-labeled SA were added to each well, and the plate was incubated at 37 °C for 1 h. The plate was washed four times with 0.05 M Tris-HCl buffer of pH 7.8 containing 0.05% Tween 20, and then subjected to the solid-phase time-gated luminescence measurement on Perkin-Elmer Victor 1420 multilabel counter under the conditions of excitation wavelength, 340 nm; emission wavelength, 615 nm; delay time, 0.2 ms; and window time (counting time), 0.4 ms. Time-Gated Luminescence Microscopy Imaging of Cryptosporidium muris. A suspension of 10 μL Cryptosporidium muris (2.5 × 105 oocysts/mL in water) was mixed with 8 μL of anti-cryptosporidium monoclonal antibody (mouse IgM, ∼50 μg/mL), 8 μL of biotinylated rabbit antimouse IgM antibody (∼44 μg/mL), and 8 μL of the BHHBCB-Eu3+labeled SA in a tube. After incubation for 10 h at room temperature, the oocysts were separated by centrifugation at 500 rpm, and washed three times each with 50 μL of distilled water. The oocysts were resuspended in 20 μL of water or an autofluorescence-rich fruit juice sample, and then spotted on a glass slide for the luminescence microscopy imaging measurement. The luminescence microscope, equipped with a 100 W Hg lamp, UV-2A filters (excitation filter, 330−380 nm; dichroic mirror, 400 nm; emission filter, >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 steady-state luminescence imaging measurement with an exposure time of 5 s. The microscope, equipped with a 30 W xenon flashlamp, UV-2A and V-2A filters, and a time-resolved digital black-and-white CCD camera system (Imagex-TGi, Photonic Research Systems Ltd.), was used for the time-gated luminescence imaging measurement with the conditions of delay time, 100 μs; gate time, 1 ms; lamp pulse width, 6 μs; and exposure time, 180 s. The time-gated luminescence images are shown in pseudocolor treated by a SimplePCI software.62

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ASSOCIATED CONTENT

S Supporting Information *

Two supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. 1249

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AUTHOR INFORMATION

Corresponding Author

* T e l / F a x : + 8 6 - 4 1 1- 8 4 9 8 6 0 4 1 . E - m a i l a d d r e ss e s : [email protected] (Z.Ye); jingliyuan@yahoo. com.cn (J.Yuan). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial supports from the National Natural Science Foundation of China (Grant No. 20835001) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 200801410003) are gratefully acknowledged.

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ABBREVIATIONS USED: BHHBCB, 1,2-bis[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″- yl)benzyl]-4-chlorosulfobenzene; BPPBCB, 1,2-bis[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″- pentanedion-5″-yl)benzyl]4-chlorosulfobenzene; BTBBCB, 1,2-bis[4′-(1″,1″,1″-trifluoro2″,4″- butanedion-4″-yl)benzyl]-4-chlorosulfobenzene; BHHCT, 4,4′-bis(1″,1″,1″,2″,2″,3″,3″- heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl; BPPCT, 4,4′-bis(1″,1″,1″,2″,2″- pentafluoro-3″,5″-pentanedion-5″-yl)-chlorosulfo-o-terphenyl; BTBCT, 4,4′-bis(1″,1″,1″-trifluoro- 2″,4″-butanedion-4″-yl)-chlorosulfo-o-terphenyl; BCDOT, 1,10-bis(4″chlorosulfo-1′,1″-diphenyl- 4′-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone; BCOT, 1,10-bis(8′-chlorosulfo- dibenzothiophene-2′-yl)-4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone; BCTOT, 1,10-bis(5′- chlorosulfothiophene-2′-yl)4,4,5,5,6,6,7,7-octafluorodecane-1,3,8,10-tetraone; TR-FIA, time-gated luminescence immunoassay; BSA, bovine serum albumin; SA, streptavidin; PSA, prostate specific antigen; TOPO, tri-n-octylphosphine oxide; EDTA, ethylenediamine tetraacetic acid.

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