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Novel Intramolecular Energy Transfer Probe for the Detection of Benzo[a]pyrene Metabolites in a Homogeneous Competitive Fluorescence Immunoassay Annette Kupstat,† Dietmar Knopp,‡ Reinhard Niessner,‡ and Michael U. Kumke*,† Department of Chemistry (Physical Chemistry), UniVersity of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany, and Chair of Analytical Chemistry and Institute of Hydrochemistry, Technische UniVersita¨t Mu¨nchen, Marchioninistrasse 17, 81377 Mu¨nchen, Germany ReceiVed: June 26, 2009; ReVised Manuscript ReceiVed: December 10, 2009
The spectroscopic properties of a novel intramolecular energy transfer probe (ET probe)sconsisting of 3-hydroxybenzo[a]pyrene (3OH-BaP) as the donor covalently linked to sulforhodamine B (SRB) as the acceptorsfor the detection of polycyclic aromatic hydrocarbons (PAH) antibody binding were characterized. Absorption and fluorescence spectra as well as fluorescence decay curves were recorded in methanol and aqueous solution, respectively. For comparison, the parent chromophores 3OH-BaP and SRB were investigated as well. In the case of the ET probe, a very strong fluorescence quenching of the BaP-moiety-related emission due to an efficient energy transfer (energy transfer efficiency of about 0.95 for methanol) to the SRB moiety was observed. Upon addition of the PAH antibody, the fluorescence intensity and anisotropy of the BaP moiety was drastically increased. On the other hand, the fluorescence anisotropy of the SRB moiety did not change. The anisotropy results clearly indicate the binding of the antibody. On the basis of these findings, we concluded the following model: the BaP moiety is incorporated in the antibody binding site, whereas the SRB moiety sticks out from the binding site, restricting the motion of the BaP moiety, but leaving the SRB moiety uninfluenced. More important, this structure results in a disruption of the intramolecular energy transfer. The antibody-induced disruption of the intramolecular energy transfer is envisaged as a detection scheme in a future homogeneous competitive fluorescence immunoassay (FIA). This may provide a novel general detection principle for the immunodetection of low-molecular analytes (haptens) in a homogeneous competitive FIA format. Introduction Polycyclic aromatic hydrocarbons (PAH) are serious environmental pollutants formed during incomplete combustion of organic matter.1 PAH exposure is related to cancer diseases of different organs, e.g., lungs, nose, oral cavity, pancreas, and kidney.2 Benzo[a]pyrene (BaP) was the first PAH for which the ability to cause cancer was directly proven.3 BaP metabolites (hydroxy-BaP, OH-BaP) can be detected in human urine.4 From this, individual BaP exposure is defined,4 specifying the health hazard more precisely than the determination of the environmental pollution. The established detection techniques contain several tedious sample preparation steps followed by chromatographic separation.4,5 With separation-free (homogeneous) fluorescence immunoassays (FIA) expensive instrumentation, extensive sample pretreatment, and large consumption of chemicals, could be overcome. Common homogeneous FIA detect the analyte via Foerster resonance energy transfer (FRET) between donor- and acceptorlabeled antibodies recognizing the analyte at two different binding sites (commercially available for different cancer markers, e.g., total prostate specific antigen from Brahms AG). Because this approach requires that the analyte can bind two antibodies simultaneously, it is mostly used for the detection of proteins (antigens). Although there are a few exceptions (e.g., Pulli et al. presented a morphine assay employing two antibod* To whom correspondence should be addressed. Tel: ++493319775209. Fax: ++493319775058. E-mail:
[email protected]. † University of Potsdam. ‡ Technische Universita¨t Mu¨nchen.
ies, one recognizing the analyte and the other the immune complex6), low-molecular-weight analytes (haptens) are mostly detected using competitive FIA. In this assay format the detection is based on a fluorophore-conjugated hapten (tracer, fluorescence probe), which is used as a competitor for the antibody binding site. Since it was observed that the fluorescence of fluorescein is changed by antibody binding, fluorescein conjugates are used in many competitive FIA.7-10 This assay format is simple and cost-effective, because only one antibody has to be generated and no antibody labeling is required. Further homogeneous FIA for hapten detection have been reported which are based on FRET between different donor-acceptor pairs conjugated to antibody and hapten.11-14 Direct measurements in complex biological samples (e.g., urine, whole blood) are challenging because of autofluorescence and absorption of tracer fluorescence by matrix components. Therefore, novel fluorescence probes generating, e.g., upconversion FRET15 or two-photon excitation FRET,16 are needed to tackle these problems. Within the development of a competitive homogeneous fluorescence immunoassay for the detection of polycyclic aromatic hydrocarbon (PAH) metabolites in human urine, a novel fluorescence probe (ET probe) was designed, in which sulforhodamine B (SRB) was covalently linked via a short aliphatic chain to 3-hydroxybenzo[a]pyrene (3OH-BaP). A fluorescence enhancement upon antibody binding of the ET probe was observed. The present study was performed to elucidate the observed phenomenon on a molecular level. The basic photophysical properties such as fluorescence decay times
10.1021/jp906014j 2010 American Chemical Society Published on Web 01/07/2010
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Figure 1. Chemical structure of the ET probe (BaP moiety, left; SRB moiety, right).
τ, fluorescence anisotropy r, and fluorescence quantum yield φ of the novel ET probe were determined in methanol and aqueous buffer (PBS), respectively. Special emphasis was drawn on induced changes in the photophysics of the ET probe upon binding to the PAH antibody. Experimental Methods Reagents and Chemicals. The parent compounds 3-hydroxybenzo[a]pyrene (3OH-BaP) and sulforhodamine B (SRB) were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). The ET probe was synthesized by the Biochemical Institute for Environmental Carcinogens (Grosshansdorf, Germany). Its chemical structure is shown in Figure 1 (M ) 895 g/mol). The stock solutions were prepared in methanol ([3OHBaP] ) 0.7 mM, [SRB] ) 2.4 mM, [ET probe] ) 0.3 mM). All chemicals and solvents were of analytical grade. The monoclonal PAH antibody (Mab13) was obtained as described in the literature.17 Mab13 was dissolved in Phosphate-buffered saline (PBS, pH 7.4, in double distilled water) in the presence of 0.01% Thimerosal as antimicrobial agent ([Mab13] ) 1 mg/mL). The spectroscopic characterization of the ET probe and the parent chromophores 3OH-BaP and SRB was carried out in methanol and PBS, respectively. For the fluorescence measurements diluted solutions of the chromophores were used (optical density τ
yes no no
298 0.17 ( 0.01 >τ 77 0.30 ( 0.01 24 ( 1 (βD) 298 0.05 ( 0.01 0.27 ( 0.02
yes no
298 0.05 ( 0.01 0.28 ( 0.03 77 0.29 ( 0.01 25 ( 1 (βA) -
0.11 ( 0.02
anisotropy of the ET probe’s BaP moiety at room temperature was close to zero (Table 3). This was measured in methanol because of the complete fluorescence quenching of the BaP moiety in PBS (assuming the rotational diffusion in both media to be equal). In presence of Mab13 (in PBS), the fluorescence anisotropy was drastically increased to r ) 0.24 ( 0.01 (Table 3). In order to judge this value, it was compared with the fundamental anisotropy r0 measured in a rigid glassy matrix (EPA) at 77 K () nearly complete restriction of the rotational diffusion). For parent 3OH-BaP r0 ) 0.30 ( 0.01 was determined (in this case, parent 3OH-BaP was used as reference, because the value of r0 for the ET probe’s BaP moiety is distorted by the intramolecular energy transfer, see Table 3). These findings clearly point to a strongly restricted rotation of the BaP moiety due to the antibody binding. Furthermore, it was observed that the fluorescence anisotropy was independent of the Mab13 concentration. This is due to the fact that only the Mab13-bound ET probe molecules show the BaP related fluorescence. The fluorescence anisotropy of parent 3OH-BaP in presence of Mab13 was investigated as well. Like for the BaP moiety of the ET probe, the fluorescence anisotropy of 3OH-BaP in methanol at room temperature was close to zero due to fast free rotation (Table 3). Upon Mab13 addition the fluorescence anisotropy was increased to r ) 0.17 ( 0.01 (Table 3). This indicates a restricted rotation of the 3OH-BaP molecules in presence of Mab13. But still, this value r is less than the
anisotropy of ET probe’s BaP moiety in presence of Mab13 (vide supra). The reason is that, in contrast to the BaP moiety, the unbound as well as the Mab13-bound 3OH-BaP molecules are fluorescent. The measured fluorescence anisotropy represents an average value of both. In addition, the steady-state fluorescence anisotropy of the SRB moiety was determined. In absence of Mab13 r ) 0.05 ( 0.01 at room temperature was determined and this did not change upon antibody addition (Table 3). In a control experiment at 77 K, the fundamental fluorescence anisotropy of SRB moiety was determined to r0 ) 0.29 ( 0.01 (Table 3). These findings indicate that the rotation of the SRB moiety is not restricted due to the binding of the ET probe to Mab13. In further studies on the interaction between Mab13 and the ET probe, time-resolved fluorescence anisotropy measurements were performed. In Figure 4 the anisotropy decays of the ET probe’s BaP moiety as well as the parent compound 3OH-BaP are shown in the absence and presence of Mab13. The corresponding fluorescence decays were measured at λex ) 375 nm and λem ) 466 nm. In case of the ET probe, the fluorescence anisotropy of the BaP moiety in the absence of antibody showed a fast single-exponential decay (Figure 4, left side, gray line) and a rotational correlation time θ ) 0.18 ( 0.01 ns was determined for the BaP moiety in methanol (Table 3). In the presence of Mab13 in PBS, the rotation of the BaP moiety was slowed down (Figure 4, left side, black line). The corresponding rotational correlation time was much larger than the fluorescence decay time and as a result the observed anisotropy appears almost constant in the time window of the measurement (see Table 2). In a control experiment, parent 3OH-BaP in the absence and presence of Mab13 was investigated as well (Figure 4, right side). Without antibody the fluorescence anisotropy of parent 3OH-BaP showed a fast, single-exponential decay (gray line). Similar to the ET probe, in methanol a rotational correlation time θ ) 0.11 ( 0.02 ns was found (Table 3). This is in excellent agreement with θ ) 0.12 ns calculated according to eq 8 (for this we assumed that the 3OH-BaP chromophore rotates like a sphere and therefore the specific volume ν was roughly estimated from the volume of a sphere with the radius of 530 pm). In contrast to the ET probe’s BaP moiety, the fluorescence anisotropy decay of 3OH-BaP in presence of Mab13 (black line) showed a fast and a very slow component. The two components were attributed to two populations of 3OHBaP: unbound 3OH-BaP molecules showing a fast, unrestricted rotational diffusion and the Mab13-bound 3OH-BaP molecules, with their slowed, restricted rotation as it was also observed in case of the ET probe bound to Mab13. Binding of the antibody to the ET probe did not restrict the rotation of the SRB moiety, the rotational correlation time in both casessabsence and presence of Mab13 (in PBS)swas
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Figure 4. Fluorescence anisotropy decay of the hapten-antibody complexes. On the left side the fluorescence anisotropy decay of the ET probe’s BaP moiety in the absence (in methanol) and in the presence of PAH antibody Mab13 (in PBS) is shown. On the right side the fluorescence anisotropy decay of the parent chromophore 3OH-BaP in the absence and presence of Mab13 (in PBS) is presented.
determined (λex ) 560 nm and λem ) 580 nm) to be about θ ) 0.3 ns (Table 3). From these results the following binding model was deduced: the antibody Mab13 binds specifically to the ET probe by incorporating the BaP moiety in its binding site, where the rotation of the BaP moiety is restricted. At the same time, the SRB moiety sticks out from the antibody binding site, maintaining the free and fast rotation. This structure results in a disruption of the intramolecular energy transfer of the ET probe. We discussed short-range interactions such as Dexter electron exchange or charge transfer to be potentially responsible for the complete fluorescence quenching of the BaP moiety in PBS. We attributed this to a “folded” conformation of the ET probe induced by the hydrophobic character of the BaP moiety (vide supra). It is obvious, that the incorporation of the BaP moiety in the antibody binding site impedes a “folded” conformation of the ET probe resulting in longer DA distances and an inhibition of short-range interactions. Furthermore, we did not observe intramolecular FRET in presence of Mab13. This was supported by the comparable fluorescence decay times of the BaP moiety and parent 3OH-BaP when bound to Mab13 (see Table 2). A further evidence was found in the fluorescence excitation spectrum of the SRB moiety in presence of Mab13 (λem ) 640 nm): we did not observe the characteristic 3OHBaP absorption bands in the spectral range of 330 nm < λex < 450 nm detected in methanol (vide supra and Supporting Information, Figure 2S). From these findings we conclude that the observed disruption of the intramolecular energy transfer is caused by a Mab13-induced change of the ET probe’s molecular conformation resulting in (i) longer DA distances (“unfolding”) impeding Dexter or charge transfer and (ii) improper mutual orientation of the chromophore’s transition dipoles inhibiting FRET. Conclusions An in-depth spectroscopic characterization of the novel ET probe was performed. The observed fluorescence quenching of the BaP moiety (D) was analyzed in methanol and PBS. For methanol, the well-established Foerster resonance energy transfer formalism was successfully applied. For methanol the Foerster distance between 3-OH-BaP (D) and SRB (A) in the ET probe was calculated to R0 ) 3.7 nm. Theoretically calculated and experimentally determined energy transfer efficiencies were in excellent agreement, from both approaches an energy transfer efficiency E > 0.9 was obtained. For PBS the observed fluorescence quenching was even further increased. The reason for the solvent-dependent amplification of the fluorescence quenching was attributed to changes in the overall conformation of the ET probe leading to a further decrease in the effective distance between the BaP and the SRB
moiety (“folding”) and permitting short-range energy transfer (Dexter or charge transfer). It was found that the binding of the PAH-specific antibody Mab13 to the ET probe inhibits short-range as well as longrange intramolecular (Foerster) energy transfer resulting in a drastically fluorescence enhancement of the BaP moiety. This was attributed to a Mab13-induced change of the ET probe’s conformation leading to (i) longer DA distances (“unfolding”) impeding Dexter or charge transfer and (ii) improper mutual orientation of the chromophore’s transition dipoles inhibiting FRET. Fluorescence anisotropy experiments were done to elucidate the ET probe-Mab13 interaction on a molecular level. For the BaP moiety the fluorescence anisotropy and the corresponding rotational correlation time were significantly increased compared to the unbound state, whereas the SRB moiety showed no change in the fluorescence anisotropy and the related rotational correlation time upon binding of the ET probe to Mab13. This indicates that the SRB moiety is not incorporated in or strongly interacting with the antibody. From these findings it was concluded that the antibody binds specifically to the BaP moiety of the ET probe, whereas the SRB moiety sticks out from the antibody binding site. The antibody-induced disruption of the intramolecular energy transfer of the ET probe competing with the analyte for the antibody binding site is envisaged as a promising novel detection scheme for low-molecular analytes (haptens) in a future homogeneous competitive FIA. Specific ET probes mimicking the haptens could be used as competing agents. Work is in progress to design improved ET probes with different acceptor molecules in order to use the acceptor related fluorescence as an additional analytical parameter. Since the acceptor emission is found at longer emission wavelength, interferences due to matrix components (e.g., absorption, autofluorescence) could be further reduced. Acknowledgment. We thank Thomas Ritschel for the quantum mechanical calculations. The financial support of the Federal Ministry of Education and Science (Bundesministerium fu¨r Bildung and Forschung, BMBF, contract no. FKZ 03IP515) is acknowledged by the authors. Supporting Information Available: Benesi-Hildebrand plot for the ET probe-Mab13 binding; fluorescence excitation spectra of the ET probe’s SRB moiety compared to parent SRB (λem ) 640 nm); fluorescence decay of the ET probe’s BaP moiety compared to parent 3OH-BaP; and absorption spectrum of the ET probe in the presence of Mab13 (in PBS buffer). This material is available free of charge via the Internet at http:// pubs.acs.org.
Detection of Benzo[a]pyrene Metabolites References and Notes (1) Beloglazova, N. V.; Goryacheva, I. Y.; Mikhirev, D. A.; de Saeger, S.; Niessner, R.; Knopp, D. Anal. Sci. 2008, 24, 1613. (2) Lodovici, M.; Bigagli, E Int. J. EnViron. Res. Public Health 2009, 6, 874. (3) Denissenko, M. F.; Pao, A.; Tang, M.; Pfeifer, G. P. Science 1996, 274, 430. (4) Vatsavai, K.; Goicoechea, H. C.; Campiglia, A. D. Anal. Biochem. 2008, 376, 213. (5) Goryacheva, I. Y.; Eremin, S. A.; Shutaleva, E. A.; Suchanek, M.; Niessner, R.; Knopp, D. Anal. Lett. 2007, 40, 1445. (6) Pulli, T.; Ho¨yhtya¨, M.; So¨derlund, H.; Takkinen, K. Anal. Chem. 2005, 77, 2637. (7) Kobayashi, Y.; Tsubota, N.; Miyai, K.; Watanabe, F. Steroids 1980, 36, 177. (8) Matveeva, E. G.; Popova, V. A.; Eremin, S. A. J. Fluoresc. 1997, 7, 251. (9) Nichkova, M.; Feng, J.; Sanchez-Baeza, F.; Marco, M.-P.; Hammock, B. D.; Kennedy, I. M. Anal. Chem. 2003, 75, 83. (10) Smith, D. S. FEBS Lett. 1977, 77, 25. (11) Kokko, T.; Kokko, L.; Soukka, T. J. Fluoresc. 2009, 19, 159. (12) Kokko, T.; Kokko, L.; Lo¨vgren, T.; Soukka, T. Anal. Chem. 2007, 79, 5935. (13) Kokko, T.; Kokko, L.; Soukka, T.; Lo¨vgren, T. Anal. Chim. Acta 2007, 585, 120. (14) Mayilo, S.; Ehlers, B.; Wunderlich, M.; Klar, T. A.; Josel, H.-P.; Heindl, D; Nichtl, A.; Ku¨rzinger, K.; Feldmann, J. Anal. Chim. Acta 2009, 646, 119.
J. Phys. Chem. B, Vol. 114, No. 4, 2010 1673 (15) Kuningas, K.; Ukonaho, T.; Pa¨kkila¨, H.; Rantanen, T.; Rosenberg, J.; Lo¨vgren, T.; Soukka, T. Anal. Chem. 2006, 78, 4690. (16) Liu, L.; Shao, M.; Dong, X.; Yu, X.; Liu, Z.; He, Z.; Wang, Q. Anal. Chem. 2008, 80, 7735. (17) Matschulat, D.; Deng, A. P.; Niessner, R.; Knopp, D. Analyst 2005, 130, 1078. (18) Fo¨rster, T. 10th Spiers Memorial Lect. 1959. (19) Stryer, L. Annu. ReV. Biochem. 1978, 47, 819. (20) Wang, X. F.; Herman, B. Fluoresc. Imaging Spectrosc. Microsc. 1996. (21) Rodrı´guez-Ca´ceres, M. I.; Agbaria, R. A.; Warner, I. M. J. Fluoresc. 2005, 15, 185. (22) Frisch, M. J.; et al. Gaussian 09, ReVision A.02; Gaussian, Inc.: Wallingford, CT, 2009. (23) Stewart, J. J. P. J. Mol. Model. 2007, 13, 1173. (24) Becke, A. D. J. Chem. Phys. 1993, 98, 1372. (25) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1998, 37, 785. (26) Lefevre, C.; Kang, H. C.; Haugland, R. P.; Malekzadeh, N.; Arttamangkul, S.; Haugland, R. P. Bioconjugate Chem. 1996, 7, 482. (27) Brismar, H.; Trepte, O.; Ulfhake, B. J. Histochem. Cytochem. 1995, 43, 699. (28) Dietrich, A.; Buschmann, V.; Mu¨ller, C.; Sauer, M. ReV. Mol. Biotechnol. 2002, 82, 211. (29) Ka¨ppel, N. D. Immunoassay-Optimierung fu¨r Verschiedene Probenmatrices; Rhombos: Berlin, 2008.
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