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A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions
Chemosphere xxx (2016) 1e7
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A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions Taihua Li a, 1, Yo Han Choi b, c, 1, Yong-Beom Shin d, Hwa-Jung Kim c, Min-Gon Kim b, e, * a
College of Biology and the Environment, Nanjing Forestry University, Nanjing 210-037, China Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science & Technology (GIST), 261 Cheomdan-gwagiro, Gwangju 500-712, South Korea c Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea d Biomedical Translational Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, South Korea e Advanced Photonics Research Institute, Gwangju Institute of Science & Technology (GIST), 261 Cheomdan-gwagiro, Gwangju 500-712, South Korea b
h i g h l i g h t s Fluorescence enhancement and FRET-based immunoassay of benzo[a]pyrene (BaP) was developed. Binding to the anti-BaP resulted in a 3.12-fold increase in the fluorescence intensity of BaP. The assay can be used to detect BaP specifically with a limit of detection of 0.06 ng mL1.
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Article history: Received 3 December 2014 Received in revised form 15 December 2015 Accepted 4 January 2016 Available online xxx
A fluorescence enhancement-based immunoassay has been developed for the detection of the polycyclic aromatic hydrocarbons (PAH), benzo[a]pyrene (BaP), in aqueous solutions. The results of this study show that BaP, which inefficiently fluoresces in aqueous solution, displays enhanced fluorescence when bound to the anti-BaP antibody (anti-BaP), as part of a label-free immunoassay system. Binding to anti-BaP results in a 3.12-fold increase in the fluorescence intensity of BaP, which emits at 435 nm when excited at 280 nm, due to the hydrophobic interaction and fluorescence resonance energy transfer (FRET) between antibody and antigen. As result of this phenomenon, the antibody-based fluorescence immunoassay system can be used to detect BaP specifically with a limit of detection (LOD) of 0.06 ng mL1. Finally, extraction recoveries of BaP from spiked wheat and barley samples were found to be in the range of 80.5e87.0% and 92.9e92.1%, respectively. © 2016 Published by Elsevier Ltd.
Handling Editor: Prof. I. Cousins Keywords: Fluorescence immunoassay FRET Fluorescence enhancement Polycyclic aromatic hydrocarbons Benzo[a]pyrene
1. Introduction The relatively high environmental levels of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), coupled with its high toxicity reflected mainly by its carcinogenicity, creates a large detrimental impact on human health (Barhoumi et al., 2000; Chang et al., 1987; Wattenburg and Leong, 1970; Weyand and Bevan,
* Corresponding author. Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science & Technology (GIST), 261 Cheomdangwagiro, Gwangju 500-712, South Korea. E-mail address: [email protected] (M.-G. Kim). 1 These authors contributed equally to this work.
1986). Natural sources of PAHs are forest fires, volcanic eruptions, incomplete combustion of petroleum and coal, tobacco smoking, and biosynthetic routes to a lesser extent (IARC, 2000). PAHs result from incomplete combustion of organic materials such as motor oils, gasoline, cooking oils, butter, margarine, and other foods. Motor vehicle exhaust is the major contributor to the presence of PAHs in the atmosphere and, as a result, BaP is on the priority pollutant list published by the U.S. Environmental Protection Agency (EPA) (EPA, 2006). Many methods utilizing various analytical techniques have been developed for the detection and quantification of PAHs, including those that employ high performance liquid (HPLC), gas (GC), thinlayer and paper chromatography, as well spectrophotometry
http://dx.doi.org/10.1016/j.chemosphere.2016.01.008 0045-6535/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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Scheme 1. Schematic illustration of the fluorescence enhancement of benzo[a]pyrene (BaP) upon addition of anti-BaP in the aqeous solution based on the immunoreaction and FRET between antibody and antigen (excitation wavelength: 280 nm).
(Barry et al., 1996; Bogusz et al., 2004; Davis et al., 1966; Hua et al., 2007; Kazerouni et al., 2001; Kershaw, 1996; Kruijf et al., 1987; Lin et al., 2005; Olsen et al., 2005; Troche et al., 2000; Vo-Dinh et al., 2000). Procedures that use HPLC in conjunction with fluorescence detection and GC in conjunction with mass spectrometry have been developed as a quantitative assay for the determination of BaP in cigarette smoke and crude or refined edible oils. These instrumentation based analytical techniques have given automated, sensitive and highly accurate results. However, the BaP assay procedures developed to date are highly time-consuming and they require sophisticated costly instrumentation. Moreover, many of the methods require the use of a large amount of a sample,
hazardous and toxic organic solvents, and several clean-up steps to remove co-extractive impurities. Immunoassay techniques that rely on antibodyeantigen interactions have become powerful tools to detect and/or monitor target substances with high levels of specificity and sensitivity (Sadana et al., 1996). But here also, heterogeneous immunoassays are limited by the fact that they require labor-intensive and timeconsuming steps including antibody immobilizations, immune reactions and washing cycles (Matveeva et al., 1996). In this regard, the development of homogeneous immunoassay systems is an important goal in the molecular target detection field because these types of assays do not require multiple washing steps to
Fig. 1. Fluorescence emission and excitation spectra of benzo[a]pyrene (BaP): excitation spectrum (solid) of 50 ng mL1 BaP in 10 mM NaHCO3 buffer with 10% MeOH (pH 9.0) when emitted at 435 nm; emission spectra of 50 ng/mL BaP in same buffer upon excitation at 280 nm (dashed) and at 380 nm (dotted).
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
T. Li et al. / Chemosphere xxx (2016) 1e7
separate of bound and unbound complexes. Consequently, homogeneous immunoassays are much more rapidly performed than their heterogeneous counterparts (Kupstat et al., 2010; Li et al., 2011, 2012, and 2013; Vo-Dinh et al., 1987). As part of considerations leading to the design of a new BaP immunoassay system, we became interested in the results of several previous studies, which show that fluorescence enhancements occur upon binding of certain ligands to their corresponding antibodies (Kobayashi et al., 1983; Kupstat et al., 2010; Li et al., 2011, 2012; McClure and Edelman, 1966; Nakamura et al., 1970; Winkler, 1962; Yoo et al., 1970; Yoo, 1975a). The increases seen in the emission efficiencies of these ligands are likely the result of the fact that antibody complex formation causes a decrease in the excited state deactivation/quenching pathways available to the ligand (Wade et al., 1978). For example, fixation in the binding site of the antibody brings the ligand into a more hydrophobic environment and prevents excimer formation and quenching. Furthermore, fluorescence resonance energy transfer (FRET) can occur between antibody and ligand to lead fluorescence enhancement of ligand when the absorption wavelengths of ligand overlap with antibody (Li et al., 2011, 2013). The phenomenon of ligand fluorescence enhancement, which has been shown to be associated with binding of antibodies, was used to develop a novel assay for BaP (shown in Scheme 1). Specifically, a fluorescence-enhancement homogeneous immunoassay method for detecting and quantifying BaP with a level of high sensitivity and specificity was designed to operate on the basis of the specific binding of BaP with its antibody. The results of an investigation probing this strategy, described below, show that upon addition of the anti-BaP antibody (anti-BaP) to an aqueous solution of BaP, the fluorescence intensity of the polycyclic hydrocarbon increases several fold. In addition, the feasibility of the new assay system was demonstrated by its use in quantitatively assessing the amounts of BaP in spiked grain samples.
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analysis.
2.3. Preparation of spiked wheat and barley grain samples To determine BaP concentrations in wheat and barley grain samples that are previously spiked with 20 and 100 ng mL1 of BaP, the samples were extracted using 80% methanol in water (v/v) and the extracts were filtered using a 0.45 mm syringe filter. The extracts were then diluted ten times with pH 9.0 10 mM NaHCO3 buffer to yield BaP containing solutions having a final methanol concentration of 10%. The extract derived solutions were mixed with anti-BaP solutions, incubated at room temperature for 20 min and subjected to fluorescence measurements. Total 18 samples were employed in the spiking and recovery experiments.
2.4. Instrumentation Fluorescence intensity measurements were made by using a LS 55 Fluorescence Spectrometer (PerkinElmer, Waltham, MA). Emission spectra were recorded in the wavelength range of 300e530 nm with excitation at 280 nm and slit widths for excitation and emission of 10 nm.
2. Materials and methods 2.1. Reagents and materials Benzo[a]pyrene (BaP), dibenzo(a,h)anthracene (DBA), pyrene, bovine serum albumin (BSA) and anti-mouse IgG antibody (antiMouse IgG) were purchased from SigmaeAldrich Chemical Co. (Yongin, Korea). Anti-BaP antibody (anti-BaP: Mab-13) and anti-C reactive protein antibody (anti-CRP) were purchased from Thermo Fisher Scientific Inc. HPLC-grade methanol was obtained from Merck Chemical Corp. (Darmstadt, Germany). The 0.45 mm syringe filter was obtained from Gelman Science (MI, USA). All other chemicals and organic solvents were of reagent grade or better. 2.2. Preparation of the fluorescence immunoassay system for BaP detection For carrying out the BaP immunoassay, 1.5 mL of the anti-BaP stock solutions (1 mg mL1 in pH 7.4 PBS) was added to vials along with 135 mL of 10 mM NaHCO3 buffer (pH 9.0). Following addition of 15 mL of stock solutions containing various concentrations BaP (varied from 0 to 50 mg mL1) in methanol to each of these vials, incubation was carried out at room temperature for 20 min and fluorescence measurements were made. In order to determine the specificity of the fluorescence immunoassay system, independent solutions were prepared containing 100 mg mL1 of antiMouse IgG, anti-CRP and 15 mg mL1 anti-BaP along with BaP (0, 2 and 10 ng mL1). In addition, solutions containing 10 mg mL1 anti-BaP and 10 ng mL1 BaP, pyrene and DBA were subjected to
Fig. 2. Fluorescence emission (excitation at 280 nm) (A) and excitation (emission at 435 nm) (B) spectra of 20 ng mL1 BaP, 10 mg mL1 BSA, 10 mg mL1 anti-BaP, and BaP coupled with BSA and anti-BaP.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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3. Results and discussion 3.1. Characterization of BaP and BaP/anti-BaP complexes In Fig. 1 are shown the fluorescence emission and excitation spectra of a BaP solution in 10 mM NaHCO3 buffer containing 10% methanol at pH 9.0. Under these conditions, BaP displays fluorescence excitation bands monitored by emission at 435 nm from 250 to 300 nm and from 360 to 380 nm. In addition, fluorescence emission peaks of BaP occur at 410 nm and 435 nm when excitation takes place at 280 nm or 380 nm (Davis et al., 1966; Kershaw, 1996). As shown in the Fig. S1 (see Supplementary Materials for details), the fluorescence intensity of BaP in 100% methanol is higher than it is in the aqueous buffer solution containing 10% methanol.
A number of other PAHs and substances that are either nonfluorescent or fluoresce with low efficiencies in aqueous solution are known to become highly fluorescent when bound to certain proteins. For example, several members of the anilinonaphthalene sulfonate (ANS) and anilinoacridine families show marked enhancements of their fluorescence efficiencies upon binding to BSA and their specific antibodies, as well as mycotoxins, especially ochratoxin A (OTA) (Kobayashi et al., 1983; Li et al., 2011, 2012; Nakamura et al., 1970; Yoo et al., 1970; Il'ichev et al., 2002). As the spectra presented in Fig. 2 show, in contrast to these substances BaP only displays a fluorescence enhancement when it is bound to its specific antibody. Specifically, the spectra show that the fluorescence of BaP centered at 435 nm (280 nm excitation) is enhanced when it is bound to anti-BaP and not to BSA. At the same
Fig. 3. (A) Fluorescence spectra (excitation at 280 nm) of solutions containing various concentrations of BaP under the conditions in which the BaP/anti-BaP complex is formed. (B) Plot of relative fluorescence intensities at 435 nm of the BaP/anti-BaP complexes from (A) as a function of concentration of BaP. The fluorescence intensity of each sample was calculated relative to the sample without any analyte. Inset figure in Figure (B): linear portion of the plot in Figure (B). Fluorescence spectra were recorded with excitation at 280 nm. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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time, the fluorescence of anti-BaP at ca. 350 nm decreases upon binding BaP as a consequence of fluorescence resonance energy transfer (FRET) from the antibody to the antigen (Li et al., 2011, 2013). Recently, Kupstat et al. have investigated the homogeneous competitive fluorescence immunoassay for the detection of BaP (Kupstat et al., 2010). In their study, they have stated the intramolecular energy between BaP and sulforhodamine B (SRB) were disrupted by the anti-BaP, and the fluorescence intensity of BaP was enhanced in the presence of the anti-BaP upon excitation at 380 nm. In comparison, the fluorescence enhancement of BaP upon the excitation at 280 nm is much higher than that of the excitation at 380 nm (shown Fig. 2B).
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3.2. Analytical performance The sensitivity and limit of detection (LOD) of the new fluorescence enhancement-based immunoassay system were determined next. The spectra and plot displayed in Fig. 3 display the changes in fluorescence intensities determined by using the immunoassay system as a function of concentrations BaP in the 0e50 ng mL1 region. As the concentration of BaP increases the fluorescence intensity increases owing to the formation of increasing amounts of the BaP/anti-BaP complex. In addition, antibody binding results in a 3.12-fold increase in the fluorescence intensity of BaP. Furthermore, in contrast to that of the BaP/anti-BaP complex, the fluorescence
Fig. 4. (A) Relative fluorescence intensities (excitation at 280 nm) of the free BaP (light gray) and BaP/anti-BaP complex (dark gray) at 435 nm as a function of BaP concentrations. The fluorescence intensity of each sample was calculated relative to the sample without any analyte. (B) Linear portion of the plot in Figure (A) corresponding to BaP concentrations of 0, 0.1, 0.2, 0.5, 1, 2, and 5 ng mL1. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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intensities of free BaP at concentrations lower than 0.5 ng mL1 cannot be distinguished from a control (Fig. 4 and S2) (Fig. S2 in Supplementary Materials). Linear ranges for plots of fluorescence intensities versus BaP and BaP/anti-BaP complex concentrations are found to be 0.1e5 ng mL1 with respective correlation coefficients (R2) of 0.9929 and 0.9972, respectively (Fig. 4). Finally, the LOD of the fluorescence immunoassay was determined to be ca. 0.06 ng mL1 of BaP, a value that is comparable to those obtained using other detection methods (Fig. 3B) (Troche et al., 2000; Bogusz et al., 2004; Hua et al., 2007; Hagestuen et al., 2000; Scharnweber et al., 2001). The antibody specificity of the immunoassay system was examined by using BSA and the nonspecific antibodies anti-mouse IgG antibody (anti-Mouse-IgG) and anti-C reactive protein antibody (anti-CRP). Importantly, no significant increase in the fluorescence intensity takes place when BSA, anti-Mouse-IgG and anti-CRP are added to solutions containing 5 and 20 ng mL1 of BaP (Fig. 5A, S3 and S4) (see Supplementary Materials). In addition, the PAH specificity of the new immunoassay system was explored using dibenzo(a,h)anthracene (DBA) and pyrene. Inspection of the results
displayed in Fig. 5A, S3 and S4 demonstrate that significant fluorescence intensity increases are not observed when anti-BaP is added to DBA and pyrene in concentrations ranges that match those used for the BaP assay. Moreover, the presence of DBA in the solution of BaP and anti-BaP has no significant effect on the fluorescence intensity arising from the BaP/anti-BaP complex (Fig. 5B, and S5) (see Supplementary Materials). This finding suggests that fluorescence enhancement phenomenon is highly specific for interaction of anti-BaP with BaP. A key to the new immunoassay technology is the fact that fluorescence enhancement is produced by binding BaP with antiBaP and that this enhancement is specific for BaP and sufficiently large to detect BaP at sub-nanomolar levels. The physical basis for this phenomenon is associated with hydrophobic interaction that take place between the analyte and antibody, which reduce the degree of molecular motion and prevent bimolecular quenching interactions (McClure and Edelman, 1966; Nakamura et al., 1970; Winkler, 1962; Yoo et al., 1970; Yoo, 1975b). Furthermore, FRET taking place partially from the antibody to the bound BaP results in enhanced fluorescence emission at 435 nm upon excitation at 280 nm (Li et al., 2011, 2013). The high degree of PAH and antibody selectivity of the BaP immunoassay system is a consequence of fact that BaP and not other PAHs is accommodated in the binding fold of anti-BaP to a greater extent than it is in the binding sites of other non-specific antibodies and BSA. 3.3. Analytical applications Wheat and barley grain samples, spiked with specific concentrations (2 and 10 ng mL1) of BaP were assayed using the newly developed system in order to demonstrate a real time application. Solutions obtained by extracting BaP from the spiked grains were mixed with proper ratios of anti-BaP and incubated at room temperature for 20 min. Fluorescence intensities of the solutions were then determined. The respective extraction recoveries of BaP from spiked wheat and barley samples were found to be in the ranges of 80.5e87.0% and 92.9e92.1% (Fig. 6 and S6) (Fig. S6 in Supplementary Materials). 4. Conclusion The investigation described above has led to the development of
Fig. 5. Specificity of the fluorescence immunoassay: (A) Relative fluorescence intensities of 5 and 20 ng mL1 BaP and mixtures with anti-BaP (10 mg mL1), antiMouse IgG (20 mg mL1), anti-CRP (10 mg mL1), and BSA; 10 mg mL1 anti-BaP with 5 and 20 ng mL1 pyrene and DBA; (B) Relative fluorescence intensities of 4 ng mL1 BaP, DBA, and mixtures with 10 mg mL1 anti-BaP. The x-axis corresponds to BaP, pyrene or DBA concentrations. The fluorescence intensities of each sample were calculated relative to the sample without any analyte. The error bars indicate standard deviations of data from experiments performed in triplicate.
Fig. 6. Relative fluorescence intensities of grain samples, spiked with BaP (2 and 10 ng mL1) and containing anti-BaP. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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a novel, label-free and direct homogeneous fluorescence immunoassay system for the sensitive and specific detection of BaP. The procedure, which relies on fluorescence enhancements that occur when BaP binds to anti-BaP, displays a high specificity for the target PAH and a lower than nano-molar sensitivity. Furthermore, the procedure employed does not involve washing steps and, consequently, it can be performed in a 20 min period. In addition, the fluorescence immunoassay system has been successfully applied to the analysis of BaP in spiked wheat and barley grain samples. Acknowledgment This work was financially supported by grants from the NLRL Program (2011-0028915) through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Science, ICT & Future Planning, the Technology Innovation Program (10053302, Development of mesoporous magnetic seramic material for diagnotics of pathogenic virus and bacteria), and the Priority Academic Program Development of Jiangsu higher Education Institutions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.01.008. References Barhoumi, R., Mouneimne, Y., Ramos, K.S., Safe, S.H., Phillips, T.D., 2000. Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line. Toxicol. Sci. 53, 264e270. Barry, J.P., Norwood, C., Vouros, P., 1996. Detection and identification of benzo[a] pyrene diol epoxide adducts to DNA utilizing capillary electrophoresiselectrospray mass spectroscopy. Anal. Chem. 68, 1432e1438. Bogusz, M.J., Hajj, S.A.E., Ehaideb, Z., Hassan, H., Al-Tufail, M., 2004. Rapid determination of benzo[a]pyrene in olive oil samples with solid-phase extraction and low-pressure, wide-bore gas chromatography-mass spectrometry and fast liquid chromatography with fluorescence detection. J. Chromatogr. A 1026, 1e7. Chang, R.L., Wood, A.W., Conney, A.H., Yagi, H., Sayer, J.M., Thakker, D.R., Jerina, D.M., Levin, W., 1987. Role of diaxial versus diequatorial hydroxyl groups in the tumorigenic activity of a benzo[a]pyrene bay-region diol epoxide. Proc. Natl. Acad. Sci. U. S. A. 84, 8633e8636. Davis, H.J., Lee, L.A., Davidson, T.R., 1966. Fluorometric determination of benzo(a) pyrene in cigarette smoke condensate. Anal. Chem. 38, 1752e1755. Hagestuen, E.D., Arruda, A.F., Campiglia, A.D., 2000. On the improvement of solidphase extraction room-temperature phosphorimetry for the analysis of polycyclic aromatic hydrocarbons in water samples. Talanta 52, 727e737. Hua, G., Broderick, J., Semple, K.T., Killham, K., Singleton, I., 2007. Rapid quantification of pilycyclic aromatic hydrocarbons in hydroxypropy-b-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS). Environ. Pollut. 148, 176e181. Il'ichev, Y.V., Perry, J.L., Simon, J.D., 2002. Interaction of ochratoxin a with human serum albumin. Preferential binding of the dianion and pH effects. J. Phys. Chem. B 106, 452e459. International Agency for Research on Cancer (IARC), 2000. Monogr. Eval. Carcinog. Risks Hum. 92, 1. Kazerouni, N., Sinha, R., Hsu, C.H., Greenberg, A., Rothman, N., 2001. Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study. Food Chem. Toxicol. 39, 423e436. Kershaw, J.R., 1996. Fluorescence spectroscopic analysis of benzo[a]pyrene in coal tar and related products. Fuel 75, 522e524. Kobayashi, N., Saito, R., Hino, H., Hino, Y., Ueno, A., Osa, T., 1983. Fluorescence and induced circular dichroism studies on host-guest complexation between g-
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Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions Taihua Li a, 1, Yo Han Choi b, c, 1, Yong-Beom Shin d, Hwa-Jung Kim c, Min-Gon Kim b, e, * a
College of Biology and the Environment, Nanjing Forestry University, Nanjing 210-037, China Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science & Technology (GIST), 261 Cheomdan-gwagiro, Gwangju 500-712, South Korea c Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, South Korea d Biomedical Translational Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, South Korea e Advanced Photonics Research Institute, Gwangju Institute of Science & Technology (GIST), 261 Cheomdan-gwagiro, Gwangju 500-712, South Korea b
h i g h l i g h t s Fluorescence enhancement and FRET-based immunoassay of benzo[a]pyrene (BaP) was developed. Binding to the anti-BaP resulted in a 3.12-fold increase in the fluorescence intensity of BaP. The assay can be used to detect BaP specifically with a limit of detection of 0.06 ng mL1.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2014 Received in revised form 15 December 2015 Accepted 4 January 2016 Available online xxx
A fluorescence enhancement-based immunoassay has been developed for the detection of the polycyclic aromatic hydrocarbons (PAH), benzo[a]pyrene (BaP), in aqueous solutions. The results of this study show that BaP, which inefficiently fluoresces in aqueous solution, displays enhanced fluorescence when bound to the anti-BaP antibody (anti-BaP), as part of a label-free immunoassay system. Binding to anti-BaP results in a 3.12-fold increase in the fluorescence intensity of BaP, which emits at 435 nm when excited at 280 nm, due to the hydrophobic interaction and fluorescence resonance energy transfer (FRET) between antibody and antigen. As result of this phenomenon, the antibody-based fluorescence immunoassay system can be used to detect BaP specifically with a limit of detection (LOD) of 0.06 ng mL1. Finally, extraction recoveries of BaP from spiked wheat and barley samples were found to be in the range of 80.5e87.0% and 92.9e92.1%, respectively. © 2016 Published by Elsevier Ltd.
Handling Editor: Prof. I. Cousins Keywords: Fluorescence immunoassay FRET Fluorescence enhancement Polycyclic aromatic hydrocarbons Benzo[a]pyrene
1. Introduction The relatively high environmental levels of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), coupled with its high toxicity reflected mainly by its carcinogenicity, creates a large detrimental impact on human health (Barhoumi et al., 2000; Chang et al., 1987; Wattenburg and Leong, 1970; Weyand and Bevan,
* Corresponding author. Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science & Technology (GIST), 261 Cheomdangwagiro, Gwangju 500-712, South Korea. E-mail address: [email protected] (M.-G. Kim). 1 These authors contributed equally to this work.
1986). Natural sources of PAHs are forest fires, volcanic eruptions, incomplete combustion of petroleum and coal, tobacco smoking, and biosynthetic routes to a lesser extent (IARC, 2000). PAHs result from incomplete combustion of organic materials such as motor oils, gasoline, cooking oils, butter, margarine, and other foods. Motor vehicle exhaust is the major contributor to the presence of PAHs in the atmosphere and, as a result, BaP is on the priority pollutant list published by the U.S. Environmental Protection Agency (EPA) (EPA, 2006). Many methods utilizing various analytical techniques have been developed for the detection and quantification of PAHs, including those that employ high performance liquid (HPLC), gas (GC), thinlayer and paper chromatography, as well spectrophotometry
http://dx.doi.org/10.1016/j.chemosphere.2016.01.008 0045-6535/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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Scheme 1. Schematic illustration of the fluorescence enhancement of benzo[a]pyrene (BaP) upon addition of anti-BaP in the aqeous solution based on the immunoreaction and FRET between antibody and antigen (excitation wavelength: 280 nm).
(Barry et al., 1996; Bogusz et al., 2004; Davis et al., 1966; Hua et al., 2007; Kazerouni et al., 2001; Kershaw, 1996; Kruijf et al., 1987; Lin et al., 2005; Olsen et al., 2005; Troche et al., 2000; Vo-Dinh et al., 2000). Procedures that use HPLC in conjunction with fluorescence detection and GC in conjunction with mass spectrometry have been developed as a quantitative assay for the determination of BaP in cigarette smoke and crude or refined edible oils. These instrumentation based analytical techniques have given automated, sensitive and highly accurate results. However, the BaP assay procedures developed to date are highly time-consuming and they require sophisticated costly instrumentation. Moreover, many of the methods require the use of a large amount of a sample,
hazardous and toxic organic solvents, and several clean-up steps to remove co-extractive impurities. Immunoassay techniques that rely on antibodyeantigen interactions have become powerful tools to detect and/or monitor target substances with high levels of specificity and sensitivity (Sadana et al., 1996). But here also, heterogeneous immunoassays are limited by the fact that they require labor-intensive and timeconsuming steps including antibody immobilizations, immune reactions and washing cycles (Matveeva et al., 1996). In this regard, the development of homogeneous immunoassay systems is an important goal in the molecular target detection field because these types of assays do not require multiple washing steps to
Fig. 1. Fluorescence emission and excitation spectra of benzo[a]pyrene (BaP): excitation spectrum (solid) of 50 ng mL1 BaP in 10 mM NaHCO3 buffer with 10% MeOH (pH 9.0) when emitted at 435 nm; emission spectra of 50 ng/mL BaP in same buffer upon excitation at 280 nm (dashed) and at 380 nm (dotted).
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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separate of bound and unbound complexes. Consequently, homogeneous immunoassays are much more rapidly performed than their heterogeneous counterparts (Kupstat et al., 2010; Li et al., 2011, 2012, and 2013; Vo-Dinh et al., 1987). As part of considerations leading to the design of a new BaP immunoassay system, we became interested in the results of several previous studies, which show that fluorescence enhancements occur upon binding of certain ligands to their corresponding antibodies (Kobayashi et al., 1983; Kupstat et al., 2010; Li et al., 2011, 2012; McClure and Edelman, 1966; Nakamura et al., 1970; Winkler, 1962; Yoo et al., 1970; Yoo, 1975a). The increases seen in the emission efficiencies of these ligands are likely the result of the fact that antibody complex formation causes a decrease in the excited state deactivation/quenching pathways available to the ligand (Wade et al., 1978). For example, fixation in the binding site of the antibody brings the ligand into a more hydrophobic environment and prevents excimer formation and quenching. Furthermore, fluorescence resonance energy transfer (FRET) can occur between antibody and ligand to lead fluorescence enhancement of ligand when the absorption wavelengths of ligand overlap with antibody (Li et al., 2011, 2013). The phenomenon of ligand fluorescence enhancement, which has been shown to be associated with binding of antibodies, was used to develop a novel assay for BaP (shown in Scheme 1). Specifically, a fluorescence-enhancement homogeneous immunoassay method for detecting and quantifying BaP with a level of high sensitivity and specificity was designed to operate on the basis of the specific binding of BaP with its antibody. The results of an investigation probing this strategy, described below, show that upon addition of the anti-BaP antibody (anti-BaP) to an aqueous solution of BaP, the fluorescence intensity of the polycyclic hydrocarbon increases several fold. In addition, the feasibility of the new assay system was demonstrated by its use in quantitatively assessing the amounts of BaP in spiked grain samples.
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analysis.
2.3. Preparation of spiked wheat and barley grain samples To determine BaP concentrations in wheat and barley grain samples that are previously spiked with 20 and 100 ng mL1 of BaP, the samples were extracted using 80% methanol in water (v/v) and the extracts were filtered using a 0.45 mm syringe filter. The extracts were then diluted ten times with pH 9.0 10 mM NaHCO3 buffer to yield BaP containing solutions having a final methanol concentration of 10%. The extract derived solutions were mixed with anti-BaP solutions, incubated at room temperature for 20 min and subjected to fluorescence measurements. Total 18 samples were employed in the spiking and recovery experiments.
2.4. Instrumentation Fluorescence intensity measurements were made by using a LS 55 Fluorescence Spectrometer (PerkinElmer, Waltham, MA). Emission spectra were recorded in the wavelength range of 300e530 nm with excitation at 280 nm and slit widths for excitation and emission of 10 nm.
2. Materials and methods 2.1. Reagents and materials Benzo[a]pyrene (BaP), dibenzo(a,h)anthracene (DBA), pyrene, bovine serum albumin (BSA) and anti-mouse IgG antibody (antiMouse IgG) were purchased from SigmaeAldrich Chemical Co. (Yongin, Korea). Anti-BaP antibody (anti-BaP: Mab-13) and anti-C reactive protein antibody (anti-CRP) were purchased from Thermo Fisher Scientific Inc. HPLC-grade methanol was obtained from Merck Chemical Corp. (Darmstadt, Germany). The 0.45 mm syringe filter was obtained from Gelman Science (MI, USA). All other chemicals and organic solvents were of reagent grade or better. 2.2. Preparation of the fluorescence immunoassay system for BaP detection For carrying out the BaP immunoassay, 1.5 mL of the anti-BaP stock solutions (1 mg mL1 in pH 7.4 PBS) was added to vials along with 135 mL of 10 mM NaHCO3 buffer (pH 9.0). Following addition of 15 mL of stock solutions containing various concentrations BaP (varied from 0 to 50 mg mL1) in methanol to each of these vials, incubation was carried out at room temperature for 20 min and fluorescence measurements were made. In order to determine the specificity of the fluorescence immunoassay system, independent solutions were prepared containing 100 mg mL1 of antiMouse IgG, anti-CRP and 15 mg mL1 anti-BaP along with BaP (0, 2 and 10 ng mL1). In addition, solutions containing 10 mg mL1 anti-BaP and 10 ng mL1 BaP, pyrene and DBA were subjected to
Fig. 2. Fluorescence emission (excitation at 280 nm) (A) and excitation (emission at 435 nm) (B) spectra of 20 ng mL1 BaP, 10 mg mL1 BSA, 10 mg mL1 anti-BaP, and BaP coupled with BSA and anti-BaP.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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3. Results and discussion 3.1. Characterization of BaP and BaP/anti-BaP complexes In Fig. 1 are shown the fluorescence emission and excitation spectra of a BaP solution in 10 mM NaHCO3 buffer containing 10% methanol at pH 9.0. Under these conditions, BaP displays fluorescence excitation bands monitored by emission at 435 nm from 250 to 300 nm and from 360 to 380 nm. In addition, fluorescence emission peaks of BaP occur at 410 nm and 435 nm when excitation takes place at 280 nm or 380 nm (Davis et al., 1966; Kershaw, 1996). As shown in the Fig. S1 (see Supplementary Materials for details), the fluorescence intensity of BaP in 100% methanol is higher than it is in the aqueous buffer solution containing 10% methanol.
A number of other PAHs and substances that are either nonfluorescent or fluoresce with low efficiencies in aqueous solution are known to become highly fluorescent when bound to certain proteins. For example, several members of the anilinonaphthalene sulfonate (ANS) and anilinoacridine families show marked enhancements of their fluorescence efficiencies upon binding to BSA and their specific antibodies, as well as mycotoxins, especially ochratoxin A (OTA) (Kobayashi et al., 1983; Li et al., 2011, 2012; Nakamura et al., 1970; Yoo et al., 1970; Il'ichev et al., 2002). As the spectra presented in Fig. 2 show, in contrast to these substances BaP only displays a fluorescence enhancement when it is bound to its specific antibody. Specifically, the spectra show that the fluorescence of BaP centered at 435 nm (280 nm excitation) is enhanced when it is bound to anti-BaP and not to BSA. At the same
Fig. 3. (A) Fluorescence spectra (excitation at 280 nm) of solutions containing various concentrations of BaP under the conditions in which the BaP/anti-BaP complex is formed. (B) Plot of relative fluorescence intensities at 435 nm of the BaP/anti-BaP complexes from (A) as a function of concentration of BaP. The fluorescence intensity of each sample was calculated relative to the sample without any analyte. Inset figure in Figure (B): linear portion of the plot in Figure (B). Fluorescence spectra were recorded with excitation at 280 nm. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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time, the fluorescence of anti-BaP at ca. 350 nm decreases upon binding BaP as a consequence of fluorescence resonance energy transfer (FRET) from the antibody to the antigen (Li et al., 2011, 2013). Recently, Kupstat et al. have investigated the homogeneous competitive fluorescence immunoassay for the detection of BaP (Kupstat et al., 2010). In their study, they have stated the intramolecular energy between BaP and sulforhodamine B (SRB) were disrupted by the anti-BaP, and the fluorescence intensity of BaP was enhanced in the presence of the anti-BaP upon excitation at 380 nm. In comparison, the fluorescence enhancement of BaP upon the excitation at 280 nm is much higher than that of the excitation at 380 nm (shown Fig. 2B).
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3.2. Analytical performance The sensitivity and limit of detection (LOD) of the new fluorescence enhancement-based immunoassay system were determined next. The spectra and plot displayed in Fig. 3 display the changes in fluorescence intensities determined by using the immunoassay system as a function of concentrations BaP in the 0e50 ng mL1 region. As the concentration of BaP increases the fluorescence intensity increases owing to the formation of increasing amounts of the BaP/anti-BaP complex. In addition, antibody binding results in a 3.12-fold increase in the fluorescence intensity of BaP. Furthermore, in contrast to that of the BaP/anti-BaP complex, the fluorescence
Fig. 4. (A) Relative fluorescence intensities (excitation at 280 nm) of the free BaP (light gray) and BaP/anti-BaP complex (dark gray) at 435 nm as a function of BaP concentrations. The fluorescence intensity of each sample was calculated relative to the sample without any analyte. (B) Linear portion of the plot in Figure (A) corresponding to BaP concentrations of 0, 0.1, 0.2, 0.5, 1, 2, and 5 ng mL1. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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intensities of free BaP at concentrations lower than 0.5 ng mL1 cannot be distinguished from a control (Fig. 4 and S2) (Fig. S2 in Supplementary Materials). Linear ranges for plots of fluorescence intensities versus BaP and BaP/anti-BaP complex concentrations are found to be 0.1e5 ng mL1 with respective correlation coefficients (R2) of 0.9929 and 0.9972, respectively (Fig. 4). Finally, the LOD of the fluorescence immunoassay was determined to be ca. 0.06 ng mL1 of BaP, a value that is comparable to those obtained using other detection methods (Fig. 3B) (Troche et al., 2000; Bogusz et al., 2004; Hua et al., 2007; Hagestuen et al., 2000; Scharnweber et al., 2001). The antibody specificity of the immunoassay system was examined by using BSA and the nonspecific antibodies anti-mouse IgG antibody (anti-Mouse-IgG) and anti-C reactive protein antibody (anti-CRP). Importantly, no significant increase in the fluorescence intensity takes place when BSA, anti-Mouse-IgG and anti-CRP are added to solutions containing 5 and 20 ng mL1 of BaP (Fig. 5A, S3 and S4) (see Supplementary Materials). In addition, the PAH specificity of the new immunoassay system was explored using dibenzo(a,h)anthracene (DBA) and pyrene. Inspection of the results
displayed in Fig. 5A, S3 and S4 demonstrate that significant fluorescence intensity increases are not observed when anti-BaP is added to DBA and pyrene in concentrations ranges that match those used for the BaP assay. Moreover, the presence of DBA in the solution of BaP and anti-BaP has no significant effect on the fluorescence intensity arising from the BaP/anti-BaP complex (Fig. 5B, and S5) (see Supplementary Materials). This finding suggests that fluorescence enhancement phenomenon is highly specific for interaction of anti-BaP with BaP. A key to the new immunoassay technology is the fact that fluorescence enhancement is produced by binding BaP with antiBaP and that this enhancement is specific for BaP and sufficiently large to detect BaP at sub-nanomolar levels. The physical basis for this phenomenon is associated with hydrophobic interaction that take place between the analyte and antibody, which reduce the degree of molecular motion and prevent bimolecular quenching interactions (McClure and Edelman, 1966; Nakamura et al., 1970; Winkler, 1962; Yoo et al., 1970; Yoo, 1975b). Furthermore, FRET taking place partially from the antibody to the bound BaP results in enhanced fluorescence emission at 435 nm upon excitation at 280 nm (Li et al., 2011, 2013). The high degree of PAH and antibody selectivity of the BaP immunoassay system is a consequence of fact that BaP and not other PAHs is accommodated in the binding fold of anti-BaP to a greater extent than it is in the binding sites of other non-specific antibodies and BSA. 3.3. Analytical applications Wheat and barley grain samples, spiked with specific concentrations (2 and 10 ng mL1) of BaP were assayed using the newly developed system in order to demonstrate a real time application. Solutions obtained by extracting BaP from the spiked grains were mixed with proper ratios of anti-BaP and incubated at room temperature for 20 min. Fluorescence intensities of the solutions were then determined. The respective extraction recoveries of BaP from spiked wheat and barley samples were found to be in the ranges of 80.5e87.0% and 92.9e92.1% (Fig. 6 and S6) (Fig. S6 in Supplementary Materials). 4. Conclusion The investigation described above has led to the development of
Fig. 5. Specificity of the fluorescence immunoassay: (A) Relative fluorescence intensities of 5 and 20 ng mL1 BaP and mixtures with anti-BaP (10 mg mL1), antiMouse IgG (20 mg mL1), anti-CRP (10 mg mL1), and BSA; 10 mg mL1 anti-BaP with 5 and 20 ng mL1 pyrene and DBA; (B) Relative fluorescence intensities of 4 ng mL1 BaP, DBA, and mixtures with 10 mg mL1 anti-BaP. The x-axis corresponds to BaP, pyrene or DBA concentrations. The fluorescence intensities of each sample were calculated relative to the sample without any analyte. The error bars indicate standard deviations of data from experiments performed in triplicate.
Fig. 6. Relative fluorescence intensities of grain samples, spiked with BaP (2 and 10 ng mL1) and containing anti-BaP. Error bars indicate standard deviations of data from experiments performed in triplicate.
Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008
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a novel, label-free and direct homogeneous fluorescence immunoassay system for the sensitive and specific detection of BaP. The procedure, which relies on fluorescence enhancements that occur when BaP binds to anti-BaP, displays a high specificity for the target PAH and a lower than nano-molar sensitivity. Furthermore, the procedure employed does not involve washing steps and, consequently, it can be performed in a 20 min period. In addition, the fluorescence immunoassay system has been successfully applied to the analysis of BaP in spiked wheat and barley grain samples. Acknowledgment This work was financially supported by grants from the NLRL Program (2011-0028915) through the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Science, ICT & Future Planning, the Technology Innovation Program (10053302, Development of mesoporous magnetic seramic material for diagnotics of pathogenic virus and bacteria), and the Priority Academic Program Development of Jiangsu higher Education Institutions. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.chemosphere.2016.01.008. References Barhoumi, R., Mouneimne, Y., Ramos, K.S., Safe, S.H., Phillips, T.D., 2000. Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line. Toxicol. Sci. 53, 264e270. Barry, J.P., Norwood, C., Vouros, P., 1996. Detection and identification of benzo[a] pyrene diol epoxide adducts to DNA utilizing capillary electrophoresiselectrospray mass spectroscopy. Anal. Chem. 68, 1432e1438. Bogusz, M.J., Hajj, S.A.E., Ehaideb, Z., Hassan, H., Al-Tufail, M., 2004. Rapid determination of benzo[a]pyrene in olive oil samples with solid-phase extraction and low-pressure, wide-bore gas chromatography-mass spectrometry and fast liquid chromatography with fluorescence detection. J. Chromatogr. A 1026, 1e7. Chang, R.L., Wood, A.W., Conney, A.H., Yagi, H., Sayer, J.M., Thakker, D.R., Jerina, D.M., Levin, W., 1987. Role of diaxial versus diequatorial hydroxyl groups in the tumorigenic activity of a benzo[a]pyrene bay-region diol epoxide. Proc. Natl. Acad. Sci. U. S. A. 84, 8633e8636. Davis, H.J., Lee, L.A., Davidson, T.R., 1966. Fluorometric determination of benzo(a) pyrene in cigarette smoke condensate. Anal. Chem. 38, 1752e1755. Hagestuen, E.D., Arruda, A.F., Campiglia, A.D., 2000. On the improvement of solidphase extraction room-temperature phosphorimetry for the analysis of polycyclic aromatic hydrocarbons in water samples. Talanta 52, 727e737. Hua, G., Broderick, J., Semple, K.T., Killham, K., Singleton, I., 2007. Rapid quantification of pilycyclic aromatic hydrocarbons in hydroxypropy-b-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS). Environ. Pollut. 148, 176e181. Il'ichev, Y.V., Perry, J.L., Simon, J.D., 2002. Interaction of ochratoxin a with human serum albumin. Preferential binding of the dianion and pH effects. J. Phys. Chem. B 106, 452e459. International Agency for Research on Cancer (IARC), 2000. Monogr. Eval. Carcinog. Risks Hum. 92, 1. Kazerouni, N., Sinha, R., Hsu, C.H., Greenberg, A., Rothman, N., 2001. Analysis of 200 food items for benzo(a)pyrene and estimation of its intake in an epidemiologic study. Food Chem. Toxicol. 39, 423e436. Kershaw, J.R., 1996. Fluorescence spectroscopic analysis of benzo[a]pyrene in coal tar and related products. Fuel 75, 522e524. Kobayashi, N., Saito, R., Hino, H., Hino, Y., Ueno, A., Osa, T., 1983. Fluorescence and induced circular dichroism studies on host-guest complexation between g-
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Please cite this article in press as: Li, T., et al., A fluorescence enhancement-based label-free homogeneous immunoassay of benzo[a]pyrene (BaP) in aqueous solutions, Chemosphere (2016), http://dx.doi.org/10.1016/j.chemosphere.2016.01.008