Analytica Chimica Acta 815 (2014) 51–56
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
“Orange alert”: A fluorescent detector for bisphenol A in water environments Liyun Zhang a,b , Jun Cheng Er a,c , Wang Xu a , Xian Qin a , Animesh Samanta d , Santanu Jana d , Chi-Lik Ken Lee e , Young-Tae Chang a,c,d,∗ a
Department of Chemistry, National University of Singapore, 3 Science Drive 2, 117543 Singapore, Singapore Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, PR China c Graduate School for Integrative Sciences and Engineering, National University of Singapore, Centre for Life Sciences, #05-01, 28 Medical Drive, 117456 Singapore, Singapore d Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), 138667 Singapore, Singapore e Centre for Biomedical and Life Sciences, Singapore Polytechnic, 139651 Singapore, Singapore b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• We report a BODIPY-based turn-on fluorescent bisphenol A sensor.
• We tested the superior selectivity toward BPA against several bisphenol analogs and phenol. • We demonstrated the stability and robustness of this probe for analyzing BPA in real, complex water samples.
a r t i c l e
i n f o
Article history: Received 19 October 2013 Received in revised form 18 December 2013 Accepted 13 January 2014 Available online 24 January 2014 Keywords: Bisphenol A Fluorescent sensor Boron dipyrromethene Water environments
a b s t r a c t Due to the prevalent use of polycarbonate plastics and epoxy resins in packaging materials and paints for ships, there has been a widespread global contamination of environmental water sources with bisphenol A (BPA). BPA, an endocrine disruptor, has been found to cause tremendous health problems. Therefore, there is an urgent need for detecting BPA in a convenient and sensitive manner to ensure water safety. Herein, we develop a fluorescent turn-on BPA probe, named Bisphenol Orange (BPO), which could conveniently detect BPA in a wide variety of real water samples including sea water, drain water and drinking water. BPO shows superior selectivity toward BPA and up to 70-fold increase in fluorescence emission at 580 nm when mixed with BPA in water. Mechanistic studies suggest a plausible water-dependent formation of hydrophobic BPA clusters which favorably trap and restrict the rotation of BPO and recover its inherent fluorescence. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bisphenol A (BPA) is a widely used chemical in high production manufacturing of polycarbonate plastics and epoxy resins, with about 3 million tons produced and over 100 tons released
∗ Corresponding author at: Department of Chemistry, National University of Singapore, 3 Science Drive 2, 117543 Singapore, Singapore. Tel.: +65 65166774. E-mail address: [email protected] (Y.-T. Chang). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.038
into the atmosphere by yearly production [1]. The pollutant can be found in numerous consumable items, including bottles, reusable food containers, polyvinyl chloride stretch films, paper products and money [2,3]. BPA is an estrogen antagonist and exposure to trace levels of the pollutant is associated with several debilitating illnesses such as cardiac disorder [4], diabetes [5], cancer [6], hormonal [7] and reproductive function [8]. Therefore, to assess the risk and ensure environmental safety and health, the measurement of BPA remains as one of the most widely studied topics.
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Various analytical methods have been employed for the direct detection and quantification of BPA, including liquid chromatography–mass spectrometry (LC–MS) [9] and gas chromatography–mass spectrometry (GC–MS) [10,11], fluorescence [12], electrochemical detection [13], surface plasmon resonance [14] as well as immunochemical methods [15]. Intriguingly, a small molecule fluorescent sensor for the convenient, selective and sensitive detection of BPA has not been reported. Currently, fluorescent sensors for detecting BPA are based on a Cy5.5-labeled antibody [16] or a DNA aptamer [17]. However, these biological macromolecules lack the robustness and stability required for practical environmental sensing. In contrast, small molecule fluorescent probes are more attractive due to their low molecular weight, high chemical stability, and relatively high sensitivity [18]. Herein, we report a triazole-derivatized boron dipyrromethene (BODIPY) dye, BPO as a turn-on fluorescent sensor for detecting BPA. BPO is capable of distinguishing BPA from various bisphenols in aqueous solution. Owing to its unique features, BPO represents a convenient and practical tool for the selective detection of BPA in real water environments and other BPA-related analyses. 2. Materials and methods
All commercially available reagents and solvents were purchased from Sigma Aldrich, Alfa Aesar, Fluka, Merck or Acros, and used as received unless otherwise stated. Dichloromethane (DCM) (Fisher Scientific, analytical grade) was freshly distilled from phosphorus pentoxide (P2 O5 ) under nitrogen. Anhydrous tetrahydrofuran (THF) was purchased from Alfa Aesar and used without further purification. Sea water and drain water samples were collected from three separate locations in Singapore (West Coast, East Coast and Punggol (North)), combined and used as such. Drinking water samples were purchased from Evian, Ice Mountain and Dasani, combined and used as such. 2.2. Fluorescence The fluorescence measurements were carried out on a SpectraMax M2 plate reader in 96-well plates by scanning the emission spectra between 520 and 700 nm. All measurements were performed under an excitation wavelength of 460 nm unless otherwise stated. Fluorescence of the dye was measured in deionized water, containing 1% dimethylsulfoxide (DMSO). The intrinsic fluorescence of BPA was measured on a Horiba JobinYuon fluoromax-4 spectrofluorometer by using a path length of 10 mm and excitation at 285 nm. The bandwidth was 5 nm. All experiments were repeated three times. The data analysis was conducted using Origin 8.0 (OriginLab Corporation, MA). 2.3. Quantum yield measurements Quantum yields were calculated by measuring the integrated emission area of the fluorescent spectra in its respective solvents and comparing to the area measured for Rhodamine B (reference) (10 M, ˚F = 0.31, ex = 500 nm) in water ( = 1.333). Quantum yields were calculated using the equation:
sample
reference
= ˚F
UV–vis absorption spectra of the dye and analyte in deionized water were recorded from 300 to 700 nm using a SpectraMax M2 spectrophotometer. 2.5. Compound characterization Analytical characterization was performed on a LC-MS (Agilent1200 series) system equipped with a DAD detector and a single quadrupole mass spectrometer (6130 series) with an ESI probe. Analytical LC method: eluents, A: H2 O (0.1% HCOOH), B: CH3 CN (0.1% HCOOH), gradient 5% B–95% B (10 min). Reverse-phase Phenomenex C18 Luna column (4.6 mm × 50 mm, 3.5 m particle size), flow rate: 1 mL min−1 . 1 H NMR, 19 F NMR and 13 C NMR spectra were recorded on Bruker ACF300 (300 MHz) and AMX500 (500 MHz) spectrometers and reported in ı (ppm). 1 H NMR spectra were referenced to residual proton signals in acetonitrile-d3 (CD3 CN) (ı = 1.94 ppm); 13 C NMR spectra were referenced to solvent resonances of CD3 CN (ı = 1.32 ppm); 19 F NMR spectra were referenced to external TFA (ı = −76.55 ppm vs. CFCl3 at 0.00 ppm). High resolution mass spectra (ESI) were obtained on a Finnigan/MAT 95XL-T spectrometer. 2.6. Pre-concentration of BPA from contaminated tap water samples
2.1. Materials
˚F
2.4. Absorbance
F sample F reference
sample reference
Absreference
Abssample
where F represents the area of fluorescent emission, is the refractive index of the solvent, and Abs is absorbance at the excitation wavelength. Emission was integrated between 520 and 700 nm.
100 mL of BPA contaminated (0, 10, 20, 40, 60, 80 and 100 nM) tap water solutions were extracted with DCM (30 mL ×3). The combined organic extracts were evaporated and redissolved in 100 L deionized water for fluorescence and LC–MS test. 2.7. Synthesis N-(4-(5,5-difluoro-3-(4-phenethyl-1H-1,2,3-triazol1-yl)-7-(piperidin-1-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2 ,1 f][1,3,2]diazaborinin-10-yl)phenyl)acetamide (BPO). 3,5-Dichloro-8-(4 -aminophenyl)-4,4-difluoro-4-bora-3a,4adiaza-s-indacence (7.5 mg, 21.3 mol) was synthesized and loaded onto 2-chlorotrityl chloride resin following the procedure described in reference [19]. The loaded resin was resuspended in DCM and shaken at room temperature for 10 min and washed with dimethylformamide (DMF). A suspension of sodium azide (NaN3 ) (12.5 mg, 0.19 mmol) in DMF (1.0 mL) was added and the reaction mixture further shaken at room temperature for 30 min. After washing with DMF (4× 5 mL), a solution of DMF:piperidine (4:1) (1.0 mL) was added and the reaction shaken for 1 h. 4-Phenyl-1butyne (30 L, 0.21 mmol), cuprous iodide (16.3 mg, 0.21 mmol) and ascorbic acid (15 mg, 0.21 mmol) were subsequently added and the reaction mixture shaken for another 30 min. The resin was filtered, washed with DMF (4× 5 mL) and DCM (2× 5 mL) following which cleavage was performed with 0.5% trifluoroacetic acid (TFA) in DCM (3× 5 mL, 10 min each). The organic extracts were recovered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (eluent:DCM to DCM:MeOH (98:2)) to afford an orange solid which was redissolved in DCM (1 mL), mixed with 6 drops of saturated aqueous NaHCO3 and cooled to 0 ◦ C. Acetyl chloride (10 L, 0.11 mmol) was added in portions over 1 min and the resulting mixture stirred at room temperature and monitored by analytical thin-layer chromatography (TLC). Upon reaction completion, the reaction was diluted with DCM (15 mL), washed with water (2× 15 mL), saturated NaHCO3 (1× 15 mL), saturated brine (1× 15 mL) and dried over anhydrous Na2 SO4 . The organic extract was evaporated to afford BPO as an orange solid (3 mg, 5.18 mmol, 25% yield). 1 H NMR (500 MHz, CD3 CN) ı 8.4–8.45 (m, 1H), 7.91 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.42
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(d, J = 8.6 Hz, 2H), 7.30–7.22 (m, 4H), 7.21–7.14 (m, 1H), 6.98 (d, J = 5.4 Hz, 1H), 6.61 (d, J = 5.4 Hz, 1H), 6.38 (d, J = 3.8 Hz, 1H), 6.26 (d, J = 3.8 Hz, 1H), 3.89–3.81 (m, 4H), 3.09–2.98 (m, 4H), 2.08 (s, 3H), 1.73–1.65 (m, 6H); 13 C NMR (128 MHz, CD3 CN) 169.8, 163.4, 146.9, 142.5, 141.0, 137.4, 136.9, 135.0, 132.1, 131.7, 130.8, 130.2, 129.5, 129.3, 128.9, 126.9, 125.4, 119.7, 115.9, 110.6, 53.0, 28.1, 27.2, 24.5, 24.4; 19 F NMR (282 MHz, CD3 CN) ı −132.13 (dd, J = 68.5, 34.3 Hz); HRMS (C28 H27 BF2 N7 ): Calc. [M+Na]+ : 602.2627, Found [M+Na]+ : 602.2644. 3. Results and discussion 3.1. Sensor identification We began by analyzing the fluorescence responses of a recently reported BODIPY-triazole library toward BPA [19]. These BODIPY-triazoles are fluorescent molecular rotors; under specific environments, the inherent fluorescence of the BODIPY core can be recovered, making them an ideal starting point for the development of a sensitive BPA turn-on fluorescent sensor. From the screening, we identified one compound (BPO) showing a remarkable 70-fold increase in fluorescence emission at 580 nm and an approximate 10 nm blue shift in emission maximum (Fig. 1a and Fig. S1). The data plot of fluorescent titration of BPO with serial concentrations of BPA was fitted according to a reported method, and we determined the apparent affinity constant (Ka ) as 513 ± 5 M−1 (Fig. 1b) [20]. In addition, we found that Ka was generally unaffected by the ionic strength of the solution (Table S1). In the range of 5–100 M BPA, the plot displays a good linear relationship with a R2 of 0.99. The limit of detection (LOD) for BPA was determined to be approximately 5 M (S/N = 3) (Fig. 1b, inset). 3.2. Selectivity Next, we examined the selectivity of BPO toward other bisphenols. Several bisphenols including Bisphenol AF, F, Z, AP and P were tested and their structures are shown in Fig. 2a. As shown in Fig. 2b and c, and Figs. S2 and S3, BPO showed weak response toward other bisphenols and phenol reinforcing its remarkable selectivity toward BPA. Previous reports of fluorescent BPA sensors generally do not display high selectivity between the bisphenols [17,21]. To the best of our knowledge, BPO is the first turn-on BODIPY fluorescent dye that displays remarkable selectivity toward BPA with weak response to other bisphenols. 3.3. Applications for water analysis After examining the excellent selectivity of BPO toward BPA, we evaluated its application to quantify BPA in real water samples. Due to the lightweight, shatter-resistant and transparent properties of polycarbonate plastics, these materials are popular choices for water bottles [14,22]. Therefore, BPA is a common contaminant found in bottled drinking water. Researchers have also reported widespread global contamination of sea water with BPA mainly originating from epoxy paint used on ships and hard plastic trash discarded into the oceans [23,24]. Due to these reasons, the detection and quantification of BPA in environmental water samples is of significant importance. We examined the fluorescence response of BPO in bottled drinking water, drain water and sea water that were spiked with increasing concentrations of BPA up to 100 M and observed excellent linear correlation between the fluorescence of the dye and BPA concentration (Fig. 3). This result demonstrates the stability and robustness of BPO for analyzing BPA even in real, complex water samples.
Fig. 1. (a) Fluorescence response of BPO (10 M) upon mixing with serial dilutions of BPA (from 0 to 10 mM) in deionized water (pH = 6.9). Inset: structures of BPO and BPA. (b) Data plot of fluorescence emission intensity at 580 nm upon interaction with serial dilutions of BPA in deionized water. Inset: standard linear curve derived from the emission intensity at 580 nm upon addition of 0–100 M BPA. Linear regression: R2 = 0.99. ex : 460 nm; ˚F (in water): 0.9%; ˚F (in 10 mM BPA): 32%. Values are represented as means and error bars as standard deviations (n = 3); measurements were taken at room temperature (RT).
We further explored the ability of BPO to accurately quantify the concentration of BPA in contaminated tap water samples. According to American legislations, BPA concentrations in the nanomolar range is classified as the risk alert limit [8]. Hence, tap water spiked with known nanomolar concentrations of BPA (0–100 nM) was analyzed. As shown in Fig. 1b (inset), the linear detection range of BPO falls within 5–100 M. Hence, to achieve nanomolar detection limits, a series of liquid–liquid extractions were performed on the spiked water samples to concentrate the BPA 1000-fold before applying BPO. LC–MS is a validated method for quantifying BPA in contaminated water samples [9]. To put the performance of BPO in perspective, the same samples were concurrently quantified using LC–MS (standard linear curve shown in Fig. S4). As shown in Table 1, the concentrations measured using BPO matched closely with that from the LC–MS method. These results demonstrated the reliability of BPO in quantifying BPA concentrations in contaminated water samples. Coupled with a simple liquid–liquid extraction, a 10 nM LOD was achieved. Together, these results confirmed that BPO could be a valuable tool for the convenient and sensitive detection of BPA in various water environments.
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Fig. 2. Selectivity of BPO. (a) Structures of bisphenol analogs. (b) Fluorescence response of BPO (10 M) with various bisphenol analogs and phenol. Conditions: BPO (10 M), bisphenols (0.5, 1.0, 2.0 and 4.0 mM) in deionized water (pH = 6.9). ex : 460 nm; em : 580 nm; values are represented as means (n = 3); measurements were taken at RT. (c) Photographic image of BPO (10 M) mixed with various bisphenols. Irradiation with a hand-held UV lamp at 365 nm.
3.4. Sensing mechanism studies Subsequent experiments were aimed at understanding the sensing mechanism of BPO. First, we analyzed the photophysical properties of BPO. As shown in Fig. 4a and Table 2, BPO showed low quantum yields in most organic solvents and water. However, in poly(ethylene glycol) 400 (PEG 400), a significantly higher quantum yield was observed. Systematic additions of PEG 400 to an aqueous
solution of BPO similarly increased the fluorescence of the dye proportionately (Fig. 4b). These observations are consistent with BPO’s behavior as fluorescent molecular rotors. The increasing viscosity of the solvent system systematically restricts bond rotation at the 3, 5 and meso positions of the BODIPY core of BPO [19,25]. Consequently, a greater proportion of energy is channeled from the non-radiative processes into fluorescence. As fluorescent molecular rotors, BPO’s turn-on response in the presence of BPA is likely
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Fig. 3. Fluorescence response of BPO (10 M) in various environmental water samples. BPA (0–100 M) was added to sea water, drain water and drinking water respectively. ex : 460 nm, em : 580 nm; values are represented as means and error bars as standard deviations (n = 3); measurements were taken at RT. Linear regres2 2 2 = 0.99, Rdrain-water = 0.99 and Rdrinking-water = 0.99. sion: Rsea-water
Table 1 Quantification of BPA in spiked tap water samples. Values were taken as means and uncertainties as SD (n = 3). Sample
Actual [BPA] (nM)
Theoretical [BPA] after pre-concentration (M)
Measured (M) BPOa
1 2 3 4 5 6
10 20 40 60 80 100
10 20 40 60 80 100
6.2 14.2 32.5 57.5 73.6 91.2
LC–MSb ± ± ± ± ± ±
0.1 0.1 0.2 0.1 0.3 0.3
7.1 16.1 35.3 52.9 71.1 84.8
± ± ± ± ± ±
0.2 0.5 0.2 0.1 0.4 0.2
a Standard linear equation for quantifying BPA concentration using the fluorescence intensity of BPO at 580 nm: y = 1.1 + 0.02x, R2 = 0.99 (Fig. 1b, inset). b Standard linear equation for quantifying BPA concentration using the integrated areas in the LC–MS spectra: y = 3.2 + 820x, R2 = 0.99 (Fig. S5).
to involve a similar rotational restriction mechanism induced by a mutual interaction between the two compounds. To investigate their interaction further, we analyzed the sensing capability of BPO to aqueous BPA in the presence of increasing proportions of organic solvents. As shown in Fig. 5a, when the organic solvent was above 20–30%, the fluorescence of BPO was completely quenched demonstrating that sensing of BPA was highly dependent on an aqueous environment. This result suggested that the interaction between BPO and BPA might be hydrophobic in nature. We studied the response of BPO to BPA under different pH and found Table 2 Photophysical properties of BPO in various solvents. Solvent
Dielectric constant, ε
Viscosity, (mPa s)
abs,max (nm)
fluo,max (nm)
˚F (%)
Water DMSO DMF EtOH Acetone PEG 400 DCM EA Toluene
80.1 46.7 38.0 24.3 21.0 12.4 9.1 6.0 2.4
0.89 2.00 0.92 1.14 0.31 90.00 0.41 0.43 0.55
470 470 470 470 470 475 475 480 495
590 580 580 580 580 580 585 585 585
0.9 2.1 2.3 2.3 2.4 18.1 3.2 1.5 1.6
DMSO: dimethyl sulfoxide; EtOH: ethanol; EA: ethyl acetate; DMF: dimethyl formamide; DCM: dichloromethane.
Fig. 4. Photophysical properties of BPO in various solvents. (a) Fluorescence spectra of BPO (10 M) in various organic solvents. (b) Fluorescence spectra of BPO (10 M) in various mixtures of PEG (0, 5, 10, 20, 40, 60, 80 and 100%) in water. ex : 460 nm; values are represented as means (n = 3); measurements were taken at RT.
that the emission intensity of BPO was independent of pH (Fig. S5). However, upon addition of BPA, a significant decrease in the dye emission intensity was observed above pH 9.0 (Fig. 5b); below pH 9.0, only a gradual decrease in fluorescence intensity was detected. The pKa of BPA is 9.6 and 10.2 [22]. This pH dependent trend thus suggests that BPO largely interacts with the de-ionized form of BPA and further supports hydrophobic interactions between BPO and BPA. Similar trends were observed in the interaction BPO and other bisphenols (Fig. S6); for example, BPAF and BPAP are estimated to have slightly lower pKa than BPA, therefore, the BPO fluorescence response was observed to decrease significantly beyond pH 8.0. To achieve maximum stability, the hydrophobic BPA probably associates spontaneously into large clusters surrounded by water cages. BPO may then be favorably trapped within these hydrophobic clusters resulting in restriction of bond rotation and recovering the intrinsic fluorescence of BODIPY. Formation of these BPA clusters that wrap around BPO is probably facilitated by a small degree of BPA deprotonation thereby accounting for the maximum fluorescence response at pH 9.0. In addition, the increased fluorescence response of BPO in the presence of increasing amounts of BPA in the aqueous solution is probably because of the increased density of the hydrophobic clusters as more BPA is added. On the contrary, addition of organic solvents disrupts these clusters and increases the rotational freedom of BPO resulting in its requenching.
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identification in environmental science as well as in analytical chemistry. Therefore, following work will explore improved methods for BPA enrichment to be incorporated into a BPO-based BPA detection kit that can be used on-site. Acknowledgements The authors acknowledge the financial support from the Singapore-Peking-Oxford Research Enterprise (SPORE, COY-15EWI-RCFSA/N197-1). Liyun Zhang acknowledges the financial support from Natural Science Foundation of China (No. 11105150), China Postdoctoral Science Foundation (No. 2012M510163). Jun Cheng Er acknowledges the receipt of an NGS scholarship. The authors thank Jun Jie Heng for offering BPA samples and his great help in discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.01.038. References
Fig. 5. Interaction of BPO with BPA. (a) Fluorescence response of BPO (10 M) upon addition of BPA in different organic solvent aqueous solution. (b) Fluorescence response of BPO (10 M) upon addition of BPA (1 mM) in aqueous solution under different pH. ex : 460 nm; em : 580 nm; values are represented as means (n = 3); measurements were taken at RT.
4. Conclusion In summary, we have developed a triazole-derivatized BODIPY compound as a BPA turn-on sensor in aqueous solution, which shows a selective recognition for BPA from other bisphenol analogs and phenol. BPO displays the high stability and robustness for analyzing BPA even in real, complex water samples. In addition, mechanistic studies suggest that BPO may be favorably trapped within BPA hydrophobic clusters resulting in restriction of bond rotation and recovery of the intrinsic fluorescence of BODIPY. For analytical applications, it has potential as a sensor for BPA
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
“Orange alert”: A fluorescent detector for bisphenol A in water environments Liyun Zhang a,b , Jun Cheng Er a,c , Wang Xu a , Xian Qin a , Animesh Samanta d , Santanu Jana d , Chi-Lik Ken Lee e , Young-Tae Chang a,c,d,∗ a
Department of Chemistry, National University of Singapore, 3 Science Drive 2, 117543 Singapore, Singapore Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, PR China c Graduate School for Integrative Sciences and Engineering, National University of Singapore, Centre for Life Sciences, #05-01, 28 Medical Drive, 117456 Singapore, Singapore d Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), 138667 Singapore, Singapore e Centre for Biomedical and Life Sciences, Singapore Polytechnic, 139651 Singapore, Singapore b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• We report a BODIPY-based turn-on fluorescent bisphenol A sensor.
• We tested the superior selectivity toward BPA against several bisphenol analogs and phenol. • We demonstrated the stability and robustness of this probe for analyzing BPA in real, complex water samples.
a r t i c l e
i n f o
Article history: Received 19 October 2013 Received in revised form 18 December 2013 Accepted 13 January 2014 Available online 24 January 2014 Keywords: Bisphenol A Fluorescent sensor Boron dipyrromethene Water environments
a b s t r a c t Due to the prevalent use of polycarbonate plastics and epoxy resins in packaging materials and paints for ships, there has been a widespread global contamination of environmental water sources with bisphenol A (BPA). BPA, an endocrine disruptor, has been found to cause tremendous health problems. Therefore, there is an urgent need for detecting BPA in a convenient and sensitive manner to ensure water safety. Herein, we develop a fluorescent turn-on BPA probe, named Bisphenol Orange (BPO), which could conveniently detect BPA in a wide variety of real water samples including sea water, drain water and drinking water. BPO shows superior selectivity toward BPA and up to 70-fold increase in fluorescence emission at 580 nm when mixed with BPA in water. Mechanistic studies suggest a plausible water-dependent formation of hydrophobic BPA clusters which favorably trap and restrict the rotation of BPO and recover its inherent fluorescence. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Bisphenol A (BPA) is a widely used chemical in high production manufacturing of polycarbonate plastics and epoxy resins, with about 3 million tons produced and over 100 tons released
∗ Corresponding author at: Department of Chemistry, National University of Singapore, 3 Science Drive 2, 117543 Singapore, Singapore. Tel.: +65 65166774. E-mail address: [email protected] (Y.-T. Chang). 0003-2670/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2014.01.038
into the atmosphere by yearly production [1]. The pollutant can be found in numerous consumable items, including bottles, reusable food containers, polyvinyl chloride stretch films, paper products and money [2,3]. BPA is an estrogen antagonist and exposure to trace levels of the pollutant is associated with several debilitating illnesses such as cardiac disorder [4], diabetes [5], cancer [6], hormonal [7] and reproductive function [8]. Therefore, to assess the risk and ensure environmental safety and health, the measurement of BPA remains as one of the most widely studied topics.
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Various analytical methods have been employed for the direct detection and quantification of BPA, including liquid chromatography–mass spectrometry (LC–MS) [9] and gas chromatography–mass spectrometry (GC–MS) [10,11], fluorescence [12], electrochemical detection [13], surface plasmon resonance [14] as well as immunochemical methods [15]. Intriguingly, a small molecule fluorescent sensor for the convenient, selective and sensitive detection of BPA has not been reported. Currently, fluorescent sensors for detecting BPA are based on a Cy5.5-labeled antibody [16] or a DNA aptamer [17]. However, these biological macromolecules lack the robustness and stability required for practical environmental sensing. In contrast, small molecule fluorescent probes are more attractive due to their low molecular weight, high chemical stability, and relatively high sensitivity [18]. Herein, we report a triazole-derivatized boron dipyrromethene (BODIPY) dye, BPO as a turn-on fluorescent sensor for detecting BPA. BPO is capable of distinguishing BPA from various bisphenols in aqueous solution. Owing to its unique features, BPO represents a convenient and practical tool for the selective detection of BPA in real water environments and other BPA-related analyses. 2. Materials and methods
All commercially available reagents and solvents were purchased from Sigma Aldrich, Alfa Aesar, Fluka, Merck or Acros, and used as received unless otherwise stated. Dichloromethane (DCM) (Fisher Scientific, analytical grade) was freshly distilled from phosphorus pentoxide (P2 O5 ) under nitrogen. Anhydrous tetrahydrofuran (THF) was purchased from Alfa Aesar and used without further purification. Sea water and drain water samples were collected from three separate locations in Singapore (West Coast, East Coast and Punggol (North)), combined and used as such. Drinking water samples were purchased from Evian, Ice Mountain and Dasani, combined and used as such. 2.2. Fluorescence The fluorescence measurements were carried out on a SpectraMax M2 plate reader in 96-well plates by scanning the emission spectra between 520 and 700 nm. All measurements were performed under an excitation wavelength of 460 nm unless otherwise stated. Fluorescence of the dye was measured in deionized water, containing 1% dimethylsulfoxide (DMSO). The intrinsic fluorescence of BPA was measured on a Horiba JobinYuon fluoromax-4 spectrofluorometer by using a path length of 10 mm and excitation at 285 nm. The bandwidth was 5 nm. All experiments were repeated three times. The data analysis was conducted using Origin 8.0 (OriginLab Corporation, MA). 2.3. Quantum yield measurements Quantum yields were calculated by measuring the integrated emission area of the fluorescent spectra in its respective solvents and comparing to the area measured for Rhodamine B (reference) (10 M, ˚F = 0.31, ex = 500 nm) in water ( = 1.333). Quantum yields were calculated using the equation:
sample
reference
= ˚F
UV–vis absorption spectra of the dye and analyte in deionized water were recorded from 300 to 700 nm using a SpectraMax M2 spectrophotometer. 2.5. Compound characterization Analytical characterization was performed on a LC-MS (Agilent1200 series) system equipped with a DAD detector and a single quadrupole mass spectrometer (6130 series) with an ESI probe. Analytical LC method: eluents, A: H2 O (0.1% HCOOH), B: CH3 CN (0.1% HCOOH), gradient 5% B–95% B (10 min). Reverse-phase Phenomenex C18 Luna column (4.6 mm × 50 mm, 3.5 m particle size), flow rate: 1 mL min−1 . 1 H NMR, 19 F NMR and 13 C NMR spectra were recorded on Bruker ACF300 (300 MHz) and AMX500 (500 MHz) spectrometers and reported in ı (ppm). 1 H NMR spectra were referenced to residual proton signals in acetonitrile-d3 (CD3 CN) (ı = 1.94 ppm); 13 C NMR spectra were referenced to solvent resonances of CD3 CN (ı = 1.32 ppm); 19 F NMR spectra were referenced to external TFA (ı = −76.55 ppm vs. CFCl3 at 0.00 ppm). High resolution mass spectra (ESI) were obtained on a Finnigan/MAT 95XL-T spectrometer. 2.6. Pre-concentration of BPA from contaminated tap water samples
2.1. Materials
˚F
2.4. Absorbance
F sample F reference
sample reference
Absreference
Abssample
where F represents the area of fluorescent emission, is the refractive index of the solvent, and Abs is absorbance at the excitation wavelength. Emission was integrated between 520 and 700 nm.
100 mL of BPA contaminated (0, 10, 20, 40, 60, 80 and 100 nM) tap water solutions were extracted with DCM (30 mL ×3). The combined organic extracts were evaporated and redissolved in 100 L deionized water for fluorescence and LC–MS test. 2.7. Synthesis N-(4-(5,5-difluoro-3-(4-phenethyl-1H-1,2,3-triazol1-yl)-7-(piperidin-1-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2 ,1 f][1,3,2]diazaborinin-10-yl)phenyl)acetamide (BPO). 3,5-Dichloro-8-(4 -aminophenyl)-4,4-difluoro-4-bora-3a,4adiaza-s-indacence (7.5 mg, 21.3 mol) was synthesized and loaded onto 2-chlorotrityl chloride resin following the procedure described in reference [19]. The loaded resin was resuspended in DCM and shaken at room temperature for 10 min and washed with dimethylformamide (DMF). A suspension of sodium azide (NaN3 ) (12.5 mg, 0.19 mmol) in DMF (1.0 mL) was added and the reaction mixture further shaken at room temperature for 30 min. After washing with DMF (4× 5 mL), a solution of DMF:piperidine (4:1) (1.0 mL) was added and the reaction shaken for 1 h. 4-Phenyl-1butyne (30 L, 0.21 mmol), cuprous iodide (16.3 mg, 0.21 mmol) and ascorbic acid (15 mg, 0.21 mmol) were subsequently added and the reaction mixture shaken for another 30 min. The resin was filtered, washed with DMF (4× 5 mL) and DCM (2× 5 mL) following which cleavage was performed with 0.5% trifluoroacetic acid (TFA) in DCM (3× 5 mL, 10 min each). The organic extracts were recovered and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (eluent:DCM to DCM:MeOH (98:2)) to afford an orange solid which was redissolved in DCM (1 mL), mixed with 6 drops of saturated aqueous NaHCO3 and cooled to 0 ◦ C. Acetyl chloride (10 L, 0.11 mmol) was added in portions over 1 min and the resulting mixture stirred at room temperature and monitored by analytical thin-layer chromatography (TLC). Upon reaction completion, the reaction was diluted with DCM (15 mL), washed with water (2× 15 mL), saturated NaHCO3 (1× 15 mL), saturated brine (1× 15 mL) and dried over anhydrous Na2 SO4 . The organic extract was evaporated to afford BPO as an orange solid (3 mg, 5.18 mmol, 25% yield). 1 H NMR (500 MHz, CD3 CN) ı 8.4–8.45 (m, 1H), 7.91 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.42
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(d, J = 8.6 Hz, 2H), 7.30–7.22 (m, 4H), 7.21–7.14 (m, 1H), 6.98 (d, J = 5.4 Hz, 1H), 6.61 (d, J = 5.4 Hz, 1H), 6.38 (d, J = 3.8 Hz, 1H), 6.26 (d, J = 3.8 Hz, 1H), 3.89–3.81 (m, 4H), 3.09–2.98 (m, 4H), 2.08 (s, 3H), 1.73–1.65 (m, 6H); 13 C NMR (128 MHz, CD3 CN) 169.8, 163.4, 146.9, 142.5, 141.0, 137.4, 136.9, 135.0, 132.1, 131.7, 130.8, 130.2, 129.5, 129.3, 128.9, 126.9, 125.4, 119.7, 115.9, 110.6, 53.0, 28.1, 27.2, 24.5, 24.4; 19 F NMR (282 MHz, CD3 CN) ı −132.13 (dd, J = 68.5, 34.3 Hz); HRMS (C28 H27 BF2 N7 ): Calc. [M+Na]+ : 602.2627, Found [M+Na]+ : 602.2644. 3. Results and discussion 3.1. Sensor identification We began by analyzing the fluorescence responses of a recently reported BODIPY-triazole library toward BPA [19]. These BODIPY-triazoles are fluorescent molecular rotors; under specific environments, the inherent fluorescence of the BODIPY core can be recovered, making them an ideal starting point for the development of a sensitive BPA turn-on fluorescent sensor. From the screening, we identified one compound (BPO) showing a remarkable 70-fold increase in fluorescence emission at 580 nm and an approximate 10 nm blue shift in emission maximum (Fig. 1a and Fig. S1). The data plot of fluorescent titration of BPO with serial concentrations of BPA was fitted according to a reported method, and we determined the apparent affinity constant (Ka ) as 513 ± 5 M−1 (Fig. 1b) [20]. In addition, we found that Ka was generally unaffected by the ionic strength of the solution (Table S1). In the range of 5–100 M BPA, the plot displays a good linear relationship with a R2 of 0.99. The limit of detection (LOD) for BPA was determined to be approximately 5 M (S/N = 3) (Fig. 1b, inset). 3.2. Selectivity Next, we examined the selectivity of BPO toward other bisphenols. Several bisphenols including Bisphenol AF, F, Z, AP and P were tested and their structures are shown in Fig. 2a. As shown in Fig. 2b and c, and Figs. S2 and S3, BPO showed weak response toward other bisphenols and phenol reinforcing its remarkable selectivity toward BPA. Previous reports of fluorescent BPA sensors generally do not display high selectivity between the bisphenols [17,21]. To the best of our knowledge, BPO is the first turn-on BODIPY fluorescent dye that displays remarkable selectivity toward BPA with weak response to other bisphenols. 3.3. Applications for water analysis After examining the excellent selectivity of BPO toward BPA, we evaluated its application to quantify BPA in real water samples. Due to the lightweight, shatter-resistant and transparent properties of polycarbonate plastics, these materials are popular choices for water bottles [14,22]. Therefore, BPA is a common contaminant found in bottled drinking water. Researchers have also reported widespread global contamination of sea water with BPA mainly originating from epoxy paint used on ships and hard plastic trash discarded into the oceans [23,24]. Due to these reasons, the detection and quantification of BPA in environmental water samples is of significant importance. We examined the fluorescence response of BPO in bottled drinking water, drain water and sea water that were spiked with increasing concentrations of BPA up to 100 M and observed excellent linear correlation between the fluorescence of the dye and BPA concentration (Fig. 3). This result demonstrates the stability and robustness of BPO for analyzing BPA even in real, complex water samples.
Fig. 1. (a) Fluorescence response of BPO (10 M) upon mixing with serial dilutions of BPA (from 0 to 10 mM) in deionized water (pH = 6.9). Inset: structures of BPO and BPA. (b) Data plot of fluorescence emission intensity at 580 nm upon interaction with serial dilutions of BPA in deionized water. Inset: standard linear curve derived from the emission intensity at 580 nm upon addition of 0–100 M BPA. Linear regression: R2 = 0.99. ex : 460 nm; ˚F (in water): 0.9%; ˚F (in 10 mM BPA): 32%. Values are represented as means and error bars as standard deviations (n = 3); measurements were taken at room temperature (RT).
We further explored the ability of BPO to accurately quantify the concentration of BPA in contaminated tap water samples. According to American legislations, BPA concentrations in the nanomolar range is classified as the risk alert limit [8]. Hence, tap water spiked with known nanomolar concentrations of BPA (0–100 nM) was analyzed. As shown in Fig. 1b (inset), the linear detection range of BPO falls within 5–100 M. Hence, to achieve nanomolar detection limits, a series of liquid–liquid extractions were performed on the spiked water samples to concentrate the BPA 1000-fold before applying BPO. LC–MS is a validated method for quantifying BPA in contaminated water samples [9]. To put the performance of BPO in perspective, the same samples were concurrently quantified using LC–MS (standard linear curve shown in Fig. S4). As shown in Table 1, the concentrations measured using BPO matched closely with that from the LC–MS method. These results demonstrated the reliability of BPO in quantifying BPA concentrations in contaminated water samples. Coupled with a simple liquid–liquid extraction, a 10 nM LOD was achieved. Together, these results confirmed that BPO could be a valuable tool for the convenient and sensitive detection of BPA in various water environments.
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Fig. 2. Selectivity of BPO. (a) Structures of bisphenol analogs. (b) Fluorescence response of BPO (10 M) with various bisphenol analogs and phenol. Conditions: BPO (10 M), bisphenols (0.5, 1.0, 2.0 and 4.0 mM) in deionized water (pH = 6.9). ex : 460 nm; em : 580 nm; values are represented as means (n = 3); measurements were taken at RT. (c) Photographic image of BPO (10 M) mixed with various bisphenols. Irradiation with a hand-held UV lamp at 365 nm.
3.4. Sensing mechanism studies Subsequent experiments were aimed at understanding the sensing mechanism of BPO. First, we analyzed the photophysical properties of BPO. As shown in Fig. 4a and Table 2, BPO showed low quantum yields in most organic solvents and water. However, in poly(ethylene glycol) 400 (PEG 400), a significantly higher quantum yield was observed. Systematic additions of PEG 400 to an aqueous
solution of BPO similarly increased the fluorescence of the dye proportionately (Fig. 4b). These observations are consistent with BPO’s behavior as fluorescent molecular rotors. The increasing viscosity of the solvent system systematically restricts bond rotation at the 3, 5 and meso positions of the BODIPY core of BPO [19,25]. Consequently, a greater proportion of energy is channeled from the non-radiative processes into fluorescence. As fluorescent molecular rotors, BPO’s turn-on response in the presence of BPA is likely
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Fig. 3. Fluorescence response of BPO (10 M) in various environmental water samples. BPA (0–100 M) was added to sea water, drain water and drinking water respectively. ex : 460 nm, em : 580 nm; values are represented as means and error bars as standard deviations (n = 3); measurements were taken at RT. Linear regres2 2 2 = 0.99, Rdrain-water = 0.99 and Rdrinking-water = 0.99. sion: Rsea-water
Table 1 Quantification of BPA in spiked tap water samples. Values were taken as means and uncertainties as SD (n = 3). Sample
Actual [BPA] (nM)
Theoretical [BPA] after pre-concentration (M)
Measured (M) BPOa
1 2 3 4 5 6
10 20 40 60 80 100
10 20 40 60 80 100
6.2 14.2 32.5 57.5 73.6 91.2
LC–MSb ± ± ± ± ± ±
0.1 0.1 0.2 0.1 0.3 0.3
7.1 16.1 35.3 52.9 71.1 84.8
± ± ± ± ± ±
0.2 0.5 0.2 0.1 0.4 0.2
a Standard linear equation for quantifying BPA concentration using the fluorescence intensity of BPO at 580 nm: y = 1.1 + 0.02x, R2 = 0.99 (Fig. 1b, inset). b Standard linear equation for quantifying BPA concentration using the integrated areas in the LC–MS spectra: y = 3.2 + 820x, R2 = 0.99 (Fig. S5).
to involve a similar rotational restriction mechanism induced by a mutual interaction between the two compounds. To investigate their interaction further, we analyzed the sensing capability of BPO to aqueous BPA in the presence of increasing proportions of organic solvents. As shown in Fig. 5a, when the organic solvent was above 20–30%, the fluorescence of BPO was completely quenched demonstrating that sensing of BPA was highly dependent on an aqueous environment. This result suggested that the interaction between BPO and BPA might be hydrophobic in nature. We studied the response of BPO to BPA under different pH and found Table 2 Photophysical properties of BPO in various solvents. Solvent
Dielectric constant, ε
Viscosity, (mPa s)
abs,max (nm)
fluo,max (nm)
˚F (%)
Water DMSO DMF EtOH Acetone PEG 400 DCM EA Toluene
80.1 46.7 38.0 24.3 21.0 12.4 9.1 6.0 2.4
0.89 2.00 0.92 1.14 0.31 90.00 0.41 0.43 0.55
470 470 470 470 470 475 475 480 495
590 580 580 580 580 580 585 585 585
0.9 2.1 2.3 2.3 2.4 18.1 3.2 1.5 1.6
DMSO: dimethyl sulfoxide; EtOH: ethanol; EA: ethyl acetate; DMF: dimethyl formamide; DCM: dichloromethane.
Fig. 4. Photophysical properties of BPO in various solvents. (a) Fluorescence spectra of BPO (10 M) in various organic solvents. (b) Fluorescence spectra of BPO (10 M) in various mixtures of PEG (0, 5, 10, 20, 40, 60, 80 and 100%) in water. ex : 460 nm; values are represented as means (n = 3); measurements were taken at RT.
that the emission intensity of BPO was independent of pH (Fig. S5). However, upon addition of BPA, a significant decrease in the dye emission intensity was observed above pH 9.0 (Fig. 5b); below pH 9.0, only a gradual decrease in fluorescence intensity was detected. The pKa of BPA is 9.6 and 10.2 [22]. This pH dependent trend thus suggests that BPO largely interacts with the de-ionized form of BPA and further supports hydrophobic interactions between BPO and BPA. Similar trends were observed in the interaction BPO and other bisphenols (Fig. S6); for example, BPAF and BPAP are estimated to have slightly lower pKa than BPA, therefore, the BPO fluorescence response was observed to decrease significantly beyond pH 8.0. To achieve maximum stability, the hydrophobic BPA probably associates spontaneously into large clusters surrounded by water cages. BPO may then be favorably trapped within these hydrophobic clusters resulting in restriction of bond rotation and recovering the intrinsic fluorescence of BODIPY. Formation of these BPA clusters that wrap around BPO is probably facilitated by a small degree of BPA deprotonation thereby accounting for the maximum fluorescence response at pH 9.0. In addition, the increased fluorescence response of BPO in the presence of increasing amounts of BPA in the aqueous solution is probably because of the increased density of the hydrophobic clusters as more BPA is added. On the contrary, addition of organic solvents disrupts these clusters and increases the rotational freedom of BPO resulting in its requenching.
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identification in environmental science as well as in analytical chemistry. Therefore, following work will explore improved methods for BPA enrichment to be incorporated into a BPO-based BPA detection kit that can be used on-site. Acknowledgements The authors acknowledge the financial support from the Singapore-Peking-Oxford Research Enterprise (SPORE, COY-15EWI-RCFSA/N197-1). Liyun Zhang acknowledges the financial support from Natural Science Foundation of China (No. 11105150), China Postdoctoral Science Foundation (No. 2012M510163). Jun Cheng Er acknowledges the receipt of an NGS scholarship. The authors thank Jun Jie Heng for offering BPA samples and his great help in discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.01.038. References
Fig. 5. Interaction of BPO with BPA. (a) Fluorescence response of BPO (10 M) upon addition of BPA in different organic solvent aqueous solution. (b) Fluorescence response of BPO (10 M) upon addition of BPA (1 mM) in aqueous solution under different pH. ex : 460 nm; em : 580 nm; values are represented as means (n = 3); measurements were taken at RT.
4. Conclusion In summary, we have developed a triazole-derivatized BODIPY compound as a BPA turn-on sensor in aqueous solution, which shows a selective recognition for BPA from other bisphenol analogs and phenol. BPO displays the high stability and robustness for analyzing BPA even in real, complex water samples. In addition, mechanistic studies suggest that BPO may be favorably trapped within BPA hydrophobic clusters resulting in restriction of bond rotation and recovery of the intrinsic fluorescence of BODIPY. For analytical applications, it has potential as a sensor for BPA
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