A sensitive enzyme biosensor for catecholics detection via the inner filter effect on fluorescence of CdTe quantum dots

Sensors and Actuators B 173 (2012) 477–482

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

A sensitive enzyme biosensor for catecholics detection via the inner filter effect on fluorescence of CdTe quantum dots Qiang Zhang, Yuanyuan Qu ∗ , Meng Liu, Xinliang Li, Jiti Zhou, Xuwang Zhang, Hao Zhou Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian 116024, PR China

a r t i c l e

i n f o

Article history: Received 4 May 2012 Received in revised form 6 July 2012 Accepted 12 July 2012 Available online 21 July 2012 Keywords: Fluorescent biosensor Catecholics detection Extradiol dioxygenase Quantum dots

a b s t r a c t In this paper, a new type of fluorescent biosensing system for catechol and 2,3-dihydroxybiphenyl (DHB) was described based on the inner filter effect (IFE), with the combination of CdTe quantum dots (QDs) and an extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC). In the presence of BphC and its substrates, catechol and DHB, the excitation of QDs was partially weakened by the competitive absorption of the enzymatic reaction products, resulting in the quenching of QDs. The dependence of the emission intensity quenching on “quencher” concentration obeyed the quadratic equation, and the detection limits for catechol and DHB were 0.02 and 2 ␮M, respectively. The present QD–BphC system also exhibited good selectivity to either catechol or DHB in the mixtures of other phenolic compounds. However, when catechol and DHB coexisted, the QD–BphC system could not separate the signals of these two compounds from each other due to the inherent substrate range of BphC. Nevertheless, the developed BphC-based fluorescent biosensor via the IFE on fluorescence of QDs could be employed as a robust support for the construction of other enzyme-based biosensors. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Catechol and its derivatives are common compounds derived from microbial metabolic processes of most aromatic compounds [1]. Furthermore, as one of the most major intermediate products involved in the industrial and agricultural activities, catechol and its derivatives have been demonstrated to accumulate in the environment and even associate with the damage to physical health. Therefore, it is highly desirable to develop feasible methods to realize convenient and high-throughput determination of catecholic compounds. Traditional techniques developed for the determination of catechol are mainly based on chemical–physical methods, such as high performance liquid chromatography [2], gas chromatography/mass spectrometry [3], electrochemical methods [4,5], and chemiluminescence [6], which realize their performances at the expense of time, cost and tedious procedures for sample pretreatment or preconcentration. To date, considerable efforts have been devoted to the design of enzyme-based biosensors due to their unique advantages, especially for their highly sensitive, selective, relatively inexpensive and ready-to-use performance [7]. It has been reported that some

∗ Corresponding author. Tel.: +86 411 84706251; fax: +86 411 84706252. E-mail address: [email protected] (Y. Qu). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.07.055

enzymes, such as tyrosinase [8], laccase [9], and horseradish peroxidase [10], are capable of catalyzing catecholic compounds, which lays a powerful foundation for the design of electro- or optical biosensors for catechol detection [11–13]. Although much progress has been made, further development of these enzymebased sensing protocols is seriously hampered because it is very difficult to separate the signal of catecholics from that of other phenolic compounds [14]. In order to break the limitation, we previously demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), a typical extradiol dioxygenase that performed the ring cleavage of catecholic compounds [15], could be successfully used to construct the catechol biosensor with high sensitivity and specific selectivity [6]. However, the combination of electro- and enzymatic catalysis makes the reaction process very complicated on the electrode, and the reaction mechanism remains to be explored. Inspired by the great progress in fluorescent sensors [16–19], a new attempt is now being made to apply BphC in the optical biosensor for catechol detection in combination with luminescent nanomaterials. Quantum dots (QDs) are extremely excellent candidates for fluorescence (FL)-based biosensors due to their size-tunable emission with broad excitation spectra [20,21]. To date, most studies on the fluorescent biosensors have focused on the FL quenching through energy transfer or charge transfer mechanism induced by quinone and quinine intermediates [22–25], hydroxyl group [26],

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NAD [27,28], biomoleculars [19,29–31], metal ions [32–36], and so on. Quenching effect of QDs resulted from the pH-dependent electron-transfer process was used for the detection of dopamine [25]. Extensive testing has also been undergone for the detection of catecholic compounds based on the quenching of QDs owing to enzyme catalysis [22,23]. Most of the reported quenching effectbased strategies for catecholic detection focused on the oxidation of catechols to quinones which functioned as electron acceptors. As is well-known, the fluorescent process typically originates from electron transitions between the ground states and excited states. In other words, the FL intensity can be significantly affected by photon absorption and electron transition processes. In fact, quenching effects in the current QD-based biosensors principally derive from the electron transition process. However, the effects of targets on the photon absorption process of QDs, such as the inner filter effect (IFE) of fluorescence, are often neglected. The IFE results from the absorption of the excitation and/or emission light of fluorophore by absorbers, which can convert the changes of absorbance signals into the exponential changes of fluorescence signals [37]. Up to now, some studies focused on the application of the IFE in developing novel fluorescence assays [38–42]. Though much progress has been made, it is still a challenge to find the suitable absorber–fluorophore pair to meet the complementary overlap between the absorption band of the absorber and the excitation and/or emission wavelength of the fluorophore. Herein, we described a feasible and rapid method for the quantitative detection of catechol and 2,3-dihydroxybiphenyl (DHB) via the IFE on fluorescence of QDs, which resulted from the competitive absorption of the exciting light between the QDs and the products of enzymatic reaction. To the best of our knowledge, it is the first report about the IFE-based fluorescent assay caused by an enzyme reaction. The combination of the efficient FL quenching of QDs and the effective enzymatic reactions should afford a novel and facile platform for the application of the IFE and various enzymes in fluorescent biosensing systems. 2. Experiments 2.1. Reagents Tellurium powder, CdCl2 , sodium borohydride, and thioglycolic acid (TGA) were purchased from the Kemiou Agent Co., Tianjin, China. NaH2 PO4 ·2H2 O, Na2 HPO4 ·12H2 O, phenol, catechol, resorcinol, hydroquinone, and all the other chemicals were of analytical grade and used without further purification. The 8 mM phosphate buffer solution (PBS) with different pH values was prepared by mixing the stock solution of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O. Double distilled water was used throughout the experiments. 2.2. Apparatus UV–vis absorption spectra were recorded with a JASCO V-560 spectrophotometer. Fluorescence spectra were acquired using a Hitachi F-4500 spectrofluorometer. TEM images were obtained from a transmission electron microscopy (TEM, FEI-Tecnai G2 20) equipped with an energy-dispersive X-ray (EDX) spectrometer. 2.3. Preparation of BphC and BphD crude extracts BphC crude extracts (1.772 U/mg) were obtained according to the method of Li et al. with some modifications [43]. Briefly, the accumulation-culture Escherichia coli BL21 (carrying pET-bphC) was harvested and suspended in PBS. After ultrasonication and centrifugation, the supernatant was taken as the crude extracts. BphD crude extracts were also obtained as mentioned above.

Fig. 1. UV–vis absorbance and FL spectra of CdTe QDs (excitation at 375 nm). Inset (A): TEM images of CdTe QDs and (B): UV–vis absorbance of 2-hydroxy-6-oxohexa2,4-dienoic acid.

2.4. Preparation of QDs A photoillumination approach was employed to prepare highly fluorescent water-soluble CdTe quantum dots (QDs) [35]. In brief, freshly prepared NaHTe was quickly injected into N2 -saturated CdCl2 solution at pH 11.2 in the presence of TGA. The molar ratio of Cd2+ :NaHTe:TGA was fixed at 8:1:1.2. At ambient temperatures, 100 mL CdTe precursor solution was then directly transferred into a sealed quartz vessel, and subjected to illumination for 6 h under vigorous stirring. The intensity of the light was approximately 150 mW/cm2 . 2.5. Fluorescence detection The total volume for fluorescence detection was 500 ␮L, which contained 100 ␮L QDs, 10 ␮L BphC crude extracts (1.772 U/mg), 10 ␮L substrate with some concentration, and 380 ␮L PBS (0.2 M) with some pH value. Before fluorescence detection, the mixtures were incubated for 5 min. 3. Results and discussion 3.1. Optical characteristics of CdTe QDs The UV/vis absorption and FL spectra of CdTe QDs are displayed in Fig. 1, as well as the typical transmission electron microscopy (TEM) image of QDs (Fig. 1, inset A). It was obvious that the as-prepared CdTe QDs presented uniform diameter and emitted strong FL at 512 nm upon excitation at 375 nm. Several compounds produced from the enzymatic reactions had maximal absorption wavelengths among the excitation waves of CdTe QDs. Fig. 1 (inset B) shows the absorbance peak of BphC–catechol reaction product, 2-hydroxy-6-oxohexa-2,4-dienoic acid (HODA), at 375 nm, which was absolutely overlapped with the excitation wavelength of QDs. Therefore, a strategy was proposed to fabricate a biosensor based on the point mentioned above. Taking catechol for example, Scheme 1 depicts the principle of our strategy. Original CdTe QDs emit intense FL. However, when the enzymatic reaction system, BphC with catechol or DHB, is introduced, a portion of exciting light will be absorbed by the reaction products of the enzymatic system at their absorbance wavelengths, which will cause the IFE on the fluorescence of QDs, thus resulting in the quenching. In this biosensing system, BphC is used as the recognition element, and QDs can act as a transducer to convert the recognition information into a detectable signal via the IFE. Furthermore, the overall

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FL response was dependent on the effective amount of catecholic substrates. 3.2. Optimization of sensing conditions of QD–BphC system The effects of pH on the FL of QD–BphC in the presence of catechol were investigated by mixing and incubating the hybrids for 5 min in buffers with different pH, i.e., 5.0, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0, respectively. The FL intensity of QDs decreased a lot both under the acid and basic conditions (Fig. 2 inset, light bars). The reason for this result might be that QDs itself is unstable under acid conditions (Fig. 2 inset, dark bars), and a part of catechol can be oxidated to o-benzoquionone under basic conditions due to the decrease of the BphC activity, resulting in the quenching. The response (FL) was defined as the difference between the FL emission intensity of CdTe QDs before (F0 ) and after incubated with BphC–catechol system (F). From Fig. 2, we can see that the FL emission intensity decreased a lot at all the tested pH levels, while the most prominent response was obtained at pH 7.0, which might be due to the better activity of enzyme in the physiological buffer with neutral pH. Therefore, phosphate buffer solution (PBS, 8 mM) with pH 7.0 was adopted in the further experiments.

Fig. 3. Effects of excitation wavelengths on the FL intensity of CdTe QD–BphC system in the presence of 0.2 mM catechol (line a) and 0.1 mM DHB (line b). 375 nm and 434 nm were the maximal absorption wavelengths of HODA and HOPDA, respectively.

As the method described here was based on the coincidence of the receptor absorbance and QDs excitation, the excitation wavelength exerted a strong influence on the results. When the excitation wavelength was changed in the range of 340–450 nm, the FL intensity tended to decrease gradually from 340 to 375 nm, and then increased rapidly at the longer wavelengths (Fig. 3, line a). The wavelength of 375 nm, where the most significant decrease was obtained, was exactly in accordance with the maximal absorption peak of HODA, which provided a powerful evidence for our proposed mechanism. In respect to DHB (Fig. 3, line b), similar results were obtained, and the weak absorbance was observed at 423 nm, which was due to the competitive absorption of the reaction product of DHB and BphC, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). Therefore, the excitation wavelength of 375 nm and 423 nm was applied to excite the QDs for catechol and DHB detection, respectively. 3.3. QDs FL quenching by the products of BphC and catecholic compounds To further confirm the effects of BphC–catechol reaction on the QDs FL emission, time-dependent FL responses in five different systems were recorded. As shown in Fig. 4, the addition of BphC or catechol had no obvious influence on the QDs FL except for a little weak bleaching of FL intensity caused by themselves (lines b and c). However, the FL intensity of QDs decreased dramatically once QDs was mixed with BphC and catechol simultaneously (Fig. 4, lines d and e). The time-dependent FL response of QD–BphC–catechol system seemed similar to the kinetics of BphC–catechol reaction, which depended on the ratio of BphC and catechol. It can be seen that the enzymatic catalysis was fast and finished within 5 min even with a small amount of enzyme (2 ␮L). When 10 ␮L BphC (1.772 U/mg) was added, the reaction with 0.2 mM catechol was much faster than that of the system with 2 ␮L BphC and 0.1 mM catechol (Fig. 4, lines e and d). The quenching effects on QDs FL in the presence of 10 ␮L BphC and 0.2 mM catechol with different incubation time were also determined (Fig. 4, inset). To make sure that catechol could be consumed completely by BphC, the volume of BphC was taken as 10 ␮L, and the incubation time for the mixtures was 5 min in the following study. Besides catechol, the quenching effect of DHB was also studied. Similar to catechol, DHB itself could only cause slight bleaching of QDs FL intensity, while in the simultaneous presence of

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3h BphC and DHB, the quenching effect was highly enhanced instantaneously (Fig. 5A). In order to illuminate the IFE of HOPDA on the FL of QDs further, 2-hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase (BphD) was introduced into the system. BphD is a meta-cleavage product hydrolase which can hydrolyze HOPDA generated in the biphenyl degradation pathway of bacteria into benzoic acid and 2-hydroxypenta-2,4-dienoic acid (HDA) [44]:

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Wavelength (nm) Excess BphD was added to make sure that HOPDA was hydrolyzed completely. Fig. 5A depicts that the QDs FL intensity could almost totally recover as soon as the addition of BphD, indicating that HOPDA played a key role in the quenching of QDs FL. Another interesting phenomenon was observed in the experiment. The QDs FL intensity decreased rapidly with the formation of HODA and then reached equilibrium basically within 5 min (Fig. 4, lines d and e). A rapid quenching also occurred once HOPDA was produced, however, different from HODA, the QDs FL intensity recovered subsequently upon excitation at 423 nm, and after 3 h, it could nearly recover to the original intensity of QDs (Fig. 5B). Moreover, the yellow color of HOPDA faded obviously. A blank control group was taken in air under the same conditions without light excitation. As a result, the color of the system had no obvious change (data not shown). The recovery of QDs FL might be related to the photodegradation of HOPDA. Compared with HODA, a benzene ring was contained in the structure of HOPDA, which made it more easily and efficiently absorb light energy resulting in photodegradation. 3.4. Detection of catechol and DHB based on QD–BphC system Fig. 6 illustrates the FL spectra as well as F0 /F of CdTe QDs in the presence of different concentrations of catechol and DHB. With the increase of catechol or DHB concentration, the IFE caused by the formation of enzymatic reaction products was enhanced (Fig. 6). It was observed that the relationship between F0 /F and catechol or DHB concentration obeyed the quadratic equation in a large range of target concentrations. For catechol (from 0.02 to 600 ␮M), it can be detected according to the quadratic

Fig. 5. (A) FL intensity representing quenching effects of BphC, BphD, DHB, mixture of DHB and BphC, and DHB in the presence of BphC and BphD and (B) the kinetic study of quenching effects of 0.1 mM DHB on QDs FL intensity with the incubation time from 0 to 3 h.

equation (y = 0.003x2 − 0.049x + 2.816, R2 = 0.997); while DHB (from 50 ␮M to 300 ␮M), the equation, for y = 0.0003x2 − 0.018x + 1.971 (R2 = 0.995) was used, which was similar to the quenching behavior of CdTe QDs by dopamine and phenol in the presence of H2 O2 /hydrogen peroxide [24]. However, at low concentrations of DHB (from 2 to 50 ␮M), the dependence of the fluorescence quenching on DHB concentration can be expressed by Stern–Volmer equation (y = 0.017x + 1.016, R2 = 0.993). As noted, this phenomenon was also observed in other fluorescent assays based on IFE [45–48]. The detection limit for catechol was 0.02 ␮M at signal-to-noise ratio of 3, which was much lower than that of our previous study based on the SiO2 sol–gel modified BphC electrode [14], indicating the higher sensity of this method. The reason of nonlinear relationship between F0 /F and catechol concentration has not been illuminated, and it might be due to the complexity of enzyme catalytic reaction and the nonlinear spectral overlap between the excitation wavelength and the absorbance of the “quencher” [24,49,50]. Since the products of enzymatic reaction were the quenching units in this study, the extent of FL quenching was actually related to the BphC enzymatic activity. Therefore, another response (F = F0 − F) was applied to evaluate the quenching kinetics. The value of (F0 − F) almost leveled off when the DHB concentration increased to 80 ␮M (Fig. S1), similar to the Michaelis–Menten kinetics. As a result, the Lineweaver–Burk plots

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Fig. 7. (A) Effects of different compounds (0.2 mM) on the FL intensity of original QDs (dark bars) and QD–BphC system (light bars) and (B) FL intensity representing quenching effects of different mixtures on the FL of QDs. A, phenol; B, hydroquinone; C, resorcinol; D, catechol.

of FL changes and DHB concentration (Fig. S2) obeyed the equation as follows:. 1 0.006 = + 1.914 × 10−4 , F0 − F CDHB

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Therefore, the kinetics of quenching can be fitted by enzyme–substrate kinetics, further indicating that the enzymatic reaction played the key role in this IFE-based quenching mechanism. 3.5. Selectivity of the QD–BphC system To demonstrate the selective detection of catechol or DHB, we compared the FL of QDs and QD–BphC hybrids in the presence of phenol (0.2 mM), hydroquinone (0.2 mM), resorcinol (0.2 mM), and catechol (0.2 mM) or DHB (0.02 mM), respectively. Fig. 7A shows the selectivity for catechol of this QD–BphC system. Only slight FL changes were observed when phenol, hydroquinone, resorcinol, or catechol was added into the QDs solution in the absence of BphC. While in the presence of BphC, only catechol could cause remarkable decrease in the FL intensity in comparison with the other interferents, which suggested the good selectivity of our system. The effects of different mixtures on the FL intensity of the QD–BphC system were also investigated, including mixtures

of phenol/catechol, hydroquinone/catechol, resorcinol/catechol, as well as phenol/hydroquinone/resorcinol/catechol (Fig. 7A). It was found that the responses of all these interferents with the same concentration of 0.2 mM did not interfere with that of 0.2 mM catechol. Only the presence of catechol can cause remarkable FL decrease, which should be related to the selectivity of extradiol dioxygenase for catecholic compounds. The selectivity for DHB of the QD–BphC system is depicted in Fig. 7B. Because of the high specificity of BphC, other phenolic compounds, including phenol, hydroquinone, resorcinol, exhibited little influence on DHB detection even at concentrations of 10 times higher than that of DHB. Prominent decrease of QDs FL intensity only occurred in the simultaneous presence of BphC and DHB. This proposed photon absorption quenching protocol presented a better selectivity than that of electron or energy transfer quenching route in traditional QD-based bosensors, which offered a versatile sensing scheme for a lot of substrates [24,51,52]. Nevertheless, as shown in the FL spectra, catechol also caused the remarkable FL quenching of QD–BphC system upon excitation at 423 nm. In addition, when catechol and DHB coexisted in the QD–BphC system, it could not separate the signals of these two compounds due to their synergistic effects on the FL quenching.

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4. Conclusions In summary, a simple fluorescent method for catechol and DHB detection with high sensitivity and specific selectivity was developed based on QD–BphC system. Compared with other QD- and enzyme-based sensors, the present fluorescent biosensing system requires no complicated surface modification of QDs and no enzyme immobilization. Taking advantage of the substrate specificity of enzymes and the facility of the IFE, we hope that the concept and strategy described here could be extended to the development of other QD–enzyme-based biosensors for various compounds. Acknowledgement This work was supported by the National Natural Science Foundation of China (Nos. 51078054; 21176040; 20923006). 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.snb.2012.07.055. References [1] Y. Takahashi, M. Shintani, L. Li, H. Yamane, H. Nojiri, Applied and Environmental Microbiology 75 (2009) 3920. [2] Y. Mizukami, Y. Sawai, Y. Yamaguchi, Journal of Agricultural and Food Chemistry 55 (2007) 4957. [3] S.C. Moldoveanu, M. Kiser, Journal of Chromatography A 1141 (2007) 90–97. [4] H. Zhang, J.S. Zhao, H.T. Liu, R.M. Liu, H.S. Wang, J.F. Liu, Microchimica Acta 169 (2010) 277. [5] H.G. Lin, T. Gan, K.B. Wu, Food Chemistry 113 (2009) 701. [6] E.B. Liu, J.K. Cheng, Chromatography 61 (2005) 619. [7] S.P. Song, H. Xu, C.H. Fan, International Journal of Nanomedicine 1 (2006) 433. [8] P.E.M. Siegbahn, Journal of Biological Inorganic Chemistry 9 (2004) 577. [9] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chemical Reviews 96 (1996) 2563. [10] A. Sadler, V.V. Subrahmanyam, D. Ross, Toxicology and Applied Pharmacology 93 (1988) 62. [11] R.P. Singh, Analyst 136 (2011) 1216. [12] J. Abdullah, M. Ahmad, L.Y. Heng, N. Karuppiah, H. Sidek, Sensors 7 (2007) 2238. [13] J. Orozco, C. Jiménez-Jorquera, C. Fernández-Sánchez, Bioelectrochemistry 75 (2009) 176. [14] Q. Zhang, Y.Y. Qu, X.W. Zhang, J.T. Zhou, H.T. Wang, Biosensors and Bioelectronics 26 (2011) 4362. [15] F.H. Vaillancourt, J.T. Bolin, Critical Reviews in Biochemistry and Molecular Biology 41 (2006) 241. [16] H.B. Li, C.P. Han, Chemistry of Materials 20 (2008) 6053. [17] R. Freeman, L. Bahshi, T. Finder, R. Gill, I. Willner, Chemical Communications 7 (2009) 764. [18] P. Wu, Y. He, H.F. Wang, X.P. Yan, Analytical Chemistry 82 (2010) 1427. [19] L. Dyadyusha, H. Yin, S. Jaiswal, T. Brown, J.J. Baumberg, F.P. Booy, T. Melvin, Chemical Communications 25 (2005) 3201. [20] M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science 281 (1998) 2013. [21] W.C.W. Chan, S. Nie, Science 281 (1998) 2016. [22] J.P. Yuan, W.W. Guo, E.K. Wang, Analytical Chemistry 80 (2008) 1141. [23] R. Gill, R. Freeman, J.P. Xu, I. Willner, S. Winograd, I. Shweky, U. Banin, Journal of the American Chemical Society 128 (2006) 15376. [24] J.P. Yuan, W.W. Guo, E.K. Wang, Biosensors and Bioelectronics 23 (2008) 1567. [25] I.L. Medintz, M.H. Stewart, S.A. Trammell, K. Susumu, J.B. Delehanty, B.C. Mei, J.S. Melinger, J.B. Blanco-Canosa, P.E. Dawson, H. Mattoussi, Nature Materials 9 (2010) 676. [26] L. Jia, J.P. Xu, D. Li, S.P. Pang, Y. Fang, Z.G. Song, J. Ji, Chemical Communications 46 (2010) 7166.

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Biographies Qiang Zhang is currently a PhD candidate in School of Environmental Science and Technology from Dalian University of Technology. Her research interests are concerned with enzyme biosensor. Yuanyuan Qu, PhD, Associate Professor, Doctoral Supervisor of Environmental Engineering in School of Environmental Science and Technology, Dalian University of Technology, China. Her current research interests include discovery of aromatic oxygenase based on metagenomic technology, Bioinformatics-based protein design, multi-step conversion of various aromatics, and enzyme biosensor for the detection of catecholic compounds. Meng Liu is currently pursuing his PhD degree in School of Environmental Science and Technology from Dalian University of Technology. His research focuses on new nanomaterials and their application in biosensor. Xinliang Li is a master candidate in School of Environmental Science and Technology from Dalian University of Technology. His research interests are concerned with the characteristics of extradiol dioxygenases. Jiti Zhou, Professor, PhD supervisor of Dalian University of Technology, Member of America Chemical Society, Member of International Water Association. His research interests focus on Enviromental Biological Engineering, Water Science and Technology, and Water Quality Simulation. Xuwang Zhang is currently pursuing his PhD degree in School of Environmental Science and Technology from Dalian University of Technology. His research focuses on new functions of aromatic oxygenases. Hao Zhou is a PhD candidate in School of Environmental Science and Technology from Dalian University of Technology. His research interests are concerned with the structure and function of aromatic hydrolase.