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ScienceDirect Procedia Technology 27 (2017) 137 – 138
Biosensors 2016
Phenol biosensor based on glassy carbon electrode directly absorbed Escherichia coli cells with surface-displayed bacterial laccase Zhen Zhanga, Zhongming Zhanga, Yonggang Hua, Jin Liua, Hong Nib, Lin Lia,* a
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070,China b School of Life Sciences, Hubei University, Wuhan 430062, China
Abstract In this study, a new biosensor was developed based on a bacterial laccase immobilizing on Escherichia coli surface and direct glassy-carbon electrode adsorption of live cells. Expression and surface localization of laccases in target cells were confirmed by assays of Western blot, immunofluorescence microscopy and flow cytometry. The engineered cells served as a highly active whole cell laccase-catalytic system with an enzyme activity of 32.7 U/mL cells. Under optimized pH condition, electrochemical response of the biosensor was linear within concentration ranges of 5.0 PM to 500.0 PM for several phenolics (catechol, caffeic acid, dopamine, gallic acid, and 2-amino phenol) with a detection limit of 1.0 PM to 5.0 PM, which was comparable to those based on chemically-modified purified laccases. The system exhibited good stability and reproducibility. It also offered considerable level of accuracy for determination of the phenolic compound contents of wed wine, pharmaceutical and wastewater samples. © 2017 2016The TheAuthors. Authors.Published Publishedbyby Elsevier Ltd. © Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016. Peer-review under responsibility of the organizing committee of Biosensors 2016 Keywords: Biosensor; Bacterial laccase; Cell surface display; Adsorption; Phenolic compounds.
Biosensors based on microbial laccases have received an increasing amount of attention because laccase do not require an additional mediator for the non-bypass electrochemical detection of the electron transfer from substrates to the corresponding oxides and laccases exhibit broad substrate specificity. To date, immobilizing enzymes using live microbial cell platforms have appeal due to the ease and effectiveness of this method. Several previous biosensors through adsorption of living bacterial cells on an electrode have proven the activity sustainability of the adsorbed cells within several weeks or months [1]. This significantly stable performance and simple immobilization
* Corresponding author. Tel.: +86-27-8728 6952; fax: +86-27-8728 0679. E-mail address:
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2212-0173 © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the organizing committee of Biosensors 2016 doi:10.1016/j.protcy.2017.04.060
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Zhen Zhang et al. / Procedia Technology 27 (2017) 137 – 138
procedures is an attractive approach for developing bacterial biosensors. In the current study, we engineered a mutated bacterial laccase WlacD [2] onto the Escherichia coli cell surface using a previously optimized bacterial cell surface display system [3]. The recombinant E. coli MB275 cells surface-expressing the fusion protein (InaQN)3/WlacD were directly deposited and adsorbed by a glassy carbon (GC) bare electrode to develop a reliable and easily regenerated biosensor system (WlacD–GC) for determining phenolic compounds (Fig. 1). Fig. 1 Schematic diagram of the construction of the engineered E. coli MB275 adsorbed on a GC electrode (step 1 to step 2) and its use in the electrochemical detection of several phenolic compounds (steps 3 to 4).
E. coli MB275 exhibited a stable whole-cell enzymatic activity at pH 2.0 to 3.0, with a highest enzymatic activity of 32.7 U/mL at pH 2.5. Eight representative phenolic compounds, including catechol, gallic acid, guaiacol, ferulic acid, caffeic acid, dopamine, 1,4-hydroquinone, and phenol, were selected to investigate the responses of the WlacD–GC electrode. The oxidation current of catechol at the potential 0.54 V in comparison with those of caffeic acid at 0.46 V, dopamine at 0.65 V, and gallic acid at 0.64 V indicated the apparently distinguishable potentials for catechol, caffeic acid, dopamine, and gallic acid, respectively. However, the WlacD–GC electrode failed to detect the potential of the other four phenolic compounds. The electrochemical response under optimized pH conditions was linear in the concentration range of 5.0 PM to 300.0 PM. Metal ions (Mn2+, Fe3+, Cu2+, Mg2+, Al3+, and Zn2+) at concentrations of 1 mg/L to 10 mg/L, bovine serum albumin and glucose at 0.1 g/L to 10 g/L, and ascorbic acid at 0.01 g/L to 0.1 g/L failed to cause a noticeable interference effect. The detection limit of 1.0 PM to 5.0 PM was comparable with the detection limits of the other biosensors based on purified chemically modified laccases. When used to detect phenolic compound contents of red wine, tea, urea, and wastewater samples, the biosensor showed a considerable level of accuracy that was comparable to the accuracy of high performance liquid chromatography. High recovery rates of all tested phenolic substances were obtained using the developed biosensor. Compared with other biosensors (i.e., those based on purified chemically modified laccases), the developed biosensor had comparable LOD and showed high stability, reproducibility, and accuracy. Given that the distinctive features of this system such as high enzymatic activity, elimination of mass transfer limits, and simple electrode preparation without using matrix or cross-linking reagent, the proposed biosensor showed significant potential for easy, accurate, and cost-effective analysis of phenolic compounds. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (item no. 31270158). References [1] Reshetilov AN, Iliasov PV, Reshetilova TA. The microbial cell based biosensors. in: Somerset, V. S. (Ed.), Intelligent and Biosensors, InTech, Croatia, pp. 289322. 2010. [2] Shao X, Gao Y, Jiang M, Li L. Deletion and site-directed mutagenesis of laccase from Shigella dysenteriae results in enhanced enzymatic activity and thermostability. Enzyme Microb Technol 2009;44:274280. [3] Li Q, Yan Q, Chen J, He Y, Wang J, Zhang H, Yu Z, Li L. Molecular characterization of an ice nucleation protein variant (InaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli. Int J Biol Sci 2012; 8:10971108.
