Horseradish peroxidase-catalyzed synthesis of poly(thiophene-3-boronic acid) biocomposites for mono-/bi-enzyme immobilization and amperometric biosensing

We report here on a facile enzymatic polymerization protocol to prepare enzyme poly(thiophene-3-boronic acid) (PTBA) polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Horseradish peroxidase (HRP)-catalyzed polymerization of thiophene-3-boronic acid (TBA) monomer was conducted in aqueous solution containing HRP (or plus glucose oxidase (GOx)) by either directly added or GOx-glucose generated oxidant H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting the dialysis-isolated PBCs on Au-plated Au electrode (Au-plate/Au), followed by coating with an outer-layer chitosan (CS) film. The boronic acid residues are capable of covalent bonding with enzyme at the glycosyl sites (boronic acid-diols interaction), which should less affect the enzymatic activity as compared with the common cases of covalent bonding at the peptide chains, and UV-vis spectrophotometric tests confirmed that the encapsulated HRP almost possesses its pristine enzymatic specific activity. The enzyme electrodes were studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry in the presence of Fe(CN)(6)(4-) mediator. The CS/HRP-PTBA/Au-plate/Au electrode responded linearly to H2O2 concentration from 1 to 300 mu M with a sensitivity of 390 mu A mM(-1) cm(-2) and a limit of detection (LOD) of 0.1 mu M. The bienzyme CS/GOx-HRP-PTBA(H2O2)/Au-plate/Au electrode responded linearly to glucose concentration from 5 mu M to 0.83 mM with a sensitivity of 75.1 mu A mM(-1) cm(-2) and a LOD of 1 mu M, and it is found here that the use of Fe(CN)(6)(4-) that can only efficiently mediate HRP favorably avoids the "unusual amperometric responses" observed when other mediators that can efficiently turn over both HRP and GOx are used. (C) 2013 Elsevier B.V. All rights reserved.

我们在此报道一种简便的酶促聚合方案，用于制备酶聚（噻吩 - 3 - 硼酸）（PTBA）聚合物生物复合材料（PBCs），以用于高性能单酶/双酶安培生物传感。在含有辣根过氧化物酶（HRP）（或加上葡萄糖氧化酶（GOx））的水溶液中，通过直接添加或由GOx - 葡萄糖产生的氧化剂H₂O₂，进行辣根过氧化物酶（HRP）催化的噻吩 - 3 - 硼酸（TBA）单体聚合。单酶/双酶安培生物传感器的制备方法很简单，即将透析分离的PBCs浇铸在镀金的金电极（Au - plate/Au）上，然后涂上一层外层壳聚糖（CS）膜。硼酸残基能够在糖基位点与酶共价键合（硼酸 - 二醇相互作用），与在肽链上共价键合的常见情况相比，这种键合对酶活性的影响较小，并且紫外 - 可见分光光度测试证实，包封的HRP几乎具有其原始的酶比活性。在Fe(CN)₆⁴⁻介体存在的情况下，通过循环伏安法、电化学阻抗谱和计时电流法对酶电极进行了研究。CS/HRP - PTBA/Au - plate/Au电极对1至300 μM的H₂O₂浓度呈线性响应，灵敏度为390 μA mM⁻¹cm⁻²，检测限（LOD）为0.1 μM。双酶CS/GOx - HRP - PTBA(H₂O₂)/Au - plate/Au电极对5 μM至0.83 mM的葡萄糖浓度呈线性响应，灵敏度为75.1 μA mM⁻¹cm⁻²，检测限为1 μM，并且在此发现，使用只能有效介导HRP的Fe(CN)₆⁴⁻，有利地避免了使用能有效转换HRP和GOx的其他介体时观察到的“异常安培响应”。（C）2013 Elsevier B.V.保留所有权利。