Fu Yingchun, Chen Chao, Xie Qingji, Xu Xiahong, Zou Can, Zhou Qingmei, Tan Liang, Tang Hao, Zhang Youyu, Yao Shouzhuo
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China.
Anal Chem. 2008 Aug 1;80(15):5829-38. doi: 10.1021/ac800178p. Epub 2008 Jul 2.
A protocol of one-pot chemical preoxidation and electropolymerization of monomers (CPEM) in enzyme-containing aqueous suspensions (or solutions) was proposed as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors, which was proven to be effective for the monomer of 1,4-benzenedithiol (BDT), 1,6-hexanedithiol, o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. As a case examined in detail, a well-dispersed aqueous suspension of the poorly soluble BDT was obtained through its dispersion assisted by ultrasonication and coexisting GOx, which was then subject to chemical preoxidation through adding K3Fe(CN)6, yielding many composites of insoluble BDT oligomers with lots of high-activity enzyme molecules entrapped. Some insoluble composites were then electrochemically codeposited with poly(1,4-benzenedithiol) on an Au electrode, yielding an enzyme film with high-load and high-activity enzyme immobilized. The glucose biosensor prepared here from the CPEM protocol showed much better performance than that from the preoxidant-free conventional electropolymerization (CEP) protocol, with a detection sensitivity increase by a factor of 32 in this case. The GOx-based and AP-based first-generation biosensors developed from the present CPEM protocol all exhibited notably improved performance compared with the analogues from the preoxidant-free CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate various electrode modification processes. The values of quantity and enzymatic specific activity (ESA) of the immobilized enzymes were evaluated through the EQCM and the conventional UV-vis spectrophotometric method, given that the CPEM protocol notably improved the quantity and the ESA of immobilized enzymes as compared with the preoxidant-free CEP protocol. The proposed CPEM protocol may be interesting in a number of fields, including biosensing, biocatalysis, biofuel cells, bioaffinity chromatography, and biomaterials, and the successful electropolymerization of dithiols in aqueous suspensions (two-phase electropolymerization) may open a new avenue for many monomers that are poorly soluble in neutral aqueous solutions to in situ immobilize biomolecules for bioapplications.
提出了一种在含酶水悬浮液（或溶液）中进行单体一锅法化学预氧化和电聚合（CPEM）的方案，作为一种将酶高活性和高负载固定化以构建电流型生物传感器的通用策略，该策略已被证明对1,4 - 苯二硫醇（BDT）、1,6 - 己二硫醇、邻苯二胺、邻氨基酚或吡咯等单体，铁氰化钾（K3Fe(CN)6）或对苯醌等预氧化剂，以及葡萄糖氧化酶（GOx）或碱性磷酸酶（AP）等酶有效，可用于开发基于GOx的葡萄糖生物传感器或基于AP的苯基磷酸二钠生物传感器。作为详细研究的一个案例，通过超声分散和共存的GOx辅助，获得了难溶性BDT的良好分散水悬浮液，然后通过加入K3Fe(CN)6进行化学预氧化，得到许多不溶性BDT低聚物的复合材料，其中 entrapped 了大量高活性酶分子。然后将一些不溶性复合材料与聚（1,4 - 苯二硫醇）在金电极上进行电化学共沉积，得到固定有高负载和高活性酶的酶膜。由此CPEM方案制备的葡萄糖生物传感器表现出比无预氧化剂的传统电聚合（CEP）方案好得多的性能，在这种情况下检测灵敏度提高了32倍。由本CPEM方案开发的基于GOx和基于AP的第一代生物传感器与无预氧化剂的CEP方案的类似物相比，均表现出显著改善的性能。采用电化学石英晶体微天平（EQCM）技术研究了各种电极修饰过程。通过EQCM和传统紫外 - 可见分光光度法评估了固定化酶的数量和酶比活性（ESA）值，因为与无预氧化剂的CEP方案相比，CPEM方案显著提高了固定化酶的数量和ESA。所提出的CPEM方案在包括生物传感、生物催化、生物燃料电池、生物亲和色谱和生物材料等许多领域可能具有吸引力，并且在水悬浮液中成功进行二硫醇的电聚合（两相电聚合）可能为许多在中性水溶液中难溶的单体开辟一条原位固定生物分子用于生物应用的新途径。
