Tuned Amperometric Detection of Reduced β-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer,Analytical Chemistry

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Tuned Amperometric Detection of Reduced β-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-04-10 00:00:00 , DOI: 10.1021/acs.analchem.7b05467
Somjai Teanphonkrang ₁ , Salome Janke ₂ , Pimchai Chaiyen ₃ , Jeerus Sucharitakul ₄ , Wipa Suginta _{1,

5} , Panida Khunkaewla ₁ , Wolfgang Schuhmann ₂ , Adrian Ruff ₂ , Albert Schulte ₃

Affiliation

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid (p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.

中文翻译：

通过变构调节固定在氧化还原聚合物中的对羟基苯乙酸乙酸羟化酶的还原酶组分的变构调节的安培电流法测定还原的β-烟酰胺腺嘌呤二核苷酸。

我们报告制造的安培NADH生物传感器系统的制造，该系统在适应性（（III）-复合物修饰的氧化还原聚合物膜中采用变构调节细菌还原酶进行分析物定量。复杂的Os（III）中心链沿着基质聚合物链在固定的氧化还原蛋白和石墨电极盘之间建立电连接，将NADH的酶促氧化转化为生物传感器电流。可持续的阳极信号传递需要（1）具有与嵌入的还原酶的氧化还原开关相匹配的形式电位的氧化还原聚合物，并避免干扰氧化还原相互作用，以及（2）形成交联的酶/聚合物膜以稳定地捕获生物催化剂。选定的还原酶的活性在效应子（即p）结合后得到增强-羟基-苯乙酸（p -HPA），可通过原位添加或与p -HPA预温育来加快传感器表面上的底物转化率。与没有变构调节剂的操作相比，NADH氧化的加速放大了生物传感器的响应，分析物检测的灵敏度提高了1.5倍。对已知NADH浓度的溶液进行的重复定量测试，验证了性能的可靠性和分析物回收率。我们据此建立了变构酶调节和基于氧化还原聚合物的酶电极布线用于底物生物传感的概念，该概念可能适用于其他变构酶。

更新日期：2018-04-10

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