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基于共价固定化乳酸氧化酶到叶形石墨烯纳米带的乳酸生物传感 通过重氮化偶联反应。

Lactate biosensing based on covalent immobilization of lactate oxidase onto chevron-like graphene nanoribbons via diazotization-coupling reaction.

机构信息

Departamento de Química Analítica y Análisis Instrumental, Facultad de Ciencias, c/ Francisco Tomás y Valiente, Nº7. Campus de Excelencia de la Universidad Autónoma de Madrid, 28049, Madrid, Spain.

Instituto de Ciencia de Materiales de Madrid (CSIC), c/ Sor Juana Inés de la Cruz Nº3. Campus de Excelencia de la Universidad Autónoma de Madrid, 28049, Madrid, Spain.

出版信息

Anal Chim Acta. 2022 May 22;1208:339851. doi: 10.1016/j.aca.2022.339851. Epub 2022 Apr 20.

Abstract

We have designed and prepared an electrochemical biosensor for lactate determination. Through a diazotation process, the enzyme lactate oxidase (LOx) is anchored onto chevron-like graphene nanoribbons (GNR), previously synthesized by a solution-based chemical route, and used as modifiers of glassy carbon electrodes. In a first step, we have performed the grafting of a 4-carboxyphenyl film, by electrochemical reduction of the corresponding 4-carboxyphenyl diazonium salt, on the GNR-modified electrode surface. In this way, the carboxylic groups are exposed to the solution, enabling the covalent immobilization of the enzyme through the formation of an amide bond between these carboxylic groups and the amine groups of the enzyme. The biosensor design was optimized through the morphological and electrochemical characterization of each construction step by atomic force microscopy, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy.The cyclic voltammetric response of the biosensor in a solution of hydroxymethylferrocene in presence of l-lactate evidenced a clear electrocatalytic effect powered by the specific design of the biosensing platform with LOx covalently attached to the GNR layer. From the calibration procedures employed for l-lactate determination, a linear concentration range of 3.4 · 10- 2.8 · 10 M and a detection limit of 11 μM were obtained, with relative errors and relative standard deviations less than 6.0% and 8.4%, respectively. The applicability of the biosensor was tested by determining lactate in apple juices, leading to results that are in good agreement with those obtained with a well-established enzymatic spectrophotometric assay kit.

摘要

我们设计并制备了一种用于乳酸测定的电化学生物传感器。通过重氮反应过程,酶乳酸氧化酶(LOx)被锚定在先前通过基于溶液的化学途径合成的 Chevron 型石墨烯纳米带(GNR)上,并用作玻璃碳电极的修饰剂。在第一步中,我们通过电化学还原相应的 4-羧基苯基重氮盐,在 GNR 修饰电极表面上进行了 4-羧基苯基膜的接枝。这样,暴露在溶液中的羧酸基团可以通过这些羧酸基团与酶的胺基团之间形成酰胺键,将酶共价固定。通过原子力显微镜、扫描电子显微镜、循环伏安法和电化学阻抗谱对每个构建步骤的形态和电化学特性进行了优化。在存在 L-乳酸的羟甲基二茂铁溶液中,生物传感器的循环伏安响应表明了通过将 LOx 共价附着到 GNR 层的生物传感平台的特定设计实现的明显电催化效应。从用于 l-乳酸测定的校准程序中,获得了 3.4·10-2.8·10 M 的线性浓度范围和 11 μM 的检测限,相对误差和相对标准偏差分别小于 6.0%和 8.4%。通过在苹果汁中测定乳酸来测试生物传感器的适用性,得到的结果与使用成熟的酶分光光度测定试剂盒得到的结果非常吻合。

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