Department of Physical Chemistry, University of Sevilla , Profesor García González 1, 41012, Sevilla, Spain.
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences , Leninsky Prospect 33/2, Moscow, 119071, Russia.
Anal Chem. 2015 Nov 3;87(21):10807-14. doi: 10.1021/acs.analchem.5b01710. Epub 2015 Oct 13.
Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.
通过酰胺键形成将酶共价固定在电极上通常通过两步法进行，其中首先用相应的偶联试剂激活表面羧酸基团，然后与蛋白质的胺基团反应。本文表明，对上述方案进行修改，涉及同时孵育烟草过氧化物酶和热解石墨电极与偶联试剂，产生比根据通常的固定化方案获得的物理吸附酶或共价固定化酶更高和更稳定的电催化电流。在 0.3 V ≤ E ≤ 0.8 V（相对于 SHE）电位范围内工作的本过氧化物酶生物传感器具有显著改善的电催化性能，这归因于烟草过氧化物酶和石墨之间的有效电子偶联以及形成的分子内和分子间酰胺键，稳定了蛋白质结构并提高了提供与电极进行电子交换的适当取向的锚固基团的百分比。优化的烟草过氧化物酶传感器具有 10-900 μM 的工作浓度范围、0.08 A M(-1) cm(-2)（RSD 0.05）的灵敏度、2 μM（RSD 0.09）的检测限和良好的长期稳定性，只要在低温下运行即可。这些参数值是迄今为止在简单直接电子转移条件下工作的过氧化物酶生物传感器的最佳报告值之一。
