Domínguez R, Serra B, Reviejo A J, Pingarrón J M
Department of Analytical Chemistry, Complutense University of Madrid, Madrid, 28040, Spain.
Anal Biochem. 2001 Nov 15;298(2):275-82. doi: 10.1006/abio.2001.5371.
The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.
报道了用于选择性测定L-或D-氨基酸的双酶安培复合生物传感器的构建与性能。通过简单的物理包埋将D-或L-氨基酸氧化酶、辣根过氧化物酶和媒介体二茂铁共固定在70%聚四氟乙烯的石墨电极基体中。优化了包括氨基酸氧化酶负载量和pH值在内的工作条件。对在+0.00 V下获得的安培响应的重复性进行了研究，包括电极表面经抛光再生和未经抛光再生的情况，研究了单个生物传感器的使用寿命以及不同生物传感器制备过程中的重现性，这些都说明了生物电极设计的稳健性。通过搅拌溶液中的安培法和流动注射安培检测法分别获得了L-精氨酸、L-苯丙氨酸、L-亮氨酸、L-蛋氨酸、L-色氨酸、D-亮氨酸、D-蛋氨酸、D-丝氨酸和D-缬氨酸的校准曲线。从底物和电极表面的疏水性角度讨论了灵敏度的差异。该双酶复合电极应用于外消旋样品中L-和D-氨基酸的测定，以及麝香葡萄中L-氨基酸含量的估算。
