Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran; Young Research Society, Shahid Bahonar University of Kerman, Kerman, Iran.
Young Research Society, Shahid Bahonar University of Kerman, Kerman, Iran.
Bioelectrochemistry. 2019 Dec;130:107323. doi: 10.1016/j.bioelechem.2019.06.008. Epub 2019 Jun 26.
Appropriate enzyme immobilization on the electrode surface in order to access its active site has always been an important strategy for electrode modification. In this report, lactate dehydrogenase enzyme was appropriately immobilized on the glassy carbon electrode via hydrophobin (HFB1) and graphene oxide nanocomposite. The step-by-step modification was successfully confirmed by water contact analysis, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimum conditions, this biosensor demonstrated a detection limit of 8.69 nM and RSD of 4.3% and 3.6% (n = 5) for reproducibility and repeatability. The effect of scan rate on the oxidation behavior of NADH was investigated by cyclic voltammetry; and diffusion coefficient for NADH was estimated at 6.27 × 10 cm.s. The apparent Michaelis-Menten constant (K) was amperometrically determined and it was lower than K for the free enzyme. Also, the modified electrode represented good stability after nine days with 6% decrease in current. The proposed assay was successfully used in real sample-serum-analysis and the obtained recoveries were between 93% and 104.0%.
为了接近酶的活性位点，将合适的酶固定在电极表面上一直是电极修饰的重要策略。在本报告中，通过疏水蛋白（HFB1）和氧化石墨烯纳米复合材料将乳酸脱氢酶酶适当地固定在玻碳电极上。通过水接触分析、循环伏安法和电化学阻抗谱成功地证实了逐步修饰。在最佳条件下，该生物传感器的检测限为 8.69 nM，重现性和再现性的 RSD 分别为 4.3%和 3.6%（n=5）。通过循环伏安法研究了扫描速率对 NADH 氧化行为的影响；并估计 NADH 的扩散系数为 6.27×10 cm.s。通过安培法测定了表观米氏常数（K），其值低于游离酶的 K。此外，修饰后的电极在 9 天内具有 6%的电流下降，表现出良好的稳定性。该测定法成功地用于实际样品-血清分析，获得的回收率在 93%至 104.0%之间。
