In this study, we demonstrate the first report of bienzyme nanoparticles (BENP)–based cholesterol electrochemical sensor. The developed BENP consists of cholesterol oxidase (ChOx) and horseradish peroxidase (HRP), and these enzymes were crosslinked by glutaraldehyde treatment. An alternative expression system of ChOx is also firstly introduced through this work by constructing its production system in Pichia pastoris exploiting ChOx expressing gene taken from Streptomyces sp. The obtained ChOx is then applied to synthesize BENP with HRP at different ratios to attain the best condition that can yield in enhanced enzymatic activity. To utilize the material for electrochemical sensors, the BENP is furtherly modified with cysteamine to graft thiol (-SH) group that facilitates BENP immobilization on gold electrode through AuS bond. The kinetic study of the BENP was conducted using cholesterol and H₂O₂, which are substrates of the BENP constituents, ChOx and HRP, respectively. Moreover, its characterizations, catalytic activity, specificity, and selectivity toward cholesterol also has been investigated. The developed BENP-based cholesterol electrochemical sensor exhibited a long dynamic range (0–675 mg/dL) with a low detection limit (0.18 mg/dL), high selectivity, and long-term stability (> 98% of its initial activity over 25 days).

在这项研究中，我们展示了基于双酶纳米颗粒 (BENP) 的胆固醇电化学传感器的第一份报告。开发的 BENP 由胆固醇氧化酶 (ChOx) 和辣根过氧化物酶 (HRP) 组成，这些酶通过戊二醛处理交联。ChOx 的替代表达系统也通过这项工作首次引入，通过利用从链霉菌中提取的 ChOx 表达基因在毕赤酵母中构建其生产系统sp. 然后将获得的 ChOx 用于合成具有不同比例的 HRP 的 BENP，以获得可产生增强酶活性的最佳条件。为了将这种材料用于电化学传感器，BENP 用半胱胺进一步修饰以接枝硫醇 (-SH) 基团，从而促进 BENP 通过 Au S 键固定在金电极上。使用胆固醇和 H ₂ O ₂进行 BENP 的动力学研究，分别是 BENP 成分 ChOx 和 HRP 的底物。此外，还研究了其对胆固醇的表征、催化活性、特异性和选择性。开发的基于 BENP 的胆固醇电化学传感器表现出长动态范围 (0–675 mg/dL)、低检测限 (0.18 mg/dL)、高选择性和长期稳定性（> 98% 的初始活性超过25 天）。