School of Chemistry and Chemical Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Biosensors (Basel). 2024 Aug 21;14(8):403. doi: 10.3390/bios14080403.
The convenient and sensitive detection of metabolites is of great significance for understanding human health status and drug development. Solid-phase electrochemiluminescence (ECL) enzyme electrodes show great potential in metabolite detection based on the enzyme-catalyzed reaction product hydrogen peroxide (HO). Herein, a solid-phase ECL enzyme sensor was fabricated based on a confined emitter and an immobilized enzyme using electrostatic nanocage array, constructing a platform for the sensitive detection of cholesterol. The electrostatic cage nanochannel consists of a bipolar and bilayer vertically aligned mesoporous silica film (bp-VMSF). The upper layer of bp-VMSF is an amino-modified, positively charged VMSF (p-VMSF), and the lower layer is a negatively charged VMSF (n-VMSF). The most commonly used ECL probe tris(bipyridine)ruthenium(II) (Ru(bpy)) is fixed in n-VMSF by electrostatic adsorption from n-VMSF and electrostatic repulsion from the upper p-VMSF, generating significantly enhanced and stable ECL signals. The successful preparation of the electrostatic cage was characterized by scanning electron microscopy (SEM) and electrochemical methods. After amino groups on the outer surface of bp-VMSF were derivatized with aldehyde, cholesterol oxidase (ChOx) molecules were covalently immobilized. The successful construction of the enzyme electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). When the corresponding enzyme substrate, cholesterol, was present in the solution, the ECL signal of Ru(bpy) was quenched by the enzyme-catalyzed reaction product HO, enabling the high-sensitivity detection of cholesterol. The linear range for detecting cholesterol was from 0.05 mM to 5.0 mM, with a limit of detection (LOD) of 1.5 μM.
基于酶催化反应产物过氧化氢 (HO)，固相亲水电化学发光 (ECL) 酶电极在代谢物检测方面显示出巨大的潜力。本文通过静电纳米笼阵列构建了一种基于受限发射体和固定化酶的固相亲水 ECL 酶传感器，为胆固醇的灵敏检测构建了一个平台。静电笼纳米通道由双极和双层垂直排列的介孔硅薄膜 (bp-VMSF) 组成。bp-VMSF 的上层是氨基修饰的带正电荷的 VMSF (p-VMSF)，下层是带负电荷的 VMSF (n-VMSF)。最常用的 ECL 探针三(联吡啶)钌(II) (Ru(bpy)) 通过静电吸附固定在 n-VMSF 中，同时受到来自上层 p-VMSF 的静电排斥，从而产生显著增强且稳定的 ECL 信号。通过扫描电子显微镜 (SEM) 和电化学方法对静电笼的成功制备进行了表征。在 bp-VMSF 的外表面的氨基被衍生化形成醛基后，胆固醇氧化酶 (ChOx) 分子被共价固定。通过循环伏安法 (CV) 和电化学阻抗谱 (EIS) 对酶电极的成功构建进行了表征。当溶液中存在相应的酶底物胆固醇时，酶催化反应产物 HO 会猝灭 Ru(bpy) 的 ECL 信号，从而实现胆固醇的高灵敏度检测。检测胆固醇的线性范围为 0.05 mM 至 5.0 mM，检测限 (LOD) 为 1.5 μM。
