组成图示
示意图生成中
传感器类型
电化学生物传感器
检测对象
胆固醇（cholesterol, Chol）；样品基质：合成血清、人血清参考样品（Accutrol，含胆固醇酯需胆固醇酯酶预处理）
检测原理
胆固醇在 ChOx 催化下与溶解氧反应生成 4-胆甾烯-3-酮和 H2O2。H2O2 扩散至 PB 层，在 −50 mV 下被 PB 电催化还原，产生与 H2O2 浓度相关的阴极电流；由于酶反应动力学控制，电流随胆固醇浓度增加而增大并符合 Michaelis–Menten 行为。PPy 层包埋 ChOx，提供酶微环境并初步排斥干扰物；外层 Nf 通过电荷/尺寸排斥屏蔽抗坏血酸、尿酸、对乙酰氨基酚等阴离子干扰，同时保护酶。SAM 改善 PB 在 Pt 表面的附着与电荷转移，提高信号稳定性。
检测灵敏度
Pt/PB/PPy-ChOx（−50 mV）: LOD: 0.008 mM；线性范围: 0.025–0.300 mM；灵敏度: 3253 nA mM−1 cm−2；r > 0.9990
Pt/SAM/PB/PPy-ChOx（−50 mV）: LOD: 0.008 mM；线性范围: 0.025–0.300 mM；灵敏度: 5714 nA mM−1 cm−2；r > 0.9990
Pt/SAM/PB/PPy-ChOx/Nf（−50 mV）: LOD: 0.012 mM（表3）/8 μM（摘要）；线性范围: 0.050–0.300 mM；灵敏度: 8572 nA mM−1 cm−2；r > 0.9990；摘要灵敏度范围: 600–8500 nA mM−1 cm−2
效应效果
三种构型在 −50 mV 下灵敏度较高，Nf 构型最高（8572 nA mM−1 cm−2），寿命约 25 天（730 次进样）；无 SAM 构型仅 9–10 天，含 SAM 构型超过 2 周并在 17–20 天后下降。重现性 CV 为 2.3–3.8%（0.20 mM 胆固醇）。PPy 与 Nf 显著抑制 AA、UA、对乙酰氨基酚等干扰。合成血清（3.98 mM 胆固醇，含 50 mM AA、0.50 mM UA、0.01 mM Pmol、5 mM Gl、0.8 g/100 mL BSA）相对误差 1.2%–5.05%；Accutrol 人血清参考样品（3.96 mM 总胆固醇，经胆固醇酯酶水解）与认证值一致。作者认为可用于临床血清胆固醇检测。
传感器的构成
- 基底/工作电极：Pt 电极，导电基底与换能器
- 自组装单分子层：1-propanethiol（C3 mercaptane）SAM，改善 PB 附着与电荷转移
- 普鲁士蓝层：Prussian-Blue（PB），电催化还原 H2O2
- 聚吡咯层：polypyrrole（PPy），固定酶并允许胆固醇扩散
- Nafion 保护层：Nafion（Nf），屏蔽阴离子干扰并保护酶
中文摘要
本文构建了三种基于铂电极上普鲁士蓝（PB）层电催化检测过氧化氢的安培式胆固醇生物传感器。胆固醇氧化酶（ChOx）包埋于电聚合到 PB 膜上的聚吡咯（PPy）层中。研究比较了 Pt/PB/PPy-ChOx、Pt/SAM/PB/PPy-ChOx 和 Pt/SAM/PB/PPy-ChOx/Nf 三种构型，考察铂表面自组装单分子层（SAM）对 PB 层附着与稳定性的影响，以及外层 Nafion（Nf）对选择性的改善。三种传感器灵敏度为 600–8500 nA mM−1 cm−2，并能在参考血清和合成血清样品中测定胆固醇。Pt/SAM/PB/PPy-ChOx/Nf 的检出限为 8 μM。SAM 和 Nf 层显著提高了稳定性与寿命，PB 层保留更久，Nf 保护酶免受流动相影响，寿命可达 25 天。SAM 还影响电荷转移和 PB 层形成。
英文摘要
Three cholesterol biosensor configurations based on the formation of a layer of Prussian-Blue (PB) on a Pt electrode for the electrocatalytic detection of the H(2)O(2) generated during the enzymatic reaction of cholesterol with cholesterol oxidase (ChOx) were constructed. The enzyme was entrapped within a polypyrrole (PPy) layer electropolymerized onto the PB film. The influence of the formation of self-assembled monolayers (SAMs) on the Pt surface on the adherence and stability of the PB layer and the formation of an outer layer of nafion (Nf) as a means of improving selectivity were both studied. A comparative study was made of the analytical properties of the biosensors corresponding to the three configurations named: Pt/PB/PPy-ChOx, Pt/SAM/PB/PPy-ChOx and Pt/SAM/PB/PPy-ChOx/Nf. The sensitivity (from 600 to 8500nAmM(-1)cm(-2)) and selectivity of the developed biosensors permitted the determination of the cholesterol content in reference and synthetic serum samples. The detection limit for the Pt/SAM/PB/PPy-ChOx/Nf biosensor was 8muM. Formation of the SAM on the electrode surface and covering with a Nf film considerably improved the stability and lifetime of the biosensor based on the catalytic effect of the PB layer (as the PB layer was retained longer on the electrode), and the Nf layer protects the enzyme from the external flowing solutions. Lifetime is up to 25 days of use. The formation of the SAM also has an effect on the charge transfer and the formation of the PB layer.