组成图示
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传感器类型
电化学生物传感器
检测对象
对氯酚（p-chlorophenol, p-CP）为主，兼及邻/间氯酚、二氯酚及甲酚等酚类化合物；样品基质：水样/标准溶液（0.1 mol/L 磷酸盐缓冲液，pH 7.0）
检测原理
TYR具有酚酶和儿茶酚酶双重活性。p-氯酚等酚类进入CF多孔电极后，被吸附TYR催化氧化为邻醌；邻醌在-50 mV下发生电化学还原生成儿茶酚，儿茶酚又被TYR反复氧化为邻醌，形成酶-电催化循环，使电子转移次数增加，阴峰电流随酚类浓度升高而增大。AO与TYR共吸附可稳定酶构象，CF的高比表面积和低扩散阻力促进传质，从而获得高灵敏度安培响应。
检测灵敏度
LOD: 2.13×10−8 mol/L (S/N = 3)；线性范围: 0.1–10 μmol/L；灵敏度: 1.41(mA·L)/mmol；采样率: 30 samples/h
效应效果
传感器对p-氯酚选择性最高，30 μmol/L下相对响应为100%，p-甲酚47%，m-氯酚1.3%，o-氯酚0.23%，2,3-二氯酚0.28%，3,4-二氯酚0.22%，2,6-二氯酚0.17%，2,4-二氯酚0.09%。峰电流响应高度可重复，采样率约30样品/h。在含0.2 mmol/L AO的磷酸盐缓冲液中4°C保存1个月保留约80%初始活性；无AO时20天损失99%。未报告RSD、实际样品回收率及与色谱/免疫方法对比。作者认为方法简单、灵敏，可用于生物传感器、生物反应器和生物燃料电池。
传感器的构成
- 基底/换能器电极：碳毡（CF），由约7 μm微碳纤维构成三维多孔微电极集合，提供高比表面积、导电性和低扩散阻力，作为流动检测工作电极。
- 识别/催化元件：酪氨酸酶（TYR, EC 1.14.18.1），物理吸附于CF表面，催化酚类氧化生成邻醌，并反复氧化儿茶酚，实现识别与信号放大。
- 稳定/保护剂：吖啶橙（AO），与TYR从混合水溶液共吸附于CF，防止TYR在疏水CF表面变性并维持催化活性。
- 参比电极：Ag/AgCl（3 mol/L NaCl），用于三电极体系电位参考。
- 对电极：铂丝（直径1 mm），用于完成电化学回路。
- 流动注射系统：双柱塞泵（DMX-2000T）与六通进样阀，以0.1 mol/L磷酸盐缓冲液（pH 7.0）为3.0 mL/min载体，注入200 μL样品。
- 信号读出：电化学分析仪（CHI 6122A），在-50 mV vs Ag/AgCl下安培检测邻醌还原阴峰电流。
中文摘要
将酪氨酸酶（TYR, EC 1.14.18.1）与吖啶橙（AO）共同物理吸附于碳毡（CF）表面，AO的共吸附对防止TYR在CF表面变性至关重要。所得TYR/AO-CF被用作新型高灵敏度安培流动生物传感器的检测单元，用于检测有毒氯酚化合物。注入200 μL酚类标准溶液后，在-50 mV（vs Ag/AgCl）下检测由TYR催化氧化（酚酶活性）产生的邻醌还原电流所形成的阴峰。在该反应中，由邻醌电化学生成的儿茶酚化合物又被TYR的儿茶酚酶活性反复氧化，从而实现充分信号放大。TYR/AO-CF对p-氯酚的选择性明显高于其他氯酚化合物。当以0.1 mol/L磷酸盐缓冲液（pH 7.0）为3.0 mL/min载体时，p-氯酚阴峰在0.1–10 μmol/L范围内线性（灵敏度：1.41 (mA·L)/mmol），采样率为30样品/h，检出限为2.13×10−8 mol/L（S/N=3）。在含0.2 mmol/L AO的0.1 mol/L磷酸盐缓冲液（pH 7.0）中4°C保存后，TYR/AO-CF保留80%以上初始活性。
英文摘要
Tyrosinase (TYR, EC 1.14.18.1) was physically adsorbed onto a carbon felt (CF) together with acridine orange (AO). Coadsorption of AO was essential to prevent the denaturation of the TYR at the CF surface. The resulting TYR and AO-coadsorbed CF (TYR/AO-CF) was successfully utilized as a detection unit of novel and highly sensitive amperometric flow-biosensor for toxic chlorophenol compounds. Standard solutions of phenolic compounds (200 μL) were injected, and the cathodic peak currents due to the reduction current of o-quinones produced by the TYR-catalyzed oxidation (phenolase activity) were detected at the applied potential of -50 mV vs. Ag/AgCl. In this reaction, the electrochemically generated catechol compounds from o-quinones are re-oxidized repeatedly by catecholase activity of the TYR, leading to a sufficient amplified signal. The TYR/AO-CF exhibited much higher selectivity toward p-chlorophenol as compared with other chlorophenol compounds. When 0.1 mol/L phosphate buffer (pH 7.0) was used as a carrier at flow rate of 3.0 mL/min, cathodic peaks for p-chlorophenol was linear in the concentration range between 0.1 and 10 μmol/L (sensitivity: 1.41(mA·L)/mmol) with sampling rate (30 samples/h), and the detection limit of p-chlorophenol was found to be 2.13 × 10(8) mol/L (S/N = 3. The ratio of signal and noise is 3). The TYR/AO-CF kept more than 80% of original activity after the storage in 0.1 mol/L phosphate buffer (pH 7.0) containing 0.2 mmol/L AO at 4°C.