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
乙醇（ethanol, EtOH，实际饮料样品，稀释10倍 PBS pH 8.8）、NADH（NADH，PBS 缓冲液）
检测原理
该传感器基于酶促反应与电催化介体再生。乙醇脱氢酶（ADH）催化乙醇氧化，同时NAD+被还原为NADH；生成的NADH扩散至MWCNTs/IL/Ox-P(NAD+)修饰电极表面，被电生NAD+氧化产物（Ox-P(NAD+)，含醌二亚胺等氧化还原活性物种）电催化氧化，并再生NAD+。MWCNTs提供导电通道，离子液体分散MWCNTs并稳定界面，促进电子转移。在+0.05 V低电位下，NADH氧化产生安培电流；由于表面NAD+和Ox-P(NAD+)浓度基本恒定，电流随乙醇浓度增加而增加。低电位和介体再生循环减少电极污染并提高选择性。
检测灵敏度
NADH: LOD: 2 × 10−8 mol L−1；线性范围: 2 × 10−7–2 × 10−5 mol L−1，R^2 = 0.9926，灵敏度: 0.44 (±0.02) A mol−1 L cm−2；2 × 10−5–4.2 × 10−5 mol L−1，R^2 = 0.9905，灵敏度: 0.2 (±0.01) A mol−1 L cm−2；CV催化电流线性范围: 5 × 10−5–3 × 10−4 mol L−1，R^2 = 0.9981，方程: Ip(µA) = 2.4214[NADH](×10−3 mol L−1) + 0.0202 µA。乙醇: LOD: 5 × 10−7 mol L−1；线性范围: 5 × 10−6–6 × 10−5 mol L−1，方程: I (nA) = 1.7 [EtOH](×10−6 mol L−1) + 8.3 nA，R^2 = 0.9981，灵敏度: 13.1 mA mol−1 L cm−2；6 × 10−5–9 × 10−4 mol L−1，方程: I (nA) = 1.0 [EtOH](×10−6 mol L−1) + 77.2 nA，R^2 = 0.9948，灵敏度: 7.7 mA mol−1 L cm−2。
效应效果
该传感器抗干扰能力强：1×10−4 mol L−1的抗坏血酸、尿酸、葡萄糖和对乙酰氨基酚几乎不干扰；尿酸、葡萄糖和对乙酰氨基酚达乙醇100倍时响应仍可忽略，仅抗坏血酸高于1×10−4 mol L−1时产生可比响应。稳定性好：Ox-P(NAD+)经250次循环峰电流仅降10%，室温空气保存4周保留95%活性；NADH安培响应1800 s仅降15%，100次连续测量无变化，保存15天降10%。实际饮料样品加标回收率为97.20%–98.10%，偏差2.40%–2.90%。与文献乙醇生物传感器相比，其10 s响应、0.10 V低电位和较宽动态范围具有优势，适合脱氢酶型安培生物传感器。
传感器的构成
- 基底/换能器电极：玻璃碳电极（GC electrode），抛光后作为工作电极，提供电子转移界面
- 纳米材料修饰层：多壁碳纳米管（MWCNTs）与N-丁基-N-甲基吡咯烷鎓双(三氟甲磺酰)亚胺离子液体（[C4mpyr][NTf2], IL）混合涂覆，形成导电、稳定、低背景电流的纳米复合膜
- 电催化介体层：电生NAD+氧化产物（Ox-P(NAD+)），由NAD+在MWCNTs/IL表面电位循环氧化生成并吸附，作为氧化还原介体催化NADH氧化
- 酶促反应组分：乙醇脱氢酶（ADH, EC 1.1.1.1）与辅因子NAD+，在溶液中催化乙醇氧化并生成NADH
- 信号转换元件：NADH作为酶促产物，被Ox-P(NAD+)电催化氧化再生NAD+，产生与乙醇浓度相关的安培电流
- 信号读出：三电极体系（工作电极MWCNTs/IL/Ox-P(NAD+)/GC、对电极Pt丝、参比Ag/AgCl/KCl 3 M），通过循环伏安和安培法读取电流
中文摘要
本文报道了一种基于多壁碳纳米管/N-丁基-N-甲基吡咯烷鎓双(三氟甲磺酰)亚胺离子液体（MWCNTs/[C4mpyr][NTf2]）修饰玻璃碳电极的电化学生物传感器。通过电位循环将NAD+氧化产物（Ox-P(NAD+)）固定在MWCNTs/IL纳米复合层表面，形成稳定的氧化还原活性介体。该修饰电极可在0.05 V（vs. Ag/AgCl）低电位下高效电催化NADH氧化，较裸玻璃碳电极降低约600 mV过电位，并显著减少电极污染。安培法检测NADH的检出限为2×10−8 mol L−1，线性范围可达4.2×10−5 mol L−1。以乙醇脱氢酶（ADH）为模型酶，该体系用于乙醇检测，在5×10−6–6×10−5和6×10−5–9×10−4 mol L−1范围内呈线性响应，检出限为5×10−7 mol L−1，响应时间约10 s。抗坏血酸、尿酸、葡萄糖和对乙酰氨基酚等干扰可忽略，并成功用于实际饮料样品中乙醇的检测。
英文摘要
The multiwalled carbon nanotubes/N-butyl-N-methyl-pyrolydinium-bis(trifluoromethylsulfonyl)imide [C(4)mpyr][NTf(2)] ionic liquid (MWCNTs/IL) modified glassy carbon (GC) electrode has been utilized as a platform to immobilize electrogenerated NAD(+) oxidation products (Ox-P(NAD(+))). During potential cycling, the adenine moiety of NAD(+) molecule is oxidized and gives rise to generation of a redox active system that shows great electrocatalytic activity toward NADH oxidation. The cyclic voltammetric results indicated the ability of MWCNTs/IL/Ox-P(NAD(+)) modified GC electrode to catalyze the oxidation of NADH at a very low potential (0.05 V vs. Ag/AgCl) and subsequently, a substantial decrease in the overpotential by about 600 mV compared with the bare GC electrode. This modified electrode thus allowed highly sensitive amperometric detection of NADH with a very low limit of detection (2 × 10(-8) mol L(-1)), low applied potential (+0.05 V) at concentration range up to 4.2 × 10(-5) mol L(-1) and minimum of surface fouling. High ability of MWCNTs/IL/Ox-P(NAD(+)) to promote electron transfer between NADH and the electrode suggested a new promising biocompatible platform for development of dehydrogenase-based amperometric biosensors. With alcohol dehydrogenase (ADH) as a model enzyme, ethanol sensing ability of the proposed system was examined. The amperometric response of the biosensor increased linearly with increasing ethanol concentration in two concentration ranges, 5 × 10(-6)-6 × 10(-5) and 6 × 10(-5)-9 × 10(-4) mol L(-1) with detection limit of 5 × 10(-7) mol L(-1) and rapid response of 10s. Furthermore, the interference effects of redox active species, such as ascorbic acid, uric acid, glucose and acetaminophen for the proposed biosensor are negligible. Finally, the ability of the proposed biosensor for detection of ethanol in real complex samples was successfully demonstrated.