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
甘油（glycerol），样品基质为葡萄酒（wines）
检测原理
两种传感器均通过酶促级联反应将甘油转化为可被TTF介导的电子信号。GDH/DP构型中，GDH催化甘油氧化为二羟基丙酮，同时NAD+还原为NADH；DP催化NADH被TTF+再氧化，生成TTF，TTF在+150 mV下于金电极上发生安培氧化，电流随甘油浓度增加。GK/GPOx/HRP构型中，GK在ATP和Mg2+存在下将甘油磷酸化为甘油-3-磷酸；GPOx以O2氧化甘油-3-磷酸生成二羟基丙酮磷酸和H2O2；HRP催化H2O2还原，TTF介导电子再生并形成TTF+，TTF+在0 mV下被还原产生电流。TTF作为电子介质降低工作电位，提高选择性；酶级联实现信号放大，稳态电流与甘油浓度成正比。
检测灵敏度
GDH/DP: LOD: 4.0 × 10−7 M；线性范围: 1.0 × 10−6 to 2.0 × 10−5 M (0.092–1.84 mg L−1)；灵敏度: 1214 ± 21 μA M−1；r = 0.999。GK/GPOx/HRP: LOD: 3.1 × 10−7 M；线性范围: 4.0 × 10−7 to 1.0 × 10−5 M (0.037–0.92 mg L−1)；灵敏度: 1460 ± 34 μA M−1；r = 0.999
效应效果
两种传感器重复性良好：校准斜率RSD为7.5%和7.0%，重复测量RSD为3.2%和4.9%，不同传感器RSD为11.7%和10.1%。GDH/DP使用51天后保留87%灵敏度，GK/GPOx/HRP 8天后保留46%。干扰试验中，葡萄糖、果糖、半乳糖、阿拉伯糖及多种有机酸无明显干扰，仅抗坏血酸有响应，但葡萄酒甘油浓度远高于抗坏血酸，实际影响可忽略。12种葡萄酒测定RSD均<10%，分析约3 min，短于商品酶试剂盒30 min；与R-Biopharm试剂盒比较，回归斜率0.95±0.07和1.02±0.07，r=0.995，无显著系统差异，且操作更简单、成本更低。
传感器的构成
- 基底/换能器电极：BAS金盘电极（AuE，Ø 3 mm），提供电子传导与安培检测
- 自组装单分子层：3-巯基丙酸（MPA）SAM修饰金电极，形成有序界面并锚定酶与介质
- 电子介质层：四硫富瓦烯（TTF）与酶共固定，介导酶反应电子转移并在电极上氧化/还原产生电流
- 双酶识别/催化层：甘油脱氢酶（GDH）与二氢吡啶核苷酸还原酶（DP），催化甘油氧化并传递NADH电子
- 三酶识别/催化层：甘油激酶（GK）、甘油-3-磷酸氧化酶（GPOx）与辣根过氧化物酶（HRP），级联催化甘油转化并产生H2O2
- 固定/限域膜：透析膜（10 K MWCO）覆盖电极表面，固定酶与TTF并限制干扰物
- 辅因子/电子受体：NAD+（GDH/DP）或ATP与Mg2+（GK/GPOx/HRP），参与酶促反应并维持催化循环
中文摘要
本文报道了用于测定甘油的集成安培生物传感器。作者评价了两种构型：甘油脱氢酶/二氢吡啶核苷酸还原酶（GDH/DP）双酶体系和甘油激酶/甘油-3-磷酸氧化酶/辣根过氧化物酶（GK/GPOx/HRP）三酶体系。酶与电子介质四硫富瓦烯（TTF）共同固定在3-巯基丙酸（MPA）自组装单分子层（SAM）修饰的金电极上，并用透析膜覆盖。双酶构型在+150 mV下通过TTF氧化监测反应，三酶构型在0 mV下通过TTF+还原监测反应。优化组成与条件后，两种传感器重复性良好且无需清洗。GDH/DP使用51天后保留87%灵敏度，GK/GPOx/HRP 8天后保留46%。甘油线性范围分别为1.0×10−6–2.0×10−5 M和1.0×10−6–1.0×10−5 M，灵敏度为1214±21和1460±34 μA M−1，检出限为4.0×10−7和3.1×10−7 M。传感器灵敏度高，葡萄酒分析中无明显干扰，并用于12种葡萄酒测定，结果与商品酶试剂盒比较具有优势。
英文摘要
The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150mV (vs. Ag/AgCl), and the reduction of TTF(+) at 0mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0x10(-6) to 2.0x10(-5) or 1.0x10(-6) to 1.0x10(-5)M glycerol and sensitivities of 1214+/-21 or 1460+/-34microAM(-1) were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0x10(-7) and 3.1x10(-7)M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.