Development of highly integrated enzyme switches as a multi-parameter sensing device

酶开关是一种新型的生物传感器件，它是在聚苯胺（PAn）膜表面涂敷酶的基础上，通过电导法测量其电导率而得到的。与传统的酶传感器相比，酶开关具有以下优点：1）传感芯片本身具有记忆功能; 2）响应与电极尺寸无关; 3）结构简单，易于小型化和集成化。4)由于电阻变化是指数，因此易于与执行器连接。在本研究中，这些优点通过使用制备的酶开关得到了验证。PAn通过氧化失去导电性，通过还原恢复导电性。如果用PAn制备氧化酶-过氧化物酶（HRP）共固定化电极，当生成的化合物I和II在PAn表面被还原时，PAn可能被氧化而失去导电性。通过监测PAn的电导率，可以构建“酶开关”，即通过特定底物的存在发出“关闭”信号。然后用戊二醛交联法将葡萄糖氧化酶（或乳酸氧化酶）和HRP固定在聚苯胺膜上。通过添加底物，可以观察到由于酶反应导致的PAn电导率的降低。通过施加相对于Ag/AgCl的0.1V，氧化的PAn恢复其导电性。在流动体系中，从“开”到“关”，或“关”到“开”的转换时间约为3 min，峰高与底物浓度相对应。所制备的酶开关兼具酶试纸条和酶传感器的优点。该酶反应过程中不需要外加电压，且反应可保持至少1 h，在多通道电极上构建了葡萄糖和乳酸的集成多通道酶开关。整合的酶开关显示出对葡萄糖或乳酸盐的独立响应，并且没有观察到化学串扰。

Enzyme switch is a novel biosensing device based on the conductometric measurements of polyaniline(PAn) film covered with enzymes. The enzyme switch might have following advantages over conventional enzyme sensors : 1)Sensing chip itself has a memory function, 2)response might be independent from the electrode size, 3)Miniaturization and integration are easy because of its simple structure. 4)Easy connection with actuators, since the resistance change is index. In this study, these advantages were verified by using prepared enzyme switches.PAn loses its electroconductivity by oxidation and recovers it by reduction. If the oxidase-peroxidase(HRP) co-immobilized electrodes are prepared with PAn, when the produced compounds I and II are reduced at the surface of PAn,PAn might be oxidized and may lose its conductivity. By monitoring conductivity of PAn, "enzyme-switch", which gives "off" signal by the existence of specific substrate, might be constructed.PAn film was prepared electrochemically over the two micro-patterned thin film electrodes under the acidic conditions. Then glucose oxidase(or lactate oxidase) and HRP were immobilized onto the PAn film by glutaraldehyde crosslinking method. By the addition of substrates, the decrease of PAn conductivity due to the enzyme reaction could be observed. The oxidized PAn recovered its conductivity by applying 0.1 V vs.Ag/AgCl. In flow system, it took approximately 3 min for switching from "on" to "off", or "off" to "on", and peak heights were correspond to substrate concentration. The prepared enzyme switch had advantages both as enzyme test strips and enzyme sensors. Voltage application was not necessary during the enzyme reaction, and the response could be preserved for at least 1h.An integrated multi-channel enzyme switch for glucose and lactate was constructed onto the multi-channel electrodes. The integrated enzyme switch showed responses to glucose or lactate independently and the chemical cross-talk was not observed.