Selective Enzyme Immobilization Methods for the Preparation of Amperometric Microelectrode Array Glutamate Sensor

• Main Authors: Wen-chin Chan, 詹文進
• Other Authors: Tina T.-C. Tseng
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/11165633040939869081

碩士 === 國立臺灣科技大學 === 化學工程系 === 101 === In this study, the electrodeposited chitosan is used as the substrate for the enzyme adsorption for the selective immobilization of glutamate oxidase on the arrayed microelectrode surface in order to prepare implantable glutamate sensor probe. In the thesis, we compare figures of merit of glutamate sensors on the micromachined probes prepared by two different enzyme immobilization methods – adsorption with electrodeposited chitosan (Method 1) and crosslinking with glutaraldehyde crosslinker and the stabilizing agent bovine serum albumin (Method 2). The permselective films overoxidized polypyrrole and Nafion® were used for rejecting common interferents. Glutamate sensors prepared by Method 1 have response time ~1 sec, linear detection range 20-217 μM, and sensitivity 38.1 ± 5.4 nA/μM• cm2 (n = 9), detection limit 2.5 ± 1.2 μM, and storage stability that the sensor remains 70% of its initial activity after 9 days of storage and that prepared by Method 2 have response time 2~3 sec, linear detection range 20-352 μM, and sensitivity 95.0 ± 8.8 nA/μM• cm2 (n = 12), detection limit 6.5 ± 1.7 μM, and storage stability that the sensor remains 50% of its initial activity after 9 days of storage. Sensors prepared by both methods can reject common interferents (ascorbic acid and dopamine) very effectively; little interferent current was observed. In general, glutamate sensors prepared by Method 1 have higher storage stability and their performance is more reproducible due to smaller standard deviations of sensor sensitivities after continuous repeating operations; on the other hand, closely arrayed control sensors without the chitosan film showed no signal upon the addition of glutamate. Successful selective glutamate oxidase immobilization on closely packed microelectrodes is demonstrated. Glutamate sensors prepared by Method 2 have higher sensor sensitivity and selectivity; however, its slower response time may due to the thicker enzyme layer prepared by Method 2. This glutamate sensor (prepared by Method 2) was implanted into the rat’s brain for real-time monitoring the release of glutamate after giving the pain stimulation (preliminary data will be shown).