植入應用之無氧葡萄糖感測技術探討

Studies of oxygen independent glucose sensing technologies used for implant applications

曾達峯(Ta-Feng Tseng)

指導教授 ： 婁世亮

中原大學/工學院/生物醫學工程研究所/博士(2011年)

https://doi.org/10.6840/cycu201101116

摘要

本研究旨在發展植入式無氧葡萄糖感測器之關鍵技術，藉無氧/無酵素電極和無氧/酵素電極做為研發平台，所探討的技術則以電極介質修飾、電極加熱和電極電位處理(potential treatment, PT)為主，其中電極電位處理是本研究之獨特創意技術。細胞實驗是用以探討電位處理及電極材料對於肌肉細胞(C2C12)生長的影響，以資植入生物體可行性之考量。無氧/無酵素電極的探討，是以開路電位法分析各種電極材料如白金(Pt)、黃金(Au)、玻璃碳(GC)及石墨(GP)於電位處理後之效應，結果顯示，電位處理有增益電極氧化或還原反應之進行。修飾有含鉻黃血鹽(CrHCF)之白金電極(CrHCF/Pt)是以安培法獲得其最佳操作電位及電位處理時間，並比較電位處理及電極加熱前後對於葡萄糖的反應訊號，結果顯示，電位處理及電極加熱後之葡萄糖訊號可有518%的增益。無氧/酵素電極的探討是以CrHCF/Pt為基底，再修飾以Nafion®和葡萄糖去氫酶修飾於白金電極上。由於本研究使用含鉻黃血鹽為中間介質，其價態原為還原態，吾人以X-射線光電子能譜(XPS)確認該電極在經電位處理後之價態是由還原態轉變為氧化態。最佳參數探討則是著重於線性濃度範圍、氧氣的干擾及穩定度，其結果顯示，電極線性範圍可達20 mM (R = 0.9923)、Km = 25.9 mM，並且於氧氣飽和狀況下無任何氧氣干擾情況；電極在保存272天後，酵素仍保有88 %的活性。細胞實驗是以裸白金電極、修飾白金電極及修飾白金生物電極三種材料探討電位處理前、後對C2C12細胞生長的影響，除了MTT測試細胞存活率，並以LDH含量試驗、TUNEL試驗及細胞週期分析佐證細胞死亡方式，結果顯示，裸白金電極、修飾白金電極及修飾白金生物電極三種材料不具有毒性，但是，由AFM分析結果得知，修飾白金電極及修飾白金生物電極之電極表面粗糙度會造成細胞死亡，再由LDH含量試驗之結果顯示細胞是經壞死的途徑死亡。總言之，運用電位處理、中間介質修飾及電極加熱可有效提高偵測靈敏度，具有應用於植入式微電極系統之潛能。

關鍵字

葡萄糖去氫酶 ； 含鉻黃血鹽 ； 無氧式葡萄糖感測器 ； 電位處理

並列摘要

This dissertation aims to develop key technologies specifically for implantable oxygen-independent glucose sensors. Electrodes modified with and without enzyme were used in the studies. Intermediate coating on electrodes, heating on electrodes, and potential treatment on electrodes were the main technologies studied. In particular, the potential treatment technology is an innovation of this work. C2C12 cells were used to determine the possible negative effects that potential treatment and electrode substrates would have on cell growth. Future usage of potential treatment and electrode substrates in implant applications is contingent upon the results of the in vitro studies mentioned above. The open circuit potentiometry technique was used in the oxygen-independent non-enzyme electrode study to investigate potential treatment effects of various electrode substrates, including platinum (Pt), gold (Au), glassy carbon (GC), and graphite (GP). Results indicate that the proposed potential treatment method on the eletrode can enhance redox reactions. The study of mediator effect was based on a modified chromium hexacyanoferrate (CrHCF) Pt electrode. The optimal operational potential and potential treatment time length of the CrHCF/Pt electrode were determined via amperometry. Amperometric measurements of the electrode before and after the potential treatment and electrode heating assessed the glucose response enhancement of the electrodes. Results indicate that the potential treatment and electrode heating increased the glucose response by 518 %. The studies of the oxygen-independent enzyme glucose electrode were based on the CrHCF/Pt electrode modified with Nafion® and pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH). To assure that CrHCF will be able to accept the electrons from PQQ-GDH, CrHCF must be in oxidation stage. Because the initial form of CrHCF was in the reduction stage, the potential treatment effect of converting CrHCF from the reduction stage to the oxidation stage was confirmed by X-ray photoelectron spectroscopy (XPS). The characteristics of the bio-electrode (Nafion/PQQ-GDH/CrHCF/Pt), such as linearity, oxygen interference, and stability, were studied. Results show that the linearity feature of the bio-electrode was 20 mM (R = 0.9923). There was no signal response detected from the bio-electrode even when saturated oxygen was applied in the tested solution, and the residual activity of the bio-electrode did not fall below 88% after 272 days. In vitro study was to evaluate cell damage effect by cisplatin, which may be produced when a power voltage was given to platinum in a solution containing chloride and NH3. In this study, a bare Pt electrode, a CrHCF/Pt electrode, and a Nafion/PQQ-GDH/CrHCF/Pt bio-electrode were individually co-cultured with a cell line of C2C12 and then followed by potential treatment. The MTT assay was used to test the viability of the C2C12 cells. In addition, LDH assay, TUNEL assay, and cell cyclic assay were used to analyze cell death mechanism. Results show that the electrode materials of bare Pt, CrHCF/Pt and Nafion/PQQ-GDH/Nafion/CrHCF/Pt are not cytotoxic. It is the surface roughness, in particular the CrHCF/Pt and Nafion/PQQ-GDH/Nafion/CrHCF/Pt, from the AFM analysis leading C2C12 cells die. The LDH releases from the C2C12 cells co-cultured with the electrode materials are high enough to conclude that the cells death is through necrosis mode. In conclusion, the techniques of OPT, mediation and electrode heating can enhance glucose response sensitivity, hence have potential to be implemented in implantable micro-electrode systems.

並列關鍵字

Chromium hexacyanoferrate ； Oxygen-independent glucose sensor ； Glucose dehydrogenase ； Potential treatment

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