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研究生:曾貴聖
研究生(外文):Tseng Kuei-Sheng
論文名稱:鐵氰化銦修飾電極感測半胱胺酸反應機制之研究
論文名稱(外文):A Study of the Detection Mechanism of Cysteine on Indium Hexacyanoferrate Modified Electrode
指導教授:何國川
指導教授(外文):Ho Kuo-Chuan
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:201
中文關鍵詞:吸附半胱胺酸電化學感測電化學石英震盪微量天平鐵氰化銦
外文關鍵詞:adsorptioncysteineelectrochemical detectionEQCMInHCF
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本研究主要探討利用鐵氰化銦(indium hexacyanoferrate,簡稱InHCF)修飾電極電催化半胱胺酸(cysteine,簡稱Cys)之反應機制以及其在Cys氧化感測之應用。在本研究中以循環伏安法,分別於玻璃碳電極(glassy carbon,簡稱GC)以及白金電極上,製備InHCF修飾電極。在感測Cys之前,本研究首先探討InHCF修飾電極在水溶液系統中老化之因素,以增進電極之穩定度。本研究中使用循環伏安以及定電位兩種電化學方法感測Cys,並且配合電化學石英震盪微量天平(EQCM)同步觀察Cys以及其氧化產物胱胺酸(cystine)在InHCF修飾電極表面之吸、脫附行為。最後根據循環伏安、定電流、以及EQCM之實驗結果推導出感測模型,並與實驗結果相互印證。本研究之主要成果將分別說明如下。
由EQCM之實驗結果顯示,在水溶液中加入In3+離子,可以有效的抑制氧化態InHCF薄膜之溶解反應,並大幅地提升InHCF修飾電極之穩定性。以循環伏安方式感測Cys時,電位之掃瞄範圍為0.6 ~ 0.95 V(vs. Ag/AgCl),掃瞄速率為50 mV/sec。電位0.95 V處之氧化電流對Cys濃度之線性感測範圍為50μM ~ 1 mM。以定電位方式感測Cys時,感測電位為1.2 V (vs. Ag/AgCl),感測之取樣時間為30秒,感測之線性範圍為100μM ~ 1 mM。感測再現性之實驗結果顯示,循環伏安法以及定電位法皆為可逆之感測過程,其感測電流之相對標準偏差分別為3.95 %以及2.79 %。本研究中,兩種電化學感測法其感測線性範圍都包含了Cys在人體內之正常濃度範圍(152.8 ~ 378.0μM)。
由結合EQCM之同步電化學實驗結果顯示,Cys在進行氧化反應之前,會吸附於氧化態之InHCF薄膜上;但不會吸附於還原態之InHCF薄膜上。Cys於氧化態InHCF薄膜上之吸附等溫線形式,與Langmuir isotherm方程式相符,其吸附平衡常數KA = 4.48×105 (cm3/mol)。Cys於InHCF修飾電極表面上進行氧化反應後,生成之cystine不但能夠吸附在氧化態InHCF薄膜上,也能吸附於還原態之InHCF薄膜上。吸附之cystine,會對InHCF氧化過程中遷出晶格之K+構成立體障礙,影響InHCF之電化學行為,並造成在連續感測Cys之過程中,InHCF修飾電極電量逐漸地衰減。在含有In3+之溶液中進行循環伏安掃瞄時,能夠將吸附之cystine還原為Cys,進而脫離還原態InHCF薄膜表面。推測其反應之機制為In3+還原成In2+(電位約 —0.8 V)後,催化吸附於電極表面之cystine的還原反應。在還原吸附於InHCF修飾電極上之cystine後,薄膜之電量有明顯的回復。
本研究藉由反應時,表面反應阻力與質傳阻力串連之概念,結合反應控制以及質傳控制之情形,推導定電位感測模型,並求出感測電流與感測濃度以及時間之關係式。由感測模型配合電化學感測以及EQCM兩方面之實驗數據,求出Cys在InHCF表面上之反應速率常數以及在溶液中之質傳速率常數的比值(θ0)為1.72。由此可知在定電位感測過程中,Cys於InHCF修飾電極上為反應控制以及質傳控制兩者混合之反應機制。由理論模型求出Cys之吸附平衡常數KA為4.86×105(cm3/mol),此結果與之前由吸附等溫線實驗所求出之KA相當接近。由理論模型模擬InHCF修飾電極感測Cys時,電流與感測濃度以及電流與時間之關係,結果顯示理論模型與實驗數據具有合理之一致性。
綜合上述研究成果可知,本論文不僅有助於瞭解Cys於普魯士藍類似物(Prussian blue analog,簡稱PBA)修飾電極上之反應機制,也為建立化學修飾電極感測生化物質之感測理論模式提供了有利的線索。有關提升InHCF修飾電極於水溶液系統中之穩定性方面的研究,相信對PBA修飾電極在水溶液系統中的應用有所幫助。
關鍵字:吸附、半胱胺酸、電化學感測、電化學石英震盪微量天平、
鐵氰化銦
The detection of cysteine (Cys) at the indium hexacyanoferrate (InHCF) modified electrode and the mechanism of Cys’s oxidative electrocatalysis were researched in this study. The InHCF modified electrode was prepared by the cyclic voltammetric (CV) deposition. During the deposition, an InHCF thin film was grown on either a glassy carbon (GC) electrode or a Pt electrode. Before performing the Cys detection, the stability of the InHCF modified electrode was improved and assured in aqueous solution. Then, the Cys detection at the InHCF modified electrode was carried out using either the CV or amperometric method. Also, an electrochemical quartz crystal microbalance (EQCM) was employed to simultaneously monitor the adsorption/desorption behaviors of Cys and its oxidative product, cystine, on the InHCF surface. By comparing the results obtained from the voltammetric, amperometric, and EQCM experiments, a model for the Cys detection at InHCF was derived and compared with the experimental results. Major findings of this study are summarized in the following paragraphs.
It was found that the stability of the InHCF modified electrode in the aqueous solution could be improved upon adding In3+ in the solution. EQCM experiments verified that such an In3+ addition greatly inhibits an oxidized InHCF thin film’s decomposition and thus considerably stabilizes the InHCF modified electrode. By applying a dynamic potential from 0.6V to 0.95V (vs. Ag/AgCl) at a rate of 50 mV/s, Cys was detected voltammetrically at the InHCF modified electrode. The voltammetric response at 0.95V was linearly dependent on the Cys concentration (designated as [Cys]) when [Cys] was ranged from 50μM to 1 mM. In comparison, the amperometric detection was performed by the application of a static potential at 1.2 V, with the sampling time at 30s. The sampled current was linearly dependent on [Cys] when [Cys] was ranged from 100μM to 1 mM. Both voltammetric and amperometric detections were reversible and produced signals with relative standard deviations of 3.95% and 2.79%, respectively. Besides, both methods show a linear correlation between the current response and [Cys] within the normal concentration range of Cys in a healthy person (152.8 ~ 378.0μM).
On the basis of the in situ EQCM experiment, it was discovered that the adsorption of Cys on the oxidized InHCF film occurs before Cys oxidation, whereas the adsorption of Cys was not observed on the reduced InHCF film. The adsorption isotherm of Cys on the oxidized InHCF film, obtained from the EQCM experiment, obeyed the Langmuir behavior. The adsorption constant, KA, was calculated to be 4.48×105 (cm3/mol). In addition to Cys, the adsorption of cystine (the product from the Cys oxidation) on the InHCF film was also observed. Different from Cys, cystine could be adsorbed on both the oxidized and reduced InHCF surfaces. It was found that the adsorbed cystine molecules served as a steric hindrance for the removal of K+ from the InHCF lattice during the InHCF oxidation. As a result, the coulometric capacity of the InHCF modified electrode faded gradually during the continuous Cys detection. On the other hand, it was demonstrated that the adsorbed cystine molecules could be reduced voltammetrically to Cys and then desorbed from the reduced InHCF surface in the presence of In2+, which were produced from the reduction of In3+ at ca. —0.8 V vs. Ag/AgCl. Such an In2+-induced reduction of cystine was observed to rejuvenate the post-detected InHCF films.
Concerning the mathematical model for the amperometric detection, two limiting cases were derived at first: the case of surface-reaction control and that of diffusion control. Since the resistances of surface reaction and diffusion are in series, the general expression for the detected current, which is functions of [Cys] and time, could be modeled. By combining the model with the results obtained from the amperometric and EQCM experiments, the ratio of the surface reaction rate constant to the diffusion rate constant (denoted asθ0) was determined to be 1.72. This implies that the oxidative electrocatalysis of Cys at the InHCF modified electrode was under a mixed surface reaction/diffusion control during the amperometric detection. The adsorption constant, KA, determined from the model, was 4.86×105 (cm3/mol), which was very close to the value calculated from the Cys adsorption isotherm mentioned earlier. In addition to the satisfactory fitness of the current-concentration curve, the model also predicts the current-time data well.
With the above findings, this thesis not only depicts a better picture for the oxidative electrocatalysis of Cys at a Prussian blue analog (PBA) modified electrode but also presents a general kinetic model for the amperometric detection of biosubstances at a chemically modified electrode. Furthermore, the InHCF stabilizing approach itself can be used in other PBA systems and will possibly promote the applications of PBA electrodes in the relevant fields, which add value to this study.
Keywords: Adsorption, cysteine, electrochemical detection,
EQCM, InHCF
中文摘要………………………………………………………………………I英文摘要…………………………………………………………………….IV致謝…………………………………………………………………………VII目錄……………………………………….………………………….….VIII表目錄………………………………………….……………………….….XI圖目錄………………………………………………………………………XII符號說明…………………………………………………………………XVIII
第一章 緒言………………………………………………………………….1
1-1 生化感測技術之簡介…………………………………………….1
1-2 感測器原理以及電化學感測器簡介…………………………….4
1-3 感測半胱胺酸之重要性及其應用……………………………….8
1-4 普魯士藍類似物之簡介以及其在生化感測上之應用………..15
1-5 研究動機與方向………………………………………………..23
1-6 研究架構………………………………………………………..26
第二章 原理…………………………………………………………………28
2-1 Michaelis — Menten 方程式……………………………………….28
2-2 InHCF修飾電極感測半胱胺酸之反應機制…………………………..33
2-3 電化學石英晶體微量天平(EQCM)之操作原理……………………...35
第三章 實驗設備與方法…………………………………………………..38
3-1 實驗儀器設備………………………………………………………….38
3-2 實驗藥品………………………. ……………………. …………….39
3-3 Cys之電化學分析………………………………………………………40
3-4 Cys在InHCF修飾電極上之反應機制………………………………….42
第四章 利用InHCF修飾電極感測Cys…..…………………………………47
4-1 InHCF修飾電極之製備…………………………………………………47
4-1-1 鍍液穩定性之提升……………………………………………….47
4-1-2 InHCF薄膜之電化學析鍍…………………………………………49
4-2 InHCF修飾電極在水溶液系統中穩定性之提升………………………52
4-2-1 物理性固定法--在電極表面塗佈Nafion®..................52
4-2-2 化學性固定法─抑制薄膜之溶解反應.…………………………55
4-3 InHCF修飾電極在Cys溶液中之循環伏安行為……………………….64
4-3-1 Cys濃度對循環伏安圖形的影響….…………………………….64
4-3-2 電位掃瞄範圍以及Cys濃度對電極老化的影響…………………71
4-3-3 掃瞄速率的影響………………………………………………….75
4-4 InHCF修飾電極在Cys溶液中之定電位行為………………………….77
4-4-1電流-電位關係圖………………………………………………….77
4-4-2 感測取樣時間之決定…………………………………….80
4-5 Cys之電化學感測結果………………..………………………………84
4-5-1感測電流-濃度曲線 (感測檢量線)…………...……………….84
4-5-2 感測之再現性……………………………………...……………91
第五章 Cys電化學感測機制與感測理論模型…………………………….95
5-1 Cys於InHCF修飾電極上之吸附之行為………………………….…..95
5-1-1 Cys在氧化/還原態InHCF修飾電極上之吸附…………………....96
5-1-2 以階梯電位法觀察Cys之吸附…………………………………….100
5-2 Cystine在InHCF薄膜表面之電化學行為………………………….104
5-2-1 Cystine之吸附行為……………………………………….…..104
5-2-2 Cystine之還原反應…………………………………………….107
5-2-3 In3+/In2+之催化效應………………………………………….116
5-2-4 Cystine還原反應對電極活性的影響………………………….118
5-3 Cys於InHCF修飾電極上之吸附等溫線……………………………..123
5-4 Cys於InHCF修飾電極上之感測理論模型…………………………..131
5-4-1 感測理論模型之推導……………………………………….….132
5-4-2 感測理論模型與實驗結果之比較…………………………..…142
5-4-3 感測理論模型與Michaelis — Menten方程式之比較……….154
5-5以感測理論模型模擬感測電流值…………………………………….158
5-5-1感測電流-濃度曲線……………………………………………..158
5-5-2感測電流-時間曲線……………………………………………..161
5-5-3感測電流-電位曲線……………………………………………..164
第六章 結論與建議……….………………………………………………169
6-1 結論……………………………………………………………………169
6-2 建議……………………………………………………………………173
第七章 參考文獻………………………………………………………….174
附錄A InHCF鍍液穩定度之提升………………………………………...178
附錄B 薄膜穩定性提升之原始CV數據…………….…………………...194
附錄C 定電位感測下擴散控制與表面反應控制之探討…………………199
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