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研究生:陳婉君
研究生(外文):Wan-Chun Chen
論文名稱:製備CdS-Chi/Gel、MWCNT及PLC/AuNPs修飾電極偵測DNA鹼基、藥物與大腸桿菌
論文名稱(外文):Fabrication of CdS-Chi/Gel, Multiwalled Carbon Nanotube and PLC/AuNPs Modified Electrode for Detection of DNA Base, Medicine and E. coli
指導教授:陳生明
口試委員:連萬福曾添文張聰慧張文雄
口試日期:2012-07-16
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:化學工程研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:90
中文關鍵詞:硫化鎘甲殼素凝膠鳥嘌呤腺嘌呤異煙肼奈米碳管奈米金顆粒聚L-半胱氨酸25-二羥基苯甲酸抗壞血酸大腸桿菌修飾電極
外文關鍵詞:Cadmium sulfide (CdS)Chitosan (Chi)Gelatine (Gel)Guanine (G)Adenine (A)Modified electrodesf-MWCNTIsoniazidGold nanoparticles (AuNPs)Poly-L-cysteine25-dihydroxybenzoic acid (25-DHBA)Ascorbic acid (AA)Escherichia coli (E. coli)
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第一部分:結合硫化鎘、甲殼素和凝膠的硫化鎘-甲殼素/凝膠(CdS-Chi/Gel)修飾電極,由於甲殼素帶正電荷所以可以和偏負電的硫化鎘均勻混合。而膠體有以下特點─高機械伸展力、非抗原生物聚合物以及對哺乳動物細胞毒性極低。CdS-Chi的親水性測試,測得的接觸角較小,顯示了其親水性較好。電化學阻抗譜(EIS)可探討氧化還原反應時的擴散效應以及關於電子轉移的動能資訊。此修飾電極的表面形態用SEM及AFM探討之,揭示了CdS-Chi和Gel平整塗佈於電極表面。微分脈衝伏安法(DPV)用來檢測分析物,不只可增加電催化電流的線性濃度範圍,也降低了測量時氧化反應產生的干擾過電位。CdS對分析物展現了極好的電催化活性,而Gel則增加了附著性及穩定性。在本篇論文中,CdS-Chi/Gel修飾電極可同時氧化偵測guanine和adenine。
第二部分:製備了以官能基化多層奈米碳管(f-MWCNT)修飾電極為主的電化學感測器偵測Isoniazid (ISN)。藉由酸化MWCNT及超音波震盪合成f-MWCNT。由電化學阻抗譜可知f-MWCNT有極小的電子轉移阻抗。在比較了一些奈米碳材

對ISN的電化學特性後,可知道f-MWCNT修飾電極對ISN的電流反應最大,比
MWCNT修飾電極大上7倍。在pH4的環境下,f-MWCNT電極對ISN在0.4V有一不可逆氧化波峰,可被用來做電分析用途。計時安培法實驗顯示了f-MWCNT修飾電極對ISN反應很快且有很好的線性偵測範圍,範圍為1~70 μM。f-MWCNT膜有很穩定的背景電流,不只對藥品樣本偵測有很好的表現,再現性也很好。
第三部分:利用金奈米顆粒再電聚合上L-cysteine形成PLC/AuNPs電極以用來偵測大腸桿菌,及放大訊號。PLC利用其硫醇基(-SH)與奈米金顆粒交互作用電聚合於電極上。PLC/AuNPs修飾電極的表面覆蓋率(Γ)比其他修飾電極大,為4.5 × 10-9 mol cm−2¬¬¬,可歸因為奈米材料的總體表面積比積體材料大。從SEM結果可知道,因為有奈米金粒子使得PLC更加的規則均勻。電化學阻抗譜可得知AuNPs非常適用於修飾電極,因為其可使導電度增加,電子轉移加快。PLC/AuNPs修飾電極在pH 1 ~ pH 11都非常穩定。在大腸桿菌代謝水楊酸的過程中,會產生多酚類成分,例如2,5-dihydroxybenzoic acid (2,5-DHBA),測量2,5-DHBA便可推得大腸桿菌濃度。本實驗使用循環伏安法及線性掃描伏安法(LSV)了解PLC/AuNPs修飾電極對2,5-DHBA及AA的電催化反應,可以觀察到此修飾電極能分開兩種成分的訊號。PLC/AuNPs修飾電極對2,5-DHBA的電流反應顯示了更高的電催化活性。我們也對大腸桿菌真實樣本的複合系統進行實驗。實驗結果顯示了PLC/AuNPs修飾電極對偵測真實樣本非常有效率。


Part I:The Cadmium sulfide (CdS) was combined with Chitosan (Chi) and Gelatine (Gel) to prepare CdS-Chi/Gel modified electrode. Chi with high positive charge density can homogenize CdS. Chitosan has the characteristic of high mechanical strength, non-antigenic biopolymer and low toxicity toward mammalian cells. Hydrophilicity test of CdS-Chi showed lower contact angle indicates that the sample had more hydrophilic on surface. Electrochemical impedance spectroscopy (EIS) applied diffusion coefficient values and some information about the kinetics of electron transfer during the redox reactions. Surface morphology of the modified electrode is investigated by using scanning electron microscopy (SEM) and atomic force microscopy (AFM), which

revealed that CdS-Chi and Gel were coated on electrode. Differential pulse voltammetry (DPVs) was used for the determination of analytes. DPVs not only increased the electrocatalytic current linear concentration range, also lowered the overpotential to oxidation the interferences in the measurements. The CdS exhibited a promising enhanced electrocatalytic activity towards analytes. The presence of Gel enhances the loaded and stability. In this paper, the electrochemical oxidation of guanine (G) and adenine (A) is at the same time.
Part II:In this work we report the fabrication of an electrochemical sensor for the amperometric determination of isoniazid (ISN) based on functionalized multiwalled carbon nanotube (f-MWCNT) modified glassy carbon electrode (GCE). f-MWCNT has been synthesized by acid treatment of MWCNT by ultrasonication. The electrochemical impedance studies showed that f-MWCNT shows less electron transfer resistance. The electrochemical properties of ISN at various carbon nanomaterials such as MWCNT, f-MWCNT, graphene oxide, reduced graphene oxide, bare GCE, etc have been studied by cyclic voltammetry (CV). We found that ISN undergoes electrochemical oxidation at all the aforementioned nanomaterials modified electrodes. However, f-MWCNT film modified GCE shows the maximum peak current for ISN among the various carbon nanomaterials, which is 7 times higher than that obtained at MWCNT film. ISN undergoes an irreversible oxidation at f-MWCNT film in pH 4 at 0.4V which is well defined and it can be utilized for electroanalytical purposes. ISN showed fast amperometric response at f-MWCNT modified GCE with a good linear range of detection 1 to 70 μM ISN. The film showed good background current stability and excellent performance in pharmaceutical sample analysis with good reproducibility.


Part III:A sensitive bacteria enrichment and detection system forviable Escherichia coli (E.coli) was developed using a amperometric biosensor with gold nanoparticles (AuNPs) as detection verifiers and amplifiers. L-cysteine is one of the twenty common amino acids, which can be easily electropolymerized on electrode surface to form poly-L-cysteine (PLC). Electrochemical polymerization might be performed by the interaction between the thiol groups of PLC (-SH) and AuNPs. The surface coverage concentration (Γ) of AuNPs modified electrode is ≈ 1.2 × 10-10 mol cm−2, PLC modified electrode is ≈ 1.8 × 10-10 mol cm−2 and PLC/AuNPs modified electrode is ≈ 4.5 × 10-9 mol cm−2 respectively. The Γ increase can be explained by the reason that geometric area of nanomaterials is higher than the bulk materials. SEM result shows more thin, regulation and homogenized structure of PLC in present of AuNPs. Electrochemical Impedance Spectroscopy (EIS) shows Faradaic impedance spectra, presented by Nyquist plots (Z′′vs. Z′) for the bare, PLC, AuNPs and PLC/AuNPs modified electrodes. AuNPs is suitable for the modification electrodes due to its high electronic conductivity for the electron transfer reactions and better electrochemical property. PLC/AuNPs modified electrode had highly stable in the pH range of 1 to 11. In the course of E. coli metabolism, the polyphenolic compounds are formed from salicylic acid, such as 2,5-dihydroxybenzoic acid (2,5-DHBA) could be associated with metabolic rates to quantitatively determine E.coli concentration. The electrocatalytic reaction of 2,5-DHBA was examined at PLC/AuNPs modified electrode by cyclic voltammetry and linear sweep voltammetry (LSV) in presence of various concentration mixture. The separation of the current peaks was well for 2,5-DHBA and AA. Amperometric response of 2,5-DHBA at PLC/AuNPs modified electrode shows higher electrocatalytic activity. We simulated more complex system of real samples for determination of E.coli.

The results show that PLC/AuNPs modified electrode was efficient for real sample detection.


摘 要 I
Abstract III
誌 謝 VII
目 錄 VIII
圖目錄 XII
第一章 緒論 1
1.1 感測器簡介 1
1.1.1 感測器定義 1
1.1.2 生物感測器 2
1.2 薄膜修飾電極簡介 3
1.3 電化學分析法 5
1.4 電催化方法 7
1.5 藥品簡介 7
1.5.1 硫化鎘(Cadmium sulfide) 7
1.5.2 甲殼素(Chitosan) 8
1.5.3 DNA (Deoxyribonucleic acid) 8
1.5.4 鳥嘌呤(Guanine)及腺嘌呤(Adenine) 9
1.5.5 奈米碳管(Carbon nanotube) 9
1.5.6 異煙肼(Isoniazid) 10
1.5.7 L-半胱氨酸(L-cysteine) 11
1.5.8 龍膽酸(2,5-dihydroxybenzoic acid) 11
1.5.9 抗壞血酸(Ascorbic acid) 12

1.5.10 大腸桿菌(Escherichia coli ) 12
第二章 實驗藥品、器材與分析方法 14
2.1 實驗藥品 14
2.2 實驗器材 15
2.3 分析方法 17
2.3.1 循環伏安法 17
2.3.2 計時安培法 20
2.3.3 微分脈衝伏安法(Differential pulse voltammetry, DPV) 20
2.3.4 掃描式電子顯微鏡(SEM) 20
2.3.5 原子力顯微鏡(AFM) 21
2.3.6 電化學阻抗譜(Electrochemical spectroscopy, EIS) 23
第三章 以CdS-Chi/Gel修飾電極偵測真實樣本DNA 32
3.1 前言 32
3.2 製備CdS-Chi/Gel 修飾電極 34
3.2.1 製備CdS奈米顆粒 34
3.2.2 製備甲殼素(Chi)、凝膠及DNA溶液 34
3.2.3 製備修飾電極 35
3.3 結果與討論 35
3.3.1 CdS-Chi/Gel膠體之物理特性 35
3.3.2 不同電極的電化學特性 36
3.3.3 CdS-Chi/Gel修飾電極的表面形態分析 37
3.3.4 DNA鹼基在CdS-Chi/Gel修飾電極上的電催化反應 38
3.3.5 CdS-Chi/Gel修飾電極對實際樣本DNA的電催化反應 39


第四章 以官能基化的多層奈米碳管修飾電極電化學氧化及偵測Isoniazid 47
4.1 前言 47
4.2 製備f-MWCNT修飾電極 48
4.3 結果與討論 49
4.3.1 修飾電極之表面型態特性 49
4.3.2 修飾電極的EIS研究 49
4.3.3 不同修飾電極對ISN的電化學特性 50
4.3.4 不同pH情況下修飾電極對ISN的電催化特性 51
4.3.5 f-MWCNT修飾電極在不同掃描速率的研究 51
4.3.6 f-MWCNT修飾電極對ISN的電催化特性 52
4.3.7 f-MWCNT修飾電極對ISN的電流偵測 52
4.3.8 f-MWCNT修飾電極的選擇性研究及偵測真實樣本 53
第五章 以奈米金顆粒結合Poly-L-cysteine修飾電極間接偵測微生物種群 61
5.1 前言 61
5.2 製備PLC/AuNPs修飾電極 64
5.3 結果與討論 64
5.3.1 不同材料修飾電極之電化學特性比較 64
5.3.2 PLC/AuNPs修飾電極的表面形態研究 65
5.3.3 PLC/AuNPs修飾電極的電化學阻抗研究 66
5.3.4 PLC/AuNPs修飾電極在不同pH情況下的電化學特性 66
5.3.5 PLC/AuNPs修飾電極電催化龍膽酸及抗壞血酸 67
5.3.6 PLC/AuNPs修飾電極對2,5-DHBA的電流測定 68
5.3.7 偵測複合系統及真實樣本大腸桿菌 69


第六章 總結論 79
參考文獻 80


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