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研究生:簡柏育
研究生(外文):CHIEN,PO-YU
論文名稱:利用低損傷電漿系統製備石墨烯氧化物/石墨烯結構作為電化學生物感測器電極應用於MicroRNA-21之偵測
論文名稱(外文):Preparation of Graphene Oxide/Graphene Structure by Low Damage Plasma System Acts As Electrochemical Biosensor Electrode for MicroRNA-21 Detect
指導教授:黃啟賢黃啟賢引用關係
指導教授(外文):HUANG,CHI-HSIEN
口試委員:蘇莉真李穎
口試委員(外文):SU,LI-CHENLI,YING
口試日期:2019-03-04
學位類別:碩士
校院名稱:明志科技大學
系所名稱:材料工程系碩士班
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:97
中文關鍵詞:電化學石墨烯應用石墨烯氧化物羧基低損傷電漿系統miRNA-21生物感測器
外文關鍵詞:ElectrochemicalGraphene applicationGraphene oxideCarboxyl groupLow damage plasma treatment (LDPT)miRNA-21biosensor
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在本研究中,提出一種新穎的電化學生物感測器,將化學氣相沉積所成長出的高品質單層石墨烯,利用濕式轉印的方式並經熱退火處理獲得雙層石墨烯,再藉由低損傷電漿系統(low damage plasma treatment, LDPT)的可控性,使其成為上層氧化且具官能化作為生物接收面,下層仍保有原結構特性之石墨烯氧化物/石墨烯電極。接著透過化學共價鍵-醯胺鍵固定microRNA-21(miRNA-21)的探針端於上層氧化石墨烯,再與濃度範圍為100 nM至10 fM的miRNA-21進行雜合。本研究主要以電化學方式分析,利用計時庫侖法探討氧電漿製程時間對於探針端固定化之影響,經過計算我們發現製程時間越長其固定化的表面探針密度越高(最多可達1×1012 molecules cm-2)。利用DPV量測所得的電流變化,計算由氧電漿(O-LDPT)所製備之生物感測器之偵測極限為236 fM、線性度R2=0.9822。而本研究室先前的研究指出,在氧電漿加入氫氣(O/H-LDPT)能夠有效提升COOH的含量,我們觀察到表面探針密度能夠有效地提升至1.42×1012 molecules cm-2,因此以氫氧電漿製備生物感測器電極發現能有效地降低偵測濃度,經由計算氫氧電漿所製備之感測器之偵測極限 24.6 fM、線性度為0.9742,此外,我們將氫氧電漿所製備之電極於固定化製程後做封閉的動作確保其對目標物之專一性,之後於牛血清的環境下進行不同濃度的miRNA-21雜合,在這些條件下仍保有相當好的結果(偵測極限19.83 fM、線性度 0.981),此外石墨烯氧化物具有相當好的生物相容性,利用檢測快速、高靈敏度以及環境友善的優勢,此生物感測器極具應用於臨床上的潛力。且因miRNA-21是一種循環在人體血漿中,可不需要經過樣品選取或擴增等步驟,能夠有效地縮短檢測時間,且此結構說明了只需要改變探針端的設計就有望能進行不同的mircoRNAs量測。
In this study, a novel electrochemical biosensor was prepared. High-quality single-layer graphene, grown by chemical vapor deposition, was wet-transferred onto a substrate, followed by thermal annealing to obtain bilayer graphene, which was then subjected to oxygen low-damage plasma treatment (O-LDPT), to obtain the target electrodes. While the top layer of graphene was oxidized, the bottom layer remained a transmission layer. Till date, this electrode structure had never been used for electrochemical detection of miRNA-21. Here, the miRNA-21 probe was immobilized on the upper graphene oxide layer by the formation of a covalent amino bond, and hybridized with the target miRNA-21 after immobilization. Chronocoulometry was used determime the surface density of the probe; the latter was found to increases with increasing processing time. The probe density was estimated to be 1 × 1012 molecules/cm2. Differential pulse voltammetry (DPV) measurements have been performed to evaluate the sensitivity of this biosensor. Peak currents obtained from the DPV spectra increased linearly as the target RNA concentration decreased from 100 nM to 1 pM; the limit of detection (LOD) of this biosensor was calculated to be 236 fM (R2=0.9822). We had previously confirmed using X-ray photoelectron spectroscopic analysis that addition of hydrogen during the oxygen plasma treatment (O/H-LDPT) can increase the carboxyl group content. We observed that probe density can be effectively increased to 1.42 × 1012 molecules/cm2. We also compared the LOD values under two different plasma conditions (O-LDPT and O/H LDPT). Preparation of biosensor electrodes by O/H-LDPT was found to effectively reduce the LOD to 24.6 fM (R2=0.9742). In order to achieve specificity of the biosensor we added a blocking step after immobilization to remove the active site, and then hybridized with different concentrations of miRNA-21 in BSA. The measured LOD was 19.83 fM (R2=0.981). Graphene oxide has excellent hydrophilicity and biocompatibility, along with the advantages of rapid detection, high sensitivity and environmental friendliness; therefore, graphene oxide/graphene structure has great potential for use in electrochemical biosensor applications.
目錄
明志科技大學碩士學位論文指導教授推薦書 i
明志科技大學碩士學位論文口試委員審定書 ii
誌謝 iii
中文摘要 iv
英文摘要 vi
第一章、緒論 2
1-1前言 2
1-2石墨烯的簡介及特性 4
1-3石墨烯氧化物的簡介及特性 8
第二章、文獻回顧 11
2-1石墨烯氧化物的製備方式 11
2-1-1化學合成氧化石墨烯 11
2-1-2氧氣電漿製備石墨烯氧化物 13
2-2熱退火 14
2-3低損傷電漿 15
2-4 microRNAs 背景與應用 20
2-5電化學生物偵測元件 23
2-6研究動機及目的 28
3-1電極製備與分析流程 30
3-2實驗藥品及材料 32
3-3實驗機台介紹 33
3-3-1低壓化學氣相沈積系統 (Low Pressure Chemical Vapor Deposition, LP-CVD) 33
3-3-2烘箱退火 35
3-3-3低損傷電漿系統 (Low Damage Plasma Treatment System, LDPT) 36
3-3-4拉曼光譜儀 (Raman Spectroscopy, Raman) 39
3-3-5紫外光-可見光光譜儀 (Ultraviolet-visible spectroscopy, UV-vis) 44
3-3-6四點探針 (Four-point probe) 45
3-3-7 CHI611E 電化學分析儀 46
3-4實驗方法 47
3-4-1成長單層石墨烯 47
3-4-2清洗ITO基板 48
3-4-3轉印單層石墨烯至氧化銦錫(ITO)基鈑 49
3-4-4烘箱退火 50
3-4-5利用低損傷電漿系統製備單層氧化石墨烯 50
3-4-6固定化DNA與雜合miRNA-21 51
3-4-7電化學量測 52
第四章、實驗結果與討論 61
4-1低壓化學氣相沈積成長之單層石墨烯特性分析 61
4-2雙層石墨烯特性分析 63
4-3探討電漿製程對石墨烯與雙層石墨烯特性影響之研究 66
4-4電化學生物感測器電極結構測試 69
4-5探討電漿製程對於探針端(Probe)固定化影響 75
4-6不同濃度之microRNA-21之偵測 77
4-6-1氧電漿製程之電極應用於生物感測器 77
4-6-2氫氧電漿製程之電極應用於生物感測器 81
4-7探討氫氧電漿製程所製備之電極的專一性與選擇性 83
第五章、結論 87
第六章、未來展望 88
參考文獻 89


圖目錄
圖1- 1,碳材成員 4
圖1- 2,石墨烯的合成方式 6
圖1- 3,石墨烯的品質與製備成本關係圖[25] 7
圖1- 4,石墨烯濕式轉印流程圖[27] 8
圖1- 5,石墨烯氧化物結構示意圖 9
圖1- 6,石墨烯氧化物與SINGLE STRAIN DNA鍵結之機制示意圖 10
圖2- 1,HUMMER法製備氧化石墨烯之分析 12
圖2- 2,HUMMER法製備氧化石墨烯示意圖 12
圖2- 3,利用電漿製程製備石墨烯氧化物之拉曼光譜圖[44] 13
圖2- 4,退火對於殘留光阻之影響[45] 14
圖2- 5,退火前後之拉曼分析[45] 14
圖2- 6,離子轟擊造成的缺陷深度關係圖[46] 16
圖2- 7,真空紫外光產生的缺陷密度關係圖[47] 16
圖2- 8,真空紫外光造成的缺陷示意圖 16
圖2- 9,電漿改質單層(A)與雙層(B)石墨烯的差異[48] 17
圖2- 10,單層石墨烯純氧電漿改質之XPS分析[53] 18
圖2- 11,單層石墨烯氫/氧混氣電漿改質之XPS分析[53] 19
圖2- 12,MICRORNAS的形成 20
圖2- 13,生物感測元件機制 23
圖2- 14,石墨烯氧化物經由EDC/NHS活化 24
圖2- 15,在一時間段中,持續加入生物標的物之電流輸出曲線[61] 25
圖2- 16,不同雜合溫度與時間之電話學交流阻抗量測[62] 25
圖2- 18,微分脈衝伏安法量測不同濃度之人體免疫缺乏病毒[64] 26
圖3- 1電極製備流程圖 30
圖3- 2 石墨烯特性分析 30
圖3- 3 電漿改質分析流程 30
圖3- 4雜合TARGET MIRNA-21後將試片放置於工作電極分析 31
圖3- 5,低壓化學氣相沈積系統 34
圖3- 6,烘箱 35
圖3- 7,低損傷電漿系統 36
圖3- 8,高密度感應耦合低損傷電漿系統示意圖 37
圖3- 9,電漿系統內部放大示意圖 37
圖3- 10,不鏽鋼材質之互補式過濾片 38
圖3- 11,透過放光光譜圖來觀測互補式過濾片的過濾效果[68] 38
圖3- 12,拉曼光譜儀系統 39
圖3- 13,單層石墨烯與石墨之拉曼光譜圖 41
圖3- 14,不同的石墨烯層數與G PEAK的關係圖 42
圖3- 15,不同的石墨烯層數與2D PEAK的關係圖 43
圖3- 16,UV-VIS 紫外光可見光光譜儀 44
圖3- 17,四點探針機台 45
圖3- 18,CHI611E 外觀 46
圖3- 19,化學氣相沉積示意圖 47
圖3- 20,成長單層石墨烯之製程圖 48
圖3- 21,濕式轉印流程圖 49
圖3- 22,在有吸附反應的情況下,電解通過之電荷與時間關係圖 53
圖3- 23,循環伏安法時間與電壓之關係[65] 54
圖3- 24,循環伏安法時間與電壓之關係 54
圖3- 25,濃度與距離之關係[65] 56
圖3- 26,EIS裝置示意圖 57
圖3- 27,改變電位與電流之向量圖[65] 57
圖3- 28,典型的電化學阻抗圖譜[65] 58
圖3- 29,電化學交流阻抗圖譜的含意 58
圖3- 30,電位波形與時間關係圖 60
圖3- 31,典型的DPV圖 60
圖4- 1,單層石墨烯之拉曼光譜圖 61
圖4- 2,單層石墨烯之紫外光-可見光光譜圖 62
圖4- 3,單層石墨烯之平均片電阻 62
圖4- 4,單層石墨烯與堆疊雙層石墨烯比較圖 64
圖4- 5,堆疊雙層石墨烯與雙層石墨烯比較圖 64
圖4- 6,雙層石墨烯之紫外光-可見光光譜圖 65
圖4- 7,雙層石墨烯之平均片電阻 65
圖4- 8,單層石墨烯經由不同電漿時間改質拉曼圖 66
圖4- 9,單層石墨烯經由不同電漿改質時間之水接觸角 66
圖4- 10,單層與雙層石墨烯於電漿改質後之拉曼圖 67
圖4- 11,拉曼MAPPING之ID/IG分布圖 68
圖4- 12,拉曼MAPPING之I2D/IG分布圖 68
圖4- 13,石墨烯氧化物/ITO及石墨烯氧化物/石墨烯結構/ITO之電化學阻抗圖譜 70
圖4- 14,接觸電阻量測試片 71
圖4- 15,石墨烯氧化物/ITO之接觸電阻量測 72
圖4- 16,石墨烯氧化物/石墨烯/ITO之接觸電阻量測 73
圖4- 17,不同掃描速率之CV量測 73
圖4- 18,不同掃描速率與電位變化之趨勢圖 74
圖4- 19,固定化後之計時庫侖法量測 76
圖4- 20,探針固定化前後及不同濃度MIRNA-21雜合化之CV量測 78
圖4- 21,MIRNA-21濃度範圍為0 M至10-6M雜合化之DPV量測 79
圖4- 22,不同濃度MIRNA-21雜合化之DPV量測 79
圖4- 23,不同濃度MIRNA-21之DPV量測趨勢圖 80
圖4- 24,不同濃度MIRNA-21雜合化之DPV量測 81
圖4- 25,不同濃度MIRNA-21之DPV量測趨勢圖 82
圖4- 26,不同濃度MIRNA-21雜合化之DPV量測 83
圖4- 27,不同濃度MIRNA-21之DPV量測趨勢圖 84
圖4- 28,完全互補、部分不互補及完全不互補之DPV量測 86
圖4- 29,完全互補、部分不互補及完全不互補之電流變化量 86

表目錄
表1- 1,石墨烯的特性表 5
表2- 1,人體中各種MICRORNAS與疾病的關係表[56] 21
表2- 2,MICRORNA-21與人類疾病整理表 22
表2- 3,電化學生物感測器及量測結果[67] 27
表4- 1,石墨烯氧化物/ITO及石墨烯氧化物/石墨烯/ITO之電荷轉移電組 70
表4- 2,石墨烯氧化物/ITO及石墨烯氧化物/石墨烯/ITO電極電阻量測 71
表4- 3,O-LDPT 不同時間之庫侖差及表面探針密度 76


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