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研究生:武高恩
研究生(外文):VU CAO AN
論文名稱:利用核適體作為訊號放大器於矽奈米線場效電晶體免疫感測器對生物標記物進行定量分析
論文名稱(外文):Aptamer as Signal Amplifier for Biomarker Quantification by Silicon Nanowire Field-Effect Transistor Immunosensors
指導教授:陳文逸陳文逸引用關係
指導教授(外文):CHEN WEN YIH
學位類別:博士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:160
中文關鍵詞:場效應晶體管生物傳感器適體兔抗體Amyloid β 1-42信號增強免疫測定
外文關鍵詞:Field-Effect Transistor BiosensorAptamerRabbit AntibodyAmyloid β 1-42Signal EnhancementImmunoassay
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為了對疾病進行早期診斷,需要一個高靈敏度的檢測技術來檢測在生物樣品中極低濃度的生物標記物。矽奈米線場效電晶體因為其對多種目標物擁有高靈敏、免標定、即時檢測等優勢,已經被證明為一個有力的醫學診斷工具。但是,由於德拜長度屏蔽效應的影響以及非專一性吸附的干擾,使用奈米線場效電晶體生物傳感器來定量在生理環境下極低濃度的蛋白質生物標記物會受到嚴重的阻礙。本研究將會提供一個方法,透過使用核適體作為訊號放大器在矽奈米線場效電晶體之免疫測定檢測法,可以提高矽奈米線場效電晶體對生物標記物檢測能力,並且將之應用在β-澱粉樣蛋白(1-42)的定量檢測以進行對阿茲罕默症的早期診斷。
在本篇論文中,會先使用核適體作為訊號放大器於矽奈米線場效電晶體以直接和三明治免疫檢測模型進行蛋白質檢測。直接免疫測定法是將六組氨酸固定在矽奈米線場效電晶體之感測表面上後,以對應之兔源抗體(IgG)識別來進行檢測;而三明治免疫測定法是將抗β-澱粉樣蛋白之鼠源抗體(IgG1)固定在感測表面上後,以專一性免疫結合的方法抓取β-澱粉樣蛋白來,再以兔源抗體結合被抓取的β-澱粉樣蛋白。在兩種免疫檢測法,最後都會加入對兔源抗體具有專一性結合的核適體R18。從加入R18進行結合後的實驗結果可以發現,直接和三明治免疫檢測法產生的電變異均得到顯著穩定和放大。
而後,這種方法被用於(3-氨基丙基)三乙氧基矽烷修飾和混合的自組裝單層修飾的矽奈米線場效電晶體生物傳感器上,對在150 mM BTP和人類血清這些具高離子強度和嚴重干擾的環境中稀釋不同濃度之β-澱粉樣蛋白進行定量分析。在兩種表面改質方式中皆是固定抗β-澱粉樣蛋白(1-42)之鼠源抗體在矽奈米線上,前者是用於三明治免疫測定法檢測β-澱粉樣蛋白;而後者是於矽奈米線上使用混和自組裝單層膜(組成: 矽氧烷-聚乙二醇-胺基和矽氧烷-聚乙二醇-醇基)進行表面改質。R18 核適體成功地用作這兩種改質方式之生物傳感器的信號放大器,由R18引起的電訊號變化量隨著生物樣品中β-澱粉樣蛋白(1-42)濃度的升高而增加,我們可以依此測定在150 mM BTP和人類血清中的β-澱粉樣蛋白(1-42)。實驗結果中共有3條檢量線,顯示了檢測到的β-澱粉樣蛋白(1-42)濃度與適體增強信號之間具有良好的線性關係。 由兩種免疫傳感器建立的三個定量範圍都非常符合Langmuir等溫吸附線模型,並且根據經驗數據計算的它們各自的Ka值(結合親和力)都在抗體-抗原結合親和力範圍內。在兩種改質方法中,尤其建議使用混和自組裝單層膜來修飾矽奈米線場效電晶體生物感測器,此方式可以在人類血清環境中測定100 fg/mL ~100 pg/mL的β-澱粉樣蛋白(1-42) ,計算檢量線的p值可以進一步證明數據有很高的顯著性。與最近的β-澱粉樣蛋白檢測技術相比,本研究可以檢測到的100 fg/ mL為最低的檢測極限濃度,並且獲得的定量範圍涵蓋了阿茲罕默症的早期診斷所需的β-澱粉樣蛋白濃度範圍。因此,本論文中描述的方法對應用於阿茲罕默症的早期診斷具有相當大的潛力,甚至可能可以在阿茲罕默症發展的更初期進行診斷,並且此檢測技術也可以應用在血液樣本中其他的生物標記物的極低濃度定量檢測。
Early-stage diagnosis plays an essential role in identifying diseases for proper treatment and therapy. However, it requires ultra-sensitive techniques to recognize biomarkers, which are associated with and alerts of various diseases, at ultra-low concentrations in bio-samples. Silicon nanowire field-effect transistor (SiNWFET) biosensors has been demonstrated as a powerful tool in diagnosis because it can yield high sensitivity for label-free and real-time detection of plentiful molecules. Nevertheless, determining protein biomarkers by SiNWFET immunosensors at ultra-low levels in physiological environments is severely hindered due to the limitation of screening effect and interference of non-specific bio-species. This thesis presents a strategy for protein sensing on SiNWFETs with aptamer as signal amplifier and apply it for protein biomarker quantification by SiNWFET immunosensors with Amyloid β 1-42 (Aβ 1-42), a biomarker for early-stage diagnosis of Alzheimer disease (AD), as a case study.
Initially, aptamer as signal enhancement for protein sensing by SiNWFET transducers are investigated via direct and sandwich immunoassays. The direct type was implemented by anchoring Hexahistidine on SiNWFET surface to be recognized by its respective rabbit immunoglobulin G (IgG) whereas the sandwich type was developed by immobilizing mouse antibodies on the sensing channels to capture Aβ 1-42 following with rabbit IgG. Both types of the sensors were then incubated with R18, an RNA aptamer specifically targeting rabbit IgG. Empirical results reveal that electrical variation produced from both direct and sandwich immunoassays is stabilized and remarkably amplified after incubating with R18 despite of inconsistent signal recorded in both models after detecting the corresponding proteins.
Subsequently, this approach is exploited for APTES-modified SiNWFET and mSAM-modified SiNWFET immunosensors (mSAM: mixed self-assembled monolayers) to quantify AB 1-42 at different concentrations in 150 mM Bis-Tris propane (BTP) and human serum (HS). The former are the ones used in sandwich immunoassay of Aβ 1-42 whereas the latter are developed from the NW surface modified with mSAMs of silane-PEG-NH2 and silane-PEG-OH. Aptamer as signal amplifier is successfully applied for both fabricated sensors to determine Aβ 1-42 in 150 mM BTP and HS with three calibration lines presenting well-linear relationship between detected concentrations of Aβ 1-42 and enhanced signal by aptamer. The linear ranges achieved by two kinds of immunosensors are well-fitted with Langmuir adsorption model and their Ka values (binding affinity), calculated from empirical data, are within the range of antibody-antigen binding affinity. Especially, the suggested solution exhibits an outstanding performance in combination with mSAM-SiNWFET immunosensors to quantify Aβ 1-42 in HS at level down to 100 fg/mL within a linear range of 100 fg/mL – 100 pg/mL, accredited the significance by p value. 100 fg/mL is the lowest limit of detection (LOD) in comparison with the most up-to-date sensing technologies for Aβ 1-42 and the linear range obtained covers the required range of Aβ 1-42 content for early-stage diagnosis of AD. The strategy described in this dissertation is therefore applicable for early-stage diagnosis of AD as well as potential for determining other biomarkers with ultra-low concentration in blood.
摘要 i
Abstract iv
Acknowledgements vi
Table of Contents vii
List of Figures x
List of Tables xv
Chapter 1: Introduction 1
1.1 Motivations 1
1.2 Objectives 4
1.3 Structure 5
Chapter 2: Research Background and Literature Review 7
2.1 SiNWFET Biosensors 7
2.2 Debye Length 12
2.3 Aptamers 15
2.4 Dementia and AD 19
2.5 Fouling Resistance for SiNWFET Immunosensors 27
Chapter 3: Materials & Methods 30
3.1 Materials 30
3.1.1 Chemicals 30
3.1.2 Apparatuses and instruments 31
3.2 Fabrication of SiNWFET Immunosensors 31
3.2.1 Preparation of 6xHis-immobilized SiNWFET immunosensors 31
3.2.2 Preparation of APTES-modified SiNWFET immunosensors (IgG1-immobilized SiNWFET immunosensors) 33
3.2.3 Preparation of mSAM-modified SiNWFET immunosensors 34
3.3 Performance of Biosensing and Signal Enhancing 35
3.3.1 Direct immunoassay and signal enhancing of 6xHis-immobilized SiNWFET imunosensors in 150 mM BTP 35
3.3.2 Sandwich immunoassay and signal enhancing of APTES-modified SiNWFET immunosensors (IgG1-immobilized SiNWFET immunosensors) 36
3.3.3 Detection of Aβ 1-42 in HS by mSAM-modified SiNWFET immunosensors and signal amplification by R18 aptamer 38
3.4 Data Analysis 40
Chapter 4: Electrical Responses of Direct and Sandwich Immunoassays by SiNWFET and Signal Enhancement by RNA Aptamer 41
4.1 Direct Immunoassay of Hexahistidine on SiNWFET and Signal Amplification by RNA Aptamer 41
4.2 Sandwich Immunoassay of Aβ 1-42 on SiNWFET and Signal Amplification by RNA Aptamer 44
4.3 Discussions on Aptamer as Signal Amplifier for SiNWFET Immunosensors 46
4.4 Summary 50
Chapter 5: Signal Enhancement from RNA Aptamer Improves Biomarker Quantification by SiNWFET Immunosensors 51
5.1 Amplified Signal from RNA Aptamer for Quantifying Aβ 1-42 in 150 mM BTP by APTES-modified SiNWFET Immunosensors 52
5.2 Amplified Signal from RNA Aptamer for Quantifying Aβ 1-42 in HS by APTES-modified SiNWFET Immunosensors 53
5.3 Amplified Signal from RNA Aptamer Improves Quantifying Aβ 1-42 in HS by mSAM-modified SiNWFET Immunosensors 55
5.4 Langmuir Adsorption Model in Further Analysis on Signal Enhancement of Aptamer for Aβ 1-42 Quantification by SiNWFET Immunosensors 58
5.5 Review and Discussions on Recent Aβ 1-42 Sensing Technologies 59
5.6 Summary 62
Chapter 6: Concluding Remarks and Future Prospects of Aptamers in FET Biosensors 63
6.1 Conclusions 63
6.2 Contributions 65
6.3 Future Prospects of Aptamers in FET Biosensors 66
Bibliographies 68
Appendixes 95
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