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系統識別號 U0026-0309201316411300
論文名稱(中文) 利用去氧核醣核酸適合體化學冷光免疫分析法測定C反應蛋白與甲型胎兒蛋白在大型系統與微流體系統之比較
論文名稱(英文) DNA aptamer-based chemiluminescence immunoassay for C-reactive protein and alpha fetoprotein compared with batch system and microfluidic system
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 101
學期 2
出版年 102
研究生(中文) 林欣億
研究生(英文) Hsin-I Lin
學號 s58921531
學位類別 博士
語文別 英文
論文頁數 89頁
口試委員 指導教授-謝淑珠
召集委員-謝達斌
召集委員-莊偉哲
召集委員-吳華林
口試委員-高照村
口試委員-李國賓
中文關鍵字 適合體  系統性配分子指數增益演繹程序  C反應蛋白  甲型胎兒蛋白  微機電系統技術晶片  微流體系統 
英文關鍵字 Aptamer  SELEX  C-reactive protein  alpha fetoprotein  MEMS chip  Microfluidic system 
學科別分類
中文摘要 系統性配分子指數增益演繹程序是從隨意組成之去氧核醣核酸庫或核醣核酸庫中,藉由其特異性結合能力篩選特定之分子目標,其經由篩選後最終產物被稱之為適合體,適合體為單股之寡核苷酸序列,可做為一些生醫用途包含純化技術、臨床診斷、生醫感測以及治療用途。微機電系統技術可藉由微製程系統微小化並整合感測器、作動器和機電系統在單一晶片,運用在生物技術、通訊、光學以及其他領域上, 近年來微機電系統技術在分子生物、生物醫學以及分析化學上亦有相當可觀的研發性。在此研究中我們發展出利用去氧核醣核酸適合體化學冷光免疫分析法來偵測血清中的C反應蛋白以及甲型胎兒蛋白,並比較在大型系統與微製程晶片上的差異。C反應蛋白不只是典型的急性期反應蛋白,也是心血管疾病如動脈粥狀硬化中關鍵的危險因子;對於全球疫情因B型或C型肝炎所造成的肝癌,甲型胎兒蛋白常被視為是重要的篩選生物標誌。適合體是利用PCR試管或微製程晶片,從72個序列的去氧核醣核酸庫中以系統性配分子指數增益演繹程序技術進行篩選;所篩選出的適合體二級結構以線上軟體DNA mfold version 2.3 software來預測;C反應蛋白適合體1、2與甲型胎兒蛋白的分子結合係數利用表面等離子體共振儀測量分別為9.93 nM、3.51 nM及 2.37 nM;化學冷光分析法的設計是以一適合體做為捕捉目標,並以一吖啶酯標誌的單株抗體做為訊號追蹤偵測,適合體在5端修飾生物素並接合上表面有鏈黴親和素的微量盤或磁珠上,並利用微量盤、塑膠試管或微製程晶片方式建立測定校正曲線用以量測臨床檢體,C反應蛋白適合體校正曲線偵測範圍從0.01 to 5.00 mg/L,甲型胎兒蛋白校正曲線偵測範圍從12.5 to 800 ng/mL, C反應蛋白微量盤法利用C反應蛋白適合體1的偵測變異是13.9% (R2=0.9610, n=5);C反應蛋白塑膠試管法利用C反應蛋白適合體1與C反應蛋白適合體2的偵測變異分別是20.8% 以及5.7% (C反應蛋白適合體 1: R2=0.9340; C反應蛋白適合體 2: R2=0.9694; n=3);甲型胎兒蛋白的偵測變異則是8.5%。總括而言在此研究中我們成功的篩選出C反應蛋白與甲型胎兒蛋白的適合體,並建立可以適用於臨床診斷用途的免疫分析法。而且我們利用微流體系統平台成功的篩選並鑑別出血紅蛋白與醣化血紅蛋白適合體,依據這些結果,微流體系統結合系統性配分子指數增益演繹程序與化學冷光免疫分析法,在用於臨床生物指標適合體的篩選與偵測上是一個強而有力的工具。
英文摘要 The systematic evolution of ligands by exponential enrichment (SELEX) is an experimental process that allows screening of given molecular targets by specific binding affinities from a random DNA or RNA library. The final products of SELEX are usually referred as aptamers, Aptamers are the single-strained DNA or RNA oligonucleotides, recognized as promising molecules for a variety of biomedical applications such as purification technology, clinical diagnostics, biosensors, and therapeutics. Micro-electro-mechanical-systems (MEMS) technology has been a promising approach in a variety of fields, including biotechnology, communications, optics and many others by integrating miniaturized sensors, actuators and electronics onto a single chip through micromachining techniques. Among them, the applications of MEMS technology for molecular biology, biomedicine and analytical chemistry have attracted considerable interest recently. In this study, we aimed to develop DNA aptamer-based chemiluminescence immunoassays for the measurement of serum C-reactive protein (CRP) and alpha fetoprotein (AFP) compared with batch system and MEMS chip. The aptamer was selected from a 72-base DNA library through several SELEX rounds in PCR tubes or MEMS chip. The secondary structures of selected aptamers were predicted by using DNA mfold version 2.3 software. The dissociation constant (KD) of the selected CRP aptamer 1, CRP aptamer 2, and AFP aptamer, measured by surface plasmon- resonance analysis, were about 9.93 nM, 3.51 nM, and 2.37 nM, respectively. The chemiluminescence immunoassays were designed using one aptamer as the capturer and an acridinium ester-labeled monoclonal antibody as the signal tracer. The aptamer was modified with 5’-biotin and immobilized on streptavidin-coated plate or magnetic beads. The chemiluminescence signals were measured to establish a calibration curve and compared with results of clinical samples on system of microtiter plate method, tube method or MEMS chip method. The calibration range of CRP and AFP measurement was from 0.01 to 5.00 mg/L and from 12.5 to 800 ng/mL, respectively. The coefficient of variation (CV) for CRP measurement with CRP aptamer 1 by plate method was measured to be about 13.9% (R2=0.9610, n=5). The CV for CRP measurement with CRP aptamer 1 and CRP aptamer 2 by tube method was measured to be about 20.8% and 5.7%, respectively (CRP aptamer 1: R2=0.9340; CRP aptamer 2: R2=0.9694; All n=3). The variation of the AFP aptamer measurements was 8.5%. In conclusions, a specific CRP and AFP DNA aptamer was selected and suitable for clinical applications as immunoassay to measure serum CRP and AFP. Furthermore, we used microfludic system as platforms to successfully screen and characterize the hemoglobin and hemoglobin A1c aptamers. According to these results, microfludic system combined with SELEX process and chemiluminescence immunoassays was a powerful tool for aptamer selection and measurement for clinical biomarkers.
論文目次 Abstract……………………………………………………………………………I
摘要…………………………………………………………………………………III
致謝……………………………………………………………………………………V
Table of Contents……………………………………………………………VII
Lists of Figures………………………………………………………………X
Lists of Supplementary Figures………………………………………XI
Abbreviation……………………………………………………………………XII
Introduction
Protein recognition………………………………………………………………………1
Aptamers compared with antibodies…………………………………………………………………………1
Application of aptamers……………………………………………………3
Systematic evolution of ligands by exponential enrichment (SELEX)……………………………………………………………………………3
Aptamer-based immunoassay…………………………………………………4
Micro-electro-mechanical-systems (MEMS) technology…………5
C-reactive protein (CRP) and clinical applications…………6
Alpha-fetoprotein (AFP) and clinical applications……………7
Hemoglobin and hemoglobin A1c……………………………………………8
Specific aims……………………………………………………………………9
Materials and methods
CRP purification………………………………………………………………10
Design of radom DNA library……………………………………………10
SELEX selection of aptamers by PCR tubes………………………11
CRP competitive assay by manual method……………………………11
Dissociation constant of aptamer to CRP and AFP……………12
Labeling of the signaling antibody for CRP, AFP, Hb or HbA1c………………………………………………………………………………13
Immobilization of the aptamer on streptavidin-modified microtiter plate………………………………………………………………13
Plate assay procedure for CRP…………………………………………13
Preparation of aptamer magnetic beads for CRP, AFP Hb or HbA1c…………………………………………………………………………………14
Tube assay procedure for CRP, AFP, Hb or HbA1c………………………………………………………………………………14
Preparation of CRP, AFP, Hb or HbA1c coated magnetic beads………………………………………………………………………………14
MEMS chip design for CRP…………………………………………………15
CRP measurement by MEMS chip……………………………………………16
Chip SELEX design for CRP…………………………………………………18
On-chip SELEX and on-chip PCR for CRP………………………………19
Design of the integrated microfluidic chip for CRP…………20
Custom-made optical and control systems……………………………21
Automatic on-chip measurement for CRP………………………………21
On-Chip SELEX design for AFP……………………………………………23
On-chip competitive assay for AFP……………………………………24
AFP MEMS-Chip design…………………………………………………………24
Tube assay procedure for AFP……………………………………………26
On-Chip SELEX design for Hb and Chip design……………………26
Tube assay procedure for Hb………………………………………………27
On-Chip SELEX design for HbA1c…………………………………………28
Tube assay procedure for HbA1c…………………………………………28
Results
CRP purification from clinical specimens…………………………30
SELEX results for CRP aptamer……………………………………………30
The dissociation constant of CRP aptamer 1………………………31
The recovery performance of acridinium ester-labeled antibody……………………………………………………………………………31
CRP measurement by microtiter plate method………………………31
CRP measurement by tube method…………………………………………32
On-chip SELEX for CRP………………………………………………………33
The dissociation constant of CRP aptamer 2………………………33
CRP measurement by tube method compared with CRP aptamer 1 and 2…………………………………………………………………………………34
An automatic microfluidic system for CRP measurement………34
On-chip SELEX for AFP………………………………………………………34
AFP measurement by tube method…………………………………………35
Undergoing works………………………………………………………………36
Discussion…………………………………………………………………………38
Conclusions………………………………………………………………………43
References…………………………………………………………………………44
Publication Lists…………………………………………………………………………………54
Lists of Figures
Fig.1. A schematic representation of the tube based SELEX process………………………………………………………………………………55
Fig.2. Results of CRP purification.…………………………………………………………………………………………56
Fig.3. The electrophoresis of SELEX analysis……………………57
Fig.4. The competitive assay from different clones and CRP aptamer structure………………………………………………………………59
Fig.5. The recovery performance of acridinium ester-labeled antibody………………………………………………………………………………60
Fig.6. A schematic representation of the CRP assay by plate method…………………………………………………………………………………61
Fig.7. The standard curve of CRP was established by microtiter plate method……………………………………………………62
Fig.8. A schematic representation of the CRP assay by tube method…………………………………………………………………………………63
Fig.9. Standard curve compared by tube and MEMS chip method for CRP………………………………………………………………………………64
Fig.10. The signal on magnetic beads was obtained by microscope …………………………………………………………………………65
Fig.11. Results of CRP aptamer 2 and structures………………67
Fig.12. CRP measurement compared with CRP apatamer 1 and 2…………………………………………………………………………………………68
Fig.13. Results of AFP aptamer and structures.…………………………………………………………………………………………70
Fig.14. AFP measurement by tube method.……………………………71
Lists of Fupplementary Figures
Supplementary Fig.1.
Schematic illustration of the microfluidic chip for CRP detection aptamers………………………………………………………………72
Supplementary Fig.2.
Schematic illustration of the on-chip detection for CRP with CRP aptamer…………………………………………………………………73
Supplementary Fig.3. Schematic illustration of chip design for CRP on-chip SELEX…………………………………………………………74
Supplementary Fig.4. Schematic illustration of on-chip SELEX for CRP……………………………………………………………………75
Supplementary Fig.5.
Schematic illustration of automatic microfluidic chip design for CRP……………………………………………………………………76
Supplementary Fig.6. Custom-made optical and control system for CRP detection………………………………………………………………77
Supplementary Fig.7. Illustration of the automatic procedure for CRP detection………………………………………………78
Supplementary Fig.8. Schematic illustration of on-chip SELEX for AFP……………………………………………………………………79
Supplementary Fig.9.
Schematic illustrations of the on-chip competitive assay process for AFP…………………………………………………………………80
Supplementary Fig.10. Schematic illustration of AFP MEMS chip design………………………………………………………………………81
Supplementary Fig.11. Schematic illustration of the on-chip SELEX for Hb………………………………………………………………………82
Supplementary Fig.12. Schematic illustration of the on-chip SELEX chip design………………………………………………………………83
Supplementary Fig.13.
Modified competitive indirect ELISA test for dissociation constant of Hb……………………………………………………………………84
Supplementary Fig.14. One antibody and one Hb aptamer-based assay for Hb………………………………………………………………………85
Supplementary Fig.15. Two-antibody assay for HbA1c.…………………………………………………………………………………………86
Supplementary Fig.16. Haptoglobin-antibody assay for HbA1c.…………………………………………………………………………………………87
Supplementary Fig. 17. One antibody and one HbA1c aptamerassay for HbA1c………………………………………………………88
Supplementary Fig. 18. Comparison of Hb and HbA1c antibody with HbA1c aptamer……………………………………………………………89

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