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系統識別號 U0026-2308201719363000
論文名稱(中文) 以光電動技術檢測糖尿病視網膜病變之雙指標性蛋白Lipocalin 1 與 Tumor Necrosis Factor-α
論文名稱(英文) Optoelectrokinetically-Enabled Diagnosis of Diabetic Retinopathy with Dual Biomarkers Lipocalin 1 and Tumor Necrosis Factor-α
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 105
學期 2
出版年 106
研究生(中文) 林孝能
研究生(英文) Hsiao-Neng Lin
學號 P86041053
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-莊漢聲
口試委員-莊漢聲
口試委員-許聖民
口試委員-涂庭源
口試委員-張國恩
中文關鍵字 生醫檢測  糖尿病視網膜病變  指標性蛋白  微珠式三明治免疫分析  光電動技術  快速光電動圖紋法  濃縮  分選 
英文關鍵字 Optoelectrokinetic technique  Bead-based immunosensing  Diabetic retinopathy  biomarker  lipocalin 1  tumor necrosis factor-α 
學科別分類
中文摘要 糖尿病視網膜病變(DR)通常發生於慢性糖尿病患者之視網膜上。如未接受適當之醫學治療,此疾病最終將導致失明。目前臨床上的診斷主要以螢光血管造影以及眼部斷層掃描,但目前這些技術皆受限於非定量性檢測以及依賴眼科醫師之臨床經驗判斷。為了突破這些限制,本文發展一套光電動式微珠免疫法來同時偵測兩種指標性蛋白Lipocalin 1 (LCN1)以及腫瘤壞死因子-α (TNF-α) 。由之前文獻已知此兩種指標性蛋白之濃度在糖尿病視網膜病變患者淚液中會隨著DR嚴重的程度而增加。我們藉由修飾有抓取式抗體之微珠來連結指標性蛋白,以微珠式三明治免疫法(Bead-based Sandwiched Immunoassay)之形式來表現,此三明治免疫結構包含抓取式抗體-抗原-探測性抗體。此外藉由外加的螢光二抗與探測性抗體接合以螢光訊號來表示指標性蛋白之接合是否存在。指標性蛋白之濃度可藉由量測螢光訊號之強度來表示。本文所使用之光電動技術稱為Rapid electrokinetic patterning (REP) 快速光電動圖紋法。此技術可濃縮修飾有抗體之微珠成一小簇,有放大螢光訊號之效。這套技術目前的The limit of detection (LOD)偵測極限目前可達到110 pg/mL。操作REP必須同時施加光以及電場。而微珠之表現行為與提供電場之頻率以及微珠尺寸有密切相關。根據這項機制,目前在同時混合3種不同粒徑微珠(1, 3 and 5 μm)之液滴中,可依尺寸順利分離。藉由以不同顏色螢光來標定不同粒徑微珠上之個別的指標性蛋白LCN1 與TNF-α,糖尿病視網膜病變之雙指標蛋白可以在同時被偵測出。藉由交叉比對糖尿病視網膜病變患者淚液中之多種指標性蛋白,疾病檢測的精準度以及效率皆得以提升。於國立成功大學眼科部取得臨床上之淚液,只需要1 μL 的樣品就能做分析量測。結果顯示增生性糖尿病視網膜病變(PDR)患者淚液中LCN1之濃度約大於健康人或其他疾病患者一萬倍左右。此技術與一般常規眼科診斷方法相比,目前取樣總人數32人中84.85%在PDR患者組中相同。基於上述之結果,此光電動技術已提供定量數據之方式期望能提供眼科醫師在判斷或治療上之協助。更甚者,在未來能延伸至其他眼部疾病之診斷。
英文摘要 Diabetic retinopathy (DR) is a morbidity that usually occurs in the retina of patients with chronic diabetes. Without proper medical intervention, DR eventually leads to blindness. The clinical application of current diagnostic techniques, such as fluorescence angiography and optical coherence tomography, remains limited because they employ non-quantitative examinations and their results vary depending on the individual experience of ophthalmologists. To address these limitations, this study developed an immunosensing technique based on optoelectrokinetically enabled beads for simultaneously detecting two biomarkers, namely, lipocalin 1 (LCN1) and tumor necrosis factor-α (TNF-α). The concentrations of these biomarkers in the tear of patients with DR increase with increasing disease severity. A sandwiched structure comprising capture antibody–antigen probe antibody was formed by conjugating particles with capture antibodies. The additional dye-labeled secondary antibodies were eventually attached to the probe antibodies to determine the presence of the target biomarkers. The concentration of the target biomarkers was measured based on fluorescence intensity. The optoelectrokinetic technique, named as rapid electrokinetic patterning (REP), was used to enhance the fluorescence signal by concentrating the functionalized particles into a cluster. REP exhibited limit of detection as low as 110 pg/mL and was operated with simultaneous applications of optical illumination and electric field. A phase diagram was plotted to reveal the particle behavior in response to electrical frequency and particle size. The diagram showed the successful separation of the mixture with different particle sizes (1, 3, and 5 μm). Particles of different sizes were labeled with different dye colors to detect the DR biomarkers. The efficiency and accuracy of the proposed technique were increased by cross-matching multiple biomarkers in the tear of patients with DR. Tears were obtained by capillary from the Department of Ophthalmology at the NCKU Hospital and used for clinical examination (1 μL of the sample fluid). The concentration of LCN-1 in tears collected from patients with proliferative diabetic retinopathy (PDR) is approximately 10,000 times higher than that in tears from healthy subjects. The accuracy of REP reached 84.85 % (N=33) in PDR compared with conventional diagnosis by opthalmoscopy. Hence, the optoelectrokinetic system can be used for treatments performed by ophthalmologists and can be extended to diagnose other oculopathies in the future.
論文目次 摘要 ……………………………………………………………………...……......I
ABSTRACT ……………………………………………………………….………..II
誌謝 ………………………………………...…………………………………...IV
CONTENTS ………………………………………………………………...………V
LIST OF TABLE ………………………………………………………………....VII
LIST OF FIGURES …………………………………………………………...VIII
NOMENCLATURE ………………………………………………………….…..X
CHAPTER 1 INTRODUCTION ……………………………………………………1
1.1 Motivation and Overview…………………………………………………...1
1.2 Diabetic Retinopathy………………………………………………………..3
1.2.1 Types of Diabetic Retinopathy……………………………………4
1.2.2 Diagnostics for Diabetic Retinopathy…………………………….6
1.3 Biological Markers In tears From Diabetic Retinopathy…………………….7
1.4 Biomarker Diagnosis in Human Tear Fluid………………………………….8
1.4.1 Bead-based Sandwiched Immunoassay…………………………..9
1.5 Rapid Electrokinetic Patterning……………………………………………10
1.6 Aims of the Thesis…………………………………………………………13
CHAPTER 2 MATERIALS AND METHODS …………………………………..14
2.1 Clinical Tear sample……………………………………………………….14
2.2 Bead-based Sandwiched Immunosensing…………………………….........15
2.3 Sample Preparation and Protocol…………………………………………..17
2.3.1 Preparation of the Antibody on Particles…………………….......17
2.3.2 PEG Blocking and Target Antigen…………………....................18
2.3.3 Probe Antibody and Secondary Antibody……………………….18
2.4 Rapid Electrokinetic Patterning System …………………………………..19
2.4.1 Chip Fabrication………………………………………………...19
2.4.2 Experimental Setup ……………………………………………..21
2.4.3 Mechanism of the Optoelectrokinetic Method…………………..22
2.4.4 Mechanism of Particle Sorting…………………………………..24
2.5 Data Analysis………………………………………………………………25
CHAPTER 3 RESULTS AND DISCUSSIONS …………………………………..26
3.1 Effect of Frequency ………………………………………..………………26
3.1.1 Sorting Two Kinds of Different Particle Size……………………29
3.1.2 Sorting Three Kinds of Different Particle Size…………………..33
3.2 Modification of Substrate Surface………………………………………....35
3.3 Results of Bead-based Immunosensing……………………………………36
3.3.1 Bead-based Immunosensing with QDs………………………….37
3.3.2 Bead-based Immunosensing with Dyes…………………………39
3.3.2.1 Detection of LCN1…………………………………………40
3.3.2.2 Detection of TNF-α ………………………………………..41
3.3.2.3 Simultaneous Detection of Dual Biomarkers ……………..43
3.4 Clinical Pre-Screening…...………………………………………………...45
3.5 Comparison with other fluorescence-based technique……………………..53
CHAPTER 4 CONCLUSION …………………………………………………..55
CHAPTER 5 FUTURE WORK …………………………………………………..57
REFERENCES …………………………………………………………………..58
APPENDICES………………………………………………………………………..61
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