基於整合型微流體平台偵測甲基化之游離DNA利用於卵巢癌之伴隨式診斷與預後監測

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研究生：	鄭右壬 Cheng, Yu-Jen
論文名稱：	基於整合型微流體平台偵測甲基化之游離DNA利用於卵巢癌之伴隨式診斷與預後監測 Detection of Methylated Cell-Free DNA for Companion Diagnosis and Prognosis of Ovarian Cancer on an Integrated Microfluidic System
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	陳致真 Chen, Chih-Chen 許耿福 Hsu, Keng-Fu
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	73
中文關鍵詞：	甲基化、抑癌基因、游離DNA 、微流體、渦旋混合、液體活檢
外文關鍵詞：	methylation, tumor suppressor gene, cell-free DNA, microfluidics, vortex-mixing, liquid biopsy
相關次數：	點閱：2 下載：0
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甲基化的游離DNA被視為是一種新穎的生物標記物，它提供了一種微創方法針對卵巢癌進行伴隨式診斷，藥物選擇和預後。但是，由於甲基化的游離DNA濃度極低、分析的耗時長且需要複雜的人為操作流程，因此直接在游離DNA中分析抑癌基因的甲基化程度仍然具有挑戰性。在這項研究中，展示了一個整合型的微流體平台使用接有甲基CpG結合蛋白的磁珠和微流控設備（如：微型幫浦、微型閥門、微型混合器和微型聚合酶連鎖反應平台），這些設備可以實現（1）血漿中甲基化游離DNA的捕獲並達到72%的抓取率和（2）定量分析抑癌基因之甲基化程度。檢測極限可達到0.2 pg/µL的甲基化游離DNA，比先前的研究低三個數量級。此外，可以實現0.2到2000 pg/µL甲基化游離DNA的身理濃度檢測範圍。整個檢測過程可在2小時內自動化完成，且僅需20 µL的血漿，證實此整合型微流體平台可使用液體活檢完成卵巢癌的藥物選擇和預後。

Methylated cell-free DNA (cfDNA) has been seen as one of promising indicators that provides a minimally invasive tool for companion diagnosis, prognosis and therapy selection of ovarian cancer. However, exploring the methylation profile of specific tumor suppressor genes in cfDNA remains challenging due to its random fragments of genes, extremely low concentration and complex labor-intensive processes. In this presented study, an integrated microfluidic system was developed to utilized microfluidic devices (such as microvalves, micropumps, micromixers and micro polymerase chain reactor) and magnetic beads coated with methyl CpG binding domain proteins (MBD) which could implement (1) capture of methylated cfDNA in plasma with 72% of capture rate and (2) quantify methylated tumor suppressor genes in isolated methylated cfDNA. The limit of detection could be as low as 0.2 pg/µL, which is three orders of magnitudes lower than the benchtop method. Furthermore, a dynamic range from 0.2 to 2000 pg/µL of methylated cfDNA could cover the physiological range of methylated cfDNA. The whole on-chip assay could be automated within 2 hr while only required 20 µL of plasma, showing great potential for using liquid biopsy to perform companion medicine (i.e. therapy selection) and prognosis of ovarian cancer.

Abstract...............................................................2
摘要...................................................................3
誌謝...................................................................4
List of figures........................................................8 List of tables........................................................13 Nomenclature and abbreviations........................................15 Chapter 1 Introduction ...............................................16
1 Methylation of tumor suppressor genes.............................16
2 Tumor suppressor gene BRCA1.......................................18
3 Methylated cell-free DNA..........................................19
4 Methylation detection assays in microfluidic systems..............20
4.1 Bisulfite-based assay..........................................20
4.2 Bisulfite-free assay...........................................22
5 Motivation and novelty............................................23
Chapter 2 Materials and methods.......................................25
1 Sample preparation................................................25
2 Materials and reagents............................................27
3 Chip design and microfabrication..................................28
4 Experimental procedure and setup..................................33
Chapter 3 Results and discussion......................................39
1 Characterizations of the microfluidic devices.....................39
1.1 Pumping volume of the micropump................................39
1.2 Mixing efficiency of the vortex-type micromixer................42
2 On-chip assay of methylated cfDNA.................................44
2.1 Optimization of the on-chip enrichment of methylated cfDNA.....44
2.2 Specificity tests..............................................47
2.3 Limit of detection (LOD).......................................53
2.4 Tests of clinical samples......................................61
Chapter 4 Conclusions and Future perspectives.........................67
1 Conclusions.......................................................67
2 Future perspectives...............................................67
References............................................................69

                                

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