用於快速抗菌藥敏性試驗基於並行納米陣列的微流控設備

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研究生：	歐薩伊 Osaid, Mohammad
論文名稱：	用於快速抗菌藥敏性試驗基於並行納米陣列的微流控設備 Parallel Nanoliter Arrays Based Microfluidic Device for Quick Antimicrobial Susceptibility Testing
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	張晃猷 Chang, Hwan-You 李炫昇 Lee, Shiuann-Sheng
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	66
中文關鍵詞：	微陣列、微流體、抗菌藥敏試驗、最低抑菌濃度
外文關鍵詞：	Microarrays, Microfluidics, Antimicrobial susceptibility testing, Minimum inhibitory concentration
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由於市場上可用的抗生素數量有限，耐藥菌的出現確實是一個巨大的挑戰。快速診斷細菌感染和細菌對抗生素的抗藥性測試（AST）有助於遏制細菌感染。用於執行AST的常規方法取決於細菌的生長，這是一個相對較長的過程，可能需要幾天的時間。近年來，微流體技術在快速診斷領域中很有前途，因為它可以顯著減少通過小體積進行AST所需的時間，因為所需的細菌數量較少，從而減少了從血液中提取細菌的時間。因此，這項研究提出了一種基於納升陣列的多重微流控平台，可在單個芯片上同時使用多種濃度或類型的抗生素進行AST。這裡開發的微流控系統由兩個微流控芯片組成，一個用於執行AST，由納升體積的微陣列組成，另一個用於稀釋抗生素，處理微升液體。將兩個芯片適當地集成以彼此轉移流體。用於執行AST的芯片由7條生產線組成，每條生產線都具有多個體積等於1納升的微陣列。該設備僅需一個加載過程即可生成所有微陣列，以使用由稀釋芯片製備的七種不同濃度的抗生素對AST進行檢測，從而使該設備實現了並行化和高效。該設備的獨特之處在於它可以執行納升量的AST。同時，它與用於稀釋抗生素的設備集成在一起，該設備可以處理微升體積的液體，以在6小時內確定MIC值。在這項工作中，使用七個不同濃度的氨芐西林和細菌（大腸桿菌ATCC 25922）在微流體平台上進行了AST驗證。由於所有抗生素均在同一設備上測試過，因此它們要經受完全相同的條件，因此適合比較。此外，採用合適的加載過程，以確保細菌在所有微陣列中同時填充和均勻分佈，以進行AST。該設備可以同時在芯片上測試7種抗生素的這種能力可以確定最小抑菌濃度（MIC）值，這將有助於臨床醫生進行早期診斷和治療。

Emergence of antibiotic-resistant bacteria is indeed a great challenge because of limited number of antibiotics available in the market. Quick diagnosis of bacterial infection and antimicrobial susceptibility testing (AST) of bacteria to antibiotics can be useful for curbing it. The conventional method employed for performing AST relies on bacterial growth, which is a relatively lengthy process, and may take a couple of days. In recent years, microfluidic technology has been promising in the field of fast diagnosis as it substantially decreases the time required by performing AST in small volume as less number of bacteria are required, which reduces the time required in bacterial culture derived from blood. This study therefore presents a nanoliter array-based multiplexed microfluidic platform to perform AST using multiple concentrations or types of antibiotics simultaneously on a single chip. The microfluidic system developed here was consisted of two microfluidic chips, one for performing AST, which was consisted of microarrays of nanoliter volume, and another chip for antibiotic dilution, which handled the fluid of microliter. The two chips were suitably integrated to transfer the fluid from one another. The chip for performing AST consisted of seven lines, each having multiple microarrays of volume equal one nanoliter. The device involved a single loading process to generate all the microarrays to perform AST with seven different concentrations of antibiotics, prepared by the dilution chip, which makes the device parallelized and efficient. The unique feature of the device is that it can perform AST in nanoliter volume. At the same time, it is integrated with a device for performing dilution of antibiotics, which could handle fluids in a micro-liter volume, to determine the MIC value in 6 hours. In this work, AST was performed using seven different concentrations of ampicillin with bacteria (E. coli ATCC 25922) was demonstrated on the microfluidic platform. Since all the antibiotics were tested on the same device, they were subjected to exactly the same conditions which makes them suitable for comparison. Furthermore, a suitable loading process was employed which ensured simultaneous filling and equal distribution of bacteria in all microarrays to perform the AST. This capability of device to test seven antibiotics simultaneously on the chip allows the determination of minimum inhibitory concentration (MIC) value, which will help clinicians for early diagnostics and treatment.

Table of contents
Abstract------------------------------------------------------3
List of Tables------------------------------------------------8
List of Figures-----------------------------------------------9
Chapter 1 Introduction---------------------------------------14
1    Bio-MEMS and microfluidic system---------------------14
2    Antimicrobial resistance-----------------------------15
3    Antimicrobial susceptibility testing (AST)-----------17
4    Methodology for AST----------------------------------18
5    Objective and novelty--------------------------------20
6    Structure of thesis----------------------------------22
Chapter 2 Materials and Methods------------------------------24
1 Design of microfluidic chip for AST----------------------24
2 Microfabrication of microfluidic chip--------------------25
3 Design and fabrication of chip for diluting antibiotics--28
4: Operation of microfluidic chip--------------------------32
5 Processes for droplet generation-------------------------34
6 Experimental setup for bacteria counting-----------------37
7 Sample preparation for on bench AST using color----------38
8 Experimental setup for on bench AST using fluorescence---38
9 Experimental setup for AST on chip-----------------------39
Chapter 3   Results and Discussion---------------------------41
1 Distribution of bacteria in microarrays------------------41
2 Computational analysis of antibiotics diffusion in the microarrays    -------------------------------------------------------------42
3 Optimized concentration of resazurin for AST-------------43
4 On-bench AST results using color-------------------------45
5 On-bench AST results using fluorescence intensity--------46
6 On-chip AST results and algorithm to find MIC------------48
7 Characterization of antibiotics dilution chip------------53
8 Integration of microfluidic devices----------------------54
9 Discussion-----------------------------------------------56
Chapter 4   Conclusions and Future Works---------------------58
1 Conclusions----------------------------------------------58
2 Future Works---------------------------------------------59
References---------------------------------------------------60


                                

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