應用往復式微流體於適體篩選、電化學感測與電鍍效率增強之研究

Efficiency enhancements of aptamer selection, electrochemical sensing and electrodeposition by microfluidic shuttling

翁瑞鴻(Jui-Hong Weng)

指導教授 ： 陳林祈

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/博士(2019年)

https://doi.org/10.6342/NTU201903493

摘要

微流體平台是近年來廣為利用的化學、生物分析技術，其優點在於具備微量樣本與試劑的精準操控與自動化控制的發展潛力，可將傳統的各種分析技術優化並整合與微小晶片中。但隨著研究需求的增加，微流體平台的複雜度快速提升，許多新穎的結構設計與操作元件被附加上去。雖然有助於繁複的實驗操作，但也產生複雜的晶片製作程序與不友善的操作需求，這將不利於微流體分析技術與實場應用的拓展。因此，近年來有許多研究針對微流體平台的系統進行簡化，像是利用paper-based或pump-free等技術發展微型化感測應用。然而，如何有效的利用微流體的優勢並改善其層流限制仍是相當重要的議題。本研究提出以往復式微流體的操作策略進行層流效應與混合效應的探討。以簡單的微流道結構設計搭配彈性的微流體操作系統來取代繁雜的晶片設計，此微流體平台將具有更靈活的應用空間。針對此平台活用之微流體特性，將其應用於三種技術進行驗證與探討：適體篩選、電化學感測、電鍍製程。在適體篩選中，整合single-bead SELEX與微流體平台發展微珠陣列微流體晶片用於蛋白質適體篩選。往復式的流體輸送的確能增進結合速率與分離強度，使篩選效能提升。以STAT3作為標的，成功的篩選出具專一性與親和力的適體，且具有癌細胞基因調控的能力。在電化學感測應用中，微電極表面的氧化還原反應受到流動操作的影響，產生流動極化(flow-polarization)與促進質傳(facilitated mass transfer)的效果。此現象將增幅電化學訊號響應，並提升其感測靈敏度。在電鍍製程應用中，原先不穩定的普魯士藍(Prussian blue, PB)電鍍薄膜，在溶液流動狀態造成的表面物質更新與薄膜沖刷下，結構穩定的PB薄膜可以更牢固的貼附於電極上，以利後續的生化感測應用。針對微流體輸送產生之效應加以利用並改善微流體技術之限制，本平台之發展將有利於微流體應用之拓展。

關鍵字

適體 ； 電化學感測 ； 電鍍 ； 往復式微流體 ； 流動效應

並列摘要

Microfluidic platform is a widely-used technology for chemical and biological analysis in the recent years. Its advantages include the precise modulation on micro-scale of samples and reagents and the potential of automatic controlling. The traditional analysis techniques can be optimized and integrated into a tiny chip. But, with the increasing needs of researches, the microfluidic platform becomes more complex, and a variety of novel structure design and operation units are embedded into the chip. Although the delicate chips benefit to operate the complicated experiment, the drawbacks like the difficult chip fabrication and un-friendly operation requirement still limit the development of microfluidic analysis in on-site application. Therefore, many researches focus on the simplification of microfluidic platform, for example, using paper-based or pump-free technology to develop miniaturized sensors. However, how to efficiently utilize the superiority of microfluidics and improve the limitation of laminar flow is still an attractive issue. In our study, the strategy of microfluidic shutting is proposed to explore the laminar flow effect and the mixing effect. Combining a simple microchannel structure and a convenient microfluidic operating system to replace the complicated chip design, this platform have a more flexible space of application. Aiming to the features of microfluidics which can be wisely used in this platform, three kinds of technology is applied to verify the assumption: aptamer selection, electrochemical sensing and electrodeposition. In the aptamer selection, the single-bead SELEX and microfluidic platform are integrated to develop a microbeads-array microfluidic chip for aptamer selection. Shuttling liquid can enhance the binding rate and partitioning strength that improve the selection performance. STAT3-specific aptamers are identified with high specificity and affinity, and they show the ability of gene regulation in the cancer cell apoptosis. In the electrochemical sensing, the redox reaction on the microelectrode surface is influenced by flowing operation, and it produces the flow-polarization and facilitated mass transfer effects. This phenomenon can increase the electrochemical response and improve the sensitivity. In the electrodeposition, the Prussian blue film formed by electrodeposition is unstable under flowing condition. With the component refreshment on surface and film polishing which are caused by flowing liquid, the structure-stable PB film can firmly attach on the electrode that is beneficial to the following biosensing applications. Taking advantage of the effect generated by microfluidic transportation to improve the limitation of microfluidic technology, this platform shows the potential of expanding the current microfluidic applications.

並列關鍵字

aptamer ； electrochemical sensing ； electrodeposition ； microfluidic shuttling ； flow effect

參考文獻

Abolhasani, M., Jensen, K.F., 2016. Oscillatory multiphase flow strategy for chemistry and biology. Lab Chip 16, 2775-2784.

Al Khatib, M., Bellini, M., Pogni, R., Giaccherini, A., Innocenti, M., Vizza, F., Lavacchi, A., 2018. Effect of Electrode Shape and Flow Conditions on the Electrochemical Detection with Band Microelectrodes. Sensors 18, 3196.

Ali, M.A., Jiang, H., Mahal, N.K., Weber, R.J., Kumar, R., Castellano, M.J., Dong, L., 2017. Microfluidic impedimetric sensor for soil nitrate detection using graphene oxide and conductive nanofibers enabled sensing interface. Sensors and Actuators B: Chemical 239, 1289-1299.

Amatore, C., Da Mota, N., Sella, C., Thouin, L., 2008. General concept of high-performance amperometric detector for microfluidic (bio) analytical chips. Analytical chemistry 80, 4976-4985.

Anderson, R.C., Bogdan, G.J., Puski, A., Su, X., 1998. Advances in integrated genetic analysis. Micro Total Analysis Systems’ 98. Springer, pp. 11-16.