生物分子檢測是生醫領域重要的發展技術。該類技術的提昇可推動疾病預測、精準醫療等願景之實現。傳統生物分子檢測技術多被認為存在檢測時間太長、操作須由實驗專業人員進行、設備昂貴、受干擾物影響大等缺點。而一般生醫感測器雖然有檢測物免標記、靈敏性高、體積小等優勢，然而當檢測物濃度極低或是需要精準計算待測分子數量時，則已不具有足夠之空間解析度來獲取資訊。
　　固態式奈米孔感測器因具備接近單分子等級之空間解析能力，陸續在蛋白質檢測、細菌型態辨別、DNA定序等方面屢獲實驗驗證應用可行性。本研究以開發應用於生物分子檢測之固態式奈米孔微流體晶片為目標，延續本實驗室過去論文所建立之穩健固態式奈米孔元件製程，並彌補學長論文階段不足之面向，例如：微流體封裝方法優化、奈米孔試片流體狀態與孔徑之追蹤、本實驗室之精密電性量測系統建立與應用、以及他種生物分子易位螢光觀測與電流訊號量測。
　　本研究最終成功發展出可減少過去90 % 製程時間之多層模組化微流體封裝結構，並完全改善填充液體或空氣洩漏問題。利用微流體晶片之IV定性結果，確立奈米孔試片孔徑之追蹤方法。本研究建立了可應用於奈米孔感測器之精密電性量測系統，確認在受測試片電阻為50 MΩ下之電流雜訊為16 pA，也探討使用本系統進行奈米孔感測器量測時之雜訊變化模式，幫助本實驗室未來在精密電性量測相關研究之發展。細菌易位螢光觀測部分，本研究觀察大腸桿菌於1.5 μm ~ 5 μm微米孔周圍受電場驅動而表現出聚集、驅散、堆積、穿孔等不同的特徵行為，並在最後完成大腸桿菌微米孔易位電流訊號量測，單個訊號峰值屆於0.5~5 nA，時間長度約30 ms，並與螢光觀測之趨勢相互驗證。本論文是首篇描述了微流體在奈米孔試片中之場控導通及導通現象、以及描述細菌螢光觀測結果與電流訊號關係之研究。

Solid-state nanopore sensors have been experimentally validated in terms of protein detection, drug molecular detection, bacterial type discrimination, and DNA sequencing, due to their near-single-molecule-level spatial resolution. This research aims to develop solid-state nanopore microfluidic chips for biomolecule detection, and is expected to improve the shortcomings of the past work from our group, such as optimization of packaging methods, tracking of fluid state and pore size of nanopore test strips, establishment and application of precision electrical measurement systems in this laboratory, and biochemical molecular translocation fluorescence observation and current signal measurement.
Eventually, this study develops a multi-layer modular microfluidic package that reduced the 90% of the process time and completely solved the leakage issue of PDMS mocrofluidic chip. Also, the tracking method of the pore size of the nanopore test piece has been established. A precision electrical measurement system that can be applied to nanopore sensors has been established, helping the development of nanopore-related research in our group. This study observed the translocation behavior of Escherichia coli around the nanopore using fluorescence observation. It was found that the bacteria were driven by the electric field and showed different characteristic behaviors such as aggregation, dispersal, accumulation and perforation around the nanopore. At the end, translocation current signal of E.coli was measured and mutually verified with the result of fluorescence observation. This paper is the first to describe the open circuit and conduction phenomenon of nanopore-embedded microfluidic chip, and also to describe the relationship between fluorescence observations and translocation signals of E. coli.

[1] M. S. Pepe, R. Etzioni, Z. Feng, J. D. Potter, M. L. Thompson, M. Thornquist, M. Winget and Y. Yasui, "Phases of biomarker development for early detection of cancer," Journal of the National Cancer Institute, 2001, 93, 1054-1061.
[2] B. Clyne and J. S. Olshaker, "The C-reactive protein," The Journal of emergency medicine, 1999, 17, 1019-1025.
[3] S. G. Paquette, D. Banner, Z. Zhao, Y. Fang, S. S. Huang, A. J. Leomicronn, D. C. Ng, R. Almansa, I. Martin-Loeches, P. Ramirez, L. Socias, A. Loza, J. Blanco, P. Sansonetti, J. Rello, D. Andaluz, B. Shum, S. Rubino, R. O. de Lejarazu, D. Tran, G. Delogu, G. Fadda, S. Krajden, B. B. Rubin, J. F. Bermejo-Martin, A. A. Kelvin and D. J. Kelvin, "Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection," PLoS One, 2012, 7, e38214.
[4] C. L. Sawyers, "The cancer biomarker problem," Nature, 2008, 452, 548-52.
[5] S. F. Kingsmore, "Multiplexed protein measurement: technologies and applications of protein and antibody arrays," Nat Rev Drug Discov, 2006, 5, 310-20.
[6] X. Luo and J. J. Davis, "Electrical biosensors and the label free detection of protein disease biomarkers," Chem Soc Rev, 2013, 42, 5944-62.
[7] M. I. Azzam, S. M. Ezzat, B. A. Othman and K. A. J. W. S. El-Dougdoug, "Antibiotics resistance phenomenon and virulence ability in bacteria from water environment," 2017, 31, 109-121.
[8] O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, "Protein measurement with the Folin phenol reagent," Journal of biological chemistry, 1951, 193, 265-275.
[9] B. J. Olson and J. Markwell, "Assays for determination of protein concentration," Current protocols in protein science, 2007, 3.4. 1-3.4. 29.
[10] M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Analytical biochemistry, 1976, 72, 248-254.
[11] S. Lee, K. M. Song, W. Jeon, H. Jo, Y. B. Shim and C. Ban, "A highly sensitive aptasensor towards Plasmodium lactate dehydrogenase for the diagnosis of malaria," Biosens Bioelectron, 2012, 35, 291-6.
[12] K. C. Lin, V. Kunduru, M. Bothara, K. Rege, S. Prasad and B. L. Ramakrishna, "Biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarkers proteins," Biosens Bioelectron, 2010, 25, 2336-42.
[13] J. J. Kasianowicz, E. Brandin, D. Branton and D. W. Deamer, "Characterization of individual polynucleotide molecules using a membrane channel," Proceedings of the National Academy of Sciences, 1996, 93, 13770-13773.
[14] F. Haque, J. Li, H. C. Wu, X. J. Liang and P. Guo, "Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA," Nano Today, 2013, 8, 56-74.
[15] I. M. Derrington, T. Z. Butler, M. D. Collins, E. Manrao, M. Pavlenok, M. Niederweis and J. H. Gundlach, "Nanopore DNA sequencing with MspA," Proc Natl Acad Sci U S A, 2010, 107, 16060-5.
[16] P. Jing, F. Haque, D. Shu, C. Montemagno and P. Guo, "One-way traffic of a viral motor channel for double-stranded DNA translocation," Nano Lett, 2010, 10, 3620-7.
[17] D. Wendell, P. Jing, J. Geng, V. Subramaniam, T. J. Lee, C. Montemagno and P. Guo, "Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores," Nat Nanotechnol, 2009, 4, 765-72.
[18] F. Haque, J. Lunn, H. Fang, D. Smithrud and P. Guo, "Real-time sensing and discrimination of single chemicals using the channel of Phi29 DNA packaging nanomotor," ACS nano, 2012, 6, 3251-3261.
[19] F. Traversi, C. Raillon, S. M. Benameur, K. Liu, S. Khlybov, M. Tosun, D. Krasnozhon, A. Kis and A. Radenovic, "Detecting the translocation of DNA through a nanopore using graphene nanoribbons," Nat Nanotechnol, 2013, 8, 939-45.
[20] G. F. Schneider, S. W. Kowalczyk, V. E. Calado, G. Pandraud, H. W. Zandbergen, L. M. Vandersypen and C. Dekker, "DNA translocation through graphene nanopores," Nano Lett, 2010, 10, 3163-7.
[21] D. Fologea, B. Ledden, D. S. McNabb and J. Li, "Electrical characterization of protein molecules by a solid-state nanopore," Appl Phys Lett, 2007, 91, 539011-539013.
[22] S. Garaj, W. Hubbard, A. Reina, J. Kong, D. Branton and J. Golovchenko, "Graphene as a subnanometre trans-electrode membrane," Nature, 2010, 467, 190-193.
[23] P. Xie, Q. Xiong, Y. Fang, Q. Qing and C. M. Lieber, "Local electrical potential detection of DNA by nanowire-nanopore sensors," Nat Nanotechnol, 2011, 7, 119-25.
[24] B. Song, G. F. Schneider, Q. Xu, G. Pandraud, C. Dekker and H. Zandbergen, "Atomic-scale electron-beam sculpting of near-defect-free graphene nanostructures," Nano Lett, 2011, 11, 2247-50.
[25] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva and A. A. Firsov, "Electric field effect in atomically thin carbon films," science, 2004, 306, 666-669.
[26] P. K. Kannan, D. J. Late, H. Morgan and C. S. Rout, "Recent developments in 2D layered inorganic nanomaterials for sensing," Nanoscale, 2015, 7, 13293-312.
[27] M. Wanunu, W. Morrison, Y. Rabin, A. Y. Grosberg and A. J. N. n. Meller, "Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient," 2010, 5, 160.
[28] K. Kant, C. Priest, J. Shapter and D. J. S. Losic, "The influence of nanopore dimensions on the electrochemical properties of nanopore arrays studied by impedance spectroscopy," 2014, 14, 21316-21328.
[29] M. I. Rudenko, M. R. Holmes, D. N. Ermolenko, E. J. Lunt, S. Gerhardt, H. F. Noller, D. W. Deamer, A. Hawkins, H. J. B. Schmidt and Bioelectronics, "Controlled gating and electrical detection of single 50S ribosomal subunits through a solid-state nanopore in a microfluidic chip," 2011, 29, 34-39.
[30] S. P. Wang, "Fabrication and Characterization of Solid-State Nanopores on Molybdenum Disulfide Nanosheets for DNA Translocation," Master Degree Thesis, 2017,
[31] I. Yanagi, T. Ishida, K. Fujisaki and K. Takeda, "Fabrication of 3-nm-thick Si3N4 membranes for solid-state nanopores using the poly-Si sacrificial layer process," Sci Rep, 2015, 5, 14656.
[32] B. M. Venkatesan, A. B. Shah, J. M. Zuo and R. Bashir, "DNA Sensing using Nano-crystalline Surface Enhanced Al(2)O(3) Nanopore Sensors," Adv Funct Mater, 2010, 20, 1266-1275.
[33] J. Feng, K. Liu, R. D. Bulushev, S. Khlybov, D. Dumcenco, A. Kis and A. Radenovic, "Identification of single nucleotides in MoS2 nanopores," Nat Nanotechnol, 2015, 10, 1070-6.
[34] K. Liu, J. Feng, A. Kis and A. Radenovic, "Atomically thin molybdenum disulfide nanopores with high sensitivity for DNA translocation," ACS nano, 2014, 8, 2504-2511.
[35] Oxford Nanopore Technologies, MinION.,
[36] M. Wanunu, T. Dadosh, V. Ray, J. Jin, L. McReynolds and M. Drndic, "Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors," Nat Nanotechnol, 2010, 5, 807-14.
[37] A. P. Ivanov, E. Instuli, C. M. McGilvery, G. Baldwin, D. W. McComb, T. Albrecht and J. B. Edel, "DNA tunneling detector embedded in a nanopore," Nano Lett, 2011, 11, 279-85.
[38] A. B. Farimani, K. Min and N. R. Aluru, "DNA base detection using a single-layer MoS2," ACS nano, 2014, 8, 7914-7922.
[39] M. Tsutsui, T. Yoshida, K. Yokota, H. Yasaki, T. Yasui, A. Arima, W. Tonomura, K. Nagashima, T. Yanagida and N. J. S. r. Kaji, "Discriminating single-bacterial shape using low-aspect-ratio pores," 2017, 7, 17371.
[40] M. Tsutsui, M. Tanaka, T. Marui, K. Yokota, T. Yoshida, A. Arima, W. Tonomura, M. Taniguchi, T. Washio and M. J. A. c. Okochi, "Identification of individual bacterial cells through the intermolecular interactions with peptide-functionalized solid-state pores," 2018, 90, 1511-1515.
[41] A. Arima, M. Tsutsui, I. H. Harlisa, T. Yoshida, M. Tanaka, K. Yokota, W. Tonomura, M. Taniguchi, M. Okochi and T. J. S. r. Washio, "Selective detections of single-viruses using solid-state nanopores," 2018, 8, 16305.
[42] K. Rogers, " "Facts about E. coli: dimensions, as discussed in bacteria: Diversity of structure of bacteria: – Britannica Online Encyclopedia" " Britannica.com, 2015,
[43] M. Tsutsui, K. Yokota, T. Yoshida, C. Hotehama, H. Kowada, Y. Esaki, M. Taniguchi, T. Washio and T. J. A. s. Kawai, "Identifying single-particles in air using a 3D-integrated solid-state pore," 2019,
[44] H. P. Erickson, "Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy," Biol Proced Online, 2009, 11, 32-51.
[45] E. B. d. Oliveira, C. Gotschlich and T. Liu, "Primary structure of human C-reactive protein," Journal of Biological Chemistry, 1979, 254, 489-502.
[46] Sigma-aldrich, "Product Information : Interleukin-6 human,"
[47] Sigma-aldrich, "Product Information : ALBUMIN FROM BOVINE SERUM,"
[48] T. Xu, V. Pagadala and D. M. Mueller, "Understanding structure, function, and mutations in the mitochondrial ATP synthase," Microb Cell, 2015, 2, 105-125.