本論文以定向且單層的Halobacterium salinarum 紫膜 (purple membrane, PM)作為生物感測器之訊號轉換器來探討一般去氧核醣核酸與甲基化去氧核醣核酸分別作為基因探針來進行miR21-RNA之檢測並探討其差異，在將其應用於exosome細胞之檢測；紫膜中存在具有單方向光驅動質子幫浦之細菌視紫質蛋白(bacteriorhodopsin, BR)，不僅可以做為光電訊號感測器其單層膜晶片根據先前研究還能作為阻抗分析感測之基底。
本論文一共進行4種紫膜為基底之生物感測平台之檢測應用，第1種是以雷射作為光源並配上比量槽之微分光電流檢測，第2種是使用LED作為光源並配上靜態檢測槽來進行靜態光電流檢測，第3種也是使用LED作為光源且配上靜態檢測槽來進行動態即時檢測，第4種是使用EIS系統來進行電阻抗之檢測；針對兩種目標物分別為純miR21-RNA以及含有miR21-RNA之exosome溶液做檢測。首先於雷射光源比量槽系統之檢測我們分別使用了1 mM及10 mM之KCl電解液進行檢測，結果發現使用n-probe之生物光電檢測晶片擁有較高的晶片解析度，且由於n-probe帶有較少的負電，因此其負電排斥效應較一般probe少，所以我們將緩衝溶液之鹽離子濃度從10 mM降低到1 mM時使用n-probe的晶片依然能夠成功進行雜合。第二部分，我們透過比對4種不同系統之檢測結果我們發現其最低miR21-RNA可檢測濃度皆為5 aM且其檢量線之靈敏度一致，且透過動態即時檢測之光電流圖我們清楚看出其在動態吸附過程使用nDNA之晶片與使用一般DNA之晶片其動態吸附過程有明顯之差異，並且對exosome溶液進行檢測時溶液內雜質對於光電感測晶片之影響也能夠清楚的觀察，最後我們前面提到的四種方法對exosome溶液檢測後進行濃度反推計算，觀察其所有檢測結果後我們得知不管使用哪種系統其所得之檢測結果均相同，因此若之後要進行micro RNA之檢測可以依據實驗用途及目的挑選最適合的系統。

This study investigates the application of a biosensing platform based on oriented and single-layered Halobacterium salinarum purple membrane (PM) for the detection of miR21-RNA. Two types of gene probes, unmodified DNA (DNA) and methylated DNA (nDNA), were used to compare their performance in detecting pure miR21-RNA solution and miR21-RNA contained in exosome solutions. Four different detection methods were employed: (1) laser-based differential photocurrent detection with a cuvette, (2) LED-based static photocurrent detection with a measurement cell, (3) LED-based real-time detection with a measurement cell, and (4) electrochemical impedance spectroscopy, EIS detection.
The results showed that the n-probe biosensing chip exhibited higher chip resolution and less negative charge repulsion effect than the DNA probe chip. The minimum detectable concentration of miR21-RNA was 5 aM for all four detection methods. The real-time photocurrent density plot clearly showed the difference in the dynamic adsorption process between the nDNA and DNA probes. The influence of impurities in the exosome solution on the photoelectric sensing chip could also be clearly observed. The concentration back-calculation results of the exosome solution detected by the four methods were all the same. Therefore, the most suitable system can be selected according to the experimental purpose and requirements for future microRNA detection.

Alice V, Wei-Lin Ou, Birgit P. Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum. 2017,10.1038.

Aharon O. Molecular ecology of extremely halophilic Archaea and Bacteria. 2001, 39, 1-7.

Ana E. Jenike. Marc K. Halushka. miR21-: a non-specific biomarker of all maladies. 2021, 9:18.

Cecilia Wickstrand, Robert Dods. Bacteriorhodopsin: Would the real structural intermediates please stand up. 2015, 536-553.

Chen H-M, Lin C-J, Jheng K-R, Kosasih A, Chang J-Y. Effect of graphene oxide on affinity-immobilization of purple membranes on solid supports. 2014, 116, 482-488

Chen, H.-M., Jheng, K.-R., Yu, A.-D., Hsu, C.-C., & Lin, J. H. Intercalating purple membranes into 2D β-alanine crystals to enhance photoelectric and nonlinear optical properties. Journal of the Taiwan Institute of Chemical Engineers, 2016, 64, 1-8.

Chen, H.-M., Jheng, K.-R., & Yu, A.-D. Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor. Biosensors Bioelectronics, 2017, 91, 24-31.

Hadeel Khamis, Sergei R, Philippa M, Ariel K. Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation. 2021, 10975-10987.

Hampp,N. Bacteriorhodopsin as a photochromic retinal protein for optical memories. Chemical Reviews, 2000, 100(5), 1755-1776

James D, Francis H-C-C. Molecular structure of nucleic acids-A structure for deoxyribose nucleic acids. 2007, 10.1097

Janos K, Lanyi. Proton transfers in the bacteriorhodopsin photocycle. 2006, 1012-1018.

Jerry Eichler. Halobacterium salinarum: Life with more than a grain of salt. 2023, 169:001327.

Kenjiro Y, Hiroyuki F. Probing multiple binding modes of DNA hybridization: A comparison between single-molecule observation and ensemble measurements. 2018, 3, 2084-2092.

Markus R, Norbert S. Real-time analysis of specific protein-DNA interactions with surface plasmon resonance. 2011, 816032, 19.

Matthew D, Tom P, Yu chen, Gabriele V. A macrocyclic peptide ligand binds the oncogenic microRNA-21 precusor and suppresses Dicer processing. 2017, 12(6): 1611-1620.

N. Tommerup, S. Kauppinen, A. Silahtaroglu, H Pfundheller. LNA-modified oligonucleotides are highly efficient as FISH probes. 2004, 107:32-37.

Roy markham, J. D. Smith. Structure of ribonucleic acid. 1951, NATURE vol.168.

S. Ikuta, K. Takagi, R. Bruce Wallace, K. Itakura. Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs. 1987, Nucleic Acids Research, Volume 15 .2.

Spudich, John L. Retinylidene proteins: Structures and funcions from archaea to humans. 2000, 16:365-92.

W.Y. Chen, Wen-Pin Hu, Chih-Chin Tsai, Yuh-Shyong Yand, Hardy Wai-Hong Chan. Synergetic improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing neutralized DNA as probe. 2018, 8:12598.

W.Y Chen, C.J. Huang, Z.E. Lin, Y.S. Yang, Hardy Wai-Hong Chan. Neutralized chimeric DNA probe for detection of single nucleotide polymorphism on surface plasmon resonance biosensor. 2018, 170-175.

Y. you, B.G. Moreira, M.A. Behlke, R. Owczarzy. Desing of LNA probes that improve mismatch discrimination. 2006, 10.1093.

H. Zipper, H. Brunner, J. Bernhagen, F. Vitzhum. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. 2004, 10.1093.

蕭奕岷，細菌視紫質單層塗覆光電感測晶片的光控制自旋過濾特性探討，國立台灣科技大學化學工程研究所碩士論文，2022

曾聖翔，紫膜複合生物光電晶片應用於腦癌血清miRNA與免疫檢測之探討，國立台灣科技大學化學工程研究所碩士論文，2021

廖清德，紫膜生物光電晶片於朝鮮薊抑菌研究之應用，國立台灣科技大學化學工程研究所碩士論文，2021

王翔禾，紫膜生物光電晶片之DNA檢測條件再適化與Micro-DNA之初步檢測，國立台灣科技大學化學工程研究所碩士論文，2020