Halobacterium salinarum紫色細胞膜 (purple membrane, PM) 簡稱紫膜，內含有細菌視紫質 (bacteriorhodopsin, BR) 以三聚體形式形成六角晶格。BR內含有一視黃醛分子，當BR受光激發後，視黃醛結構開始改變使BR將質子由細胞內側推送至細胞外側，因此BR為單一方向之光驅動質子幫浦，利用此特性再外加電極與導線可產生光電流訊號，藉此我們已開發出以紫膜為訊號轉換器之光電生物感測晶片。本論文分為兩部分，第一部分可檢測腦癌病患血清中miR17-RNA晶片之開發，首先將單股miRNA之miR17-probe DNA固定化於PM晶片上，再分別與互補的miR17-DNA和miR17-RNA以及修飾有奈米金 (gold nanoparticle, AuNPs) 的miR17-DNA和miR17-RNA雜合。結果發現固定有miR17-probe的PM晶片對miR17-DNA及miR17-RNA檢測時，最低可檢測濃度為50 aM，檢測範圍為50 aM ~ 500 fM，晶片光電流值分別下降5.7% ~ 28.7%及4.1% ~ 29.7%。對於修飾有AuNPs之miR17-DNA和miR17-RNA進行檢測，最低可檢測濃度同樣為5 aM，檢測範圍為5 aM ~ 500 fM，晶片光電流值分別下降15.0% ~ 60.2%及24.8% ~ 63.1%。最後對血清檢測初步進行可行性探討，發現將修飾有AuNPs之miR17-DNA與血清混合後進行檢測時，血清至少需要稀釋200倍才不影響晶片檢測結果。本研究結果可提供未來對腦癌血清檢測的參考。第二部分為延續本實驗室已建立的紫膜生物光電免疫晶片製作技術，開發serum amyloid A1 (SAA1) 蛋白的光電PM膜免疫感測晶片，首先利用protein A/G將SAA1單株抗體固定化於PM晶片上後，即可檢測SAA1蛋白。此外，可繼續以另一個SAA1多株抗體進行三明治檢測後，再使用經奈米金修飾後的二次抗體進行訊號放大以提高檢測靈敏度。僅對SAA1蛋白進行檢測時，最低可檢測濃度為1 ng/mL，檢測範圍為1 ng/mL~10 μg/mL，晶片光電流值下降5.3% ~ 24.0%；若以三明治方法加上奈米金訊號放大進行第二種檢測方法時，SAA1蛋白的最低可檢測濃度為10 pg/mL，檢測範圍為10 pg/mL~10 μg/mL，晶片光電流下降 21.4 % ~ 67.9 %。因此以後者三明治法加上奈米金訊號放大，可有效提升SAA1抗體-bPM膜複合晶片的SAA1蛋白檢測靈敏度。此晶片對glutathione S-transferase並無非特異性吸附。對病患血清先進行初步可行性檢測，發現血清需要稀釋200倍以上才不會對晶片檢測造成影響；對於高SAA1血清濃度的檢體，血清甚至需要稀釋到2000或4000倍。最後，對27個病患血清以所開發的光電免疫感測晶片進行檢測，並與Western blot electrogenerated chemiluminescence (ECL) 分析相結果比較，發現若以血清稀釋2000倍及4000倍進行晶片檢測後的SAA1平均值做比較，則與ECL結果間具有良好相關值(r=0.97)，顯示本研究所開發的免疫感測晶片適合用以臨床檢測。

The purple membrane (PM) of Halobacterium salinarum contains bacteriorhodopsin (BR), which exists in the form of trimers packing as hexagonal lattices. Each BR contains a retinal chromophore. When BR is excited by light, the structure of retinal changes, making BR pump a proton from the intracellular side to the extracellular side of PM. Therefore, BR is a unidirectional light-driven proton pump capbale to generate photocurrents through electric circuits. We have previously developed several different photoelectric biosensors using PM as the signal transducer. There are two parts in this thesis. The first part is to develop a PM-based miRNA biosensor to detect serum miR17-RNA in glioma patients. The single-stranded miR17-probe DNA is first immobilized on a PM-coated chip to detect the complementary miR17-DNA and miR17-RNA, as well as another miR17-DNA and miR17-RNA each conjugated with gold nanoparticles (AuNPs). For both of miR17-DNA and miR17-RNA detections, the lowest detection concentration is 50 aM and the dynamic range is 50 aM ~ 500 fM, with photocurrent reduction ranging 5.7% ~ 28.7% and 4.1% ~ 29.7%, respectively. For both detections of miR17-DNA and miR17-RNA conjugated with AuNPs, the lowest detection concentration is 5 aM and the dynamic range is 5 aM ~ 500 fM, with photocurrent reduction ranging 15.0% ~ 60.2% and 24.8% ~ 63.1%, respectively. Through a preliminary study, at least 200 folds of serum dilution is suggested to minimize interference. Those results suggest the feasibility of applying the developed miRNA biosensor in glioma detect. The second part is to device a PM-based immunosensor to detect serum amyloid A1 (SAA1) in glioma patients using the preparation technique we previously developed. Monoclonal antibodies against SAA1 are first immobilized on a PM-coated chip using protein A/G as the linker to detect SAA1. Furthermore, polyclonal antibodies against SAA1 are employed for sandwich assay, which are subsequently labeled with secondary antibodies conjugated with AuNPs to amplify the detection signal. For the detection without further sandwich assay, the lowest detection concentration on SAA1 is 1 ng/mL and the dynamic range is 1 ng/mL~10 μg/mL, with photocurrent reduction ranging 5.3% ~ 24.0%. On the other hand, when the sandwich assay is employed along AuNPs signal amplification, the lowest detection concentration on SAA1 is 10 pg/mL and the dynamic range is 10 pg/mL~10 μg/mL, with photocurrent reduction ranging 21.4% ~ 67.9%, indicating a significant increase of SAA1 detection sensitivity. This immunosensor hardly exhibits any unspecific binding to glutathione S-transferase. Upon the feasibility test with patient serum, at least 200-fold dilution on serum is suggested to minimize interference, and 2000/4000-fold dilutioin is suggested for serum containing high concentrations of SAA1. Finally, the immunosensor is used to analyze 27 patient sera. In comparison with the analysis with the Western-blot electrochemiluminescence immunoassay, a good correlation (r=0.97) is achieved when the averaged SAA1 values, which are obtained from the immunosensor assays on 2000- and 4000-fold diluted samples, are used. This study suggests the applicability of the developed immunosensor in clinical diagnosis.

Al-Aribe KM, Knopf GK, Bassi AS. Photoelectric monolayers based on
self-assembled and oriented purple membrane patches. Journal of
microelectromechanical systems. 2011, 20(4). 800-810.

Bartosik M, Hrstka R, Palecek E, Vojtesek B. Magnetic bead-based hybridization
assay for electrochemical detection of microRNA. Analytica chimica acta. 2014, 813,
35-40.

Calboli FC, Cox DG, Buring JE, Gaziano JM, Ma J, Stampfer M, Willett WC,
Tworoger SS, Hunter DJ, Camargo Jr CA. Prediagnostic plasma IgE levels and risk of
adult glioma in four prospective cohort studies. Journal of the National Cancer
Institute. 2011, 103(21). 1588-1595.

Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL,
Mahuvakar VR, Andersen MR. Real-time quantification of microRNAs by stem–loop
RT–PCR. Nucleic acids research. 2005, 33(20). e179-e179.

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.

Chen H-M, Jheng K-R, Yu A-D, Hsu C-C, Lin JH. 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, Lin C-J, Jheng K-R, Kosasih A, Chang J-Y. Effect of graphene oxide on
affinity-immobilization of purple membranes on solid supports. Colloids Surfaces B:
Biointerfaces. 2014, 116, 482-488.

Deng H, Liu Q, Wang X, Huang R, Liu H, Lin Q, Zhou X, Xing D. Quantum
dots-labeled strip biosensor for rapid and sensitive detection of microRNA based on
target-recycled nonenzymatic amplification strategy. Biosensors Bioelectronics. 2017,
87, 931-940.

Dong H, Jin S, Ju H, Hao K, Xu L-P, Lu H, Zhang X. Trace and label-free microRNA
detection using oligonucleotide encapsulated silver nanoclusters as probes. Analytical
chemistry. 2012, 84(20). 8670-8674.

Elstner A, Stockhammer F, Nguyen-Dobinsky T-N, Nguyen QL, Pilgermann I, Gill A,
Guhr A, Zhang T, Von Eckardstein K, Picht T. Identification of diagnostic serum
protein profiles of glioblastoma patients. Journal of neuro-oncology. 2011, 102(1).
71-80.

Fang S, Lee HJ, Wark AW, Corn RM. Attomole microarray detection of microRNAs
by nanoparticle-amplified SPR imaging measurements of surface polyadenylation
reactions. Journal of the American Chemical Society. 2006, 128(43). 14044-14046.

Gruszka R, Zakrzewska M. The oncogenic relevance of miR-17-92 cluster and its
paralogous miR-106b-25 and miR-106a-363 clusters in brain tumors. International
journal of molecular sciences. 2018, 19(3). 879.

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

Jones TA, Jeyapalan JN, Forshew T, Tatevossian RG, Lawson AR, Patel SN, Doctor
GT, Mumin MA, Picker SR, Phipps KP. Molecular analysis of pediatric brain tumors
identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-κB
pathways. Acta neuropathologica communications. 2015, 3(1). 86.

Kim YJ, Gallien S, El‐Khoury V, Goswami P, Sertamo K, Schlesser M, Berchem G,
Domon B. Quantification of SAA1 and SAA2 in lung cancer plasma using the
isotype‐specific PRM assays. Proteomics. 2015, 15(18). 3116-3125.

Knebel FH, Uno M, Galatro TF, Bellé LP, Oba-Shinjo SM, Marie SKN, Campa A.
Serum amyloid A1 is upregulated in human glioblastoma. Journal of neuro-oncology.
2017, 132(3). 383-391.

Kosaka N, Iguchi H, Ochiya TJCs. Circulating microRNA in body fluid: a new
potential biomarker for cancer diagnosis and prognosis. Cancer science. 2010,
101(10). 2087-2092.

Li F, Zhang H, Dever B, Li X-F, Le XC. Thermal stability of DNA functionalized
gold nanoparticles. Bioconjugate chemistry. 2013, 24(11). 1790-1797.

Lin CY, Yang ST, Shen SC, Hsieh YC, Hsu FT, Chen CY, Chiang YH, Chuang JY,
Chen KY, Hsu TI. Serum amyloid A1 in combination with integrin αVβ3 increases
glioblastoma cells mobility and progression. Molecular oncology. 2018, 12(5).
756-771.

Lin Y-C, Lin C-Y, Chen H-M, Kuo L-P, Hsieh C-E, Wang X-H, Cheng C-W, Wu C-Y,
Chen Y-S. Direct and label-free determination of human glycated hemoglobin levels
using bacteriorhodopsin as the biosensor transducer. Sensors. 2020, 20(24). 7274.

Linhares P, Carvalho B, Vaz R, Costa BM. Glioblastoma: is there any blood
biomarker with true clinical relevance? International journal of molecular sciences.
2020, 21(16). 5809.

Lu S, Wang S, Geng S, Ma S, Liang Z, Jiao B. Increased expression of microRNA-17
predicts poor prognosis in human glioma. Journal of Biomedicine Biotechnology.
2012, 2012.

Lusi E, Passamano M, Guarascio P, Scarpa A, Schiavo L. Innovative electrochemical
approach for an early detection of microRNAs. Analytical chemistry. 2009, 81(7).
2819-2822.

Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL,
Peterson A, Noteboom J, O'Briant KC, Allen A. Circulating microRNAs as stable
blood-based markers for cancer detection. Proceedings of the National Academy of
Sciences of the United States of America. 2008, 105(30). 10513-10518.

Moshkovskii SA, Serebryakova MV, Kuteykin‐Teplyakov KB, Tikhonova OV,
Goufman EI, Zgoda VG, Taranets IN, Makarov OV, Archakov AI. Ovarian cancer
marker of 11.7 kDa detected by proteomics is a serum amyloid A1. Proteomics. 2005,
5(14). 3790-3797.

Nogly P, Weinert T, James D, Carbajo S, Ozerov D, Furrer A, Gashi D, Borin V,
Skopintsev P, Jaeger K. Retinal isomerization in bacteriorhodopsin captured by a
femtosecond x-ray laser. Science. 2018, 361(6398).

Northcott PA, Fernandez-L A, Hagan JP, Ellison DW, Grajkowska W, Gillespie Y,
Grundy R, Van Meter T, Rutka JT, Croce CM. The miR-17/92 polycistron is
up-regulated in sonic hedgehog–driven medulloblastomas and induced by N-myc in
sonic hedgehog–treated cerebellar neural precursors. Cancer research. 2009, 69(8).
3249-3255.

Oesterhelt F, Oesterhelt D, Pfeiffer M, Engel A, Gaub H, Müller D. Unfolding
pathways of individual bacteriorhodopsins. Science. 2000, 288(5463). 143-146.

Onufriev A, Smondyrev A, Bashford D. Proton affinity changes driving unidirectional
proton transport in the bacteriorhodopsin photocycle. Journal of Molecular Biology.
2003, 332(5). 1183-1193.

Piccioni DE, Achrol AS, Kiedrowski LA, Banks KC, Boucher N, Barkhoudarian G,
Kelly DF, Juarez T, Lanman RB, Raymond VM. Analysis of cell-free circulating
tumor DNA in 419 patients with glioblastoma and other primary brain tumors. CNS
oncology. 2019, 8(2). CNS34.

Quaranta M, Divella R, Daniele A, Di Tardo S, Venneri MT, Lolli I, Troccoli G.
Epidermal growth factor receptor serum levels and prognostic value in malignant
gliomas. Tumori Journal. 2007, 93(3). 275-280.

Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. Simple,
quantitative primer-extension PCR assay for direct monitoring of microRNAs and
short-interfering RNAs. Rna. 2005, 11(11). 1737-1744.

Shan Y, He X, Song W, Han D, Niu J, Wang J. Role of IL-6 in the invasiveness and
prognosis of glioma. International journal of clinical experimental medicine. 2015,
8(6). 9114.

Spudich JL, Yang C-S, Jung K-H, Spudich EN. Retinylidene proteins: structures and
functions from archaea to humans. Annual review of cell developmental biology.
2000, 16(1). 365-392.

Tanwar MK, Gilbert MR, Holland EC. Gene expression microarray analysis reveals
YKL-40 to be a potential serum marker for malignant character in human glioma.
Cancer research. 2002, 62(15). 4364-4368.

Tolson J, Bogumil R, Brunst E, Beck H, Elsner R, Humeny A, Kratzin H, Deeg M,
Kuczyk M, Mueller GA. Serum protein profiling by SELDI mass spectrometry:
detection of multiple variants of serum amyloid alpha in renal cancer patients.
Laboratory Investigation. 2004, 84(7). 845-856.

Wang J-p, Song L, Yoo S-k, El-Sayed MA. A comparison of the photoelectric current
responses resulting from the proton pumping process of bacteriorhodopsin under
pulsed and CW laser excitations. The Journal of Physical Chemistry B. 1997, 101(49).
10599-10604.

Wang J-P, Yoo S-K, Song L, El-Sayed MA. Molecular mechanism of the differential
photoelectric response of bacteriorhodopsin. The Journal of Physical Chemistry B
1997, 101(17). 3420-3423.

Wang J. Vectorially oriented purple membrane: characterization by photocurrent
measurement and polarized-Fourier transform infrared spectroscopy. Thin Solid Films
2000, 379(1-2). 224-229.

Wen Y, Pei H, Shen Y, Xi J, Lin M, Lu N, Shen X, Li J, Fan C. DNA
nanostructure-based interfacial engineering for PCR-free ultrasensitive
electrochemical analysis of microRNA. Scientific reports. 2012, 2, 867.

Wrensch M, Wiencke JK, Wiemels J, Miike R, Patoka J, Moghadassi M, McMillan A,
Kelsey KT, Aldape K, Lamborn KR. Serum IgE, tumor epidermal growth factor
receptor expression, and inherited polymorphisms associated with glioma survival.
Cancer research. 2006, 66(8). 4531-4541.

Wu H-H, Liao X-Q, Wu X-Y, Lin C-D, Jheng K-R, Chen H-R, Wang Y-Y, Chen H-M.
Versatile protein-a coated photoelectric immunosensors with a purple-membrane
monolayer transducer fabricated by affinity-immobilization on a graphene-oxide
complexed linker and by shear flow. Sensors. 2018, 18(12). 4493.

Yeh Y-C, Creran B, Rotello VM. Gold nanoparticles: preparation, properties, and
applications in bionanotechnology. Nanoscale. 2012, 4(6). 1871-1880.

Zakrzewska M, Fendler W, Zakrzewski K, Sikorska B, Grajkowska W,
Dembowska-Bagińska B, Filipek I, Stefańczyk Ł, Liberski PP. Altered microRNA
expression is associated with tumor grade, molecular background and outcome in
childhood infratentorial ependymoma. PloS one. 2016, 11(7).

Zhang Y, Zhang C-y. Sensitive detection of microRNA with isothermal amplification
and a single-quantum-dot-based nanosensor. Analytical chemistry. 2012, 84(1).
224-231.

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. 2019.

吳欣穎. 細菌視紫質泛用型免疫光電晶片製備與原子力顯微鏡分析. 國立台灣科技大學化學工程研究所碩士論文. 2015.

林承德. 氧化石墨烯濃度對以親合吸附作用製備細菌視紫質光電晶片之影響. 國立台灣科技大學化學工程研究所碩士論文. 2013.

郭力彬. 紫膜生物光電晶片應用於糖化血紅素(HbA1c)檢測之探討. 國立台灣科技大學化學工程研究所碩士論文. 2017.

陳冠辰. 適體-細菌視紫質生物光電感測晶片之探討. 國立台灣科技大學化學工程研究所碩士論文. 2015.

廖信銓. 微流體於細菌視紫質光電晶片製備之應用. 國立台灣科技大學化學工程研究所碩士論文. 2015.