本研究是以專一性核酸適體 (aptamer) 為辨識分子，開發以Halobacterium salinarum紫膜 (purple membrane, PM)作為訊號轉換器的生物光電感測器，用於定量檢測ampicillin和kanamycin兩種不同抗生素。首先開發ampicillin檢測晶片，製備方式為將可與ampicillin結合的核酸適體 (Amp aptamer)，和另一修飾有奈米金 (gold nanoparticle, AuNPs) 的互補序列 (簡稱AuNPs-oligo)，依序固定於塗覆有 PM 膜的電極晶片上，此時晶片會因AuNPs遮光性而使所產生的光電流訊號降低；接續檢測ampicillin時，ampicillin會與固定於晶片上之 Amp aptamer結合，使AuNPs-oligo脫附而造成晶片光電流訊號回升，而得以定量檢測ampicillin濃度。在最適化晶片製程下，晶片可檢測ampicillin濃度範圍為0.1 nM至10 µM，當ampicillin濃度10 µM時，光電流回升率可達66%。接著開發kanamycin檢測晶片，同樣於最適化製程與條件下，晶片可檢測kanamycin濃度範圍為50 pM至0.1 mM，當kanamycin 濃度0.1 mM時，光電流回升率可達83 %。這兩種晶片各自對其目標抗生素皆具有高度專一性，並不受彼此干擾，且都不受chloramphenicol與sulfadimethoxine干擾 (p<0.01)。最後初步進行抗生素混合檢測，結果顯示所開發晶片將可應用於實際混合樣品的檢測。

Using specific aptamers as the recognition elements and purple membrane (PM) from Halobacterium salinarum as the signal transducer, we developed two photoelectric biosensors for ampicillin and kanamycin detection, respectively. The ampicillin sensing chip was prepared by sequentially immobilizing the ampicillin specific aptamer (Amp aptamer) and another gold nanoparticles (AuNPs) conjugated complementary oligonucleotide (AuNPs-oligo) onto a PM-coated electrode, resulting in a reduction of photocurrent generation by PM due to the light-sheilding effect of AuNPs. Subsequent binding of ampicillin with the Amp aptamer coated on the sensing chip led to AuNPs-oligo dissociation and hence photocurrent recovery. The ampicillin concentration was determined with the photocurrent recovery level. Under the optimal production and detection conditions, the ampicillin sensing chip exhibited a dynamic range of 0.1 nM to 10 µM, with a 66% photocurrent recovery upon 10 µM ampicillin. With a similar prepation scheme and under the optimal production and detection conditions, the kanamycin sensing chip exhibited a dynamic range of 50 pM to 0.1 mM, with a 83% photocurrent recovery upon 0.1 mM kanamycin. High selectivity without crossactivity was observed with both sensing chips, as well as no interference from chloramphenicol and sulfadimethoxine (p < 0.01). Finally, a preliminary analysis on mixed samples was conducted, demonstrating the feasibility of the application of those two biosensors on field sample detection.

Ahmed, S., Ning, J., Cheng, G., Ahmad, I., Li, J., Mingyue, L., Qu, W., Iqbal, M., Shabbir, M., & Yuan, Z. Receptor-based screening assays for the detection of antibiotics residues–A review. Talanta. 2017, 166, 176-186.

Al-Aribe, K. M., Knopf, G. K., & Bassi, A. S. Photoelectric monolayers based on self-assembled and oriented purple membrane patches. Journal of microelectromechanical systems. 2011, 20(4), 800-810.

Al-kadmy, E. M. S. Detection of 16S rRNA Methylation Gene between Two Species of Gram Negative with High Level Resistance to Aminoglycosides. Diss. M. Sc. Thesis. College of Science. Al-Mustansiryah University. 2012.

Balashov, S. P. Protonation reactions and their coupling in bacteriorhodopsin. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2000, 1460(1), 75-94.

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 and Surfaces B: Biointerfaces. 2014, 116, 482-488.

Chen, Y., Wang, Y., Liu, L., Wu, X., Xu, L., Kuang, H., Li, A., & Xu, C. A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams. Nanoscale. 2015, 7(39), 16381-16388.

Cho, H., Uehara, T., & Bernhardt, T. G. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell. 2014, 159(6), 1300-1311.

Gopinath, S. C. B. Methods developed for SELEX. Analytical and bioanalytical chemistry. 2007, 387(1), 171-182.

Gai, P., Gu, C., Hou, T., & Li, F. Ultrasensitive self-powered aptasensor based on enzyme biofuel cell and DNA bioconjugate: a facile and powerful tool for antibiotic residue detection. Analytical chemistry. 2017, 89(3), 2163-2169.

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

Khabbaz, L. S., Hassanzadeh-Khayyat, M., Zaree, P., Ramezani, M., Abnous, K., & Taghdisi, S. M. Detection of kanamycin by using an aptamer-based biosensor using silica nanoparticles. Analytical Methods. 2015, 7(20), 8611-8616.

Kurihara, M., & Sudo, Y. Microbial rhodopsins: wide distribution, rich diversity and great potential. Biophysics and physicobiology. 2015, 12, 121-129.

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

Li, F., Guo, Y., Wang, X., & Sun, X. Multiplexed aptasensor based on metal ions labels for simultaneous detection of multiple antibiotic residues in milk. Biosensors and Bioelectronics. 2018, 115, 7-13.

Li, M., Liu, Q., Teng, Y., Ou, L., Xi, Y., Chen, S., & Duan, G. The resistance mechanism of Escherichia coli induced by ampicillin in laboratory. Infection and Drug Resistance. 2019, 12, 2853.

Li, Y. T., Tian, Y., Tian, H., Tu, T., Gou, G. Y., Wang, Q., Qiao, Y. C., Yang, Y., & Ren, T. L. A review on bacteriorhodopsin-based bioelectronic devices. Sensors. 2018, 18(5), 1368.

Liu, B., & Liu, J. Methods for preparing DNA-functionalized gold nanoparticles, a key reagent of bioanalytical chemistry. Analytical Methods. 2017, 9(18), 2633-2643.

Luo, Z., Wang, Y., Lu, X., Chen, J., Wei, F., Huang, Z., Zhou. C., & Duan, Y. Fluorescent aptasensor for antibiotic detection using magnetic bead composites coated with gold nanoparticles and a nicking enzyme. Analytica chimica acta. 2017, 984, 177-184.

Ma, X., Qiao, S., Sun, H., Su, R., Sun, C., & Zhang, M. Development of structure-switching aptamers for kanamycin detection based on fluorescence resonance energy transfer. Frontiers in Chemistry. 2019, 7, 29.

Mehlhorn, A., Rahimi, P., & Joseph, Y. Aptamer-based biosensors for antibiotic detection: a review. Biosensors. 2018, 8(2), 54.

Neutze, R., Pebay-Peyroula, E., Edman, K., Royant, A., Navarro, J., & Landau, E. M. Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2000, 1565(2), 144-167.

Pang, S., & He, L. Understanding the competitive interactions in aptamer–gold nanoparticle based colorimetric assays using surface enhanced Raman spectroscopy (SERS). Analytical Methods. 2016, 8(7), 1602-1608.

Peng, J., Cheng, G., Huang, L., Wang, Y., Hao, H., Peng, D., Liu, Z., Yuan, Z. Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of β-lactam antibiotics in foods. Analytical and bioanalytical chemistry. 2013, 405(27), 8925-8933.

Saha, K., Agasti, S. S., Kim, C., Li, X., & Rotello, V. M. Gold nanoparticles in chemical and biological sensing. Chemical reviews. 2012, 112(5), 2739-2779.

Song, K. M., Cho, M., Jo, H., Min, K., Jeon, S. H., Kim, T., Han, M. S., Ku, J.K., & Ban, C. Gold nanoparticle-based colorimetric detection of kanamycin using a DNA aptamer. Analytical biochemistry. 2011, 415(2), 175-181.

Song, K. M., Jeong, E., Jeon, W., Cho, M., & Ban, C. Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods. Analytical and bioanalytical chemistry. 2012, 402(6), 2153-2161.

Song, S., Wang, L., Li, J., Fan, C., & Zhao, J. Aptamer-based biosensors. TrAC Trends in Analytical Chemistry. 2008, 27(2), 108-117.

Sharma, A., Istamboulie, G., Hayat, A., Catanante, G., Bhand, S., & Marty, J. L. Disposable and portable aptamer functionalized impedimetric sensor for detection of kanamycin residue in milk sample. Sensors and Actuators B: Chemical. 2017, 245, 507-515.

Stoltenburg, R., Reinemann, C., & Strehlitz, B. SELEX—a (r) evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular engineering. 2007, 24(4), 381-403.

Uehara, K., Kawai, K., & Kouyama, T. Photoelectric response of oriented purple membrane electrodeposited onto poly (vinyl alcohol) film. Thin Solid Films. 1993, 232(2), 271-277.

Wang, J. P., Song, L., Yoo, S. K., & El-Sayed, M. A. 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, M. A. 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.

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.

Wang, J., Ma, K., Yin, H., Zhou, Y., & Ai, S. Aptamer based voltammetric determination of ampicillin using a single-stranded DNA binding protein and DNA functionalized gold nanoparticles. Microchimica Acta. 2018, 185(1), 68.

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

Youn, H., Lee, K., Her, J., Jeon, J., Mok, J., So, J. I., Shin S., & Ban, C. Aptasensor for multiplex detection of antibiotics based on FRET strategy combined with aptamer/graphene oxide complex. Scientific reports. 2019, 9(1), 1-9.

Zhou, N., Zhang, J., & Tian, Y. Aptamer-based spectrophotometric detection
of kanamycin in milk. Analytical Methods. 2014, 6(5), 1569-1574.

蘇志溫.Bacteriorhodopsin 生物晶片之光電響應. 國立臺灣科技大學化學工程研究所碩士論文, 2005.

許培鈞.以循序批式培養Halobacterium salinarum及其紫膜之大量純化研究. 國立臺灣科技大學化學工程研究所博士論文, 2013.

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

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

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

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. 國立臺灣科技大學化學工程研究所碩士論文, 2017.

陳泓任. 檢測三磷酸線苷、革藍氏陽性菌和白血球之紫膜生物光電晶片開發. 國
立臺灣科技大學化學工程研究所碩士論文, 2018.

鄭智文. 以紫膜生物光電晶片檢測遷離子及糖化血紅素, 國立台灣科技大學化
學工程研究所碩士論文, 2018.

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.

賴銀德. 檢測真菌、革蘭氏陽性與陰性菌之紫膜生物光電晶片開發, 國立台灣科
技大學化學工程研究所碩士論文, 2019