免標定電容式核酸適體生物感測器運用於凝血酶的檢測

Label-Free Capacitive Aptasensor for Thrombin

陳欣儒(Hsin-Ju Chen)

指導教授 ： 鄭宗記

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/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10852

摘要

電容式生物感測器具有免標定的優點，具潛力發展成為手持式裝置應用於居家定點照護。傳統電容式感測器多半使用指插式電極，然而此種電極需要較繁複的微機電製程，以及具有電場不均勻分布的缺點。此外，利用電化學阻抗分析量測電容時，通常需要額外添加氧化還原物質以提高檢測靈敏度。因此，基於簡便之設計概念，本研究利用平行對電極以開發免標定與高靈敏度之電容式核酸適體生物感測器。此電極對為系統中的電容式傳感器，使用光碟片自製之金電極與氧化銦錫(indium tin oxide, ITO)薄膜作為對極，電極間以聚酯(PET)雙面膠帶貼合達到50 um之電極間距。於金電極上，藉由分子自組裝修飾核酸適體(aptamer)作為生物分子辨識元件，再修飾正十二烷基硫醇(1-dodecanethiol, DDT)以防止非特異性蛋白質吸附。此分子自組裝層的電化學特性顯示其亦能形成有效的絕緣層，適合用於開發電容式生物感測器。而本研究選擇凝血酶作為檢測標的物以驗證系統的有效性，使用自製電容式感測電路量測aptamer辨識到thrombin與其結合時產生之電容變化。在人體血清中，凝血酶檢測的濃度線性範圍為10-11 ~ 10-6 M，相關係數達0.97。於牛血清白蛋白(bovine serum albumin, BSA)與人血清白蛋白(human serum albumin, HSA)中，並無明顯的非特異性蛋白質吸附發生。因此，本研究成功利用電極對建構免標定電容式核酸適體生物感測器並應用於thrombin檢測。未來或許能透過置換不同的aptamer以檢測不同的目標蛋白質。

關鍵字

電容式 ； 電極對 ； 核酸適體 ； 分子自組裝 ； 凝血酶

並列摘要

Capacitive biosensors are label-free and promising for developing hand-held devices and point-of-care applications. However, conventional micro-fabricated capacitive sensors such as those based on interdigitated electrodes faced the challenges including delicate manufacture and non-uniform distribution of electric field. Furthermore, electrochemical impedance spectroscopy for capacitance measurements usually required extra addition of redox species to enhanced sensitivity. A label-free and sensitive capacitive aptasensor was developed with simple design based on electrode pairs. The electrode pair was the transducer of the capacitive aptasensor, which was composed of a gold electrode (from compact discs) and an indium tin oxide (ITO) film with micrometer separation by a double-side PET tape. On the gold electrode was layered with aptamers as the bio-recognition elements and then a self-assembled monolayer of 1-dodecanethiol to prevent non-specific protein binding. Electrochemical characterization proved that the self-assembly monolayer also formed effective insulating layer adequate for capacitive sensing. Thrombin was selected as a model analyte to validate the assay. The capacitance change originated from thrombin binding with aptamers was measured with a home-made capacitance sensing circuit by switched capacitor technology. The linear range for thrombin detection in human serum was from 10-11 to 10-6 M with a regression coefficient of 0.97. Non-specific binding of bovine serum albumin (BSA) and human serum albumin (BSA) were not observed in the proposed aptasensor. This work demonstrated the successful application of label-free capacitive aptasensor for thrombin detection. It could be applied in the detection of other target proteins by replacing different aptamers.

並列關鍵字

Capacitive ； Electrode pair ； Aptamer ； Self-assembly monolayer ； thrombin

參考文獻

Akkerman, H. B., P. W. M. Blom, D. M. de Leeuw and B. de Boer. 2006. Towards molecular electronics with large-area molecular junctions. Nature 441:69-72.

Aldaz-Carroll, L. Tallet, B. Dausse, E. Yurchenko, L. Toulme and Jean-Jacques. 2002. Apical loop−internal loop interactions:  a new RNA−RNA recognition motif identified through in vitro selection against RNA hairpins of the Hepatitis C virus mRNA. Biochemistry 41:5883-5893.

Allara, D. L. and R. G. Nuzzo. 1985. Spontaneously organized molecular assemblies. 1. Formation, dynamics, and physical properties of n-alkanoic acids adsorbed from solution on an oxidized aluminum surface. Langmuir 1:45-52.

Alleman, K. S., K. Weber and S. E. Creager. 1996. Electrochemical rectification at a monolayer-modified electrode. The Journal of Physical Chemistry 100:17050-17058.

Angnes, L., E. M. Richter, M. A. Augelli and G. H. Kume. 2000. Gold electrodes from recordable CDs. Analytical Chemistry 72:5503-5506.