探討以自組裝單層修飾之銅電極發展阻抗式適體感測器:以四環黴素類抗生素為例

Study of Self-Assembled Monolayers of Thiolates on Copper Electrodes for Impedimetric Aptasensor Development: Tetracycline Detection as an Example

沈柏亨(Po-Heng Shen)

指導教授 ： 鄭宗記

國立臺灣大學/生物資源暨農學院/生物機電工程學系/碩士(2021年)

https://doi.org/10.6342/NTU202103317

摘要

電化學生物感測器一般以較昂貴的惰性材料（例如:金電極）做為修飾感測元件之基材，電極成本可能阻礙實務的普及與應用。故本研究旨在以銅箔為材料製備銅電極，並利用硫醇基與銅的自組裝將適體與硫醇分子固定於電極表面以做為感測電極使用。本研究從市售銅箔材料加工製作銅電極，以二價與三價的氯化六氨合釕（Ru(NH3)6Cl2/Ru(NH3)6Cl3）氧化還原對，在-400 mV至-100 mV（vs. Ag/AgCl）的電位區間可有效地進行電化學方法（循環伏安法、電化學阻抗頻譜法、定頻阻抗量測法）分析銅電極界面的定性與半定量分析。以50%硝酸清洗25秒做為前處理程序可有效去除銅電極表面抗氧化層與雜質之效果，亦可以低濃度硝酸於較長時間清洗下達相同的前處理效力。適體固定化上展現了相當於金電極的阻抗變化，顯示有效的適體固定化成果，而以適體與1-己硫醇以依序修飾方式可製備共修飾的感測界面。評估長鏈分子（正十二烷基硫醇）與短鏈分子（1-己硫醇）所形成自組裝單層的界面電性，100 mM正十二烷基硫醇修飾9小時的電荷轉移電阻Rct大於500 kΩ，可於銅電極形成緻密的自組裝單層並依感測器量測需求調整對應單層緻密度的修飾時間。在交流電阻抗量測的體系下，電荷轉移電阻Rct變化遠較電雙層電容Cdl顯著，故以阻抗分析界面時有較佳解析力，然而經修飾短鏈1-己硫醇的銅電極於接觸不同離子強度溶液時顯示其界面的不穩定性，呈現適體和1-己硫醇修飾於銅電極做為感測界面輔以電化學阻抗譜法量測的困難性，若能在銅電極修飾緻密且穩定的感測界面輔以電容式量測是較可能的感測方案。

關鍵字

適體感測器 ； 硫醇 ； 自組裝單層 ； 電化學阻抗頻譜法

並列摘要

The materials of working electrode in electrochemical biosensors are generally used by some expensive noble metal (such as gold) as the base material for biorecognition elements immobilized on it. In the light of this, this research aims to use copper foil to manufacture copper electrodes and use the self-assembled strategy based on strong bonding between thiols and copper, immoboilizing aptamers and blocking agents (such as 1-mercaptohexane, MCH and 1-dodecanethiol, DDT) on the surface of the electrode as a biosensor. Copper electrodes were fabricated from commercially available copper foil, and more suitable redox couples: hexaammineruthenium(II) chloride (Ru(NH3)6Cl2) and hexaammineruthenium(III) chloride (Ru(NH3)6Cl3) were selected as the reporters in the potential range of -400 mV to -100 mV (vs. Ag/AgCl), which can effectively perform qualitative and semi-quantitative analysis on copper electrodes by electrochemical methods (cyclic voltammetry, electrochemical impedance spectroscopy, and real-time impedance measurement). The pretreatment of copper electrodes with 50% nitric acid cleaning for 25 seconds as a cleaning procedure can effectively remove the anti-oxidation layers and impurities on the surface of copper electrodes. The same cleaning effect can also be achieved by lower concentration of nitric acid for a longer reaction time. The aptamer immobilization on copper electrodes exhibits an impedance change equivalent to gold electrodes, showing effective aptamer immobilization results, and co-immobilization sensing interface can be prepared by subsequent modification of 1-hexanthiol. On the self-assembled monolayers, we evaluated the electrical properties of the interface formed by long-chain molecules (1-dodecanethiol) and short-chain molecules (1-mercaptohexane). The charge transfer resistance (Rct) of 100 mM 1-Dodecanethiol modification for 9 hours is greater than 500 kΩ, which can form a dense self-assembled monolayers on the copper electrode and we can adjust the modification time to fit the biosensor’s need. Under the system of AC electrical impedance measurement, the change of charge transfer resistance (Rct) is much more significant than the change of electric double layer capacitance (Cdl). Therefore, evaluation by the change of Rct will have better resolution. However, the copper electrode modified with short-chain 1-Mercaptohexane after contacting with different ionic strength solutions shows the instability of the interface, indicating the difficulty of aptamer and 1-hexanthiol modification on the copper electrode as the sensing interface under electrochemical impedance spectroscopy. If the copper electrode can be modified with a dense and stable sensing interface which measured by capacitance, it seems to be a more possible strategy.

並列關鍵字

aptasensors ； thiol ； self-assembled monolayers (SAMs) ； electrochemical impedance spectroscopy (EIS)

參考文獻

農委會。 2015。動物用藥品使用準則。農防字第 1051472565 號。

衛生福利部。2019。動物用藥殘留標準。衛授食字第 1081302402 號。

Abd el Haleem, S. M., and B. G. Ateya. 1981. Cyclic voltammetry of copper in sodium hydroxide solutions. J. Electroanal. Chem. 117: 309-319.

Alleman, K. S., K. Weber, and S. E. Creager. 1996. Electrochemical Rectification at a Monolayer-Modified Electrode. J. Phys. Chem. 100(42): 17050-17058.

Arya, S. K., P. Zhurauski, P. Jolly, M. R. Batistuti, M. Mulato, and P. Estrela. 2018. Capacitive aptasensor based on interdigitated electrode for breast cancer detection in undiluted human serum. Biosens. Bioelectron. 102: 106-112.