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標題: | 應用即時指叉狀電極阻抗分析於適體-肌鈣蛋白I結合動力學之研究 Study of Aptamer-Troponin I Binding Kinetics Using Real-time Interdigitated Array Impedance Analysis |
作者: | 易昌旻 Chang-Min Yi |
指導教授: | 陳林祈 Lin-Chi Chen |
關鍵字: | 肌鈣蛋白I,指叉狀電極,阻抗,動力學分析,微流體,適體感測, cardiac troponin I,interdigitated array electrode,impedance,kinetic analysis,microfluid,aptasensing, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 指叉狀電極 (interdigitated array electrode;IDA electrode) 是一種電極呈交叉狀設計的共平面電極,其可藉由微機電技術中的光微影製程 (photolithography) 進行開發。輔以電化學阻抗頻譜 (electrochemical impedance spectroscopy;EIS) 的量測方法,基於指叉狀電極的電化學阻抗式生物感測裝置已多被應用於生物標的物檢測上。在本研究中,基於指叉狀電極晶片之二極式電化學量測系統被使用於評估肌鈣蛋白I與其DNA核酸適體間的結合反應動力學,欲分析適體選用Jo等人的研究團隊於2015年所篩選之對肌鈣蛋白I具有高親和性的Tro4適體 (解離常數KD = 270 pM) 做為適體感測平台的辨識元件。首先,基於Jo等人研究團隊所篩選之Tro4和Tro6適體進行酶聯寡核苷酸試驗 (enzyme-linked oligo-nucleotide assay;ELONA),驗證適體對於肌鈣蛋白I之親和性 (affinity) 與特異性 (specificity)。接著為探討肌鈣蛋白I與Tro4適體於穩態和即時態 (real-time) 的結合反應,由指叉狀電極晶片組成之靜態式和流動式系統被建構以量測結合反應穩態與即時態的阻抗響應。藉由靜態式量測系統決定2.5 nM至40 nM肌鈣蛋白I在穩態的校正電荷轉移阻抗響應 (normalized ΔRct),以Langmuir的吸附方程式作為數學模型,其所求得之結合響應最大值Bmax = 1.33,KD = 1.40 nM,其調整後迴歸係數平方值 (adjusted R2 ) 為0.725。在流動式量測系統中,電荷轉移阻抗 (Rct) 和即時阻抗響應 (Z-t) 被作為評估流動式結合反應穩態和即時態阻抗響應之方法:在穩態阻抗響應分析中,normalized ΔRct同樣以Langmuir模型進行回歸,其Bmax = 2.53,KD = 360 nM,其adjusted R2 為0.957,而Δ|Z|則對肌鈣蛋白I濃度進行一次方線性迴歸,其方程式為Δ|Z| = 8.40 "×" 10-3 + 1.06 "×" 10-4 CcTnI,R2 為0.987,由結果顯示ΔRct和Δ|Z| (交流頻率100Hz) 的穩態響應對於肌鈣蛋白I濃度具有劑量效應 (dose dependence),且由其相對於人類血清蛋白 (human serum albumin;HSA) 的阻抗響應差異驗證出Tro4適體對於肌鈣蛋白I在流動式感測之專一性。而在結合反應動力學上,本研究針對在0.2 μL/s流率下,流動300 nM的肌鈣蛋白I與固定於指叉狀電極之1 μM Tro4適體的結合反應進行探討,由類比於表面電漿共振(surface plasmon resonance;SPR)基於質量作用原理(law of mass action)之即時動力學響應方程式,即時阻抗響應被應用於迴歸計算反應速率常數,其計算之結合反應速率常數為2.31×109 (M-1s-1),解離反應速率常數為7.18×10-4 (s-1),而R2=0.959,展現快速結合和緩慢解離的動力學特徵。綜上所述,本研究應用指叉狀電極阻抗分析於肌鈣蛋白I和Tro4適體靜態式和流動式結合反應動力學之探討,展現藉由此裝置發展肌鈣蛋白I阻抗式適體感測器之可行性,亦顯示指叉狀電極與阻抗響應計算用於不同分子反應動力學分析之可能性,此將有利於電化學式生物檢測裝置與方法學發展,更有潛力用於定點照護相關產品的開發上。 Interdigitated Array Electrode (IDA electrode) is a type of coplanar electrode designed with an interdigitated pattern. It can be developed using photolithographic techniques in microelectromechanical technology. With the measurement method of electrochemical impedance spectroscopy (EIS), EIS-based biosensors have been extensively applied in the detection of biomarkers. In this study, a two-electrode electrochemical measurement system based on IDA electrode chips was used to evaluate the binding kinetics between DNA aptamers and cardiac troponin I (cTnI). The Tro4 aptamer with high affinity to cTnI (dissociation constant KD = 270 pM), selected by Jo et al.'s team in 2015, was used as the recognition element for the aptamer sensing platform. First of all, Tro4 and Tro6 aptamers, both selected by Jo et al., were analyzed with enzyme-linked oligo-nucleotide assay (ELONA) to validate their affinity and specificity for cTnI. To investigate the binding kinetics between cTnI and the Tro4 aptamer in both steady and real-time states of reactions, static and flowing systems composed of IDA electrode chips were constructed to measure the impedimetric responses in steady and real-time states of binding reactions. By the static measurement system, the steady-state response of normalized charge transfer resistance (normalized ΔRct) of cTnI was determined in the concentration range from 2.5 nM to 40 nM. The Langmuir adsorption equation was used as the mathematical model, and the obtained maximum binding response (Bmax) was 1.33 with a dissociation constant (KD) of 1.40 nM. The adjusted R-squared value was 0.725. In the flowing system, charge transfer resistance (Rct) and real-time impedimetric response (Z-t) were used to evaluate the steady-state and real-time impedimetric responses. The normalized ΔRct was also analyzed using the Langmuir model. The obtained Bmax was 2.53, KD was 360 nM, and the adjusted R-squared value was 0.957. Δ|Z| was linearly regressed with cTnI concentration, and the equation was Δ|Z| = 8.40×10-3 + 1.06×10-4CcTnI with an R-squared value of 0.987. The results indicated dose dependence of ΔRct and Δ|Z| at an AC frequency of 100Hz to cTnI. The specificity of the Tro4 aptamer to cTnI in the flowing system was also confirmed by the difference in impedimetric response relative to human serum albumin (HSA) based on ΔRct and Δ|Z|. Regarding the binding kinetics, the study investigated the binding reaction between 300 nM cTnI flowing at a rate of 0.2 μL/s and 1 μM Tro4 aptamer immobilized on the IDA electrode. The real-time kinetics response equation based on the principles of the law of mass action similar with surface plasmon resonance (SPR) was used to calculate the reaction rate constants. The calculated association rate constant was 2.31×109 (M-1s-1), and the dissociation rate constant was 7.18×10-4 (s-1) with an R-squared value of 0.959, which shows fast binding and slow dissociation kinetics. In conclusion, this study applied impedance analysis with IDA electrodes to investigate the steady-state and real-time binding kinetics between cTnI and the Tro4 aptamer. The feasibility to develop cTnI impedimetric aptasensors using this device and the potential of using IDA electrodes and impedance response calculations for analyzing different molecular reaction kinetics were demonstrated. This will be beneficial for the development of electrochemical biosensing devices and methodologies, potentially leading to the development of related products of point-of-care-healthcare. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90675 |
DOI: | 10.6342/NTU202303332 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 生物機電工程學系 |
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