應用於 CRISPR/Cas12a 之電化學阻抗量測方法研究

Abstract

本論文旨在建立一套結合 CRISPR/Cas 系統與以金為電極材料搭配 hairpin DNA 探針之電化學量測方法，並探討實驗中可能影響訊號表現的潛在干擾因子。研究中發現，當以電化學阻抗分析(Electrochemical Impedance Spectroscopy, EIS)量測 Cas12a 系統的剪切反應時，商用反應緩衝液中的重組白蛋白(recombinant albumin)可能造成非特異性吸附與介面阻擋效應，導致背景訊號上升，進而干擾目標訊號的辨識與解析。 為解決此問題，本研究調整量測流程，改以蛋白質預先覆蓋後的電極狀態作為新的基線條件，以降低蛋白質成分對 EIS 量測的干擾。此外，在使用排除蛋白質成分的緩衝液條件下進行剪切反應時，我們亦觀察到 hairpin DNA 探針的二級結構穩定性可能影響其作為 Cas12a 反式剪切底物的效果。因此，我們進一步參考相關文獻，整理過往 hairpin DNA 做為 Cas12a 反式剪切目標之設計，並歸納出在結構穩定性與反應效率間可進一步優化之方向。 綜合本研究結果，初步測試以hairpin DNA做為金電極表面探針之CRISPR/Cas12a 電化學生物感測方法，並針對其訊號準確性之干擾來源與潛在解決方案進行探討，期望能作為後續相關系統優化之參考依據。

This thesis aims to establish an electrochemical sensing platform that integrates the CRISPR/Cas system with gold electrodes modified by hairpin DNA probes, and to investigate potential interfering factors that may affect signal performance. During measurements of the Cas12a cleavage reaction using Electrochemical Impedance Spectroscopy (EIS), we found that recombinant albumin, commonly present in commercial reaction buffers, may lead to nonspecific adsorption and interfacial blocking effects. These effects result in elevated background signals, thereby interfering with the identification and interpretation of target signals. To address this issue, we modified the measurement workflow by using the electrode surface pre-coated with proteins as a new baseline condition, which helped reduce the interference caused by protein components during EIS detection. In addition, under buffer conditions without albumin, we observed that the secondary structure stability of hairpin DNA probes could also influence their suitability as trans-cleavage substrates for Cas12a. Therefore, we further reviewed relevant literature and summarized prior designs of hairpin DNA used as Cas12a trans-cleavage targets, identifying possible directions for structural optimization to balance stability and reaction efficiency. In summary, this study provides preliminary testing of a CRISPR/Cas12a electrochemical biosensing approach based on hairpin DNA probes immobilized on gold electrodes, and discusses key sources of signal interference as well as potential solutions. The findings are expected to serve as a reference for future optimization of related sensing systems.