利用CRISPR/Cas12a提升電化學分子檢測技術

Abstract

本論文旨在探索使用金電極來測量CRISPR系統的剪切能力，並進一步研究其靈敏度和選擇性。CRISPR技術自發現以來，由於其在基因編輯和生物感測領域的巨大潛力，以成為一個備受關注的研究焦點。本研究選用Hairpin DNA和Linear DNA作為報告基因(reporter gene)。

實驗過程中我們使用Hairpin DNA和Linear DNA分別固定在金電極表面上，這些電極與不同濃度的目標DNA(target DNA)進行反應，測量CRISPR系統在不同條件下的剪切活性。透過電化學阻抗圖譜(electrochemical impedance spectroscopy, EIS)和循環伏安法(cyclic voltammetry, CV)，我們能夠定量分析CRISPR系統對Hairpin DNA和Linear DNA的剪切效率。

為了評估CRISPR系統的靈敏度(sensitivity)，我們測量了在不同濃度目標DNA下，金電極上的電化學訊號變化，也測試了CRISPR系統對非目標DNA(non-target DNA)的反應，以評估其選擇性(selectivity)。

本論文展現了使用金電極來測量CRISPR系統剪切能力的可行性，並證實了CRISPR系統在不同濃度的目標DNA下高靈敏度以及對非目標DNA的高選擇性，也就是說，以CRISPR系統可作為高靈敏度及高選擇性的生物感測器，對於基因診斷、疾病檢測等領域具有重要的應用價值。未來的研究可以進一步探討不同的電極材料及不同DNA的修飾端對CRISPR系統剪切能力的影響。

The CRISPR system has become an emerging research field due to its immense potential in gene editing and biosensing fields. It is important to promote the applications of CRISPR detection technologies. Therefore, this work aims to explore the use of gold electrodes in measuring the cleavage activity of the CRISPR system. Furthermore, we will also investigate its sensitivity and selectivity. In this study, we also explored hairpin DNA and linear DNA as reporter genes.

In this experiment, hairpin DNA and linear DNA were separately immobilized on the surface of gold electrodes. These electrodes were then subjected to reactions with target DNA at varying concentrations to measure the CRISPR system’s cleavage activity under different conditions. By employing electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), we quantitatively analyzed the CRISPR system’s cleavage efficiency on hairpin DNA and linear DNA.

To evaluate the sensitivity of the CRISPR system, we measured changes in electrochemical signals on the gold electrode at different target DNA concentrations. Additionally, we tested the CRISPR system’s response to non-target DNA to assess its selectivity.

This work demonstrates the feasibility of using gold electrodes to measure the cleavage ability of the CRISPR system. It confirms that the CRISPR system exhibits high sensitivity to target DNA at various concentrations and maintains high selectivity even in the presence of non-target DNA. In other words, the CRISPR system can serve as a highly sensitive and selective biosensor, with significant applications in genetic diagnostics and disease detection. Future research could explore the impact of different electrode materials and modifications of DNA ends on the CRISPR system’s cleavage efficiency.