利用循環鏈置換策略結合三股螺旋 DNA 奈米開關開發對食道癌具特異性的微小核糖核酸之電化學感測平台

Abstract

食道癌具有隱形殺手之稱，近十年來蟬聯國人十大癌症死因之一。早期食道癌並無任何症狀，且診斷方式多以侵入式為主，導致國人檢查之意願大幅降低，也因此錯過黃金治療期。根據研究顯示，血液中的miR-365、miR-22可作為早期食道癌之生物標記。由此，我們利用循環鏈置換放大策略 (Circular Strand Displacement Amplification, CSDA)結合三螺旋DNA奈米開關 (Triplex DNA nanoswitch)，開發不須酵素參與且恆溫之電化學核酸感測平台，作為一種非侵入式之早期食道癌診斷工具。miR-365及miR-22具有啟動循環鏈置換放大反應之功能，我們透過膠體電泳分析發現在1小時即有足夠大量的單股觸發子 (Triplex reporter, TR) 被生成釋出，達到有效循環放大之成效。此外，我們透過反向平行三螺旋核酸結構 (Antiparallel triplex) 之設計，能有效降低非專ㄧ性的雜訊，藉此提升反應之訊噪比。接著，被釋出之兩種TR分別可與修飾在電極表面之核酸探針 (Capture probe, CP) 形成60% TAT及80% TAT之平行三螺旋核酸結構 (Parallel triplex)。由miR-365主導之60% TAT三螺旋核酸只能於酸性環境下穩定生成，而miR-22所主導之80% TAT三螺旋核酸則可於酸性至中性環境下穩定形成。透過pH值調控可於3分鐘內使核酸探針折彎，與TR穩定形成三螺旋核酸結構，促使核酸探針末端修飾之甲基藍分子 (Methylene blue, MB) 靠近電極表面而產生更高的電化學訊號。本研究利用反應液pH的調控作為平台的開關，在平台上只需標定一種電化學活性分子，透過不同的pH值調控Triplex nanoswitch構型改變便能雙重偵測不同的目標待測物。經計算後miR-365、miR-22之偵測極限可分別下達6.2 pM及1.14 pM。此電化學偵檢平台具有快速、低成本、高靈敏性且具雙重偵測特性之優勢。我們相信其具有潛力被應用於食道癌或其他臨床疾病之檢測。

Esophageal cancer has been considered as the silent killer and ranked among the top ten leading causes of cancer death for more than a decade. Previous studies showed that circulating miR-365 and miR-22 were found to be potential biomarkers for early Esophageal cancer detection. Herein, we established an electrochemical platform with enzyme-free amplification that can be used as a non-invasive diagnostic tool for early-stage Esophageal cancer. Our detection scheme was initiated with a miR-365 and miR-22-induced circular strand replacement amplification (CSDA). It was followed by the production of a large number of triplex reporters (TR) in one hour that was confirmed by gel electrophoresis. We also found that anti-parallel triplex configuration of our probe design was able to reduce the non-specific hybridization significantly. Subsequently two different releasing TR hybridized with methylene blue (MB)-sensitized CP (MB-CP) modified on the electrode to form 60% TAT parallel triplex and 80% TAT parallel triplex, respectively. The formation of 60% TAT triplex triggered by miR-365 was found to yield steadily in an acidic environment only; however, the 80% TAT triplex controlled by miR-22 was generated in both acidic or neutral conditions. After the re-configuration of MB-CP to form triplex structure within three minutes, enabling MB to face toward the electrode that turned the signal-switch on. This newly developed biosensing platform is capable of detecting dual targets via pH control, offering the advantages of simplicity, cost effectiveness, and rapid detection. It holds great promise in diagnosis of liquid biopsy samples collected from EC patients, and sheds new light on the development of point-of-care (POC) diagnostics.