瞬間訊號放大方法用於超靈敏快速偵測肺結核中單核甘酸多態性

Abstract

近幾年，由於許多傳染性疾病的盛行與變異，使得對快速、靈敏且簡單的DNA診斷方法需求日益漸增。本研究目標為開發一種瞬間放大方法結合電化學偵測用於無須聚合酶鏈鎖反應的肺結核分子診斷。我們透過丁純與水溶液相互微溶的特性，使得捕獲探針DNA、單股錯配DNA與金奈米粒子溶液瞬間濃縮至微小體積，以大幅減少在溶液中所需要的反應與擴散時間。此外，由於兩段單股DNA會雜交並以超高密度結合至金奈米粒子表面，使電化學訊號顯著增加。為了降低非特異性吸附的影響與辨認錯配DNA，我們依序修飾自組裝單層與MutS蛋白質在工作電極表面。這種靈敏且專一的方法可以檢測阿托莫爾濃度程度的DNA目標物，並成功應用於檢測骨關節液中的結核病相關之單核苷酸多態性。更重要的是，由於此生物感測策略可在幾秒鐘完成，因此它在定點照護和分子診斷的應用中有巨大的潛力。

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications.