以非酵素型訊號放大策略結合電化學分析平台應用於研發液體活檢中之胃癌標記核酸分子

Abstract

近年來，微型核醣核酸 (miRNA) 被指出為多種疾病的新興生物標誌 (Biomarker)，並被視有取代傳統侵入式活檢手術的發展潛力。而miR-223在文獻中被提出為一可靠的早期胃癌之癌症標誌，因此我們擬發展一個對於胃癌病患體內異常表現之miRNA-223具有高專一性、高靈敏性的檢測平台。此實驗中，我們以核酸迴路 (Nucleic acid circuits) 搭配電化學分析平台設計了一種非酵素型的檢測方法。首先，目標miRNA會被雙探針系統 (Binary probe) 專一性的辨識，以此誘使一條單股的DNA initiator被釋放並啟動下一階段的雜合連鎖反應 (Hybridization Chain Reaction, HCR)，讓訊號得以被放大。在此HCR反應中，髮夾結構的DNA hairpins自發性的組裝形成DNA奈米線 (Nanowires)，並能夠與修飾在電極表面上的核酸探針進行雜合。此時，位於hairpin上被截成兩段的G-四聯體 (G-quadruplex) 序列會由於形成長鏈的雙股結構而互相靠近，並且在加入氯化血紅素 (Hemin) 時形成具有類似辣根過氧化物酶 (Horseradish peroxidase, HRP) 活性的DNAzyme。當環境中有對苯二酚 (Hydroquinone，HQ) 作為中介物質時，具HRP活性的DNAzyme便可催化過氧化氫的還原，並產生電化學上的訊號。此種不需酵素的檢測策略能在溶液系統中靈敏且專一性的檢測出微量的miRNA，可在合理的反應時間內達成且耗費相對低的實驗成本。此系統之偵測極限 (Limit of detection) 達到2.15 pM，線性範圍在1 pM~1000 pM之間，涵跨3個數量級。預期在未來能夠將我們發展出的篩檢策略應用於常規性的身體檢查，於胃癌的早期階段將其篩檢出並輔助醫生進行即時且準確的臨床診斷及治療，藉此提升胃癌病患的存活率。

MicroRNAs have been reported intensively as novel biomarkers for varieties of diseases providing alternatives to replace traditional invasive biopsy procedures. MiR-223 was found previously to be a reliable gastric cancer marker in early stage. We therefore aimed to develop an analytical method with high specificity and sensitivity to detect miR-223 that expressed abnormally in patients with gastric cancer. A non-enzymatic detection method was herein designed and developed, combining nucleic acid circuits and electrochemical detection platform. Firstly, the target miRNA was selectively recognized by one of the binary probes to induce a release of ssDNA initiators, and subsequently promoting a Hybridization Chain Reaction (HCR) to occur for amplification. It was followed by the generation of the DNA nanowires by autonomous assembly of hairpins that could hybridize with capture probes modified on the electrode surface. Due to the approaching of two split sequences of G-quadruplex on the hairpins, a horseradish peroxidase (HRP)-mimicking DNAzyme was formed simultaneously, upon addition of hemin. With presence of hydroquinone as mediator, HRP-mimicking DNAzyme catalyzed redox reaction of hydrogen peroxide, producing an electrochemical signal. This enzyme-free strategy enables not only sensitive but selective detection of trace amount of miRNA in buffer system with reasonable assay time and relatively low cost. Our detection platform offers a detection limit of 2.15 pM, and a linear dynamic range of 1 pM~1000 pM, covering 3 orders of magnitude. Our assay design may be further extended to be used in routine physical checkup, leading to an early detection procedure for gastric cancer that aids doctors to make prompt and accurate clinical decision to improve survival rate.