利用催化髮夾組合放大結合暗場顯微鏡影像開發偵測對卵巢癌具特異性的微小核糖核酸感測器

卵巢癌佔臺灣地區婦科癌症的第二位，由於缺乏典型臨床徵狀，且當前的篩檢方法缺乏精準度，因此 75 %卵巢癌患者被診斷出來時通常已屬晚期，有遠端轉移和擴散的現象，然而患者若能被早期偵測並接受治療，則5年存活率可從10 %提升至 80 %，因此開發一種新型且非侵入性早期偵檢方法為亟欲達成的目標。先前研究指出，miR-205為典型卵巢癌之生物標誌，而miR-93被發現在CA-125出現前就有上調現象，兩者均可有效作為卵巢癌的生物標誌。於此，我們結合核酸循環放大系統 (nucleic acid circuits) 並搭配暗場顯微鏡偵測不同形狀之金奈米粒子，開發可同時偵測兩種目標物，不需酵素參與之等溫放大核酸檢測平台。在催化髮夾組裝(Catalytic Hairpin Assembly, CHA) 系統中，兩種目標 miRNA 是雜合反應中的啟動子。當目標 miRNA 被 Hairpin DNA (I) 進行專一辨認後，第二條 Hairpin DNA (II) 則啟動自發性組裝的功能，接續與前者雜合並取代目標 miRNA，使之可被循環利用。藉由暗場顯微鏡對攜有金奈米粒子之 CHA 產物進行成像，粒子會分別呈現綠色及藍色，並以程式計數作為訊號定量的基礎。本研究針對 Hairpin DNAs 序列及反應比例、緩衝溶液條件、聚乙二醇 (Polyethylene glycol, PEG) 濃度與反應時間進行實驗條件之優化，並透過電泳膠圖選定CHA 鏈置換反應的最佳條件。利用此種無需酵素參與反應的等溫放大策略，結合暗場顯微鏡計數的方法，我們建立一個新型 miRNA 的偵測平台。期望未來能應用此平台所建立好的序列設計原則，結合更多具不同散射光特性之奈米粒子，達到多因子檢測之成效，以增加早期篩檢卵巢癌或其他癌病的精準度。

關鍵字

卵巢癌；微小核糖核酸；非酵素；核酸等溫擴增技術；催化型髮夾組裝；金奈米粒子；暗場顯微鏡

並列摘要

Ovarian cancer is one of the leading causes of death among gynecological cancers due to its lack of specific clinical symptom and low accuracy diagnosis tests. Previous studies reported that the five-year survival rate has increased from 10% to 80% for patients who were diagnosed and received proper treatment at early stage. Therefore, there is an urgent need to develop a non-invasive and effective method for early ovarian cancer diagnosis. MiR-205 and miR-93 have been found to be upregulated significantly in the serum of cancer patients, thus both can serve as potential biomarkers. The combination of nucleic acid circuits and plasmonic nanoparticles enables us to detect two target miRNAs simultaneously using dark-field microscopic technology. The single strand initiator (target miRNA) can interact with a toehold of the hairpin DNA (I) on the gold nanomaterials and open the structure, resulting in the formation of a liner H (I) / T duplex with a newly exposed single-stranded region. Subsequently, hairpin DNA (II) as a fuel can hybridize with the toehold region of the H (I) /T duplex to form the “H (I) / H (II)” duplex and release the target through the displacement reaction. The optimized reaction conditions for the operation of catalytic hairpin assembly, including modification of DNA sequences for various hairpins, ratio of hairpins, type of working buffer, polyethylene glycol concentration, and reaction time were investigated by polyacrylamide gel electrophoresis. The results demonstrated that the target-induced cross-opening of these hairpin reactants led to an effective generation of products, gold nanomaterials-tagged hpDNAs. With the aid of dark-field microscope, two distinct light scattering signals [green (G), and blue (B)] were exhibited. The direct counting of plasmonic particles was carried out for signal quantification. This enzyme-free, isothermal amplification strategy based on spectral-resolved dark-field images enables simultaneous detection of multiple miRNA targets in a label-free, simple and straightforward manner within 2 hours for the detection of ovarian cancer-associated miRNAs. The sensing platform was confirmed to hold great potential for early diagnosis and prognosis of ovarian cancer.

並列關鍵字

ovarian cancer ； miRNA ； nucleic acid circuits ； catalytic hairpin assembly ； gold nanomaterials ； dark-field microscope

參考文獻

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利用催化髮夾組合放大結合暗場顯微鏡影像開發偵測對卵巢癌具特異性的微小核糖核酸感測器

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