微小核醣核酸（miRNA）為具有分析價值的癌症相關生物標記，惟現行檢測技術常受限於對外源性酵素的依賴、繁複的溫控需求及微量樣本分析能力不足等限制。為此，本研究旨在建立一套「數位液滴級聯催化髮夾擴增檢測流程」，發展具備穩定度與分析效率之無酶等溫檢測平台。針對傳統催化髮夾自組裝（Catalytic Hairpin Assembly， CHA）反應動力學受限之物理特性，本研究設計了級聯催化髮夾擴增系統。該設計利用二級CHA產物作為一級反應之次級觸發因子（Secondary Trigger），形成自催化反饋迴路，具提升訊號生成速率與光學判讀可讀性之特性。同時，本研究進一步結合數位液滴技術，將反應體系分割為單分散微液滴，藉由微觀空間之區隔化效應降低背景干擾，並實現miRNA之數位濃度推算。本研究以miRNA-21作為模型標的進行平台驗證，實驗結果顯示，整合級聯擴增與數位液滴區隔化技術之檢測流程，能有效改善無酶反應之信號累積表現，成功對飛莫耳（fM）等級之微量miRNA-21進行分析。綜上所述，本研究初步建構之檢測流程，不僅提供非酵素核酸檢測新的可行方案，亦為未來生物標記分析與定點照護應用奠定基礎。;MicroRNAs (miRNAs) serve as important cancer-related biomarkers holding clinical analytical value; however， current detection methodologies are often constrained by their reliance on exogenous enzymes， complex thermal cycling requirements， and limited performance for low-abundance targets. To address these challenges， this study establishes a "Droplet Digital Cascade Catalytic Hairpin Assembly quantitative detection workflow"， developing an enzyme-free isothermal sensing platform. To improve the kinetic rates inherent in traditional Catalytic Hairpin Assembly (CHA)， a dual-hairpin cascade amplification system was designed. This design employs the product of the secondary CHA as a secondary trigger for the primary reaction， establishing an autocatalytic feedback loop to enhance signal generation rates and readability. Furthermore， by integrating Droplet Digital technology， the reaction system is partitioned into monodisperse microdroplets， utilizing spatial compartmentalization to suppress background interference and enable Poisson-based miRNA concentration estimation. Utilizing miRNA-21 as a model target， experimental results demonstrate that this integrated workflow enhances the analytical performance of non-enzymatic assays， achieving femtomolar (fM) detection limits. In conclusion， by synergizing the cascade strategy with droplet partitioning， this study provides a reliable methodology for non-enzymatic nucleic acid sensing， offering technical support for potential point-of-care applications.
