Rolling circle amplification (RCA) has been widely used for nucleic acid and other biomarkers analysis, however, the limited amplification efficiency and low specificity in traditional RCA hinder its further application. Here, a toehold mediated feedback rolling circle amplification (TMFRCA) strategy with exponential signal amplification was proposed for label-free nucleic acids sensing. The rational engineered TH and CT ensured exponential signal amplification by utilizing the feedback of amplicons to the reactant TH for circularly initiating new RCA. Introduction of toehold strand displacement and G-quadruplex (G4) dimers in RCA reaction allowed enhanced specific recognition and further improved fluorescence signal due to higher quantum yield of G4 dimers/ThT. Taking classic swine fever virus (CSFV) as model target, this TMFRCA method exhibited great sensitivity and high specificity with a limit of detection down to 0.29 fM and enabled to discriminate target with single base differences. In addition, the proposed TMFRCA could be used for detecting CSFV RNA in actual samples with great accuracy comparable to RT-qPCR, and possessed great universality for detecting nucleic acid without length limitation. We believe this method can promote the development of nucleic acid signal amplification and be an effective tool for nucleic acid sensing-based fundamental research and clinical diagnosis.

滚环扩增（RCA）已广泛用于核酸和其他生物标志物的分析，但传统RCA的扩增效率有限且特异性低，阻碍了其进一步应用。在这里，提出了一种用于无标记核酸传感的具有指数信号放大的立足点介导的反馈滚环放大 (TMFRCA) 策略。合理设计的 TH 和 CT 通过利用扩增子对反应物 TH 的反馈来循环启动新的 RCA，确保了指数信号放大。由于 G4 二聚体/ThT 的更高量子产率，在 RCA 反应中引入立足点链置换和 G-四链体 (G4) 二聚体可以增强特异性识别并进一步改善荧光信号。以经典猪瘟病毒（CSFV）为模型靶点，这种 TMFRCA 方法表现出极高的灵敏度和高特异性，检测限低至 0.29 fM，并且能够区分具有单碱基差异的目标。此外，所提出的TMFRCA可用于检测实际样本中的CSFV RNA，其准确度可与RT-qPCR相媲美，并且在检测核酸方面具有很强的普适性，不受长度限制。我们相信这种方法可以促进核酸信号放大的发展，成为基于核酸传感的基础研究和临床诊断的有效工具。对核酸检测具有很强的普适性，不受长度限制。我们相信这种方法可以促进核酸信号放大的发展，成为基于核酸传感的基础研究和临床诊断的有效工具。对核酸检测具有很强的普适性，不受长度限制。我们相信这种方法可以促进核酸信号放大的发展，成为基于核酸传感的基础研究和临床诊断的有效工具。