Abstract：
Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.
摘要：
这里,通过靶标诱导的链置换效应，然后激活多组分核酸酶（MNAzyme）介导的裂解，设计了一种无酶侧流aptasensor，以实现快速，便携式的曲霉毒素A（OTA）检测。将底物制备为用磁珠(MB)和人绒毛膜促性腺激素(hCG)修饰的寡核苷酸链。OTA与适体的相互作用诱导阻断DNA的释放，与三个分离的DNA亚基杂交，形成序列特异性MNAzyme催化核心。该核心随后在Mg2辅因子存在下引发无酶MNAzyme裂解反应，切割特殊底物并释放不完全的MNAzyme催化核心和hCG-DNA探针。然后，底物再次识别不完整的MNAzyme催化核心，触发级联循环裂解，并导致产生大量的hCG-DNA探针。磁富集后，游离的hCG-DNA探针通过妊娠试纸（PTS）流向T线，生成明确确认目标OTA存在的比色读数。这项工作利用了MNAzyme的高效无酶切割扩增和基于PTS的便携式检测设备，提出了一种生物传感策略，具有灵敏和便携式OTA检测的巨大潜力。该方法对OTA检测具有显著的灵敏度和选择性，具有5nM的检测限。本研究成功地证明了该方法在实际样品上的实际应用。为快速和便携式检测真菌毒素提供了可行的替代方法。
