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首页> 外文期刊>The Analyst: The Analytical Journal of the Royal Society of Chemistry: A Monthly International Publication Dealing with All Branches of Analytical Chemistry >Applying biosensor development concepts to improve preamplification-free CRISPR/Cas12a-Dx
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Applying biosensor development concepts to improve preamplification-free CRISPR/Cas12a-Dx

机译：应用生物传感器的开发概念来改进免费的免费CRISPR / CAS12A-DX

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摘要

Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to <46 min, representing the most sensitive LOD without preamplification and the fastest time-to-positive for this LOD to date. More broadly, our work provides a roadmap for further advancing Cas12a-Dx and perhaps other classes of CRISPR/Cas-Dx.
机译：基于CRISPR / CAS的体外诊断装置或CRISPR / CAS-DX的开发已成为一个强烈的研究区域。在不同类别的CRISPR / CAS-DX中，基于CAS12A酶（即CRISPR / CAS12A-DX或简单的CAS12A-DX）的类主要用于检测DNA靶标。 CAS12A-DX的当前研究专注于将CAS12A-DX附加到前置放大器技术或耦合CAS12A-DX到不同的检测方式，这不可避免地透过了CAS12A-DX的检测性能，并忽略了其内在检测能力，而无需预先放大。我们认识到Cas12A-DX，它依赖于DNA活化的CAS12A切割单链DNA，与其他基于核酸酶的DNA生物传感器共享显着的相似性，其性能可以受到从反应缓冲液到反应温度的参数的影响。因此，我们通过探索和系统地评估可能影响其检测灵敏度和时间的若干潜在参数来探讨无放大的CAS12A-DX的极限。使用先前报告的基于荧光的CAS12A-DX作为试验台，我们已经确定了CAS12A酶，反应缓冲液，底物标记，底物浓度和反应温度可以优化，以显着改善信号 - 到 - 背景比率与CAS12A-DX的反应速率。基于这些发现，我们改善了CAS12A-DX至100 FM的检测极限（LOD），同时将时间变为阳性至<46分钟，表示最敏感的床位，没有更放大器和最快的时间 - 迄今为止为此划分的呈现性。更广泛地，我们的工作为进一步推进CAS12A-DX提供了一种路线图，也许是其他类别的CRISPR / CAS-DX。

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