We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units─a peptide and a peptide nucleic acid (PNA), fused together. The peptide presents the substrate of MMP2, while the PNA serves as a nucleic acid output for subsequent processing. This chemical translator was immobilized on micrometer magnetic beads as a physical support for an activity-based assay. We incorporated into our design a single-stranded DNA partially hybridized with the PNA sequence and bearing a region complementary to the RNA guide of CRISPR-Cas12a. The target-induced nuclease activity of Cas12a results in the degradation of FRET-labeled DNA reporters and amplified fluorescence signal, enabling the detection of MMP2 in the low picomolar range, showing a limit of detection of 72 pg/mL. This study provides new design principles for a broader applicability of CRISPR-Cas-based biosensing.

中文翻译：

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使用 CRISPR-Cas12a 进行蛋白酶活性检测的合成蛋白质到 DNA 起始量交换


我们提出了一种针对基质金属蛋白酶 2 （MMP2） 的新型基于活性的检测策略，MMP2 是一种关键的癌症蛋白酶生物标志物，利用一种响应 MMP2 蛋白水解活性的机制及其与 CRISPR-Cas12a 辅助信号放大的整合。我们设计了一种化学翻译器，由两个功能单元——肽和肽核酸 （PNA） 融合在一起组成。肽呈递 MMP2 的底物，而 PNA 作为后续加工的核酸输出。该化学翻译器固定在微米磁珠上，作为基于活性的测定的物理支持。我们将与 PNA 序列部分杂交的单链 DNA 整合到我们的设计中，并带有与 CRISPR-Cas12a 的 RNA 向导互补的区域。Cas12a 的靶标诱导的核酸酶活性导致 FRET 标记的 DNA 报告基因降解并放大荧光信号，从而能够在低皮摩尔范围内检测 MMP2，显示 72 pg/mL 的检测限。本研究为基于 CRISPR-Cas 的生物传感的更广泛适用性提供了新的设计原则。