Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze 17/A, 43124 Parma, Italy.
Department of Chemistry, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy.
Anal Chem. 2024 Nov 26;96(47):18645-18654. doi: 10.1021/acs.analchem.4c02622. Epub 2024 Nov 14.
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.
我们提出了一种新颖的基于活性的基质金属蛋白酶 2（MMP2）检测策略，MMP2 是一种关键的癌症蛋白酶生物标志物，利用对 MMP2 的蛋白水解活性有响应的机制及其与 CRISPR-Cas12a 辅助信号放大的整合。我们设计了一种包含两个功能单元的化学翻译器 - 肽和肽核酸（PNA），融合在一起。肽呈现 MMP2 的底物，而 PNA 作为随后处理的核酸输出。这种化学翻译器固定在微米级的磁性珠上，作为基于活性的测定的物理支撑。我们将部分与 PNA 序列杂交的单链 DNA 纳入我们的设计中，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 报告分子的降解和扩增的荧光信号，从而能够在低皮摩尔范围内检测 MMP2，检测限为 72 pg/mL。这项研究为基于 CRISPR-Cas 的生物传感的更广泛适用性提供了新的设计原则。
