Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis

Jul 17, 2025Methods (San Diego, Calif.)

Highly sensitive detection of MMP-2 using CRISPR-enhanced sensor for diagnosing COPD

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Abstract

The biosensor achieved a detection limit as low as 62.05 fM for matrix metalloproteinase-2 (MMP-2).

The biosensor utilizes T7 RNA polymerase amplification and CRISPR/Cas13a for signal enhancement.
A peptide nucleic acid (PNA) probe specifically targets MMP-2 cleavage to release DNA templates.
The generated RNA activates CRISPR/Cas13a, significantly amplifying the electrochemiluminescence (ECL) signal.
The biosensor demonstrated excellent specificity and resistance to interference in complex biological samples.
This detection method may provide an efficient strategy for monitoring biomarkers related to chronic inflammatory diseases.

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In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor's surface was constructed using a AuNPs/TiCTx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring. 3 2

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Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis

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