# Engineering a CRISPR-Mediated Dual Signal Amplification-Based Biosensor for miRNA Determination

## TL;DR

This paper describes a new CRISPR-based biosensor for detecting miRNA-21, a biomarker for cancer, with high sensitivity and accuracy.

## Contribution

A novel CRISPR-mediated dual signal amplification method for miRNA detection using SIAM and TdT labeling is introduced.

## Key findings

- The biosensor detects miRNA-21 with a detection limit of 9.2 fM.
- The method successfully quantifies miRNA-21 in commercial serum and biological lysates.
- The system uses a single probe and suppresses nonspecific reactions for improved accuracy.

## Abstract

MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO43− modified single-stranded DNAs (ssDNAs); the resulting 3′ hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 108 fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.