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

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Analytical Chemistry

Background:

• MicroRNAs (miRNAs) are crucial gene regulators and reliable biomarkers for early cancer detection and treatment.
• miRNA-21 is a significant biomarker providing critical information for cancer diagnosis, prognosis, and therapy.

Purpose of the Study:

• To develop a sensitive and straightforward electrochemical biosensor for miRNA-21 detection.
• To integrate CRISPR technology with isothermal amplification and enzymatic labeling for enhanced detection.

Main Methods:

• Utilized CRISPR-Cas12a system with target-induced self-priming hairpin isothermal amplification (SIAM) and terminal transferase (TdT) polymerization labeling.
• Designed a single-probe assay to suppress nonspecific reactions and enable unidirectional signal amplification.
• Employed Cas12a/crRNA to trans-cleave modified ssDNAs, followed by TdT labeling and SA-HRP catalyzed signal amplification.

Main Results:

• Achieved sensitive detection of miRNA-21 with a logarithmic relationship between cathodic current and concentration (20 fM to 5.0 × 10⁸ fM).
• Established a low detection limit of 9.2 fM for miRNA-21.
• Successfully quantified miRNA-21 in commercial serum samples and biological lysates.

Conclusions:

• The developed electrochemical biosensor offers a facile and sensitive method for miRNA-21 detection.
• Demonstrated the potential of this biosensor for early cancer diagnostics and therapy monitoring.
• Highlighted the synergistic integration of CRISPR, SIAM, and TdT for robust biosensing applications.