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Electrochemical-Fluorescent Bimodal Biosensor Based on Dual CRISPR-Cas12a Multiple Cascade Amplification for ctDNA
Hehua Zhang1, Hongmin Gao2, Wendi Mu2
1Collaborative Research Center, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China.
Analytical Chemistry
|August 12, 2024
Summary
This study presents a novel bimodal biosensor for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). The sensor accurately quantifies circulating tumor DNA (ctDNA) using dual CRISPR-Cas12a systems for early cancer diagnosis.
Area of Science:
- Biotechnology
- Molecular Diagnostics
- Oncology
Background:
- Circulating tumor DNA (ctDNA) is a vital biomarker for early cancer detection.
- Accurate quantification of low-abundance ctDNA in serum presents a significant analytical challenge.
- Epidermal growth factor receptor (EGFR) mutations, like L858R, are key targets in non-small cell lung cancer (NSCLC).
Purpose of the Study:
- To develop and validate a sensitive bimodal biosensor for detecting the EGFR L858R mutation in NSCLC patients.
- To address the challenge of low-abundance ctDNA quantification in human serum.
- To enable early tumor detection through precise biomarker analysis.
Main Methods:
- Utilized dual CRISPR-Cas12a systems for signal amplification and detection.
- Integrated fluorescence and electrochemical detection methods for signal verification.
- Leveraged the differential activity of CRISPR-Cas12a in response to the EGFR L858R mutation and wild-type EGFR (wt EGFR) in the presence of the restriction enzyme MscI.
Main Results:
- Achieved a dynamic detection range from 10 fM to 1 μM for EGFR L858R.
- Established a low limit of detection of 372 aM.
- Demonstrated excellent specificity, reproducibility, stability, and recovery rates, validating the sensor's performance.
- Successfully quantified EGFR L858R using both fluorescence and electrochemical signals.
Conclusions:
- The developed bimodal biosensor accurately quantifies EGFR L858R ctDNA, overcoming previous limitations in sensitivity.
- The dual-signal approach enhances reliability and accuracy in biomarker detection.
- This technology holds significant promise for early disease diagnosis and biosensing applications in oncology.
