A CRISPR/dCas9 mediated electrochemical impedimetric biosensor for sensitive mtDNA detection

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Biosensors

Background:

• Mitochondrial DNA (mtDNA) mutations are linked to human diseases.
• Detecting single-nucleotide variations (SNVs) in mtDNA is crucial for understanding disease mechanisms.
• Existing sequencing methods are complex, and CRISPR-based methods risk contamination.

Purpose of the Study:

• To develop a sensitive and specific method for SNV detection in mtDNA.
• To overcome limitations of existing sequencing and CRISPR-based approaches.
• To enable trace DNA analysis for disease-related biomarkers.

Main Methods:

• Developed a CRISPR/dCas9-mediated electrochemical impedimetric biosensor.
• Utilized dCas9/sgRNA for specific mtDNA sequence recognition.
• Employed hybridization chain reaction (HCR) for signal amplification.
• Used PEI-Ag NPs for signal enhancement and EIS for quantification.

Main Results:

• Achieved sensitive and specific detection of mtDNA SNVs without pre-amplification.
• Demonstrated a low detection limit of 67 fM.
• Showcased excellent anti-interference ability in 10% human plasma.
• Successfully detected mtDNA mutations in cell lysates.

Conclusions:

• The developed strategy offers a highly sensitive tool for mtDNA mutation detection.
• The method exhibits excellent anti-interference in complex biological samples.
• Presents a viable strategy for electrochemical detection of cancer biomarkers with diagnostic potential.