Digital CRISPR-Powered Biosensor Concept without Target Amplification Using Single-Impact Electrochemistry.

Area of Science:

• Biotechnology and Biosensing
• Molecular Diagnostics
• Nanotechnology

Background:

• Accurate nucleic acid detection is vital for diagnostics like infectious diseases and cancer.
• Conventional methods (e.g., quantitative polymerase chain reaction) require complex lab setups and sophisticated equipment, limiting their accessibility.
• There is a need for rapid, reliable, and amplification-free nucleic acid quantification methods suitable for diverse applications.

Purpose of the Study:

• To develop a novel amplification-free digital sensing strategy for nucleic acid detection and quantification.
• To combine the collateral cleavage activity of the Cas12a enzyme with single-impact electrochemistry.
• To create stable DNA-nanoparticle conjugates for biosensor applications.

Main Methods:

• Utilized the Cas12a enzyme's collateral cleavage activity.
• Employed single-impact electrochemistry for detection.
• Modified silver nanoparticles (AgNPs) via temperature-assisted co-functionalization to create DNA-AgNP conjugates.
• Detected particle collision events as current spikes on a microelectrode array.

Main Results:

• Successfully developed stable DNA-AgNP conjugates through a straightforward co-functionalization process.
• Demonstrated the detection of Cas12a-activated particle release as distinct current spikes.
• Achieved amplification-free quantification of nucleic acids.

Conclusions:

• The study presents a novel, amplification-free digital sensing strategy for nucleic acid quantification.
• The integration of Cas12a-based diagnostics with impact-based digital sensing shows significant potential.
• The developed DNA-AgNP conjugates are suitable for various biosensor applications, enabling rapid and accessible diagnostics.