Detection of Tetracycline with a CRISPR/Cas12a Aptasensor Using a Highly Efficient Fluorescent Polystyrene...

Area of Science:

• Molecular Biology
• Biotechnology
• Biosensing

Background:

• CRISPR-Cas diagnostics offer specific, sensitive, and rapid detection for medical applications.
• Current CRISPR/Cas systems face limitations in reporter stability and sensitivity, hindering complex biological analyses.

Purpose of the Study:

• To investigate CRISPR/Cas12a trans-cleavage activity using fluorescent microspheres for tetracycline detection.
• To develop and validate stable and sensitive microsphere-based CRISPR/Cas aptasensors.

Main Methods:

• Utilized fluorescent polystyrene microspheres as substrates for CRISPR/Cas12a trans-cleavage activity.
• Developed microsphere-based fluorescent probes by attaching single-stranded DNA (ssDNA) reporters to microspheres.
• Investigated the impact of ssDNA reporter concentration on Cas12a activity.

Main Results:

• Cas12a system demonstrated robust and sensitive trans-cleavage activity on microsphere-bound ssDNA reporters.
• Microsphere probes significantly improved reporter stability and overall biosensor sensitivity.
• Achieved a highly sensitive tetracycline detection limit of 0.1 microM.
• Successfully applied the microsphere-based CRISPR/Cas aptasensor to spiked food samples.

Conclusions:

• Microsphere-based CRISPR/Cas aptasensors overcome limitations of traditional ssDNA reporters, offering enhanced stability and sensitivity.
• This innovative approach holds significant potential for sensitive and reliable biological research and medical diagnostics.
• The developed aptasensor is practical for real-world applications, including food safety testing.