Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device

Area of Science:

• Biotechnology
• Biosensing
• Molecular Diagnostics

Background:

• Nucleic acid testing (NAT) is crucial but often complex, requiring specialized equipment and reagents.
• Existing NAT methods face limitations in user-friendliness, speed, and accessibility for field applications.

Purpose of the Study:

• To develop a fully integrated, 3D-printed biosensing device for user-friendly and highly sensitive nucleic acid detection.
• To utilize a CRISPR/Cas12a-based dual-enzymatic mechanism for amplification-free detection.
• To enable both visual and quantitative DNA analysis with smartphone compatibility.

Main Methods:

• A 3D-printed device incorporating a probe stick with gold nanoparticles for enhanced enzyme labeling.
• CRISPR/Cas12a system with alkaline phosphatase (ALP) conjugated via single-stranded DNA for signal generation.
• Development of a smartphone accessory for quantitative analysis and integration of all reagents.

Main Results:

• Achieved a limit of detection (LOD) as low as 10 pM for amplification-free colorimetric nucleic acid detection using the probe stick.
• The integrated device demonstrated a visual detection limit of approximately 100 pM.
• The system exhibited high specificity, successfully distinguishing between matched and mismatched nucleic acid sequences.

Conclusions:

• The developed 3D-printed biosensor offers a simple, portable, and sensitive platform for nucleic acid detection.
• This technology shows promise for rapid screening in clinical diagnostics, environmental monitoring, and food safety.
• The study advances the development of practical biosensors for point-of-care testing (POCT) applications.