A novel universal small-molecule detection platform based on antibody-controlled Cas12a switching

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensor Technology

Background:

• Molecular diagnostics are crucial for disease detection, prevention, and treatment, especially at the point-of-care.
• CRISPR/Cas12a systems offer precise molecular detection capabilities.
• Developing sensitive and specific assays for small molecules remains a challenge.

Purpose of the Study:

• To develop a novel CRISPR/Cas12a-based platform for small-molecule detection.
• To create an Antibody-Controlled Cas12a Biosensor (ACCBOR) that utilizes antibody binding to regulate Cas12a activity.
• To demonstrate the platform's versatility and sensitivity for detecting various small molecules, including 25-hydroxyvitamin D3 (25-OH-VD3).

Main Methods:

• Designed an ACCBOR system where antibodies control CRISPR/Cas12a trans-cleavage activity.
• Labeled no target sequences (NTS) with small molecules, preventing Cas12a binding upon antibody interaction.
• Validated the platform using biotin, digoxin, and 25-OH-VD3, and developed a one-pot assay for 25-OH-VD3 detection.

Main Results:

• The ACCBOR platform demonstrated broad applicability across different small-molecule targets.
• A one-pot assay for 25-OH-VD3 achieved high sensitivity and specificity.
• The limit of detection for 25-OH-VD3 in serum was determined to be 259.86 pg/mL within 30 minutes.

Conclusions:

• The developed ACCBOR platform provides a sensitive, specific, and rapid method for small-molecule detection.
• The assay is cost-effective, easy to operate, and suitable for point-of-care applications.
• This platform opens new possibilities for the highly sensitive detection of diverse small-molecule targets.