Origami Paper-Based Immunoassay Device with CRISPR/Cas12a Signal Amplification

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Assay Development

Background:

• Conventional enzyme-linked immunosorbent assays (ELISA) are time-consuming for clinical diagnosis.
• Paper-based ELISA offers faster, cheaper analysis but often lacks sensitivity.
• Limited sensitivity in paper-based assays hinders clinical application for low-concentration targets.

Purpose of the Study:

• To develop a simple, rapid, and highly sensitive detection method for non-nucleic acid targets.
• To integrate the CRISPR/Cas12a system into a paper-based ELISA platform.
• To enhance the sensitivity of paper-based immunoassays for clinical diagnosis.

Main Methods:

• Designed an origami-type paper-based device for assay operation.
• Utilized CRISPR/Cas12a enzyme with a fluorophore/quencher-labeled ssDNA probe for fluorescence detection.
• Employed antibodies labeled with DNA to activate CRISPR/Cas12a for enhanced signal amplification.

Main Results:

• Achieved significantly boosted detection sensitivity for human IgG and hepatitis B virus surface antigen (HBsAg).
• Demonstrated a limit of detection (LOD) of 12 pg/mL for HBsAg, over 10-fold improvement compared to commercial ELISA kits (200 pg/mL).
• Validated fluorescence detection in porcine whole blood samples using smartphone imaging for quantitative analysis.

Conclusions:

• The developed CRISPR/Cas12a-integrated paper-based ELISA platform offers high sensitivity and rapid detection.
• The platform shows potential for point-of-care clinical diagnostics, overcoming limitations of existing paper-based assays.
• This approach facilitates sensitive detection of non-nucleic acid targets in complex biological samples.