One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Traditional protein biomarker detection methods like ELISA and mass spectrometry are complex and multi-step.
• Existing CRISPR-Cas12a protein detection assays often retain workflow complications, limiting point-of-care applications.

Purpose of the Study:

• To develop a simplified, single-step, one-pot, proximity-based isothermal immunoassay for homogeneous protein detection.
• To integrate CRISPR-Cas12a technology with a simplified workflow for high-sensitivity protein analysis.

Main Methods:

• Designed a proximity-based isothermal immunoassay using CRISPR-Cas12a for protein detection.
• Utilized probes with various binders (small molecule, aptamer, antibody) conjugated to oligonucleotides.
• Achieved two-way extension upon target binding, initiating DNA amplification for Cas12a signal generation in a single buffer system.

Main Results:

• Demonstrated a single-step, one-pot, isothermal assay for protein detection within a 10 μL reaction volume.
• Proved the successful integration of target binding, DNA amplification, and Cas12a signal generation.
• Showcased plug-and-play applicability with four diverse protein targets (streptavidin, PDGF-BB, antidigoxigenin antibody, IFNγ) with limits of detection from fM to pM.

Conclusions:

• The developed assay offers a simplified workflow for homogeneous protein detection.
• This method achieves high sensitivity and broad applicability for various protein targets.
• The assay holds potential for advancing point-of-care diagnostic tools.