A Non-Label and Enzyme-Free Sensitive Detection Method for Thrombin Based on Simulation-Assisted DNA Assembly.

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biochemistry

Background:

• Thrombin is a key biomarker in various diseases, necessitating sensitive detection methods.
• Existing thrombin detection methods often rely on enzymes or labels, limiting their practicality.
• Aptamer-based assays offer high specificity but require efficient signal amplification.

Purpose of the Study:

• To develop a novel, enzyme-free, and label-free fluorescent and colorimetric strategy for sensitive thrombin detection.
• To leverage catalyzed hairpin assembly (CHA) for signal amplification in aptamer-based assays.
• To validate the assay's performance through theoretical simulation and experimental testing.

Main Methods:

• Utilized aptamers for specific thrombin recognition and a designed DNA hairpin for CHA initiation.
• Employed a label-free approach where target binding liberates an initiation strand to trigger CHA.
• Incorporated G-quadruplex formation for signal generation, detected using N-methyl-mesoporphyrin IX (fluorescent) and TMB (colorimetric).
• Performed thermodynamic simulations of DNA interactions and strand displacement processes.

Main Results:

• Achieved highly sensitive thrombin detection down to 2.4 pM.
• Generated distinct fluorescent and colorimetric signals, with the latter visible to the naked eye.
• Simulations confirmed the feasibility of the toehold strand displacement-driven assembly process.
• Demonstrated a label-free and enzyme-free amplification strategy with high specificity.

Conclusions:

• The developed aptamer-CHA strategy offers a sensitive, rapid, and cost-effective method for thrombin detection.
• The integration of theoretical simulation simplifies experimental design and enhances understanding.
• This approach holds significant potential for biomarker detection and quantitative monitoring in disease diagnosis.