Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protein biomarker assays are crucial for diagnostics and research.
• Monoclonal antibodies, commonly used, have production and variability issues.
• Nucleic acid aptamers offer easier synthesis but often lack affinity.

Purpose of the Study:

• To evaluate aptamers as detection probes in SiMREPS for sensitive biomarker detection.
• To demonstrate the advantages of aptamers in SiMREPS compared to traditional methods.
• To optimize aptamer kinetics for enhanced SiMREPS performance.

Main Methods:

• Systematic evolution of ligand by exponential enrichment (SELEX) for aptamer generation.
• Single molecule recognition through equilibrium Poisson sampling (SiMREPS) platform.
• Aptamer sequence optimization through single nucleotide mutation and shortening.

Main Results:

• Achieved low femtomolar limits of detection (3-9 fM) for VEGF165 and IL-8 using aptamers in SiMREPS.
• Demonstrated rational optimization of aptamer kinetics for improved SiMREPS probe performance.
• Successfully detected endogenous IL-8 in human serum below ELISA detection limits.

Conclusions:

• Aptamers are effective detection probes for SiMREPS, offering high sensitivity and overcoming antibody limitations.
• SiMREPS with optimized aptamers provides a sensitive, reproducible, and precise method for biomarker quantification.
• This approach holds promise for advanced clinical diagnostics and biomedical research applications.