Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique

Area of Science:

• Biophysics
• Molecular Biology
• Surface Science

Background:

• Accurate detection of nucleic acid hybridization is vital for biomedical applications.
• Current methods face challenges in rapid, quantitative, and label-free surface detection.
• Understanding DNA-surface interactions is key to developing advanced biosensors.

Purpose of the Study:

• To evaluate the Reflective Phantom Interface (RPI) for quantifying DNA hybridization.
• To investigate the impact of sequence defects on DNA-surface binding kinetics and equilibrium.
• To compare DNA-DNA binding on a surface versus in bulk solution.

Main Methods:

• Utilized the Reflective Phantom Interface (RPI), a label-free multiplexed detection technique.
• Measured reflected light intensity to quantify hybridization of DNA oligomers.
• Analyzed hybridization of complementary and defected 12-base DNA oligomers.

Main Results:

• RPI achieved sensitive detection (10 pg mm(-2)) of DNA hybridization in real-time.
• Single-base mismatches and their location significantly affected hybridization kinetics and binding.
• DNA-DNA binding affinity was found to be lower on a surface compared to bulk solution.

Conclusions:

• RPI is a viable technology for sensitive, quantitative, label-free detection of nucleic acid hybridization.
• Surface-based DNA binding is influenced by sequence fidelity and probe-surface interactions.
• Weak nonspecific probe binding to the surface contributes to reduced binding affinity in surface assays.