Quantification of DNA and protein adsorption by optical phase shift

Area of Science:

• Biophysics
• Biochemistry
• Surface Science

Background:

• Label-free detection offers quantitative analysis of biomolecular interactions, surpassing fluorescent methods.
• Interferometric techniques link optical phase to surface biomolecular density, but lack accurate conversion factors.
• Accurate calibration is crucial for quantifying biomolecular binding kinetics.

Purpose of the Study:

• To develop and validate a calibration method for phase shift measurements in label-free detection.
• To establish a precise relationship between biolayer thickness and surface density.
• To enable accurate determination of absolute molecule numbers for various biomolecules.

Main Methods:

• A novel calibration method for phase shift measurements was developed.
• Known concentrations of bovine serum albumin, immunoglobulin G, and single-stranded DNA were precisely spotted.
• Evaporation of solvent allowed calculation of deposited mass, enabling comparison with measured thickness.

Main Results:

• A linear dependence between calculated mass and measured thickness was observed over four orders of magnitude.
• The conversion factor of 1 nm thickness ≈ 1 ng/mm² surface density was validated.
• The calibration method allows precise determination of absolute molecule numbers for proteins and DNA.

Conclusions:

• The developed calibration method accurately relates phase shift to biomolecular surface density.
• This technique enhances quantitative analysis of biomolecular interactions using label-free interferometry.
• Precise quantification of biomolecules is now achievable for diverse biological applications.