Quantification of DNA and protein adsorption by optical phase shift

Emre Özkumur, Ayça Yalçin, Marina Cretich, Carlos A. Lopez, David A. Bergstein, Bennett B. Goldberg, Marcella Chiari, M. Selim Ünlü*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to ∼1 ng/mm2 surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.

Original languageEnglish (US)
Pages (from-to)167-172
Number of pages6
JournalBiosensors and Bioelectronics
Volume25
Issue number1
DOIs
StatePublished - Sep 15 2009

Keywords

  • Biolayer density
  • Interferometry
  • Label-free detection
  • Mass quantification
  • Microarray
  • Optical biosensor

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry

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