Quantitative analysis of the number of antigens immobilized on a glass surface by AFM

Ultramicroscopy. 2004 Aug;100(3-4):203-10. doi: 10.1016/j.ultramic.2004.01.015.

Abstract

To develop force measurements using an atomic force microscope (AFM) in a quantitative manner, it is necessary to estimate the number density of target molecules on a sample surface, and for this, the sensitivity of detection should be known. In this study, the AFM was used as a mechanical detector and an antigen and its antibody were used as a model to evaluate the sensitivity of detection. Antigens were immobilized on a glass surface and number density was estimated by monitoring optical absorbance due to product formation by the reaction of crosslinkers. The concentration of antigen was controlled by mixing control peptides. A microbead was used as a probe and antibodies were immobilized on the bead. AFM force measurements were then made for a range of number densities in the order of 10-10(6) antigen molecules per square micrometer of surface and were compared to evaluate the sensitivity of detection. Our result establishes the reliability of estimating a number of molecules like receptors on the cell surface, and indicates that the AFM is useful as a mechanical detector with high sensitivity.

MeSH terms

  • Amino Acid Sequence
  • Antigens / analysis*
  • Microscopy, Atomic Force / methods*
  • Molecular Sequence Data

Substances

  • Antigens