Advantages of aptamers as ligands upon protein detection by AFM-based fishing.

A combined AFM/MS method was employed for protein registration in solution. This method is based on reversible specific capturing of a target protein from a large volume of analyzed solution onto a small sensor area of a chip with immobilized aptamer ligands. Fishing of the core antigen of hepatitis C virus (HCVcoreAg) from 10 M solution of this protein in buffer was carried out. It has been shown that the AFM/MS method allows the detection of HCVcoreAg in the form of a protein conjugate as well as in the presence of protein matrix (components of human serum). At the first stage, aptamer/antigen complexes were AFM-registered on the chip surface after its incubation in 10 M solution of antigen in buffer. A technique for the analysis was developed, and criteria for the evaluation of AFM analysis data based on the counting of the aptamer/antigen complexes were proposed. At the second stage, the mass spectrometric identification of protein objects on the chip surface was accomplished; this allowed the reliable identification of the target protein (the core antigen of hepatitis C virus, HCVcoreAg) and confirmed the AFM analysis data.