Detection of hepatitis C virus core protein in serum by atomic force microscopy combined with mass spectrometry.

A method for detection and identification of core antigen of hepatitis C virus (HCVcoreAg)-containing particles in the serum was proposed, with due account taken of the interactions of proteotypic peptides with Na(+), K(+), and Cl(-) ions. The method is based on a combination of reversible biospecific atomic force microscopy (AFM)-fishing and mass spectrometry (MS). AFM-fishing enables concentration, detection, and counting of protein complexes captured on the AFM chip surface, with their subsequent MS identification. Biospecific AFM-fishing of HCVcoreAg-containing particles from serum samples was carried out using AFM chips with immobilized antibodies against HCVcoreAg (HCVcoreAgim). Formation of complexes between anti-HCVcoreAgim and HCVcoreAg-containing particles on the AFM chip surface during the fishing process was demonstrated. These complexes were registered and counted by AFM. Further MS analysis allowed reliable identification of HCVcoreAg within the complexes formed on the AFM chip surface. It was shown that MS data processing, with account taken of the interactions between HCVcoreAg peptides and Na(+), K(+) cations, and Cl(-) anions, allows an increase in the number of peptides identified.

SOI nanowire for the high-sensitive detection of HBsAg and α-fetoprotein.

Silicon-on-isolator-nanowires (SOI-NWs) were used for the label-free, real-time biospecific detection of the hepatitis B marker HBsAg and cancer marker α-fetoprotein (AFP). Specific protein-protein recognition was carried out using individual NWs that were functionalized with antibodies. To solve the problem of non-specific binding of target protein molecules to the sensor element the use of a reference NW with immobilized antibodies against non-target proteins was proposed. Using individual SOI-NW surface functionalization allowed the fabrication of a NW array, containing working NWs and reference NWs within one chip. It was shown that this approach allows us to reach a detection limit up to 10(-14) and 10(-15) M for HBsAg and AFP, respectively. Our investigations also allowed us to reveal the influence of the charged state of the target protein molecules and antibodies in solutions with various pH values on the target protein detection limit. A high sensitivity NW-detector is of interest for the creation of diagnosticums for hepatitis B and for the early stages of cancer diseases.