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Yuri D Ivanov,1 Anna L Kaysheva,1,2 Pavel A Frantsuzov,1 Tatyana O Pleshakova,1 Nikolay V Krohin,1 Alexander A Izotov,1 Ivan D Shumov,1 Vasiliy F Uchaikin,1 Vladimir A Konev,1 Vadim S Ziborov,1 Alexander I Archakov11Institute of Biomedical Chemistry, 2PostgenTech Ltd, Moscow, RussiaAbstract: 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.Keywords: hepatitis C virus, molecular detector, biospecific fishing

Yuri D Ivanov,1 Natalia S Bukharina,1 Tatyana O Pleshakova,1 Pavel A Frantsuzov,1 Elena Yu Andreeva,1 Anna L Kaysheva,1,2 Victor G Zgoda,1 Alexander A Izotov,1 Tatyana I Pavlova,1 Vadim S Ziborov,1 Sergey P Radko,1 Sergei A Moshkovskii,1 Alexander I Archakov1 1Department of Personalized Medicine, Orekhovich Institute of Biomedical Chemistry of the Russian Academy of Medical Sciences, Moscow, Russia; 2PostgenTech Ltd., Moscow, Russia Abstract: Atomic force microscopy (AFM) was applied to carry out direct and label-free detection of gp120 human immunodeficiency virus type 1 envelope glycoprotein as a target protein. This approach was based on the AFM fishing of gp120 from the analyte solution using anti-gp120 aptamers immobilized on the AFM chip to count gp120/aptamer complexes that were formed on the chip surface. The comparison of image contrasts of fished gp120 against the background of immobilized aptamers and anti-gp120 antibodies on the AFM images was conducted. It was shown that an image contrast of the protein/aptamer complexes was two-fold higher than the contrast of the protein/antibody complexes. Mass spectrometry identification provided an additional confirmation of the target protein presence on the AFM chips after biospecific fishing to avoid any artifacts. Keywords: gp120 HIV-1 envelope glycoprotein, aptamer, atomic force microscopy, mass spectrometry