Atomic force microscopy fishing and mass spectrometry identification of gp120 on immobilized aptamers

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.

Aptamer-Sensitized Nanoribbon Biosensor for Ovarian Cancer Marker Detection in Plasma

“…This AFM image indicates that objects with heights of up to 4 nm were visualized on the substrate surface with immobilized aptamers after its incubation in the CA 125 solution. These objects have been attributed to aptamer-antigen complexes, since complexes of gp120 (whose molecular weight is 110 kDa, i.e., close to that of CA 125) with immobilized aptamers were demonstrated to have similar heights [39].…”

“…Atomic force microscopy (AFM) and mass spectrometry (MS) were employed in order to confirm the efficiency of the technique used for the sensitization of the NRs. Such an approach was shown to be an efficient tool in studying antibody-antigen [38] and aptamer-antigen [39,40] complexes on the surface of solid substrates sensitized with molecular probes. Since immobilized aptamers are barely distinguishable on the AFM substrate surface, the efficiency of the sensitization of the substrate surface with aptamers is estimated after the formation of aptamer-antigen complexes on it [39].…”

“…Such an approach was shown to be an efficient tool in studying antibody-antigen [38] and aptamer-antigen [39,40] complexes on the surface of solid substrates sensitized with molecular probes. Since immobilized aptamers are barely distinguishable on the AFM substrate surface, the efficiency of the sensitization of the substrate surface with aptamers is estimated after the formation of aptamer-antigen complexes on it [39]. Via AFM and MS, the successful formation of aptamer-antigen complexes on the surface of SOI substrates with covalently immobilized aptamers against CA 125 was revealed.…”

Nanowire Aptamer-Sensitized Biosensor Chips with Gas Plasma-Treated Surface for the Detection of Hepatitis C Virus Core Antigen

“…Therefore, the characteristic Debye screening length in 1 mM potassium phosphate buffer makes up ~5 nm [28], which is non-optimal for the detection of relatively large protein complexes formed on the SOI-NW sensor surface. This problem can be solved by using smaller probe molecules-for instance, aptamers instead of relatively large antibodies, as was demonstrated in a number of studies [24,[29][30][31]. NW biosensors with aptamer-sensitized sensor surfaces are called NW aptasensors [25].…”