This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed.

Atomic force microscopy (AFM) study of the oligomeric state of CYP102A1 was carried out in three different environmental conditions – liquid, air and vacuum. It was shown that usage of a standard probe with a radius of curvature of 10 nm allowed determination of the monomers-to-oligomers ratio, α ≈ 0.5 : 0.5, in these three media, but it wasn't possible to estimate the oligomerization degree more precisely. Application of a supersharp probe with a radius of curvature of 2 nm made it possible not only to obtain data on the ratio of monomers-to-oligomers but also to obtain the approximate ratio between dimers, trimers and tetramers 0.3 : 0.1 : 0.1, but only in vacuum.

Atomic force microscopy with two types of probes—standard (radius of curvature R ∼ 10 nm) and supersharp (R ∼ 2 nm)—was used to determine an oligomeric state of CYP102A1. Using the standard probes CYP102A1 images were obtained in liquid, air and vacuum environments, and a CYP102A1 monomer: oligomer ratio α ≈ 1 was also determined. However, the use of standard probes did not allow to resolve structures of these oligomers. Using the supersharp probes it was possible to determine not only the monomer: oligomer ratio, but also to evaluate the dimer: trimer: tetramer ratio in vacuum. Thus, the ratio α for CYP102A1 in liquid can be determined by the standard probes in liquid, air, and vacuum, while oligomeric states of this protein can be specified by using the supersharp probes in vacuum.

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

Change in temperature is one of the factors affecting the activity of enzymes. In this work, the thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. The specific temperature transitions were studied by fluorescence analysis. In the low melting temperature range (10–33°C), a decrease in the fluorescence intensity of the aromatic residues was observed simultaneously with an increase in the fluorescence intensity of the flavin groups. The protein melting in this range indicated three narrow S-shaped cooperative transitions at 16, 22, and 29°C. Atomic force microscopy analysis in this temperature range showed that the BM3 molecules retained a globular shape as compact objects (heights, h < 7 nm; lateral dimensions, d < 50 nm), but the protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one by its increase.