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.

The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications-for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10 −12 W/cm 2 at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10 −12 W/cm 2) radiation power density; at the same time, the enzymatic activity remained unchanged. the results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects-for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops. It is known that electromagnetic radiation of various intensity can have different influence on human body. Electromagnetic fields can have various topology, such as transverse and knotted one (knotted electromagnetic field, KEMF) 1,2. The simplest and most common electromagnetic waves are transverse ones, and, to date, their effects have been widely studied in various frequency and intensity ranges. As regards biomedical applications, microwave radiation is interesting in that, depending on its intensity, it is employed in biological research, and in both medical diagnostics and therapy. In this way, upon exposure of biological tissues to high-intensity radiation (~90 W/cm 2), their temperature increases to ~90 °С, and denaturation of biological objects is observed. It was shown that, under such conditions, partial loss of functional activity of proteins (for instance, peroxidase) is observed 3. At lower radiation intensity (10 μW/cm 2 4), both positive therapeutic effects (which usually take place owing to local heating 5) and negative effects are observed. Here, it should be noted that studies on the application of non-thermal effects of low-power microwave r...

The influence of liquid motion in flow-based systems on the aggregation state of an enzyme and on its enzymatic activity was studied, with horseradish peroxidase (HRP) as an example. Our experiments were carried out in a setup modeling the flow section of the biosensor communication with a measuring cell containing a protein solution. Studies were conducted for a biosensor measuring cell located along the axis of a spiral-moving liquid flow. The aggregation state of the protein was determined with an atomic force microscopy-based sensor (AFM sensor). It has been demonstrated that upon flowing of water through silicone biosensor communications, an increased aggregation of HRP protein was observed, but, at the same time, its enzymatic activity did not change. Our results obtained herein are useful in the development of models describing the influence of liquid flow in biosensor communications on the properties of enzymes and other proteins. This is particularly important for the development of serologic protein biosensors, which are beginning to be used for the early diagnosis of oncological diseases (such as brain cancer, prostate cancer, breast cancer etc.). The results obtained herein should also be taken into account when considering possible changes in hemodynamics due to increased protein aggregation.

An approach combining atomic force microscopy (AFM) fishing and mass spectrometry (MS) analysis to detect proteins at ultra‐low concentrations is proposed. Fishing out protein molecules onto a highly oriented pyrolytic graphite surface coated with polytetrafluoroethylene film was carried out with and without application of an external electric field. After that they were visualized by AFM and identified by MS. It was found that injection of solution leads to charge generation in the solution, and an electric potential within the measuring cell is induced. It was demonstrated that without an external electric field in the rapid injection input of diluted protein solution the fishing is efficient, as opposed to slow fluid input. The high sensitivity of this method was demonstrated by detection of human serum albumin and human cytochrome b5 in 10−17–10−18 m water solutions. It was shown that an external negative voltage applied to highly oriented pyrolytic graphite hinders the protein fishing. The efficiency of fishing with an external positive voltage was similar to that obtained without applying any voltage.

In the present study, we demonstrate atomic force microscopy (AFM)-based detection of hepatitis C virus (HCV) particles in serum samples using a chip with aptamer-functionalized surface (apta-based AFM chip). The target particles, containing core antigen of HCV (HCVcoreAg protein), were biospecifically captured onto the chip surface from 1 mL of test solution containing 10 µL of serum collected from a hepatitis C patient. The registration of aptamer/antigen complexes on the chip surface was performed by AFM. The aptamers used in the present study were initially developed for therapeutic purposes; herein, these aptamers have been successfully utilized as probe molecules for HCVcoreAg detection in the presence of a complex protein matrix (human serum). The results obtained herein can be used for the development of detection systems that employ affine enrichment for protein detection.

No abstract

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, a z 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. Results and discussion PCS study of CYP102A1 oligomeric stateUsing PCS, it is possible to obtain the size distribution of macromolecules directly in solution. 8 In our work, this method

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.