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An approach to measure the activity of single oligomers of the heme-containing enzyme cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young’s modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.

Electron microscopy is widely used in biological and medical research, enabling to obtain images of the investigated objects with nanometer resolution. Exposure of biological objects under electron beam during the visualization can, however, cause their damage. Destructive effect of the electron beam can be significantly decreased by formation of metal coating on the surface of the objects to be studied. In the present study, an approach for the formation of ultra-thin metal coatings on the surface of protein-containing structures is proposed. Magnetron sputtering in argon plasma in DC mode was employed to form 8 A-thick tungsten films on the surface of model protein-containing objects— tobacco mosaic virus particles — for their subsequent visualization by electron microscopy.

It is known that a charge is generated in water and protein solutions upon their motion; this phenomenon is also observed in analytical systems for atomic force microscopy (AFM)-based fishing. At that, the efficiency of protein fishing correlates with the value of charge, generated upon the motion of the analyzed solution. Earlier, we demonstrated that a pulsed electric field can well be used for the enhancement of the efficiency of AFM-based fishing of low-abundant protein. In this paper, we have demonstrated the influence of a pulsed electric field on the stimulation of the electric charge generation in a solution of low-abundant proteins observed in the injector part of an AFM-based fishing system at 23 °C and 38 °C. Taking this effect into account is important for the development of novel highly sensitive flow-based diagnostic systems, as well as for the development of models describing the influence of a pulsed electric field on pathological processes in the body, hemodynamics, and physicochemical properties of solutions.

The nanowire (NW) detection is one of fast-acting and high-sensitive methods allowing to reveal potentially relevant protein molecules. A NW biosensor based on the silicon-on-insulator (SOI)-structures was used for biospecific label-free detection of NFAT 1 (D-NFAT 1) oncomarker in real time. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFAT 1 used as molecular probes. It was shown that using this biosensor it is possible to reach the sensitivity of ~10(-15) M. This sensitivity was comparable with that of the NW biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate promising approaches used to form the sensor elements for high-sensitive disease diagnostics.Odnim iz bystrodeĭstvuiushchikh i vysokochuvstvitel'nykh metodov, pozvoliaiushchikh vyiavliat' potentsial'no vazhnye belkovye molekuly, iavliaetsia metod nanoprovolochnoĭ (NP) detektsii. Nanoprovolochnyĭ biosensor na osnove struktur kremniĭ na izoliatore (KNI), byl ispol'zovan dlia biospetsificheskoĭ detektsii NFAT 1 (D-NFAT 1)-onkomarkera v real'nom vremeni bez metok. Dlia étogo KNI-nanoprovoloki byli modifitsirovany aptamerami k NFAT 1, ispol'zuemymi v kachestve makromolekuliarnykh zondov. Pokazano, chto takoĭ biosensor pozvoliaet dostigat' chuvstvitel'nost' poriadka 10-15 M. Éta chuvstvitel'nost' sopostavima s chuvstvitel'nost'iu, poluchennoĭ na nanoprovolochnom biosensore s immobilizovannymi antitelami, ispol'zuemymi v kachestve makromolekuliarnykh zondov. Rezul'taty demonstriruiut perspektivnost' ispol'zovannykh v rabote podkhodov formirovaniia sensornykh élementov dlia vysokochuvstvitel'noĭ diagnostiki zabolevaniĭ.

Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10(-17)-10(-18) M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes.Dostizhenie kontsentratsionnogo predela detektsii belkov na urovne obratnogo chisla Avogadro opredeliaet sovremennoe razvitie proteomiki. V nastoiashcheĭ rabote analiziruiutsia vozmozhnosti priblizheniia k dostizheniiu obratnogo chisla Avogadro s pomoshch'iu nanotekhnologicheskikh metodov: ASM-fishinga s mekhanicheskim i élektricheskim vozbuzhdeniem sredy, nanoprovodnykh detektorov, i drugikh metodov. Prodemonstrirovana vozmozhnost' ASM-fishinga dlia vyiavleniia, podscheta, vizualizatsii i kharakterizatsii fiziko-khimicheskikh svoĭstv belkov v kontsentratsiiakh vplot' do 10-17–10-18 M. Kombinirovanie ASM-fishinga s mass-spektrometricheskimi metodami pozvoliaet identifitsirovat' takim obrazom vyiavlennye belki ne tol'ko v chistykh rastvorakh, no i v mnogokomponentnoĭ biologicheskoĭ zhidkosti (syvorotke krovi). Analiziruiutsia vozmozhnosti ispol'zovaniia SOMAmerov dlia povysheniia éffektivnost i biospetsificheskogo fishinga kak v ASM, tak i v nanoprovolochnykh sistemakh. V rabote privodiatsia kriterii otsenki chuvstvitel'nosti detektiruiushchikh sistem na osnove fishinga. Daetsia otsenka éffektivnosti fishinga v zavisimosti ot parametrov detektiruiushcheĭ sistemy. Analiziruiutsia osobennosti prakticheskoĭ realizatsii fishinga belkov v zavisimosti ot sootnosheniia ob"ema analiziruemogo rastvora i poverkhnosti detektiruiushcheĭ sistemy; obsuzhdaiutsia preimushchestva i nedostatki sovremennykh perspektivnykh nanotekhnologicheskikh metodov obnaruzheniia belkov, realizuemykh na baze étikh skhem.

Development of atomic force microscopy (AFM)-based nanotechnological approaches to highly sensitive detection of proteins is a perspective direction in biomedical research. These approaches use AFM chips to concentrate the target proteins from the test solution volume (buffer solution, diluted biological fluid) onto the chip surface for their subsequent registration on the chip surface by AFM. Atomic force microscope is a molecular detector that enables protein detection at ultra-low (subfemtomolar) concentrations in single-molecule counting mode. Due to extremely high sensitivity of AFM, its application for multiplexed protein detection is of great interest for use in proteomics and diagnostic applications. In this study, AFM chips containing an array of sensor areas have been fabricated. Magnetron sputtering of chromium and tungsten nanolayers has been used to form optically visible metallic marks on the AFM chip surface to provide necessary precision of AFM probe positioning against each sensor area for scanning. It has been demonstrated that the marks formed by magnetron sputtering of Cr and W are stable on the surface of the AFM chips during the following activation and intensive washing of this surface. The results obtained in our present study allow application of the developed chips for multiplexed protein analysis by AFM.

The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IF ) has been developed for the detection of proteins at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10 17 M) was detected in solution. A theoretical model that allows the description of obtained experimental data, is proposed. This model takes into consideration both the transport of the protein from the bulk solution onto the AFM-chip surface and its irreversible binding to the activated area.

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.

The development of genome and proteomic technologies is related, first of all, to the progress recently achieved in developing of high-sensitivity, rapid methods for registration and analysis of nucleic acids and proteins. One of such methods, combining high sensitivity and high response speed, is the nanowire (NW) detection technique. This technique allows one to register analyte species in real time without using labels. Nowadays, the concentration sensitivity of such systems in registering proteins and DNAs reaches the femtomolar level. In the present chapter, we consider nanowire sensor systems based on silicon nanowire field-effect transistors. We discuss the achieved potential in detecting low-copy proteins with the help of nanowire systems. We show that it is possible to use not only antibodies, but also aptamers, as probing molecules for biospecific protein detection. We also discuss the possibility of using nanowire detectors in genome studies.

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.

CD-based optico-acoustical biosensor (OAB) was used for detection of various types of proteins represented by bovine serum albumin (BSA), heme-containing myoglobin (Mb), monoclonal antibody against viral protein marker of hepatitis B (anti-HBsAg) and membrane-bound cytochrome P450scc (P450scc). We applied standard compact disc reader (CD-ROM) as an optical analyzer and a standard compact disc (CD) as a biochip containing immobilized protein molecules. This biosensor can translate into a digital code the changes of optical signal from the proteins and their complexes immobilized on the CD surface. Then, the digital code is translated into an acoustic series or, in other words, into a "music of proteins". We demonstrate the use of the OAB for direct detection of proteins with different molecular weights, such as BSA, Mb, P450scc, anti-HBsAg with the concentration detection limit (DL) about 10(-7)M. By signal amplification achieved with autometallography, a higher sensitivity level (DL∼10(-9)M) for the detection of myoglobin was obtained. The method of OAB-detection of proteins is cheap: it requires no special equipment like spectrometers, refractometers and other devices. Due to the fact that acoustic series of the protein complexes antigen/antibody differs from that of single proteins, the OAB-detection is of particular interest for rapid assay in yes/no data type and for home diagnostics. Combination of the OAB with a mass spectrometer allowed the detection and identification of the target proteins fished out directly onto a standard CD surface.

Possibility of detection and identification of hepatitis C viral particles with mass spectrometry (MS) in combination with atomic force microscopy (AFM) had been investigated. AFM/MS approach is based on two technologies: 1. AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on a surface of AFM-nanochip; 2. mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcoreim complexes were formed onto AFM-nanochips in quantity sufficient for mass spectrometric identification. Thus, AFM/MS approach allows to identify fragments of hepatitis C virus fished onto a surface of AFM-nanochip from serum.

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.

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 possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by a new atomic force microscopy (AFM)-based nanotechnological approach has been demonstrated.