Selection of Aptamers for Use as Molecular Probes in AFM Detection of Proteins.

Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors-highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope-the cantilever-the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe-target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore.

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

The Study of Performance of a Nanoribbon Biosensor, Sensitized with Aptamers and Antibodies, upon Detection of Core Antigen of Hepatitis C Virus.

The development of highly sensitive diagnostic systems for the early revelation of diseases in humans is one of the most important tasks of modern biomedical research, and the detection of the core antigen of the hepatitis C virus (HCVcoreAg)-a protein marker of the hepatitis C virus-is just the case. Our study is aimed at testing the performance of the nanoribbon biosensor in the case of the use of two different types of molecular probes: the antibodies and the aptamers against HCVcoreAg. The nanoribbon sensor chips employed are based on "silicon-on-insulator structures" (SOI-NR). Two different HCVcoreAg preparations are tested: recombinant ß-galactosidase-conjugated HCVcoreAg ("Virogen", Watertown, MA, USA) and recombinant HCVcoreAg ("Vector-Best", Novosibirsk, Russia). Upon the detection of either type of antigen preparation, the lowest concentration of the antigen detectable in buffer with pH 5.1 was found to be approximately equal, amounting to ~10-15 M. This value was similar upon the use of either type of molecular probes.

Nanoribbon Biosensor-Based Detection of microRNA Markers of Prostate Cancer.

Prostate cancer (PC) is one of the major causes of death among elderly men. PC is often diagnosed later in progression due to asymptomatic early stages. Early detection of PC is thus crucial for effective PC treatment. The aim of this study is the simultaneous highly sensitive detection of a palette of PC-associated microRNAs (miRNAs) in human plasma samples. With this aim, a nanoribbon biosensor system based on "silicon-on-insulator" structures (SOI-NR biosensor) has been employed. In order to provide biospecific detection of the target miRNAs, the surface of individual nanoribbons has been sensitized with DNA oligonucleotide probes (oDNA probes) complementary to the target miRNAs. The lowest concentration of nucleic acids, detectable with our biosensor, has been found to be 1.1 × 10-17 M. The successful detection of target miRNAs, isolated from real plasma samples of PC patients, has also been demonstrated. We believe that the development of highly sensitive nanotechnology-based biosensors for the detection of PC markers is a step towards personalized medicine.

Stopped Flow of Glycerol Induces the Enhancement of Adsorption and Aggregation of HRP on Mica.

Glycerol is a usable component of heat-transfer fluids, and is thus suitable for the use in microchannel-based heat exchangers in biosensors and microelectronic devices. The flow of a fluid can lead to the generation of electromagnetic fields, which can affect enzymes. Herein, by means of atomic force microscopy (AFM) and spectrophotometry, a long-term effect of stopped flow of glycerol through a coiled heat exchanger on horseradish peroxidase (HRP) has been revealed. Samples of buffered HRP solution were incubated near either the inlet or the outlet sections of the heat exchanger after stopping the flow. It has been found that both the enzyme aggregation state and the number of mica-adsorbed HRP particles increase after such an incubation for 40 min. Moreover, the enzymatic activity of the enzyme incubated near the inlet section has been found to increase in comparison with that of the control sample, while the activity of the enzyme incubated near the outlet section remained unaffected. Our results can find application in the development of biosensors and bioreactors, in which flow-based heat exchangers are employed.

Mass Spectrometric Identification of BSA Covalently Captured onto a Chip for Atomic Force Microscopy.

Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.

Analysis of Single Biomacromolecules and Viruses: Is It a Myth or Reality?

The beginning of the twenty-first century witnessed novel breakthrough research directions in the life sciences, such as genomics, transcriptomics, translatomics, proteomics, metabolomics, and bioinformatics. A newly developed single-molecule approach addresses the physical and chemical properties and the functional activity of single (individual) biomacromolecules and viral particles. Within the alternative approach, the combination of "single-molecule approaches" is opposed to "omics approaches". This new approach is fundamentally unique in terms of its research object (a single biomacromolecule). Most studies are currently performed using postgenomic technologies that allow the properties of several hundreds of millions or even billions of biomacromolecules to be analyzed. This paper discusses the relevance and theoretical, methodological, and practical issues related to the development potential of a single-molecule approach using methods based on molecular detectors.

MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface.

This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow "irreversible" binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (102X, 104X, and 106X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.

AFM Investigation of the Influence of Steam Flow through a Conical Coil Heat Exchanger on Enzyme Properties.

The present study is aimed at the revelation of subtle effects of steam flow through a conical coil heat exchanger on an enzyme, incubated near the heat exchanger, at the nanoscale. For this purpose, atomic force microscopy (AFM) has been employed. In our experiments, horseradish peroxidase (HRP) was used as a model enzyme. HRP is extensively employed as a model in food science in order to determine the influence of electromagnetic fields on enzymes. Adsorption properties of HRP on mica have been studied by AFM at the level of individual enzyme macromolecules, while the enzymatic activity of HRP has been studied by spectrophotometry. The solution of HRP was incubated either near the top or at the side of the conically wound aluminium pipe, through which steam flow passed. Our AFM data indicated an increase in the enzyme aggregation on mica after its incubation at either of the two points near the heat exchanger. At the same time, in the spectrophotometry experiments, a slight change in the shape of the curves, reflecting the HRP-catalyzed kinetics of ABTS oxidation by hydrogen peroxide, has also been observed after the incubation of the enzyme solution near the heat exchanger. These effects on the enzyme adsorption and kinetics can be explained by alterations in the enzyme hydration caused by the influence of the electromagnetic field, induced triboelectrically by the flow of steam through the heat exchanger. Our findings should thus be considered in the development of equipment involving conical heat exchangers, intended for either research or industrial use (including miniaturized bioreactors and biosensors). The increased aggregation of the HRP enzyme, observed after its incubation near the heat exchanger, should also be taken into account in analysis of possible adverse effects from steam-heated industrial equipment on the human body.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

Atomic Force Microscopy Study of the Effect of an Electric Field, Applied to a Pyramidal Structure, on Enzyme Biomolecules.

The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.

Atomic Force Microscopy Study of the Temperature and Storage Duration Dependencies of Horseradish Peroxidase Oligomeric State.

This paper presents an investigation of the temperature dependence of the oligomeric state of the horseradish peroxidase (HRP) enzyme on the temperature of its solution, and on the solution storage time, at the single-molecule level. Atomic force microscopy has been employed to determine how the temperature and the storage time of the HRP solution influence its aggregation upon direct adsorption of the enzyme from the solution onto bare mica substrates. In parallel, spectrophotometric measurements have been performed in order to estimate whether the HRP enzymatic activity changes over time upon the storage of the enzyme solution. The temperature dependence of the HRP oligomeric state has been studied within a broad (15-40 °C) temperature range. It has been demonstrated that the storage of the HRP solution for 14 days does not have any considerable effect on the oligomeric state of the enzyme, neither does it affect its activity. At longer storage times, AFM has allowed us to reveal a tendency of HRP to oligomerization during the storage of its buffered solution, while the enzymatic activity remains virtually unchanged even after a 1-month-long storage. By AFM, it has been revealed that after the incubation of a mica substrate in the HRP solution at various temperatures, the content of the mica-adsorbed oligomers increases insignificantly owing to a high-temperature stability of the enzyme.

The Effect of a Dodecahedron-Shaped Structure on the Properties of an Enzyme.

In this research, the influence of a dodecahedron-shaped structure on the adsorption behavior of a horseradish peroxidase (HRP) enzyme glycoprotein onto mica substrates was studied. In the experiments, samples of an aqueous HRP solution were incubated at various distances (0.03 m, 2 m, 5 m, and control at 20 m) from the dodecahedron surface. After the incubation, the direct adsorption of HRP onto mica substrates immersed in the solutions was performed, and the mica-adsorbed HRP particles were visualized via atomic force microscopy (AFM). The effect of the increased HRP aggregation was only observed after the incubation of the enzyme solution at the 2 m distance from the dodecahedron. In addition, with respect to the control sample, spectrophotometric measurements revealed no change in the HRP enzymatic activity after the incubation at any of the distances studied. The results reported herein can be of use in the modeling of the possible influences of various spatial structures on biological objects in the development of biosensors and other electronic equipment.

Radiothermometric Study of the Effect of Amino Acid Mutation on the Characteristics of the Enzymatic System.

The radiothermometry (RTM) study of a cytochrome-containing system (CYP102 A1) has been conducted in order to demonstrate the applicability of RTM for monitoring changes in the functional activity of an enzyme in case of its point mutation. The study has been performed with the example of the wild-type cytochrome (WT) and its mutant type A264K. CYP102 A1 is a nanoscale protein-enzymatic system of about 10 nm in size. RTM uses a radio detector and can record the corresponding brightness temperature (Tbr) of the nanoscale enzyme solution within the 3.4-4.2 GHz frequency range during enzyme functioning. It was found that the enzymatic reaction during the lauric acid hydroxylation at the wild-type CYP102 A1 (WT) concentration of ~10-9 M is accompanied by Tbr fluctuations of ~0.5-1 °C. At the same time, no Tbr fluctuations are observed for the mutated forms of the enzyme CYP102 A1 (A264K), where one amino acid was replaced. We know that the activity of CYP102 A1 (WT) is ~4 orders of magnitude higher than that of CYP102 A1 (A264K). We therefore concluded that the disappearance of the fluctuation of Tbr CYP102 A1 (A264K) is associated with a decrease in the activity of the enzyme. This effect can be used to develop new methods for testing the activity of the enzyme that do not require additional labels and expensive equipment, in comparison with calorimetry and spectral methods. The RTM is beginning to find application in the diagnosis of oncological diseases and for the analysis of biochemical processes.

Prediction of Monomeric and Dimeric Structures of CYP102A1 Using AlphaFold2 and AlphaFold Multimer and Assessment of Point Mutation Effect on the Efficiency of Intra- and Interprotein Electron Transfer.

The three-dimensional structure of monomers and homodimers of CYP102A1/WT (wild-type) proteins and their A83F and A83I mutant forms was predicted using the AlphaFold2 (AF2) and AlphaFold Multimer (AFMultimer) programs, which were compared with the rate constants of hydroxylation reactions of these enzyme forms to determine the efficiency of intra- and interprotein electron transport in the CYP102A1 hydroxylase system. The electron transfer rate constants (ket), which determine the rate of indole hydroxylation by the CYP102A1 system, were calculated based on the distances (R) between donor-acceptor prosthetic groups (PG) FAD→FMN→HEME of these proteins using factor ß, which describes an exponential decay from R the speed of electron transport (ET) according to the tunnelling mechanism. It was shown that the structure of monomers in the homodimer, calculated using the AlpfaFold Multimer program, is in good agreement with the experimental structures of globular domains (HEME-, FMN-, and FAD-domains) in CYP102A1/WT obtained by X-ray structural analysis, and the structure of isolated monomers predicted in AF2 does not coincide with the structure of monomers in the homodimer, although a high level of similarity in individual domains remains. The structures of monomers and homodimers of A83F and A83I mutants were also calculated, and their structures were compared with the wild-type protein. Significant differences in the structure of all isolated monomers with respect to the structures of monomers in homodimers were also found for them, and at the same time, insignificant differences were revealed for all homodimers. Comparative analysis for CYP102A1/WT between the calculated intra- and interprotein distances FAD→FMN→HEME and the rate constants of hydroxylation in these proteins showed that the distance between prosthetic groups both in the monomer and in the dimer allows the implementation of electron transfer between PGs, which is consistent with experimental literature data about kcat. For the mutant form of monomer A83I, an increase in the distance between PGs was obtained, which can restrict electron transportation compared to WT; however, for the dimer of this protein, a decrease in the distance between PGs was observed compared to the WT form, which can lead to an increase in the electron transfer rate constant and, accordingly, kcat. For the monomer and homodimer of the A83F mutant, the calculations showed an increase in the distance between the PGs compared to the WT form, which should have led to a decrease in the electron transfer rate, but at the same time, for the homodimer, the approach of the aromatic group F262 with heme can speed up transportation for this form and, accordingly, the rate of hydroxylation.

"Silicon-On-Insulator"-Based Nanosensor for the Revelation of MicroRNA Markers of Autism.

MicroRNAs (miRNAs), which represent short (20 to 22 nt) non-coding RNAs, were found to play a direct role in the development of autism in children. Herein, a highly sensitive "silicon-on-insulator"-based nanosensor (SOI-NS) has been developed for the revelation of autism-associated miRNAs. This SOI-NS comprises an array of nanowire sensor structures fabricated by complementary metal-oxide-semiconductor (CMOS)-compatible technology, gas-phase etching, and nanolithography. In our experiments described herein, we demonstrate the revelation of ASD-associated miRNAs in human plasma with the SOI-NS, whose sensor elements were sensitized with oligonucleotide probes. In order to determine the concentration sensitivity of the SOI-NS, experiments on the detection of synthetic DNA analogues of autism-associated miRNAs in purified buffer were performed. The lower limit of miRNA detection attained in our experiments amounted to 10-17 M.

"Silicon-On-Insulator"-Based Biosensor for the Detection of MicroRNA Markers of Ovarian Cancer.

Ovarian cancer is a gynecological cancer characterized by a high mortality rate and tumor heterogeneity. Its early detection and primary prophylaxis are difficult to perform. Detecting biomarkers for ovarian cancer plays a pivotal role in therapy effectiveness and affects patients' survival. This study demonstrates the detection of microRNAs (miRNAs), which were reported to be associated with ovarian cancer tumorigenesis, with a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). The advantages of the method proposed for miRNA detection using the SOI-NW biosensor are as follows: (1) no need for additional labeling or amplification reaction during sample preparation, and (2) real-time detection of target biomolecules. The detecting component of the biosensor is a chip with an array of 3 µm wide, 10 µm long silicon nanowires on its surface. The SOI-NW chip was fabricated using the "top-down" method, which is compatible with large-scale CMOS technology. Oligonucleotide probes (oDNA probes) carrying sequences complementary to the target miRNAs were covalently immobilized on the nanowire surface to ensure high-sensitivity biospecific sensing of the target biomolecules. The study involved two experimental series. Detection of model DNA oligonucleotides being synthetic analogs of the target miRNAs was carried out to assess the method's sensitivity. The lowest concentration of the target oligonucleotides detectable in buffer solution was 1.1 × 10-16 M. In the second experimental series, detection of miRNAs (miRNA-21, miRNA-141, and miRNA-200a) isolated from blood plasma samples collected from patients having a verified diagnosis of ovarian cancer was performed. The results of our present study represent a step towards the development of novel highly sensitive diagnostic systems for the early revelation of ovarian cancer in women.

Nanoribbon Biosensor in the Detection of miRNAs Associated with Colorectal Cancer.

A nanoribbon biosensor (NRBS) was developed to register synthetic DNAs that simulate and are analogous to miRNA-17-3p associated with colorectal cancer. Using this nanoribbon biosensor, the ability to detect miRNA-17-3p in the blood plasma of a patient diagnosed with colorectal cancer has been demonstrated. The sensing element of the NRBS was a nanochip based on a silicon-on-insulator (SOI) nanostructure. The nanochip included an array of 10 nanoribbons and was designed with the implementation of top-down technology. For biospecific recognition of miRNA-17-3p, the nanochip was modified with DNA probes specific for miRNA-17-3p. The performance of the nanochip was preliminarily tested on model DNA oligonucleotides, which are synthetic analogues of miRNA-17-3p, and a detection limit of ~10-17 M was achieved. The results of this work can be used in the development of serological diagnostic systems for early detection of colorectal cancer.

Detection of Circulating Serum microRNA/Protein Complexes in ASD Using Functionalized Chips for an Atomic Force Microscope.

MicroRNAs, which circulate in blood, are characterized by high diagnostic value; in biomedical research, they can be considered as candidate markers of various diseases. Mature microRNAs of glial cells and neurons can cross the blood-brain barrier and can be detected in the serum of patients with autism spectrum disorders (ASD) as components of macrovesicles, macromolecular protein and low-density lipoprotein particles. In our present study, we have proposed an approach, in which microRNAs in protein complexes can be concentrated on the surface of AFM chips with oligonucleotide molecular probes, specific against the target microRNAs. MicroRNAs, associated with the development of ASD in children, were selected as targets. The chips with immobilized molecular probes were incubated in serum samples of ASD patients and healthy volunteers. By atomic force microscopy (AFM), objects on the AFM chip surface have been revealed after incubation in the serum samples. The height of these objects amounted to 10 nm and 6 nm in the case of samples of ASD patients and healthy volunteers, respectively. MALDI-TOF-MS analysis of protein components on the chip surface allowed us to identify several cell proteins. These proteins are involved in the binding of nucleic acids (GBG10, RT24, RALYL), in the organization of proteasomes and nucleosomes (PSA4, NP1L4), and participate in the functioning of the channel of active potassium transport (KCNE5, KCNV2).

Nanoribbon-Based Electronic Detection of a Glioma-Associated Circular miRNA.

Nanoribbon chips, based on "silicon-on-insulator" structures (SOI-NR chips), have been fabricated. These SOI-NR chips, whose surface was sensitized with covalently immobilized oligonucleotide molecular probes (oDNA probes), have been employed for the nanoribbon biosensor-based detection of a circular ribonucleic acid (circRNA) molecular marker of glioma in humans. The nucleotide sequence of the oDNA probes was complimentary to the sequence of the target oDNA. The latter represents a synthetic analogue of a glioma marker-NFIX circular RNA. In this way, the detection of target oDNA molecules in a pure buffer has been performed. The lowest concentration of the target biomolecules, detectable in our experiments, was of the order of ~10-17 M. The SOI-NR sensor chips proposed herein have allowed us to reveal an elevated level of the NFIX circular RNA in the blood of a glioma patient.