The limit of mass determination with an AFM cantilever-based system

This study was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013-2020. Shumov I.D. is a recipient of Russian Federation President scholarship for young scientists for 2016-2018 (project identificator SP-4280.2016.4)

Magnetron sputtering deposition of ultra-thin tungsten coatings onto amorphous graphite for enhancement of horseradish peroxidase adsorption

The present study was performed in the framework of the Program for Basic Research of State Academies of Sciences for 2013-2020

Combination of atomic force microscopy and mass spectrometry for the target protein in the serum samples of children with autism spectrum disorders

Показана возможность детекции целевых белков, ассоциированных с развитием аутистических расстройств у детей, с помощью комбинированного метода АСМ/МС. Метод основан на комбинации аффинного обогащения белков из биообразцов, их АСМ-визуализации и МС-анализе с количественной детекцией целевых белков.Possibility of detection of target proteins associated with development of autistic disorders in children with use of combined AFM/MS method is demonstrated. The proposed method is based on the combination of affine enrichment of proteins from biological samples, visualization of these proteins by AFM and MS analysis with quantitative detection of target proteins.Работа выполнена при поддержке гранта РНФ 14-25-00132. Шумов И.Д. является получателем стипендии Президента РФ молодым ученым и аспирантам, осуществляющим перспективные научные исследования и разработки по приоритетным направлениям модернизации российской экономики на 2016-2018 годы (СП-4280.2016.4)

Detection of Hepatitis C Virus Core Protein in Serum Using Aptamer-Functionalized AFM Chips

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

“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

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase

Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning

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)

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase

Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme’s secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications—particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning

The Impact of Fast-Rise-Time Electromagnetic Field and Pressure on the Aggregation of Peroxidase upon Its Adsorption onto Mica

Our present study concerns the influence of the picosecond rise-time-pulsed electromagnetic field, and the impact of nanosecond pulsed pressure on the aggregation state of horseradish peroxidase (HRP) as a model enzyme. The influence of a 640 kV/m pulsed electromagnetic field with a pulse rise-time of ~200 ps on the activity and aggregation state of an enzyme is studied by the single-molecule atomic force microscopy (AFM) method. The influence of such a field is shown to lead to aggregation of the protein and to a decrease in its enzymatic activity. Moreover, the effect of a shock wave with a pressure front rise-time of 80 ns on the increase in the HRP aggregation is demonstrated. The results obtained herein can be of use in modeling the impact of electromagnetic and pressure pulses on enzymes and on whole living organisms. Our results are also important for taking into account the effect of pulsed fields on the body in the development of drugs, therapeutic procedures, and novel highly sensitive medical diagnosticums