Investigation of the Influence of Liquid Motion in a Flow-Based System on an Enzyme Aggregation State with an Atomic Force Microscopy Sensor: The Effect of Glycerol Flow

Ziborov, Vadim S.; Pleshakova, Tatyana O.; Shumov, Ivan D.; Kozlov, Andrey F.; Ivanova, Irina A.; Valueva, Anastasia A.; Tatur, Vadim Yu.; Negodailov, Andrey N.; Lukyanitsa, Andrei A.; Ivanov, Yuri D.

doi:10.3390/app10144825

Cited by 15 publications

(48 citation statements)

References 47 publications

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“…This fact indicates that the flow-induced field only impacts the spatial structures of the protein globule near its surface, while not affecting its active site. The effects of HRP aggregation in the presence of electromagnetic fields were previously reported in our recent studies [ 8 , 35 , 36 ] and by Sun et al [ 37 , 38 ].…”

Section: Discussionsupporting

confidence: 69%

“…The AFM experiments were carried out using the direct surface adsorption method [ 8 , 15 , 35 , 36 ], similar to [ 8 ]. Briefly, muscovite mica sheets (SPI, West Chester, PA, USA) were used as AFM substrates.…”

Section: Methodsmentioning

confidence: 99%

“…The total number of imaged objects in each sample was no less than 200, and the number of frames for each sample was no less than 10. Analogously to [ 36 ], the density of relative distribution of the imaged objects with height ρ(h) and the absolute number N 400 of surface-adsorbed particles, normalized per 400 μm 2 area, was calculated as described elsewhere [ 36 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionsupporting

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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“…Previously, we demonstrated that the incubation of a protein solution over the coiled pipe with flowing liquid leads to a change in adsorption properties of the protein macromolecules [5]. We discussed that the flow of a non-aqueous liquid (such as glycerol) generates an electric charge [5,6]. This charge induces an electric field, which, in turn, can affect proteins' properties.…”

Section: Introductionmentioning

confidence: 99%

“…The use of HRP protein in our experiments was justified in our previous papers [5,7,8]. Briefly, this protein was studied in much detail, and this is why it is often employed as a model in studies of enzymes, including peroxidases which play important roles in living systems [9].…”

Section: Introductionmentioning

confidence: 99%

AFM Study of the Influence of Glycerol Flow on Horseradish Peroxidase near the in/out Linear Sections of a Coil

et al. 2021

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Flow-based coiled systems, through which a heat transfer fluid (such as glycerol) is pumped, are widely used for thermal stabilization of bioreactors and biosensor cuvettes and cells. Previously, using horseradish peroxidase (HRP) as a model protein, we have demonstrated that the incubation of a protein solution in a flow-based system over coiled pipe with flowing glycerol leads to a change in the adsorption properties of the protein macromolecules. Herein, we have studied the effect of the glycerol flow on the properties of HRP, the solution of which was placed differently: i.e., near either the inflow or the outflow linear sections of the pipe, while the coiled section of the pipe was shielded with a grounded metallic cover. Atomic force microscopy (AFM) has been employed in order to visualize the HRP protein macromolecules adsorbed from its solution onto the mica substrate surface. The quantity of adsorbed protein was estimated based on the AFM data. The enzymatic activity of HRP was estimated by spectrophotometry. We demonstrate that a change in the properties of HRP enzyme was observed after the incubation of its solution near the inflow/outflow linear sections of the pipe with flowing glycerol. Namely, after the incubation of HRP solution near the inflow section, a decrease in the protein adsorption onto mica was observed, but its enzymatic activity remained unchanged in comparison to the control sample. In another case, when the HRP solution was incubated near the outflow section, an increased protein adsorption was observed, while the enzyme exhibited considerably lower activity.

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AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure

Ivanov

Pleshakova

Shumov

et al. 2021

Sci Rep

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In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid’s apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.

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Investigation of the Influence of Liquid Motion in a Flow-Based System on an Enzyme Aggregation State with an Atomic Force Microscopy Sensor: The Effect of Glycerol Flow

Cited by 15 publications

References 47 publications

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

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

AFM Study of the Influence of Glycerol Flow on Horseradish Peroxidase near the in/out Linear Sections of a Coil

AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure

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