Abstract
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.
Highlights
The corresponding ρ(h) plot (Figure 3, blue line) indicates that the maximum number of the mica-adsorbed molecules is of (1.2 ± 0.2) nm in height, and can be attributed to monomeric horseradish peroxidase (HRP). These data are in agreement with the height values (1.6 ± 0.1 nm) obtained for atomic force microscopy (AFM)-visualized HRP captured onto chemically activated amino mica from its ultra-low-concentration (10−17 M) aqueous solution [4]
The results reported indicate the effect of an electromagnetic field, induced by water flow through a polymeric pipe coil, on a solution of HRP enzyme incubated near this coil
With the example of studying the change in the aggregation state of the HRP protein by AFM, it has been shown that a water flow, formed in flow sections of biosensors, has an effect on an enzyme
Summary
Diagnosis of oncological diseases in humans requires the detection of cancerassociated marker proteins at femtomolar (10−15 M) or, better, even lower concentrations [1]. The enzymatic activity of HRP has been monitored by conventional spectrophotometry In our research, these three methods have been employed together to study the influence of a flow-induced electromagnetic field on the properties of the HRP enzyme protein. A standard Eppendorf-type polypropylene test tube, modeling a biosensor’s measuring cell, placed near and outside the coil, contained the test solution of the HRP model protein (Figure 1). By AFM, an increased adsorbability of the HRP protein structures has been revealed after the incubation of the HRP solution near the coil with flowing water. A change in the mutual intensity of Amide II to Amide I has been observed in the ATR-FTIR spectra This indicates a change in the HRP secondary structure, occurring during its incubation near the coil. These data can be of use in studying hemodynamics in the human body
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