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

Abstract

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.

AFM Study of the Influence of Stopped Glycerol Flow in a Coil on Peroxidase Enzyme

“…In other words, the glycerol flow has been stopped prior to the incubation of the enzyme. The enzymatic activity of HRP has been found unaffected -as opposed to the previously reported case with the incubation of the enzyme near the inlet section of the heat exchanger [19]. Nevertheless, a considerably increased content of aggregated enzyme has been revealed by AFM on the surface of mica substrates after the incubation of the enzyme in our experimental setup.…”

“…Our present study reveals a considerable 40-minute aftereffect (the so-called long-term effect [19]) of the glycerol flow in a cylindrically coiled heat exchanger on the aggregation state of HRP after incubation of its solution near the outer side of the coiled section. In other words, the glycerol flow has been stopped prior to the incubation of the enzyme.…”

“…One of possible effects of electromagnetic fields on an enzyme is exhibited in a form of a change in its aggregation state on upon adsorption onto a solid substrate surface [18][19][20]. This is an important point, since surface-adsorbed enzymes are widely employed in biotechnology [1].…”

“…Accordingly, enzyme aggregation and external factors, influencing this process, require further thorough investigation. In this respect, ultrasensitive methods such as atomic force microscopy (AFM) are of use [18,19,[29][30][31], since they allow the researchers to reveal even subtle effects on the enzyme aggregation [31].…”

“…Furthermore, HRP has found many industrial applications in food technology [36], wastewater purification [37] and biofuel cells [38][39][40]. At that, aggregation state of HRP was shown to be influenced by external magnetic [29,30] and electromagnetic fields [18][19][20]. Electromagnetic fields are ubiquitous in industry [41].…”