1. Institute of Biomedical Chemistry, Moscow

2. Joint Institute for High Temperatures of the Russian Academy of Sciences, Moscow

3. Foundation of Perspective Technologies and Novations, Moscow

4. Moscow State Academy of Veterinary Medicine and Biotechnology Named after Skryabin, Moscow

5. Faculty of Computational Mathematics and Cybernetics, Moscow State University, Moscow

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.

Keywords: glycerol flow; enzyme adsorption; horseradish peroxidase; atomic force microscopy

Glycerol is a trihydric alcohol, which is widely used in industry [1,2]. Pauling noted the usability of glycerol as a component of antifreezes [1]; in this regard, glycerol represents a safe alternative to ethylene glycol, which is known to be toxic for animals and humans [3]. With regard to its application in biomedical research, the use of glycerol for modulation of the viscosity of solutions to be analyzed [4,5], and for preliminary treatment of surface of biosensor chips [6] was reported. Moreover, Yang et al. emphasized good suitability of glycerol for its use as a component of heat transfer fluids in microchannel-based miniaturized heat exchangers, which are of use in biotechnology and microelectronics [2]. In this regard, it should be emphasized that modern nanotechnology-based biosensors comprise microelectronic components [7–9]. Microelectronic chips are characterized by high heat flux densities [10]. Accordingly, providing efficient heat transfer becomes a crucial task of the development of novel biosensor systems. In this connection, apart from traditional heat transfer fluids, one should also mention the so-called nanofluids [11,12], which consist of conventional heat transfer fluids with added metallic or oxide nanoparticles [11]. Akkurt et al. reported that the use of nanofluids allows one to additionally increase the efficiency of heat transfer rate [11].

The above-mentioned biosensors [4,6] and heat exchangers [2] pertain to flow-based systems, in which a flow of glycerol-containing working fluids is organized. In this connection, one should emphasize the possible occurrence of the so-called triboelectric effect, which consists in the generation of electric charge upon the flow of liquid media along solid surfaces [13]. Such an effect was reported for both aqueous [14–19] and non-aqueous fluids [20–22], including glycerol [23,24]. The triboelectric generation of charge results in an occurrence of electromagnetic fields [18,23,24]. Electric fields and electromagnetic waves can influence biological macromolecules [25]. This circumstance is particularly important in flow-based systems intended for operation with biological macromolecules, such as the above-mentioned biosensors and bioreactor heat exchangers.

Electric [26–31], magnetic [32–36] and electromagnetic [37–47] fields are known to affect enzymes. In many papers, the effects of pulsed electric [26–31] and electromagnetic [42] fields on enzymes are considered. It is to be emphasized that the effect of pulsed electric field (PEF) can be quite different, and depends on the enzyme type [27,31] and treatment conditions [26,27]. On the one hand, high-voltage (approximately 27 to 42 kV/cm) 126-µs-long PEF treatment was reported to lead to a considerable (by tens of %) inactivation of many enzymes, such as pepsin, peroxidase, and polyphenol oxidase [31], and lipase [26], while lysozyme was virtually unaffected by 38 kV/cm PEF after a 126-µs-long treatment [31]. On the other hand, Ohshima et al. [27] revealed an enhancing effect of ≤12 kV/cm PEF on peroxidase and β-galactosidase. These authors observed a 20% increase in the activity of peroxidase and β-galactosidase after exposure of these enzymes to 12 and 13 kV/cm PEF for 30 and 60 s, respectively [27]. At that, these enzymes were found to lose their activity after treatment with stronger PEF [27]. Regarding magnetic fields, Wasak et al. [32] demonstrated that the action of a low-frequency rotating magnetic field can either enhance or suppress the enzymatic activity of horseradish peroxidase (HRP) against o-dianisidine depending on the solution pH, field parameters, and exposure time. Earlier, by atomic force microscopy (AFM), Sun et al. [35] found that HRP forms extended structures on mica after exposure to alternating magnetic field, while the enzymatic activity was hardly affected. In this way, these authors demonstrated the advantages of using AFM for studying the interaction of applied magnetic fields with an enzyme [35,36].

Similar to the case with PEFs, the effect of electromagnetic fields (EMFs) on the activity of various enzymes was also reported to be either positive (enhancing) or negative (suppressing) [41,45,46] depending on the EMF frequency and the enzyme type. Morelli et al. observed that membrane-associated enzymes (phopsphoglycerate kinase, alkaline phosphatase, adenylate kinase) lose their activity after the exposure to 75 Hz EMF, while other enzymes (for instance, succinic dehydrogenase) were found to be insensitive to such an EMF [45]. In contrast, Thumm et al. revealed a stimulation of cAMP-dependent protein kinase by a very low-frequency (20 Hz) EMF [46]. HRP was found to lose its catalytic activity after the exposure to a 50 Hz, 2.7 mT EMF, while being insensitive to the EMF with 2-times higher (100 Hz; 5.5 mT) frequency [41]. Regarding EMFs of higher frequencies (radiofrequency EMFs, RF EMFs), interesting results were reported for HRP enzyme. Fortune et al. did not reveal any non-thermal effect of RF EMFs of various frequencies (13.56 MHz, 915 MHz, and 2.45 GHz) on HRP [38]. Yao et al. observed a slight activation of HRP upon 27.12 MHz RF heating at high power (6 kW) and 50 ^◦C temperature, while treatment at the same frequency but at higher temperatures resulted in the loss of enzymatic activity of HRP [44]. Latorre et al. observed a dramatic inactivation of peroxidase from red beet and polyphenoloxidase by microwave treatment [39].

The data listed above clearly indicate that the effect of electric and electromagnetic fields enzymes can vary, and further investigation is required in order to better understand the phenomenon of the interaction of external fields with enzymes. Here, we must emphasize that in the majority of studies listed above, macroscopic methods in solution (such as spectrophotometry) were employed for the determination of changes in the properties of enzymes. In the macroscopic methods, the signal from a large ensemble of the molecules under study is acquired. This leads to the fact that subtle effects of external fields on enzyme macromolecules can well remain unrevealed by macroscopic methods [35,36,48]. In this regard, high-resolution methods are of use, and AFM is one of them. In a number of papers, it was demonstrated that AFM allows one to investigate changes in physicochemical properties of enzymes at the level of single molecules [35,36,49,50], thus revealing even subtle effects (which are often indistinguishable by macroscopic methods [35,36,48]) of external fields on the enzymes under study, including the effects caused by action of weak EMFs [47].

Herein, we demonstrate how our well-established approach, which consists in a combined use of AFM-based adsorption studies and spectrophotometry-based estimation of enzymatic activity, has allowed us to reveal a long-term effect of glycerol flow in a heat exchanger on the properties of HRP, which has been used as a model enzyme, as was proposed in a number of published papers [41,44,47,48]. Previously, we demonstrated that the electromagnetic field induced by the flow of glycerol can affect both the adsorption properties and the enzymatic activity of HRP [24]. That is, the effect of glycerol flow is so strong that the changes in the enzyme properties can be revealed by the macroscopic method (spectrophotometry)—as opposed to the subtle effects [48]. Our present study is aimed at further investigating the influence of glycerol flow on enzymes with the example of HRP. In this connection, the first question is whether the electromagnetic field, induced by glycerol flow, can have a long-term effect on the enzyme. This has not yet been studied. Another question is whether this effect will be evident at the macroscopic level. With this purpose, herein, the glycerol flow through the heat exchanger had been stopped prior to the incubation of HRP in the experimental setup. Only after stopping the flow was the HRP solution incubated near the linear inlet and outlet sections of the heat exchanger. This is the major difference with our experiments reported previously [24], when glycerol was pumped continuously without stopping its flow before the experiment. In this previous paper, electromagnetic fields, generated by the flow of glycerol through the linear sections of a heat exchanger (coiled polymeric pipe), were found to induce an increased aggregation of HRP enzyme upon its adsorption onto mica. Herein, for the first time, we have revealed that there is a long-term effect on an enzyme incubated near these sections of the heat exchanger after stopping the glycerol flow. Our recent results reported herein should thus be taken into consideration in the development of biosensors and bioreactors containing flow-based heat exchangers, in which non-aqueous heat-transfer fluids are utilized.

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