1. Introduction
One of the problems of further development of proteomics and medical diagnostics is to increase the sensitivity of protein registration methods. This is particularly important in the case of early diagnosis of diseases (including oncological and viral ones), when the analysis sensitivity must be at the level of femtomolar concentrations, corresponding to the concentrations of disease-associated marker proteins [
1].
Nanobiosensor systems for proteomics and medical diagnostics, such as those employing atomic force microscope (AFM)-based fishing and nanowire detectors (i.e., the systems, in which molecular detectors are employed), allow one to register single viral particles and protein molecules with ultra-high sensitivity in counting mode [
2,
3,
4,
5]. These systems use an approach based on the introduction of an aqueous solution of an analyte (protein or virus) through an injector using either a pipette or flow system into a measuring cell containing a nanochip; analyte particles are captured onto the nanochip surface (i.e., fishing of the analyte occurs), where they are registered with molecular detectors in counting mode. In our previous paper [
6] it was reported that by using systems for AFM-based fishing (that is, the systems, in which protein particles are captured from a large volume onto a small area of a chip for an atomic force microscope) it is possible to detect proteins at femtomolar and subfemtomolar concentrations upon feeding the protein solution into the measuring cell using an injector. As one of the factors of such a high efficiency of protein detection, the effect of charge generation in the analyte solution during its flow through an injector is considered. It is known that an electric charge is generated during the injection of a solution through a pipette tip [
7]. At the same time, according to Reference [
2], in a nanobiosensor system for AFM-based fishing at femtomolar and subfemtomolar protein concentrations, we observed a tendency for it to increase the number of captured protein molecules with the increase in the charge generated upon injection of analyzed solution into a measuring cell. External pulsed voltage applied to the measuring cell, was previously used by us to induce electric fields in flow-based systems for AFM-based fishing in order to enhance their sensitivity (up to the subfemtomolar level), as was demonstrated in Reference [
6]. Thus, to enhance the efficiency of protein capturing in an AFM-based fishing system, it is important to study the phenomenon of charge generation during the flow of an aqueous solution through a flow-based system in various conditions. It is to be noted that the generation of charge during the flow of water and aqueous solutions along various surfaces [
8], as well as changes in the physicochemical properties of water upon its flowing through polymer injectors [
9], were long and widely discussed in the literature. In our previous paper [
10], we demonstrated that negative pulsed voltage applied to parallel metal plates, between which an injector of the AFM-based fishing system was located, led to an increase in the efficiency of charge generation in the injector. At the same time, we demonstrated that an external AC electric field (with 50 Hz mains frequency, which is often present in biosensor devices) caused an increase in efficiency of charge generation in water flowing through an injector at a low-grade fever temperature (38 °C) [
11]; this temperature is higher than that of phase transition of water related with heat capacity (36.6 °C, [
9]) and corresponds to a pathology in human.
Attention to the effect of materials, which are not directly contacting with flowing aqueous solutions, is usually given from the viewpoint of controlling the charge generation in a flowing solution with an external electric field. In this way, in Reference [
12], the impact of the external electric potential of a closed metal ring on the generation of charge that occurs upon injection of a solution through this ring (which is not in contact with the liquid) is discussed. At the same time, the influence of materials of chips and communications on charge generation that are not in contact with the solution on the generation of charge in the solution flowing through the injector of the AFM-based fishing system is virtually not discussed in the literature. Also, it is known that the presence of macromolecules at low concentrations (including femtomolar ones) can influence the physicochemical properties of water (particularly, electrical conductivity, pH, surface tension, [
13,
14]. In the literature, it is discussed that these effects can be connected with the transitions between ortho- and para-states of water owing to pumping of mixed quantum states of ortho-/para-H
2O with natural and anthropogenic electromagnetic radiation (at which analytical biochemical measurements are usually carried out) [
15]. In this connection, it is interesting to study not only the influence of the presence of proteins at low concentrations on charge generation of the AFM-based fishing system, but also the influence of external electromagnetic fields.
In systems for AFM-based fishing, AFM chips fabricated from mica or graphite are commonly employed. Communications fabricated from polymer materials (particularly polypropylene) are also often used in nanobiosensors. For this reason, in our present work, the influence of materials of chips and communications on charge generation in a biosensor based on a system for AFM-based fishing in its flow section (described in Reference [
6]) was studied.
Stimulating influence of AFM chips, fabricated from mica and graphite, on charge generation in the injector section of a system for AFM-based fishing was demonstrated. The effect of an increase in the efficiency of charge generation in the flow section of nanobiosensors can well be influenced by polymer materials (particularly, those used in fluidic communications). At that, this efficiency depends on the completeness of filling of these communications with analyzed aqueous medium (water and/or aqueous protein solutions) used throughout the operation of nanobiosensors. The possibility of influence of highly dilute protein solutions (with concentrations in the range of 10
−15 M, what is the range of operation of highly sensitive biosensors) on the stimulation of charge generation in polymeric injector of flow-based biosensor systems has been noted. The influence of external low-frequency (50 Hz) AC electric field, applied to the injector of the system for AFM-based fishing, has also been studied. Similar to References [
10,
11,
16], this field has been induced by negative AC voltage applied to electrodes, between which the injector was located. The temperature of T = 35 °C was selected due to the following considerations. Firstly, this temperature is lower than the temperature point of phase transition related to heat capacity [
9]; secondly, this temperature is within the range of physiological temperatures of the human body, and this is the temperature of operation of biosensors upon studying biochemical processes in the human body in near-native conditions. These effects should be taken into account in the development of highly sensitive systems, whose operation is influenced by the charge state of analyzed solution.
4. Discussion
It is known that electrokinetic phenomena, such as charge generation, are observed upon flow of water and aqueous solutions along the surface of polymer materials (for instance, polymethylmetacrylate, polytetrafluoroethylene, polystyrene, polyethylene, and polypropylene) [
7,
17].
In the literature, this fact is explained by the formation of an electric double layer and the motion of ions upon displacement of the liquid phase relative to the stationary one, when a pressure gradient is applied.
In Reference [
7], a ~0.1 nC positive charge was registered upon pipetting of water through a plastic tip with an automatic pipette. It was reported that the pipette tip charged negatively, while the charge of the water drops was positive [
7].
In our present study, an accumulation of a positive charge in the measuring cell has been observed upon flow of water at its continuous pumping with a peristaltic pump through the injector.
In our work, not only linear, but also linear-stepwise
∆q(t) dependencies have been observed in water. Examples of linear dependencies in water are demonstrated in
Figure 2. Examples of linear-stepwise
∆q(t) dependencies are shown in
Figure 9 and
Figure 10. Insignificant variation is observed between the data obtained in the series with linear
∆q(t) dependencies. Such slight variations between
∆q(t) curves can be caused by insignificant oscillations of the time dependence of charge accumulation due to the possible manifestation of electrokinetic effects associated with quantum-mechanical phenomena of spontaneous transitions of ortho-para states of water. The possibility of such transitions was discussed in Reference [
15]. These transitions occur due to the fact that water is a non-equilibrium liquid in terms of spin temperature; and the equilibrium in water is shifted towards increasing the number of para-isomers of water. Thus, water is capable of changing the ortho-para ratio towards the equilibrium state [
9]—particularly upon external influences. The efficiency of occurrence of these transitions should be more probable near water phase transition point at T = 35 °C to 37 °C [
9,
18]. These spontaneous transitions can be stimulated by the influence of a peristaltic pump on the water, flowing through the communications, as well as by other electrokinetic phenomena (such as flow of water through a tapering tip), as was noted in References [
9]. Significant variations within one and the same series were observed for the cases when
∆q(t) dependencies were described by linear-stepwise curves. Occurrence of these linear-stepwise dependencies can be caused by an additional influence of external electromagnetic fields, which induce a change in the ratio between ortho- and para-isomers of water. Such influence was noted in the study by Pershin et al. [
19], and possibly, also by other factors. Let us point out that linear-stepwise dependencies are observed not only in water, but also in aqueous protein solutions.
The data on the stimulation of the generation and accumulation of charge in relative units (i.e., relatively to the charge generated in the absence of the investigated material), obtained when the investigated material was placed near the injector nozzle, are summarized in
Table 1.
As seen from
Table 1, the mica chip placed near the injector caused an increase in efficiency of generation and accumulation of charge in the measuring cell (by 28% at
l = 4 mm). It is known that the mica surface was charged negatively, and our experiments demonstrated that this charge was about −16 nC. The differences between
∆q(t) curves obtained in the presence of mica can be connected with causes of quantum-mechanical effects of transitions of ortho-para states of water near the critical point in external electromagnetic field. Upon the motion of water, an additional interaction of charged particles in water with the electric field occurs. This can lead to a number of effects, including additional effects on the motion of charged particles in water, and additional stimulation of transitions between ortho- and para-states of water. At that, with increasing
l from 4 mm to 1 cm, this efficiency decreases twofold, to 14% (
Figure 3).
We have measured the charge of an empty polypropylene tube, and it appeared to be of the same order of magnitude, −24 nC. As seen from
Table 1, the presence of a polypropylene tube also causes an increase in efficiency of generation and accumulation of charge (by 204% at
l = 1 cm). The diameter and the length of the polypropylene tube were 28 mm and 120 mm, respectively, while the dimensions of the mica chip were 25 mm × 75 mm. That is, despite the dimensions of these materials being similar, the presence of the polypropylene tube caused a more pronounced effect. This is possibly connected with different electric field distribution near the injector caused by the difference in the materials’ geometry, and accordingly, by the different capacity of the material/water-filled injector system. It is interesting to point out that filling of the polypropylene tube with water led to a decrease in the efficiency of charge accumulation in the measuring cell by almost an order of magnitude. Charge measurements in the system with the polypropylene tube have indicated that the value of charge accumulated in the cell also decreased by about an order of magnitude (from −24 nC to −5 nC). Accordingly, with decrease in the charge, which induces external electric field, from −24 nC to −5 nC, the influence of the electric field on the motion of charged particles in water decreases, and this leads to the observed effects. The system with the ethyl alcohol-filled polypropylene tube had a charge about −5 nC, and the effect of such tube was approximately equal to that caused by water-filled tube.
When the tube was filled with 10
−4 M protein solution (whose concentration is approximately equal to the protein concentration in human blood [
20]), the increase in the efficiency of charge accumulation in the measuring cell was approximately of the same level, as in the case of the tube filled with pure water. Here, it is to be noted that the charge of the polypropylene tube filled with protein solution was at the same level (−6 nC).
Thus, one can conclude that the efficiency of generation and accumulation of charge in the measuring cell during the flow of liquid through the injector is influenced by the charge state of neighboring non-conductive mica and polymer surfaces: the efficiency of the generation of charge and its accumulation in the measuring cell increases with the increase in the value of negative charge of these surfaces. At that, the change in the capacity of the system in the presence of these surfaces also matters.
Comparative experiments with protein solution and with water were carried out in eight sets. In all these sets, an effect of stimulation of the charge accumulation in the measuring cell has been observed in the presence of protein in water. The averaged data obtained in these eight sets of experiments have indicated that at 35 °C, the efficiency of charge generation in the pumped protein solution exceeded that in pure water by ~80 ± 40%. This increase in charge generation efficiency is possibly caused by the fact that in protein solution, an increase in the fraction of ortho-state should be observed, what has resonant character at ~35 °C, as was noted in Reference [
18]. Possibly, in this case protein molecules at very low (femtomolar) concentration serve as catalysts of the transition of water in the interface layer from para- to ortho-state. The possibility of such transitions in aqueous heterogeneous structures in the presence of proteins was discussed in [
21].
The influence of an external electromagnetic field (50 Hz) on the generation and accumulation of charge has also been investigated. As noted above, the influence of an external electric field (including the external electromagnetic environment) is supposed to be (as was noted above in this section) one of the causes of the variation between
∆q(t) curves. Industrial electrical networks typically operate at 50 Hz frequency. Upon the influence of an external low-frequency AC electric field at physiological temperatures of human body (35 °C), in all experiments, a tendency of the increase in the efficiency of generation and accumulation of charge was observed (
Figure 11). The value of the increase in the generation and accumulation of charge, averaged over all the experimental sets, was ~38%. This increase in charge accumulation can be caused by a number of effects connected with the stimulating influence of an external AC electric field on the motion of charged particles in water, as well as on the quantum mechanical effects associated with the ratio between ortho- and para-isomers in moving water [
19].