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Citation: Penkov, N.V. Influence of
the Combined Magnetic Field and
High Dilution Technology on the
Intrinsic Emission of Aqueous
Solutions. Water 2023,15, 599.
https://doi.org/10.3390/
w15030599
Received: 30 December 2022
Revised: 27 January 2023
Accepted: 31 January 2023
Published: 3 February 2023
Copyright: © 2023 by the author.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
water
Article
Influence of the Combined Magnetic Field and High Dilution
Technology on the Intrinsic Emission of Aqueous Solutions
Nikita V. Penkov
Institute of Cell Biophysics RAS, Federal Research Center “Pushchino Scientific Center for Biological Research of
the Russian Academy of Sciences”, Pushchino 142290, Russia; nvpenkov@rambler.ru
Abstract:
Liquids prepared by sequential multiple dilutions with mechanical action (highly diluted
or HD solutions) are able to influence certain properties of adjacent solutions without direct contact,
which is mediated by their emission in the infrared (IR) frequency range. These properties do not
manifest when HD solutions are prepared in a geomagnetic field-free chamber. Here we studied the
influence of a magnetic field and the intensity of mechanical treatment on the intrinsic emission of HD
solutions of antibodies (Ab) to IFN
γ
and their effect on the adjacent water. IR-emission spectra were
recorded using a Fourier-transform IR spectrometer. Magnetic field treatment reduced the intrinsic
emission intensity of all HD samples; non-contact incubation with HD Ab prepared with intense
(iHD Ab) shaking or gentle (gHD Ab) mixing reduced the emission intensity of HD water as well.
The emission intensity of intact water was affected only by iHD Ab. Pre-treatment of HD Ab with a
magnetic field did not modify their non-contact effect on intact water. We confirmed the presence of
a non-contact effect and determined what factors it depends on (treatment with a magnetic field and
the intensity of shaking when preparing HD solutions). The intensity of water emission both in the
presence of HD Ab and in the presence of a magnetic field changes in a similar way.
Keywords:
infrared spectroscopy; emission spectroscopy; water; protein solution; aqueous solution;
emission of solution; antibodies to IFNγ; high dilutions; magnetic field treatment; intense shaking
1. Introduction
Water possesses many unusual properties that distinguish it from other liquids. Physi-
cal parameters such as boiling and melting points, features such as shrinkage on melting,
unusually low compressibility, etc., make this substance unique [
1
]. Physical influences
(i.e., temperature, pressure, solutes, and external fields) have been found to alter water
characteristics by disturbing or rearranging its hydrogen bond network. Besides that, due
to the delocalization of electrons held among water molecules, water can interact with
external fields, i.e., electric field, magnetic field (MF), and electromagnetic field (EMF).
Subsequently, under the effect of external fields, the hydrogen bond network is reshaped,
which can be detected by various methods [
2
]. For example, radiofrequency EMF treat-
ment of distilled water has been shown to alter its power density spectrum for tens of
minutes after exposure [
3
]. Chibowski et al. [
4
] revealed the effect of EMF on the rate of
water evaporation and surface tension. The electric field, in turn, increases gas uptake and
stabilizes nanobubbles in water [
5
]. Several studies have shown the effect of an MF on
the physicochemical properties of aqueous solutions. For example, Sronsri et al. [
6
] found
changes in electron distribution, molecular dipole moment, molecular polarization, heat
capacity, and salt solubility upon application of an MF; Wu et al. [
7
] noted the effect of
an MF on pH and electrical conductivity; and Wang et al. [
8
] determined a change in the
thermodynamic characteristics of the boiling process.
The characteristics of external fields capable of exerting an effect on aqueous solutions
can vary greatly. For example, impacts can be high-intensity [
5
] or super-weak [
9
], fields
can be constant [
10
], variable [
11
], or even pulsating [
12
]. However, among a variety of field
Water 2023,15, 599. https://doi.org/10.3390/w15030599 https://www.mdpi.com/journal/water
Water 2023,15, 599 2 of 18
parameters, it is possible to single out some common characteristics. For example, special
frequencies of a weak MF were revealed, which surprisingly coincided with the frequencies
of the cyclotron resonance of various ions: Ca, K, Mg, etc. [
13
]. Even though the cyclotron
resonance model raises serious doubts from the point of view of physics [
14
], no alternative
versions have been presented in the literature. In addition, treatment of samples with
an alternating MF with the cyclotron resonance frequency of H
9
O
4+
is of interest, as this
frequency was reported to change the refractive index of water, i.e., influence the structure
of water [
15
]. Thus, water is sensitive to magnetic and electromagnetic field action, which
may be due to the effect on the structural organization of water on clusters with a dipole
moment or on bulk nanobubbles [16].
It is logical to assume that the above-mentioned alterations of physical or physico-
chemical properties of water and aqueous solutions, persisting for at least tens of minutes
and even hours [3], also influence biological and biochemical processes taking place in an
aqueous environment. Indeed, several studies have established the effect of EMF and MF
on the physical characteristics of aqueous solutions of biological molecules [
17
,
18
] and on
the functioning of ion channels [
19
]. Other works showed the influence of these fields on
complex biological systems: cells, tissues, and multicellular organisms [
20
,
21
]. For example,
MF-treated water was shown to have positive effects on microorganisms resulting in higher
biogas production [
22
], on plants enhancing the germination of seeds, plant growth and
development, the ripening and yield of field crops [
23
–
28
], and on animals [
24
,
29
–
32
].
However, Fesenko et al. [
19
] clearly showed that the main target of the fields (or one of
the targets) is the water itself, through which the effect of the field on the biological object
is realized.
Another process that modifies the properties of water is the technology of preparation
of highly diluted (HD) solutions [
33
,
34
]. This technology utilizes a series of dilutions (up to
concentrations below Avogadro’s limit) accompanied by mechanical stress application such
as controlled hydrodynamic treatment/shaking (with defined amplitude and frequency).
HD solutions are different from water used for their preparation in such physicochemical
properties as electrical conductivity, pH, and surface tension [
35
]. Interestingly, HD solu-
tions acquire specific biological properties that are inherent neither to the solvent nor to the
diluted substance [
33
,
34
]. These unique properties of HD solutions found implementation
in biomedicine and in pharmaceuticals [
36
–
39
]. In addition, the latest advances in this field
seem to expand possible applications of HD solutions in materials science [40,41].
One of the enigmatic features attributed to HD solutions is their ability to influence
certain properties of adjacent solutions without direct contact [
42
]. For example, immersion
of a microtube with interferon-
γ
(IFN-
γ
) dissolved in phosphate-buffered saline into a vial
filled with HD solution of antibodies (Ab) to IFN-
γ
alters the physicochemical properties
of the former [
43
]. Nevertheless, no physical explanation of this phenomenon has been
provided yet.
In a recent work, Novikov et al. [
44
] showed that HD solutions prepared in a geomag-
netic field-free chamber do not acquire modifying properties. This fact considered together
with the influence of an MF on aqueous solutions and non-contact influence property of
HD solutions can hint that all the aforementioned phenomena may have a common nature.
Therefore, it is plausible to assume that the ability of HD solutions to transfer properties to
another solution without contact is at least partially due to electromagnetic radiation. If
that is the case, it is expected to detect a non-contact effect of HD solutions on water, similar
to the direct effect of a field on water.
IR-emission spectroscopy is a highly sensitive, reliable, and non-invasive method for
recording emitted spectra, which has already made it possible to detect the specific emission
of aqueous solutions that differ in solute concentration [
42
–
44
]. This work is a continuation
of a previous study on the characterization of HD solutions, including HD solution of Ab
to IFN-
γ
, also performed using IR-emission spectroscopy [
43
]. HD solution of Ab to IFN-
γ
was chosen as an object of investigation because a number of its characteristic properties
Water 2023,15, 599 3 of 18
have been described in previous works [
45
]. The information can be helpful in interpreting
the study results.
2. Materials and Methods
2.1. Sample Preparation
To prepare all solutions, we used deionized water obtained from a Milli-Q purification
system (Millipore, Germany) with a resistivity of 18.2 M
Ω•
cm at 25
◦
C. Affinity-purified
rabbit polyclonal Ab (IgG) to recombinant human IFN
γ
(containing 144 amino acids) in
glycine buffer (pH 7.2) at 2.3 mg/mL, 99% purity (AB Biotechnology, UK) were used for
HD Ab solutions preparation.
The following types of aqueous solutions were prepared for comparison:
1. Highly diluted Ab made with intense shaking (iHD Ab).
Ab to IFN
γ
had undergone a gradual reduction of their initial concentration (2.3 mg/mL)
under specific conditions. Namely, Ab to IFN
γ
was mixed with a solvent (water) at a ratio
of 1:100 and shaken vigorously with impact by hand with a controlled frequency of about
4 Hz (21 strokes in about 4.8 s) to produce the first centesimal dilution. All subsequent
dilutions consisted of one part of the previous dilution and 99 parts of the solvent, with
vigorous shaking with impact between each dilution. Thus, the final solution contained a
12th centesimal dilution of antibodies to IFN
γ
. A theoretical concentration reduction of the
original antibodies was at least 10
24
times, i.e., the theoretical concentration of the Ab in
the solution was 2.3 ×10−24 mg/mL.
2. Highly diluted Ab made with gentle mixing (gHD Ab)
The preparation procedure for the gHD Ab was similar to that for the iHD Ab, except
that gentle mixing (10 circular motions with a frequency of about 2 Hz) between each
dilution was performed.
3. Highly diluted water made with intense shaking (HD water).
The method for preparing HD water was the same as for preparing iHD Ab to IFN
γ
,
except that Milli-Q water was used instead of Ab solution.
4. Intact Milli-Q water.
All samples (1–4) were prepared by OOO “NPF “MATERIA MEDICA HOLDING” in
sterile glass vials with screw caps (Glastechnik Gräfenroda, Germany) and stored in the
dark at room temperature for no more than 2 weeks. Samples of the same type were stored
together, and samples of different types were stored in separate boxes. The samples were
tested blindly and decoded after the results have been obtained.
2.2. Experimental Setup for Magnetic Field Treatment
We assembled an installation consisting of a magnetically shielded chamber, two coax-
ial insulated solenoids, a control unit, and an electrical power generator. A magneti-
cally shielded ZG-209 MuMETAL Zero Gauss Chamber (Magnetic Shield Corporation,
Bensenville, IL, USA) consists of three coaxial cylinders with end caps made of permalloy
(an alloy of nickel, iron, copper, and molybdenum or chromium), which has a high mag-
netic permeability for weak fields. Near the midpoint of the central axis of this setup (the
area of sample placement), weak low-frequency MFs, as well as the geomagnetic field, are
suppressed by more than 1400 times.
The installation has two coaxial insulated solenoids of equal length wound one over the
other. Each solenoid is made from a single layer of polyester enameled copper wire (diameter
0.8 mm, coating thickness 0.1 mm) closely wound over a hollow cylinder of 160 mm external
diameter and 152 mm internal diameter, with 675 mm winding length. The internal space
of the hollow cylinder allows for placement of samples inside. One solenoid is connected
to an E36312A DC power supply (Keysight, Santa Rosa, CA, USA) and used to generate
a constant MF. Another solenoid is connected to a 33511B waveform generator (Keysight,
Santa Rosa, CA, USA), which allows creating an alternating MF. In this work, a collinear
combination of constant and alternating MFs was used to treat the samples. The value of the
induction of a constant MF was chosen close to the Earth’s natural MF of 60
µ
T. The magnetic
Water 2023,15, 599 4 of 18
induction amplitude of an alternating (in the form of a harmonic signal) field was 0.15
µ
T
with a frequency of 12.6 Hz. This corresponds to the cyclotron resonance frequency of the
hydrated hydronium ion (H9O4+) in the constant MF of 60 µT [15].
For treatment with an MF of defined parameters, the sample was placed in the center
of the installation (at the midpoint of the central axis of the hollow cylinder), since the
distribution of the field was most stable in this area. The sample was kept in the MF for 1 h
followed by the spectral analysis of the emitted radiation.
2.3. Non-Contact Effect
We studied the non-contact effect of Ab solutions (iHD Ab and gHD Ab—untreated
and having undergone MF treatment). Thus, samples of water and HD of water acted as
sensors of external influences, and Abs samples underwent various technological process-
ing as effectors. Specifically, a 0.5 mL microcentrifuge polypropylene tube (Eppendorf, EU)
with the sensor solution (water or HD water) was immersed in a glass vial (Glastechnik
Gräfenroda, Geratal, Germany) with 5 mL of one of the effector solutions: iHD Ab or gHD
Ab for 1 h at room temperature (21–23
◦
C) followed by the spectral analysis of the radiation
emitted by the sensor solution. There was no direct contact between the sensor and effector
solutions. They were separated from each other by the wall of the test tube, which was
0.45 mm thick. The experiment was repeated 9 times.
2.4. Emission Spectra Recording
A custom-built system for recording the spectra of intrinsic emission was assembled
based on a vacuum IR spectrometer with Fourier transform Vertex 80 v (Bruker, Hamburg,
Germany). The spectrometer was adjusted in such a way that the specimen, placed in the
focus of the optical system from the outside, served as a radiation source. The necessary
condition for detecting radiation from a specimen at ambient temperature is the presence of
a cold background, which does not allow thermal radiation from the environment to enter
the detector. For this, a black plate immersed in liquid nitrogen was placed behind the
specimen. Thus, we replaced the background thermal radiation with blackbody radiation
at a temperature of
−
196
◦
C. According to the Stefan-Boltzmann law, the integral emission
intensity of a black body is proportional to the fourth power of the absolute temperature.
Therefore, we managed to reduce the thermal background by more than 200 times [46].
To record emission, we used a highly sensitive mercury cadmium telluride (MCT)
detector cooled with liquid nitrogen. Before measurements, the vacuum jacket of the
detector was evacuated with a vCube Turbo Station turbomolecular vacuum pump (Turbo
Vacuum, Orlando, FL, USA) to 10
−6
Torr, and the vacuum integrity was checked after
the experiment. In this work, we analyzed the emission spectra in the range from 400 to
3000 cm
−1
, with a spectral resolution of 4 cm
−1
. To operate the spectrometer with Fourier
transform in this frequency range, a transparent KBr beam splitter was installed. The cell
windows were made of KRS-5 material. An annular Teflon spacer was placed between the
windows, which set the thickness of the liquid samples to 5 µm.
The details of the experimental setup used for measuring the intrinsic emission of
solutions, as well as the scheme of the cuvette and the method for processing the spectra,
have been described by us previously [43,47].
For each type of sample, 9 emission spectra were obtained.
2.5. Data Analysis and Statistics
The spectral region of 470–2000 cm
−1
exhibiting the most pronounced emission was
separated from the total absorption spectrum. This spectral region’s dimension was reduced
to 3 dimensions using the principal component analysis (PCA) [
48
–
51
]. The spectra within
the analyzed range (470–2000 cm
−1
) were separated in the principal component space using
a K-means clustering algorithm. According to the elbow method, 5 clusters were chosen,
as for a given number of clusters the best separation of the samples was observed. While
samples belonging to the same cluster are suggested as similar, the method does not allow
Water 2023,15, 599 5 of 18
obtaining p-values for pairwise spectra comparison. To overcome this, a bootstrap-like
procedure involving several steps was developed.
(1)
For each sample, a normality assumption, a mean, and a standard deviation of the
distribution of repeated measurements for the given wavelength were assessed.
(2)
The assumption of the repeated measurements being distributed normally was not
violated. This allows performing the generation of new spectra points at each wave-
length, using the normal distribution with parameters obtained at the first step. By
generating a curve point-by-point, a whole synthetic spectrum was obtained for
each sample.
(3)
The generated batch of samples’ spectra were also processed with PCA and clustered
by the K-means as the original spectra. For each pair of samples, their co-occurrence
in the same cluster was recorded.
(4)
Steps 2 and 3 were repeated 1000 times.
(5)
For each pair of samples, the ratio of co-occurrence in one cluster [
49
,
51
] among the
1000 simulations was considered as a bootstrapped p-value. The pairwise comparison
p-values were adjusted using Holm’s procedure for multiplicity of comparisons.
Differences were considered statistically significant at p< 0.05.
The integral emission intensity was defined as the area under the spectral curve within a
range of 470–2000 cm−1. The area under the curve was calculated using the trapezoidal rule.
The statistical significance of the differences between the samples in terms of the
integral emission intensity was determined by pairwise comparison of areas using the
Welch test with Holm’s correction for the multiplicity of comparisons. Differences were
considered statistically significant at p< 0.05. Additionally, Cohen’s D effect size was
calculated: a D-value above 0.8 was considered as a large effect size and above 1.2 as a very
large effect size.
Statistical analysis was performed using R (version 4.0.2; R Core Team (2020). R: A lan-
guage and environment for statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. URL: https://www.R-project.org/ (accessed on 1 January 2020)).
3. Results
3.1. Emission Spectrum of Water in the Infrared Range
A characteristic spectrum of the intrinsic emission of water is shown in Figure 1. The
spectra of the rest of the studied solutions were very similar. A typical emission spectrum
contains two well-defined peaks at wave numbers of about 700 and 1637 cm
−1
. Each of
these emission bands has a corresponding band in the absorption spectrum of water [
52
],
reflecting librational (720 cm−1) and bending (1647 cm−1) modes of water molecules.
When zooming in on the spectra, two more weak emission bands can be observed at
about 2100 and 3280 cm
−1
(Figure 1c), which correspond to two bands in the absorption
spectrum: the combination of librational and bending vibrations band (2200 cm
−1
) and the
band of stretching vibrations of water molecules (3400 cm
−1
). However, in this work, the
integral emission was analyzed only in the region of 470–2000 cm
−1
, including the band
of librational and bending vibrations, where it is possible to record the spectrum with a
sufficiently high signal-to-noise ratio.
In subsequent experiments, the following effects were assessed:
(1) MF effect on intrinsic emission of water, HD water, iHD Ab, and gHD Ab;
(2) Non-contact influence of iHD Ab and gHD Ab on intrinsic emission of water and
HD water;
(3) Non-contact influence of HD Ab, treated with an MF, on intrinsic emission of water.
3.2. Impact of a Magnetic Field on Samples
Figure 2shows the spectra of the samples recorded in the range of 470–2000 cm
−1
before and after treatment with a magnetic field. We found that an MF had a greater effect
on some samples than on others (Figure 2). The emission spectrum of iHD Ab to IFNg
(Figure 2c) underwent the strongest changes after the action of an MF.
Water 2023,15, 599 6 of 18
Water 2023, 15, x FOR PEER REVIEW 6 of 19
(a)
(b) (c)
Figure 1. The spectrum of intrinsic emission of water. (a) full recorded emission spectrum in the
range of 430–7000 cm
−1
, (b) zoomed spectral region in the range of 470–2000 cm
−1
, and (c) zoomed
spectral region in the range of 1810–4100 cm
−1
.
In subsequent experiments, the following effects were assessed:
(1) MF effect on intrinsic emission of water, HD water, iHD Ab, and gHD Ab;
Figure 1.
The spectrum of intrinsic emission of water. (
a
) full recorded emission spectrum in the
range of 430–7000 cm
−1
, (
b
) zoomed spectral region in the range of 470–2000 cm
−1
, and (
c
) zoomed
spectral region in the range of 1810–4100 cm−1.
Water 2023,15, 599 7 of 18
Water 2023, 15, x FOR PEER REVIEW 7 of 19
(2) Non-contact influence of iHD Ab and gHD Ab on intrinsic emission of water and
HD water;
(3) Non-contact influence of HD Ab, treated with an MF, on intrinsic emission of wa-
ter.
3.2. Impact of a Magnetic Field on Samples
Figure 2 shows the spectra of the samples recorded in the range of 470–2000 cm
−1
before and after treatment with a magnetic field. We found that an MF had a greater effect
on some samples than on others (Figure 2). The emission spectrum of iHD Ab to IFNg
(Figure 2c) underwent the strongest changes after the action of an MF.
(a) (b)
Water 2023, 15, x FOR PEER REVIEW 8 of 19
(с) (d)
Figure 2. Emission spectra of samples in the range of 470–2000 cm
−1
before and after magnetic field
treatment. ‘+MF’ indicates that the solution has been exposed to a magnetic field. (a) water, (b) HD
water, (c) iHD Ab, and (d) gHD antibody.
We determined the integral emission intensity of the samples, calculated from the
area under the emission spectrum curve in the considered spectral range. The results are
shown in Figure 3. A statistically significant decrease of 3% in the integral emission inten-
sity of iHD Ab to IFNg sample after the action of an MF was found. At the same time,
Cohen’s D-value was 1.3, which is considered as a very large effect size. Under the action
of an MF, the intensity of the emission of HD water decreased by 2%, and in the remaining
samples, it decreased by less than 1% (statistical significance was absent).
Figure 2.
Emission spectra of samples in the range of 470–2000 cm
−1
before and after magnetic field
treatment. ‘+MF’ indicates that the solution has been exposed to a magnetic field. (
a
) water, (
b
) HD
water, (c) iHD Ab, and (d) gHD antibody.
Water 2023,15, 599 8 of 18
We determined the integral emission intensity of the samples, calculated from the area
under the emission spectrum curve in the considered spectral range. The results are shown
in Figure 3. A statistically significant decrease of 3% in the integral emission intensity of
iHD Ab to IFNg sample after the action of an MF was found. At the same time, Cohen’s
D-value was 1.3, which is considered as a very large effect size. Under the action of an MF,
the intensity of the emission of HD water decreased by 2%, and in the remaining samples,
it decreased by less than 1% (statistical significance was absent).
Water 2023, 15, x FOR PEER REVIEW 9 of 19
Figure 3. Emission intensities of samples in the spectral range of 470–2000 cm
−1
before and after
exposure to a magnetic field. ‘+MF’ indicates that the solution has been exposed to a magnetic field.
Data are presented as mean ± SD. *—statistically significant difference in the integral emission in-
tensity of the sample after the action of the magnetic field and the corresponding sample before the
action of the magnetic field (p < 0.05, Welch test with the Holm correction).
We assumed that despite the absence of statistically significant differences between
the above-described samples before and after the MT treatment in terms of the integral
intensity of their intrinsic emission, the shape of the emission spectra was likely to change.
Therefore, to assess whether the shape of the emission spectrum changes after treatment
of the sample with an MF, we applied the principal component analysis and grouped
samples in the space of the principal components using K-means clustering followed by a
bootstrap-like procedure to obtain the ratio of co-occurrence in one cluster (p-value) (Fig-
ure 4).
As can be seen in Figure 4, the distribution of spectra of samples in the space of the
principal components is clearly different before and after treatment with an MF. The shape
of the spectra of iHD Ab to IFNg and iHD Ab to IFNg samples treated with an MF differ
statistically significantly (p < 0.05), and spectra are clearly separated in the space of the
principal components. The shape of the spectra of HD water and gHD Ab to IFNg also
differ significantly (p < 0.05) from the corresponding samples treated with an MF, but
these groups are less separated in the space of the principal components. At the same time,
the treatment of water with an MF does not affect the shape of the emission spectra be-
cause no statistically significant difference in the location of spectra in the space of the
principal components was found.
Thus, based on the analysis of the distribution of spectra in the space of the principal
components, it can be concluded that iHD Ab is the most sensitive sample, HD water and
gHD Ab to IFNg are less sensitive, while water is insensitive to the MF of the specific
characteristics used in this study. An analysis of the spectra indicates a change in the shape
of the spectrum for the HD water and gHD Ab to IFNg samples treated with MF (Figure
4), while the evaluation of the integral emission intensity did not reveal such differences
for these samples (Figure 3).
Figure 3.
Emission intensities of samples in the spectral range of 470–2000 cm
−1
before and after
exposure to a magnetic field. ‘+MF’ indicates that the solution has been exposed to a magnetic
field. Data are presented as mean
±
SD. *—statistically significant difference in the integral emission
intensity of the sample after the action of the magnetic field and the corresponding sample before the
action of the magnetic field (p< 0.05, Welch test with the Holm correction).
We assumed that despite the absence of statistically significant differences between the
above-described samples before and after the MT treatment in terms of the integral intensity
of their intrinsic emission, the shape of the emission spectra was likely to change. Therefore,
to assess whether the shape of the emission spectrum changes after treatment of the sample
with an MF, we applied the principal component analysis and grouped samples in the
space of the principal components using K-means clustering followed by a bootstrap-like
procedure to obtain the ratio of co-occurrence in one cluster (p-value) (Figure 4).
As can be seen in Figure 4, the distribution of spectra of samples in the space of the
principal components is clearly different before and after treatment with an MF. The shape
of the spectra of iHD Ab to IFNg and iHD Ab to IFNg samples treated with an MF differ
statistically significantly (p< 0.05), and spectra are clearly separated in the space of the
principal components. The shape of the spectra of HD water and gHD Ab to IFNg also
differ significantly (p< 0.05) from the corresponding samples treated with an MF, but these
groups are less separated in the space of the principal components. At the same time, the
treatment of water with an MF does not affect the shape of the emission spectra because
no statistically significant difference in the location of spectra in the space of the principal
components was found.
Water 2023,15, 599 9 of 18
Water 2023, 15, x FOR PEER REVIEW 10 of 19
Figure 4. The influence of a magnetic field (+MF) on the distribution of spectra in the wavelength
range of 470–2000 cm
−1
in the space of the principal components.
Double-sided arrows depict statis-
tically significant differences in the emission spectra between the parenthetical groups (p < 0.05,
comparison of the K-means with the Holm correction).
3.3. Non-Contact Effect of iHD Ab and gHD Ab on Water Samples and HD Water
Figure 5 shows the spectra of the samples before and after the non-contact action of
iHD Ab and gHD Ab on water and HD water recorded in the range of 470–2000 cm
−1
.
We estimated the integral emission intensity of the samples in the studied range (Fig-
ure 6). Although both gHD Ab and iHD Ab reduced the integral emission intensities of
both water (by less than 1% or about 1.5%, respectively) and HD water (by about 2% in
both cases), these changes did not reach statistical significance (p > 0.05). However, when
comparing HD water and HD water exposed to gHD Ab, Cohen’s D-value was 0.81, indi-
cating a large effect size.
To evaluate the changes in the shape of the spectra after non-contact exposure to HD
samples, we compared the spectra arrangement in the space of the principal components
(Figure 7). The data indicate a change in the emission spectrum of water that has experi-
enced a non-contact effect of iHD Ab, but not gHD Ab. At the same time, HD water emis-
sion spectrum after non-contact incubation with both iHD Ab and gHD Ab changed.
Thus, we have shown that there is a non-contact effect on the properties of water and HD
water samples, estimated by the shape of their emission spectra.
Figure 4.
The influence of a magnetic field (+MF) on the distribution of spectra in the wavelength
range of 470–2000 cm
−1
in the space of the principal components. Double-sided arrows depict
statistically significant differences in the emission spectra between the parenthetical groups (p< 0.05,
comparison of the K-means with the Holm correction).
Thus, based on the analysis of the distribution of spectra in the space of the principal
components, it can be concluded that iHD Ab is the most sensitive sample, HD water and
gHD Ab to IFNg are less sensitive, while water is insensitive to the MF of the specific
characteristics used in this study. An analysis of the spectra indicates a change in the shape
of the spectrum for the HD water and gHD Ab to IFNg samples treated with MF (Figure 4),
while the evaluation of the integral emission intensity did not reveal such differences for
these samples (Figure 3).
3.3. Non-Contact Effect of iHD Ab and gHD Ab on Water Samples and HD Water
Figure 5shows the spectra of the samples before and after the non-contact action of
iHD Ab and gHD Ab on water and HD water recorded in the range of 470–2000 cm−1.
We estimated the integral emission intensity of the samples in the studied range
(Figure 6). Although both gHD Ab and iHD Ab reduced the integral emission intensities
of both water (by less than 1% or about 1.5%, respectively) and HD water (by about 2%
in both cases), these changes did not reach statistical significance (p> 0.05). However,
when comparing HD water and HD water exposed to gHD Ab, Cohen’s D-value was 0.81,
indicating a large effect size.
Water 2023,15, 599 10 of 18
Water 2023, 15, x FOR PEER REVIEW 11 of 19
(a) (b)
Figure 5. Emission spectra of samples ((a) water, (b) HD water) before and after non-contact action
of iHD Ab and gHD Ab in the range of 470–2000 cm
−1
. In sample labels, ‘exp’ means ‘ exposed to’.
Figure 6. Integral emission intensities of water samples and HD water before and after the non-
contact impact of iHD Ab and gHD Ab in the spectral range of 470–2000 cm
−1
. In sample labels, ‘exp’
means ‘exposed to’. Data are presented as mean ± SD.
Figure 5.
Emission spectra of samples ((
a
) water, (
b
) HD water) before and after non-contact action of
iHD Ab and gHD Ab in the range of 470–2000 cm−1. In sample labels, ‘exp’ means ‘ exposed to’.
Water 2023, 15, x FOR PEER REVIEW 11 of 19
(a) (b)
Figure 5. Emission spectra of samples ((a) water, (b) HD water) before and after non-contact action
of iHD Ab and gHD Ab in the range of 470–2000 cm
−1
. In sample labels, ‘exp’ means ‘ exposed to’.
Figure 6. Integral emission intensities of water samples and HD water before and after the non-
contact impact of iHD Ab and gHD Ab in the spectral range of 470–2000 cm
−1
. In sample labels, ‘exp’
means ‘exposed to’. Data are presented as mean ± SD.
Figure 6.
Integral emission intensities of water samples and HD water before and after the non-
contact impact of iHD Ab and gHD Ab in the spectral range of 470–2000 cm
−1
. In sample labels, ‘exp’
means ‘exposed to’. Data are presented as mean ±SD.
Water 2023,15, 599 11 of 18
To evaluate the changes in the shape of the spectra after non-contact exposure to HD
samples, we compared the spectra arrangement in the space of the principal components
(Figure 7). The data indicate a change in the emission spectrum of water that has expe-
rienced a non-contact effect of iHD Ab, but not gHD Ab. At the same time, HD water
emission spectrum after non-contact incubation with both iHD Ab and gHD Ab changed.
Thus, we have shown that there is a non-contact effect on the properties of water and HD
water samples, estimated by the shape of their emission spectra.
Water 2023, 15, x FOR PEER REVIEW 12 of 19
Figure 7. The influence of non-contact impact of iHD Ab and gHD Ab on the distribution of water
and HD water samples spectra in the wavelength range of 470–2000 cm−1 in the space of the principal
components. In sample labels, ‘exp’ means ‘exposed to’. Double-sided arrows depict statistically
significant differences in the emission spectra between the parenthetical groups (p < 0.05, compari-
son of the K-means with the Holm correction).
3.4. Non-Contact Effect of HD Ab Treated with a Magnetic Field on Water
In this section, we present the results of the determination of the integral emission
intensities (in the range of 470–2000 cm−1) of samples of water after non-contact exposure
to different effector solutions: iHD Ab and gHD Ab without and with their preliminary
treatment with an MF (spectra not shown) (Figure 8), and the results of analysis of the
shape of the spectra from the distribution of spectra in the space of the principal compo-
nents (Figure 9).
As shown in Figure 8, iHD Ab treated with an MF (iHD Ab + MF) have a similar non-
contact effect on water as iHD Ab; the integral emission intensity of water decreases (for
1 and 1.5%, correspondingly, p > 0.05). In this case, the shape of the spectra, compared to
the distribution of samples in the space of the principal components, differ statistically
significantly from the spectra of water (Figure 9).
The integral emission intensity of water after its non-contact incubation with gHD
Ab or gHD Ab + MF decreased by less than 1% (p > 0.05). Based on the sample distribution
data in the principal component space, it can also be concluded that neither gHD Ab nor
gHD Ab + MF have a non-contact effect on the properties of water emission. Therefore, an
MF does not have any effect on the ability of iHD Ab and gHD Ab to influence water in a
non-contact manner.
Figure 7.
The influence of non-contact impact of iHD Ab and gHD Ab on the distribution of water
and HD water samples spectra in the wavelength range of 470–2000 cm
−1
in the space of the principal
components. In sample labels, ‘exp’ means ‘exposed to’. Double-sided arrows depict statistically
significant differences in the emission spectra between the parenthetical groups (p< 0.05, comparison
of the K-means with the Holm correction).
3.4. Non-Contact Effect of HD Ab Treated with a Magnetic Field on Water
In this section, we present the results of the determination of the integral emission
intensities (in the range of 470–2000 cm
−1
) of samples of water after non-contact exposure
to different effector solutions: iHD Ab and gHD Ab without and with their preliminary
treatment with an MF (spectra not shown) (Figure 8), and the results of analysis of the shape
of the spectra from the distribution of spectra in the space of the principal components
(Figure 9).
Water 2023,15, 599 12 of 18
Water 2023, 15, x FOR PEER REVIEW 13 of 19
Figure 8. Integral intensity of emission in the range of 470–2000 cm
−1
of samples of water and water
exposed to iHD Ab and gHD Ab, without and with their preliminary treatment with a magnetic
field. In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with a magnetic field’. Data
are presented as mean ± SD.
Figure 9. The influence of non-contact impact of iHD Ab and gHD Ab treated with a magnetic field
on the distribution of water samples spectra in the wavelength range of 470–2000 cm
−1
in the space
of the principal components. In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with
a magnetic field’. Data are presented as mean ± SD. Double-sided arrows depict statistically signif-
icant differences in the emission spectra between the parenthetical groups (p < 0.05, comparison of
the K-means with the Holm correction).
Figure 8.
Integral intensity of emission in the range of 470–2000 cm
−1
of samples of water and water
exposed to iHD Ab and gHD Ab, without and with their preliminary treatment with a magnetic field.
In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with a magnetic field’. Data are
presented as mean ±SD.
Water 2023, 15, x FOR PEER REVIEW 13 of 19
Figure 8. Integral intensity of emission in the range of 470–2000 cm
−1
of samples of water and water
exposed to iHD Ab and gHD Ab, without and with their preliminary treatment with a magnetic
field. In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with a magnetic field’. Data
are presented as mean ± SD.
Figure 9. The influence of non-contact impact of iHD Ab and gHD Ab treated with a magnetic field
on the distribution of water samples spectra in the wavelength range of 470–2000 cm
−1
in the space
of the principal components. In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with
a magnetic field’. Data are presented as mean ± SD. Double-sided arrows depict statistically signif-
icant differences in the emission spectra between the parenthetical groups (p < 0.05, comparison of
the K-means with the Holm correction).
Figure 9.
The influence of non-contact impact of iHD Ab and gHD Ab treated with a magnetic field
on the distribution of water samples spectra in the wavelength range of 470–2000 cm
−1
in the space
of the principal components. In sample labels, ‘exp’ means ‘exposed to’, ‘+MF’ means ‘treated with a
magnetic field’. Data are presented as mean
±
SD. Double-sided arrows depict statistically significant
differences in the emission spectra between the parenthetical groups (p< 0.05, comparison of the
K-means with the Holm correction).
Water 2023,15, 599 13 of 18
As shown in Figure 8, iHD Ab treated with an MF (iHD Ab + MF) have a similar
non-contact effect on water as iHD Ab; the integral emission intensity of water decreases
(for 1 and 1.5%, correspondingly, p> 0.05). In this case, the shape of the spectra, compared
to the distribution of samples in the space of the principal components, differ statistically
significantly from the spectra of water (Figure 9).
The integral emission intensity of water after its non-contact incubation with gHD Ab
or gHD Ab + MF decreased by less than 1% (p> 0.05). Based on the sample distribution
data in the principal component space, it can also be concluded that neither gHD Ab nor
gHD Ab + MF have a non-contact effect on the properties of water emission. Therefore, an
MF does not have any effect on the ability of iHD Ab and gHD Ab to influence water in a
non-contact manner.
4. Discussion
Currently, HD technology is successfully used in medicine [
53
,
54
] and technology [
40
,
41
],
and other areas of its application are being pursued [
55
]. It is important to understand
and consider the factors that affect the final characteristics of the product (such as activity,
stability, etc.) during its development and production. Therefore, the properties of HD
solutions must be carefully and comprehensively studied.
During the first stage of our work, we studied the influence of an MF on the properties
of HD samples. We have shown that the influence of an MF on the spectrum and intensity of
the intrinsic emission of the HD sample depends on the technology used in its preparation.
In particular, the applied MF has a greater effect on the emission spectrum of the samples
obtained by the repeated dilution procedure with intense mechanical action, and to a lesser
extent on the spectrum of the samples obtained by the multiple dilution procedure with
gentle mixing. This result is comparable with the conclusion of the work [
56
], which shows
the dependence of the impedance measurement result not on the concentration of the
dissolved substance, but on the procedure for sample preparation. Thus, the preliminary
mechanical treatment of both an aqueous solution and the solvent itself (water) leads to a
change in the investigated properties. The fact that an MF can affect the properties of HD
samples must be considered during their production and quality control, and MF treatment
should be applied to obtain HD solutions with desired characteristics.
Long-term (from tens of minutes to several days) effects of MF and EMF on the
properties of solutions of complex molecules, electrolytes, and water are already known in
the literature [
3
,
19
,
57
–
60
]. One of the mechanisms for realizing the effects of an MF is its
influence on the structural organization of water: on clusters with a dipole moment or on
bulk nanobubbles [
16
,
60
]. In [
60
] it is argued that the more complex the structure of water,
the greater the influence of an MF. The long-term effects of mechanical action on water,
which are expressed in a change in the level of water saturation with gas and a change in
the formation of a submicron nanobubble phase, are shown in [61].
The results of our work are consistent with the conclusions of the above-mentioned
studies: MF causes alterations in the aqueous structure of solutions. It is likely that intense
shaking during the preparation of iHD Ab and HD water solutions leads to a more active
formation of bulk nanobubbles, compared with gentle mixing during the preparation of
gHD Ab. At least at the initial stages of the preparation, nucleating centers (molecules)
are present in iHD Ab samples and absent in HD water solution. Such nucleating centers
contribute to the formation of a larger number of inhomogeneities (nanobubbles, water
clusters) in iHD Ab compared to water HD. Considering all of the above, we believe that
it is water clusters, as well as the gas phase (represented by dissolved air molecules or
stable bubbles formed as a result of mechanical action), that are the target for an MF. The
change in the structural characteristics of water is the most likely reason for the change in
its emissivity.
We have previously shown that HD solutions are capable of emission in the mid-IR
range [
47
], which may determine their effect on the aqueous environment of the target
without direct contact [
42
]. In a recent study, the presence of a distant effect of HD Ab to
Water 2023,15, 599 14 of 18
INF
γ
on the solution of INF
γ
was also demonstrated [
62
]. The authors showed that the
non-contact effect of HD Ab to INF
γ
on the INF
γ
solution and water varies with different
duration of their joint incubation, and also depends on the presence and parameters of
the MF in which the sensor and effector samples are incubated. We assume that it is the
emission of HD solutions, without their chemical interaction with the target, that determines
their effects used in medicine and technology. In this regard, for the manufacturing process
of products based on HD solutions, it is important to understand what factors will affect
the implementation of their mechanism of action.
In continuation of this study, during the second stage of our work, we investigated
the role of HD preparation technology in the implementation of the non-contact effect.
In particular, we evaluated how the sensor sample emission changed after non-contact
incubation with an effector sample prepared using different technologies. It turned out
that the IR emission of HD water changed both after exposure to iHD Ab and to gHD Ab.
At the same time, only iHD Ab but not gHD Ab changed intact water emission. Thus,
solutions obtained using the multiple dilution procedure with intense mechanical action
have a stronger non-contact effect on water than solutions obtained by multiple dilutions
with gentle shaking. In addition, HD water is more sensitive to non-contact exposure than
intact water. Therefore, for the manifestation of the non-contact effect, at least one of the
solutions (sensor or effector) must undergo the HD procedure with intense shaking. This
result must be considered both in the development and in the manufacturing of products
based on HD solutions.
The fact that a system with a higher concentration of bulk nanobubbles (HD water),
which are themselves susceptible to electromagnetic effects, is more sensitive to the non-
contact effect of the effector solution, confirms the electromagnetic nature of the non-contact
effect of the latter. Moreover, both the MF and the effector solution reduce the emission
intensity of the sensor solutions.
The changes in IR-emission spectra are due to spontaneous transitions of molecules
from excited vibrational energy levels with a nonzero population to the ground state.
Thus, the possibility of emitting radiation by a substance at certain frequencies depends
on the presence of the corresponding energy levels and their population. The position of
vibrational energy levels is determined by the molecular structure of the substance, and
their population depends on temperature according to the Boltzmann distribution:
NE
N0
=exp−hν
kT
where N
E
—number of molecules at a level with energy E, N
0
—number of molecules at the
ground state,
ν
—frequency of a photon emitted during the transition between these levels,
h—Planck constant, k—Boltzmann constant, T—absolute temperature.
The presence of energy levels and their population determines the emissivity of the
substance at the corresponding frequencies. Thus, the emission spectrum is completely
determined by the molecular structure of the substance, which may differ for various solutions.
In this study, we consider the frequency range that corresponds to spontaneous
transitions of water molecules from excited librational (440–1000 cm
−1
) and deformation
(1550–1750 cm
−1
) levels. Librational vibrations are of an intermolecular nature, while
deformation vibrations are intramolecular. It is important to note that intramolecular
vibrations depend on intermolecular binding [52].
To interpret the obtained differences in the integral emission intensity of the samples,
one can refer to the well-known two-structure model of water [
1
,
63
–
66
], according to which
water can be divided into two fractions: low-density water (LDW) and high-density water
(HDW). Summarizing the data obtained by different studies of water in the context of
considering these two structures, it can be argued that their equilibrium coexistence is
determined by fundamentally different thermodynamic criteria underlying their stability:
the striving for the minimum enthalpy for LDW and the striving for the maximum entropy
for HDW. Apparently, the observed changes in the emissivity of the analyzed solutions
Water 2023,15, 599 15 of 18
exposed to the MF and effector solutions are associated with a change in the ratio of these
two fundamental phases of water.
Note that our data and conclusions about the effect of an MF and effector solutions
on the water structure of the sensor solutions are consistent with data from other authors,
in whose works the changes in the physical properties of water were found using various
methods. For example, Liboff [
15
] described the change in the refractive index of water as
a result of exposure to a weak MF of 0.05
µ
T with the cyclotron resonance frequency of
the H
9
O
4+
ion. In [
67
], the authors studied the effect of electric current passing through
water with frequencies of 0.1–45 Hz and strength of the order of
µ
A. Using an IR camera,
a decrease in water temperature by 2
◦
K was registered after these impacts. It is known
that alternating current, according to the Ampere–Maxwell law, generates an alternating
MF. Therefore, the phenomena discussed by the authors belong to the same class as those
discussed by us in this article. In [
68
], the authors also studied the effect of an MF on the
structure of water. Based on theoretical and experimental data, it was concluded that the
effect of an MF is limited to a redistribution of hydrogen bonds, namely, weakening of
bonds within large cluster structures and strengthening of bonds within smaller structures.
During the third stage of our work, we evaluated the influence of an MF applied
to the effector solution on the presence and magnitude of the non-contact effect. We
demonstrated that an MF treatment did not affect the ability of iHD samples to cause a
non-contact effect on water. In contrast, gHD samples, whether treated with an MF or not,
had no detectable effect on water. The emission spectrum of iHD Ab or gHD Ab potentially
has some specificity, which is not disturbed by the MF. The characteristics of the spectra of
electromagnetic emission, which determine such specificity, have yet to be clarified. On the
other hand, water itself seems to have a different sensitivity to exposure to electromagnetic
radiation with a particular spectrum (i.e., exposure to different samples).
Future studies are needed to investigate other factors that could potentially affect the
non-contact effect, namely whether it depends on the distance between the effector and
the sensor solutions, on the area of contact between the effector and the sensor, and on the
concentration of the sensor and the effector. In addition, it is important to determine for
how long the changed properties of the sensor persist after the end of joint incubation with
the effector, as well as whether the properties of the effector would change afterwards.
5. Conclusions
1.
An MF with the cyclotron resonance frequency of H
9
O
4+
changes the emission prop-
erties of highly diluted aqueous solutions.
2.
Exposure to an MF and non-contact exposure to HD solution effectors similarly change
the emission properties of solution sensors.
3.
The manifestation of the non-contact effect of HD solutions depends on the technology
of preparation of both the effector and sensor solutions. To implement the non-contact
effect, at least one of the HD solutions must be prepared using intense shaking.
4.
Pre-treatment of HD solution effectors with an MF does not affect the presence and
magnitude of their non-contact effect on the IR-emission properties of water.
Funding: This research received no external funding.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
This work was performed in part on instruments of the Optical Microscopy and
Spectrophotometry core facility, ICB RAS, Federal Research Center “Pushchino Scientific Center for
Biological Research of the Russian Academy of Sciences” (https://www.pbcras.ru/services/tskp/).
All samples for testing were provided by OOO “NPF “MATERIA MEDICA HOLDING” as a company
for manufacturing drugs based on highly diluted antibodies.
Conflicts of Interest:
Different versions of highly diluted antibodies to IFN-
γ
are the substances
(single or one among other components) for commercial drugs produced by OOO “NPF “MATE-
Water 2023,15, 599 16 of 18
RIA MEDICA HOLDING”. Patents on this substance belong to OOO “NPF “MATERIA MEDICA
HOLDING”. OOO “NPF “MATERIA MEDICA HOLDING” was not involved in the study design,
collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for
publication. The author declares no other conflict of interest.
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