HK1131661B - Method for characterising a biologically active biochemical element by analysing low frequency electromagnetic signals - Google Patents

Description

Method for characterizing biologically active biochemical elements by analyzing low frequency electromagnetic signals

The present invention relates to the field of characterization of biochemical material characteristics of microorganisms or their structural or molecular components by analysis of electromagnetic signals generated after filtration, preferably after dilution.

Following the work taught by the Jacques Benzenist, it is well known to record and digitize the specific activity of biologically active molecules using a computer sound card. The molecules analyzed in the prior art are natural substances (histamine, caffeine, nicotine, epinephrine.) or drugs.

In the prior art it is proposed to receive or transmit this signal in analog or preferably in digital form.

In these studies, european patent EP0701695 describes a device and a method for the transmission of signals characterized by biological activities or biological characteristics that show a known substance specificity. It also describes the processing of such signals from a first carrier material to a second material having the above-mentioned biological activity, said second material being physically separated from said first material and initially being free of the physical presence of said known substance, and the material obtained by such a method. Such methods in the prior art include amplifying the electrical or electromagnetic signal emitted by the first material and received by a sensor and transmitting a signal characteristic of exhibiting a biological activity or biological property of the first material to an emitter, and then detecting in a second material a signal characteristic of exhibiting a biological activity specific to the known substance and transmitted to the second material in a high gain amplification.

It is known that French patent FR2811591 describes a method for generating a signal, in particular an electrical signal, which is characteristic of the biological and/or chemical activity of a substance of interest, in order to treat a receiving substance (une substentine laceration), in particular water, which initially has no specific biological activity, so that it is biologically active after being treated. The processed received material is hereinafter referred to as "processed material" (or material (matiere Inform) to be investigated). When the receiving substance is water, the substance being treated is referred to as "water being treated" (or water being investigated). The biologically active substance may also be in the form of preparations or homeopathic granules (granualemouthiques).

International patent application WO0001412 describes a method for activating an inactive solution with very low concentrations of chemically and/or biologically known substances in a solvent, which comprises placing the solution in a mechanical excitation field (un champ d' excitation mecanum), agitating the solution to generate the mechanical excitation field. The concentration of the known substance in the solution is less than 10^-6And (4) mol/L.

It is an object of the present invention to provide improvements to the technology to extend the range of applications and their performance.

To this end, in its broadest sense, the invention relates to a method for characterising a biochemical element having biological activity by analysing low-frequency electromagnetic signals emitted by a solution prepared from a sample of biological material to be analysed, characterised in that it comprises a pre-filtering stage (une betaable prable able de filtering).

Preferably, the sample is filtered before the analysis phase through a filter having a porosity of less than or equal to 150 nm and more particularly a porosity comprised between 20 nm and 100 nm.

Advantageously, the dilution in the dilution stage is at 10^-2To 10^-20In particular at 10^-2To 10^-9In the meantime.

According to a preferred embodiment, the method comprises a vigorous agitation phase and/or a centrifugation phase.

According to a preferred embodiment, the solution is excited (excitation) by white noise when an electromagnetic signal is obtained.

The invention relates in particular to the use of said characterization method for the analysis of microorganisms.

It also relates to a biological analysis comprising recording a characteristic signal obtained by applying said characterization method to a known biochemical element, and comparing the characteristic signal obtained with a characteristic signal of the biochemical element to be characterized of a pre-recorded characteristic signal.

The invention also relates to a biological inhibition method comprising recording at least one characteristic signal obtained by applying said characterization method to at least one known biochemical element and applying an inhibition signal to the sample according to said characteristic signal.

The invention also relates to a device for biological analysis comprising a sensor for obtaining an electromagnetic signal emitted by a solution by applying the characterization method of the invention and a circuit for processing said signal to calculate a characteristic signal of the sample being analyzed and a circuit for comparing the characteristic signal thus calculated with a base (une base) of a pre-recorded characteristic signal.

The invention may be better understood by reading the following description and by referring to the accompanying drawings, which correspond to non-limiting embodiments, in which:

figure 1 is a general diagram of a signal receiving apparatus;

FIG. 2 is a schematic view of the electrical signal generated by the solenoid in the absence of any emission source (background noise);

figures 3, 4 show schematic diagrams of the electrical signal generated by the solenoid in the presence of the emission source (m.pirum) after filtration with 0.02 micron and 0.1 micron, respectively;

FIG. 5 is a three-dimensional histogram of the wavelength distribution detected by the solenoid in the absence of any emission source (background noise);

FIG. 6 shows a three-dimensional bar graph of the wavelength distribution detected by the solenoid in the presence of the emission source (Mycoplasma pyricularis) after 0.02 micron filtration;

figure 7 shows a fourier analysis (harmonics of the unfiltered source current) under the same background noise;

figure 8 shows a fourier analysis of the signal generated by the solenoid in the presence of the emission source (mycoplasma pyriformis);

fig. 9 shows an overview of the amplification means used to pre-record the signal.

Hereinafter, it should be noted that:

^*living organisms are suspended in an in vitro or in vivo culture medium of a blood sample, more particularly in plasma extracted in the presence of an anticoagulant, preferably heparin.

^*The signal-emitting nanostructures are isolated from the culture medium or plasma filtered to remove all living organisms (0.45, 0.1 or 0.02 microns for bacteria and 0.45, 0.02 microns for viruses).

^*The electromagnetic signals are recorded in a computer and can be represented in different ways:

in total, two recordings every 6 seconds, a signal being considered positive when it is at least 1.5 times background noise

Analysis in a three-dimensional histogram

Analysis by fourier transform.

The present specification discloses the implementation of the method of the invention by way of an example of the characterization of three microorganisms by analysis of the emission signals:

mycoplasma pyriformis

HIV (human immunodeficiency Virus), strain IIIB (LAI)

Escherichia coli (Escherichia coli) K12 bacterium (E.coli)

Plasma of HIV-infected patients.

Experiment 1: cultures of Mycoplasma pyriformis applied to CEM cells

A Mycoplasma pyriformis culture of CEM cells was prepared in 10% fetal bovine serum RPMI1640 medium. These healthy cells show the presence of typical aggregates (agregastypiques) associated with the presence of mycoplasma pyriformis.

The suspension was centrifuged at low speed to remove the cells. The supernatant was filtered through a 0.45. mu. PEVD Millipore (trade name) filter, and the filtrate was further filtered through a 0.02. mu. Whatman Anotop (trade name) filter or a 0.1. mu. Millipore (trade name) filter.

Compared to the supernatant of uninfected CEM cells filtered under the same conditions. The solution was diluted 10-fold to 10-fold in complete RPMI in a laminar flow hood^-7. Each solution was treated at the vortexer (max power) for 15 seconds prior to subsequent dilution.

Signal detection is performed by the apparatus shown in the schematic diagram of fig. 1. The device comprises a solenoid unit (1) with a sensitivity comprised between 0 and 20000 hertz, placed on a platform made of insulating material. The solution to be read was dispensed in a 1.5 ml conical plastic tube (2) Eppendorf (trade name). The liquid volume is typically 1ml, in some cases 0.3 to 0.5 ml, without any noticeable difference in response. Each sample was taken twice in 6 seconds and each reading was entered separately.

The electrical signal emitted by the solenoid is amplified by the sound card (4) and transmitted to the computer (3), where appropriate software gives a visual image of the recorded elements:

the original overall amplitude image is shown in fig. 2, 3 and 4. Background noise (-) can be observed (fig. 2), which is averaged. A positive signal, defined as (+), can be detected when the amplitude exceeds the background noise by at least a factor of 1.5. Typically, the detected amplitude is twice (++), sometimes three times, the background noise: the detected signal will be referred to as the SEM electromagnetic signal.

The 3d histogram analysis of the signal in the presence of the background noise and the sample is shown in figures 5 and 6, respectively.

The decomposition of the background noise and the signal in the presence of the sample by fourier transformation into the respective frequencies is shown in fig. 7 and 8, respectively.

As a result:

1) SEM emission

Unfiltered suspension: background noise (-) was observed in uninfected controls and infected suspensions. Fig. 2 is a representation of the original overall amplitude of the detected signal.

0.02 micron filtered solution. Fig. 3 is a representation of the original overall amplitude of the detected signal: a clear difference can be observed. Solutions from Mycoplasma suspensions were diluted to 10^-7Are all (++). The uninfected CEM control is (-). From 10^-6A supplementary experiment carried out for several hours at the beginning of the dilution makes it possible to carry out the dilution 10^-14Are all positive (++), to a dilution of 10^-15Is (+) at this point. Dilution 10 of the first experiment^-6And 10^-7Remains (++) -after several hours at 20 ℃.

0.1u of the filtered solution. Fig. 4 is a representation of the original overall amplitude detected. Mycoplasma pear filtrate until dilution 10^-7Are all (++). Control except at dilution 10^-2Are negative except 1 reading. It is noted that there were 8 control tubes close to the M.pyriformis tubes, all of which were placed on the same plastic support. The positive values of one of the pipes can be interpreted by the transmission of signals from one pipe to the other through the pipe wall.

Fourier analysis of the positive frequency shows different peaks in decreasing order of intensity: for 10^-6And 10^-71000, 2000, 3000, 1999, 999, 2999, 500, 399, 300, 900 (all filtered 0.02).

The 3D analysis (fig. 6) shows a shift in the positive signal (+) towards the highest frequency.

Experiment 2: density gradient equilibrium centrifugation of 20-70% sucrose in Ca + + and Mg + + free PBS Behavior of the Medium SEM Source

Centrifugation was carried out at 35000 rpm at 4 ℃. Starting from the first 0.02 μ filtrate stored overnight at +4 ℃. It was confirmed to be positive before centrifugation.

After centrifugation, 12 fractions were collected from the bottom of the tube. The refractive index was measured to determine the density gradient.

Then the fractions are re-fractionated in pairsAnd (4) grouping. Diluted to 10% in RPMI16/40+ 10% fetal bovine serum^-7。

The negative values of the less diluted fractions can be explained by the self-interference (auto-interference) of the signals emitted by many sources. This self-inhibition can be detected as follows: mix 0.1ml undiluted element with 0.4ml 10^-4The diluent (2): after the vortex, signal quenching that becomes negative can be effectively observed.

Sample cell 5-6, density 1.21-1.225	7-8 sample cells with density of 1.165-1.194
		Undiluted (-)	Undiluted (-)
10-1(-)	10-1(-)
		10-2(-)	10-2(-)
10-3(-)	10-3(-)
		10-4(-)	10-4(-)
10-5(++)	10-5(++)
		10-6(++)	10-6(++)
10-7(+)	10-7(++)

Sample cell 9-10, density 1.112-1.114	Sample cell 11-12-13 (high)
		10-7Undiluted (-)	10-7Undiluted (-)

Note that the electromagnetic signal source behaves like a polymer with a large size (but <0.02 μ) and a density between 1.16 and 1.26.

There was a span effect (un effect de zone) not seen in the non-centrifuged raw product. Up to dilution 10^-4Self-interference with peaks (5-6 and 7-8) occurs.

Experiment 3: CEM cell cultures for HIV1/IIIB infection

This experiment involved CEM cell cultures infected with HIV1/IIIB, which were prepared by two cultures.

-4 days: onset of cytopathic effect (CPE)

-6 days: CPE + + Effect

Compared to uninfected CEM control cultures.

The operation method comprises the following steps:

-filtering the supernatant with 0.45 micron

Then filtered with 0.02 micron

10-fold dilution of the filtrate in RPMI Medium + bovine serum to 10^-7

Vortex vigorous stirring for 15 seconds at each dilution step.

As a result:

1) with the 4 day culture, no signal above background noise was observed. Until the dilution degree is 10^-7There was no difference from the uninfected CEM control.

2) Cultures infected with 6 days:

10^-1to 10^-5 (-)

10^-6 (++)

10^-7 (++)

10^-8 (++)

10^-9To 10^-15 (-)

And (3) performing a self-interference experiment again:

0.1ml of 10^-1Solution (minus) +0.4ml of 10^-7Solution (positive): the latter becomes negative. Therefore, self-interference can also occur at low dilution.

3) Analysis of Density gradient

The supernatant of the positive culture filtered through 0.02 microns was centrifuged using a BECKMANN (trade name) SW56 rotor at 35000 rpm at 4 ℃ with a density gradient of 20-70% sucrose.

Control supernatants of uncontaminated CEM cells were treated in a similar manner.

After centrifugation, 13 fractions were collected, grouped in pairs. The refractive indices of some fractions were determined using an ABBE (trade name) refractometer to determine the density gradient.

The 400ml fractions were diluted in RPMI16/40 medium and bovine serum. Serial dilutions were performed 10-fold starting from these fractions.

Note the groups with densities of 1.23-1.24 and 1.19-1.21 up to dilution 10^-7Are all positive. Group to dilution 10 with a density of 1.15-1.16^-7A positive signal is given. No signal was present in the top set of tubes regardless of dilution.

The group at the bottom of the tube (density 1.25-1.28) gave a positive signal only at some dilutions.

In contrast to M.pyriformis, self-interference occurred in the initial filtrate and no self-interference occurred in the gradient fractions.

As with Mycoplasma pyriformis, most of the signal in this case is concentrated in the density fraction 1.19-1.26, with a shoulder (une repaule) appearing near the lower density fraction 1.16.

Experiment 5: SEM source inactivation test of Mycoplasma pyriformis

Placing 1ml of 0.02 micron filtered 10 of Mycoplasma pyricularis in an Eppendorf tube^-1And (4) diluting the solution. This tube was placed in an active solenoid for 10 minutes and the original electrical signal of a Mycoplasma pyriformis preparation with the same dilution was pre-recorded after amplification.

Fig. 9 is an overview of the apparatus, including a computer 3 with a sound card (4) whose output is connected to an amplifier (10) with a maximum power of 60 watts, as described in the examples. The amplified signal is applied to a flexible solenoid (un) disposed in the Eppendorf tube (12)flex) (11). The applied signal is measured with a device (13).

Different types of amplified signals were applied for 10 minutes to positive-signaling mycoplasma pyrimethanii suspensions.

a) Amplified same signal: the start signal is positive. In contrast, the control tube containing a negative 0.02 micron filtrate of uninfected CEM cells became positive. This suggests that an electromagnetic signal can be delivered in an inactive medium as long as its initial spectrum is not altered.

b) If the highest intensity frequencies (179, 374, 624, 1000, 2000 hz) are chosen in the spectrum of the electromagnetic signal emitted by the mycoplasma pirium nanostructure, the signal remains positive after these amplified frequencies are applied.

c) Conversely, if the same signal with phase reversal is applied, the SEM positive values disappear.

This is also true when all SEM cells emitted by phase-reversed mycoplasma pyriae are used.

d) The signal can also be neutralized by self-interference, i.e. the signal of another microorganism (E.coli).

Test 4: analysis with different infections (HIV, ureolytic ureaplasma urealyticum (Urapilasma) urolyticum) urethritis and rheumatoid arthritis)

This analysis shows that these once filtered and suitably diluted plasma transmit signals similar to those transmitted by the same microorganisms in vitro, except that the cause of the infection of polyarthritis has not yet been identified.

In particular, when AIDS infected patients are treated by antiretroviral triple therapy, this signal consists of a high dilution of plasma (up to 10)^-16) It was emitted, suggesting that it was present in large amounts after the disappearance of the plasma virus challenge and had an effect on the residual infection remaining after treatment.

Summary of the invention

Microorganisms of different nature such as retroviruses (HIV), bacteria without rigid cell walls like Gram + (mycoplasma pyriformis), bacteria with rigid cell walls (escherichia coli) produce persistent nanostructures in aqueous solution.

These nanostructures (less than 100 nm in size) emit complex low frequency electromagnetic signals that can be recorded and digitized after the necessary filtering step to remove the microbial physical particles.

The same results were obtained from the plasma of patients infected with these microorganisms.

These nanostructures of microorganisms differ, which allows these microorganisms to produce a broad density spectrum and sensitivity to freezing. The signal it emits may be neutralized by self-interference with a pre-recorded phase-reversed signal or by self-interference with signals of other microorganisms.

Claims (14)

1. A method for characterizing a bio-active bio-chemical element by analyzing a low frequency electromagnetic signal emitted by a solution prepared from a sample of an analytical material, characterized in that it comprises a pre-filtration phase, wherein said sample is filtered before the analysis phase through a filter having a porosity of less than 150 nm.

2. The method for characterizing biochemical elements according to claim 1, wherein the sample is filtered through a filter having a porosity between 20 nm and 100 nm before the analysis phase.

3. A method of characterising a biochemical element according to claim 1 or 2, characterised in that the method includes a dilution stage with a dilution of 10^-2To 10^-20In the meantime.

4. A method of characterising a biochemical element according to claim 3, characterised in that the dilution is at 10^-2To 10^-9In the meantime.

5. Method for characterizing biochemical elements according to claim 1, characterized in that it comprises a phase of intense agitation.

6. Method for characterizing a biochemical element according to claim 1, characterized in that it includes a centrifugation phase.

7. The method of characterizing a biochemical element according to claim 1, wherein the solution is excited with white noise while the electromagnetic signal is obtained.

8. Use of a characterization method according to at least one of the preceding claims for the analysis of microorganisms.

9. A method of characterizing a biochemical element, comprising:

(a) recording a characteristic signal obtained by analysis of the low-frequency electromagnetic signal emitted by a solution prepared from a known biological sample after a pre-filtering phase, said pre-filtering being carried out before the analysis phase with a filter having a porosity lower than or equal to 150 nm,

(b) recording a characteristic signal obtained by analysis of the low-frequency electromagnetic signal emitted by a solution prepared from the biological sample to be characterized after a pre-filtering phase, said pre-filtering being carried out before the analysis phase, with a filter having a porosity of less than or equal to 150 nm, and

(c) the characteristic signal of the component to be characterized is compared with a prerecorded characteristic signal.

10. The method of characterizing a biochemical element according to claim 9, wherein the porosity of the filter in (a) and/or (b) is between 20 nanometers and 100 nanometers.

11. Use of the characterization method according to claim 1 for biological inhibition, characterized in that it comprises a phase of recording at least one characteristic signal of a biologically active biochemical element, comprising the analysis of a low-frequency electromagnetic signal emitted by a solution prepared from a known analytical material after a pre-filtration phase with a filter having a porosity of less than or equal to 150 nm before the analysis phase, and then the application of an inhibition signal to the sample according to said characteristic signal.

12. Use according to claim 11, wherein the porosity of the filter is between 20 nm and 100 nm.

13. Apparatus for characterizing biochemical elements according to the method of claim 1, the apparatus comprising means for preparing a solution with a sample filtered through a filter having a porosity of less than or equal to 150 nm before an analysis phase, a sensor for receiving an electromagnetic signal emitted from the solution, a circuit for processing the signal to calculate a characteristic signal of the sample being analyzed and a comparison circuit for comparing the characteristic signal thus calculated with a pre-recorded characteristic signal base.

14. The device of claim 13, wherein the porosity of the filter is between 20 nanometers and 100 nanometers.