RU2408280C2 - Device for diagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: device for analysis of biological fluid contains laser module, containing source of laser radiation, optic unit, which contains flask element, module of photoreceiving device and analogue-digital converter. Flask element is optically connected with module of photoreceiving device, and analogue-digital converter is electrically connected with photoreceiving device and computer. Additionally module of photoreceiving device contains low-noise amplifier and normalising amplifier connected with low-noise amplifier. Optic unit contains element of optic tract formation and light-dividing shevron, optically connected with source of laser radiation and flask element, and laser module and module of photoreceiving device are rigidly fixed on optic unit.

EFFECT: device makes it possible to carry out multi-parametre disease diagnostics.

11 cl, 7 dwg, 1 tbl

Description

The invention relates to medicine, namely to the field of cancer diagnosis, in particular, by using the methods of molecular physics and is industrially applicable in devices intended for use both during examination (screening) and monitoring the course of treatment (monitoring), and when selecting in vitro necessary for the treatment of drugs and their doses.

A diagnostic device is known in which the preparation of a biological fluid solution is carried out, laser radiation is applied to the solution and the physical parameters of radiation emitted from the biological fluid solution are recorded, which are used to judge the patient’s condition, and when registering the physical parameters of radiation emanating from the biological fluid solution, analog-to-digital signal conversion and its computer processing [RF patent No. 2219549, IPC G01N 33/52]. This device measures the physical parameters of a weak aqueous solution of the patient’s native blood serum using laser correlation spectroscopy. For this, two solutions are prepared, alkali is added to one of the mentioned solutions, and acid to the other. For each solution mentioned, the probability density distribution of the amplitude of the fluctuation of the light scattering intensity is determined.

The disadvantage of this analogue is not a large number of monitored parameters, which are used for diagnostics.

Closest to the claimed is a device containing a laser module that contains a laser source, an optical unit that contains a cuvette assembly, at least one module of a photodetector that contains a photodetector, and an analog-to-digital converter, wherein the cuvette assembly is optically coupled with the photodetector unit, and the analog-to-digital converter is electrically connected to the photodetector and computer [RF patent No. 2219549, IPC G01N 33/52]. Using this device, the physical parameters of a weak aqueous solution of the patient’s native blood serum are measured by laser correlation spectroscopy. For this, two solutions are prepared, alkali is added to one of the mentioned solutions, and acid to the other. For each solution mentioned, the probability density distribution of the amplitude of the fluctuation of the light scattering intensity is determined.

The disadvantage of this closest analogue is not a large number of monitored parameters, which are used for diagnostics.

Using the claimed invention, the technical problem of providing multi-parameter diagnostics is solved.

This goal is achieved by the fact that in the known diagnostic device containing a laser module that contains a laser radiation source, an optical unit that contains a cuvette assembly, at least one photodetector module containing a low-noise amplifier and a normalizing amplifier electrically coupled to low-noise an amplifier, as well as an analog-to-digital converter, moreover, the cuvette assembly is optically connected to the photodetector module, and the analog-to-digital converter is electrically connected en with the photodetecting device and the computer, the optical unit further comprises a chevron beam splitter, optically coupled to a laser radiation source and the cuvette assembly and the laser module and a photodetector module is rigidly secured to the optical block.

In particular, the beam splitting chevron can be rigidly fixed in the optical unit.

In particular, the device may further comprise a control board electrically connected to the laser module and configured to define a cyclogram for turning the laser module on and off.

In particular, the optical unit may further comprise an optical path forming unit optically coupled to the cuvette assembly and rigidly fixed to the optical unit.

In particular, the module of the photodetector may further comprise a unit of the forming input optics optically coupled to the photodetector and rigidly fixed in this module.

In particular, the analog-to-digital converter may further comprise a filter defining a timer, a buffer memory, made on one board.

In particular, the device can record a beat spectrum between Rayleigh scattered radiation of a biological fluid solution and reference deterministic laser radiation.

In this case, the laser radiation and the reference deterministic laser radiation can be obtained from the same source.

Moreover, when exposed to a solution of a biological fluid, the laser radiation and the reference deterministic laser radiation can be directed at an angle of 90 ° to each other,

In this case, Rayleigh scattered radiation can be detected in the direction of propagation of the reference deterministic laser radiation.

In this case, Rayleigh scattered radiation can be recorded in two directions oriented at an angle of 180 ° to each other.

When using the claimed invention for analysis, 1 ml of serum or blood plasma is sufficient, which remains unclaimed during a standard biochemical polyclinic blood test. The average analysis time is from 3 to 15 minutes, depending on the speed of the computer. The invention allows for mass express diagnostics, while cancer patients can be detected in the early stages of the disease, when traditional diagnostic methods are ineffective. At the same time, patients at risk can be identified in advance, when a predisposition to the occurrence of neoplasms only appears in the body. The claimed invention can be used to monitor the effectiveness of treatment using all modern methods of treating tumors and subsequent dispensary monitoring. It allows reliable control of individual patient treatment.

The development of pathological processes in the body is accompanied by changes in a number of molecular parameters in cells and tissues, as well as in blood serum. Proteins with different weights and in various proportions enter the blood serum. The main serum proteins include albumin, weight ~ 70,000 (55%), α ₁ -globulins, average weight ~ 40,000 (6%), α ₂ -globulins, average weight ~ 50,000 (11%), β-globulins, average weight ~ 70,000 (7%), γ-globulins ~ 150,000 (16%). Estimates give a value of ~ 100000 for the weight average molecular weight of serum particles.

Protein molecules carry an electric charge on their surface, which also leads to the presence of a large dipole moment of the protein of the order of several hundred D units (Debye). The surface charge of a protein molecule in solutions can be changed over a wide range, varying the concentration of free protons (pH value). Due to these features, there is a strong electrostatic interaction between the charges of protein molecules, as well as between the molecules of the polar solvent and the charged surface groups of the protein, which, in turn, affects the nature of the Brownian motion of the molecules.

Features of light scattering in solutions of blood serum and lymph are determined by the physical parameters of the dissolved molecules. As models of biological fluids such as blood or lymph, aqueous solutions of proteins can be used.

The most direct and effective method for studying intermolecular interaction, mobility, and polarization properties of solutions of macromolecules, including aqueous solutions of proteins, is the Rayleigh light scattering method. The method makes it possible to directly determine the molecular mass M, for which it is necessary to measure the Rayleigh coefficient or turbidity R ₉₀ at several concentrations of solution C and extrapolate the obtained dependence to C = 0, plotting the value of CH / R ₉₀ as a function of C. The slope of this straight line, equal to 2 V , allows you to calculate the second virial coefficient B, which characterizes the degree of deviation of the behavior of the solution from ideal and serves as a measure of intermolecular interaction in the solution. For solutions of charged protein molecules, the effect of the charge on the surface of the molecule on its behavior in solution, in particular, on the intermolecular interaction parameter, is very significant. The appearance of a cancer or a predisposition to it is the result of a change in the surface charge on the protein molecules. With a decrease in charge, the Coulomb repulsive forces between the protein molecules weaken, and the attractive forces between them begin to prevail. This leads to a change in the magnitude and sign of coefficient B and to the formation of complexes of molecules having a greater mass than individual proteins.

The dynamic parameters of Rayleigh light scattering are determined by the method of photon correlation with the Fourier analysis of the spectral composition of the scattered light. This method investigates the correlation function of the fluctuations in the intensity of the scattered light due to the Brownian motion of the particles of the solution. In this case, the coefficients of translational diffusion of particles and their hydrodynamic radii can be determined. The concentration dependences of the translational diffusion coefficient and the scattering parameter are determined by the same virial coefficient. The dynamic parameters of charged molecules in solutions also significantly depend on the surface charge of the protein molecule. The presence of an oncological disease leads to a change in the surface electric charge of the blood protein molecules, which leads to a change in the intensity of the spectral components of the scattered light and their half-widths.

The development of cancer has an initial autocatalytic stage, which is associated with the formation of free radical states in the lipid phase of cells and tissues. These processes can be reliably detected using fluorescence analysis using appropriate probes. The initial stage of the aggregation of serum proteins can also be detected by studying the polarization of fluorescence of various probes connected to serum proteins, including chelates.

When a fluorescent volume containing a solution of protein molecules is irradiated with monochromatic linearly polarized light, anisotropy of the fluorescence of the solution particles takes place. Rotational diffusion, which occurs during the lifetime of the excited state and changes the direction of the emitting dipole of the fluorophore, can reduce the measured value of anisotropy to values below the maximum.

Protein fluorescence is determined by the amino acid composition and is observed in the ultraviolet region. In this regard, when studying proteins in aqueous solutions, compounds are specially selected that, when adsorbed on the surface of protein molecules, fluoresce in the visible part of the spectrum. Fluorescent probes or dyes are used as such compounds. Having a large quantum yield, the probes intensively fluoresce in the visible part of the spectrum, and information on the molecular mobility of the protein is extracted from the parameters of this fluorescence. Using probes, you can study the surface charge of a protein molecule.

The invention is further illustrated by drawings, a description of specific options for its implementation with reference to the accompanying drawings, in which:

figure 1 depicts a diagram of the inventive device;

figure 2 depicts a diagram of a laser module;

figure 3 depicts a diagram of an optical unit;

figure 4 depicts a module diagram of a photodetector;

figure 5 depicts a one-parameter histogram of the coefficient of intermolecular interaction for three groups of patients (healthy patients, risk group and cancer patients);

6 depicts the dependence of translational diffusion coefficient on pH for aqueous solutions of BSA and γ-globulin;

Fig.7 displays the results of a clinical examination of healthy and sick patients in two ways.

The diagnostic device (Fig. 1) contains a laser module 1 and two modules of photodetector devices 2, rigidly mounted on the optical unit 3. The signals from the photodetector devices 2 are fed to the board of the analog-to-digital converter 4, which also serves as a communication card with the computer through matching board, installed on the computer motherboard. The functions of monitoring and controlling the operation of the device are carried out by the control board 5, and its power is supplied from the built-in power supply 6.

The laser module 1 (figure 2) consists of a laser diode 7 and its power and control circuit 8, which ensures the stability of the output radiation of the laser diode 7 and controls its operation according to the signals from the control board 5.

The module of the photodetector 2 (Fig. 3) is designed to detect the Rayleigh scattering signal and bring it to the level necessary for applying to the analog-to-digital converter 4. It contains a block of forming input optics 9, a photodetector 10, a low-noise amplifier 11, and a normalizing amplifier 12 The input forming optics unit 9 provides the formation of a wavefront of scattered radiation, which gives the most useful signal at the photodetector 10. Low-noise amplifier 11 provides amplification of the received Igna lowest possible deterioration of its noise characteristics. The normalizing amplifier 12 is designed to bring the signal to a value that ensures optimal use of the dynamic range of the analog-to-digital Converter 4.

The optical unit 1 (Fig. 4) consists of a beam-splitting chevron 13, a cuvette assembly 14 and an optical path forming unit 15. The cuvette assembly 14 fixes the cuvette with the test solution with the necessary accuracy with respect to the beam of the laser diode 7 and the input apertures of the photodetector 10. The forming unit optical path 15 provides adjustment and fixation of the position of the laser diode 7, photodetector devices 10 and the elements of the forming optics in the measurement process. The forming unit of the optical path 15 consists of a rigid guide structure on which are mounted apertures, quotation units and fastening elements.

An analog-to-digital converter 4 is intended for limiting the frequency band (filtering) of the analyzed signal, converting the filtered analyzed signal from an analog form to digital, storing the results in its buffer memory before writing them to random access memory, controlling the digital accumulation of the analyzed signal, and providing a signal transmission protocol to the computer through the matching board installed in the ISA slot of the motherboard. The board of the analog-to-digital converter 4 is made in the form of a printed circuit board and is equipped with a connector for docking with a computer and contains two inputs of the analog signal and allows you to simultaneously store 1048 samples. The number of bits of the analog-to-digital converter is 12. The board of the analog-to-digital converter 4 can operate in the accumulation mode and the data transfer mode. It contains a master timer, a buffer memory, a digital-to-digital signal conversion circuit, buffer registers for storing the high and low bytes of a number to provide a protocol for transferring it to a computer.

The control board 5 is designed to ensure the operation of the device in normal and emergency modes of operation. It sets the cyclogram of turning the device on and off, indicating the state of readiness or unavailability of the device for operation, monitoring the operability of the device’s functional blocks, controlling access and archiving information about the device’s status, performing service functions, for which there is a connection between the device and an external computer via the serial port.

The power supply 6 is designed to convert the input voltage of 220 V at a frequency of 50 Hz to a constant voltage with parameters that ensure optimal operation of the device.

The principle of operation of the device (figure 1) is based on the method of laser correlation spectroscopy, which consists in measuring the power spectra of quasi-elastic scattered light. It is known that the scattering of light by particles performing Brownian motion is accompanied by an increase in the width of the spectrum of the initial radiation — diffusion broadening. Since the intrinsic width of the spectrum of laser radiation is very small, the width of the spectrum of the scattered light is proportional to the coefficient of translational diffusion, analytically associated with the size of the scattering particles according to the well-known Stokes-Einstein formula,

In the case of a polydisperse system, when particles with different diffusion coefficients contribute to the scattering, the problem of determining the size distribution function of scattering particles reduces to a complex mathematical processing of the experimental spectra, during which it is necessary to solve a poorly conditioned inverse spectral problem. The stability of the solution to this problem is achieved using the mathematical method of regularization. The distribution functions of scattering particles obtained after the regularization procedure are entered into a computer database for further statistical multi-parameter analysis.

The inventive device, which uses coherent exciting radiation and analyzes the scattered radiation in an aqueous solution of blood serum by correlation spectroscopy, including methods of analysis of integrated and dynamic light scattering, provides multi-parameter diagnosis of common diseases, mainly oncological by determining static and dynamic molecular parameters (mass, shape, intermolecular interaction coefficients, depolarization coefficients and, the coefficients of translational and rotational diffusion, intrinsic viscosity and hydrodynamic radii). Unlike the closest analogue, the claimed invention allows the simultaneous measurement of static and dynamic parameters of particles (proteins or their aggregates) of an aqueous solution of blood serum, while both examination (screening) and monitoring of the course of treatment (monitoring) and selection in vitro necessary for the treatment of drugs and their doses.

Rayleigh scattering was used to diagnose cancer. We measured the relative intensity of the integral Rayleigh scattering R (at an angle of 90 ° to the incident beam), the mass of scattering particles M, and the intermolecular interaction coefficient B. It turned out that all three of these parameters for blood serum solutions of healthy people and cancer patients differ significantly (see table) .

Table Healthy patients Cancer patients No. R M (10 ⁴ ) B (10 ⁴ ) R M (10 ⁴ ) B (10 ⁴ ) Disease one 1.12 7.5 3.5 7.14 - -1.7 cervical cancer 2 0.8 thirty 1.0 5.14 - -0.65 uterine cancer 3 0.7 twenty 5.8 4.2 - 0.00 cervical cancer four 0.6 3 4.0 2.0 - -0.5 uterine cancer 5 0.9 - 5.3 1.4 - -0.8 mammary cancer 6 1.0 3 4.4 1.0 - -7.0 uterine cancer 7 0.5 10 7.0 1.0 - -3.5 uterine cancer 8 1.06 thirty 6.7 1.2 - -3.2 stomach cancer 9 1.06 fifteen 2.5 1.0 - -0.3 mammary cancer 10 1.06 fifty 0.4 1.9 - -4.0 mammary cancer eleven 0.8 - 1.0 1.7 200 0.00 mammary cancer 12 1.7 twenty 3.5 1.7 - -4.0 uterine cancer 13 2.3 twenty 5.0 3,5 - -0.2 esophageal carcinoma fourteen 0.82 40 0.6 fifteen 0.95 twenty 1.0 16 1.35 twenty 5.0 17 0.91 10 3.2 eighteen 1.8 fifty 2.5 19 0.8 thirty 6.4 twenty 0.8 13 0.7 21 1.0 fifty 1.5 22 1.5 45 5.0 23 0.7 8 1.7

For healthy patients and cancer patients, the values of B differ most strongly (see table). For cancer patients, B becomes negative, and the mass of scattering molecules increases. For patients with non-oncological diseases, the values of B and M differ little from the corresponding parameters for healthy patients. Figure 5 shows the histograms of the coefficient of intermolecular interaction B for healthy patients 16, risk groups 17 and cancer patients 18. The histograms for healthy patients 16 and cancer patients 18 practically do not overlap (figure 5).

The translational diffusion coefficient D _t obtained by dynamic light scattering using a Malvern correlometer for protein 19 and γ-globulin 20 depends on the surface charge of the molecule in a nonlinear manner with a minimum at the isoelectric point (Fig. 6).

The results of a clinical examination of healthy groups 21, at risk 22 and patients 23 patients according to two parameters characterizing the ratio of the concentrations of P albumin and globulin, as well as the spectrum width of albumin W are different (Fig.7).

When using the method of fluorescence analysis, a fluorophore from a number of naphthylamine sulfonic acids was used as a probe. This probe non-covalently binds to a protein; it almost does not fluoresce in water, but intensely fluoresces when it is bound to a protein molecule. The low quantum yield in the aqueous phase allows not to take into account this part of the fluorophore radiation. Anisotropy or the degree of polarization of fluorescence is also a diagnostic parameter and serves to increase the reliability of diagnosis.

Claims (11)

1. A device for the study of biological fluid containing a laser module containing a laser radiation source, an optical unit containing a cuvette assembly, at least one photodetector module and an analog-to-digital converter, the cuvette assembly being optically coupled to a photodetector module, and analog -digital converter is electrically connected to a photodetector and a computer, characterized in that the photodetector module further comprises a low noise amplifier and a nor The amplifying amplifier, electrically coupled to a low-noise amplifier, the optical unit contains an optical path forming unit and a beam splitter chevron optically coupled to a laser source and a cuvette assembly, and the laser module and photodetector module are rigidly fixed to the optical unit. 2. The device according to claim 1, characterized in that the beam splitting chevron is rigidly fixed in the optical unit. 3. The device according to claim 1, characterized in that it further comprises a control board electrically connected to the laser module and configured to set the cyclogram on and off of the laser module. 4. The device according to claim 1, characterized in that the node forming the optical path is rigidly fixed in the optical unit. 5. The device according to claim 1, characterized in that the photodetector module further comprises a forming input optics unit, optically coupled to the photodetector and rigidly fixed in this module. 6. The device according to claim 1, characterized in that the analog-to-digital Converter further comprises a filter defining a timer, a buffer memory, made on one board. 7. The device according to claim 1, characterized in that it is arranged to register a beat spectrum between Rayleigh scattered radiation of a biological fluid solution and reference deterministic laser radiation. 8. The device according to claim 7, characterized in that the laser radiation and the reference deterministic laser radiation are received from the same source. 9. The device according to claim 7, characterized in that the optical path forming unit is configured to direct the laser radiation and the deterministic reference laser radiation at an angle of 90 ° to each other. 10. The device according to claim 7, characterized in that the photodetector module is configured to detect Rayleigh scattered radiation in the direction of propagation of the deterministic reference laser radiation. 11. The device according to claim 7, characterized in that the photodetector module is configured to detect Rayleigh scattered radiation in two directions oriented at an angle of 180 ° to each other.

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