RU2672534C1 - Optical method of measurement of concentration and morphology of particles in wide range of turbidity and device for its implementation - Google Patents

Abstract

FIELD: measuring equipment.SUBSTANCE: invention relates to the field of measurement technology, allowing measurement of concentrations of suspended particles in a liquid, and more specifically to coherent fluctuation nephelometers (hereinafter CFN), in particular microbiological analyzers designed on their basis, which allow to measure the growth of microbiological flora including in biological samples in a wide range of turbidity values, including for the purpose of diagnosis, and to evaluate the morphological composition of suspended microbial particles. Claimed method and apparatus allow measuring the concentrations and morphology of suspended particles in a liquid in a wide range of concentrations and sizes due to the simultaneous recording of the intensity of the direct transmitted radiation (T) and fluctuations in the intensity of the scattered light (FISL) at two or more scattering angles, calculation of the linear combination of the initial FISL signals at different angles and the signal of the turbidimeter (T), preferably to a power of 2 to 4 with different weighting factors, which are determined by calibration for a suspension of each type of analyte. According to the calculated linear combination of signals, the particle concentration is judged, the FIRS signals at different angles judge the particle morphology in the sample, which makes it possible to obtain a monotonic dependence of the resulting signal over a wide range of concentrations (10–10CFU/ml) and in a wide range of sizes (10 nm – 100 mcm). Also, the ratio of FISL signals at different angles makes it possible to evaluate the morphology of the particles under study.EFFECT: expansion of the dynamic range of the measured turbidity and evaluation of the morphological composition of particles in the sample under study.6 cl, 7 dwg

Description

The invention relates to the field of measuring technology, which allows to measure the concentration of particles suspended in a liquid, and more specifically to coherent fluctuation nephelometers (hereinafter - FSC), in particular to microbiological analyzers designed on their basis, which allows to measure the growth of microbiological flora, including in biological samples in a wide range of turbidity values, including for the purpose of diagnosis and to evaluate the morphological composition of suspended microbial particles.

The invention also relates to the creation of immunochemical and hematological analyzers, the use of which involves obtaining information not only about the absolute concentration values in a wide range of values, but also about the morphological parameters of the light scattering particles, in particular about changes in these parameters both at small and at high concentrations particles. An example of such biomedical applications is the registration of immunological reactions associated with aggregation and disaggregation of particles, for example, in latex agglutination, transformation or cell lysis processes.

There are three main methods for measuring the concentration of particles suspended in a liquid by detecting the turbidity of the suspension: turbidimetry (hereinafter - T), nephelometry (hereinafter - H) and CFN. All these methods are based on a general principle, namely, recording the intensity and / or intensity fluctuations of transmitted or scattered light detected by one or more detectors during elastic scattering of light by particles in the test fluid.

In T, the intensity of light transmitted through the test sample is measured; however, such a measurement is characterized by low sensitivity.

The main disadvantage of H is that in order to achieve high sensitivity it is necessary to exclude the illumination on the detector from the optical elements and the transmitted beam. That is why in multi-angle H used to measure the distribution of the intensity of scattered light over the corners, the transition to small angles is associated with solving the problem of cutting off the flare, which is possible only at a large distance from the cuvette to the detector and high quality cuvettes and other elements of the optical path. Therefore, multi-angle H cannot be used to measure concentrations in a wide range of values, are structurally complex, and therefore multichannel systems cannot be built on their basis. [Clin Chem Lab Med. 2012 Jul; 50 (7): 1253-62. Low angle light scattering analysis: a novel quantitative method for functional characterization of human and murine platelet receptors. Mindukshev I et al.]

The dynamic ranges of turbidity measurements for each of these methods are different. Specifically, to measure the concentration of bacteria in colony forming units (CFU hereinafter) these methods have respective dynamic ranges of measurement: for T is 10 ⁷ -10 ¹⁰ cfu / ml for H is 10 ⁵ -10 ⁸ CFU / ml, and for this KNF 10 ³ -10 ⁸ CFU / ml. It is important that in many applications the measurement of the concentrations and morphology of particles suspended in liquids is often necessary in a wide range of turbidity, as well as in a wide range of morphological parameters of suspended particles.

Often, the measurement process has to be interrupted due to the need to switch to an optical scheme with a reduced or increased optical path length of the probe beam, which is achieved, for example, by changing the geometric size of the optical cell, which is difficult in analytical processes in laboratory diagnostics.

Another widely used analytical approach that provides the measurement of suspension concentration in a wide range of turbidity is the dilution of the suspension in a given optical cuvette in a given optical device. However, in the study of complex processes, the indicated dilution or alteration of the size of the cuvette influence the course of the investigated processes themselves, the effectiveness of which (for example, flow rate) depends on the concentration. In addition, the system under investigation is often unique and difficult to reproduce, and any interference with the observed process leads to its destruction. In addition, in microbiological studies, additional intervention can lead to contamination by foreign microorganisms and to distort the results. Thus, the expansion of the dynamic range of measured concentrations under given observation conditions of the studied multi-parameter systems is undoubtedly relevant.

The measurement of light scattered at small angles by the H and QPS methods, being more sensitive, has the disadvantage of not being able to measure turbidity at high concentrations of scattering particles, because the intensity of the recorded signal with increasing concentration ceases to depend on the concentration of scattering particles due to the multiple scattering effect, and with a further increase in concentration, the signal begins to decrease. Those. the dependence of the recorded signal for H and QPS is nonmonotonic in a wide range of angles. Method T, which reduces to measuring the intensity of the non-scattered part of the probe light beam, allows one to detect high turbidity in a monotonic mode, however, it is not sensitive to low concentrations of scattering particles, i.e. its signal practically does not change in this concentration range.

Known devices in which, including with the aim of expanding the dynamic range of turbidity measurements, measurements with H and T are combined in one device. An example is the IMMAGE 800 specific protein analyzer (Beckman Coulter), in which simultaneously two separate detectors record H signals at an angle of 90 degrees and T. Such devices are convenient, for example, for recording the concentration of protein colloidal solutions and nanometer-sized particles, because . such particles, having a size much smaller than the length of visible light, scatter light almost uniformly at all angles, and the nephelometric registration of scattered light at a large angle makes it possible to reliably separate the light scattered by these particles from spurious illumination caused by scattering of the direct beam primarily from the inhomogeneities of the optical cell.

At the same time, the registration of turbidity by these devices in a wide dynamic range due to large particles, for example, bacteria having a size comparable to the length of visible light, becomes less effective, because large particles scatter light primarily forward and H baud detection at large angles becomes ineffective. And the combination of T and H at small angles is extremely difficult. However, the registration of turbidity for particles whose sizes are larger or comparable with the wavelength of the scattering light is of great practical interest.

Known microbiological analyzer HB&L (Alifax S.p.a.), in which, to increase the efficiency of H, the measurement of turbidity caused by a bacterial culture, scattered light intensity is recorded at several angles, which allows more reliable detection of bacterial culture growth. Meanwhile, the dynamic range of measured concentrations remains within the framework of the above boundaries inherent to the H method. Ultimately, this leads, among other things, to the fact that contamination and cell defects can affect both signals differently, which leads to a deterioration in the reproducibility of measurements.

Thus, the disadvantages of the methods considered are either insufficient sensitivity, including due to sensitivity to spurious illumination, and / or a limited dynamic range of different recording channels, which leads to the need to use various instruments and measurement conditions if it is necessary to measure turbidity in a wide range of values .

However, there are tasks in which it is necessary to observe the processes occurring with a change in turbidity over a wide range of values, for example, when recording the growth curves of microorganisms in a nutrient medium, including antibiotics, which is implemented, for example, in biotechnological and diagnostic systems related with the growth of microflora, as well as, for example, when registering latex aggregation or disaggregation involving particles with a size comparable to or greater than the length of the probe light.

None of the methods considered makes it possible to combine the registration of turbidity (i.e., the intensity of scattered and / or transmitted light) and the morphology of particles in the processes of aggregation, disaggregation, division, lysis, etc., the registration of which is complicated by the varying polydispersity of the studied systems. The ability to control the turbidity and morphology of particles simultaneously in a wide range of turbidity and morphology expands the possibilities for studying and identifying complex structures in liquids, especially living systems.

A known method of measuring particle concentration in a wide range of turbidity [Scientific journal: Instruments and experimental equipment. Related scientific article: “Coherent fluctuation nephelometry: A highly sensitive method for detecting particles in a liquid”. The author Rastopov SF], in which a probe beam of coherent radiation passes through a cell with the sample under study, after which the intensities of the direct transmitted radiation and scattered radiation are recorded using photodetectors (FPU), while the intensity of the direct transmitted through the cell with the studied sample is measured radiation T (turbidimetry) and fluctuations in the intensity of scattered light FIRS at two scattering angles. This method allows to achieve measurement sensitivities up to 103-104 particles / ml with a fairly simple device design.

The disadvantage of the prototype is that this method of detecting micron and submicron particles in a liquid is based on measuring fluctuations in the intensity of coherent radiation scattered on the particles and does not allow to evaluate the morphology of the particles. Also in the known method there is no possibility of measuring the frequency characteristics of the FIRS.

Closest to the claimed method is the analyzer [DOI: http://dx.doi.org/10.15789/2220-7619-2016-4-395-398], which simultaneously uses one channel of registration of the FSC and one channel T to expand the dynamic range, and the viewing angle is chosen optimal for bacterial cells with a size of about 1 μm and the selected concentration range (10 ⁴ -10 ⁸ CFU / ml for CFN) about 7 degrees.

The disadvantages of the prototype are the following:

- the registration of signals in the prototype is nonmonotonic in a wide dynamic range of the measured turbidity and morphological parameters of the particles, i.e. particle sizes that can be analyzed are limited.

- the impossibility of recording scattered light at different angles, which is necessary to optimize the detection of particles in a wide range of sizes;

- the prototype also does not allow to evaluate the morphology of the particles, which is important, for example, for the aggregation of particles and changes in the structure of blood cells.

- it is also difficult to separate the change in the signals of the QPS and T caused by either a change in the number of particles or a change in their morphology.

The aim of the invention is to expand the dynamic range of the measured turbidity and assess the morphological composition of the particles in the test sample, which is achieved by simultaneously registering T, fluctuations in the scattered light intensity (FIRS) at different scattering angles (at least two), calculating a linear combination of the initial FIRS signals at different scattering angles and the signal of the turbidimeter T, preferably to a degree of 2 to 4 with different weights, which are determined by calibration for the suspension uu each type of particle being analyzed and calculated by a linear combination of signals is judged on particle concentration ratio PIS signal at different angles are judged on the particles morphology in the sample. The dependence of such a resulting signal on the concentration of particles is monotonous in a wide range of concentrations and morphologies of particles. Registration of FIRS signals simultaneously at several angles allows detecting particles of various sizes with maximum sensitivity. Registration of the FIRS signal ratio allows one to evaluate the particle morphology, for example, in particle aggregation processes.

Additionally, the frequency spectra are measured, the FIRS frequency spectra are preferably in the band of more than 1 Hz and the frequency spectra of T are preferably in the band of less than 1 Hz in order to separate the changes in the morphology of the particles from the changes in their number, as well as to detect the effect of large polluting particles on the resulting signal. The indicated frequency limits are optimized based on experimental data.

The device according to the claimed method (implementation example) contains a coherent radiation source made in the form of a laser, a cuvette with a test sample and a direct transmitted and scattered radiation intensity recorder made in the form of photodetector devices (FPU), characterized in that three or more FPU are located on different angles relative to the axis of the laser beam, the first FPU is located on the axis of the radiation beam (zero angle), and its output is connected to the input of the intensity meter, the outputs of the remaining FPU are connected with inputs meters intensity fluctuations across the constant component of the signal cancellers, the output intensity and output intensity fluctuations meter gauges are connected to a computing device which calculates a linear combination of the PIS signal and the signal T is preferably a degree of from 2 to 4.

Also, in order to separate the changes in the morphology of particles from changes in their number, as well as to detect the effect of large polluting particles on the resulting signal, the device further includes a frequency spectrometer T, preferably in the band of less than 1 Hz and a FIRS frequency spectrometer in the band of more than 1 Hz.

Also, in order to increase the resistance to fluctuations in the intensity of the radiation source, FPUs that register FIRS are preferably made in the form of two photosensitive elements (PSEs) for each scattering angle located symmetrically relative to the axis of the laser beam, and the outputs of these PSEs are connected to different inputs of the corresponding differential amplifiers.

Also for detecting particles of various sizes in order to optimize the detection angles, the cuvette and / or FPU are arranged to move along the axis of the radiation beam.

The invention is illustrated by seven figures.

In FIG. 1 presents a block diagram of a device according to the claimed method, where 1 is a laser; 2 - a cuvette with a test sample; 3 - axis of the laser beam, 4 - FPU signal T; 5, 6 - FPU signals FIRS; 7 - computing device (WU); 8 - signal intensity meter T; 9, 10 - signal intensity meters FIRS; 11, 12 - suppressors of the constant component of the signals.

In FIG. 2 shows a block diagram of the subtraction of FIRS signals in a given scattering angle (top view of the device), where 5a and 5c are various photosensitive elements (PSE) of the FPU.

In FIG. 3 presents experimental data on the growth of bacteria E. coli, where 13 is the signal T; 14 - signal FIRS in one angle of scattering; 15 - the resulting signal according to the formula (1) below for this angle.

In FIG. Figure 4 schematically shows the resulting signals (1) at different scattering angles without changing the particle size during growth (solid lines) and during their aggregation (dashed lines).

In FIG. 5 presents experimental data on the registration of growth and “clumping” of bacteria Staphylococcus aureus, 16 — signal T; 17 - signal FIRS at one angle of scattering.

In FIG. Figure 6 shows the experimental FIRS frequency spectra at one scattering angle for measuring the turbidity of a suspension of particles without pollution (left) and when a large foreign particle passes through a radiation beam (right).

In FIG. Figure 7 shows the experimental FIRS signals at four different scattering angles for a suspension of red blood cells that settle and aggregate over time.

The essence of the proposed method for measuring the concentration and morphology of particles in a wide range of turbidity consists in passing a probe beam of coherent radiation through a cuvette with the sample to be studied, recording the intensities of the direct transmitted and scattered radiation using photodetector devices (FPU), while simultaneously measuring the intensity of the transmitted through the cuvette with the studied light sample T and FIRS at several scattering angles. By the ratio of the intensities of the FIRS at different angles, one can judge the sizes of the particles under study by the scattering indicatrix. For example, with an increase in particle size over time (agglutination), the scattered light is concentrated mainly at smaller angles, which is determined by the ratio of signals at different angles.

To ensure the monotonicity of the output signal, a linear combination of the initial FIRS signals at various angles and the turbidimeter signal T, taken to the power from 2 to 4, with different weighting coefficients, which are determined by calibration for suspensions of the analyzed particles of each type, is calculated.

The initial signals of the FIRS at different angles and T can be summarized by the formula:

- среднеквадратичное отклонение сигнала ФИРС на i-м ФПУ, I_T - интенсивность сигала Т, α_i - весовые коэффициенты, k - степенной коэффициент, находящийся в диапазоне значений от 2 до 4.Where,

is the standard deviation of the FIRS signal at the ith FPU, I _T is the signal strength T, α _i are weight coefficients, k is a power coefficient, which is in the range from 2 to 4.

Thus, the generated combination of signals allows you to expand the dynamic range of the measured concentrations due to the fact that the resulting signal monotonically depends on the concentration of particles. This summation of the FIRS signals also leads to an increase in the sensitivity of measurements at low concentrations. In addition, the concentration of scattering particles of a given type is judged by the resulting signal, and the particle sizes are judged by the ratio of the FIRS signals at different angles.

To determine the absolute concentration of particles, a preliminary calibration of the resulting signal from the concentration is carried out for specific sample particles of different morphology.

In a real sample, in addition to the particles under investigation, various contaminants of a relatively large size can be found, as well as air bubbles that distort the T and FIRS signals. To reduce the influence of such foreign particles on the measured concentration of the studied particles, the frequency spectra of the signals are also calculated. Registration of the FIRS signal spectra allows to increase the reliability and accuracy of measurement. For example, when a beam is crossed by large-scale contaminants, or when microbubbles of air form on the walls of a cell, the standard form of the Fourier spectrum of the FIRS signal is violated. It has been experimentally shown that such violations for T are preferably manifested at frequencies less than 1 Hz, and for FIRS, preferably at frequencies greater than 1 Hz. Moreover, as a rule, in these cases, the signal from not all FPUs located at different angles is disturbed. In such measurements, signals with a disturbed Fourier spectrum can be excluded from the resulting signal, thereby reducing the effect of contaminants and bubbles. In addition, in some processes, for example, when the growth of bacteria Staphylococcus aureus is registered, the total number of bacteria continuously increases, however, the signals of FIRS and T begin to decrease. This is due to the massive aggregation of bacteria known as "clamping." An increase in the low-frequency fluctuations of the signal T allows one to register this process and distinguish it from the death of a bacterial culture, which also leads to a decrease in the FIRS and T signals, but without an increase in the low-frequency fluctuations of the signal T. Thus, the proposed method and device allows collecting multi-parameter information about the processes taking place in the investigated suspension of particles. This information is related to the number of particles (which is determined by the FIRS and T signals), their morphology (which is determined by the ratio of the FIRS signals at different angles), and the dynamics of particle motion in the cell (which is determined by the Fourier spectral distributions of the FIRS and T signal). The indicated multiparametricity makes it possible to control the processes occurring in the studied suspension of particles in the recognition format of patterns formed by the neural network and allowing to identify the type of particles involved in the studied processes and their number.

The device according to the method is implemented as follows (example of a specific preferred implementation, Fig. 1). The device consists of a coherent radiation source made in the form of a laser, 1, a cuvette with the studied sample 2, located on the axis of the laser beam 3, several photodetector devices (FPU) for recording the T 4 signal and FIRS 5, 6 signals (the first two, the total number (typically 8) depends on the necessary detail of measurements at various angles), and the computing device (WU) 7. The output of the FPU 4 is connected to the input of the intensity meter 8 (for example, an operational amplifier (OS)), and the outputs of the remaining FPU ( 5 and 6) connected s with the inputs of intensity fluctuation meters 9 and 10 (also opamps) through the suppressors of the constant component of the signal 11 and 12 (in the simplest case, these are just isolation capacitors). The outputs of all op-amps are connected to the inputs of the VU 7. As a VU, a multi-channel analog-to-digital converter (ADC) and a computer or microcontroller can be used to perform the necessary calculations, measure the signal spectra and display the measurement results.

To increase the sensitivity of measurements of FIRS signals, in particular, to subtract fluctuations in the radiation intensity of the source, the corresponding FPUs can be made in the form of a pair of photosensitive elements (PSE), for example, 5a and 5c (Fig. 2, top view of the device), the outputs of which are connected to various the inputs of op-amp 9. In this case, the common-mode signals of the PSE are subtracted, and the FIRS signals are summed with random phases, since these phases are independent at each PSF, which leads to an increase of approximately 1.4 times the resulting FIRS signal at the output of the op-amp 9.

Since it is preferable to use different scattering angles for particles of different sizes, to change the recorded scattering angles of cell 2 and / or all the FPUs, you can fix it with the possibility of movement along the axis of the radiation beam, i.e. with the possibility of changing the distance from the cell 2 to the FPU 5, 6, 7. In this case, the range of recorded angles varies with the FPU being unchanged relative to each other, and the process conditions in the system under study are preserved.

As an illustration of the implementation of the method and device of FIG. Figure 3 shows the experimental data on the registration of growth curves of E. coli bacteria in a concentration range of 10 ³ -10 ¹⁰ CFU / ml, where 13 is the change in the T signal, 14 is the change in the FIRS signal (for one scattering angle, about 5 degrees), 15 is the resulting signal for one set of weights and degree 2 of the T signal in the measured signal.

With multi-angle recording, the larger the scattering angle, the smaller the corresponding signal 15, while with a constant morphology (particle size), the relative difference of the signals at different angles does not change with time, which is shown schematically in Fig. 4 (solid curves), however, if, for example, particle aggregation occurs during growth (aggregation into large aggregates), this is manifested in an increase in the relative difference of signals from time (dashed curves).

In microbiological practice, the growth curves of microorganisms are recorded in a liquid nutrient broth. For example, bacterial growth occurs due to the division of bacteria, thereby their number increases with time. Moreover, if bacteria do not aggregate, then the turbidity of the suspension will also increase with time. Due to certain circumstances, for example, when antibacterial drugs are added to a liquid, bacteria may begin to break down, in which case the turbidity of the suspension will begin to decrease. However, if the bacteria begin to aggregate, while continuing to multiply, the turbidity of the suspension will drop, and this can be mistakenly interpreted as destruction of bacteria by both the FIRS signal and T. The appearance of relatively large bacterial aggregates leads to the appearance of low-frequency fluctuations T by which it is possible to distinguish the death of bacteria from their aggregation. In FIG. Figure 5 presents experimental data on the growth and “clumping” of Staphylococcus aureus bacteria, when bacterial aggregation leads to a false decrease in the FIRS and T signals, and at the same time leads to the appearance of low-frequency fluctuations of the T signal, 16 - T signal; 17 - signal FIRS at one angle of scattering.

When large contaminants pass through the radiation beam, the typical shape of the frequency spectrum of the FIRS signals is distorted. In FIG. Figure 6 shows the experimental FIRS frequency spectra at one scattering angle for measuring the turbidity of a suspension of particles without pollution (6a) and when a large foreign particle passes through a radiation beam (6b). Registration of the FIRS frequency spectrum allows you to ignore the effect of large contaminants on the resulting signal.

In FIG. 7 shows FIRS signals at 4 different scattering angles (18-21, angles increase with increasing number) for a suspension of red blood cells. Cells settle and aggregate over time, the dynamics of the FIRS signals at different angles is different, their ratio can be used for calibration by the size of the aggregates of the studied cells.

Claims (6)

1. An optical method for measuring the concentration and morphology of particles in a wide range of turbidity, including the passage of a probe beam of coherent radiation through a cuvette with the studied sample, recording the intensities of the direct transmitted and scattered radiation using photodetectors (FPU), characterized in that they simultaneously record the intensity of the direct transmitted radiation (T) and fluctuations in the intensity of the scattered light (FIRS) at two or more scattering angles, calculate a linear combination of the source signals FIRS at different angles and a turbidimeter signal T, preferably from 2 to 4, with different weights, which are determined by calibration for a suspension of the analyzed particles of each type, according to the calculated linear combination of signals, the concentration of particles is judged by the ratio of the FIRS signals at different angles morphology of particles in the sample. 2. The method according to p. 1, characterized in that it further measure the frequency spectra of the FIRS preferably in the band of more than 1 Hz and the frequency spectra of T, preferably in the band of less than 1 Hz. 3. A device for measuring the concentration and morphology of particles in a wide range of turbidity, including a source of coherent radiation, made in the form of a laser, a cuvette with an investigated sample and a recorder of intensities of direct transmitted and scattered radiation, made in the form of photodetectors (FPU), characterized in that three or more FPUs are located at different angles relative to the axis of the laser beam, the first FPU is located on the axis of the radiation beam (zero angle), and its output is connected to the input of the intensity meter vnosti, the FPU outputs remaining connected to inputs meters intensity fluctuations across the constant component of the signal cancellers, the output intensity meter and outputs intensity fluctuations gauges are connected to a computing device which calculates a linear combination of the PIS signal and T signal to the power of 2 to 4. 4. The device according to p. 3, characterized in that the computing device further includes a frequency spectral meter T, preferably in the band of less than 1 Hz and a FIRS frequency spectral meter, preferably in the band of more than 1 Hz. 5. The device according to p. 3, characterized in that the FPU detecting the FIRS are preferably made in the form of two photosensitive elements (PSEs) for each scattering angle located symmetrically relative to the axis of the laser beam, and the outputs of these PSUs are connected to different inputs of the respective differential amplifiers . 6. The device according to p. 3, characterized in that the cuvette and / or FPU are arranged to move along the axis of the radiation beam.

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