WO2022231470A1 - Procédé d'analyse microbiologique utilisant un procédé optique et analyseur microbiologique portatif - Google Patents

Procédé d'analyse microbiologique utilisant un procédé optique et analyseur microbiologique portatif Download PDF

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WO2022231470A1
WO2022231470A1 PCT/RU2022/000138 RU2022000138W WO2022231470A1 WO 2022231470 A1 WO2022231470 A1 WO 2022231470A1 RU 2022000138 W RU2022000138 W RU 2022000138W WO 2022231470 A1 WO2022231470 A1 WO 2022231470A1
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microbiological
microorganisms
medium
data
analyzer
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PCT/RU2022/000138
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English (en)
Russian (ru)
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Артем Викторович БУДАЕВ
Роман Николаевич БЕЛЕНЬКОВ
Дмитрий Николаевич УКОЛОВ
Никита Александрович ЕМЕЛЬЯНОВ
Анастасия Игоревна ЛАВРОВА
Евгений Борисович ПОСТНИКОВ
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Артем Викторович БУДАЕВ
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Priority claimed from RU2021111818A external-priority patent/RU2779840C1/ru
Application filed by Артем Викторович БУДАЕВ filed Critical Артем Викторович БУДАЕВ
Publication of WO2022231470A1 publication Critical patent/WO2022231470A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/79—Photometric titration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications

Definitions

  • the technical solution claimed as an invention relates to the field of material analysis using optical means, namely to methods and devices for microbiological analyzes and can be used as the main tool for assessing the dynamics of microbial populations in the studied environment.
  • Immunochem-2100 [url: http://www. intermedika.ru/ catalo g/katalo g/laboratomoe-oborudovanie/oborudovanie-dlia-ifa/immunochem-21001 or iMark microplate photometer [url: https://all-pribors.ru/opisanie/68941-17-mark-isp - imark-784231 - designed to measure the optical density in the wells of a standard 96-well plate during various studies (enzymatic immunoassay, biochemical studies). The principle of operation of photometers is based on measuring the optical density of analyzed samples in 96-well plates. To correct the instability of the light source, a reference light channel is used.
  • the selection of the required wavelength in the light flux is carried out using narrow-band interference filters.
  • the light flux falls through the upper lens block from above, onto the well with the measured sample.
  • the attenuated breakdown of the light flux is recorded by a photodetector located under the sample.
  • the carriage with the microplate moves relative to the light flux.
  • a common feature of the photometer with the claimed technical solution is the principle of operation based on the measurement of optical density.
  • the proposed mass-produced photometers are characterized by high cost, bulkiness (characteristic overall dimensions are several tens of centimeters; weight - from several to more than ten kilograms), the need for a permanent connection to work with a 220 V network, the need for specialized proprietary software for processing the recorded signals. .
  • the express analyzer contains a light-tight casing, a cuvette or a flow optical cell, a photodetector - a television camera, a comparison unit, a dark red programmable memory unit, an electric counter of the number of bacteria of E. coli pink, an electric counter of transparent bacteria of E. coli, an adder-calculator of the coli-index, a coli-titer calculator, display unit, digital-to-analog converter.
  • the disadvantages of the technical solution according to the patent of the Russian Federation 2103369 are the use of a bulky component-photodetector - a television camera, the ability to work with only one large-volume cuvette, i.e. incompatibility with a microbiological plate, output of measurement results in digital form for subsequent computer processing, narrow specialization of the analyzer in the wavelength ranges typical for stained E. coli bacteria.
  • a known method for assessing the threat of anemia in the third trimester of gestation according to the Russian patent for the invention 2553361 dated 06/19/2014, MGPS: G01N 33/50, published on 06/10/2015, including the determination of the titer of antibodies to cytomegalovirus, content in erythrocytes 2, 3 DPG (2,3-diphosphoglycerate), oxyhemoglobin.
  • the purpose of the development is to find and develop a new method for assessing the degree of oxygenation of hemoglobin by measuring the specific optical density.
  • the electronic structure of the molecules suggests the ability to absorb electromagnetic radiation, while the absorption spectra of hemoglobin are very characteristic, which is used to quantify the degree of saturation of hemoglobin with oxygen.
  • the absorption of light by molecules is manifested in the weakening of the light flux after it passes through the object, i.e.
  • the absorption of light by an object is the higher, the higher its density. This is derived from the Bouguer-Lamberg-Beer law and is measured by the specific optical density, which is a fairly accurate characteristic.
  • the method and device of the prototype allows you to determine the photometric parameters of cells for the diagnosis of diseases, in the description of the prototype, one of the studies with this device was carried out on patients with bronchial asthma to determine hemoglobin in peripheral blood erythrocytes.
  • the technical solution of the prototype has signs of similarity with the claimed technical solution, such as the use of optical density measurement, automated operation of the cytophotometric method created on the basis of the proposed method, individual analysis of specific microbiological samples.
  • the disadvantages of the prototype are a relatively narrow range of applications, the developed formula is intended only for the analysis of hemoglobin cells in determining the threat of anemia in the third trimester of gestation.
  • the features of the spectrophotometric method criticized in the prototype do not lead to clarity regarding the essence of the proposed evaluation method.
  • the authors of the technical solution of the prototype write in the description that with a homogeneous structure of a substance (such as hemoglobin), the light intensity at all points of the optical path is the same, but this statement is incorrect, because the intensity of the light flux when passing through the substance is the same and the intensity of absorption of light by the substance will be the same in this case.
  • the rate of change in intensity will be the same under the described conditions for the analysis of a substance such as hemoglobin. Therefore, the prototype method does not allow the measurement of optical density in terms of specific parameters of a wide range of analyzed samples.
  • the application of the method used in the prototype, in particular, is difficult in the study of bacterial cultures.
  • the claimed technical solution makes it possible to make the task of microbiological analysis universal, and its use allows the operation of a portable analyzer even in the absence of the possibility of connecting the mains power supply to the measuring device, while the application of the proposed method allows both qualitative and quantitative analysis.
  • the inventive method and device operate using the resazurin test.
  • the resazurin test is based on the ability of living cells to reduce resazurin (7-hydroxy-3H-phenoxazin-3-on-10-oxide), an indicator that has a blue color of sazurin (peak absorption 605 nm) into resorufin (7-hydroxy-3H -phenoxazin-3-one), which has a pink color (peak absorption 573 nm).
  • the resazurin test in the study of pathogenic microorganisms requires regular movement of the microbiological plate from the premises in the biological hazard zone, in which direct growing crops in conditions of high temperature and humidity, to the location of the spectro- or photometer, which leads to an increased risk of contamination.
  • Examples of such processes are described in: Palomino, J. C., Martin, A., Camacho, M., Guerra, H., Swings, J., Portaels, F. 2002. V. 46. P. 2720-2722.
  • the proposed technical solution is designed to solve the above problems.
  • the purpose of the development of the proposed technical solution is to automate the process of microbiological analysis based on the optical method, creation of a portable device for microbiological study of the growth of microorganisms under conditions close to real medical and diagnostic processes.
  • the technical task of the claimed invention is to develop the optimal parameters of an effective method and a portable autonomous device that will allow microbiological studies of the vital activity and growth of microorganisms based on obtaining quantitative characteristics of changes in the optical density of the studied cultural medium containing the indicator due to a change in its color.
  • the technical result of the claimed invention is the practical application of the method of microbiological analysis based on the optical method with a qualitative and quantitative assessment of the dynamics of populations of microbiological cultures; calculation and visualization, based on the data obtained, of the population growth curve; providing the possibility of quantitative assessment of the dynamics of populations of microbiological cultures using a reductase test with resazurin, including in the absence of the possibility of connecting the mains power supply to the measuring device, as well as in the presence of weight and size restrictions to it.
  • the method of microbiological analysis based on the optical method with a qualitative and quantitative assessment of the dynamics of populations of microbiological cultures includes connecting a power source to to the control unit, setting the operating mode, transmitting a signal to turn on / off the lighting unit, emitting the initial light flux, assessing the light flux passed through the microbiological plate with samples of the medium under study, while the lighting unit generates a light flux in the emission band of 600-605 nm, while the samples of the studied medium contain microorganisms and resazurin solution, and the microbiological plate with samples of the studied medium is placed in the analyzer and, using the elements of interaction with the operator, the operating modes of the device are set, namely, the duration of the experiment, the frequency of switching on the analyzing elements per unit time, the type of microorganisms under study, the number N active cells, in which the samples of the studied medium are placed, and after setting all the initial conditions, the process of measuring the optical density of the
  • a portable microbiological analyzer includes a lighting unit connected to each other in the form of a printed circuit board with LEDs into N cells, divided into independent channels, placed between the lighting and analyzing units, a microbiological tablet with samples of the studied environment, as well as an analyzing node with sensors for estimating illumination, converting the level of transmitted radiation into a digital signal using an analog-to-digital converter that transmits the result obtained from N sensors for estimating illumination, switched into one digital signal line, to the control node that selects the active channel ; the analyzer also includes an autonomous power supply unit that provides an autonomous operation mode of the microbiological analyzer when it is disconnected from the power source, as well as elements of interaction with the operator and external data transmission interfaces, moreover, these functional elements are implemented in the format of printed circuit assemblies on which a control microcontroller, a source and optical radiation receiver, batteries, active and passive radio-electronic products.
  • the inventive method is illustrated by the graph of figure 1, which shows the dependence of the population growth curve on time. Shown in blue is the processed signal of the analyzer and in black is the smoothed population growth curve according to the Verhulst model.
  • Figure 2 shows a block diagram of the operation of the proposed device, where:
  • the proposed technical solution was tested in laboratory conditions.
  • the claimed device was used to determine the dynamics of the population of microorganisms (yeast Saccharomyces cerevisiae and lactobacilli Lactobacillus acidophilus) on the basis of obtaining quantitative characteristics of changes in optical density (and as a result of color) of a solution containing an indicator introduced into the culture medium.
  • a nutrient medium for yeast a glucose solution was used with the addition of an indicator (a working solution of resazurin-sodium salt obtained in accordance with GOST 32901-2014 Milk and dairy products. Methods of microbiological analysis). After that, the resulting solution was introduced into the cuvette cells and placed in a portable microbiological analyzer.
  • the photodetectors of the microbiological analyzer recorded the change in the light flux passing through the cells of the microcuvette during a time exceeding by several times the characteristic period of cell division of the studied microorganisms. Due to the fact that the spectral characteristic of the lighting LEDs is consistent with the absorption band in the visible light of the indicator solution during the transition of resazurin to resorufin during the life of growing and multiplying microorganisms, accompanied by a change in the color of the solution from blue to crimson, light sensors registered a decrease in voltage proportional to an increase in their illumination.
  • the resulting curves have a sigmoidal shape, which is typical for the growth curve of the density of the population of microorganisms, since the maximum value of the illumination of the sensors corresponds to the maximum number of microorganisms in the solution. This fact indicates a qualitative agreement between the obtained photometric curve and a mathematical model of the response of a biochemical process in an indicator medium to the growth dynamics of a population of microorganisms.
  • Quantitative confirmation is based on the following operations: based on the data on the change in the intensity of the light flux during the change in the population of microorganisms obtained from the analyzing unit of a portable microbiological analyzer, they are averaged over a set of curves from each of the cells of the microbiological plate, followed by processing the sample over time with a sliding filter with the purpose of removing noise components; the resulting smoothed curve is reduced to a form typical of the population growth curve; the resulting curve is regressed using functions typical of microbiological growth models (Verhulst, Gomperz models) and the corresponding model parameters corresponding to the cell division time are determined (Figure 1). It is shown that the found values are consistent with the values typical for the studied microorganisms, which is a quantitative confirmation of the adequacy of the method for studying biophysical processes for these objects.
  • the parameters of population growth found in this way make it possible to obtain the magnitude of the quantitative increase in the number of microorganism in relative units (under the condition of the introduced normalization of their maximum (stationary) number per unit volume). If necessary, the absolute value is determined by using a normalization factor found on the basis of a calibration curve generated independently by optical microscopy and/or microtitering of a sample sample from the cells of a microbiological plate.
  • a portable microbiological analyzer is implemented in the form of printed circuit assemblies on which radio-electronic products are installed and includes: lighting assembly 1; node analyzing 2; node control 3; node autonomous power supply 4; power supply 5; elements of interaction with the operator 6; external data transfer interfaces 7 and a microbiological plate with samples of the studied medium with microorganisms and resazurin solution 8.
  • Portable microbiological analyzer works as follows.
  • the user starts the measurement process.
  • any sources / receivers of visual, sound information, as well as electrical and radio frequency signals can act.
  • Node lighting 1 generates a luminous flux in the emission band 600-605 nm.
  • This element of the analyzer is implemented in the format of a printed circuit board with LEDs for N cells, divided into independent channels.
  • This node is controlled by a digital signal (coming from control node 3), which turns on / off the power of the node and selects the active channel.
  • Microbiological plate 8 placed between the lighting unit 1 and the analyzing unit 2 with samples of the studied medium with microorganisms and a solution of resazurin partially absorbs this radiation, and the intensity of absorption is directly related to the concentration of resazurin in the solution and, as a consequence, the dynamics of the population of microorganisms contained in the medium.
  • the transmitted radiation hits the sensors for illuminance evaluation placed on the analyzer node 2, which convert the level of the transmitted radiation into digital signal using an ADC (analogue-to-digital converter). Further, the result obtained from N sensors for estimating illumination is switched into one digital signal line and transmitted to control unit 3. Any hardware and software for estimating illumination can be used as sensors.
  • the illumination values received via the data transmission channel from the sensors for assessing the illumination are recorded in the internal memory of the microcontroller, which processes them and, based on the calibration data, determines the number of microorganisms contained in the medium under study.
  • the microcontroller transmits the result obtained via external data transfer interfaces 7. Any hardware-software means of data transfer, storage and visualization can act as these interfaces.
  • control node 3 performs the following functions: stabilizes the input DC voltage coming from the autonomous power supply node 4 and power source 5 to the power line for the operation of all functional nodes; processes information received from the user through the elements of interaction with the operator 6; coordinates the work of all functional units of the device; protects the input power circuits from incorrectly switching on the polarity of the supply voltage and disconnects the autonomous power supply unit 4 from the load if its batteries are too discharged.
  • the autonomous energy saving unit 4 ensures the operation of the device when it is disconnected from the AC voltage network for the time necessary for the experiment.
  • the battery cells of this unit are charged automatically using the power source 5.
  • a microbiological plate with samples of the studied medium with microorganisms and a solution of resazurin 8 is not an integral part of a portable microbiological analyzer.
  • any standard microbiological tablet As a container for the cultivation of microorganisms, any standard microbiological tablet. Therefore, microorganisms whose respiratory chain metabolites enter into redox reactions with resazurin during their vital activity can act as biological objects under study.
  • Approbation showed that the proposed device allowed to obtain quantitative estimates of the dynamics of populations of microbiological cultures at a parity level with modern professional microbiological analyzers and, at the same time, with smaller weight and size characteristics. Also, due to the fact that the claimed device is portable and self-contained, independent of the removal and output of a photometric signal from each cuvette cell, the use of the proposed method and device is feasible in practice for the widest range of microbiological measurements.
  • the claimed technical solution can find wide practical application not only in the food, chemical, flour and cereal, feed and medical industries, but also in microbiological laboratories of environmental analysis and calculation centers, specialized universities and medical institutions.

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Abstract

L'invention se rapporte au domaine de l'analyse des matériaux à l'aide de moyens optiques, concerne notamment des procédés et des dispositifs d'analyses microbiologiques, et peut être utilisée comme instrument principal pour évaluer la dynamique de populations de micro-organismes dans un milieu étudié. Le but de la présente invention est l'automatisation du processus d'analyse microbiologique sur la base d'un procédé optique, la création d'un instrument portatif pour l'étude microbiologique de la croissance de micro-organismes dans des conditions proches des processus de thérapie et de diagnostic réels. Le but de la présente invention est d'élaborer des paramètres optimaux d'un procédé efficace et un dispositif portatif autonome qui permettent d'effectuer des recherches microbiologiques du processus biologique et de la croissance de micro-organismes sur la base de l'obtention de caractéristiques quantitatives de changement de la densité optique d'une solution (suite un changement de sa teinte) contenant un indicateur ajouté dans le milieu de culture. Ce but et cette solution technique sont atteints grâce à un procédé d'analyse optique microbiologique comprenant une estimation qualitative et quantitative de la dynamique des populations de cultures microbiologiques, lequel consiste à: connecter une source d'alimentation à une unité de commande, établir un mode de fonctionnement du dispositif, transmettre un signal d'activation/désactivation d'une unité d'éclairage, émettre un flux lumineux initial, estimer le flux lumineux qui a traversé une plaque de microbiologique avec des échantillons du milieu étudié; l'unité d'éclairage génère un flux lumineux dans une plage de rayonnement de 600-605 nm, et les échantillons du milieu étudié comprennent des micro-organismes et une solution de résazurine; la plaque microbiologique est disposée dans un analyseur et, à l'aide d'éléments d'interaction avec l'opérateur, on définit les modes de fonctionnement du dispositif; on transmet ensuite les données obtenues lors de l'estimation sur un canal de transmission de données vers l'unité de commande qui va les traiter et, sur la base de données d'étalonnage, va calculer la densité optique du milieu et convertir la densité optique du milieu en nombre de micro-organismes qu'il contient; la mesure quantitative se fait sur la base de données sur le changement de l'intensité du flux lumineux au cours du changement du nombre de micro-organismes dans le milieu, ceci en établissant leur moyenne sur un ensemble de courbes depuis chacune des cellules de la plaque microbiologique, après quoi la courbe lissée est mise sous une forme typique pour une courbe de croissance de population permettant de déterminer des paramètres caractérisant la valeur de temps de division cellulaire. Ce dispositif d'analyseur microbiologique portatif comprend, connectés entre eux par un montage câblé, une unité d'éclairage au format d'une plaque d'impression avec des diodes et N cellules divisées en canaux indépendants, une plaque microbiologique disposée entre les unités d'éclairage et d'analyse et comportant des échantillons de milieu à étudier, ainsi qu'une unité d'analyse avec des capteurs pour estimer la luminosité qui convertissent le niveau de rayonnement qui est passé en un signal numérique à l'aide d'un convertisseur analogique-numérique transmettant le résultat reçu des N capteurs d'estimation de luminosité vers l'unité de commande; l'analyseur comprend également une unité d'alimentation électrique autonome assurant un mode de fonctionnement autonome de l'analyseur microbiologique portatif lors de la coupure de la source d'alimentation, ainsi que des éléments d'interaction avec l'opérateur et des interfaces externes de transmission de données; les données des éléments fonctionnels sont éditées dans le format d'unités d'impression sur lesquelles sont installés un micro-contrôleur de commande, une source et un récepteur de rayonnement optique, des accumulateurs, ainsi que des appareils radio-électroniques actifs et passifs.
PCT/RU2022/000138 2021-04-26 2022-04-25 Procédé d'analyse microbiologique utilisant un procédé optique et analyseur microbiologique portatif WO2022231470A1 (fr)

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RU2021111818 2021-04-26
RU2021111818A RU2779840C1 (ru) 2021-04-26 Способ микробиологического анализа на основе оптического метода и портативный микробиологический анализатор

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RU2103369C1 (ru) * 1996-05-06 1998-01-27 Виктор Васильевич Хохлов Экспресс-анализатор концентрации бактерий в водной среде
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2103369C1 (ru) * 1996-05-06 1998-01-27 Виктор Васильевич Хохлов Экспресс-анализатор концентрации бактерий в водной среде
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