WO2022164341A1

WO2022164341A1 - Method for detecting antibiotics in raw milk

Info

Publication number: WO2022164341A1
Application number: PCT/RU2021/050273
Authority: WO
Inventors: Игорь Сан-Сенович ДЮ
Original assignee: Игорь Сан-Сенович ДЮ
Priority date: 2021-01-26
Filing date: 2021-08-20
Publication date: 2022-08-04
Also published as: RU2757226C1

Abstract

The invention relates to the food industry. Proposed is a method for detecting antibiotics in raw milk, which includes taking samples of raw milk; preparing a buffer solution having a pH selected from within the range of 4.0 – 8.0; applying said buffer solution to electrodes of electrochemical sensors; measuring the samples of raw milk using a sensor platform consisting of a test strip and a potentiostat, where the test strip with electrodes is an electrode array, wherein a selective layer is applied to the surface of the electrodes by chemical modification of said surface using polyelectrolytes, an aptamer is used as a selective element, and the potentiostat comprises modules for applying and removing an electrical signal and modules for converting and transmitting a signal; and processing and analysing the electrical signals obtained. The invention provides a quick and accurate method for detecting the presence of antibiotics in raw milk.

Description

METHOD FOR DETERMINING ANTIBIOTICS IN RAW MILK

The invention relates to the field of the dairy industry and can be used in the analysis of raw milk to determine antibiotics dangerous to living organisms, preferably directly on the farm and / or other agricultural complex.

BACKGROUND OF THE INVENTION

From the prior art, a method is known for the determination of antibiotics containing the P-lactam cycle in a liquid of biological origin, which includes the following steps: bringing a certain volume of the specified liquid of biological origin into contact with a certain amount of a recognizing agent and thermostating the resulting mixture under conditions in which complex formation is possible antibiotics that may be present in said biological fluid, with a recognizing agent; bringing the mixture obtained in the step into contact with at least one reference antibiotic immobilized on the support under conditions that allow the formation of a complex of the reference antibiotic with the amount of recognition agent that did not react in the first step; and a certain amount of a recognition agent associated with the substrate, which is used to judge the amount of antibiotic in the test liquid, while the recognition agent is a receptor sensitive to antibiotics containing a P-lactam ring obtained from Bacillus licheniformis (RU 2213973 C2, 10.10.2003 ).

Known immunochromatographic test strip for the simultaneous determination of the presence of four groups of antibiotics and casein in milk or dairy products, which is a design made of a substrate with four membranes sequentially fixed on its surface in a row, the first of which is a filter membrane for applying the test sample, the second of which a conjugate membrane with components applied to its surface conjugated with a label and specifically binding antibiotics and casein, the third of which is a working membrane, the surface of which includes a control area, a test area for determining an antibiotic of the P-lactam group, a test area for determination of an antibiotic of the tetracycline group, a test region for the determination of an antibiotic of the streptomycin group, a test region for the determination of an antibiotic of the group of derivatives of chloramphenicol, a test region for the determination of casein, where the test region includes a carrier protein conjugate with a detectable antigen, the fourth of which is an adsorbing membrane ( RU 191660 Sh, 15.08.2019).

A known method for the quantitative determination of levomycetin by differential voltammetry, which consists in the fact that levomycetin is transferred from the sample into solution, acid hydrolysis is carried out and the protein is precipitated from the hydrolyzate, followed by a voltammetric determination, while the voltammetric determination of chloramphenicol is carried out by registering the cathodic peaks of the antibiotic on an indicator mercury-film or glassy carbon electrodes in the differential mode of shooting voltammograms at the corresponding potentials of - (0.67 ± 0.05) V and - (0.60 ± 0.03) V relative to a saturated silver chloride electrode on backgrounds of 0.1 mol / dm ³ ammonium citrate disubstituted (pH 4.7-5, 1) or 0.1 mol / dm ³ (NH4) 2SO4 With the addition of HC1 to pH 5.1 at a potential sweep rate of 10-25 mV / s, and the concentration of chloramphenicol is determined by the peak height by the method additives of certified mixtures (RU 2180748 C1 20.03.2002).

A device for chromatographic analysis used for immunoassays is known, which allows fast and convenient analyzes of biologically important analytes and perform the necessary in situ extractions without the use of separate extraction equipment. The device has a wide dynamic range and is protected from the influence of particles or colored components. In one form, the device includes a first opposable containing a sample preparation zone adapted to obtain a test sample, and a second opposable containing a chromatographic medium, the first and second opposable can be placed against each other so that by applying the preparation zone to the chromatographic medium sample to transfer the test sample into it, preferably when the analyte is detected using a visually detectable label, other versions of the device differ in the arrangement of blocks in order to ensure optimal chromatography of a number of analytes, as well as the implementation of bidirectional chromatography; subsequent options are suitable for competitive immunoassays (RU 2124729 C1 10.01.1999).

The closest analogue of the claimed invention is a system for detecting antibiotic residues and determining their concentrations in milk, containing a milk inflow channel and thermostatically parallel milk flow channels, each of which contains an oxygen sensor integrated with a different enzyme, forming a sensor, which together form an array of biosensors, and a means for separating non-standard milk connected to the signal processing device, and a milk outflow channel, while for the simultaneous detection of various antibiotics and determining their concentration during milking in real time, the system contains an additional parallel milk flow channel, which includes an oxygen sensor with an enzyme carrier, but without enzyme, and the hydrolysis device located between the milk inflow channel and the parallel milk flow channels, and said hydrolysis device contains a physical and/or chemical means for controlled hydrolysis of lactose to obtain a target given equal concentrations of glucose and galactose in each milk sample under study, and each biosensor in the array of biosensors is integrated with a different enzyme that catalyzes the oxidation of glucose and galactose by molecular dissolved oxygen, and the catalytic activity of these enzymes increases or decreases in the presence of antibiotics, and the amount of these enzymes is sufficient to generate a decrease in the transition phase of oxygen concentration, which is measured with a given accuracy before the analyzed milk is mixed with high quality milk (RU 2524624 C2, 27.07.2014).

A significant disadvantage of the known technical solutions is the duration, complexity and laboriousness of their implementation.

PURPOSE OF THE INVENTION

The problem to be solved by the present invention is to provide an effective method for determining the presence of antibiotics in raw milk, preferably directly during milking. The technical result achieved in solving the problem is to create a fast and accurate method for determining the presence of antibiotics in raw milk.

SUMMARY OF THE INVENTION

To achieve this technical result, a method for determining antibiotics in raw milk is proposed, which includes sampling raw milk; preparation of a buffer solution with a pH selected from the range of 4.0 - 8.0; applying the resulting buffer solution to the electrodes of electrochemical sensors; measuring raw milk samples using a sensor platform consisting of a test strip and a potentiostat, while test strips with electrodes are made using screen or inkjet printing on a substrate, and they represent an array of electrodes, while a selective layer is applied to the surface of the electrodes using chemical modification of the surface using polyelectrolytes, aptamers are used as a selective element, and the potentiostat includes modules for applying and removing an electrical signal, modules for converting and transmitting a signal; at the same time, the processing of the received electrical signals is carried out by the method of mathematical statistics, the obtained values are compared with the reference values, and the result of this comparison determines the presence of antibiotics in raw milk and their concentration with a certain accuracy. At the same time, antibiotics of the beta-lactam type, tetracycline group, chloramphenicol and streptomycin can be determined as antibiotics.

At the same time, the process of determining antibiotics in raw milk can be carried out directly during milking.

DETAILED DESCRIPTION OF THE INVENTION

The presence and concentration of antibiotics in raw milk is determined using a device that is a test strip with electrodes and a potentiostat connected to it, while data processing is carried out using software. In one aspect of the present invention, a method for detecting antibiotics in raw milk includes the following steps:

- preparation of raw milk samples, preferably immediately after milking or directly during milking;

- application of raw milk samples to the surface of test strips containing electrodes with electrochemical sensors;

- preliminary application of a layer of salt on the surface of the electrodes, which, when interacting with milk, creates a buffer solution with a pH of 4.0-8.0, for more accurate analysis;

- carrying out electrochemical measurements using a potentiostat, for which various voltage sweeps are applied to the electrodes of the electrochemical sensor, while oxidation or reduction of the antibiotic occurs on the electrode surface, or complexes are formed on the electrode surface;

- registration of current-voltage dependences of processes with the help of a potentiostat;

- conversion and processing of the received electrical signals by the method of mathematical statistics;

- comparison of the obtained values with reference values;

- determination of the presence of antibiotics in raw milk.

Preferably, antibiotics of the beta-lactam type, tetracycline group, chloramphenicol and streptomycin are defined as antibiotics.

It is preferable that the determination of antibiotics in milk occurs directly at the time of milking. To do this, the sensor platform is built into the automatic milking system, and the sample is analyzed in a chamber with a volume, preferably up to 2 cm ³ , into which raw milk is taken directly during milking. In the same chamber, there are directly test strips containing electrodes of electrochemical sensors, with a buffer solution previously applied to them with a pH of 4, 0-8.0 selected from the range.

As a buffer solution, you can use a sodium phosphate buffer, which is an aqueous salt solution containing sodium chloride, sodium hydrogen phosphate, potassium chloride and potassium dihydrogen phosphate, as well as a sodium acetate buffer containing sodium acetate and acetic acid. At the same time, the total time for analyzing a raw milk sample is no more than 20 minutes.

At the same time, the above method is carried out using a device that is a test system: a test strip with electrodes, a potentiostat connected to them, and software that processes the received data.

The test strip with electrodes is made by screen or inkjet printing on a substrate, while the test strip is an array of electrodes. Transition group metals and their oxides are used as electrodes, preferably copper, silver, chromium, manganese, iron, cobalt, nickel, zinc, cadmium, gallium, titanium, vanadium, as well as various modifications of carbon, preferably graphite, glassy carbon, carbon black, carbon microparticles.

A selective layer is deposited on the surface of the electrodes by chemical modification of the surface. The selective layer contains selective elements - aptamers, which are immobilized on the electrode surface.

To immobilize aptamers, the method of sequential ion layering is used, which makes it possible to increase the number of aptamers deposited on the electrode and leads to an increase in the sensitivity of the electrochemical sensor. As a co-immobilized layer, it is possible to use polyelectrolytes of different charge, molecular weight, and different strength. Polyelectrolytes are an organic material or polymer selected from the group consisting of or consisting of polyalkyleneimine, for example polyethyleneimine, polystyrenesulfonate, polyallylamine, polyvinyl alcohol, polyhydroxybutyric acid, polystyrene, polydiallyldimethylammonium chloride, polymethacrylic acid, polyalkylene glycol, for example polyethylene glycol, polyvinylpyridine and biopolymers, in particular polymers based on polysaccharides, and polyamino acids such as gelatin, chitosan, agarose, cellulose, alginic acid, dextran, casein, polyarginine, polyglycine, polyglutamic acid, polyaspartic acid and derivatives, copolymers or mixtures

The use of polyelectrolytes makes it possible to effectively immobilize aptamers on the electrode surface due to the structure of polyelectrolytes and their physicochemical properties. The last adsorbed layer on the electrode surface must ensure the prevention of non-specific adsorption. A cationic water-soluble polyelectrolyte is used as the first layer. The molecular weight of polyelectrolytes is selected for effective nanostructuring with immobilized biological molecules. The choice of a cationic polyelectrolyte is also explained by the fact that the next layer - aptamers, at given pH has a partially negative charge. Further application of oppositely charged layers of polyelectrolytes is carried out in order to create a barrier between the analyzed solution and the deposited sensitive layer - aptamers. The negatively charged polyelectrolyte is used to deposit subsequent layers together with the cationic polyelectrolyte. The molecular weight of the anionic polyelectrolyte is preferably close to that of the cationic polyelectrolyte.

Synthesis of aptamers for each antibiotic is carried out using PCR amplification. For example, selection of binding aptamers is accomplished through a process of systematic ligand evolution by exponential enrichment, in which antibiotic-coated magnetic beads are exposed to a single-stranded DNA library. The synthetic DNA library consists of a central randomized region of 43 nucleotides flanked by two constant primer binding sequences. As a result, for example, for cefkinome, the aptamer has the following structure: (5'-GCTGTGTGACTCCTGCAA-N43-GCAGCTGTATCTTGTCTCC-3').

The potentiostat connected to the electrodes includes modules for applying and removing an electrical signal, modules for converting and transmitting a signal via wireless (Bluetooth, Wi-fi) or wired signal transmission (USB).

The potentiostat allows you to convert a chemical signal into an electrical one, as well as transfer measured data to a personal computer or cloud storage. Data processing and interpretation is carried out in a cloud data storage or on a personal computer. The result of data processing is systematized information on the quantitative content of substances in the determined milk samples. This information will form the digital fingerprint of the product, which is produced from the objects of analysis, or a product that is itself an object of analysis.

The received data is processed using software. The application is written in Java for the Android mobile OS. During its development, standard libraries were used - for working with Bluetooth, as well as third-party libraries: Retrofit - for interacting with the client-server. The SQLite database is used to store data on the device.

Sensor data is processed using a microcircuit built into the potentiostat circuit, Bluetooth was chosen for wireless communication of the sensor with the user's smartphone, because BLE has low power consumption and is implemented in all modern devices as a common communication mode. Thus, the proposed device is compatible with all smartphones without additional equipment.

The application includes software for all functional modules, a user-friendly graphical interface and complete documentation for use.

The application includes neural network algorithms that are trained during the experiment, and the successful evaluation of such a neural network in predicting the given properties will signal the end of the experiment.

The search and connection of software to the potentiostat is carried out using a wireless (Bluetooth, Wi-fi) or wired (USB) connection. The software starts the determination of the presence and measurement of the concentration of antibiotics in the sample using the "Start" button. Measurement of electrical signals is carried out within 5 - 20 minutes. At the end of the measurement, the data obtained are processed and corrected using software algorithms. The result is displayed on a personal computer or smartphone in the form of a quantitative characteristic - the concentration of four types of antibiotics. Also, the result of the analysis is automatically sent for logging in the cloud data storage, as well as in third-party data accounting systems. It is possible to send the results of the analysis in the form of an automatically generated report by e-mail or as a notification to a smartphone.

Signal processing is carried out as follows: the obtained current-voltage values are subjected to probabilistic statistical processing, after which each the value is assigned statistical significance. Then, in accordance with the assigned statistical significance, the obtained signals are related to the values obtained for the current-voltage dependences of the antibiotic of various concentrations (control values). Close values allow us to attribute the test sample to a certain concentration of the antibiotic.

Probabilistic aggregation may occur, for example, as follows:

Input signals (volt-ampere values) for processing by the method of mathematical statistics are collected in an array x = [x 1, x2, x3, . . . , xN] . Each value is assigned statistical weights w = [wl, w2, w3, . . ., wN], which are also an array of values. When entering data, each value of the current-voltage dependence is assigned a random value of the statistical weight. The sum of the products of the statistical weights will create the z value:

where b is a term that increases the degree of freedom of the function z.

Next, the z value is processed according to the formula:

The next step is to compare the obtained value of o with the expected value obtained during training on model solutions of milk with an antibiotic. To do this, each value from each concentration of each antibiotic (k) is compared with each tk value obtained during training.

Then the signal error is calculated:

^S k = - tr _k - (1 - fffc)

A \u003d (A - “ ■ A ■ C ¹ - A) Further, the method of iterations updates the statistical weights w for each value of the current-voltage dependence (x):

where 1r is a multiplier (in the range from 0 to 1) obtained during training on model solutions of milk with an antibiotic.

Each point of the current-voltage dependence of the test sample is assigned a statistical weight:

The highest value of the statistical weight indicates the concentration of the corresponding antibiotic. The determination error is calculated by the formula:

As a result of the performed operations, the value of the content of the antibiotic of each group in milk is obtained, which is compared with the control value for each group of antibiotics. A lower value than the control indicates the suitability of milk for consumption.

The antibiotic group is a group consisting of amoxicillin, ampicillin, benzicillin, hetacillin, dihydrostreptomycin, dicloxacillin, doxycycline, cloxacillin, chloramphenicol, nafcillin, oxacillin, oxytetracycline, penicillin g, penicillin v, piperacillin, procaine-penicillin, streptomycin, tetracycline , ticarcillin, chlortetracycline, cefadroxil, cefazolin, cephalexin, cephalonia, cephalonium, cefapirin, cefacetril, cefkinoma, cefoxazole, cefoperazone, cefotaxime.

EXAMPLES

Example 1. Method for the determination of antibiotics in raw milk.

A sample of raw milk (sample 1) was placed in a container for analysis with a volume of up to 1 ml. The volume of the analyzed sample was from 0.01 ml to 0.5 ml. After sampling, put a test strip with electrodes into the container for analysis, pre-applied with a buffer solution and connected to the potentiostat using a connector. . Then, the software was launched on a personal computer or smartphone and the concentration of each type of antibiotic was determined by analyzing the data obtained. As a result of the study and subsequent analysis of the data obtained, it was found that the content of the antibiotic of the tetracycline group in sample 1 is 0.001 mg/l, while the content of this antibiotic in the reference milk sample is 0.003 mg/l, therefore, the content of the antibiotic in the milk sample under study contains in acceptable values. Thus, the studied milk is suitable for use.

The proposed method provides a quick and accurate determination of the presence of antibiotics and their concentration in raw milk.

Claims

Claim

1. Method for the determination of antibiotics in raw milk, characterized in that it includes the sampling of raw milk; applying the obtained buffer solution with pH 4.0 - 8.0 on the electrodes of electrochemical sensors; applying selected milk samples to test strips, including electrodes of electrochemical sensors, measuring raw milk samples using a sensor platform consisting of a test strip and a potentiostat; conversion and processing of the received electrical signals by the method of mathematical statistics using software; comparison of the obtained values with the reference values of the content of antibiotics in milk, while the test strips with electrodes are an array of electrodes, where the selective layer is applied to the surface of the electrodes using chemical surface modification using polyelectrolytes, while aptamers are used as an element of the selective layer, and a potentiostat includes modules for imposing and removing an electrical signal and modules for converting and transmitting a signal for processing.

2. The method for determining antibiotics in raw milk according to claim 1, characterized in that antibiotics of the beta-lactam type, tetracycline group, chloramphenicol and streptomycin are determined as antibiotics.

3. The method for determining antibiotics in raw milk according to claim 1, characterized in that the process of determining antibiotics in raw milk occurs directly during milking.