CN118655199B - Preparation method of cell imprinting electrochemical sensor and method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using same - Google Patents
Preparation method of cell imprinting electrochemical sensor and method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using same Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention relates to the technical field of microorganism detection, in particular to a preparation method of a cell imprinting electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using the same. The method comprises 1) immersing a cell imprinting electrochemical sensor into lactobacillus solution with known concentration, then washing with PBS, detecting current on the surface of an electrode in solution containing [ Fe (CN) 6]3‑/4‑ and KCl ] by using differential pulse voltammetry, drawing a standard curve by taking the logarithmic value of the lactobacillus solution concentration as an abscissa and the current value as an ordinate, and 2) detecting lactobacillus in a dairy product. The cell imprinting electrochemical sensor can realize rapid, sensitive and selective detection of lactic acid bacteria, and has the advantages of simple sample pretreatment, short analysis time, simple experimental operation and strong specific recognition capability. The sensor is used in dairy product production, can rapidly, accurately and quantitatively detect lactobacillus in dairy products, effectively reduces production cost, and is also suitable for detecting lactobacillus in various dairy products.
Description
Technical Field
The invention relates to the technical field of microorganism detection, in particular to a preparation method of a cell imprinting electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using the same.
Background
The yoghurt is a fermented dairy product obtained by inoculating one or more probiotics into sterilized cow milk and sheep milk and fermenting. The yogurt can generate a large amount of organic acid, amino acid, B vitamins, enzymes and other nutritional ingredients due to the action of lactobacillus in the fermentation process, wherein the lactobacillus also has the functions of improving intestinal flora, regulating gastrointestinal peristalsis, promoting digestion and the like, so that the yogurt is popular with consumers.
In yogurt, the amount of lactic acid bacteria is an important index for yogurt quality, and quantitative detection of lactic acid bacteria has become an important index for product quality and functional evaluation. At present, the traditional culture method is a common method for detecting lactobacillus in yoghurt, the operation sensitivity of the method is low, the used selective culture medium cannot count bacterial cells which are difficult to culture despite having metabolic activity, and the method is long in time consumption and is easy to be interfered by mixed bacteria. In addition, yogurt is typically co-fermented with a plurality of different lactic acid bacteria, and the number of viable bacteria in the yogurt needs to be determined for each lactic acid bacteria. Molecular biology techniques such as Polymerase Chain Reaction (PCR), real-time fluorescent quantitative PCR (qPCR), high throughput sequencing technology and the like can realize quantitative detection of different lactic acid bacteria, but the time consumption is long, the required instruments are expensive, and the data analysis is complex. Therefore, the method has the advantages of simple and rapid development and high specificity for dynamically monitoring the lactobacillus quantity during the fermentation of the yoghurt, and has important significance for the quality control of dairy products.
The biosensor is a sensor for quantitatively analyzing the matrix content in the sample liquid by taking biological materials such as microorganisms, enzymes, antibodies, DNA, RNA and the like as molecular recognition elements, has the advantages of high sensitivity, high analysis speed, strong anti-interference capability and the like, and has a wide application prospect in the aspect of food microorganism detection. Currently, immunosensors are relatively mature in their application to microbial detection, and such sensors utilize specific modifications of antibodies, aptamers, or phage to selectively recognize target bacteria. However, the complex synthesis process of the biological recognition element, the number of pretreatment steps, and the strict requirements on the environmental pH and temperature limit the further development of immunosensors to a certain extent. Molecular imprinting techniques have been widely used as a substitute for natural receptors, in which a mixture of functional monomers and cross-linking agents polymerize around template cells and are capable of specifically binding to the cells to form a cell-imprinted polymer. After the polymerization reaction is completed, eluting the template from the imprinted polymer to obtain the cell imprinted sensor. The cell imprinting sensor is provided with holes (namely imprinting sites) which are complementary with the shape, the size and the chemical functional groups of the template molecules, the imprinting sites can specifically identify and bind cells similar to the template cells in the sample to be detected, and the selective quantitative detection of target microorganisms is achieved by detecting the change of electric signals before and after the cell imprinting sensor adsorbs the cells. Current technology for molecular imprinting sensors in the detection of dairy microorganisms is not mature.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a preparation method of a cell imprinting electrochemical sensor and a method for rapidly and quantitatively detecting lactic acid bacteria in dairy products by using the same. The method is a cell imprinting electrochemical sensor method for lactobacillus detection of dairy products. In the invention, the cell imprinting electrochemical sensor can realize rapid, sensitive and selective detection of lactobacillus, and in addition, the sample pretreatment is simple, the detection sensitivity is high, the analysis time is short, the experimental operation is simple, and the specific recognition capability is strong.
In order to achieve the above object, the present invention has the following specific technical scheme:
a preparation method of a cell imprinting electrochemical sensor comprises the following steps:
1) Preparing activated biochar, namely heating biomass to obtain biomass carbon, and activating the biomass carbon to obtain activated biochar;
2) Adding PBS solution into activated biochar, stirring to dissolve to prepare a suspension with the biochar concentration of 1mg/mL, polishing a screen printing electrode, respectively flushing with ethanol and ultrapure water, and drying at room temperature;
3) The preparation of the electrode with the lactobacillus-polypyrrole polymer comprises the steps of putting a modified electrode into PBS solution containing polypyrrole, KCl and lactobacillus templates, and carrying out electrochemical polymerization by cyclic voltammetry to obtain the electrode with the lactobacillus-polypyrrole polymer;
4) The preparation of the cell imprinting electrochemical sensor comprises the steps of flushing a polymer electrode with lactobacillus-polypyrrole by PBS, immersing the polymer electrode in lysozyme, then adding NaOH, applying voltage to remove lactobacillus wrapped in the polymer, and leaving imprinting cavities to obtain the cell imprinting electrochemical sensor with specific binding capacity.
Further, 1) activated biochar is specifically prepared by heat-treating biomass at 600-800 ℃ for 1-2 h (more preferably 700 ℃) for 2 hours to obtain biomass carbon.
Further, the biomass includes all plants, microorganisms, animals and waste materials produced by the animals (specifically, bamboo leaves, straw, vinasse and the like).
Further, the biomass carbon and the potassium carbonate are mixed and heated, and the whole pyrolysis process is carried out in nitrogen, so that activated biochar is obtained.
Further, the weight ratio of the biomass carbon to the potassium carbonate is 1:2, the heating temperature is 800-900 ℃, and the heating time is 1-2 hours.
Further, the specific step of 2) modifying the electrode is to polish the Screen Printed Electrode (SPE) to a mirror surface with 0.05 μm alumina powder, rinse with ethanol and ultrapure water, and dry at room temperature. And (3) dripping biochar suspension on the surface of the electrode, and naturally drying to obtain the modified electrode.
Further, the screen printing electrode comprises a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode is made of a bare glassy carbon electrode, the diameter of the working electrode is 3mm, the auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.
Further, the volume of the biochar suspension dripped on the surface of the electrode is 6-10 mu L.
Further, the specific preparation steps of the electrode with the lactobacillus-polypyrrole polymer comprise the steps of placing the modified electrode into 10 mL PBS solution containing 0.1-0.2 mol/L polypyrrole, 0.05-0.1 mol/L KCl and 10 7~109 CFU/mL lactobacillus template, and carrying out electrochemical polymerization by a cyclic voltammetry method to obtain the electrode with the lactobacillus-polypyrrole polymer.
The lactobacillus template comprises but is not limited to lactobacillus delbrueckii subsp bulgaricus and streptococcus salivarius subsp thermophilus, and can be correspondingly selected according to different types of detected microorganisms.
The PBS solution contains NaH 2PO4 and Na 2HPO4, the concentration is 0.1 mol/L, the pH is 7.4, and the same applies.
The cyclic voltammetry is used for electrochemical polymerization, the scanning circle number is 9-11, the voltage is 0-1.2V, and the scanning speed is 40-60 mV/s.
Still further, the specific preparation steps of the 4) cell-imprinted electrochemical sensor are:
And (3) washing the electrode with the lactobacillus-polypyrrole polymer by PBS, soaking in 10-15 mg/mL lysozyme for 1-2 hours, then adding 0.1mol/L NaOH, and applying 0.98V voltage to remove lactobacillus wrapped in the polymer, and leaving a imprinting cavity to obtain the cell imprinting electrochemical sensor with specific binding capacity.
In another aspect, the invention provides the use of a cell-imprinted electrochemical sensor in the detection of lactic acid bacteria in a dairy product.
A method for rapidly and quantitatively detecting lactic acid bacteria in dairy products, which comprises the following steps:
1) Preparation of standard curve for detecting lactobacillus by using cell blotting electrochemical sensor:
Immersing a cell imprinting electrochemical sensor into lactobacillus solution with known concentration, washing with PBS, detecting the current on the surface of an electrode in a solution containing [ Fe (CN) 6]3-/4- and KCl ] by using differential pulse voltammetry, and drawing a standard curve by taking the logarithmic value of the lactobacillus solution concentration as an abscissa and the current value as an ordinate;
2) Detection of lactic acid bacteria in dairy products:
Immersing the cell-imprinted electrochemical sensor in a sample to be detected, washing with PBS, detecting the current on the surface of the electrode in a solution containing [ Fe (CN) 6]3-/4- and KCl ] by using differential pulse voltammetry, and detecting the corresponding bacteria concentration on a standard curve according to the detected current value.
Further, 1) the preparation of the standard curve of the lactobacillus for detecting the cell-imprinting electrochemical sensor comprises the specific steps of immersing the cell-imprinting electrochemical sensor in 100 mu L of lactobacillus liquid with known concentration and keeping the cell-imprinting electrochemical sensor at room temperature for 1 hour. The electrode surface was then rinsed with PBS and the current was measured using differential pulse voltammetry in a solution of 10 mL containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl. And drawing a standard curve by taking the logarithmic value of the concentration of the lactobacillus liquid as an abscissa and the current value as an ordinate.
The voltage range of the differential pulse voltammetry is 0-0.6V, the pulse amplitude is 50 mV, and the pulse width is 0.2 s, which are the same as the following.
The lactobacillus bacterial liquid comprises but is not limited to Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus salivarius thermophilus, and the standard method for determining the bacterial liquid concentration by using the bacterial liquid concentration of 1×101CFU/mL、1×102CFU/mL、1×103CFU/mL、1×104CFU/mL、1×105CFU/mL、1×106CFU/mL、1×107CFU/mL、1×108CFU/mL、1×109CFU/mL, is a plate counting method.
Further, the specific steps of 2) detecting lactic acid bacteria in the dairy product are as follows:
The cell-imprinted electrochemical sensor was immersed in 100 μl of the sample to be measured and kept at room temperature for 1 hour. After washing with PBS, the current at the electrode surface was measured by differential pulse voltammetry in 10mL solutions containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl. The corresponding bacterial concentration is found out on the standard curve according to the measured current value.
The sample to be tested comprises, but is not limited to, yoghurt, fresh cow milk, sterilized milk, milk powder and the like, and the solid sample can be prepared into a liquid sample by adding a pretreatment step according to the requirement.
The invention has the working principle that under the modification action of biological carbon, the specific surface area of the electrode surface is increased, the conductivity is increased, and higher peak current appears. Under further electropolymerization, the polymers of functional monomers and cross-linking agents are able to bind specifically to living cells to form polymers on the electrode surface, and accumulation of living cells can hinder current transport resulting in reduced current. After the template cells are removed, a imprinting cavity is left, the imprinting cavity is used as a channel for electron transfer to promote current transportation, and the current is increased. The imprinting cavity can be specifically identified and recombined with the cells according to the size, shape and chemical group structure of the cells, so that the imprinting cavity is blocked again to prevent electron transfer and current is reduced again. Therefore, the concentration of bacteria can be obtained by calculating according to the current change value before and after the cell blotting electrochemical sensor is combined with cells.
Compared with the prior art, the invention has the beneficial effects that:
(1) The method for detecting the lactobacillus by using the cell-imprinting electrochemical sensor has the advantages of high efficiency, sensitivity and high selectivity, is low in preparation cost, simple in sample pretreatment, short in analysis time and simple in experimental operation, and has the potential of being applied to fermented foods for monitoring microbial indexes in real time.
(2) The cell imprinting electrochemical sensor prepared by the invention can be applied to dairy product production, can realize the purpose of rapidly, accurately and quantitatively detecting the lactic acid bacteria in dairy products, shortens the detection of the number of the lactic acid bacteria from 1-2 days to 4-6 hours in the traditional culture method, effectively reduces the production cost, and is suitable for detecting the lactic acid bacteria in various dairy products.
(3) The invention can achieve the quantitative detection of different lactobacillus in dairy products.
Drawings
FIG. 1 is a standard curve of a cell blot electrochemical sensor for detection of Lactobacillus delbrueckii subspecies bulgaricus provided by the invention.
FIG. 2 is a standard curve of the cell blot electrochemical sensor for detecting streptococcus salivarius thermophilus subspecies.
Detailed Description
All of the features disclosed in this specification, or all of the steps in a method or process disclosed, may be combined in any combination, except for mutually exclusive features and/or steps.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. That is, each feature is one example only of a generic series of equivalent or similar features, unless expressly stated otherwise.
The features and capabilities of the present invention are described in further detail below in connection with examples.
The experimental method and the detection method in each embodiment are conventional methods unless otherwise specified, the medicament and the material are commercially available unless otherwise specified, and the index data are conventional measurement methods unless otherwise specified.
Example 1:
a method for preparing a cell imprinting electrochemical sensor based on Lactobacillus delbrueckii subspecies bulgaricus.
(1) The activated biochar is prepared by cleaning folium Bambusae with water, oven drying at 60deg.C for 12 h, and pulverizing. And pouring a proper amount of bamboo leaf powder into a crucible, and heating for 2h ℃ in a tube furnace to obtain the biochar. The biochar was rinsed with 1 mol/L hydrochloric acid and ultrapure water until its pH was neutral, and then dried in an oven 12: 12 h. The dried material was mixed with potassium carbonate in a weight ratio of 1:2 and then heated in a tube furnace at 850 ℃ for 2h, the entire pyrolysis process being carried out under nitrogen. After pyrolysis is completed, washing with 1 mol/L hydrochloric acid and ultrapure water until the pH value is neutral, then placing the mixture in an oven to dry 12: 12 h, and re-suspending the mixture with PBS solution to obtain 1mg/mL biochar suspension;
(2) Electrode pretreatment, namely polishing the screen printing electrode to a mirror surface by using 0.05 mu m alumina powder, flushing the screen printing electrode by using ethanol and ultrapure water, and drying the screen printing electrode at room temperature. Dripping 6 mu L of the biochar suspension obtained in the step (1) on the surface of an electrode, and naturally drying to obtain a modified electrode;
(3) The preparation of the lactobacillus-polypyrrole polymer comprises the steps of putting 10 mL obtained in the step (2) into a PBS solution containing 0.2 mol/L polypyrrole, 0.1 mol/L KCI and 10 9 CFU/mL lactobacillus delbrueckii subsp bulgaricus template (the PBS contains NaH 2PO4 and Na 2HPO4, the concentration is 0.1 mol/L, the pH is 7.4 and the same applies below), carrying out electrochemical polymerization by a cyclic voltammetry, wherein the scanning number is 9, the voltage is 1.1V, and the scanning rate is 50 mV/s;
(4) And (3) preparing the cell-imprinted electrochemical sensor, namely flushing the cell-imprinted electrochemical sensor with the PBS solution obtained in the step (3), soaking the cell-imprinted electrochemical sensor in 10 mg/mL of lysozyme for 2 hours, then adding 0.1 mol/L of NaOH, and applying overvoltage of 0.98V to remove the lactobacillus delbrueckii subspecies bulgaricus embedded in the polymer, thereby obtaining the cell-imprinted electrochemical sensor with specific binding capacity.
Example 2:
A method for preparing a cell-imprinting electrochemical sensor based on streptococcus salivarius thermophilus subspecies.
The preparation of this example is identical to that of example 1, except that the bacterial template used in step (3) is 10 9 CFU/mL Streptococcus salivarius subspecies thermophilus.
Example 3
A method for detecting a standard curve prepared by lactobacillus delbrueckii subspecies bulgaricus by using a cell imprinting electrochemical sensor.
(1) Lactobacillus delbrueckii subspecies bulgaricus bacterial solutions with different concentrations are prepared by inoculating lactobacillus delbrueckii subspecies bulgaricus lb31 into a sterile MRS liquid culture medium according to an inoculum size of 2% (v/v), standing and culturing at 37 ℃ for 24 h to obtain first-generation activating solution, and then inoculating the first-generation activating solution into the sterile MRS liquid culture medium according to an inoculum size of 2% (v/v), and culturing at 37 ℃ for 24 h to obtain second-generation activating solution. Centrifuging the second generation activation solution for 10 min at 9000 r/min, reserving a precipitate, continuing to resuspend the reserved precipitate with sterile purified water, adjusting the concentration of the thallus to 1.0X10 9 CFU/mL, and then carrying out gradient dilution on the resuspension to obtain 1×101CFU/mL、1×102CFU/mL、1×103CFU/mL、1×104CFU/mL、1×105CFU/mL、1×106CFU/mL、1×107CFU/mL、1×108CFU/mL、1×109CFU/mL bacterial solution;
(2) And (3) detecting bacterial liquid to be detected, namely immersing the cell imprinting electrochemical sensor obtained in the example 1 into the bacterial liquid obtained in the step (1), and keeping the cell imprinting electrochemical sensor at the room temperature to be 1 h. After washing with PBS, detecting the current on the electrode surface in 10mL solutions containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl, with the voltage range of 0-0.6V, the pulse amplitude of 50 mV, and the pulse width of 0.2 s;
(3) Drawing a standard curve, namely drawing the standard curve by taking the logarithmic value of the concentration of lactobacillus delbrueckii subspecies bulgaricus bacterial liquid as an abscissa and the corresponding current value as an ordinate.
Example 4
A method for preparing standard curve by detecting streptococcus salivarius thermophilus subspecies by using a cell-imprinting electrochemical sensor.
(1) The preparation of streptococcus salivarius thermophilus subspecies bacterial liquid with different concentrations comprises inoculating streptococcus salivarius subspecies st195 into a sterile MRS liquid culture medium according to an inoculum size of 2% (v/v), standing and culturing at 37 ℃ for 24h to obtain first-generation activating liquid, and inoculating the first-generation activating liquid into the sterile MRS liquid culture medium according to an inoculum size of 2% (v/v), and culturing at 37 ℃ for 24h to obtain second-generation activating liquid. Centrifuging the second generation activation solution for 10 min at 9000 r/min, reserving a precipitate, continuing to resuspend the reserved precipitate with sterile purified water, adjusting the concentration of the thallus to 1.0X10 9 CFU/mL, and then carrying out gradient dilution on the resuspension to obtain 1×101CFU/mL、1×102CFU/mL、1×103CFU/mL、1×104CFU/mL、1×105CFU/mL、1×106CFU/mL、1×107CFU/mL、1×108CFU/mL、1×109CFU/mL bacterial solution;
(2) And (3) detecting bacterial liquid to be detected, namely immersing the cell imprinting electrochemical sensor obtained in the example 2 into the bacterial liquid obtained in the step (1), and keeping the cell imprinting electrochemical sensor at the room temperature for 1 h. After washing with PBS, detecting the current on the electrode surface in 10mL solutions containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl, with the voltage range of 0-0.6V, the pulse amplitude of 50 mV, and the pulse width of 0.2 s;
(3) Drawing a standard curve, namely drawing the standard curve by taking the logarithmic value of the concentration of streptococcus salivarius thermophilus subspecies as an abscissa and the corresponding current value as an ordinate.
Example 5
A method for detecting lactobacillus delbrueckii subsp bulgaricus in yoghurt by using a cell-imprinting electrochemical sensor.
(1) Preparation of a Standard Curve the method for preparing the Standard Curve was the same as in example 3;
(2) Sample detection the cell-imprinted electrochemical sensor obtained in example 1 was immersed in 100. Mu.L of a yogurt (commercially available from Supermarket, lactobacillus delbrueckii subsp. Bulgaricus, live bacteria content. Gtoreq.2X10 8 CFU/100 g) sample and kept at room temperature for 1 hour. After washing with PBS, detecting the current on the electrode surface in 10mL solutions containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl, with the voltage range of 0-0.6V, the pulse amplitude of 50 mV, and the pulse width of 0.2 s;
(3) And (3) calculating a result, namely finding out the corresponding bacteria concentration on a standard curve according to the current value measured in the step (2).
Example 6
A method for detecting Lactobacillus delbrueckii subspecies bulgaricus in full-fat sterilized milk by using a cell-imprinting electrochemical sensor.
The preparation of this example is identical to example 5, except that the sample used in step (2) is sterilized milk (commercially available). Example 7
A method for detecting streptococcus salivarius thermophilus subspecies in flavored yoghurt by using a cell-imprinting electrochemical sensor.
(1) Preparation of a Standard Curve the method for preparing the Standard Curve was the same as in example 4;
(2) Sample detection the cell blot electrochemical sensor obtained in example 2 was immersed in 100. Mu.L of a flavored yoghurt (purchased in supermarket, added with Lactobacillus delbrueckii subsp. Bulgaricus of not less than 2.3X10 8 CFU/100 g and added with Streptococcus salivarius thermophilus of not less than 3.2X10 8/100 g) and kept at room temperature for 1 hour. After washing with PBS, detecting the current on the electrode surface in 10 mL solutions containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl, with the voltage range of 0-0.6V, the pulse amplitude of 50 mV, and the pulse width of 0.2 s;
(3) And (3) calculating a result, namely finding out the corresponding bacteria concentration on a standard curve according to the current value measured in the step (2).
Comparative example 1
The preparation of the sensor was performed by the method of example 1, except that lactobacillus delbrueckii subsp bulgaricus template was not added in step (3). The sensor prepared in comparative example 1 was used to detect Lactobacillus delbrueckii subspecies bulgaricus bacterial solutions at different concentrations by the method of example 3.
Comparative example 2
The sensor was prepared by the method of example 2, except that polypyrrole was not added in step (3), and the template bacteria used was 10 9 CFU/mL Streptococcus salivarius thermophilus subspecies. Using the method of example 4, different concentrations of Streptococcus salivarius subspecies thermophilus bacterial solutions were detected using the sensor prepared in comparative example 2.
Comparative example 3
The detection of Lactobacillus delbrueckii subsp bulgaricus in yogurt (commercially available from supermarket, lactobacillus delbrueckii subsp bulgaricus content. Gtoreq.2X10 8 CFU/100 g) was carried out by the method of example 5, except that the sensor used in step (2) was the method of comparative example 1.
Comparative example 4
The detection of Streptococcus salivarius thermophilus in a flavored yogurt (commercially available from Supermarket, lactobacillus delbrueckii, bulgaria subspecies bulgaricus, added in an amount of not less than 2.3X10 8 CFU/100g, streptococcus salivarius thermophilus added in an amount of not less than 3.2X10 8 CFU/100 g) was carried out by the method of example 7, except that the sensor used in step (2) was prepared by the method of comparative example 2.
Comparative example 5
The method for detecting the lactobacillus in the yoghourt (purchased in supermarket, the content of the viable bacteria of the Lactobacillus delbrueckii subspecies bulgaricus is more than or equal to 2 multiplied by 10 8 CFU/100 g) is adopted for detecting the lactobacillus by using a food microbiology inspection method of national food safety standard GB 4789.35-2023.
Experiment
Experimental example 1
The standard curves prepared in example 3, example 4, comparative example 1, and comparative example 2 are provided in this experimental example.
FIG. 1 is a standard curve obtained in example 3, and the linear regression equation is y= -2.4473x+46.789, and R 2 = 0.9986, and the detection limit is 10 CFU/mL, when the concentration of Lactobacillus delbrueckii subsp. Bulgaricus is 1×10 1~1×109 CFU/mL.
FIG. 2 is a standard curve prepared in example 4, and the linear regression equation is y= -2.6283x+48.697, R 2 = 0.9988, and the detection limit is 10 CFU/mL, when the concentration of Streptococcus salivarius subspecies thermophilus is 1X 10 1~1×109 CFU/mL.
In comparative example 1, the imprinting sites lacking the template bacteria cannot specifically bind to the target bacteria, and the current value does not change significantly with the increase of the bacterial concentration, which indicates that the lactic acid bacteria cannot be detected.
In comparative example 2, the functional monomer polypyrrole was absent, so that lactic acid bacteria could not be efficiently polymerized into the polymer to form imprinting sites, and thus the current value did not change much with the increase of the bacterial concentration.
Compared with the comparative example, the current value of the surface of the cell-imprinted electrochemical sensor decreases with the increase of the concentration of bacteria, which indicates that the cell-imprinted electrochemical sensor can successfully identify and bind to target bacteria, and the current transport is hindered with the accumulation of cells in the polymer. From the experimental results, the current value and the bacteria concentration of the cell-imprinted electrochemical sensor prepared by the invention can be in a linear relation when the lactic acid bacteria are detected, so that the cell-imprinted electrochemical sensor prepared by the invention is a lactic acid bacteria detection tool with high sensitivity and good stability.
Experimental example 2
The experimental examples provide the number of lactobacillus delbrueckii subspecies bulgaricus in the yoghurt measured in example 5, example 6, comparative example 3 and comparative example 5, and the results are shown in table 1.
The detection limit of the lactobacillus delbrueckii subsp bulgaricus amount from the sterilized milk is less than 10CFU/mL when the lactobacillus delbrueckii subsp bulgaricus amount is detected from the yogurt by adopting a cell imprinting electrochemical sensor to be 3.45 multiplied by 10 8 CFU/mL. In comparative example 3, the target bacteria could not be identified due to lack of blotting sites of the sensor, and the detection limit was less than 10CFU/mL. The number of Lactobacillus delbrueckii subspecies bulgaricus measured by plate counting in comparative example 5 was 3.3X10 8 CFU/mL. The result measured by the cell-imprinting electrochemical sensor is not greatly different from the result measured by a plate counting method, the time for detecting the cell-imprinting electrochemical sensor is 4 hours, and the time for detecting the cell-imprinting electrochemical sensor is 2 days, so that the cell-imprinting electrochemical sensor prepared by the invention can rapidly and effectively detect the quantity of lactic acid bacteria in the yoghurt.
TABLE 1
Experimental example 3
The present experimental example provides the number of streptococcus salivarius thermophilus subspecies in the yoghurt measured in example 7 and comparative example 4.
The cell-imprinted electrochemical sensor is used for measuring that the quantity of streptococcus salivarius thermophilus subspecies is 1.42 multiplied by 10 7 CFU/mL from the flavor yoghourt, and the cell-imprinted electrochemical sensor cannot be successfully prepared in comparative example 4 because the sensor lacks functional monomer polypyrrole, and lactic acid bacteria cannot be detected, so that the detection limit is less than 10CFU/mL. In a flavored yogurt sample containing a plurality of lactic acid bacteria, when the template bacteria in the cell blotting sensor are streptococcus salivarius thermophilus subspecies, the quantity of the streptococcus salivarius thermophilus subspecies in the sample can be detected, which indicates that the cell blotting electrochemical sensor prepared by the invention has specificity and can quantitatively detect the lactic acid bacteria with different cell forms.
TABLE 2
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
This background section is provided to generally present the context of the present invention and the work of the presently named inventors, to the extent it is described in this background section, as well as the description of the present section as not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present invention.
Claims (10)
1. The preparation method of the cell imprinting electrochemical sensor is characterized by comprising the following steps of:
1) Preparing activated biochar, namely heating biomass to obtain biomass carbon, and activating the biomass carbon to obtain activated biochar;
2) Adding PBS solution into activated biochar, stirring to dissolve to prepare a suspension with the biochar concentration of 1mg/mL, polishing a screen printing electrode, respectively flushing with ethanol and ultrapure water, and drying at room temperature;
3) The preparation of the electrode with the lactobacillus-polypyrrole polymer comprises the steps of putting the modified electrode into PBS solution containing polypyrrole, KCl and lactobacillus templates, and performing electrochemical polymerization by a cyclic voltammetry to obtain the electrode with the lactobacillus-polypyrrole polymer, wherein the lactobacillus templates comprise but are not limited to Lactobacillus delbrueckii Bulgaria subspecies and Streptococcus salivarius thermophilus subspecies;
4) The preparation method of the cell imprinting electrochemical sensor comprises the steps of flushing a polymer electrode with lactobacillus-polypyrrole with PBS, soaking the polymer electrode in lysozyme, adding NaOH, applying voltage to remove lactobacillus wrapped in the polymer, and leaving imprinting cavities to obtain the cell imprinting electrochemical sensor with specific binding capacity, wherein the cell imprinting electrochemical sensor is used for rapidly and quantitatively detecting the lactobacillus in dairy products.
2. The preparation method of the cell imprinting electrochemical sensor according to claim 1 is characterized by comprising the specific preparation steps of 1) heating biomass at 600-800 ℃ for 1-2 h to obtain biochar, then mixing and heating the biochar and potassium carbonate, performing the whole pyrolysis process in nitrogen to obtain activated biochar, wherein the weight ratio of the biomass carbon to the potassium carbonate is 1:2, the heating temperature after mixing is 800-900 ℃, and the heating time is 1-2 h.
3. The preparation method of the cell imprinting electrochemical sensor according to claim 1 or 2 is characterized in that the specific steps of the step 2) of modifying an electrode are that PBS solution is added into activated biochar, the mixture is stirred until the PBS solution is dissolved, a suspension with the concentration of the biochar of 1mg/mL is prepared, a screen printing electrode is polished to a mirror surface by using 0.05 mu m alumina powder, the mirror surface is washed by ethanol and ultrapure water, the solution is dried at room temperature, the activated biochar suspension is dripped on the surface of the dried screen printing electrode, the modified electrode is obtained after natural drying, the dripping volume is 6-10 mu L, the screen printing electrode comprises a working electrode, a reference electrode and an auxiliary electrode, the working electrode is prepared from a bare glass carbon electrode, the diameter of the working electrode is 3mm, the auxiliary electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode.
4. The method for preparing the cell-imprinted electrochemical sensor according to claim 3, wherein the specific preparation step of the electrode with the lactobacillus-polypyrrole polymer in the step 3) is that the modified electrode is put into 10mL PBS solution containing 0.1-0.2 mol/L polypyrrole, 0.05-0.1 mol/L KCl and 10 7~109 CFU/mL lactobacillus template, and electrochemical polymerization is carried out by a cyclic voltammetry method, so that the electrode with the lactobacillus-polypyrrole polymer is obtained.
5. The method for preparing the cell-imprinted electrochemical sensor according to claim 4, wherein the specific preparation step of the cell-imprinted electrochemical sensor in step 4) is that the lactobacillus-polypyrrole polymer electrode is washed by PBS and then soaked in 10-15 mg/mL lysozyme for 1-2 hours, then 0.1 mol/L NaOH is added, and 0.98V voltage is applied to remove lactobacillus wrapped in the polymer, so that a imprinted cavity is left, and the cell-imprinted electrochemical sensor is obtained.
6. The method for preparing the cell-imprinted electrochemical sensor according to claim 5, wherein the lactobacillus template comprises, but is not limited to, lactobacillus delbrueckii subspecies bulgaricus and streptococcus salivarius thermophilus, the lactobacillus template is correspondingly selected according to different types of detected microorganisms, the PBS contains NaH 2PO4 and Na 2HPO4, the concentration is 0.1 mol/L and the pH is 7.4, the scanning number of turns of the electrochemical polymerization by the cyclic voltammetry is 9-11, the voltage is 0-1.2V, and the scanning rate is 40-60 mV/s.
7. A method for rapid quantitative detection of lactic acid bacteria in a dairy product, characterized in that a cell-imprinted electrochemical sensor prepared by the method of claim 6 is used, the method comprising the steps of:
1) Preparation of standard curve for detecting lactobacillus by using cell blotting electrochemical sensor:
Immersing a cell imprinting electrochemical sensor into lactobacillus solution with known concentration, washing with PBS, detecting current on the surface of an electrode in a solution containing [ Fe (CN) 6]3-/4- and KCl ] by using differential pulse voltammetry, and drawing a standard curve by taking the logarithmic value of the lactobacillus solution concentration as an abscissa and the current value as an ordinate;
2) Detection of lactic acid bacteria in dairy products:
Immersing the cell-imprinted electrochemical sensor in a sample to be detected, washing with PBS, detecting the current on the surface of the electrode in a solution containing [ Fe (CN) 6]3-/4- and KCl ] by using differential pulse voltammetry, and detecting the corresponding bacteria concentration on a standard curve according to the detected current value.
8. The method for rapidly and quantitatively detecting lactobacillus in dairy products according to claim 7, wherein the specific preparation steps of the standard curve of the lactobacillus detection by the cell-imprinted electrochemical sensor in step 1) are that the cell-imprinted electrochemical sensor is immersed in 100 mu L of lactobacillus liquid with known concentration and kept at room temperature for 1 hour, the solution is washed by PBS for several times, the current on the surface of an electrode is detected in 10mL solution containing 5 mmol/L [ Fe (CN) 6]3-/4- and 0.1 mol/L KCl by using differential pulse voltammetry, the logarithmic value of the lactobacillus liquid concentration is taken as an abscissa, the current value is taken as an ordinate, the standard curve is drawn, and the standard method for determining the concentration of the lactobacillus liquid comprises but is not limited to lactobacillus delbrueckii bulgaricus subspecies and streptococcus thermophilus subspecies with the bacterial liquid concentration of 1×101CFU/mL、1×102 CFU/mL、1×103 CFU/mL、1×104 CFU/mL、1×105 CFU/mL、1×106 CFU/mL、1×107 CFU/mL、1×108 CFU/mL、1×109 CFU/mL, respectively is a plate count method.
9. The method for rapid quantitative detection of lactic acid bacteria in dairy products according to claim 7 or 8, wherein the specific step of step 2) is to immerse the cell-imprinted electrochemical sensor in 100 μl of sample to be tested, hold it for 1 hour at room temperature, then use PBS to wash, then use differential pulse voltammetry to detect the current on the electrode surface in 10 mL solution containing 5 mmol/L [ Fe (CN) 6]3-/4-, 0.1 mol/L KCl, and find the corresponding bacteria concentration on the standard curve.
10. The method for rapidly and quantitatively detecting lactobacillus in dairy products according to claim 9, wherein the differential pulse voltammetry is characterized in that the voltage range is 0-0.6V, the pulse amplitude is 50 mV, the pulse width is 0.2 s, and the sample to be detected comprises but is not limited to yoghurt, fresh cow milk, sterilized milk and milk powder.
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