CN114047241A - Electrochemical detection method for bacterial activity - Google Patents
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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Abstract
The invention discloses a bacterial activity electrochemical detection method, which belongs to the technical field of biological detection and comprises the following steps: recovering and culturing bacteria; washing bacteria and preparing a bacterial suspension; carrying out detection pretreatment on the bacterial suspension, wherein the pretreatment adopts a water bath heating method; centrifuging the bacterial suspension heated in the water bath, and sucking the supernatant; the supernatant was electrochemically detected using an electrode system. The electrochemical detection method provided by the invention has the advantages of being rapid, simple and convenient, low in cost, capable of continuously detecting the bacterial sample and providing a new idea for the use of the electrochemical method in the aspect of microbial activity detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to an electrochemical detection method for bacterial activity.
Background
Bacteria are ubiquitous in our lives, and food-borne, water-borne, animal-borne and other bacteria can be easily transmitted among individuals through ways of diet, drinking water, contact, air and the like, so that infection is caused, and the life can be threatened when the infection is serious. The fields of food manufacturing industry, pharmaceutical industry, water quality monitoring, clinical diagnosis, public health and the like need to strictly carry out bacterial activity detection according to relevant standards and control the bacterial content. Besides, the detection of bacterial activity can provide a basis for technical updating and mechanism explanation in the fields of food processing, soil detection, water quality monitoring, medical diagnosis technology, environmental protection, petroleum degradation and the like. With the development of society and science, the demand of various fields for detecting the activity of bacteria is increased, and the required detection method needs to have higher accuracy and sensitivity.
Conventional bacterial activity detection methods include plate counting, nephelometry, flow cytometry and the like. The plate counting method is the most common bacterial activity detection method, but the method has high labor intensity, wastes time and labor and is complicated to operate; the turbidity method cannot distinguish the death and activity states of bacteria, and has more influence factors; flow cytometry is expensive and requires high professional literacy for the operator. Therefore, the traditional methods have certain limitations. In recent years, electrochemical methods, such as amperometric methods and impedance methods, have been widely used in the field of microbial detection due to their characteristics of rapidness, simplicity, low cost, simple operation, etc. The current method is relatively quick, but cannot directly detect bacteria, and needs to add oxidoreductase or a proper medium to a detection sample for indirect detection, so that the result influencing factors are more, the defects of low sensitivity, poor selectivity and the like exist, and the detection time is relatively long; the impedance method is used for measuring the content of microorganisms by detecting the change of the electrical characteristics of a culture medium, and when the components of an experimental sample are complex, the method is easily interfered, so that the result accuracy is low, the electric signal monitoring performance is poor, and the factors hinder the development of the impedance method. The electrochemical detection method using purine in bacterial suspension as a marker is a direct electrochemical detection method, the method detects the activity of bacteria by detecting the content and variety change of purine released into the suspension by the bacteria, and is simpler and more reliable than a flat plate counting method and a turbidity method, but the peak signal can be detected by the method only when the purine content released after the bacteria secrete for at least 2 hours, so that the time consumption is long and the response is lower.
Disclosure of Invention
Therefore, the invention provides a bacterial activity electrochemical detection method, which aims to solve the problems of time and labor consumption, complex operation and low sensitivity and accuracy in the existing bacterial detection technology.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
the electrochemical detection method for the bacterial activity provided by the invention comprises the following steps:
firstly, recovering and culturing bacteria;
secondly, washing bacteria and preparing a bacterial suspension;
thirdly, carrying out detection pretreatment on the bacterial suspension, wherein the pretreatment adopts a water bath heating method;
fourthly, centrifuging the bacterial suspension heated by the water bath, and sucking the supernatant;
and fifthly, carrying out electrochemical detection on the supernatant by using an electrode system.
Further, in the third step, the water bath temperature of the bacterial suspension is 30-80 ℃, and the water bath time is 5-50 min.
Further, the water bath temperature is 50-55 ℃, and the water bath time is 10-20 min.
Further, in the first step, the bacteria include gram-negative bacteria and gram-positive bacteria.
Further, in the second step, the solution used for washing is PBS solution with pH of 6.5-7.5, and the number of washing times is 3-5.
Further, in the second step, in the preparation of the bacterial suspension, the liquid used for suspension is a PBS solution with the pH value of 6.5-7.5.
Further, in the fourth step, the centrifugal speed is 4000-8000 rpm, and the centrifugal time is 5-15 min.
Further, the rotation speed is 6000rpm, and the centrifugation time is 10 min.
Further, in the fifth step, the electrode system used is a three-electrode system, the three-electrode system comprises an auxiliary electrode, a reference electrode and a working electrode, and the auxiliary electrode is a platinum counter electrode or a carbon counter electrode; the reference electrode is a standard hydrogen electrode, a calomel electrode or a silver/silver chloride electrode; the working electrode is a carbon, glassy carbon, gold or platinum electrode modified by a metal nano material, a carbon nano material, a metal oxide material or a high molecular polymer material.
Further, in the fifth step, the electrochemical detection method is voltammetry, and the voltammetry is Linear Sweep Voltammetry (LSV), Cyclic Voltammetry (CV), alternating voltammetry (ACV), Square Wave Voltammetry (SWV), conventional pulse voltammetry (NPV), and Differential Pulse Voltammetry (DPV).
The invention has the following advantages:
the electrochemical detection method provided by the invention promotes the release of electroactive substances in the thallus by water bath heating, and the electrochemical method is used for direct detection, so that the electrochemical volt-ampere response of bacteria is effectively enhanced, the sensitivity and accuracy of bacterial activity detection are improved, the source of the active substances can be revealed, the electrochemical detection method has the advantages of rapidness, simplicity, convenience, low cost and capability of continuously detecting bacterial samples, and a new thought can be provided for the aspect of microbial activity detection by using the electrochemical method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a comparison of cyclic voltammograms of Staphylococcus aureus treated with or without water bath heating in the test group and the control group provided in example 1 of the present invention;
FIG. 2 is a graph plotting the growth of Staphylococcus aureus in the test group and the control group 1 provided in example 2 of the present invention;
FIG. 3 is a graph plotting the growth of Staphylococcus aureus in the test group and the control group 2 provided in example 2 of the present invention;
fig. 4 is a trend graph of the peak current value of the test group and the Log value of the viable count of the control group according to the change of the concentration of levofloxacin hydrochloride provided in example 3 of the present invention;
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1 to 4, an electrochemical detection method for bacterial activity provided by an embodiment of the present invention includes the following steps:
firstly, recovering and culturing bacteria;
secondly, washing bacteria and preparing a bacterial suspension;
thirdly, carrying out detection pretreatment on the bacterial suspension, wherein the pretreatment adopts a water bath heating method;
fourthly, centrifuging the heated bacterial suspension and sucking the supernatant;
and fifthly, carrying out electrochemical detection on the supernatant by using an electrode system.
According to a specific embodiment provided by the invention, the water bath temperature of the bacterial suspension in the step three is 30-80 ℃, and the water bath time is 5-50 min.
According to a specific embodiment provided by the invention, the water bath temperature is 50 ℃, and the water bath time is 10 min.
According to a specific embodiment provided by the invention, the water bath temperature is 55 ℃, and the water bath time is 20 min.
According to a specific embodiment of the present invention, in the first step, the bacteria include gram-negative bacteria and gram-positive bacteria.
According to a specific embodiment of the present invention, in the first step, the gram-positive bacteria represent staphylococcus aureus.
According to an embodiment of the present invention, in the second step, the solution used for washing is a PBS solution with a pH of 6.5 to 7.5, and the number of washing times is 3 to 5.
According to a specific embodiment provided by the present invention, in the second step, the solution used for washing is a PBS solution with pH 7.4, and the number of washing times is 3.
According to a specific embodiment provided by the invention, in the second step, in the preparation of the bacterial suspension, the liquid used for suspension is a PBS solution with pH of 6.5-7.5.
According to a specific embodiment provided by the invention, in the second step, in the preparation of the bacterial suspension, the liquid used for suspension is a PBS solution with the pH value of 7.4.
According to a specific embodiment provided by the invention, in the fourth step, the centrifugal rotation speed is 4000-8000 rpm, and the centrifugal time is 5-15 min.
According to a specific embodiment provided by the invention, the rotating speed is 6000rpm, and the centrifugation time is 10 min.
According to a specific embodiment provided by the present invention, in the fifth step, the electrode system used is a three-electrode system, the three-electrode system includes an auxiliary electrode, a reference electrode and a working electrode, and the auxiliary electrode is a platinum counter electrode or a carbon counter electrode; the reference electrode is a standard hydrogen electrode, a calomel electrode or a silver/silver chloride electrode; the working electrode is a carbon, glassy carbon, gold or platinum electrode modified by a metal nano material, a carbon nano material, a metal oxide material or a high molecular polymer material.
According to a specific embodiment provided by the present invention, the auxiliary electrode is a platinum counter electrode; the reference electrode is a silver/silver chloride electrode; the working electrode is a glassy carbon electrode modified by a carboxylated carbon nanotube.
According to a specific embodiment provided by the present invention, in the fifth step, the electrochemical detection method is voltammetry, and the voltammetry is Linear Sweep Voltammetry (LSV), Cyclic Voltammetry (CV), Alternating Current Voltammetry (ACV), Square Wave Voltammetry (SWV), conventional pulse voltammetry (NPV), and Differential Pulse Voltammetry (DPV).
The invention will be further illustrated with reference to specific examples:
example 1:
the pretreatment method of the bacteria for electrochemical detection of the test group is a water bath heating method.
The method comprises the following specific steps: after the bacteria to be detected are cultured, 5mL of bacterial liquid is sucked, the culture medium is removed through centrifugation, PBS with the pH value of 7.4 is used for washing for three times, 5mL of PBS with the same pH value is used for suspending the bacteria, the bacteria are placed in a water bath with the temperature of 50 ℃ for heating for 10min, and after the heating is finished, the bacteria are centrifuged to suck the supernatant.
The control group electrochemical detection bacteria are not pretreated, and the specific method comprises the following steps: after the bacteria to be detected are cultured, 5mL of bacterial liquid is sucked, the culture medium is removed by centrifugation, PBS with the pH value of 7.4 is used for washing three times, then 5mL of PBS with the same pH value is used for suspending the bacteria, and supernatant liquid is sucked by centrifugation.
And detecting the supernatants of the test group and the control group by using cyclic voltammetry, recording electrochemical peak current values, and enabling the test group to generate obvious peak signals at +0.731V and +1.077V, which shows that the pretreatment mode of the bacteria greatly improves the voltammetry response, and the control group which is not subjected to heat treatment does not generate the peak signals (figure 1).
Example 2:
the method for detecting the activity of the bacteria in the test group is to detect the activity of the bacteria by an electrochemical method after the heat treatment of the bacteria.
The method comprises the following specific steps: adding a blank liquid culture medium with the same volume into a test tube, adding bacterial liquid with the same volume, placing the test tube into a constant-temperature incubator to culture for 0, 1, 2, 4, 6, 8, 10, 12, 24, 28 and 32 hours respectively, shaking the test tube after the culture is finished, uniformly mixing the bacterial liquid, absorbing 5mL of the bacterial liquid, centrifugally removing the culture medium, washing the bacterial liquid for three times by using PBS with the pH value of 7.4, suspending the bacterial liquid by using 5mL of PBS with the same pH value, placing the bacterial liquid in a water bath with the temperature of 50 ℃ to heat for 10min, centrifugally absorbing supernatant, detecting the supernatant by using a cyclic voltammetry, and recording the peak current value.
Wherein, the method for detecting the bacterial activity of the control group 1 is a photoelectric turbidimetry method.
The method comprises the following specific steps: adding a blank liquid culture medium with the same volume into a test tube, adding bacterial liquid with the same volume, placing the test tube into a constant-temperature incubator to culture for 0, 1, 2, 4, 6, 8, 10, 12, 24, 28 and 32 hours respectively, shaking the test tube after the culture is finished, uniformly mixing the bacterial liquid, taking the bacterial liquid to measure absorbance, taking a blank reference as the blank liquid culture medium, and recording the absorbance value.
The method for detecting the bacterial activity of the control group 2 is a plate counting method.
The method comprises the following specific steps: filling blank liquid culture media with the same volume into the test tube, adding bacterial liquid with the same volume, placing the test tube into a constant-temperature incubator to culture for 0, 1, 2, 4, 6, 8, 10, 12, 24, 28 and 32 hours respectively, shaking the test tube after the culture is finished, mixing the bacterial liquid uniformly, diluting the bacterial liquid to carry out plate coating, placing the plate into the constant-temperature incubator to incubate for 24 hours, and recording the number of viable bacteria on the plate.
The growth curves were plotted based on the data of the test group and the control group 1 and 2, and the results are shown in fig. 2 and 3. As can be seen from FIG. 2, the absorbance of the bacterial liquid steadily increases within 0-28h of bacterial growth, the peak current value of the supernatant liquid steadily increases within 0-28h, the increase amplitude is smaller within 0-1, 2-4 and 8-10h, the peak current value starts to decrease within 28-32h, the integral trend of the growth curve drawn according to the electrochemical method of bacterial heat treatment and the photoelectric turbidimetry is approximately similar, but after 28h, the absorbance tends to be steady, the visible photoelectric turbidimetry cannot distinguish dead bacteria from live bacteria, so that the actual bacterial activity cannot be reflected, and the peak current value measured by the electrochemical method of bacterial heat treatment shows a decreasing trend after 28h, which is more accurate than the photoelectric turbidimetry. As can be seen from FIG. 3, the viable count increases slowly within 0-10h, increases rapidly within 10-12h, increases slowly within 12-28h, and decreases within 28-32h, the overall trend of the growth curve drawn by the electrochemical method of bacterial heat treatment and the flat plate counting method is approximately similar, and the growth curve starts to exhibit a decreasing trend at 28h of bacterial growth.
Example 3:
1. the test group is used for evaluating the bacteriostatic activity of the drug by detecting the bacteria after heat treatment by using an electrochemical method
The method comprises the following specific steps: adding 10mL of blank liquid culture medium into a test tube, adding 2mL of bacterial liquid, adding 1mL of levofloxacin hydrochloride liquid with the drug concentration of 0, 2, 4, 8, 32, 128, 192 and 384 mu g/mL, placing the test tube into a constant-temperature incubator for incubation for 3h, shaking the test tube after the culture is finished to mix the bacterial liquid uniformly, sucking 5mL of bacterial liquid, centrifugally removing the culture medium, washing the bacterial liquid three times by using PBS with the pH of 7.4, suspending the bacterial liquid by using 5mL of PBS with the same pH value, placing the suspended bacterial liquid in a water bath at 50 ℃ for heating for 10min, centrifugally sucking the supernatant after heating, detecting the supernatant by using a cyclic voltammetry, and recording the peak current value.
2. Drug bacteriostasis evaluation of control group by using plate counting method
The method comprises the following specific steps: adding 10mL of blank liquid culture medium into a test tube, adding 2mL of bacterial liquid, adding 1mL of levofloxacin hydrochloride liquid with drug concentrations of 0, 2, 4, 8, 32, 128, 192 and 384 mu g/mL, placing the test tube into a constant-temperature incubator for incubation for 3h, shaking the test tube after the incubation is finished, uniformly mixing the bacterial liquid, diluting the bacterial liquid, coating, placing the diluted bacterial liquid into the constant-temperature incubator for incubation for 24h, recording the number of viable bacteria on a flat plate after the incubation is finished, and calculating the Log value of the number of viable bacteria per mL.
3. Results and conclusions of the experiment
The data of the results of the test group and the control group are shown in FIG. 4. As can be seen from FIG. 4, the Log value of viable count rapidly decreases when the concentration of the drug is 0-32. mu.g/mL; when the concentration of the medicine is 32-192 mug/mL, the Log value of the viable count is slowly reduced; the Log value of the viable bacteria number tends to be stable and does not change greatly when the concentration of the medicament is 192-384 mu g/mL. The peak current value of the supernatant is not changed greatly when the drug concentration is 0-8 mug/mL; when the concentration of the drug is 8-192 mug/mL, the peak current value is gradually reduced; when the drug concentration is 192-. The peak current value and the viable bacteria number Log value show descending trend along with the increase of the drug concentration, and then are gradually stable, which shows that the bacteriostatic activity of the drug is gradually increased along with the increase of the drug concentration until no great change exists, and the peak current value and the viable bacteria number Log value are generally similar on the whole along with the change trend of the drug concentration, which shows that the bacteria after the electrochemical method detection heat treatment can be used for representing the activity of the bacteria and can be applied to the evaluation of the bacteriostatic activity of the drug.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. An electrochemical detection method for bacterial activity is characterized by comprising the following steps:
firstly, recovering and culturing bacteria;
step two, washing bacteria and preparing a bacteria suspension;
step three, carrying out detection pretreatment on the bacterial suspension, wherein the pretreatment adopts a water bath heating method;
step four, centrifuging the bacterial suspension heated in the water bath, and sucking the supernatant;
and step five, carrying out electrochemical detection on the supernatant by using an electrode system.
2. The electrochemical detection method for bacterial activity according to claim 1, wherein in the third step, the water bath temperature of the bacterial suspension is 30-80 ℃, and the water bath time is 5-50 min.
3. The electrochemical detection method for bacterial activity according to claim 2, wherein the water bath temperature is 50-55 ℃ and the water bath time is 10-20 min.
4. The electrochemical method for detecting bacterial activity of claim 1, wherein in step one, said bacteria comprise gram negative bacteria and gram positive bacteria.
5. The electrochemical detection method for bacterial activity according to claim 1, wherein in the second step, the solution used for washing is PBS solution with pH of 6.5-7.5, and the number of washing is 3-5.
6. The electrochemical detection method for bacterial activity according to claim 1, wherein in the second step, in the preparation of the bacterial suspension, the liquid used for suspension is a PBS solution with pH of 6.5-7.5.
7. The electrochemical detection method for bacterial activity according to claim 1, wherein in the fourth step, the centrifugal rotation speed is 4000-8000 rpm, and the centrifugal time is 5-15 min.
8. The electrochemical detection method of bacterial activity according to claim 7, wherein said rotation speed is 6000rpm and the centrifugation time is 10 min.
9. The electrochemical detection method of bacterial activity according to claim 1, wherein in step five, the electrode system used is a three-electrode system comprising an auxiliary electrode, a reference electrode and a working electrode, wherein the auxiliary electrode is a platinum counter electrode or a carbon counter electrode; the reference electrode is a standard hydrogen electrode, a calomel electrode or a silver/silver chloride electrode; the working electrode is a carbon, glassy carbon, gold or platinum electrode modified by a metal nano material, a carbon nano material, a metal oxide material or a high molecular polymer material.
10. The electrochemical detection method of bacterial activity according to claim 1, wherein in step five, the electrochemical detection method is voltammetry;
the voltammetry is linear sweep voltammetry, cyclic voltammetry, alternating voltammetry, square wave voltammetry, regular pulse voltammetry, and differential pulse voltammetry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111325120.5A CN114047241A (en) | 2021-11-10 | 2021-11-10 | Electrochemical detection method for bacterial activity |
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| Application Number | Priority Date | Filing Date | Title |
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