CN110530957B

CN110530957B - Electrochemical detection method for bacterial drug resistance

Info

Publication number: CN110530957B
Application number: CN201910794286.8A
Authority: CN
Inventors: 孙秀兰; 任奕婧; 纪剑; 孙嘉笛; 皮付伟; 张银志
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2021-01-29
Anticipated expiration: 2039-08-27
Also published as: CN110530957A

Abstract

The invention discloses a bacterial drug resistance electrochemical detection method, and belongs to the technical field of electrochemical chips and bacterial detection. The invention utilizes a specific sample solution containing resazurin and combines a differential pulse voltammetry to detect the drug sensitivity of bacteria under a specific electrochemical environment. The method can be used for directly detecting the activity of bacteria, has high accuracy and simple operation, can be used for determining the drug resistance and the minimum inhibitory concentration in a short time, and can be widely used for various researches and clinical detections in the fields of medicine and the like.

Description

Electrochemical detection method for bacterial drug resistance

Technical Field

The invention belongs to the technical field of electrochemical chips and bacterial detection, and particularly relates to a bacterial drug resistance electrochemical detection method.

Background

Resazurin (RZ), also called sodium azurin, resin azurin, is a redox dye, is dark blue or black, has green luster, and is a safe and nontoxic water-soluble dye. As redox indicators, in cytobiology research make internal disorder or usurp, it is commonly used for the functional assay of mitochondria (resazurin method) and the assessment of cell viability and cell proliferation ability or for the assay of bacteria; as an oxygen-free indicator, the method can be used for preparing a microorganism culture medium. Meanwhile, resazurin is used as a redox mediator to regulate the metabolic activity of microorganisms. Metabolically active substances are capable of irreversibly reducing resazurin (blue) to resorufin (pink), which continues to be reversibly reduced to dihydrofuran (white). Resazurin (disappearance of blue color) has been used to characterize bacterial contamination in milk, to test various biological materials, such as biochemical antioxidants, to determine cell viability, semen quality, sensitivity tests for pathogenic microorganisms, including many gram negative bacteria, enterococci, reproductive bacteria, and to quantify microbial activity in sediments. The resazurin also has electrochemical properties in the reduction process, dead bacteria lose metabolic capability and cannot reduce the resazurin, the current cannot be reduced, the viable bacteria with metabolic capability reduce electrochemistry, and the tested current can be obviously reduced, so the substance can specifically detect the viable bacteria with metabolic capability.

Over-use of broad-spectrum antibiotics and improper use of antibiotics have long led to an increase in antibiotic-resistant strains, which have posed an increasing threat to global public health. The rapid spread of antibiotic resistance and its associated problems in the areas of food safety, clinical use and bio-environmental monitoring, requires rational use of antibiotics to prevent overuse or improper use. Moreover, many genes encoding drug resistance can be transferred and spread among pathogenic bacteria of animals and human beings, thus seriously harming the breeding industry and human health. Therefore, the development of bacterial drug resistance detection is of great significance in medicine, food, public health and other aspects.

In response to this threat, there is an urgent need to perform antimicrobial drug susceptibility testing (AST) to quickly diagnose salmonella resistance to ensure rapid and accurate use of antibiotics. There are a variety of laboratory methods available for characterizing the in vitro sensitivity of microorganisms to antibiotics. Conventional AST has a macrobouillon dilution and microdilution test, a paper diffusion test and an agar dilution method. The conventional detection method for bacterial drug resistance has the advantages of simple operation and low cost, and certainly, has some detection methods with better effect, such as a test card method, an mPCR method and the like, wherein the mPCR method is a molecular detection method based on resistance genes in microorganisms and has the characteristics of rapidness, high efficiency and the like. However, the conventional bacterial drug resistance method is time-consuming and easy to interfere, the diagnostic result of the molecular biology method represented by PCR still needs to be verified by the conventional method, the drug sensitivity test based on bacterial culture cannot be replaced, and the actual field detection has great limitation. Furthermore, the development of resistance genetic mechanisms and the simultaneous testing of limits for all possible mutations indicate the utility of bacterial viability assays in antibiotic susceptibility tests.

Therefore, the existing conventional laboratory method for detecting drug sensitivity cannot quickly and conveniently obtain a drug resistance result, cannot quickly and accurately judge the drug resistance condition, and can cause great adverse consequences in practical application.

Disclosure of Invention

In order to realize rapid and accurate detection of bacterial drug resistance, the invention constructs a method based on electrochemical detection, can rapidly, accurately and specifically detect bacterial drug resistance, and solves the technical problems of long detection time, complicated steps and complicated required instruments in the prior art of drug resistance detection.

The electrochemical method used in the present invention requires only simple electronics and can perform electronic detection directly from confined droplets. The electrochemical detection is fast, simple and convenient, and importantly, the cost is low, and the electrochemical detection can be used together with other technologies, so that the electrochemical sensor has considerable prospect in practical application.

The principle of the invention is that based on the electrochemical property of the resazurin, the resazurin is reduced into the resorufin through living bacteria metabolism, the resazurin and the resorufin have different electrochemical signals, and the reduction degree of the resazurin can be expressed according to the current intensity, so that the effect of the antibiotics can be judged.

The invention aims to provide an electrochemical method for detecting bacterial drug resistance, which comprises the following steps:

(1) modifying the screen printing electrode by using the multi-walled carbon nano tube and the electrogilding nano particles to obtain a modified screen printing electrode;

(2) taking the modified screen printing electrode obtained in the step (1) as a working electrode, adding a sample solution on the working electrode, and measuring a current value by adopting a differential pulse voltammetry method; the sample solution comprises bacteria, antibiotics and resazurin, wherein the solubility of the resazurin is 0.1-0.5 mmol/L.

In one embodiment of the invention, the concentration of the multi-walled carbon nanotubes in the modified screen-printed electrode is 0.1-5 mg/mL.

In one embodiment of the invention, the gold-electroplated nanoparticles in the modified screen-printed electrode are prepared by using 10mL of a solution containing 1% chloroauric acid and 1mmol/L sulfuric acid as a gold-plating solution. And a time current method is adopted, wherein the electroplating voltage is-0.2-0.4V, preferably-0.3V; the plating time is 80-120s, preferably 100 s.

In one embodiment of the invention, the modified screen-printed electrode is prepared by immersing the screen-printed electrode in an ethanol aqueous solution (4:1, v/v) for 1min, and then rinsing the screen-printed electrode with ultrapure water.

After the screen printing electrode is modified to enhance an electric signal, the current of the resazurin solution is detected on the electrode through further cultivation of bacteria, antibiotics and resazurin. And the multi-walled carbon nanotubes and the electrogilding nanoparticles are dripped on the surface of the screen printing electrode, so that the electron transfer rate is increased, and the sensitivity and the signal intensity are increased.

In one embodiment of the present invention, the sample solution in step (2) is prepared by culturing Salmonella typhimurium and Staphylococcus aureus in NB medium at 37 deg.C in a shaker, and diluting to 7.5 × 10⁵cfu/mL; adding resazurin to a concentration of 0.2mmol/L, adding different amounts of antibiotic ofloxacin, mixing, and incubating in a metal bath with shaking at 37 deg.C for 1-6 hr.

In one embodiment of the present invention, it is also necessary to prepare a solution of the same bacteria without antibiotic addition for use in a control experiment.

Preferably, the culture time of the bacteria, the antibiotics and the resazurin is 4 hours.

In one embodiment of the present invention, the step (2) comprises filtering the sample solution through a 0.22 μm filter to remove bacteria, preventing them from affecting the measurement signal, and then measuring the electrochemical value of the solution: dropping 100-150 mul of sample solution on a screen printing electrode modified by multi-wall carbon nano tubes-gold nano particles, and measuring by adopting a differential pulse voltammetry method, wherein the voltage setting range is as follows: -0.7V-0V, the current value in the scan is recorded.

In one embodiment of the invention, a modified screen printed electrode is used as the test element, which uses PET as the substrate, carbon as the working electrode, silver/silver chloride as the reference electrode, and carbon as the counter electrode. The electrical signal of the resazurin concentration was then tested using cyclic voltammetry, Differential Pulse Voltammetry (DPV), at the following voltages: the current change of the differential pulse voltammetry is more obvious when the voltage is between-0.1 and-0.7V, and the current change of the cyclic voltammetry is not obvious, so that the differential pulse voltammetry is selected for carrying out subsequent drug resistance tests.

The detection part of the invention needs to be completed on an electrochemical detector, and the structure of the detection part is as follows: the electrochemical detector is an electrochemical workstation with set voltage, is provided with an LED display screen and is used for recording an impedance value and a current value formed when electrons flow from a counter electrode, pass through an electric conduction liquid and then flow back to the workstation from the working electrode.

The invention has the beneficial effects that:

the method monitors the metabolic activity of bacteria by utilizing the electrochemical properties of resazurin, can realize on-site detection, has high sensitivity and simple operation, can not carry out complex treatment on samples, and has relatively simple instruments and equipment. The invention directly detects the activity of bacteria, can determine the drug resistance and the minimum inhibitory concentration within a few hours, and can be popularized and applied to various researches and clinical detections in the fields of medicine and the like.

Drawings

FIG. 1 is a schematic diagram of the preparation and detection principle of a modified screen printed electrode;

FIG. 2 shows the result of detecting drug resistance of Salmonella typhimurium;

FIG. 3 shows the result of the detection of Staphylococcus aureus resistance;

FIG. 4 is an electrochemical signal of solutions of resazurin at different concentrations; wherein A is an electrochemical signal measured by cyclic voltammetry CV on an AuNP/MWCNT/SPCE substrate; b is an electrochemical signal obtained by differential pulse voltammetry DPV on an AuNP/MWCNT/SPCE substrate; c is an electrochemical signal measured on a bare SPCE base by cyclic voltammetry CV; d is the electrochemical signal obtained from differential pulse voltammetry DPV on a bare SPCE substrate.

Detailed Description

The embodiments of the present invention are illustrated below by specific examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various other respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments of the invention are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below.

Referring to fig. 1, the preparation of the electrochemical sensor is described, which includes the following steps: (1) modifying a multi-walled carbon nanotube on the surface of a screen printing electrode; (2) immersing the multiwalled carbon nanotube screen-printed electrode in a gold plating solution, and modifying the multiwalled carbon nanotube-gold nanoparticle screen-printed electrode by electroplating; (3) and then placing the reaction solution of the bacteria, the antibiotics and the resazurin on an electrode for detection.

Preparing a modified screen printing electrode:

(1) firstly, modifying a multi-wall carbon nanotube on the surface of a screen printing electrode, wherein the modification method comprises the following steps: activating a screen printing electrode in 0.5mol/L iron-sulfuric acid solution, scanning by adopting cyclic voltammetry, and controlling the voltage as follows: -0.2V to 0.6V, scan rate: 50mv/s, number of scan cycles: 10; preparing a 1mg/mL multi-walled carbon nanotube solution, carrying out ultrasonic treatment for 30min, uniformly mixing the solution, dripping the solution on the surface of the activated screen-printed electrode, and placing the activated screen-printed electrode at room temperature to naturally air-dry the activated screen-printed electrode;

(2) and then modifying the gold nanoparticles by electroplating, wherein the modification method comprises the following steps: immersing the multi-wall carbon nano tube screen printing electrode in 10mL of gold plating solution containing 1% chloroauric acid and 1mmol/L sulfuric acid solution for gold plating, and adopting a time current method, wherein the plating voltage is as follows: -0.3V, plating time: 100 s; and finally, soaking the screen printing electrode in an ethanol water solution (4:1, v/v) for 1min, and then leaching the screen printing electrode with ultrapure water to finish the preparation of the multi-wall carbon nanotube-gold nanoparticle screen printing electrode.

Example 1

(1) And (3) bacterial treatment:

salmonella typhimurium ATCC14028 was cultured in NB medium at 37 ℃ in a shaker, and then turbidity was measured after background subtraction using a bacterial turbidimeter and converted into the original concentration of the bacterial liquid. The bacterial liquid was diluted to 7.5X 10 with NB medium according to the original concentration⁵cfu/mL. Adding resazurin and antibiotics in different dosageMixing ofloxacin, and incubating for 4 hours at 37 ℃ in a metal bath with shaking; wherein the concentration of resazurin in the solution is 0.2mmol/L, and the concentration of antibiotic ofloxacin is 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125. mu.g/mL respectively. One identical bacterial solution was also prepared for the control experiment without antibiotic addition.

(2) And (3) drug resistance detection:

to detect bacterial resistance, the incubated solution was filtered through a 0.22 μm filter to remove bacteria, and then the electrochemical value of the solution was measured. And (2) dropping 150 mu l of solution on the screen printing electrode modified with the multi-wall carbon nano-tubes and the gold nano-particles prepared by the method, and measuring by adopting Differential Pulse Voltammetry (DPV), wherein the voltage setting range is as follows: and (4) recording the current value in scanning at-0.7V-0V, and drawing a detection result after calculation to obtain the minimum inhibitory concentration of the bacterium. Relative Survival (SR) is the ratio of the current of the reductive change at each concentration of antibiotic to the current of the change in the control.

As can be seen from FIG. 2, the minimal inhibitory concentration of Salmonella typhimurium is 8. mu.g/mL.

Example 2

(1) And (3) bacterial treatment:

staphylococcus aureus ATCC6538 was cultured in NB medium in a shaker at 37 ℃ and then turbidity was measured after background subtraction using a bacterial turbidimeter and converted to the original concentration of the bacterial liquid. The bacterial liquid was diluted to 7.5X 10 with NB medium according to the original concentration⁵cfu/mL. Then adding resazurin and the antibiotic ofloxacin with different dosages, mixing, and oscillating and incubating for 4 hours in a metal bath at 37 ℃; wherein the concentration of resazurin in the solution is 0.2mmol/L, and the concentration of antibiotic ofloxacin is 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125. mu.g/mL. One identical bacterial solution was also prepared for the control experiment without antibiotic addition.

(2) And (3) drug resistance detection:

to detect bacterial resistance, the incubated solution was filtered through a 0.22 μm filter to remove bacteria, and then the electrochemical value of the solution was measured. Dripping 150 mul of the solution on a multi-wall carbon nano tube-gold nano particle screen printing electrode, and measuring by adopting a differential pulse voltammetry method: voltage setting range: and (4) recording the current value in scanning at-0.7V-0V, and drawing a detection result after calculation to obtain the minimum inhibitory concentration of the bacterium. Relative Survival (SR) is the ratio of the current of the reductive change at each concentration of antibiotic to the current of the change in the control.

As can be seen from FIG. 3, the minimum inhibitory concentration of Staphylococcus aureus was 32. mu.g/mL.

Example 3 validation experiment

Determination of Minimum Inhibitory Concentration (MIC) of antibacterial drug by broth dilution method: sterile operation, adding the antibacterial drug Ofloxacin solution (Ofloxacin) with different concentrations after dilution in multiple proportions into a sterile 96-hole polystyrene plate respectively, adding the liquid medicine into the 1 st to 11 th holes, wherein each hole is 10 mu L, and the 12 th hole is not added with the liquid medicine to be used as a growth control.

Diluting different bacterial suspensions prepared by growth method or direct bacterial suspension method with concentration equivalent to 0.5 McLeod's ratio standard with NB broth 1: 1000, adding 100 μ l into each well, sealing, placing in a common air incubator at 37 deg.C, and incubating for 24h to determine the result. At this time, the drug concentrations in the 1 st to 11 th wells were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125. mu.g/mL (corresponding to Log)₂C is shown in table 1). The cultured 96-well plate was placed in an OD600 microplate reader for measurement, whereby drug resistance data were obtained, and the results are shown in Table 2.

TABLE 1 minimum inhibitory concentration and corresponding Log₂C

Table 2 results of verifying the accuracy of the detection methods of examples 1-2

As shown in Table 2, the MIC values obtained in example 1-2 are generally about 1 order of magnitude greater than those obtained in the standard test (the order of magnitude here refers to 2 orders of magnitude), and the method belongs to a narrower application range, and can indicate more accurately the appropriate MIC application conditions of the drug. Moreover, the method in example 1-2 has good consistency with the results measured by the standard method, and can effectively judge the drug resistance of bacteria.

Example 4 electrochemical assay Condition optimization

(1) Selection of different resazurin solubilities:

4, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02 and 0mM of resazurin solution is dripped on a bare screen printing electrode and a modified screen printing electrode, different signal differences are observed, and an appropriate resazurin concentration is selected as a working concentration. As a result, the high-concentration resazurin has two cathode peaks, while the low-concentration resazurin only shows one cathode peak, but the current value of the low concentration is smaller, and the prediction accuracy is lower; therefore, in consideration of obtaining clearer detection data and larger current value at the same time, a solution of resazurin of 0.1-0.5mM having only one cathodic peak and larger current is selected, and the optimum concentration is 0.2mM of resazurin as the working concentration.

(2) Selection of different electrochemical assay methods:

a series of concentration gradients of resazurin were tested for electrochemical signal in NB medium using different methods, respectively. FIG. 4-A is the electrochemical signal measured by cyclic voltammetry CV on AuNP/MWCNT/SPCE substrate, as shown in FIG. 4; FIG. 4-B is the electrochemical signal obtained from differential pulse voltammetry DPV on AuNP/MWCNT/SPCE substrate; FIG. 4-C is an electrochemical signal measured at a bare SPCE base with cyclic voltammetry CV; FIG. 4-D is the electrochemical signal obtained from differential pulse voltammetry DPV on a bare SPCE substrate. Wherein, a: 4 mM; b: 2 mM; c: 1 mM; d: 0.5 mM; e: 0.2 mM; f: 0.1 mM; g: 0.05 mM; h: 0.02 mM; i: 0 mM.

The electrochemical principle of Resazurin: the cathodic peak of high potential corresponds to the conversion of Resazurin to resorufin in the irreversible two-electron process, while the cathodic peak of low potential and the oxidation peak correspond to the conversion of resorufin to dihydrofuran in the reversible two-electron process. As can be seen from FIG. 4, the electrochemical signals of Resazurin at different concentrations showed significant differences, and the current increased significantly with increasing concentration. As the concentration increases, the total peak value of resazurin shifts to a low potential, and the peak shape becomes wider. However, when measured at low concentrations, the two cathodic peaks sometimes overlap, so only one peak appears in the CV and DPV plots. Both CV and DPV showed typical electrochemical signals of resazurin, but the signal peak representing the reduction of resazurin to resorufin was more pronounced on DPV and the current of DPV was stronger, so DPV was chosen as the detection index.

Claims

1. An electrochemical method for detecting drug resistance of salmonella typhimurium and staphylococcus aureus is characterized by comprising the following steps:

(2) taking the modified screen printing electrode obtained in the step (1) as a test element, filtering a sample solution through a filter, then dripping the sample solution on the modified screen printing electrode for electrochemical detection, and measuring a current value by adopting a differential pulse voltammetry method; the sample solution comprises bacteria, antibiotics and resazurin, wherein the solubility of the resazurin is 0.1-0.5 mmol/L; the concentration of the multi-wall carbon nano tube in the modified screen printing electrode is 0.1-5 mg/mL;

the sample solution is prepared by mixing, culturing and incubating bacteria, resazurin and antibiotics for 4 h;

the antibiotic is ofloxacin.

2. The method of claim 1, wherein the solubility of resazurin is 0.1-0.4 mmol/L.

3. The method according to claim 1, wherein the gold-electroplated nanoparticles in the modified screen-printed electrode are prepared by using 10mL of a solution containing 1% chloroauric acid and 1mmol/L sulfuric acid as a gold-plating solution.

4. The method as claimed in claim 1 or 3, wherein the plating voltage is-0.2-0.4V and the plating time is 80-120s when the gold nanoparticles are plated to modify the screen-printed electrode.

5. The method according to any one of claims 1 to 3, wherein the voltage at the time of electrochemical detection in step (2) is-0.7V to 0V.