CN110530957A - A kind of bacterial drug resistance electrochemical detection method - Google Patents
A kind of bacterial drug resistance electrochemical detection method Download PDFInfo
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- CN110530957A CN110530957A CN201910794286.8A CN201910794286A CN110530957A CN 110530957 A CN110530957 A CN 110530957A CN 201910794286 A CN201910794286 A CN 201910794286A CN 110530957 A CN110530957 A CN 110530957A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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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
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a kind of bacterial drug resistance electrochemical detection methods, belong to electrochemical chip and Bacteria Detection technical field.The present invention has carried out drug sensitivity detection to bacterium in conjunction with Differential Pulse Voltammetry using under the specifically sample solution containing resazurin again specific electrochemical environment.The method of the present invention can be detected directly against bacterial activity, had accuracy high, easy to operate, can be carried out the measurement of drug resistance, minimum inhibitory concentration in a short time, can be widely used for the various researchs and clinical detection in the fields such as medicine.
Description
Technical field
The invention belongs to electrochemical chips and Bacteria Detection technical field, and in particular to a kind of bacterial drug resistance electrochemistry inspection
Survey method.
Background technique
Resazurin (Resazurin, RZ) is also referred to as day orchidization sodium, and resin is reddish black, is redox dye, is in navy blue or black
Color has green gloss, is a kind of safe and non-toxic water-soluble dye.As oxidation-reduction indicator, grinds and make internal disorder or usurp in cell biology
In, it is usually used in the Function detection (resazurin method) of mitochondria and the assessment of cell viability and ability of cell proliferation or for bacterium
Detection and analysis；As no oxygen indicator, it can be used for the preparation of microbiological culture media.Resazurin is as a kind of redox simultaneously
Mediator can be with the metabolic activity of regulating and controlling microbial.Having the active material of metabolic capability can irreversibly restore resazurin (blue)
For resorufin (pink), resorufin will continue to reversibly be reduced to dihydrofuran (white).Resazurin (blue disappearance)
The germ contamination in characterization milk is had been used for, various biomaterials are tested, such as biochemical antioxidant measures cell viability, sperm
Quality, the sensitivity tests of pathogenic microorganism, including many Gram-negative bacterias, enterococcus, reproduction bacterium, and quantization deposit
In microbial activity.Resazurin in reduction process also have electrochemical properties, dead bacterium lose metabolic capability and cannot
Resazurin is restored, electric current would not also become smaller, and have the viable bacteria reduction electrochemistry of metabolic capability, and the electric current of test can significantly reduce,
So the substance energy specific detection has the work bacterium of metabolic capability.
For a long time, broad-spectrum antibiotic and improper use antibiotic, which is excessively used, causes antibiotics resistance bacterial strain to increase, this
Increasing threat is constituted to global public health.It the fast propagation of antibiotic resistance and its is answered in food safety, clinic
With the relevant issues with biotic environment monitoring field, Using adapted Antibios are needed to prevent excessive or improper use.And permitted
The gene of odd encoder drug resistance mutually can be shifted and be propagated between animals and humans pathogen, seriously endanger aquaculture and the mankind are strong
Health.Therefore, carry out bacterial drug resistance detection medicine, food and in terms of be all of great significance.
In order to cope with this threat, there is an urgent need to carry out antibacterial drug sensitive test (AST), with the resistance to of quick diagnosis salmonella
Pharmacological property, to ensure rapidly and accurately to use antibiotic.It can be used for characterizing microorganism to the body of antibiotic there are many laboratory method
Outer sensibility.Conventional AST has constant broth dilution and Microdilution test, scraps of paper diffusion test and agar dilution.Bacterium is resistance to
The advantages of common detection methods of pharmacological property be it is easy to operate, at low cost, certainly, there are also the preferable detection method of some effects, than
If any examination block-regulations, mPCR method etc., wherein mPCR method is a kind of molecular detecting method based on resistant gene in microorganism, is had fast
The features such as fast, efficient.But conventional bacterial drug resistance method often than it is relatively time-consuming, vulnerable to interference, using PCR as the molecule of representative
The diagnostic result of biological method need to be verified toward contact with conventional method, still cannot replace the susceptibility examination based on Bacteria Culture
It tests, has biggish limitation in actual field detection.And the development of genetics of resistance mechanism and to it is all it is possible be mutated while
The limitation of test shows effectiveness of the bacterial action measurement in antibiotics sensitivity test.
It follows that detecting drug sensitivity using existing conventional laboratory methods, drug resistance cannot be quickly and conveniently obtained
Property as a result, and be unable to fast accurate and determine Drug resistance status, will cause biggish adverse consequences in practical applications.
Summary of the invention
In order to realize that fast and accurately detection bacterium drug resistance, the present invention construct the method based on Electrochemical Detection, energy
It is enough quickly, it is accurate, without specific detection bacterial drug resistance, solving Resistance detection, detection time is long in the prior art, step is mixed
The technical problem of miscellaneous, required instrument complexity.
The electrochemical method that the present invention uses only needs simple electronic device, and can be direct from restricted drop
Carry out detection of electrons.It is the fast speed of Electrochemical Detection, simple, conveniently, it is important that cost is relatively low, can join with other technologies
It closes and uses, so the prospect of electrochemical sensor in practical applications is considerable.
The principle of the present invention is the electrochemical properties based on resazurin, by viable bacteria metabolism by resazurin reduction at examination halogen
Spirit, resazurin and resorufin have different electrochemical signals, and the reducing degree of resazurin can be indicated according to current strength,
It is possible thereby to the effect to differentiate antibiotic.
The object of the present invention is to provide a kind of electrochemical methods that can be used in detection bacterium drug resistance, including following step
It is rapid:
(1) using multi-walled carbon nanotube and plating gold nano particle modification screen printing electrode, modification silk-screen printing is obtained
Electrode；
(2) gained modification screen printing electrode is as working electrode using in step (1), by sample solution addition in work electricity
On extremely, current value is measured using Differential Pulse Voltammetry；Including bacterium, antibiotic and resazurin in the sample solution, wherein
The solubility of resazurin is 0.1-0.5mmol/L.
In one embodiment of the invention, the concentration of multi-walled carbon nanotube is in the modification screen printing electrode
0.1-5mg/mL。
In one embodiment of the invention, gold nano grain is electroplated in the modification screen printing electrode is to pass through
10mL contains 1% gold chloride and 1mmol/L sulfuric acid solution is prepared as gold plating liquid.And carried out using chronoamperometry,
Middle electroplating voltage is -0.2-0.4V, preferably -0.3V；Electroplating time is 80-120s, preferably 100s.
In one embodiment of the invention, the modification screen printing electrode is that screen printing electrode leaching is placed in second
1min is impregnated in alcohol solution (4:1, v/v), then elutes screen printing electrode with ultrapure water, completes multi-walled carbon nanotube-
The preparation of gold nano grain screen printing electrode.
After modification screen printing electrode enhancing electric signal, pass through the further cultivation of bacterium, antibiotic and resazurin, In
The electric current of resazurin solution is detected on electrode.Also, drop coating multi-walled carbon nanotube and plating Jenner on screen printing electrode surface
Rice grain increases electron transport rate, to increase sensitivity, signal strength.
In one embodiment of the invention, the sample solution in the step (2) is by salmonella typhimurium, gold
Staphylococcus aureus is cultivated in 37 DEG C of shaking tables with NB culture medium respectively, and 7.5 × 10 are diluted to after culture5cfu/mL；So
After resazurin is added, make its concentration 0.2mmol/L, be added different amounts antibiotic Ofloxacin, mixing, at 37 DEG C
Concussion is incubated for 1-6 hours in metal bath.
In one embodiment of the invention, it is also necessary to while preparing one and being not added with antibiotic, other same thin
Bacterium solution is used for check experiment.
Preferably, the incubation time of the bacterium, antibiotic and resazurin is 4 hours.
It in one embodiment of the invention, include that sample solution is passed through into 0.22 μm of filter in the step (2)
Filtering then measures the electrochemistry value of solution: taking 100~150 μ l sample solutions except degerming, to prevent it from influencing measuring signal
It drips on multi-walled carbon nanotube-gold nano particle modification screen printing electrode, is measured using Differential Pulse Voltammetry, electricity
Range: -0.7V~0V, the current value in writing scan is arranged in pressure.
It in one embodiment of the invention, is to modify screen printing electrode as testing element, using PET as base
Bottom, using carbon as working electrode, silver/silver chlorate is used as reference electrode, carbon to electrode.Then using cyclic voltammetry, show
Poor pulse voltammetry (DPV) tests the electric signal of resazurin concentration, applied voltage are as follows: -0.1V~-0.7V, test shown
The curent change of poor pulse voltammetry becomes apparent from, and cyclic voltammetry is unobvious, thus selection Differential Pulse Voltammetry come into
The subsequent drug-resistant test of row.
Detection part needs of the invention are completed on Electrochemical detector, are constructed as follows: Electrochemical detector is
The electrochemical workstation for setting voltage, equipped with LED display, for recording electronics to electrode, by conductance liquid, then from work
The impedance value and current value formed when flowing back to work station as electrode.
Beneficial effects of the present invention:
The present invention monitors the metabolic activity of bacterium using the electrochemical properties of resazurin, and on-site test may be implemented, should
Method high sensitivity, easy to operate, sample can be without complex process, and instrument equipment is relatively easy.The direct needle of the present invention
Bacterial activity is detected, the measurement of drug resistance, minimum inhibitory concentration can be carried out within a few hours, can promote and be suitable for
The various researchs in the fields such as medicine and clinical detection.
Detailed description of the invention
Fig. 1 is that the screen printing electrode of modification prepares schematic diagram and testing principle；
Fig. 2 is salmonella typhimurium Resistance detection result；
Fig. 3 is drug resistance of Staphylococcus aureus testing result；
Fig. 4 is the electrochemical signals of various concentration resazurin solution；Wherein, A is in AuNP/MWCNT/SPCE substrate
The electrochemical signals measured by cyclic voltammetry CV；B is Differential Pulse Voltammetry DPV in AuNP/MWCNT/SPCE substrate
Obtained electrochemical signals；C is the electrochemical signals measured on the naked base SPCE with cyclic voltammetry CV；D is differential pulse voltammetry
The electrochemical signals that voltammetry DPV is obtained in naked SPCE substrate.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example.The present invention can also be by different specific
Embodiment is embodied or practiced, and the various details in this specification can also not carried on the back based on different viewpoints and application
From carrying out various modifications or alterations under spirit of the invention.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to under
Belong to specific specific embodiment.
In conjunction with Fig. 1, the preparation of electrochemical sensor is illustrated, comprising the following steps: (1) in screen printing electrode table
Face modifying multiwall carbon nano-tube；(2) multi-walled carbon nanotube screen printing electrode leaching is placed in gold plating liquid, is repaired by plating
Adorn into multi-walled carbon nanotube-gold nano grain screen printing electrode；(3) reaction solution of bacterium, antibiotic and resazurin is set again
In on electrode, detected.
Modify the preparation of screen printing electrode:
(1) first in screen printing electrode surface modification multi-walled carbon nanotube, method of modifying: screen printing electrode is existed
It activates, is scanned using cyclic voltammetry, voltage in the iron sulfuric acid solution of 0.5mol/L are as follows: -0.2V~0.6V, sweep speed are as follows:
50mv/s, scanning circle number are as follows: 10；The multi-walled carbon nanotube solution of 1mg/mL is configured, ultrasonic 30min, drop coating exists after it is mixed
Screen printing electrode surface after activation, being placed on room temperature makes its natural air drying；
(2) method of modifying: the leaching of multi-walled carbon nanotube screen printing electrode is placed in by plating modified nano gold particle again
10mL contain carried out in the gold plating liquid of 1% gold chloride and 1mmol/L sulfuric acid solution it is gold-plated, using chronoamperometry, electroplating voltage
Are as follows: -0.3V, electroplating time are as follows: 100s；Finally screen printing electrode leaching is placed in ethanol water (4:1, v/v) and is impregnated
Then 1min elutes screen printing electrode with ultrapure water, complete multi-walled carbon nanotube-gold nano grain screen printing electrode
Preparation.
Embodiment 1
(1) bacterial treatment:
Salmonella typhimurium ATCC14028 is cultivated in 37 DEG C of shaking tables with NB culture medium, then uses bacterial turbidity
Instrument measures its turbidity after background correction, then is converted into bacterium solution original concentration.Bacterium solution is diluted with NB culture medium according to original concentration
At 7.5 × 105cfu/mL.Then the antibiotic Ofloxacin mixing of resazurin and different amounts is added, in metal at 37 DEG C
Concussion is incubated for 4 hours in bath；Wherein the concentration of resazurin is 0.2mmol/L in solution, and antibiotic Ofloxacin concentration is respectively
128,64,32,16,8,4,2,1,0.5,0.25,0.125μg/mL.Prepare an identical bacterial solution simultaneously, for compareing
It tests, not added with antibiotic.
(2) Resistance detection:
For detection bacterium drug resistance, the solution after above-mentioned incubation is degermed by 0.22 μm of filter filtering with removing,
The then electrochemistry value of measurement solution.150 μ l solution drop is taken to be modified with multi-walled carbon nanotube-Jenner in what the above method prepared
On the screen printing electrode of rice grain, measured using Differential Pulse Voltammetry DPV, voltage set range: -0.7V~0V,
Current value in writing scan draws testing result after calculating, obtains the minimum inhibitory concentration of the bacterium.Relative survival rate
(SR) it is the ratio between reduction variable-current and the variable-current of control group under the conditions of every kind of concentration antibiotic.
As shown in Figure 2, the minimum inhibitory concentration of salmonella typhimurium is 8 μ g/mL.
Embodiment 2
(1) bacterial treatment:
Staphylococcus aureus ATCC6538 is cultivated in 37 DEG C of shaking tables with NB culture medium, then uses bacterial turbidity
Instrument measures its turbidity after background correction, then is converted into bacterium solution original concentration.Bacterium solution is diluted with NB culture medium according to original concentration
At 7.5 × 105cfu/mL.Then the antibiotic Ofloxacin mixing of resazurin and different amounts is added, in metal at 37 DEG C
Concussion is incubated for 4 hours in bath；Wherein in solution resazurin concentration be 0.2mmol/L, antibiotic Ofloxacin concentration be 128,
64,32,16,8,4,2,1,0.5,0.25,0.125μg/mL.Prepare an identical bacterial solution simultaneously, be used for check experiment,
Not added with antibiotic.
(2) Resistance detection:
For detection bacterium drug resistance, the solution after above-mentioned incubation is degermed by 0.22 μm of filter filtering with removing,
The then electrochemistry value of measurement solution.Take 150 μ l drops in multi-walled carbon nanotube-gold nano grain screen printing electrode solution
On, measured using Differential Pulse Voltammetry: voltage set range: -0.7V~0V, the current value in writing scan pass through
Testing result is drawn after calculating, obtains the minimum inhibitory concentration of the bacterium.Relative survival rate (SR) is every kind of concentration antibiotic condition
Under reduction variable-current and the ratio between the variable-current of control group.
From the figure 3, it may be seen that the minimum inhibitory concentration of staphylococcus aureus is 32 μ g/mL.
3 confirmatory experiment of embodiment
Micro-broth dilution method antibacterials minimum inhibitory concentration (MIC): sterile working, it will be different after doubling dilution
The antibacterials Ofloxacin solution (Ofloxacin) of concentration is added separately in 96 sterile hole polystyrene plates, and the 1st to the 11st
Hole adds medical fluid, every 10 μ L of hole, and the 12nd hole not dosing is used as growth control.
Different bacteria suspensions of 0.5 Maxwell than turbid standard will be equivalent to the concentration of growth method or the preparation of direct bacteria suspension method,
It after the dilution of NB meat soup 1: 1000, to every 100 μ l of Kong Zhongjia, seals in 37 DEG C of normal air incubators of postposition, is incubated for judgement knot for 24 hours
Fruit.At this point, the 1st hole to the 11st hole drug concentration is respectively 128,64,32,16,8,4,2,1,0.5,0.25,0.125 μ g/mL
(corresponding Log2C is as shown in table 1).96 orifice plates after culture are placed in the microplate reader of OD600 and are measured, drug resistance is obtained with this
Data, the results are shown in Table 2.
Table 1 is minimum to inhibit dense and corresponding Log2C
The Accuracy Verification result of 2 embodiment 1-2 detection method of table
As shown in Table 2, the MIC value that embodiment 1-2 is obtained is generally higher than code test 1 several magnitude (numbers herein
Magnitude refers to 2 order of magnitude), belong to relatively narrow use scope, can relatively accurately indicate that the drug MIC being more suitble to is used
Condition.And the result consistency that the method in embodiment 1-2 and standard law measure is preferable, can effectively judge the drug resistance of bacterium
Property.
4 Electrochemical Detection condition optimizing of embodiment
(1) selection of different resazurin solubility:
By 4,2,1,0.5,0.2,0.1,0.05,0.02, the resazurin solution of 0mM drips in naked screen printing electrode and modification
Screen printing electrode on, observe different signal differences, select suitable resazurin concentration as working concentration.As a result it sends out
Existing, the resazurin of high concentration is there are two cathode peak, and low concentration only shows a cathode peak, but the current value of low concentration compared with
Small, the accuracy of prediction is lower；So being based on while obtaining clearer detection data and the consideration of biggish current value, therefore select
The resazurin solution at only one cathode peak and the biggish 0.1-0.5mM of electric current, wherein optimal is dense with 0.2mM resazurin
Degree is used as working concentration.
(2) selection of different electrochemical determination methods:
It is utilized respectively the electrochemical signals that distinct methods test a series of resazurin of concentration gradients in NB culture medium.Such as
Shown in Fig. 4, Fig. 4-A is the electrochemical signals to be measured in AuNP/MWCNT/SPCE substrate by cyclic voltammetry CV；Fig. 4-
B is the electrochemical signals that Differential Pulse Voltammetry DPV is obtained in AuNP/MWCNT/SPCE substrate；Fig. 4-C is with cyclic voltammetric
The electrochemical signals that method CV is measured on the naked base SPCE；Fig. 4-D is that Differential Pulse Voltammetry DPV is obtained in naked SPCE substrate
Electrochemical signals.Wherein, a:4mM；B:2mM；C:1mM；D:0.5mM；E:0.2mM；F:0.1mM；G:0.05mM；H:
0.02mM；I:0mM.
Resazurin electrochemical principle: resazurin is converted into examination during the cathode peak of high potential corresponds to irreversible bielectron
Halogen spirit, and resorufin is converted into dihydrofuran during the cathode peak and oxidation peak of low potential correspond to reversible bielectron.By Fig. 4
It is found that the electrochemical signals of the resazurin of various concentration show significant difference, and electric current is significant with the increase of concentration
Increase.With the increase of concentration, total peak value of resazurin is mobile to low potential, and peak shape broadens.However, ought measure at low concentrations
When, two cathode peaks are overlapped sometimes, therefore only occur a peak in CV and DPV figure.CV and DPV all shows the allusion quotation of resazurin
Type electrochemical signals, but it is more significant on DPV as the signal peak of resorufin to represent resazurin reduction, and the electric current of DPV is more
By force, therefore selected DPV as Testing index.
Claims (10)
1. a kind of electrochemical method of detection bacterium drug resistance, which comprises the steps of:
(1) using multi-walled carbon nanotube and plating gold nano particle modification screen printing electrode, modification screen printing electrode is obtained；
(2) gained modification screen printing electrode is as testing element using in step (1), by sample solution addition in modification screen printing
On the working electrode of brush electrode, current value is measured using Differential Pulse Voltammetry；It include bacterium, antibiotic in the sample solution
And resazurin, wherein the solubility of resazurin is 0.1-0.5mmol/L.
2. the method according to claim 1, wherein the solubility of the resazurin is 0.1-0.4mmol/L.
3. method according to claim 1 or 2, which is characterized in that multi-wall carbon nano-tube in the modification screen printing electrode
The concentration of pipe is 0.1-5mg/mL.
4. method according to claim 1 to 3, which is characterized in that Jenner is electroplated in the modification screen printing electrode
Rice grain is to contain 1% gold chloride by 10mL and 1mmol/L sulfuric acid solution is prepared as gold plating liquid.
5. method according to claim 1 to 4, which is characterized in that plating gold nano particle modification screen printing electrode
When electroplating voltage be -0.2-0.4V, electroplating time 80-120s.
6. method according to claim 1 or 2, which is characterized in that the sample solution is by bacterium and resazurin, antibiosis
Plain drug mixed culture is incubated for 1-6 hours gained.
7. according to the method described in claim 6, it is characterized in that, culture incubation time is 4h.
8. -7 any method according to claim 1, which is characterized in that in the step (2) further include: by sample solution
It is filtered, is then dripped in carrying out electrochemistry on multi-walled carbon nanotube-gold nano particle modification screen printing electrode by filter
Detection.
9. -8 any method according to claim 1, which is characterized in that the bacterium includes salmonella typhimurium, gold
Staphylococcus aureus.
10. -8 any method according to claim 1, which is characterized in that the electricity in the step (2) when Electrochemical Detection
Pressure is -0.7V~0V.
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| CN110530957B (en) | 2021-01-29 |
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Inventor after: Sun Xiulan Inventor after: Ren Yijing Inventor after: Ji Jian Inventor after: Sun Jiadi Inventor after: Pi Fuwei Inventor after: Zhang Yinzhi Inventor before: Sun Xiulan Inventor before: Ren Yijing Inventor before: Ji Jian Inventor before: Sun Jiadi Inventor before: Pi Fuwei Inventor before: Zhang Yinzhi |
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