CN107271524B - Method for applying (CNTs/PANI) n-ITO anode-based MFC biosensor to drug sensitivity test - Google Patents

Method for applying (CNTs/PANI) n-ITO anode-based MFC biosensor to drug sensitivity test Download PDF

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CN107271524B
CN107271524B CN201710513917.5A CN201710513917A CN107271524B CN 107271524 B CN107271524 B CN 107271524B CN 201710513917 A CN201710513917 A CN 201710513917A CN 107271524 B CN107271524 B CN 107271524B
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吴文果
杨达云
王士斌
牛浩
陈爱政
刘源岗
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Abstract

The invention discloses a method for applying a (CNTs/PANI) n-ITO anode-based MFC biosensor to drug sensitivity tests. The invention provides a new method for the traditional antibiotic drug sensitivity test method, avoids the problems of long detection time, complex operation, poor repeatability and the like of a microbial plate method, and the constructed microbial fuel cell biosensor is simple and convenient to operate and can realize rapid, real-time and high-sensitivity drug sensitivity test.

Description

Method for applying (CNTs/PANI) n-ITO anode-based MFC biosensor to drug sensitivity test
Technical Field
The invention belongs to the technical field of drug detection of a microbial fuel cell biosensor, and particularly relates to a method for applying an MFC biosensor based on a (CNTs/PANI) n-ITO anode to a drug sensitivity test.
Background
In recent years, the abuse of antibiotics seriously threatens the life and health of human beings. The traditional antibiotic susceptibility test methods include a paper diffusion method, an agar dilution method, an E test method, a broth dilution method and the like. However, these methods have the disadvantages of long time (24-48 h) and complicated operation, and cannot meet the requirement of clinical rapid diagnosis and treatment. Microbial Fuel Cells (MFCs) use microorganisms as catalysts and direct conversion of chemical energy to biosensors as a device for converting chemical energy to electrical energy, and are used in the fields of power generation and supply, sewage treatment, environmental bioremediation, biosensors and the like. The microbial fuel cell is used in the field of biosensors and can be used for detecting toxic substances. The basic principle is that after the toxic substances enter the MFC, the metabolism of the electrochemical active bacteria is inhibited by the toxic substances, so that the output current is reduced, and the reduction degree of the current has certain correlation with the concentration of the toxic substances. The more toxic the toxic substance is, the larger the current reduction amplitude is, and different toxicity sensors can be constructed according to the relationship between the toxic substance and the current reduction amplitude. Although biosensors have been constructed using MFC for antibiotic detection at present, detection sensitivity of the sensors and the minimum detection limit of antibiotics have not been studied.
The sensing characteristics of biosensors based on microbial fuel cells are mainly dependent on the electricity generation performance of the microbial fuel cells. The anode material is one of the important factors for directly contacting with the microorganisms and transferring the extracellular electrons. The electrode materials such as nano metal particles, carbon nano tubes, nano-structure conducting polymers and the like can improve the electrical performance when applied to the microbial fuel cell. Although the anode of the MFC nanostructured material is prepared by different methods, the method for preparing the nanostructured carbon nanotube/polyaniline compound modified ITO anode and constructing the MFC sensor to perform drug sensitivity test research and solve the problem of drug sensitivity test sensitivity does not exist.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for applying a (CNTs/PANI) n-ITO anode-based MFC biosensor to a drug sensitivity test. The invention provides a new method for the traditional antibiotic drug sensitivity test method, avoids the problems of long detection time, complex operation, poor repeatability and the like of a microbial plate method, and the constructed microbial fuel cell biosensor is simple and convenient to operate and can realize rapid, real-time and high-sensitivity drug sensitivity test.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitivity testing, comprising:
1) soaking an ITO (indium tin oxide) electrode treated by a 3-5% NaOH solution into an anhydrous toluene solution, and adding APTES (3-aminopropyltriethoxysilane), wherein the formula ratio of the APTES to the anhydrous toluene is 1.0-1.2 g: 25 mL; placing the ITO electrode in a nitrogen atmosphere at normal temperature for 15-17 h, taking out the ITO electrode, sequentially carrying out ultrasonic cleaning by using toluene, ethanol and deionized water, and finally drying by nitrogen;
2) mixing the components in a volume ratio of 1: 2.5-3.5, carrying out ultrasonic acidification treatment on CNTs (carbon nanotubes) for 23-25 h by using a mixed solution of saturated concentrated nitric acid and concentrated sulfuric acid, centrifuging, washing with ultrapure water, and repeatedly centrifuging and washing until the supernatant is neutral; freezing and storing the CNTs obtained by centrifugation, and preparing 0.9-1.1 mg/mL CNTs suspension by using ultrapure water;
3) mixing the following components in a mass ratio of 1: dissolving 0.9-1.1 of PANI (polyaniline) and HCSA (camphorsulfonic acid) into chloroform, stirring until the solvent is completely volatilized, and then ultrasonically dispersing by using ultrapure water to prepare an HCSA-doped PANI solution with the concentration of 0.9-1.1 mg/mL;
4) soaking the ITO electrode obtained in the step 1) in the HCSA-doped PANI solution obtained in the step 3) for 25-35 min, taking out, soaking and cleaning with ultrapure water for 4-6 min, soaking in the CNTs suspension obtained in the step 2) for 25-35 min, taking out, soaking and cleaning with ultrapure water for 4-6 min, and obtaining a (CNTs/PANI) n-ITO electrode, wherein n is the number of modified layers, and n is 1; repeating the steps on the (CNTs/PANI) n-ITO electrode with n being 1 to obtain (CNTs/PANI) n-ITO electrodes with different modification layers;
5) constructing a single-chamber microbial fuel cell biosensor by using the (CNTs/PANI) n-ITO electrode: the (CNTs/PANI) n-ITO electrode is used as a working electrode, Pt is used as a counter electrode, a saturated Ag/AgCl electrode is used as a reference electrode, an electrolyte containing a drug is added into the single-chamber biosensor, the constant potential is 0.18-0.22V, the single-chamber biosensor is operated for 25-35 min at the constant temperature of 36-38 ℃, then a bacterial solution is added, and the electrochemical parameter change of the single-chamber microbial fuel cell biosensor is observed to carry out a drug sensitivity test; and/or the presence of a gas in the gas,
constructing a two-chamber microbial fuel cell biosensor by using the (CNTs/PANI) n-ITO electrode: and (3) taking the (CNTs/PANI) n-ITO electrode as an anode and carbon cloth as a cathode, adding electrolyte and bacterial liquid into the anode chamber, adding 9-11 mM potassium ferricyanide solution into the cathode chamber, keeping the external resistance at 1900-2100 omega, operating at the constant temperature of 36-38 ℃ for 24-26 h, adding a medicament, and observing the change of electrochemical parameters of the two-chamber microbial fuel cell biosensor to perform a drug sensitivity test.
In one embodiment: n is 1, 3, 6, 8, 12, 15.
In one embodiment: and n is 12.
In one embodiment: the bacteria of the bacteria liquid are pathogenic microorganisms capable of generating extracellular electrons.
In one embodiment: the bacteria of the bacterial liquid are at least one of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, staphylococcus epidermidis, streptococcus pneumoniae and klebsiella.
In one embodiment: the bacteria of the bacterial liquid are Shewanella loihica PV-4 and/or Escherichia coli 25922.
In one embodiment: the medicine can kill pathogenic microorganisms and/or inhibit the growth and activity of the pathogenic microorganisms.
In one embodiment: the drug is an antibiotic.
In one embodiment: the drug is at least one of penicillin antibiotics, cephalosporin antibiotics, aminoglycoside antibiotics, macrolide antibiotics and quinolone antibiotics.
In one embodiment: the drug is gentamicin.
Compared with the background technology, the technical scheme has the following advantages:
the invention utilizes the microbial fuel cell to construct the biosensor for the study of gentamicin drug sensitivity test, and avoids the defects of long detection time and complex operation of the traditional method. The carbon nano tube/polyaniline compound modified ITO anode is prepared and used for a microbial fuel cell biosensor, so that the detection sensitivity of the sensor is further improved, and real-time online monitoring is realized.
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The invention is further illustrated by the following figures and examples.
FIG. 1 is a time-current curve of different numbers of layers of CNTs/PANI modified ITO electrodes prepared in example 1 in a microbial fuel cell.
Fig. 2 is an SEM image of the effect of the different number of layers of CNTs/PANI modified ITO electrode on the adhesion of s.loihica PV-4 prepared in example 1, in which, (a) 1 layer is modified, (b) 3 layers are modified, (c) 6 layers are modified, (d) 8 layers are modified, (e) 12 layers are modified, and (f) 15 layers are modified.
FIG. 3 shows the results of example 2 with various concentrations of gentamicin added based on (CNTs/PANI)12Current response curves of single-chamber microbial fuel cell biosensors with ITO anodes.
FIG. 4 shows the results of example 3 with various concentrations of gentamicin added based on (CNTs/PANI)12Voltage response curves of ITO anode two-chamber microbial fuel cell biosensors.
Detailed Description
The present invention will be described in detail with reference to the following examples:
example 1
1) Soaking an ITO (indium tin oxide) electrode treated by a 4% NaOH solution into 25mL of an anhydrous toluene solution, adding about 1.1g of APTES (3-aminopropyltriethoxysilane), standing at normal temperature for 16h in a nitrogen atmosphere, taking out the ITO electrode, sequentially ultrasonically cleaning the ITO electrode by using toluene, ethanol and deionized water, and finally drying the ITO electrode by using nitrogen;
2) performing ultrasonic acidification treatment on CNTs (carbon nanotubes) for 24 hours by using a mixed solution (v/v, 1:3) of saturated concentrated nitric acid and concentrated sulfuric acid, centrifuging, and washing by using ultrapure water until the supernatant is neutral; the CNTs obtained by centrifugation are freeze-dried and stored, and then the CNTs suspension with 1mg/mL is prepared by ultrapure water;
3) dissolving PANI (polyaniline) 100mg and HCSA (camphorsulfonic acid) 100mg into chloroform 20mL, stirring until the solvent is completely volatilized, and then ultrasonically dispersing with ultrapure water to prepare HCSA-doped PANI solution with the concentration of 1 mg/mL;
4) soaking the ITO electrode obtained in the step 1) in the HCSA-doped PANI solution obtained in the step 3) for 30min, taking out, soaking and cleaning with ultrapure water for 5min, soaking in the CNTs suspension obtained in the step 2) for 30min, taking out, soaking and cleaning with ultrapure water for 5min, and obtaining a (CNTs/PANI) n-ITO electrode, wherein n is 1; repeating the steps on the (CNTs/PANI) n-ITO electrode with n being 1 to obtain (CNTs/PANI) n-ITO electrodes with different modification layer numbers (n being 1, 3, 6, 8, 12 and 15);
using (CNTs/PANI) n-ITO electrode as working electrode, Pt as counter electrode, saturated Ag/AgCl electrode as reference electrode, adding electrolyte into reactor, constant potential of 0.2V, operating at 37 deg.C for 30min, adding OD600The electricity generation experiment was carried out using Shewanella loihica PV-4 bacterial solution of 2.0.
As shown in FIGS. 1 and 2, FIG. 1 is a time-current curve of the different numbers of layers of CNTs/PANI modified ITO electrodes in the microbial fuel cell, and it can be seen that the number of modified layers n is 12 (CNTs/PANI)12Maximum current density value (6.98. mu.A/cm) produced by the ITO electrode2). Fig. 2 is an SEM image showing the effect of different numbers of CNTs/PANI modified ITO electrodes on the adhesion of s.loihica PV-4, and it can be seen that s.loihica PV-4 has better adhesion on electrodes with more than 3 modified layers.
Example 2
1) Soaking the ITO electrode treated by the 4% NaOH solution into 25mL of anhydrous toluene solution, adding about 1.1g of APTES, standing at normal temperature for 16h in a nitrogen atmosphere, taking out the ITO electrode, sequentially ultrasonically cleaning the ITO electrode by using toluene, ethanol and deionized water, and finally drying the ITO electrode by using nitrogen;
2) performing ultrasonic acidification treatment on CNTs for 24h by using a mixed solution (v/v, 1:3) of saturated concentrated nitric acid and concentrated sulfuric acid, centrifuging, and washing by using ultrapure water until the supernatant is neutral; the CNTs obtained by centrifugation are freeze-dried and stored, and then the CNTs suspension with 1mg/mL is prepared by ultrapure water;
3) dissolving 100mg of PANI and 100mg of HCSA into 20mL of chloroform, stirring until the solvent is completely volatilized, and then ultrasonically dispersing by using ultrapure water to prepare an HCSA-doped PANI solution with the concentration of 1 mg/mL;
4) soaking the ITO electrode obtained in the step 1) in the HCSA-doped PANI solution obtained in the step 3) for 30min, taking out, soaking and cleaning with ultrapure water for 5min, soaking in the CNTs suspension obtained in the step 2) for 30min, taking out, soaking and cleaning with ultrapure water for 5 min; repeating the above steps for 12 times to obtain (CNTs/PANI) with 12 modified layers12-an ITO electrode;
5) using the (CNTs/PANI)12ITO electrodes single-chamber microbial fuel cell biosensors were constructed and subjected to drug sensitivity tests: with the (CNTs/PANI)12An ITO electrode is used as a working electrode, Pt is used as a counter electrode, a saturated Ag/AgCl electrode is used as a reference electrode, electrolytes of gentamicin with the concentration of 0mM, 0.25mM, 0.5mM, 1mM and 1.5mM are respectively added into a single-chamber biosensor, the constant potential is 0.2V, after the biosensor operates at the constant temperature of 37 ℃ for 30min, OD is added600The Escherichia coli25922 bacterial liquid of 2.0 is used for carrying out an electrogenesis experiment, and the electrochemical parameter change of a single-chamber microbial fuel cell biosensor is observed to carry out a drug sensitivity experiment.
FIG. 3 shows the results of (CNTs/PANI) based solutions with various concentrations of gentamicin added12Current response curves of single-chamber microbial fuel cell biosensors with ITO anodes. As can be seen from the graph, the current signal of the sensor gradually decreased with the increase of the concentration of gentamicin, and the minimum detection limit of gentamicin was 0.25 mM.
Example 3
1) Soaking the ITO electrode treated by the 4% NaOH solution into 25mL of anhydrous toluene solution, adding about 1.1g of APTES, standing at normal temperature for 16h in a nitrogen atmosphere, taking out the ITO electrode, sequentially ultrasonically cleaning the ITO electrode by using toluene, ethanol and deionized water, and finally drying the ITO electrode by using nitrogen;
2) performing ultrasonic acidification treatment on CNTs for 24h by using a mixed solution (v/v, 1:3) of saturated concentrated nitric acid and concentrated sulfuric acid, centrifuging, and washing by using ultrapure water until the supernatant is neutral; the CNTs obtained by centrifugation are freeze-dried and stored, and then the CNTs suspension with 1mg/mL is prepared by ultrapure water;
3) dissolving 100mg of PANI and 100mg of HCSA into 20mL of chloroform, stirring until the solvent is completely volatilized, and then ultrasonically dispersing by using ultrapure water to prepare an HCSA-doped PANI solution with the concentration of 1 mg/mL;
4) soaking the ITO electrode obtained in the step 1) in the HCSA-doped PANI solution obtained in the step 3) for 30min, taking out, soaking and cleaning with ultrapure water for 5min, soaking in the CNTs suspension obtained in the step 2) for 30min, taking out, soaking and cleaning with ultrapure water for 5 min; repeating the above steps for 12 times to obtain (CNTs/PANI) with 12 modified layers12-an ITO electrode;
5) using the (CNTs/PANI)12ITO electrodes a two-compartment microbial fuel cell biosensor was constructed and subjected to a drug sensitivity test: with the (CNTs/PANI)12-ITO electrode as anode, carbon cloth as cathode, electrolyte and OD being added to anode chamber6002.0 Escherichia coli25922, adding 10mM potassium ferricyanide solution into a cathode chamber, connecting with an external resistance of 2000 omega, operating at a constant temperature of 37 ℃ for 25 hours, respectively adding gentamicin with the concentration of 0mM, 0.25mM, 0.5mM, 1mM and 1.5mM, carrying out an electrogenesis experiment, and observing the change of electrochemical parameters of a two-chamber microbial fuel cell biosensor to carry out a drug sensitive test.
FIG. 4 shows the results of (CNTs/PANI) based solutions with various concentrations of gentamicin added12Voltage response curves of ITO anode two-chamber microbial fuel cell biosensors. As can be seen from the graph, the voltage was increased from 0mV after inoculation of the bacterial solution, and after 8 hours, the voltage was about 150mV and then stabilized at about 120 mV. After 25h of operation, the voltage dropped with the addition of gentamicin. The higher the gentamicin concentration is, the voltage signal of the sensor is reduced about remarkably, the minimum detection limit of the gentamicin is 0.25mM, and the gentamicin sensor has good consistency with the gentamicin drug sensitivity test result of a single-chamber microbial fuel cell biosensor.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Claims (10)

1. A method for using a (CNTs/PANI) n-ITO anode-based microbial fuel cell biosensor for drug sensitivity test is characterized in that: the method comprises the following steps:
1) immersing an ITO electrode treated by a 3-5% NaOH solution into an anhydrous toluene solution, and adding APTES, wherein the formula ratio of the APTES to the anhydrous toluene is 1.0-1.2 g: 25 mL; placing the ITO electrode in a nitrogen atmosphere at normal temperature for 15-17 h, taking out the ITO electrode, sequentially carrying out ultrasonic cleaning by using toluene, ethanol and deionized water, and finally drying by nitrogen;
2) mixing the components in a volume ratio of 1: 2.5-3.5, carrying out ultrasonic acidification treatment on the carbon nano tube CNTs for 23-25 h by using a mixed solution of saturated concentrated nitric acid and concentrated sulfuric acid, centrifuging, and washing with ultrapure water until the supernatant is neutral; freezing and storing the CNTs obtained by centrifugation, and preparing 0.9-1.1 mg/mL CNTs suspension by using ultrapure water;
3) mixing the following components in a mass ratio of 1: dissolving polyaniline PANI and camphorsulfonic acid (HCSA) of 0.9-1.1 into chloroform, stirring until the solvent is completely volatilized, and then ultrasonically dispersing by using ultrapure water to prepare an HCSA-doped PANI solution with the concentration of 0.9-1.1 mg/mL;
4) soaking the ITO electrode obtained in the step 1) in the HCSA-doped PANI solution obtained in the step 3) for 25-35 min, taking out, soaking and cleaning with ultrapure water for 4-6 min, soaking in the CNTs suspension obtained in the step 2) for 25-35 min, taking out, soaking and cleaning with ultrapure water for 4-6 min, and obtaining a (CNTs/PANI) n-ITO electrode, wherein n is 1; repeating the steps on the (CNTs/PANI) n-ITO electrode with n being 1 to obtain (CNTs/PANI) n-ITO electrodes with different modification layers;
5) constructing a single-chamber microbial fuel cell biosensor by using the (CNTs/PANI) n-ITO electrode: the (CNTs/PANI) n-ITO electrode is used as a working electrode, Pt is used as a counter electrode, a saturated Ag/AgCl electrode is used as a reference electrode, an electrolyte containing a drug is added into the single-chamber biosensor, the constant potential is 0.18-0.22V, the single-chamber biosensor is operated for 25-35 min at the constant temperature of 36-38 ℃, then a bacterial solution is added, and the electrochemical parameter change of the single-chamber microbial fuel cell biosensor is observed to carry out a drug sensitivity test; and/or the presence of a gas in the gas,
constructing a two-chamber microbial fuel cell biosensor by using the (CNTs/PANI) n-ITO electrode: and (3) taking the (CNTs/PANI) n-ITO electrode as an anode and carbon cloth as a cathode, adding electrolyte and bacterial liquid into the anode chamber, adding 9-11 mM potassium ferricyanide solution into the cathode chamber, keeping the external resistance at 1900-2100 omega, operating at the constant temperature of 36-38 ℃ for 24-26 h, adding a medicament, and observing the change of electrochemical parameters of the two-chamber microbial fuel cell biosensor to perform a drug sensitivity test.
2. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: and n is 3, 6, 8, 12 and 15.
3. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: and n is 12.
4. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the bacteria of the bacteria liquid are pathogenic microorganisms capable of generating extracellular electrons.
5. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the bacteria of the bacterial liquid are at least one of escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, staphylococcus epidermidis, streptococcus pneumoniae and klebsiella.
6. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the bacteria of the bacterial liquid are Shewanella loihica PV-4 and/or Escherichia coli 25922.
7. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the medicine can kill pathogenic microorganisms and/or inhibit the growth and activity of the pathogenic microorganisms.
8. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the drug is an antibiotic.
9. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the drug is at least one of penicillin antibiotics, cephalosporin antibiotics, aminoglycoside antibiotics, macrolide antibiotics and quinolone antibiotics.
10. The method of using a (CNTs/PANI) n-ITO anode based microbial fuel cell biosensor for drug sensitive testing according to claim 1, characterized in that: the drug is gentamicin.
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