CN109254039B - EBFC-based self-powered bacterial biosensor and application thereof - Google Patents

EBFC-based self-powered bacterial biosensor and application thereof Download PDF

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CN109254039B
CN109254039B CN201811075816.5A CN201811075816A CN109254039B CN 109254039 B CN109254039 B CN 109254039B CN 201811075816 A CN201811075816 A CN 201811075816A CN 109254039 B CN109254039 B CN 109254039B
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aptamer
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CN109254039A (en
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李峰
盖盼盼
于汶
谷成成
侯婷
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Qingdao Agricultural University
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Abstract

The invention discloses an EBFC-based self-powered bacteria biosensor and application thereof, belonging to the technical field of biosensing. PB can receive electrons from the biological anode and convert them into Prussian White (PW), and PB blue can also convert them into transparent PW by getting electrons. The rate of change from blue to colorless depends on the concentration of Vibrio parahaemolyticus. This color can be restored to the original PB state by attaching it to the bioanode. When the target Vibrio parahaemolyticus is present, the open circuit voltage generated by the EBFCs decreases, with an amplitude dependent on the Vibrio parahaemolyticus concentration identified for the aptamer of the target. Vibrio parahaemolyticus prevents fuel glucose from approaching the Glucose Dehydrogenase (GDH) active site on the bioanode due to steric hindrance effects, resulting in a reduction in prussian white color. The invention realizes simple, convenient, rapid, sensitive and efficient detection of the food pathogens.

Description

EBFC-based self-powered bacterial biosensor and application thereof
Technical Field
The invention discloses a self-powered biosensing platform based on visual repeatable enzyme biofuel cells (EBFCs) and used for detecting vibrio parahaemolyticus, which integrates Prussian Blue (PB) electrochromism and specific combination of an aptamer and an antigen, and belongs to the technical field of biosensing.
Background
The Enzyme Biological Fuel Cells (EBFCs) are a novel green energy conversion technology, and can extract biological energy from biochemical reaction due to mild operation conditions and good application prospect. An EBFC-based self-powered biosensor is a type of sensor that outputs a battery performance as an analytical detection signal that is proportional to the analyte concentration being detected. The concrete advantages are mainly shown in that: (1) the equipment is simple. In the detection process, only two electrodes, namely the anode and the cathode of the EBFC are needed to realize detection; (2) the anti-interference capability is strong. The test system is not provided with an additional power supply, so that the electroactive substances which are easy to be oxidized and reduced can be effectively prevented from reacting on the surface of the electrode, and the anti-interference capability of the sensor is improved; (3) simple, quick and real-time detection can be realized. Power supply equipment such as an electrochemical workstation is not needed in the detection process, and detection can be realized only by the simple voltmeter and the appropriate light source, so that the detection equipment is easy to carry, and real-time monitoring can be realized. Self-powered biosensors based on EBFC have been successfully applied to the determination of toxic pollutants, immunoassays, cancer markers, drug release and food safety, have become a novel biosensing platform, enabling detection of different targets.
Pathogenic bacteria in food have great threat to human life, such as salmonella typhi, staphylococcus aureus, escherichia coli and the like. There are currently a number of methods for detecting pathogenic bacteria in food, including Polymerase Chain Reaction (PCR), biochemical immunoassay, Next Generation Sequencing (NGS), DNA probes, Surface Enhanced Raman Scattering (SERS) spectroscopy, but they are expensive and complex and difficult to detect. To solve these problems, it is very urgent to develop a highly sensitive and selective biosensor to potentially detect food pathogens. In addition, aptamers with the advantages of remarkable chemical stability, easiness in chemical modification, small size, simplicity in synthesis, high specificity and the like have become ideal pathogen detection molecular receptors. Therefore, the self-powered biosensor based on the biofuel cell is designed and prepared, and the simple, convenient, high-sensitivity and high-specificity detection of the edible pathogenic bacteria such as vibrio parahaemolyticus is realized.
Disclosure of Invention
The invention constructs a novel visual and repeatable self-powered biosensing platform based on EBFCs, which is used for ultra-sensitively detecting pathogenic bacteria (food pathogens) in food. The change of the electrochromic and redox state of PB and the specific recognition of pathogenic bacteria by aptamers play an important role in biosensors. The invention fixes Glucose Dehydrogenase (GDH) and aptamer on PTH/CNTs/AuNP as a biological anode, a mediated PB electrode and a biological cathode which fixes Bilirubin Oxidase (BOD) on CNT to construct a fuel cell. On the one hand, when the bioanode is connected to the PB electrode, GDH oxidizes glucose to generate electrons that flow through an external circuit to PB to convert PB to Prussian White (PW), while the blue color of PB is also changed to transparent. On the other hand, the biocathode electrode is connected to the PW electrode, and the biocathode receives electrons from the PW electrode to oxidize them into the original PB state. In this case, the EBFC generates two high open-circuit voltages (E)OCV). When a pathogen is present, itThey are specifically recognized and bound by aptamers, and fuel glucose is kept away from the active site on the GDH surface due to steric hindrance effect, resulting in low E production by EBFCOCV. Meanwhile, when the PW electrode is connected to the biocathode, another low E is received since PB is more difficult to convert to PWOCV. Thus, the degree of color change exhibited by the electrochromic display and the two E's produced by the bioanode and PB and PW and the biocathode are comparedOCVThe detection of pathogenic bacteria in food with ultra-sensitivity is successfully realized. This efficient and simple strategy not only exhibits high sensitivity and high selectivity, but also enables visual detection and re-use. Therefore, the effective and simple strategy not only has higher sensitivity and selectivity, but also realizes the visualization and reusability of the production of the self-powered biosensing platform, and has the potential of being used as a field tool for detecting the vibrio parahaemolyticus in food safety.
The invention is realized by adopting the following technical scheme:
an EBFC-based self-powered biosensor comprising an anode, a PB electrode, a cathode and an electrolyte; the anode is an MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, the cathode is a BOD/CNTs/CP biological cathode, and the electrolyte is 1-3 mM NAD+NADH, 3-6 mM glucose pH 6.0 0 0.1M PB buffer system.
The preparation method of the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP bioanode comprises the following steps:
performing cyclic voltammetry scanning on the surface of a CP electrode modified with CNT/AuNPs to polymerize thionine, coating the electrode with a mixture containing a GDH solution and an aptamer (SH-DNA), and performing primary incubation to obtain aptamer/GDH/PTH/CNT/AuNPs/CP; and (3) washing the surface of the electrode with secondary water, dripping an MCH solution on the surface of the electrode, performing secondary incubation, closing the plate, and washing the surface of the electrode with the secondary water to obtain the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode.
The preparation method of the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP bioanode is shown in figure 1 and comprises the following steps:
step A: adding 1-6 mL of HAuCl4Adding 90-100 mL of secondary water, stirring and heating to boilingThen, quickly adding 7-15 mL of 37-40 mM sodium citrate solution, boiling for 15min, and cooling to obtain wine red transparent liquid which is AuNPs;
and B: weighing 6-10 mg of Carbon Nano Tube (CNT) and dissolving in 2-6 mL of 0.5-2% polydiallyldimethylammonium chloride (PDDA) for ultrasonic treatment for 30min, centrifuging and washing the obtained solution for 11000-15000 rpm for 15min for many times, taking supernate to obtain a black solid, fixing the volume with 0.5-2mL of secondary water, adding the solution obtained in the step A until 8-12 mL of the solution is adsorbed for one night, ultrasonically washing at 4000-6000 rpm for 4-7 min, and dispersing the obtained precipitate in 0.5-2.5 mL of secondary water to obtain a CNT/AuNPs mixture;
and C: and D, dripping 20-60 mu L of the CNT/AuNPs prepared in the step B on the surface of the CP electrode, drying at 37 ℃, and putting into a three-electrode battery. At 50mv s-1The sweep rate of (2) is determined by performing cyclic voltammetric sweep in a solution of 1 to 3mM thionine (2.2mM acetic acid). The sweeping speed can be 30-70mv s-1The concentration of the thionine solution is 1-3 mM (dissolved in 2.2mM acetic acid). The sweep ranged from-0.3V to 1.2V vs Ag/AgCl and back to-0.3V for 10 cycles. Electrode composition containing 1-4 mg/mL-1And coating a mixture of 25-60 mu L of the GDH solution and 400-700 nM aptamer (SH-DNA), incubating for 8-16 h at 3-7 ℃, and obtaining an aptamer/GDH/PTH/CNT/AuNPs modified electrode through Au-S bonds between AuNPs and SH-DNA. And (3) washing the surface of the electrode with secondary water, dripping 20-50 mu L of 1-3 mM MCH solution on the surface of the electrode, incubating for 0.5-2 h at room temperature, sealing the plate, washing the surface of the electrode with secondary water, and standing at 4 ℃ for later use.
The preparation method of the BOD/CNT/CP biological cathode comprises the following steps:
step I: dispersing a certain amount of CNT in 1-3 mL of secondary water, dripping 20-50 mu LCNT solution on the surface of an ITO electrode, and drying at 37 ℃ for 2-4 h;
step II: and dripping 10-25 mu L of 0.5-2 mg/mL BOD solution on the electrode, drying at 4 ℃ for 12-24 hours to obtain a BOD/CNT/CP biological cathode, washing with secondary water, and standing at 4 ℃ for later use.
The preparation method of the PB electrode comprises the following steps:
the method comprises the following steps: by on an ITO electrodeAnd electro-depositing a Prussian blue film to prepare an electrochromic display. Before modification, the ITO electrode was washed with ethanol and water in an ultrasonic bath. The ITO electrode was then immersed in 1:1 ethanol: NaOH (1-3M) for 30 minutes to activate the surface. After washing with the second water, applying a constant potential of 0.4V to the mixture, and washing with a solution containing 0.1-0.3M KCl, 0.05-0.2M HCl, 2-3 mM K3[Fe(CN)6]And 1.5 to 4mM FeCl3·6H2And carrying out 120-360 s electropolymerization on the ITO electrode in the newly prepared solution of O. Successful preparation of PB films was confirmed by its apparent color and absorbance change. The ITO electrode was then rinsed with secondary water and dried in air.
An EBFC-based self-powered bacterial biosensor comprising an anode, a PB electrode, a cathode and an electrolyte; the anode is an MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, the cathode is a BOD/CNT/CP biological cathode, and the electrolyte contains 1-3 mM NAD+NADH, 3-6 mM glucose pH 6.0 0 0.1M PB buffer system.
Use of an EBFC-based self-powered bacterial biosensor as described above for the detection of foodpathogens.
The detection method comprises the following steps:
step (1): the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, PB electrode, 1-3 mMNAD+Measuring E of a battery assembled by a 0.1M PB buffer system of NADH and 3-6 mM glucose with pH of 6.01 OCVIs marked as E10 OCV(ii) a BOD/CNT/CP biological cathode, PW electrode, containing 1-3 mM NAD+A 0.1MPB buffer system of NADH and 3-6 mM glucose with pH 6.0 is assembled into a battery, and the E of the battery is measured2 OCVIs marked as E20 OCV；
Step (2): placing the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode in 0.5-2mL of pathogenic bacteria with different concentrations, incubating for 60-120 minutes at 37 ℃ to obtain a pathogens/MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, and washing the surface of the electrode with secondary water;
and (3): the pathogens/MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, the PB electrode and the 1-3 mM NAD+Measuring E of a battery assembled by a 0.1M PB buffer system of NADH and 3-6 mM glucose with pH of 6.01 OCVIs marked as E1n OCV(ii) a Assembling BOD/CNT/CP biological cathode, PW electrode, and 0.1M PB buffer system containing pH 6.0 into battery, and measuring E of the battery2 OCVIs marked as E2n OCVThe color change of the PB electrode was observed.
The principle of the EBFC-based self-powered bacterial biosensor for detecting food pathogens with ultra-sensitivity is shown in FIG. 1:
when there is no target pathogens, glucose is oxidized to gluconolactone near the active site on the electrode GDH to generate a large current, and a large number of electrons flow to the PB electrode, and flow to PB through an external circuit to convert PB to Prussian White (PW), and the blue color of PB becomes transparent, and at this time, the open circuit voltage of EBFC is large. Connecting a biocathode electrode to the PW electrode, the biocathode receiving electrons from the PW electrode to oxidize it to the original PB state while generating a high open circuit voltage (E)OCV). When target pathogenic bacteria are introduced, they are specifically recognized and bound by the aptamer, glucose cannot get close to the active site on the GDH surface due to steric hindrance effect, and EBFC produces low EOCVPB can not be completely converted into PW, and PW electrodes generated by pathogenic bacteria with different concentrations have different colors. Thus, the degree of color change exhibited by the electrochromic display and the two E's produced by the bioanode and PB and PW and the biocathode are comparedOCVThe method successfully realizes the ultra-sensitive detection of the number of pathogenic bacteria in food.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides an EBFC-based self-powered bacterial biosensor, which realizes simple, convenient, rapid, sensitive and efficient detection of food pathogens, and has the following characteristics compared with the existing food pathogens detection method:
(1) according to the EBFC-based self-powered bacteria biosensor, an external power supply is not needed in the detection process, the whole detection equipment is simple, and the field real-time monitoring is convenient to realize;
(2) the PB disclosed by the invention has good electrochromic performance, high color change degree, high stability and good repeatability;
(3) the EBFC-based self-powered bacterial biosensor adopts the aptamer to identify the target object, and has the advantages of extremely high selectivity, low cost, simplicity in operation and the like;
(4) in the EBFC-based self-powered bacteria biosensor, the pathogenic bacteria have good steric hindrance effect on oxidized glucose of a biological anode, meanwhile, a biological cathode has good electrocatalytic activity on oxygen, and a PB electrode has good ability of getting lost electrons and changing color, so that ultra-sensitive detection on a target food pathogens is realized, and the detection sensitivity is improved;
(5) by constructing the self-powered biosensor without additional power supply equipment, expensive instrument equipment is not needed, and the miniaturization, portability and integration of the food pathogens detection can be realized.
Drawings
FIG. 1 is a schematic diagram of the EBFC-based self-powered bacterial biosensor for the ultra-sensitive detection of food pathogens;
FIG. 2(A) is a graph showing the combination of MCH/aptamer/GDH/PTH/CNT/AuNPs/CP bioanode and PB electrode E of various concentrations of food pathogens1 OCVThe value, FIG. 2(C), is E for BOD/CNT/CP biocathodes in combination with PW electrodes2 OCVA value;
FIGS. 2(B) and (D) are graphs showing E measured for different concentrations of food pathogens on the abscissa and for different concentrations of food pathogensn OCVThe values are plotted as logarithmically linear relationships of the ordinate.
Detailed Description
In order to make the object and technical solution of the present invention more apparent, the present invention is further described in detail by the following examples.
Example one：
EBFC-based self-powered bacterial biosensors were used for the detection of v.
(1) Preparation of AuNPs:
4mL of HAuCl4Adding 96mL of secondary water, stirring and heating to boil, quickly adding 10mL of 38.8mM sodium citrate solution, boiling for 15min, and cooling to obtain wine red transparent liquid which is AuNPs;
(2) preparation of CNT/AuNPs:
weighing 8mg of Carbon Nano Tube (CNT) to dissolve in 4mL of 1% polydiallyldimethylammonium chloride (PDDA) to carry out ultrasonic treatment for 30min, carrying out multiple centrifugal washing on the obtained solution at 15000rpm for 15min, taking supernate to obtain a black solid, fixing the volume with 1mL of secondary water, adding the solution obtained in the step A to 10mL of the solution to adsorb overnight, carrying out ultrasonic washing at 5000rpm for 6min, and dispersing the obtained precipitate in 1mL of secondary water to obtain a CNT/AuNPs mixture;
(3) preparation of MCH/aptamer/GDH/PTH/CNT/AuNPs/CP bioanode:
and (3) dripping 50 mu L of the CNT/AuNPs prepared in the step (2) on the surface of the CP electrode, drying at 37 ℃, and putting into a three-electrode battery. At 50mv s-1The cyclic voltammetric scan was performed in a 2mM thionine solution (2.2mM acetic acid). The sweep ranged from-0.3V to 1.2V vs Ag/AgCl and back to-0.3V for 10 cycles. The modified electrode contained 2mgmL of-1The GDH solution and a 40. mu.L mixture of 500nM aptamer (SH-DNA) were coated and incubated at 3-7 ℃ for 8-16 h to obtain aptamer/GDH/PTH/CNT/AuNPs/CP through Au-S bond between AuNPs and SH-DNA. And (3) washing the surface of the electrode with secondary water, dripping 30 mu L of 1mM MCH solution on the surface of the electrode, incubating for 0.5h at room temperature, sealing the plate, washing the surface of the electrode with secondary water to obtain MCH/aptamer/GDH/PTH/CNT/AuNPs/CP, and standing at 4 ℃ for later use.
(4) Preparation of BOD/CNT/CP biocathodes:
1mg of CNT was dispersed in 1mL of secondary water, and 50. mu.L of the CNT solution was applied dropwise to the surface of an ITO electrode and dried at 37 ℃ for 2 hours. And dripping 15 mu L of BOD solution with the concentration of 1mg/mL on the surface of the electrode, drying at 4 ℃ for 12 hours to obtain the BOD/CNT/CP biological cathode, washing with secondary water, and standing at 4 ℃ for later use.
(5) Preparation of a PB electrode:
an electrochromic display was prepared by electrodepositing a prussian blue film on an ITO electrode. The ITO electrode was washed with ethanol and water in an ultrasonic bath. Then soaking the ITO electrodeAdding 1:1 ethanol: NaOH (2M) for 30 minutes to activate the surface. After washing with secondary water, by applying a constant potential of 0.4V, the mixture was washed with a solution containing 0.1M KCl, 0.1M HCl, 2.5mM K3[Fe(CN)6]And 2.5mM FeCl3·6H2The ITO electrode was electropolymerized for 240s in a freshly prepared solution of O. Successful preparation of PB films was confirmed by its apparent color and absorbance change. The ITO electrode was then rinsed with secondary water and dried in air.
(6) Construction and measurement of a self-powered bacterial biosensor based on EBFC:
as shown in FIG. 1A, the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP bioanode, PB as the cathode, was transferred to a container containing 2mM NAD+Measuring E of the cell by using a two-electrode system, a working electrode and a reference electrode which are connected together to clamp a photo anode in a small cell of 0.1M PB buffer solution with pH 6.0 of 5mM glucose and NADH to carry out signal test1 OCVIs marked as E10 OCV(ii) a In a small cell which is provided with a BOD/CNT/CP biological cathode, an anode PW (converted from PB) electrode and a 0.1M PB buffer solution with pH of 6.0, a biological cathode is clamped by a two-electrode system through a working electrode, a reference electrode and a counter electrode are connected together to clamp a photo anode, signal test is carried out, and the E of the cell is measured2 OCVIs marked as E20 OCV；
Placing the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode in 1mL of V.parahaemolyticus with different concentrations, incubating for 90 minutes at 37 ℃ to obtain V.p/MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, and washing the surface of the electrode with secondary water; assembling the bioanode and PB electrode into a cell, and measuring E of the cell1 OCVIs marked as E1n OCV(ii) a Assembling BOD/CNT/CP biological cathode, PW electrode, and 0.1M PB buffer system containing pH 6.0 into battery, and measuring E of the battery2 OCVIs marked as E2n OCVThe color change of the PB electrode was observed.
Replacing the concentration of the target V.parahaemolyticus to obtain a series of E1n OCVAnd E2n OCVValues, different concentrations of vCoordinates, E measured at different concentrations of a series of v1n OCVAnd E2n OCVThe values are set as ordinate to obtain V.parahaemolyticus concentration and E1n OCVAnd E2n OCVLinear relationship between values to facilitate E by assay1n OCVAnd E2n OCVAnd obtaining the specific V.parahaemolyticus concentration according to the linear relation, thereby achieving the purpose of detecting the V.parahaemolyticus.
In this example, the DNA sequence of the aptamer was: 5' -SH- (CH)2)6-TCT AAA AAT GGG CAA AGA AACAGT GAC TCG TTG AGA TAC T-3′。
The above embodiment is only a detection method of v.parahaemolyticus, and a general platform of EBFC-based self-energized cell sensors can detect corresponding food markers by changing the type of aptamers, and is highly practical.
FIGS. 2(A) and (C) are modified with V.parahaemolyticus E at different concentrations1n OCVAnd E2n OCVValues, from left to right, were 5, 10, 50, 100, 200, 500, 1000, 5000, 10000CFU mL, respectively-1(ii) a FIGS. 2(B) and (D) are graphs showing the results of measurements of E at different concentrations of V.parahaemolyticus, with the abscissa representing the different concentrations of V.parahaemolyticus1n OCVAnd E2n OCVThe values are used as ordinate, and a logarithmic linear relation graph is obtained.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A method of detecting pathogenic bacteria in food using an EBFC-based self-powered bacterial biosensor comprising an anode, a PB electrode, a cathode and an electrolyte; the anode is an MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, the cathode is a BOD/CNTs/CP biological cathode, and the electrolyte is 1-3 mM NAD+A 0.1M PB buffer system of NADH, 3-6 mM glucose, pH 6.0;
the preparation method of the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode comprises the following steps:
performing cyclic voltammetry scanning on the surface of a CP electrode modified with CNT/AuNPs to polymerize thionine, coating the electrode with a mixture containing a GDH solution and aptamer SH-DNA, and performing primary incubation to obtain aptamer/GDH/PTH/CNT/AuNPs/CP; washing the surface of the electrode with secondary water, dripping an MCH solution on the surface of the electrode, performing secondary incubation, sealing a plate, and washing the surface of the electrode with the secondary water to obtain an MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode;
the method for detecting pathogenic bacteria in food comprises the following steps:
step (1): the MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, PB electrode, containing 1-3 mM NAD+Measuring E of a battery assembled by a 0.1M PB buffer system of pH 6.0 and NADH and 3-6 mM glucose1 OCV，E1 OCVIs the open circuit voltage between the bioanode and the PB electrode, denoted as E10 OCV(ii) a BOD/CNT/CP biological cathode, PW electrode, containing 1-3 mM NAD+Measuring E of a battery assembled by a 0.1M PB buffer system of NADH and 3-6 mM glucose with pH of 6.02 OCV，E2 OCVIs the open circuit voltage between the PW electrode and the biological cathode, and is marked as E20 OCV；
Step (2): placing an MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode in 0.5-2mL of pathogenic bacteria with different concentrations, incubating at 37 ℃ for 60-120 minutes to obtain a pathogens/MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, and washing the surface of the electrode with secondary water;
and (3): the pathogens/MCH/aptamer/GDH/PTH/CNT/AuNPs/CP biological anode, the PB electrode and the 1-3 mM NAD+Measuring E of a battery assembled by a 0.1M PB buffer system of pH 6.0 and NADH and 3-6 mM glucose1 OCVIs marked as E1n OCV(ii) a Assembling BOD/CNT/CP biological cathode, PW electrode, and 0.1M PB buffer system containing pH 6.0 into battery, and measuring E of the battery2 OCVIs marked as E2n OCVThe color change of the PB electrode was observed.
2. The method for detecting pathogenic bacteria in food by using the EBFC-based self-powered bacteria biosensor as claimed in claim 1, wherein the concentration of GDH and aptamer is 1-4 mg mL-1And 400-700 nM, the amount of the mixture to be applied by dripping is 25-60 μ L.
3. The method for detecting pathogenic bacteria in food by using the EBFC-based self-powered bacteria biosensor as claimed in claim 1, wherein the sweep rate of the polymeric thionine is 30-70mv s by cyclic voltammetry scan on the electrode surface-1The thionine is dissolved in 2.2mM acetic acid solution, so that the concentration of the thionine is 1-3 mM.
4. The method for detecting pathogenic bacteria in food by using the EBFC-based self-powered bacteria biosensor as recited in claim 1, wherein the MCH solution is 20-50 μ L and 1-3 mM MCH solution.
5. The method for detecting pathogenic bacteria in food by using the EBFC-based self-powered bacteria biosensor as claimed in claim 1, wherein the primary incubation is performed at 3-7 ℃ for 8-16 h.
6. The method for detecting pathogenic bacteria in food by using the EBFC-based self-powered bacteria biosensor as claimed in claim 1, wherein the secondary incubation is performed at room temperature for 0.5-2 h.
CN201811075816.5A 2018-09-14 2018-09-14 EBFC-based self-powered bacterial biosensor and application thereof Expired - Fee Related CN109254039B (en)

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