CN113311029A - Impedance rapid detection method of escherichia coli in food based on nano magnetic beads - Google Patents
Impedance rapid detection method of escherichia coli in food based on nano magnetic beads Download PDFInfo
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- 239000011324 bead Substances 0.000 title claims abstract description 27
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- 238000001514 detection method Methods 0.000 title claims abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000003828 vacuum filtration Methods 0.000 claims abstract description 10
- 239000002105 nanoparticle Substances 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 7
- 239000002131 composite material Substances 0.000 claims abstract description 4
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- 239000000243 solution Substances 0.000 claims description 27
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- 229940055416 blueberry extract Drugs 0.000 claims description 12
- 235000019216 blueberry extract Nutrition 0.000 claims description 12
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 12
- 240000000851 Vaccinium corymbosum Species 0.000 claims description 10
- 235000003095 Vaccinium corymbosum Nutrition 0.000 claims description 10
- 235000017537 Vaccinium myrtillus Nutrition 0.000 claims description 10
- 235000021014 blueberries Nutrition 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 229910052763 palladium Inorganic materials 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- UBEWDCMIDFGDOO-UHFFFAOYSA-N cobalt(2+);cobalt(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Co+2].[Co+3].[Co+3] UBEWDCMIDFGDOO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
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- 239000012498 ultrapure water Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
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- 102000004856 Lectins Human genes 0.000 claims description 3
- 108090001090 Lectins Proteins 0.000 claims description 3
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- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 2
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- 229910052737 gold Inorganic materials 0.000 abstract description 3
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 2
- 244000052616 bacterial pathogen Species 0.000 description 6
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- 238000003752 polymerase chain reaction Methods 0.000 description 2
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- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 229910052697 platinum Inorganic materials 0.000 description 1
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Abstract
The invention belongs to the field of food safety detection, and relates to a method for quickly detecting impedance of escherichia coli in food based on nano magnetic beads, which comprises the following steps: step one, obtaining a graphene paper flexible electrode in a vacuum filtration and reduction mode, and modifying the surface of the electrode with gold nanoparticles; modifying an element with an escherichia coli recognition function on the surface of the graphene/nano-gold composite paper electrode; step three, synthesizing corresponding nano magnetic beads and modifying recognition elements with the escherichia coli capturing capacity on the surfaces of the magnetic beads; and step four, the immunomagnetic beads capture bacteria, combine with the antibody on the surface of the electrode to form a sandwich structure and generate an impedance signal.
Description
Technical Field
The invention discloses a method for rapidly detecting impedance of escherichia coli in food based on nano magnetic beads, and belongs to the technical field of food safety detection.
Background
Pathogenic bacteria that cause infectious diseases are attracting increasing public attention. Meanwhile, huge economic loss can be caused due to infection of pathogenic bacteria, and meanwhile, a food safety alarm clock is knocked to the world. Of the many pathogenic bacteria that can cause life-threatening symptoms, enterohemorrhagic escherichia coli can cause very severe outbreaks of food-borne diseases.
At present, new requirements are put on pathogenic bacteria detection methods, such as simple, rapid and sensitive provision of abundant information of pathogenic bacteria, especially in resource-limited areas. A large number of complex methods such as Polymerase Chain Reaction (PCR) methods, DNA microarray methods, DNA sequencing techniques, enzyme-linked immunosorbent assays, staining methods, separation methods, cell culture methods, and the like, are used for the detection and analysis of pathogenic bacteria. These techniques have high sensitivity but require expensive instrumentation, complex pre-treatment steps and specialized operators.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides a method for rapidly detecting the impedance of escherichia coli in food based on nano magnetic beads, and is suitable for rapidly detecting the escherichia coli in various occasions.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for rapidly detecting impedance of escherichia coli in food based on nano magnetic beads comprises the following steps:
firstly, obtaining a graphene paper flexible electrode in a vacuum filtration and reduction mode, and modifying the surface of the electrode by nano particles and polymers to obtain a graphene/nano composite paper electrode;
secondly, modifying an element with an escherichia coli recognition function on the surface of the graphene/nano composite paper electrode;
thirdly, synthesizing corresponding nano magnetic beads and modifying the surfaces of the magnetic beads with recognition elements with the capture capacity of escherichia coli;
fourthly, the immunomagnetic beads capture bacteria, and the bacteria are combined with antibodies on the surface of the electrode to form a sandwich structure and generate an impedance signal;
and fifthly, analyzing the linear relation between the impedance value and the concentration of the added escherichia coli to obtain a correction equation of the electrode for the escherichia coli.
The reduction mode in the first step is that the extract of the fresh blueberries is used as a green reducing agent.
In the first step, nanoparticles and polymers are modified into a composite of nano palladium and polypyrrole.
The element having an E.coli recognition function in the second step is a phage against E.coli.
And in the third step, the nano magnetic beads are cobaltosic oxide.
The recognition element having an escherichia coli capturing ability in the third step is lectin.
The mass ratio of graphene to nano palladium to polypyrrole in the graphene/nano composite paper electrode is 1:2: 2.
The preparation method of the graphene/nano composite paper electrode in the first step comprises the following steps:
1) firstly, the preparation of blueberry extract aqueous solution (50%) is carried out, and because the blueberry extract is an extremely unstable reducing agent, the blueberry extract can be quickly oxidized and decomposed when being heated by light, and the blueberry extract can be used as the preparation in each time and is sealed and refrigerated in a dark place for later use. The configuration method comprises the following steps: weighing 1g of fresh blueberries, carrying out homogeneous dissolution by using 5mL of ultrapure water, filtering to obtain a clear transparent solution, and immediately refrigerating and storing in a dark place;
2) adding 5mL of LGO solution and 15mL of ultrapure water into a 50mL centrifuge tube, then adding 5mL of prepared blueberry solution, mixing and shaking up, and finally enabling the mixed solution to be dark brown;
3) carrying out ultrasonic dispersion on the preliminarily reduced GO solution for 2 hours, fully reacting, and then standing in a dark place for 24 hours until the solution is pure black;
4) building and installing a vacuum filtration platform, placing 50mm x 0.22 mu m of water-based filter paper, then pouring the solution for vacuum filtration until the solution is filtered, and forming preliminarily reduced wet GO paper on the filter paper;
5) drying the wet GO paper in a vacuum drying oven for 12h to obtain GOP (group of pictures) primarily reduced by ascorbic acid, wherein the GOP is pure black;
6) separating the dried GOP from the filter paper, putting the GOP into a beaker, adding HI until the GOP is immersed, sealing the opening of the beaker, putting the beaker into a shaking table for reaction for 6 hours, and setting parameters: the temperature is 25 ℃, and the revolution is 60 rmp;
7) and finally, washing the reacted GOP with absolute ethyl alcohol to remove HI residues, and drying with nitrogen to obtain rGOP with metallic silvery luster.
Compared with the prior art, the invention has the beneficial effects that: compared with the traditional electrode detection result, the graphene/nano composite paper electrode has higher detection sensitivity, no hydrogen is separated out in the detection process, the result is more stable, and the detection range is wider; and the electrode is more flexible in structure, easier to miniaturize and suitable for rapid detection of escherichia coli in various occasions.
Drawings
The invention is further described below with reference to the accompanying drawings.
Fig. 1 is a comparison of conductivity of a graphene/nanocomposite paper electrode and a graphene flexible electrode according to an embodiment of the present invention.
Fig. 2 is a graph of the relationship between the content of nano-palladium in the graphene/nano-composite paper electrode and the number of scanning cycles of cyclic voltammetry.
FIG. 3 is a cyclic voltammogram of E.coli at different concentrations measured with graphene/nanocomposite paper electrodes according to an embodiment of the present invention.
Fig. 4 is a linear graph of escherichia coli concentration versus concentration for different concentrations measured with graphene/nanocomposite paper electrodes according to embodiments of the present invention.
Detailed Description
The invention relates to a method for rapidly detecting impedance of escherichia coli in food based on nano magnetic beads, which comprises the following steps:
firstly, obtaining a graphene paper flexible electrode in a vacuum filtration and reduction mode, and modifying the surface of the electrode by nano particles and polymers to obtain a graphene/nano composite paper electrode;
secondly, modifying an element with an escherichia coli recognition function on the surface of the graphene/nano composite paper electrode;
thirdly, synthesizing corresponding nano magnetic beads and modifying the surfaces of the magnetic beads with recognition elements with the capture capacity of escherichia coli;
fourthly, the immunomagnetic beads capture bacteria, and the bacteria are combined with antibodies on the surface of the electrode to form a sandwich structure and generate an impedance signal;
and fifthly, analyzing the linear relation between the impedance value and the concentration of the added escherichia coli to obtain a correction equation of the electrode for the escherichia coli.
The reduction mode in the first step is that the extract of the fresh blueberries is used as a green reducing agent.
In the first step, nanoparticles and polymers are modified into a composite of nano palladium and polypyrrole.
The element having an E.coli recognition function in the second step is a phage against E.coli.
And in the third step, the nano magnetic beads are cobaltosic oxide.
The recognition element having an escherichia coli capturing ability in the third step is lectin.
The mass ratio of graphene to nano palladium to polypyrrole in the graphene/nano composite paper electrode is 1:2: 2.
The preparation method of the graphene/nano composite paper electrode in the first step comprises the following steps:
1) firstly, the preparation of blueberry extract aqueous solution (50%) is carried out, and because the blueberry extract is an extremely unstable reducing agent, the blueberry extract can be quickly oxidized and decomposed when being heated by light, and the blueberry extract can be used as the preparation in each time and is sealed and refrigerated in a dark place for later use. The configuration method comprises the following steps: weighing 1g of fresh blueberries, carrying out homogeneous dissolution by using 5mL of ultrapure water, filtering to obtain a clear transparent solution, and immediately refrigerating and storing in a dark place;
2) adding 5mL of LGO solution and 15mL of ultrapure water into a 50mL centrifuge tube, then adding 5mL of prepared blueberry solution, mixing and shaking up, and finally enabling the mixed solution to be dark brown;
3) carrying out ultrasonic dispersion on the preliminarily reduced GO solution for 2 hours, fully reacting, and then standing in a dark place for 24 hours until the solution is pure black;
4) building and installing a vacuum filtration platform, placing 50mm x 0.22 mu m of water-based filter paper, then pouring the solution for vacuum filtration until the solution is filtered, and forming preliminarily reduced wet GO paper on the filter paper;
5) drying the wet GO paper in a vacuum drying oven for 12h to obtain GOP (group of pictures) primarily reduced by ascorbic acid, wherein the GOP is pure black; wherein the GOP is graphene oxide paper.
6) Separating the dried GOP from the filter paper, putting the GOP into a beaker, adding HI until the GOP is immersed, sealing the opening of the beaker, putting the beaker into a shaking table for reaction for 6 hours, and setting parameters: the temperature is 25 ℃, and the revolution is 60 rmp;
7) and finally, washing the reacted GOP with absolute ethyl alcohol to remove HI residues, and drying the HI residues with nitrogen to obtain rGOP with metallic silvery luster, wherein the rGOP is reduced graphene oxide paper.
Compared with the conductivity of the pure graphene paper, as shown in fig. 1, it can be obviously seen that the conductivity of the graphene/nano composite paper electrode is obviously improved compared with the pure graphene paper, wherein the bGCE is a bare glassy carbon electrode, and the AuNP is nano gold nanoparticles.
The graphene flexible electrode prepared by the preparation method of the graphene/nano composite paper electrode provided by the embodiment of the invention has excellent mechanical properties and the capability of bending at a large angle without damage; after the chemical treatment of mixing the nano platinum on the surface, the conductivity of the electrode is greatly improved, and the electrode has great advantages compared with the traditional solid electrode; and the preparation method has simple process. The blueberry extracting solution is adopted to carry out primary reduction on the graphene oxide, oxygen-containing groups which cannot be reduced by hydroiodic acid in the graphene oxide can be reduced, and then the hydroiodic acid is adopted to carry out thorough reduction, so that the conductivity of the graphene is better.
The graphene/nano composite paper electrode prepared by the embodiment of the invention is applied to quantitative measurement of escherichia coli. Specifically, the concentration detection of escherichia coli according to the embodiment of the present invention includes the following steps:
1. the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode; the electrochemical working platform comprises an electrochemical working station, an electrolytic cell (containing phosphate buffer solution) and detection result display equipment;
2. connecting the graphene/nano composite paper electrode system to an electrochemical workstation, and measuring in an electrolytic cell of a phosphoric acid buffer solution (0.1mol/L, pH 2.5) by adopting an electrochemical impedance method;
3. adding 9.2X 10 of the raw materials into an electrolytic cell respectively2～9.2×107The impedance values of the E.coli solutions of different concentrations of CFU/mL, as shown in FIG. 3, increased with increasing concentration of the bacterial solution.
4. Analyzing the linear relation between the impedance value and the concentration of the added escherichia coli to obtain a correction equation of the electrode to the escherichia coli: y 355.24+106.43x, as shown in fig. 4, where the accuracy of the correction equation is R2＝0.995。
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
Claims (9)
1. A method for rapidly detecting impedance of escherichia coli in food based on nano magnetic beads is characterized by comprising the following steps:
firstly, obtaining a graphene paper flexible electrode in a vacuum filtration and reduction mode, and modifying the surface of the electrode by nano particles and polymers to obtain a graphene/nano composite paper electrode;
secondly, modifying an element with an escherichia coli recognition function on the surface of the graphene/nano composite paper electrode;
thirdly, synthesizing corresponding nano magnetic beads and modifying the surfaces of the magnetic beads with recognition elements with the capture capacity of escherichia coli;
fourthly, the immunomagnetic beads capture bacteria, and the bacteria are combined with antibodies on the surface of the electrode to form a sandwich structure and generate an impedance signal;
and fifthly, analyzing the linear relation between the impedance value and the concentration of the added escherichia coli to obtain a correction equation of the electrode for the escherichia coli.
2. The method for rapidly detecting the impedance of escherichia coli in food based on nano magnetic beads as claimed in claim 1, wherein the reduction mode in the first step is that an extract of fresh blueberries is used as a green reducing agent.
3. The method for rapidly detecting the impedance of escherichia coli in food based on nanobead according to claim 1, wherein the nanoparticles and the polymer are modified into a composite of nano-palladium and polypyrrole in the first step.
4. The method as claimed in claim 1, wherein the element having the function of identifying escherichia coli in the second step is phage for escherichia coli.
5. The method as claimed in claim 1, wherein the nano magnetic beads in the third step are cobaltosic oxide.
6. The method as claimed in claim 1, wherein the recognition element with capture ability of escherichia coli in the third step is lectin.
7. The method for rapidly detecting the impedance of escherichia coli in food based on nano magnetic beads as claimed in claim 3, wherein the mass ratio of graphene to nano palladium to polypyrrole in the graphene/nano composite paper electrode is 1:2: 2.
8. The method for rapidly detecting the impedance of escherichia coli in food based on nano magnetic beads as claimed in claim 1, wherein the method for preparing the graphene/nano composite paper electrode in the first step comprises the following steps:
1) firstly, the preparation of blueberry extract aqueous solution (50%) is carried out, and because the blueberry extract is an extremely unstable reducing agent, the blueberry extract can be quickly oxidized and decomposed when being heated by light, and the blueberry extract can be used as the preparation in each time and is sealed and refrigerated in a dark place for later use. The configuration method comprises the following steps: weighing 1g of fresh blueberries, carrying out homogeneous dissolution by using 5mL of ultrapure water, filtering to obtain a clear transparent solution, and immediately refrigerating and storing in a dark place;
2) adding 5mL of LGO solution and 15mL of ultrapure water into a 50mL centrifuge tube, then adding 5mL of prepared blueberry solution, mixing and shaking up, and finally enabling the mixed solution to be dark brown;
3) carrying out ultrasonic dispersion on the preliminarily reduced GO solution for 2 hours, fully reacting, and then standing in a dark place for 24 hours until the solution is pure black;
4) building and installing a vacuum filtration platform, placing 50mm x 0.22 mu m of water-based filter paper, then pouring the solution for vacuum filtration until the solution is filtered, and forming preliminarily reduced wet GO paper on the filter paper;
5) drying the wet GO paper in a vacuum drying oven for 12h to obtain GOP (group of pictures) primarily reduced by ascorbic acid, wherein the GOP is pure black;
6) separating the dried GOP from the filter paper, putting the GOP into a beaker, adding HI until the GOP is immersed, sealing the opening of the beaker, putting the beaker into a shaking table for reaction for 6 hours, and setting parameters: the temperature is 25 ℃, and the revolution is 60 rmp;
7) and finally, washing the reacted GOP with absolute ethyl alcohol to remove HI residues, and drying with nitrogen to obtain rGOP with metallic silvery luster.
9. The method for rapidly detecting the impedance of the escherichia coli in the food based on the nano magnetic beads as claimed in claim 1, wherein the step of detecting the concentration of the escherichia coli in the fifth step comprises the steps of:
1) the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode; the electrochemical working platform comprises an electrochemical working station, an electrolytic cell containing phosphate buffer solution and detection result display equipment;
2) connecting the graphene/nano composite paper electrode system to an electrochemical workstation, and measuring in an electrolytic cell with the concentration of a phosphoric acid buffer solution of 0.1mol/L and the pH value of 2.5 by adopting an electrochemical impedance method;
3) adding 9.2X 10 of the solution into an electrolytic cell respectively2～9.2×107The impedance value of the Escherichia coli solution with different concentrations of CFU/mL is improved along with the increase of the concentration of the bacterial solution.
4) And analyzing the linear relation between the impedance value and the concentration of the added escherichia coli to obtain a correction equation of the electrode to the escherichia coli: 355.24+106.43x, where the accuracy of the correction equation is R2＝0.995。
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