CN117607226B - Application of two-dimensional metal organic framework nano material and method for detecting salmonella - Google Patents
Application of two-dimensional metal organic framework nano material and method for detecting salmonella Download PDFInfo
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- 241000607142 Salmonella Species 0.000 title claims abstract description 69
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 60
- 239000012621 metal-organic framework Substances 0.000 title claims abstract description 36
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- -1 ferrous porphyrin Chemical class 0.000 claims abstract description 16
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- 238000004132 cross linking Methods 0.000 claims abstract description 9
- 229910001453 nickel ion Inorganic materials 0.000 claims abstract description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 29
- 229940098773 bovine serum albumin Drugs 0.000 claims description 29
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- B01J31/1805—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms the ligands containing nitrogen
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Abstract
The invention provides application of a two-dimensional metal organic framework nanomaterial and a method for detecting salmonella, and belongs to the technical field of salmonella detection. The invention provides application of a two-dimensional metal organic framework nanomaterial in detecting salmonella, wherein the two-dimensional metal organic framework nanomaterial is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking. The two-dimensional metal organic framework nano material with specific structure and composition has the characteristics of simulating enzyme action and inducing Fenton reaction, and can be used for constructing a rapid, high-sensitivity and secondary-antibody-free detection method of salmonella based on an electrochemical platform.
Description
Technical Field
The invention relates to the technical field of salmonella detection, in particular to application of a two-dimensional metal organic framework nanomaterial and a method for detecting salmonella.
Background
Salmonella is a common food-borne bacterium that causes food poisoning and intestinal infections, manifested by diarrhea, nausea, vomiting, fever and abdominal pain, where Salmonella typhi can damage the bone marrow, liver and spleen of the human body, and severe cases can lead to death. In recent years, the incidence of salmonella infection has remained high, and early diagnosis of salmonella infection has been of great importance for effective treatment and prevention of transmission. However, the conventional detection method represented by the plate identification method is time-consuming, and the new technology represented by the molecular detection technology is difficult to popularize in the basic layer due to cost and technical problems (such as relatively complex operation and strict personnel qualification requirements). Thus, there remains a great clinical need to explore the construction of new rapid, sensitive and low cost techniques for salmonella detection.
Electrochemical biosensing assays have been widely explored and applied as a rapid, sensitive and low cost detection method, and electrochemical sensors of salmonella have been reported. For example, a team of woods professor (Huang, f.; xue, l.; qi, w.; cai, g.; liu, y.; lin, j. An ultrasensitive impedance biosensor for Salmonella detection based on rotating high gradient magnetic separation and cascade reaction signal amplification. Biosens. Bioelect. 2020, 176, 112921) reported an impedance biosensor using gyromagnetic separation and cascade reactions for rapid, ultrasensitive detection of salmonella typhimurium. However, most of the biological sensing of salmonella constructed at present is used for food detection, and the components in the food are relatively single, so that the purification treatment is easy. The clinical biological sample has complex components, various protein types, easy generation of non-specific interference, high requirements on the sensitivity and specificity of the method, direct and tight connection of the diagnosis result and the subsequent treatment, and high requirements on the detection method, but the novel biological sensing strategy for the detection of the clinical biological sample is rarely reported at present.
Disclosure of Invention
The invention aims to provide application of a two-dimensional metal organic framework nano material and a method for detecting salmonella.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a two-dimensional metal organic framework nanomaterial in detecting salmonella, wherein the two-dimensional metal organic framework nanomaterial is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking.
Preferably, the sheet diameter of the two-dimensional metal organic framework nanomaterial is 0.5-2 μm.
Preferably, the two-dimensional metal organic framework nanomaterial is modified by sequentially adopting canavalin A and bovine serum albumin before detecting salmonella.
The invention provides a method for detecting salmonella based on Fenton reaction, which comprises the following steps:
assembling an electrochemical biosensor, wherein a gold film is arranged on the surface of a working electrode of the electrochemical biosensor, and the surface of the gold film is modified with a salmonella specific antibody and bovine serum albumin;
sequentially dripping a liquid to be detected on the surface of the working electrode for first incubation and dripping a dispersion liquid of the nano material compound for second incubation, then performing electrochemical detection, and obtaining the concentration of the salmonella in the liquid to be detected according to the working curve of the salmonella and the electrochemical signal intensity of the liquid to be detected;
the nano material composite is obtained by modifying a two-dimensional metal organic framework nano material sequentially through canavalin A and bovine serum albumin, and the two-dimensional metal organic framework nano material is a lamellar nano material with a network structure, wherein the lamellar nano material is formed by bonding ferrous porphyrin and nickel ions and then crosslinking.
Preferably, the working electrode is a glassy carbon electrode.
Preferably, the temperature of the specific antibody of the salmonella is 32-38 ℃ and the time is 2-4 hours.
Preferably, the temperature of the bovine serum albumin modified on the surface of the gold film is 32-38 ℃ and the time is 30-60 min.
Preferably, the temperature of the first incubation is 35-38 ℃ and the time is 20-80 min.
Preferably, the concentration of the dispersion liquid of the nano material compound is 0.4-2.0 mg/mL.
Preferably, the temperature of the second incubation is 35-38 ℃ and the time is 20-60 min.
The invention provides application of a two-dimensional metal organic framework nanomaterial in detecting salmonella, wherein the two-dimensional metal organic framework nanomaterial is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking. The two-dimensional metal organic framework nano material with specific structure and composition has the characteristics of simulating enzyme action and inducing Fenton reaction, and can be used for constructing a rapid, high-sensitivity and secondary-antibody-free detection method of salmonella based on an electrochemical platform.
Drawings
FIG. 1 shows the Ni-TCPP (Fe 2+ ) Characterization of nanoplatelets, wherein (A) is Ni-TCPP (Fe) 2+ ) TEM image of nano-sheet, (B) is Ni-TCPP (Fe) 2+ ) Analysis chart of carbon element in nano-sheet, wherein (C) is Ni-TCPP (Fe) 2+ ) Analysis chart of iron element in nano-sheet, wherein (D) is Ni-TCPP (Fe) 2+ ) Oxygen element analysis chart in nano-sheet, wherein (E) is Ni-TCPP (Fe) 2+ ) The analysis chart of nitrogen element in the nano-sheet is Ni-TCPP (Fe) 2+ ) The analysis chart of nickel element in the nano-sheet is (G) Ni-TCPP (Fe) 2 + ) TEM image of ConA-BSA bioconjugate, (H) is Ni-TCPP (Fe 2+ ) AFM mapping of nanoplatelets;
FIG. 2 shows the Ni-TCPP (Fe 2+ ) Nanoplatelets and Ni-TCPP (Fe 2+ ) -ultraviolet absorbance spectra of ConA-BSA bioconjugates;
FIG. 3 shows that Ni-TCPP (Fe 2+ ) SEM image of bacteria after incubation with ConA-BSA, wherein a corresponds to no Ni-TCPP (Fe 2+ ) Bacteria after ConA-BSA incubation, B corresponds to the addition of Ni-TCPP (Fe 2+ ) Bacteria after incubation with ConA-BSA;
FIG. 4 is a schematic diagram of a Salmonella rapid detection electrochemical biosensor;
FIG. 5 is a fluorescence spectrum for detecting hydroxyl radicals (. OH) in different reaction systems;
FIG. 6 is a graph of electrochemical signal intensity and a graph of the relationship between the electrochemical signal intensity and the logarithm of the bacterial concentration of the sensor prepared in example 2 under different bacterial concentration conditions, wherein A is a graph of electrochemical signal intensity and B is a graph of the relationship between the electrochemical signal intensity and the logarithm of the bacterial concentration of the sensor under different bacterial concentration conditions.
Detailed Description
The invention provides application of a two-dimensional metal organic framework nanomaterial in detecting salmonella, wherein the two-dimensional metal organic framework nanomaterial is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking.
In the invention, the two-dimensional metal organic framework nanomaterial (MOF) is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking, and the sheet diameter of the two-dimensional metal organic framework nanomaterial is preferably 0.5-2 mu m, and the thickness of the two-dimensional metal organic framework nanomaterial is preferably 2 nm.
In the present invention, the preparation method of the two-dimensional metal organic framework nanomaterial preferably includes the following steps:
mixing nickel chloride, pyrazine, polyvinylpyrrolidone and N, N-dimethylformamide with ethanol to obtain nickel source mixed solution; ferrous porphyrin (TCPP (Fe) 2+ ) Mixing N, N-dimethylformamide with ethanol to obtain a ferrous porphyrin solution; and mixing the ferrous porphyrin solution with the nickel source mixed solution, and then carrying out reduction reaction to obtain the two-dimensional metal organic framework nano material.
In the present invention, unless otherwise specified, all materials are commercially available or prepared by methods well known to those skilled in the art.
The invention mixes nickel chloride, pyrazine, polyvinylpyrrolidone, N-dimethylformamide and ethanol to obtain nickel source mixed solution. In the invention, the mass ratio of the nickel chloride, the pyrazine and the polyvinylpyrrolidone is preferably 2.45:0.8:20, wherein the volume ratio of the N, N-dimethylformamide to the ethanol is preferably 3:1, the dosage ratio of the nickel chloride to the N, N-dimethylformamide is preferably 2.45 and mg:9 mL. The invention mixes ferrous porphyrin, N-dimethylformamide and ethanol to obtain ferrous porphyrin solution. In the present invention, the volume ratio of N, N-dimethylformamide to ethanol is preferably 3:1, the porphyrin subclassThe ratio of iron to N, N-dimethylformamide is preferably 4.4. 4.4 mg:3 mL. In the invention, the mixing mode of the ferrous porphyrin solution and the nickel source mixed solution is preferably ultrasonic mixing; the temperature of the reduction reaction is preferably 80℃and the time is preferably 24 h. After the reduction reaction is finished, the obtained material is preferably cooled to room temperature, the solid product is washed by fresh ethanol and is centrifugally separated to obtain the two-dimensional metal organic framework nano material which is named as Ni-TCPP (Fe) 2+ ) The nanoplatelets are resuspended in ethanol and stored at 4 ℃ for later use.
In the present invention, the Ni-TCPP (Fe 2+ ) The nanosheets are preferably modified by sequentially adopting canavalin A (ConA) and Bovine Serum Albumin (BSA) before detecting salmonella, wherein the canavalin A acts as a secondary antibody of salmonella, and the bovine serum albumin acts as a blocking Ni-TCPP (Fe) 2+ ) Sites in the nanoplatelets where canavalin a is not bound. In particular, the present invention preferably uses the Ni-TCPP (Fe 2+ ) And mixing the dispersion liquid of the nano-sheets with a Canavalia gladiata solution for first modification to obtain the Canavalia gladiata modified nano-sheets. In the present invention, the Ni-TCPP (Fe 2+ ) The dispersing agent in the nano-sheet dispersion is preferably ethanol, and the Ni-TCPP (Fe 2+ ) The concentration of the nano-sheet dispersion is preferably 1 mg/mL; the concentration of the Canavalia gladiata solution is preferably 20 mug/mL; the Ni-TCPP (Fe 2+ ) The volume ratio of the nano-sheet dispersion to the canavalin a solution is preferably 1:1. in the present invention, the temperature of the first modification is preferably 4℃and the time is preferably 12 h. In the present invention, during the first modification, the Ni-TCPP (Fe 2+ ) The carboxyl in the nano-sheet and the amino of the canavalin A react to form an amide bond to realize the bonding of the carboxyl and the canavalin A.
After the first modification, the method does not need post-treatment, and the obtained product system is directly mixed with bovine serum albumin solution for second modification to obtain the nano material composite, so that the nano material composite can be used for detecting salmonella based on Fenton reaction. In the present invention, the concentration of the bovine serum albumin solution is preferably 1g/100mL, which is recorded as 1%; the Ni-TCPP (Fe 2+ ) Dispersion of nanoplateletsThe volume ratio of the bovine serum albumin solution is preferably 5:1. in the present invention, the temperature of the second modification is preferably 37℃and the time is preferably 1 h. The invention adopts bovine serum albumin to be non-specifically adsorbed on the canavalin A modified nano-sheet so as to block the site which is not combined with the canavalin A, thereby eliminating the non-specific binding site and preventing Ni-TCPP (Fe 2+ ) The nanoplatelets nonspecifically adsorb the target substance. After the second modification, the present invention preferably uses a PBS solution to centrifugally wash the obtained solid product to obtain the nanomaterial complex (redispersed in the PBS solution and stored at 4 ℃ for later use).
The Ni-TCPP (Fe) 2+ ) The nano-sheet can realize rapid and high-sensitivity detection of salmonella. MOFs nanoplatelets, as a member of an emerging family of two-dimensional nanomaterials, have the advantages of infinite assembly and ultra-thin, and have attracted tremendous research interest in the fields of nanotechnology and catalysis. Compared with 3D MOFs, the 2D MOFs nano-sheet has excellent physicochemical properties: high specific surface area, ultra-thin layer thickness, rich exposed unsaturated metal sites, high aspect ratio, adjustable chemical composition, and identifiable surface atomic structure. More importantly, the ultrathin thickness of the MOFs nano-sheet is rich in unsaturated metal sites which are easy to contact, so that the MOFs nano-sheet has a small diffusion barrier, is beneficial to contact between reactants and active sites, and has good charge transmission capability and catalytic capability. The Ni-TCPP nanosheets are two-dimensional MOFs with excellent performance, combine the catalysis advantages of porphyrin structures and transition metals, and have excellent catalysis performance. In addition, as a layered structure, it is more advantageous to construct a microbial sensor at the interface, mainly because bacteria themselves have an ultra-high specific surface area and sufficient binding sites, effectively promoting the binding amount of the mimic enzyme. Currently, electrochemical signals are mainly generated by the following catalytic systems: direct electron transfer between the electrochemically active molecular beacon and the electrode, enzymatic catalysis of the relevant substrate, nanomaterial catalysis of hydrogen peroxide. However, most of the catalytic systems are still to be perfected, for example, molecular beacons produce weaker signals, while high-concentration hydrogen peroxide catalytic systems produce stronger signalsThe above presents challenges for constructing an electrochemical biosensor. The invention uses the ferrous porphyrin nano-sheet to synthesize Ni-TCPP (Fe) 2+ ) Nanosheets and initiate Fenton reaction to enhance electrochemical catalytic reaction, fe in Fenton reaction 2+ With Fe 3+ The conversion of the catalyst generates a large amount of active oxygen such as hydroxyl free radicals, not only obviously enhances the catalytic reaction of an electrochemical system, but also reduces the concentration of hydrogen peroxide, and has the effect of improving the signal to noise ratio. The detection method provided by the invention is described in detail below.
The invention provides a method for detecting salmonella based on Fenton reaction, which comprises the following steps:
assembling an electrochemical biosensor, wherein a gold film is arranged on the surface of a working electrode of the electrochemical biosensor, and the surface of the gold film is modified with a salmonella specific antibody and bovine serum albumin;
sequentially dripping a liquid to be detected on the surface of the working electrode for first incubation and dripping a dispersion liquid of the nano material compound for second incubation, then performing electrochemical detection, and obtaining the concentration of the salmonella in the liquid to be detected according to the working curve of the salmonella and the electrochemical signal intensity of the liquid to be detected;
the nano material composite is obtained by modifying a two-dimensional metal organic framework nano material sequentially through canavalin A and bovine serum albumin, and the two-dimensional metal organic framework nano material is a lamellar nano material with a network structure, wherein the lamellar nano material is formed by bonding ferrous porphyrin and nickel ions and then crosslinking.
The invention assembles an electrochemical biosensor, a gold film is arranged on the surface of a working electrode of the electrochemical biosensor, and a salmonella specific antibody and bovine serum albumin are modified on the surface of the gold film. In the present invention, the working electrode is preferably a glassy carbon electrode (GCE, Φ=3 mm); the working electrode is preferably polished, washed and dried in sequence before use; the material used for the polishing is preferably alumina, and the working electrode has a mirror-like appearance through polishing; the washing is preferably ultrasonic washing, and the reagent used in the washing is preferably water, more preferably deionized water; the drying is preferably nitrogen blow drying. The gold film is arranged on the surface of the working electrode, so that the conductivity of the electrochemical biosensor is improved; the thickness of the gold film is preferably 0.05 μm; the gold film is preferably prepared on the surface of the working electrode by electroplating.
In the invention, the surface of the gold film is modified with a salmonella specific antibody (Ab 1) which can specifically identify and capture salmonella. In an embodiment of the invention, the salmonella-specific antibody is purchased from bio-technology limited of boaosen, beijing. In the invention, the temperature of modifying the salmonella specific antibody on the surface of the gold film is preferably 32-38 ℃, more preferably 37 ℃; the time is preferably 2 to 4 hours, more preferably 2 to 2.5 hours. The invention preferably drops the salmonella specific antibody solution onto the surface of the gold film, and then modifies the salmonella specific antibody under the above conditions; the concentration of the salmonella specific antibody solution is not particularly limited, and the concentration well known to those skilled in the art can be adopted; in an embodiment of the invention, the concentration of the Salmonella-specific antibody solution is specifically 10. Mu.g/mL. After modification of the salmonella specific antibodies, the present invention preferably involves washing the resulting working electrode to remove non-specific adsorption, followed by modification of Bovine Serum Albumin (BSA); the invention blocks nonspecific adsorption by modification of bovine serum albumin. The conditions for modifying the bovine serum albumin on the surface of the gold film are not particularly limited, and the conditions well known to the person skilled in the art can be adopted; specifically, in the invention, the temperature of the bovine serum albumin modified on the surface of the gold film is preferably 32-38 ℃, more preferably 37 ℃; the time is preferably 30 to 60 minutes, more preferably 40 minutes. In the present invention, it is preferable that a 1% concentration of bovine serum albumin solution is dropped onto the surface of the gold film modified with the antibody specific for Salmonella, and then modification of bovine serum albumin is performed under the above conditions.
The present invention is not particularly limited to other components of the electrochemical biosensor, a specific assembly method, etc., and may employ an electrochemical biosensor well known to those skilled in the art.
After the electrochemical biosensor is assembled, the invention sequentially drops the liquid to be detected on the surface of the working electrode of the electrochemical biosensor for first incubation and drops the dispersion liquid of the nano material compound for second incubation, and then electrochemical detection is carried out. In the present invention, the liquid to be tested is preferably a liquid containing salmonella, and specifically may be PBS bacterial liquid containing salmonella or serum dilution containing salmonella. In the invention, the temperature of the first incubation is preferably 35-38 ℃, more preferably 37 ℃; the time is preferably 40 to 80 minutes, more preferably 1 to h. In the invention, the nanomaterial composite is obtained by modifying a two-dimensional metal organic framework nanomaterial sequentially through canavalin A and bovine serum albumin, and the two-dimensional metal organic framework nanomaterial is a lamellar nanomaterial with a network structure formed by bonding ferrous porphyrin and nickel ions and then crosslinking; the specific structure, composition and preparation method of the nanomaterial composite as a signal material of an electrochemical biosensor are preferably consistent with the above technical schemes, and are not described herein. In the present invention, the concentration of the dispersion of the nanomaterial complex is preferably 0.4 to 2.0 mg/mL, and more preferably 1.6. 1.6 mg/mL. In the invention, the temperature of the second incubation is preferably 35-38 ℃, more preferably 37 ℃; the time is preferably 20 to 60 minutes, more preferably 50 minutes.
The electrochemical detection is preferably carried out by adopting an electrochemical workstation, the electrode system is preferably a three-electrode system, and the electrochemical detection device further comprises a counter electrode and a reference electrode, wherein the counter electrode is preferably a platinum wire counter electrode, and the reference electrode is preferably an Ag/AgCl reference electrode. The specific conditions for the electrochemical detection are not particularly limited in the present invention, and in the examples of the present invention, differential Pulse Voltammetry (DPV) is specifically used in the presence of ciprofloxacin and H 2 O 2 Preferably 10 mM, and H 2 O 2 Preferably 5 mM, and the concentration of the PBS buffer is preferably 0.1M. In embodiments of the invention, electrochemical biosensors are particularly detected using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS)Wherein CV is preferably in the range of-0.2 to 0.6V, and the scanning rate is preferably 0. V.s -1 The method comprises the steps of carrying out a first treatment on the surface of the In the presence of [ Fe (CN) 6 ] 3-/4- KCl aqueous solution ([ Fe (CN)) 6 ] 3- And [ Fe (CN) 6 ] 4- Preferably 10 mM, and KCl concentration of 0.1. 0.1M), EIS is performed under the conditions of bias potential of 0.24. 0.24V and frequency of 0.1 Hz-100 kHz.
After the electrochemical detection is finished, the concentration of the salmonella in the liquid to be detected is obtained according to the working curve of the salmonella and the electrochemical signal intensity of the liquid to be detected. In the invention, the electrochemical signal intensity (specifically, current, muA) has a good linear relation with the logarithm of the concentration (CFU/mL) of the salmonella, and the high-sensitivity measurement of the salmonella in the liquid to be measured can be realized. The method for obtaining the working curve of the salmonella is not particularly limited, and the method known to those skilled in the art can be adopted.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Will 2.45 mg NiCl 2 (0.01 mM), 0.8 mg pyrazine (0.01 mM) and 20.0 mg polyvinylpyrrolidone (PVP) are dissolved in a mixed solution of N, N-Dimethylformamide (DMF) and ethanol (12 mL, the volume ratio of DMF to ethanol is 3:1) to obtain a nickel source mixed solution; ferrous 4.4 mg porphyrin (TCPP (Fe) 2+ ) Dissolving mixed solution of DMF and ethanol (4 mL, volume ratio of DMF to ethanol is 3:1) purchased from Shanghai Bai Shun Biotechnology Co., ltd.) in water, then ultrasonically mixing with the nickel source mixed solution for 10 min, transferring into a reaction kettle, and reacting at 80 ℃ for 24 h; after the reaction was completed, cooled to room temperature, the solid product was washed 3 times with fresh ethanol and separated by centrifugation (8000 rpm,10 min) to give Ni-TCPP (F)e 2+ ) Nanoplatelets of the Ni-TCPP (Fe 2+ ) The nanosheets are resuspended in ethanol and stored at 4 ℃ for later use;
500. Mu.L of Ni-TCPP (Fe) with a concentration of 1 mg/mL 2+ ) The nanoplatelet ethanol dispersion was mixed with 500. Mu.L of ConA (available from Shanghai Biotechnology Co., ltd.) solution at a concentration of 20. Mu.g/mL, and reacted under stirring at 4℃for 12 h; subsequently, 100. Mu.L of 1% BSA solution was added to the resulting product solution, and the reaction was carried out under shaking at 37℃for 1 h to eliminate non-specific binding sites; after the reaction was completed, the solid product was centrifuged (12000 rpm,10 min) with PBS solution having a concentration of 0.01. 0.01M for 2 times to remove unbound ConA, thereby obtaining Ni-TCPP (Fe) 2+ ) -ConA-BSA bioconjugate, said Ni-TCPP (Fe 2+ ) The ConA-BSA bioconjugate was redispersed in PBS at a concentration of 0.01M at 1. 1 mL and stored at 4℃for further use.
FIG. 1 shows the Ni-TCPP (Fe 2+ ) Nanoplatelets and Ni-TCPP (Fe 2+ ) Characterization of ConA-BSA bioconjugates, wherein (A) is Ni-TCPP (Fe 2+ ) TEM image of nano-sheet, (B) is Ni-TCPP (Fe) 2+ ) Analysis chart of carbon element in nano-sheet, wherein (C) is Ni-TCPP (Fe) 2+ ) Analysis chart of iron element in nano-sheet, wherein (D) is Ni-TCPP (Fe) 2+ ) Oxygen element analysis chart in nano-sheet, wherein (E) is Ni-TCPP (Fe) 2+ ) The analysis chart of nitrogen element in the nano-sheet is Ni-TCPP (Fe) 2+ ) The analysis chart of nickel element in the nano-sheet is (G) Ni-TCPP (Fe) 2+ ) TEM image of ConA-BSA bioconjugate, (H) is Ni-TCPP (Fe 2+ ) AFM mapping of nanoplatelets; as can be seen from FIG. 1A, ni-TCPP (Fe 2+ ) The nano-sheets are lamellar, and Ni-TCPP (Fe) is obtained according to element analysis in (B) - (F) in the graph 1 2+ ) The nano-sheet consists of C, fe, O, N, ni elements; (G) in FIG. 1 is Ni-TCPP (Fe 2+ ) TEM image of nanosheet-modified ConA and blocked with BSA, FIG. 1 (H) is Ni-TCPP (Fe 2 + ) AFM image of nanoplatelets, results show Ni-TCPP (Fe 2+ ) The thickness of the nano-sheet is about 2 nm.
FIG. 2 is an embodiment1 prepared Ni-TCPP (Fe 2+ ) Nanoplatelets and Ni-TCPP (Fe 2+ ) UV absorption spectrum of ConA-BSA bioconjugate, wherein a corresponds to Ni-TCPP (Fe 2+ ) Nanosheets, b corresponds to Ni-TCPP (Fe 2+ ) -a ConA-BSA bioconjugate; FIG. 2 shows the specific peak at wavelength 412 and nm for the two materials and the specific peak at wavelength 280 and nm for the antibody protein.
Comparative example 1
Preparation of Ni-TCPP (Fe 3+ ) Nanoplatelets, in particular, are described in the literature (Gan, x., han, d., wang, j., liu, p., li, x., zheng, q.,&yan, y. (2021) A highly sensitive electrochemiluminescence immunosensor for h-FABP determination based on self-enhanced luminophore coupled with ultrathin 2D nickel metal-organic framework nanosheets, biosensors and Bioelectronics, 171, 112735).
Example 2
1. Bacteria were cultured and combined with Ni-TCPP (Fe 2+ ) ConA-BSA bioconjugate binding
(1) And (5) culturing bacteria. Separating Salmonella on SS culture medium, selecting single colony, culturing in LB culture medium, culturing at 37deg.C under multi-amplitude vibration 8 h, and counting bacteria to 2×10 by Maillard turbidimetry and plate colony counting 8 CFU/mL, centrifuged (5000 rpm/min,5 min) and washed 3 times with PBS solution at a concentration of 0.01. 0.01M, and the washed bacteria were diluted to different concentrations for detection;
（2）Ni-TCPP (Fe 2+ ) ConA-BSA bioconjugate binding to Salmonella validation experiments. Single colony bacteria were first picked from the petri dish, then cultured at 37℃and a rotation speed of 200 rpm, 0.5 McO was selected as the binding assay concentration, and 500. Mu.L of the bacterial liquid was then added to 500. Mu.L of Ni-TCPP (Fe 2+ ) In ConA-BSA solution, incubation was carried out at 37℃for 30 min at 300 rpm, after which the bacteria were immobilized by washing 3 times with PBS solution at a concentration of 0.01. 0.01M, and finally adding 200. Mu.L glutaraldehyde immobilization solution.
FIG. 3 shows that Ni-TCPP (Fe 2+ ) Incubation with ConA-BSASEM image of the bacteria after that, wherein A corresponds to a non-added Ni-TCPP (Fe 2+ ) Bacteria after ConA-BSA incubation, B corresponds to the addition of Ni-TCPP (Fe 2+ ) Bacteria after incubation with ConA-BSA, the results show that Ni-TCPP (Fe 2+ ) After incubation of the bacteria with ConA-BSA, the bacterial surface was bound with a large amount of lamellar material.
2. Construction of the sensor and electrochemical analysis
(1) The sensor is assembled. Firstly, continuously polishing a glassy carbon electrode (GCE, phi=3 mm) with aluminum oxide to enable the glassy carbon electrode to have a mirror-like appearance, carrying out ultrasonic treatment in deionized water for 3 times, then drying with nitrogen, plating gold on the surface of the glassy carbon electrode through electroplating, and forming a gold film (with the thickness of 0.05 mu m) on the surface of the glassy carbon electrode to enhance the conductivity of a sensor to obtain a first modified electrode; then 8 mu L of a Salmonella specific antibody (Ab 1, purchased from Beijing Boaosen Biotechnology Co., ltd.) solution with the concentration of 10 mu g/mL is dripped on the gold membrane surface of the first modified electrode, and incubated for 2 h at 37 ℃, after incubation, the second modified electrode is obtained by washing to remove nonspecific adsorption, then 4 mu L of a BSA solution with the concentration of 1% is dripped on the second modified electrode, and incubated for 40 min at 37 ℃ to block nonspecific adsorption, so as to obtain a third modified electrode; when detecting the target, 8. Mu.L of a series of Salmonella solution concentrations were added dropwise to the third modified electrode, incubated at 37℃for 1 h, followed by 8. Mu.L of Ni-TCPP (Fe 2+ ) The ConA-BSA dispersion was added dropwise to the third modified electrode and incubated at 37℃for 50 min.
(2) And (5) detecting an electrochemical workstation. A three electrode system consisting of a glassy carbon working electrode (i.e., a third modified electrode), a platinum wire counter electrode and an Ag/AgCl reference electrode was used, using Differential Pulse Voltammetry (DPV) in the presence of ciprofloxacin 10 mM and 5 mM H 2 O 2 In a 0.1M PBS buffer, using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS), wherein CV conditions include: the scanning range is-0.2-0.6V, and the scanning speed is 0.05V s -1 The method comprises the steps of carrying out a first treatment on the surface of the Containing 10 mM [ Fe (CN) 6 ] 3-/4- 0.1M KCl water solubleIn the liquid, EIS is performed under the conditions that the bias potential is 0.24-V and the frequency is 0.1 Hz-100 kHz.
FIG. 4 is a schematic diagram of an electrochemical biosensor for rapid detection of Salmonella, in which a gold plating film is first applied to a working electrode to enhance conductivity, then a primary antibody is modified on the surface thereof and a blank site is blocked with BSA, and when a target substance is present, the antibody binds to Salmonella and then Ni-TCPP (Fe 2+ ) The ConA-BSA bioconjugate is used for detecting the current intensity in an electrochemical system of hydrogen peroxide and ciprofloxacin by using an electrochemical workstation, and the current intensity is directly proportional to the concentration of a target, so that the rapid detection of salmonella is realized.
3. Fenton reaction detection
To evaluate Ni-TCPP (Fe 2+ ) Fenton reaction efficacy of nanoplatelets, this example compares Ni-TCPP (Fe 2+ ) Nanoplatelets and Ni-TCPP (Fe 3+ ) The Fenton reaction intensity difference of the nano-sheet is specifically determined by taking the generation amount of hydroxyl radical (OH) as an evaluation index, and Ni-TCPP (Fe) with the concentration of 50M at 1 mL respectively 2+ ) Nanosheet dispersion and Ni-TCPP (Fe 3+ ) Adding 0.5. 0.5 mM final concentration of phthalic acid (TA) and 2 μl of 10M H into the nanosheet dispersion 2 O 2 Since the solution produced OH during the process, and the OH reacted with terephthalic acid to form the fluorescent product TA-OH, the amount of produced OH was reflected by monitoring TA, and the fluorescence spectrum of the reaction solution was measured by a fluorescence spectrophotometer.
FIG. 5 is a fluorescence spectrum for detecting hydroxyl radicals in different reaction systems, wherein a corresponds to Ni-TCPP (Fe 2+ ) Nanosheet +TA +H 2 O 2 B corresponds to Ni-TCPP (Fe 3+ ) Nanosheet +TA +H 2 O 2 C corresponds to TA+H 2 O 2 D corresponds to Ni-TCPP (Fe 2+ ) Nanoplatelets+ta; the results show that Ni-TCPP (Fe 2+ ) The hydroxyl radical generated by the hydrogen peroxide catalyzed by the nano-sheet is obviously higher than that of Ni-TCPP (Fe 3+ ) Nanoplatelets, confirming that Fenton reaction generated by ferrous catalysis is stronger.
FIG. 6 shows the sensor of example 2 at different finenessAn electrochemical signal intensity diagram under the condition of bacterial concentration and a relation diagram of the electrochemical signal intensity and the logarithm of bacterial concentration, wherein A is an electrochemical signal intensity diagram (adopting a DPV method as a signal output mode) of a sensor under different bacterial concentration conditions, B is a relation diagram of the electrochemical signal intensity and the logarithm of bacterial concentration, and the concentration of salmonella corresponding to a-h in A is 0 CFU/mL and 2.5X10 in sequence 1 CFU/mL、2.5×10 2 CFU/mL、2.5×10 3 CFU/mL、2.5×10 4 CFU/mL、2.5×10 5 CFU/mL、2.5×10 6 CFU/mL、2.5×10 7 CFU/mL. As shown in FIG. 6, the current value was varied from 2.5X10 with the concentration of Salmonella 1 CFU/mL to 2.5X10 7 CFU/mL increases linearly and the log bacterial concentration appears to be a stable linear correlation with amperage. The linear equation is i= 0.8526-3.197 lgC Bacteria and method for producing same Wherein I is the amperage (. Mu.A), C Bacteria and method for producing same Represents the concentration of Salmonella (CFU/mL); r is R 2 =0.9961. Further, the detection limit was calculated to be 2 CFU/mL based on the blank detection value and the standard deviation.
According to the embodiment, the method provided by the invention can realize rapid, high-sensitivity and secondary antibody-free detection of the salmonella, and particularly, the method can be developed under the condition that bacteria are free from culture, and can be used for directly quantitatively detecting the salmonella in a sample, and the detection is completed by 2 h; meanwhile, ni-TCPP (Fe 2+ ) The nanosheets not only can play a role of mimic enzyme and catalyze hydrogen peroxide, but also can catalyze and start Fenton reaction in the reaction of ferrous iron and hydrogen peroxide, so that the oxidation-reduction reaction degree is effectively improved, and the signal-to-noise ratio of the detection method can be effectively improved; furthermore, in the present invention, the electrochemical signal is derived from two catalytic reactions, the first being Ni-TCPP (Fe 2+ ) The simulated enzyme catalytic effect of the nano-sheet is second Ni-TCPP (Fe 2+ ) The Fenton reaction of the nano-sheet is carried out, the whole method does not need protease catalysis, and the signal-to-noise ratio of the detection result can be improved on the basis of saving the cost; in addition, the invention uses the canavalin A to replace the secondary antibody, thereby saving the cost and improving the stability of detection reaction.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A method for detecting salmonella based on the Fenton reaction, comprising the steps of:
assembling an electrochemical biosensor, wherein a gold film is arranged on the surface of a working electrode of the electrochemical biosensor, and the surface of the gold film is modified with a salmonella specific antibody and bovine serum albumin;
sequentially dripping a liquid to be detected on the surface of the working electrode for first incubation and dripping a dispersion liquid of the nano material compound for second incubation, then performing electrochemical detection, and obtaining the concentration of the salmonella in the liquid to be detected according to the working curve of the salmonella and the electrochemical signal intensity of the liquid to be detected;
the nano material composite is obtained by modifying a two-dimensional metal organic framework nano material sequentially through canavalin A and bovine serum albumin, and the two-dimensional metal organic framework nano material is a lamellar nano material with a network structure, wherein the lamellar nano material is formed by bonding ferrous porphyrin and nickel ions and then crosslinking.
2. The method of claim 1, wherein the working electrode is a glassy carbon electrode.
3. The method of claim 1, wherein the temperature of the specific antibody of salmonella modified on the surface of the gold film is 32-38 ℃ for 2-4 hours.
4. The method of claim 1, wherein the temperature of the bovine serum albumin modified on the surface of the gold film is 32-38 ℃ for 30-60 min.
5. The method according to claim 1, wherein the first incubation is at a temperature of 35-38 ℃ for a time of 20-80 min.
6. The method of claim 1, wherein the concentration of the dispersion of nanomaterial complex is 0.4-2.0 mg/mL.
7. The method according to claim 1 or 6, wherein the second incubation is performed at a temperature of 35-38 ℃ for a period of 20-60 min.
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| CN108344783A (en) * | 2018-01-23 | 2018-07-31 | 东南大学 | A kind of electro-chemical cells sensor and its preparation method and application |
| CN111157599A (en) * | 2020-01-06 | 2020-05-15 | 杭州电子科技大学 | Electrochemical immunosensor for detecting escherichia coli and preparation method and application thereof |
| CN111398392A (en) * | 2020-05-18 | 2020-07-10 | 河南工业大学 | Preparation method of electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme |
| CN111458516A (en) * | 2020-06-05 | 2020-07-28 | 西北大学 | Electrochemical luminescence biosensor for detecting bacterial drug resistance and preparation method thereof |
| CN112964765A (en) * | 2021-02-05 | 2021-06-15 | 重庆医科大学 | Electrochemical immunosensor for detecting CEA and preparation and application thereof |
Non-Patent Citations (2)
| Title |
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| A highly sensitive electrochemiluminescence immunosensor for h-FABP determination based on self-enhanced luminophore coupled with ultrathin 2D nickel metal-organic framework nanosheets;Xiufeng Gan et al.;Biosensors and Bioelectronics;20210118;第171卷;112735 * |
| A novel electrochemical immunosensor based on rifter-like Ni-TCPP(Fe) nanosheets and PSS-functionalized graphene for ultrasensitive detection of H-FABP;Zixuan Zhang et al.;Journal of Solid State Electrochemistry;20230410;第28卷;389–398 * |
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