CN116203239A - Colorimetric biosensor for detecting Shewanella, preparation method and detection method thereof - Google Patents

Colorimetric biosensor for detecting Shewanella, preparation method and detection method thereof Download PDF

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CN116203239A
CN116203239A CN202310320064.9A CN202310320064A CN116203239A CN 116203239 A CN116203239 A CN 116203239A CN 202310320064 A CN202310320064 A CN 202310320064A CN 116203239 A CN116203239 A CN 116203239A
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shewanella
coooh
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周丽华
江明霞
黄尉辰
王航
袁勇
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Guangdong University of Technology
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Abstract

The invention discloses a colorimetric biosensor for detecting Shewanella, a preparation method and a detection method thereof, and aims to provide a colorimetric biosensor for detecting Shewanella, which has high sensitivity and good specificity, and provides a good platform for rapidly screening Shewanella and deeply researching extracellular respiratory mechanism and ecological environment restoration application; the technical scheme includes that the magnetic nanoparticle with the surface modified by streptavidin, the Shewanella MR-1 polyclonal antibody with the biotin modified by biotin, the immunoflower-like cobalt oxyhydroxide nano-enzyme and the Shewanella MR-1 are modified on the surface; relates to the technical field of biological detection.

Description

Colorimetric biosensor for detecting Shewanella, preparation method and detection method thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to a colorimetric biosensor for detecting Shewanella, and also relates to a preparation method of the colorimetric biosensor.
Background
Shewanella is a reducing bacterium of iron catabolite, widely existing in aquatic and terrestrial environments. It plays an important role in geochemical circulation, bioerosion, bioremediation and bioenergy.
Currently, methods for bacterial classification and identification include traditional proliferation, isolation, serum and biochemical identification methods; enzyme-linked immunosorbent assay (ELISA); polymerase Chain Reaction (PCR) and other conventional methods. Methods for detecting Shewanella have been studied mainly as follows. For example, jung schroers et al compared biochemical techniques, 16S rRNA sequencing, MALDI-TOF MS and Shellock Microbiological Identification System (MIS) based on fatty acid ethyl esters to identify 74 Shewanella strains. Recently, yu et al evaluated a method for rapid identification of Shewanella by matrix assisted laser Desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). However, these methods are either expensive, time-consuming or require complex operations. Thus, there is an urgent need to develop a simple, highly sensitive and more specific method for detection of Shewanella.
The biosensor is an instrument or system which is sensitive to biological substances and converts the concentration of the biological substances into an electric signal for detection, and the instrument or system consists of a biological recognition molecule, a signal conversion system (transducer), a signal amplification system and a signal detection system. In the past ten years, the biosensor is mainly applied to pathogen, heavy metal and toxic small molecule detection because of the advantages of wide application, low cost, sensitivity, rapidness and the like. In combination with the action mechanism and advantages of the optical sensor, besides detecting pathogenic bacteria in the environment, the optical sensor has great potential for rapid and specific detection of environmental microorganisms, and can be applied to expanding the application of the biological sensor.
In recent years, various nanometer mimic enzymes are used as new generation artificial enzymes, and have attracted extensive attention and have greatly progressed due to unique characteristics of effective catalysis, easy synthesis, good stability, low cost and the like. In many enzyme mimetic-tmb systems, hydrogen peroxide (H 2 O 2 ) Must participate as an oxidizing agent. However, due to Zhou Guo environment to H 2 O 2 Has great influence on the properties of the (B) and is difficult to find out the direct and rapid H detection 2 O 2 Therefore, a new nano-mimic enzyme detection method is especially necessary.
Disclosure of Invention
Based on the problems, the first object of the invention is to provide a colorimetric biosensor for detecting Shewanella, which has high sensitivity and good specificity, and provides a good platform for rapid screening of Shewanella and deep research on extracellular respiratory mechanism and ecological environment restoration application.
A second object of the present invention is to provide a method for preparing the colorimetric biosensor described above.
A third object of the present invention is a method of using the colorimetric biosensor
For this purpose, the first technical solution provided by the present invention is as follows:
a colorimetric biosensor for detecting Shewanella comprises magnetic nanoparticles with streptavidin modified surfaces, shewanella MR-1 polyclonal antibodies with biotin modified surfaces, immunolorenty cobalt oxyhydroxide nanoenzyme and Shewanella MR-1.
Furthermore, the colorimetric biosensor for detecting Shewanella comprises magnetic nanoparticles modified by streptavidin, a Shewanella MR-1 polyclonal antibody modified by biotin and an immunolorescence cobalt oxyhydroxide nanoenzyme to form MNP-Shewanella-CoOOH NFs complex.
The invention provides MNP-Shewanella-CoOOH NFs complex principle as follows: since the color biosensor provided by the application comprises magnetic nano-particles with the surfaces modified by streptavidin, shewanella oneidensis MR-1 polyclonal antibodies modified by biotin and immune flower-like cobalt oxyhydroxide nano-enzymes (CoOOH NFs). According to the binding of biotin and streptavidin, biotinylated capture antibodies were coupled to streptavidin-modified magnetic nanoparticles to prepare immunomagnetic nanoparticles (MNPs). Carboxylated CoOOH nanoflowers were used to couple with MR-1 antibodies to prepare immune cooohns. Fully mixing and culturing immune magnetic nano-particles (MIPs), shewanella and immune CoOOH NFs to form an MNP-Shewanella-CoOOH sandwich compound, so as to obtain an initial solution. And (3) magnetically capturing the initial solution to obtain a sensor to be detected, injecting 3,3', 5' -tetramethyl benzidine (TMB) substrate into the sensor to be detected, and catalyzing the complex by using CoOOH NFs nano enzyme in the sensor to be detected to generate a yellow catalyst. Finally, the absorbance is measured to determine the Shewanella concentration.
The second object of the present invention is to provide a method for preparing the colorimetric biosensor for detecting Shewanella, comprising the following steps in order:
1) Culturing the bacteria and determining the total number of colonies
2) Preparation of antibodies
Concentration is set to 10 9 Injecting emulsified antigens subcutaneously in different points on the back of a New Zealand female white rabbit by cfu/mL, injecting 0.2mL into each point, performing primary immunization for 30 days, performing booster immunization for 1 time, performing booster immunization for 3 times every 10 days, and performing booster immunization by using Freund's incomplete adjuvant; finally, after 10 days of immunization for 1 time, blood is taken from the ear vein, firstly, the blood is taken in a water bath at 37 ℃ for one hour, the blood is placed at 4 ℃ for standing overnight, centrifugation is carried out at 3000r/min, supernatant fluid is taken, shewanella antibody solution is obtained, and purification is carried out;
3) Preparation of immunomagnetic nanoparticles
200. Mu.L of streptavidin-modified MNPs and 10. Mu.L of biotinylated polyclonal antibody were added to a centrifuge tube containing 500. Mu.L of PBS and blocked with 1% BSA; then, the mixture was incubated at 15rpm for 45 minutes; after removal of excess polyclonal antibodies by PBST washing, immunoMNPs were formed and stored in 500. Mu.L PBS at 4 ℃;
4) Preparation of immune CoOOH nanoflower
10mL of CoOOH NFs was centrifuged at 10,000rpm for 10 minutes to remove the supernatant and resuspended in 2mL of deionized water; taking 5mL of each of the EDC and NHS dilutions, mixing the CoOOH dilutions with EDC/NHS to achieve a CoOOH/EDC/NHS ratio of 1:1:1 for 1h at 15 rpm; mixing the mixture after the reaction with 250 mu L of the antibody prepared in the step 2), and incubating for 1h at 15rpm at room temperature; the antibody is grafted through coordination complexing, so that immune CoOOH nanoflower is finally obtained, after the antibody is successfully immobilized on CoOOH NFs, 200 mu L of 10.0% BSA is added to immune CoOOH NFs, and the immune CoOOH NFs are incubated for 1h to block active sites; then resuspended by centrifugation in 500. Mu.L of 1% BSA in PBS and finally stored in a brown flask at 4 ℃;
5) Preparation of Sandwich complexes
200 mu L of immunized MNPs, 500 mu L of samples containing Shewanella with different concentrations and 100 mu L of immunized CoOOH NFs are incubated at 15rpm for 30min to form MNP-bacteria-CoOOH sandwich complexes, an initial solution is obtained, and the initial solution is enriched to obtain the sensor to be tested.
Further, the preparation method of the colorimetric biosensor for detecting Shewanella, wherein the bacterial culture comprises the following steps in sequence:
(1) Preparing a liquid culture medium: adding 4.2g of LB broth powder and 200mL of deionized water into a conical flask, stirring ultrasonically for dissolution, sealing, and placing into a sterilizing pot for high-temperature sterilization to obtain an LB liquid culture medium;
(2) Resuscitating bacteria: thawing frozen bacteria in a refrigerator at 4 ℃, adding the frozen bacteria into the LB liquid culture medium in the step (1), uniformly mixing, and placing the mixture into a shaking table for culturing for 24 hours;
(3) Preparing a solid LB culture medium: 5g of NaCl, 2.5g of yeast, 5g of tryptone and 10g of agar are dissolved in 500mL of deionized water and uniformly mixed; pouring the prepared liquid culture medium into a plurality of culture dishes to prepare an LB solid culture medium;
further, the preparation method of the colorimetric biosensor for detecting Shewanella bacteria comprises the steps of Shewanella bacteria, shewanella putrefying bacteria, geobacillus bacteria, staphylococcus aureus and escherichia coli.
Further, according to the preparation method of the colorimetric biosensor for detecting Shewanella, the volume ratio of CoOOH, EDC, NHS is 1:1:1.
Further, in the preparation method of the colorimetric biosensor for detecting Shewanella, the concentrations of the Shewanella samples are respectively as follows: 5X 10 3 cfu/mL、5×10 4 cfu/mL、5×10 5 cfu/mL、5×10 6 cfu/mL、5×10 7 cfu/mL、5×10 8 cfu/mL、5×10 9 cfu/mL。
Further, the preparation method of the colorimetric biosensor for detecting Shewanella comprises the steps of placing an initial solution on a magnet through a test tube, magnetically separating a compound, collecting substances adsorbed by the magnet, washing for 3-5 times by using 500 mu L of PBST, and removing impurities to obtain the colorimetric biosensor.
A third object of the present invention is to provide a method of using the colorimetric biosensor for detecting shiwanella as described above, comprising the steps of, in order:
1) The colorimetric biosensor was injected with 200 μl of TMB and catalyzed for 15min, terminated with 100 μl of 1.5M dilute sulfuric acid;
2) Transferring the solution after the catalytic reaction into a 96-well plate, and measuring the absorbance of the solution by using an enzyme-labeled instrument; and (3) calculating the concentration of the Shewanella in the liquid to be tested by taking the absorbance value at the peak value of the Shewanella in the series of concentrations as a standard curve.
Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:
(1) The technical scheme provided by the invention utilizes the specific interaction and immunomagnetic capture between the antigen and the antibody to construct the sandwich colorimetric sensor for Shewanella detection, so that Shewanella can be effectively enriched and separated to improve the sensitivity and the specificity of the sensor;
(2) The technical scheme provided by the invention uses the cobalt oxyhydroxide nanoflower (CoOOH NFs) with high simulated enzyme activity and good stability as a probe for amplifying biological signals, does not need any oxidant to participate in TMB reaction, and has the advantages of simple preparation, mild reaction condition, low cost, good stability, good biocompatibility and the like;
(3) According to the technical scheme provided by the invention, the absorbance is measured by ultraviolet, the relation between the absorbance and the bacterial quantity is obtained, and the quick detection of Shewanella is realized through a judgment result of a purple light absorption spectrometer;
(4) The colorimetric sensor provided by the invention has the advantages of simple preparation method, high sensor sensitivity and good specificity, and provides technical support for rapid screening of Shewanella, deep research of extracellular respiratory mechanism and ecological environment restoration application.
In summary, the colorimetric biosensor constructed by the invention utilizes the specific interaction and immunomagnetic capture between antigen and antibody to effectively enrich and separate Shewanella so as to improve the sensitivity and the specificity of the sensor; compared with peroxidase analogues or oxidase analogues, cobalt oxyhydroxide nanoflower (CoOOH NFs) does not need any oxidant to participate in TMB reaction by using CoOOH NFs with higher simulated enzyme activity and better stability as a signal probe for effectively amplifying biological signals, and has the advantages of simple preparation, mild reaction condition, low cost, good biocompatibility and the like; the sensor has simple preparation method, stable performance, good repeatability of TMB, and can be used for measuring 5×10 in 45min 3 ～5×10 9 The detection of the Shewanella in CFU/mL is expected to provide technical support for the rapid screening of the Shewanella and the deep research of the extracellular respiratory mechanism and the ecological environment restoration application thereof.
Drawings
Fig. 1 is a schematic structural diagram of a sandwich-type colorimetric sensor provided in the present application.
FIG. 2 is a diagram showing SDS-PAGE electrophoresis of the purified MR-1 antibody of the present invention;
FIG. 3 is a MALDI-TOF diagram of the MR-1 antibody used in the present invention;
FIG. 4 is a graph demonstrating the peroxidase-like properties of CoOOH nanoflower in the present invention;
wherein (a) is an ultraviolet absorption spectrum; (b) Is a graph of the relationship between the ultraviolet absorbance and the concentration of CoOOH NFs
FIG. 5 is a TEM image (a) and SEM image (b) of CoOOH nanoflower according to the present invention;
FIG. 6 is an SEM image of an MNP-bacterial-CoOOH sandwich complex according to the invention;
FIG. 7 is a sensitivity test result of the biosensor in the present invention;
wherein (a) is the visible color change of different concentrations of the Shewanella catalyst; (b) For the biosensorCalibration curve for detecting salmonella in the range of 5×10 3 ～5×10 9 CFU/mL(N＝3)
FIG. 8 is the specificity of the biosensor of the present invention for non-target bacteria (N=3)
Detailed Description
The following will make clear and complete descriptions of the technical solutions in the embodiments of the present invention in conjunction with the process of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. 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
The colorimetric biosensor for detecting Shewanella comprises magnetic nano-particles with streptavidin modified surfaces, shewanella MR-1 polyclonal antibodies with biotin modified surfaces, immunoflower-like cobalt oxyhydroxide nano-enzyme and Shewanella MR-1.
The surface of the magnetic nanoparticle modified with streptavidin, the Shewanella MR-1 polyclonal antibody modified with biotin and the immunoflower-like cobalt oxyhydroxide nanoenzyme form a MNP-Shewanella-CoOOH NFs complex with a sandwich structure, and the magnetic nanoparticle modified with streptavidin, the Shewanella MR-1 polyclonal antibody modified with biotin and the immunoflower-like cobalt oxyhydroxide nanoenzyme form a sandwich structure, and the MNP-Shewanella-CoOOH NFs complex is shown in figure 1.
Example 2
The preparation method of the colorimetric biosensor for detecting Shewanella in the embodiment 1 provided in this embodiment sequentially comprises the following steps:
first) cultivation of bacteria
(1) Preparing and culturing: a250 mL Erlenmeyer flask was cleaned, 4.2g of Luria-Bertani (LB) broth powder and 200mL of deionized water were added, dissolved by ultrasonic agitation, sealed with a sealing film, and placed in a sterilization pot for high temperature sterilization.
(2) Resuscitating bacteria: frozen bacteria (Shewanella, staphylococcus aureus, escherichia coli and Bacillus subtilis) were thawed in a refrigerator at 4 ℃. And then opening an alcohol lamp to wipe a sample with alcohol, then slightly burning the bottle mouth of the conical flask for placing the LB culture medium by using the outer flame of the alcohol lamp, finally adding bacteria into the LB liquid culture medium, uniformly mixing, and placing the mixture into a shaking table for culturing for 24 hours.
(3) Preparing a solid LB culture medium: 5g NaCl, 2.5g yeast, 5g tryptone and 10g agar were dissolved in 500mL deionized water and mixed well. Pouring the prepared liquid culture medium into a culture dish to prepare the LB solid culture medium.
Two) determination of colony count
The total number of colonies was calculated by the plating plate dilution method. The sample to be tested is made into several different 10-fold increasing dilutions, 1mL of each dilution is taken out and placed in a sterilizing dish to be mixed with nutrient agar medium, after a certain period of cultivation at a certain temperature (generally 48 hours), the number of bacterial colonies formed in each dish is recorded, and the total number of bacterial colonies contained in each gram (or each mL) of original sample is calculated according to the dilution.
Three) antibody preparation
Concentration is set to 10 9 The cfu/mL treated Shewanella liquid is injected subcutaneously with emulsified antigen 2mL (0.2 mL for each site) at different sites on the back of New Zealand female white rabbits, and after 30 days of primary immunization, the Shewanella liquid is boosted for 1 time, and then the Shewanella liquid is boosted for 1 time every 10 days, and 3 times in total, wherein Freund's incomplete adjuvant is adopted for the boosting. After the last 1 immunization for 10 days, blood is taken from the ear vein, firstly, the blood is taken in a water bath at 37 ℃ for one hour, the blood is placed at 4 ℃ for standing overnight, centrifugation is carried out at 3000r/min, and the supernatant is taken to obtain the Shewanella antibody solution.
Fourth) antibody purification
The same batch of antibodies with high titers are purified by an octanoic acid-ammonium sulfate method, and the specific experimental steps are as follows:
1) 200. Mu.L of serum was added to 800. Mu.L of acetic acid-sodium acetate (60 mM, pH=4), pH=4.6 to 4.8 was adjusted with 0.1mol/L NaOH, and the mixture was stirred at room temperature.
2) N-octanoic acid is added dropwise, stirred at room temperature for 30min, and then placed at 4 ℃ for standing for 2h, so that a white precipitate layer appears gradually in the solution.
3) Centrifuging at 10,000rpm at 4deg.C for 20min, collecting supernatant, and recording volume V 1 。
4) V at an addition level of 1/10 1 Volume V was recorded in the supernatant with 10 XPBS (0.1 mol/L, pH=7.4) 2 (V 1 ∶V 2 ＝10∶1)
5) Slowly adding saturated ammonium sulfate (within 30 min) dropwise at 4deg.C under ice bath to make its concentration 45%, adding 0.8 times V 2 Standing for 2h at 4 ℃.
6) After standing, centrifugation was performed at 10000rpm for 3min, the precipitate was collected, and the supernatant was discarded.
7) Redissolved in a small amount of PBS (0.01 m, ph=7.4), centrifuged at 10,000rpm for 20min at 4 ℃, the supernatant collected and the pellet discarded.
8) Dialysis was performed at 4℃for 3 days with PBS (0.01M, pH=7.4), with 3 changes per day.
9) After removing excessive ammonium sulfate by dialysis, the dialysate was centrifuged at 10000rpm for 15min, and the supernatant was collected.
Fifth) preparation of immunomagnetic nanoparticles
200. Mu.L of streptavidin-modified MNPs and 10. Mu.L of biotinylated polyclonal antibody were added to a 1.5mL centrifuge tube containing 500. Mu.L LPBS (10 mM, pH 7.4), blocked with 1% BSA. The mixture was then incubated at 15rpm for 45 minutes. After removal of excess polyclonal antibodies by PBST washing, immunoMNPs were formed and stored in 500. Mu.L PBS at 4 ℃.
Sixth) preparation of immune CoOOH nanoflower
10mL of CoOOH NFs was centrifuged at 10,000rpm for 10 minutes to remove the supernatant and resuspended in 2mL of deionized water. EDC and NHS dilutions were each 5mL, and CoOOH dilutions were mixed with EDC/NHS to 1:1:1 (v/v) CoOOH/EDC/NHS and mixed at 15rpm for 1h.
The mixture after the above reaction was mixed with 250. Mu.L of antibody, and incubated at 15rpm for 1 hour at room temperature. And (3) attaching an antibody through coordination complexing, finally obtaining immune CoOOH nanoflower, successfully fixing the antibody on CoOOH NFs, adding 200 mu L of 10.0% (w/v) BSA to immune CoOOH NFs, and incubating for 1h to block active sites. Then resuspended by centrifugation in 500. Mu.L of 1% BSA in PBS and finally stored in a brown flask at 4 ℃.
Seventh) preparation of Sandwich complexes
200. Mu.L of the immunoMNPs were mixed with 500. Mu.L of the immunoMNPs at different concentrations (5X 10) 3 -5×10 9 cfu/mL) of Shewanella samples and 100 μl CoOOH NFs were incubated at 15rpm for 30min to form MNP-bacteria-CoOOH sandwich complexes, giving the initial solution.
Eighth) initial solution enrichment
Placing the prepared initial solution on a magnet through a test tube, magnetically separating the compound, collecting the magnetically adsorbed compound, washing with 500 mu L of PBST for 3-5 times, and removing impurities to obtain the colorimetric biosensor.
In order to better use the technical scheme provided by the application, the following provides a colorimetric biosensor using method:
1) Catalytic reaction of sensor to be measured
The colorimetric biosensor for Shewanella detection prepared in example 2 was injected with 200. Mu.L TMB and catalyzed for 15min, and terminated with 100. Mu.L dilute sulfuric acid (1.5M).
2) Measurement of absorbance
The solution after the catalytic reaction was transferred to a 96-well plate, and its absorbance was measured with an enzyme-labeled instrument. And (3) calculating the concentration of the Shewanella in the liquid to be tested by taking the absorbance value at the peak value of the Shewanella in the series of concentrations as a standard curve.
In order to prove the effect of the technical scheme provided by the application, the detection experimental detection data of the colorimetric biosensor for detecting Shewanella provided by the application are given below;
the Shewanella is used for carrying out 5 rounds of immunization on New Zealand big rabbits, the Freund complete adjuvant fully emulsifies the antigen during the primary immunization, the flow of the boosting immunization is similar to the primary immunization, and the Freund incomplete adjuvant is used for fully emulsifing during the following 4 times of immunization. Multiple injections were performed subcutaneously at different sites visible behind the back or neck of the animals. After 5 immunizations, the rabbit polyclonal antiserum was obtained. The antibody titer is mainly expressed by the physical state of the antibody itself and the time that the antibody can stay in an organism, and is proportional to the effect of the antibody, as expressed by the degree of binding to the antigen. In order to obtain the prepared polyclonal antibody, the titer of the antibody is measured by adopting an indirect ELISA method, and if the titer of the antibody exceeds 64,000, the antibody titer is high and can be used for the next experiment.
This example uses SDS-PAGE electrophoresis experiments to determine the purification effect of MR-1 antibodies. As shown in FIG. 2, pAb has a total molecular weight of about 70kDa, wherein the heavy chain has a molecular weight of about 55kDa and the light chain has a molecular weight of 20-25 kDa. The result shows that pAb purified by the octanoic acid-ammonium sulfate method has no obvious impurity band, and has good purification effect, high purity and satisfactory result. MALDI-TOF measurements were performed on the purified antibodies. As shown in FIG. 3, shewanella antibody with a molecular weight of 66464kDa can be obtained. SDS-PAGE experiments were approximately consistent with the molecular weight size measured by MALDI-TOF. Can be further used for the next experiment.
This example uses an ultraviolet-visible spectrophotometer to verify the peroxidase-like properties of CoOOH nanoflower. See FIG. 4 (a) only when CoOOH NFs are associated with TMB and H 2 SO 4 When mixed, the color is obviously yellow, and H 2 SO 4 TMB and TMB+H 2 SO 4 Almost colorless, coOOH was brown. Thus, it was demonstrated that CoOOHNFs had good peroxidase-like enzyme activity. CoOOHNFs at different concentrations (1-500. Mu.g/mL) were used to catalyze TMB substrates in this example and absorbance was measured using UV light. As shown in FIG. 4 (b), the absorbance value increased with the concentration of CoOOH NFs, and the absorbance value (A) had a good linear relationship with the concentration of CoOOHNFs (C), ranging from 1 to 120. Mu.g/mL, indicating the feasibility of CoOOH NF as a label.
The present example characterizes the CoOOH nanoflowers prepared using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). With reference to fig. 5, an SEM image of flower-like CoOOH, it can be seen that CoOOH sheets are clustered together to form flower-like nanostructures. Preparing CoOOH nanoplatelets into flower shapes can increase surface area and couple more antibodies. The flower-like structure of CoOOH can be clearly seen from the TEM images. To further confirm the formation of sandwich complexes, TEM imaging was used to characterize the complexes. See fig. 6, the images shown verify their successful formation.
Testing the sensitivity of a biosensor
A calibration model between the absorbance value of the biosensor and the concentration of the target bacteria is established and is used for measuring the concentration of salmonella in an unknown sample. The biosensor was used to perform 3 parallel tests on Shewanella cells in pure culture at a concentration of 5×10 respectively 3 ～5×10 9 CFU/mL. Referring to FIG. 7, it can be seen that the higher the Shewanella concentration the greater the color change of the bacterial sample.
The absorbance value (a) has a good linear relationship with the logarithm of the bacterial concentration (C), which can be expressed as a=0.0373 log (C) -0.0952 (R 2 ＝0.93)。
The specificity of the biosensor of the present example was evaluated by detecting target bacteria (Shewanella MR-1), non-target bacteria (Shewanella putrefying, geobacillus, escherichia coli and Staphylococcus aureus). As is evident from FIG. 8, the saturation values of non-target bacteria (e.g., 0.1529, 0.1023 for Staphylococcus aureus, 0.2233 for Shewanella putrefying, 0.2304 for Geobacillus) were much lower than for Shewanella (0.7977), confirming that the biosensor has good specificity. Three repeated experiments are studied, and fluctuation of the solution absorbance and standard error are smaller, so that the method has better repeatability.
The foregoing is merely illustrative of the structures of this invention and various modifications, additions and substitutions for those skilled in the art can be made to the described embodiments without departing from the scope of the invention or from the scope of the invention as defined in the accompanying claims.

Claims (9)

1. A colorimetric biosensor for detecting Shewanella is characterized by comprising magnetic nano-particles with streptavidin modified surfaces, a Shewanella MR-1 polyclonal antibody modified with biotin, an immunolorenty cobalt oxyhydroxide nano-enzyme and Shewanella MR-1.
2. The colorimetric biosensor for detecting Shewanella according to claim 1, wherein the surface-modified magnetic nanoparticles, the biotin-modified Shewanella MR-1 polyclonal antibody, and the immunolorenty cobalt oxyhydroxide nanoenzyme form a sandwich-structured MNP-Shewanella CoOOH NFs complex.
3. The method for preparing a colorimetric biosensor for detecting shiva according to claim 1, comprising the following steps in order:
1) Culturing the bacteria and determining the total number of colonies
2) Preparation of antibodies
Concentration is set to 10 9 Injecting emulsified antigens subcutaneously in different points on the back of a New Zealand female white rabbit by cfu/mL, injecting 0.2mL into each point, performing primary immunization for 30 days, performing booster immunization for 1 time, performing booster immunization for 3 times every 10 days, and performing booster immunization by using Freund's incomplete adjuvant; finally, after 10 days of immunization for 1 time, blood is taken from the ear vein, firstly, the blood is taken in a water bath at 37 ℃ for one hour, the blood is placed at 4 ℃ for standing overnight, centrifugation is carried out at 3000r/min, supernatant fluid is taken, shewanella antibody solution is obtained, and purification is carried out;
3) Preparation of immunomagnetic nanoparticles
200. Mu.L of streptavidin-modified MNPs and 10. Mu.L of biotinylated polyclonal antibody were added to a centrifuge tube containing 500. Mu.L of PBS and blocked with 1wt% BSA; then, the mixture was incubated at 15rpm for 45 minutes; after removal of excess polyclonal antibodies by PBST washing, immunoMNPs were formed and stored in 500. Mu.L PBS at 4 ℃;
4) Preparation of immune CoOOH nanoflower
10mL of CoOOH NFs was centrifuged at 10,000rpm for 10 minutes to remove the supernatant and resuspended in 2mL of deionized water; taking 5mL of each of the EDC and NHS dilutions, mixing the CoOOH dilutions with EDC/NHS to achieve a CoOOH/EDC/NHS ratio of 1:1:1 for 1h at 15 rpm; mixing the mixture after the reaction with 250 mu L of the antibody prepared in the step 2), and incubating for 1h at 15rpm at room temperature; the antibody is grafted through coordination complexing, so that immune CoOOH nanoflower is finally obtained, after the antibody is successfully immobilized on CoOOH NFs, 200 mu L of 10.0% BSA is added to immune CoOOH NFs, and the immune CoOOH NFs are incubated for 1h to block active sites; then resuspended by centrifugation in 500. Mu.L of 1% BSA in PBS and finally stored in a brown flask at 4 ℃;
5) Preparation of Sandwich complexes
200 mu L of immunized MNPs, 500 mu L of samples containing Shewanella with different concentrations and 100 mu L of immunized CoOOH NFs are incubated at 15rpm for 30min to form MNP-bacteria-CoOOH sandwich complexes, an initial solution is obtained, and the initial solution is enriched to obtain the sensor to be tested.
4. A method of preparing a colorimetric biosensor for detecting shiwanella according to claim 3, wherein the culturing of the bacteria comprises the following steps in order:
(1) Preparing a liquid culture medium: adding 4.2g of LB broth powder and 200mL of deionized water into a conical flask, stirring ultrasonically for dissolution, sealing, and placing into a sterilizing pot for high-temperature sterilization to obtain an LB liquid culture medium;
(2) Resuscitating bacteria: thawing frozen bacteria in a refrigerator at 4 ℃, adding the frozen bacteria into the LB liquid culture medium in the step (1), uniformly mixing, and placing the mixture into a shaking table for culturing for 24 hours;
(3) Preparing a solid LB culture medium: 5g of NaCl, 2.5g of yeast, 5g of tryptone and 10g of agar are dissolved in 500mL of deionized water and uniformly mixed; the prepared liquid medium was poured into a plurality of dishes to prepare an LB solid medium.
5. A method of preparing a colorimetric biosensor for detecting shiva according to claim 3, wherein the bacteria comprise shiva, shiva putrefying bacteria, geobacillus, staphylococcus aureus and escherichia coli.
6. The method of claim 3, wherein the ratio of CoOOH, EDC, NHS to Shewanella colorimetric biosensor is 1:1:1.
7. A colorimetric bio-sensor for detection of shiva according to claim 3The preparation method of the sensor is characterized in that the concentration of the Shewanella sample is respectively as follows: 5X 10 3 cfu/mL、5×10 4 cfu/mL、5×10 5 cfu/mL、5×10 6 cfu/mL、5×10 7 cfu/mL、5×10 8 cfu/mL、5×10 9 cfu/mL。
8. The method for preparing a colorimetric biosensor for detecting Shewanella according to claim 3, wherein the initial solution enrichment method is to put the initial solution on a magnet through a test tube, magnetically separate the complex, collect the substances adsorbed by the magnet, wash for 3-5 times with 500 μl of PBST, and remove impurities to obtain the colorimetric biosensor.
9. The method of using a colorimetric biosensor for detecting Shewanella as claimed in claim 1, wherein,
1) The colorimetric biosensor was injected with 200 μl of TMB and catalyzed for 15min, terminated with 100 μl of 1.5M dilute sulfuric acid;
2) Transferring the solution after the catalytic reaction into a 96-well plate, and measuring the absorbance of the solution by using an enzyme-labeled instrument; and (3) calculating the concentration of the Shewanella in the liquid to be tested by taking the absorbance value at the peak value of the Shewanella in the series of concentrations as a standard curve.
CN202310320064.9A 2023-03-28 2023-03-28 Colorimetric biosensor for detecting Shewanella, preparation method and detection method thereof Pending CN116203239A (en)

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