A colorimetric biosensor for detecting Shewanella, and its preparation and detection methods.

Abstract

This invention discloses a colorimetric biosensor for detecting Shewanella, along with its preparation and detection methods. The aim is to provide a colorimetric biosensor for detecting Shewanella, which exhibits high sensitivity and specificity, offering a good platform for rapid screening of Shewanella, in-depth research into its extracellular respiration mechanism, and applications in ecological environment restoration. The technical solution includes magnetic nanoparticles surface-modified with streptavidin, a biotin-modified Shewanella MR-1 polyclonal antibody, an immunoflorescent cobalt hydroxyoxide nanozyme, and Shewanella MR-1; it pertains to the field of biodetection technology.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a colorimetric biosensor for detecting Shewanella, and also to a method for preparing the colorimetric biosensor. Background Technology

[0002] Shewanella is a heterotrophic iron-reducing bacterium widely distributed in aquatic and terrestrial environments. It plays an important role in geochemical cycles, biodegradation, bioremediation, and bioenergy.

[0003] Currently, methods used for bacterial classification and identification include traditional methods such as propagation, isolation, serological and biochemical identification; enzyme-linked immunosorbent assay (ELISA); polymerase chain reaction (PCR); and other traditional methods. To date, methods for detecting Shewanella have been studied in the following ways. For example, Jung Schroers et al. compared biochemical techniques, 16S rRNA sequencing, MALDI-TOF MS, and the fatty acid ethyl ester-based Sherlock microbial identification system (MIS) to identify 74 Shewanella strains. Recently, Yu et al. evaluated a method for rapid identification of Shewanella using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). However, these methods are either expensive and time-consuming or require complex operation. Therefore, there is an urgent need to develop simple, highly sensitive, and more specific methods for detecting Shewanella.

[0004] A biosensor is an instrument or system that is sensitive to biological substances and converts their concentration into an electrical signal for detection. It consists of a biorecognition molecule, a signal conversion system (transducer), a signal amplification system, and a signal detection system. Over the past decade, biosensors have been widely used for the detection of pathogens, heavy metals, and toxic small molecules due to their advantages such as broad applicability, low cost, and rapid sensitivity. Combining the working mechanism and advantages of optical sensors, in addition to detecting pathogens in the environment, they have great potential for rapid and specific detection of environmental microorganisms, thereby expanding the applications of biosensors.

[0005] In recent years, various nano-mimetic enzymes, as a new generation of artificial enzymes, have attracted widespread attention and made significant progress due to their unique characteristics such as efficient catalysis, ease of synthesis, good stability, and low cost. In many enzyme mimicry-TMB systems, hydrogen peroxide (H2O2) must participate as an oxidant. However, because the surrounding environment has a significant impact on the properties of H2O2, it is difficult to find a suitable storage medium for the direct and rapid detection of H2O2. Therefore, developing a new nano-mimetic enzyme detection method is particularly necessary. Summary of the Invention

[0006] Based on the above problems, the first objective of this invention is to provide a colorimetric biosensor for detecting Shewanella bacteria. This sensor has high sensitivity and good specificity, providing a good platform for rapid screening of Shewanella bacteria, in-depth research on its extracellular respiration mechanism, and ecological environment restoration applications.

[0007] A second objective of this invention is to provide a method for preparing the aforementioned colorimetric biosensor.

[0008] A third objective of this invention is the method of using the colorimetric biosensor.

[0009] Therefore, the first technical solution provided by this invention is as follows:

[0010] A colorimetric biosensor for detecting Shewanella includes magnetic nanoparticles modified with streptavidin, a biotin-modified polyclonal antibody against Shewanella MR-1, an immunofloret-like cobalt hydroxyoxide nanozyme, and Shewanella MR-1.

[0011] Furthermore, in the aforementioned colorimetric biosensor for detecting Shewanella, the surface is modified with streptavidin-modified magnetic nanoparticles, biotin-modified Shewanella MR-1 polyclonal antibody, and an immunoflorescent cobalt hydroxyoxide nanozyme to form an MNP-Shewanella-CoOOH NFs complex.

[0012] The principle of the MNP-Shewanella-CoOOH NFs complex provided in this invention is as follows: The colorimetric biosensor provided in this application includes magnetic nanoparticles modified with streptavidin, a biotin-modified Shewanella oneidensis MR-1 polyclonal antibody, and immunoflorescent cobalt hydroxyoxide nanozymes (CoOOH NFs). Based on the binding of biotin and streptavidin, the biotinylated capture antibody is coupled to the streptavidin-modified magnetic nanoparticles to prepare immunomagnetic nanoparticles (MNPs). Carboxylated CoOOH nanoflores are used to couple with the MR-1 antibody to prepare immunoCoOOH NFs. The immunomagnetic nanoparticles (MIPs), Shewanella, and immunoCoOOH NFs are thoroughly mixed and cultured to form the MNP-Shewanella-CoOOH sandwich complex, obtaining the initial solution. The initial solution was magnetically captured to obtain the sensor to be tested. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was injected into the sensor. The CoOOH NFs nanozyme in the sensor catalyzed the complex, generating a yellow catalyst. Finally, its absorbance was measured to determine the concentration of Shewanella bacteria.

[0013] A second objective of this invention is to provide a method for preparing the above-mentioned colorimetric biosensor for detecting Shewanella, comprising the following steps in sequence:

[0014] 1) Culture the bacteria and determine the total number of colonies.

[0015] 2) Preparation of antibodies

[0016] With a concentration of 10 9 Shewanella bacteria culture treated with CFU / mL was subcutaneously injected into the back of female New Zealand white rabbits at different sites using emulsified antigen, with 0.2 ml injected at each site. A booster immunization was given 30 days after the initial immunization, followed by booster immunizations every 10 days for a total of 3 times. Freund's incomplete adjuvant was used for booster immunizations. Ten days after the final immunization, blood was collected from the ear vein, incubated at 37°C for one hour, then placed at 4°C overnight, centrifuged at 3000 rpm, and the supernatant was collected to obtain the Shewanella antibody solution, which was then purified.

[0017] 3) Preparation of immunomagnetic nanoparticles

[0018] 200 μL of streptavidin-modified MNPs and 10 μL of biotinylated polyclonal antibody were added to a centrifuge tube containing 500 μL of PBS and blocked with 1% BSA. The mixture was then incubated at 15 rpm for 45 minutes. After washing with PBST to remove excess polyclonal antibody, immune MNPs were formed and stored in 500 μL of PBS at 4°C.

[0019] 4) Preparation of immune CoOOH nanoflowers

[0020] Centrifuge 10 mL of CoOOH NFs at 10,000 rpm for 10 minutes to remove the supernatant and resuspend in 2 mL of deionized water; take 5 mL each of EDC and NHS diluents, mix the CoOOH diluent with EDC / NHS to achieve a CoOOH / EDC / NHS ratio of 1:1:1, and mix at 15 rpm for 1 h; mix the above reaction mixture with 250 μL of the antibody prepared in step 2) and incubate at 15 rpm for 1 h at room temperature; attach the antibody through coordination complexation to finally obtain the immunogenic CoOOH nanoflowers. After successfully immobilizing the antibody on CoOOH NFs, add 200 μL of 10.0% BSA to immunize the CoOOH NFs and incubate for 1 h to block the active site; then centrifuge and resuspend in 500 μL of PBS containing 1% BSA, and finally store in a brown flask at 4 °C;

[0021] 5) Preparation of sandwich complex

[0022] 200 μL of immunized MNPs, 500 μL of samples containing different concentrations of Shewanella, and 100 μL of immunized CoOOHNFs were incubated at 15 rpm for 30 min to form an MNP-bacteria-CoOOH sandwich complex, which was then used to obtain the initial solution. The initial solution was then enriched to obtain the sensor to be tested.

[0023] Furthermore, in the above-mentioned method for preparing a colorimetric biosensor for detecting Shewanella, the bacterial culture sequentially includes the following steps:

[0024] (1) Preparation of liquid culture medium: Add 4.2g LB broth powder and 200mL deionized water to the conical flask, stir and dissolve by ultrasonication, seal the flask, and sterilize it in an autoclave at high temperature to obtain LB liquid culture medium;

[0025] (2) Bacterial resuscitation: After thawing the frozen bacteria in a 4°C freezer, add them to the LB liquid medium in step (1), mix well, and incubate in a shaker for 24 hours.

[0026] (3) Preparation of solid LB medium: Dissolve 5g NaCl, 2.5g yeast, 5g tryptone and 10g agar in 500mL deionized water and mix well; pour the prepared liquid medium into multiple petri dishes to make LB solid medium;

[0027] Furthermore, in the above-mentioned method for preparing a colorimetric biosensor for detecting Shewanella, the bacteria include Shewanella, Shewanella putrefactive, Geobacterium, Staphylococcus aureus, and Escherichia coli.

[0028] Furthermore, in the above-mentioned method for preparing a colorimetric biosensor for detecting Shewanella, the volume ratio of CoOOH, EDC, and NHS is 1:1:1.

[0029] Furthermore, in the above-described method for preparing a colorimetric biosensor for detecting Shewanella, the concentration of the Shewanella sample is 5 × 10⁻⁶. 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.

[0030] Furthermore, in the above-mentioned method for preparing a colorimetric biosensor for detecting Shewanella, the initial solution enrichment method involves placing the initial solution on a magnet through a test tube to magnetically separate the complex, collecting the substances adsorbed by the magnet, and then washing with 500 μL PBST 3 to 5 times to remove impurities, thereby obtaining the colorimetric biosensor.

[0031] A third objective of this invention is to provide a method for using the aforementioned colorimetric biosensor for detecting Shewanella, the method comprising the following steps in sequence:

[0032] 1) Inject 200 μL of TMB into the colorimetric biosensor, catalyze for 15 min, and terminate with 100 μL of 1.5 M dilute sulfuric acid;

[0033] 2) Transfer the solution after the catalytic reaction to a 96-well plate and measure its absorbance using an ELISA reader; plot a standard curve using the absorbance values ​​at the peak values ​​of a series of Shewanella concentrations, and calculate the concentration of Shewanella in the test solution.

[0034] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0035] (1) The technical solution provided by the present invention utilizes the specific interaction between antigen and antibody and immunomagnetic capture to construct a sandwich-type colorimetric sensor for Shewanella detection, which can effectively enrich and separate Shewanella to improve the sensitivity and specificity of the sensor.

[0036] (2) The technical solution provided by the present invention uses cobalt hydroxy oxide nanoflowers (CoOOH NFs) with high enzyme-simulating activity and good stability as probes to amplify biological signals. No oxidant is required to participate in the TMB reaction. It has the advantages of simple preparation, mild reaction conditions, low cost, good stability and good biocompatibility.

[0037] (3) The technical solution provided by the present invention utilizes ultraviolet light to measure absorbance, obtains the relationship between absorbance and bacterial count, and determines the results through a violet light absorption spectrometer, thereby achieving rapid detection of Shewanella.

[0038] (4) The colorimetric sensor provided by the present invention has a simple preparation method, high sensitivity and good specificity, and provides technical support for rapid screening of Shewanella, in-depth study of its extracellular respiration mechanism and ecological environment restoration.

[0039] In summary, the colorimetric biosensor constructed in this invention utilizes the specific interaction between antigen and antibody and immunomagnetic capture to effectively enrich and separate Shewanella, thereby improving the sensor's sensitivity and specificity. It employs CoOOH nanoflowers (NFs), which exhibit high enzyme activity and good stability, as signal probes to effectively amplify biological signals. Compared to peroxidase or oxidase analogs, CoOOH nanoflowers (NFs) offer advantages such as simple preparation, mild reaction conditions, low cost, and good biocompatibility because they do not require any oxidant to participate in the TMB reaction. The sensor is simple to prepare, exhibits stable performance, and demonstrates good TMB repeatability, capable of detecting 5 × 10⁵ cells / day within 45 minutes. 3 ~5×10 9 The detection of Shewanella at CFU / mL is expected to provide technical support for rapid screening of Shewanella, in-depth research on its extracellular respiration mechanism, and applications in ecological environment restoration. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the sandwich-type colorimetric sensor structure provided in this application.

[0041] Figure 2 This is an SDS-PAGE electrophoresis diagram of the purified MR-1 antibody in this invention;

[0042] Figure 3 This is the MALDI-TOF image of the MR-1 antibody used in this invention;

[0043] Figure 4 This invention verifies the peroxidase-like properties of CoOOH nanoflowers;

[0044] (a) is the ultraviolet absorption spectrum; (b) is the graph showing the relationship between ultraviolet absorbance and the concentration of CoOOH NFs.

[0045] Figure 5 These are TEM (a) and SEM (b) images of the CoOOH nanoflowers in this invention;

[0046] Figure 6 This is a SEM image of the MNP-bacterial-CoOOH sandwich complex in this invention;

[0047] Figure 7 These are the sensitivity test results of the biosensor in this invention;

[0048] (a) shows the visible color changes of different concentrations of Shewanella catalyst; (b) shows the calibration curve of the biosensor for detecting Salmonella, in the range of 5 × 10⁻⁶. 3 ~5×10 9 CFU / mL (N=3)

[0049] Figure 8 The biosensor in this invention exhibits specificity for non-target bacteria (N=3). Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the process of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0051] Example 1

[0052] The present invention provides a colorimetric biosensor for detecting Shewanella, comprising magnetic nanoparticles modified with streptavidin, a biotin-modified polyclonal antibody against Shewanella MR-1, an immunofloret-like cobalt hydroxyoxide nanozyme, and Shewanella MR-1.

[0053] The surface-modified magnetic nanoparticles with streptavidin, the biotin-modified Shewanella MR-1 polyclonal antibody, and the sandwich-structured MNP-Shewanella-CoOOHNFs complex formed by the immunoflorescent cobalt hydroxyoxide nanozyme are described in the reference document. Figure 1 .

[0054] Example 2

[0055] The method for preparing the colorimetric biosensor for detecting Shewanella in Example 1 provided in this embodiment includes the following steps in sequence:

[0056] 1) Bacterial culture

[0057] (1) Preparation of culture: Take a 250mL Erlenmeyer flask, wash it, add 4.2g Luria-Bertani (LB) broth powder and 200mL deionized water, sonicate and stir to dissolve, seal with sealing film, and put it into an autoclave for high-temperature sterilization.

[0058] (2) Bacterial resuscitation: Frozen bacteria (Shewanella, Staphylococcus aureus, Escherichia coli and Bacillus subtilis) were thawed in a 4°C freezer. Then, the samples were wiped with alcohol by turning on an alcohol lamp, and the mouth of the Erlenmeyer flask containing LB medium was gently heated with the outer flame of the alcohol lamp. Finally, the bacteria were added to LB liquid medium, mixed well, and incubated in a shaker for 24 hours.

[0059] (3) Preparation of solid LB medium: Dissolve 5g NaCl, 2.5g yeast, 5g tryptone and 10g agar in 500mL deionized water and mix well. Pour the prepared liquid medium into a petri dish to make solid LB medium.

[0060] (ii) Determination of total bacterial count

[0061] The total bacterial count is calculated using the plate dilution method. The sample is prepared into several different 10-fold serial dilutions. 1 mL of each dilution is then placed in a sterile Petri dish and mixed with nutrient agar medium. After incubation at a specific temperature for a certain period (usually 48 hours), the number of colonies formed in each dish is recorded. Based on the dilution factor, the total bacterial count per gram (or per mL) of the original sample is calculated.

[0062] III) Antibody Preparation

[0063] With a concentration of 10 9Shewanella bacteria culture treated with CFU / mL was subcutaneously injected at different sites on the back of female New Zealand white rabbits with 2 mL of emulsified antigen (0.2 mL per site). A booster immunization was given 30 days after the initial immunization, followed by booster immunizations every 10 days for a total of three times. Freund's incomplete adjuvant was used for booster immunizations. Ten days after the final immunization, blood was collected from the ear vein, incubated at 37°C for one hour, then stood at 4°C overnight, and centrifuged at 3000 rpm. The supernatant was collected to obtain the Shewanella antibody solution.

[0064] IV) Antibody Purification

[0065] The same batch of antibodies with high titers was purified using the octanoic acid-ammonium sulfate method. The specific experimental steps are as follows:

[0066] 1) Take 200 μL of serum and add 800 μL of acetate-sodium acetate (60 mM, pH=4). Adjust the pH to 4.6-4.8 with 0.1 mol / L NaOH and stir at room temperature.

[0067] 2) Add octanoic acid dropwise, stir at room temperature for 30 minutes, and then let stand at 4°C for 2 hours. A white precipitate layer will gradually appear in the solution.

[0068] 3) Centrifuge at 10,000 rpm for 20 min at 4℃, collect the supernatant and record the volume V1.

[0069] 4) Add 1 / 10 of V1's 10×PBS (0.1mol / L, pH=7.4) to the supernatant and record the volume V2 (V1∶V2=10∶1).

[0070] 5) Slowly add saturated ammonium sulfate (within 30 min) under an ice bath at 4℃ to make its concentration 45%, add 0.8 times V2, and let stand at 4℃ for 2 h.

[0071] 6) After standing, centrifuge at 10,000 rpm for 3 minutes, collect the precipitate and discard the supernatant.

[0072] 7) Reconstitute with a small amount of PBS (0.01M, pH=7.4), centrifuge at 10,000 rpm for 20 min at 4°C, collect the supernatant and discard the precipitate.

[0073] 8) Dialyze with PBS (0.01M, pH=7.4) at 4℃ for 3 days, changing the solution 3 times a day.

[0074] 9) After dialysis to remove excess ammonium sulfate, centrifuge the dialysate at 10,000 rpm for 15 minutes and collect the supernatant.

[0075] (v) Preparation of immunomagnetic nanoparticles

[0076] 200 μL of streptavidin-modified MNPs and 10 μL of biotinylated polyclonal antibody were added to a 1.5 mL centrifuge tube containing 500 μL PBS (10 mM, pH 7.4), which was blocked with 1% BSA. The mixture was then incubated at 15 rpm for 45 minutes. After washing with PBST to remove excess polyclonal antibody, immunogenic MNPs were formed and stored in 500 μL PBS at 4°C.

[0077] VI) Preparation of Immuno-CoOOH Nanoflowers

[0078] Centrifuge 10 mL of CoOOH NFs at 10,000 rpm for 10 minutes to remove the supernatant and resuspend it in 2 mL of deionized water. Mix 5 mL each of EDC and NHS diluents with the CoOOH diluent to achieve a CoOOH / EDC / NHS ratio of 1:1:1 (v / v) and mix at 15 rpm for 1 hour.

[0079] The mixture from the above reaction was then mixed with 250 μL of antibody and incubated at 15 rpm for 1 h at room temperature. The antibody was attached via coordination complexation to obtain immunogenic CoOOH nanoflowers. After successful antibody immobilization on CoOOH NFs, 200 μL of 10.0% (w / v) BSA was added to immunize the CoOOH NFs, and incubation was carried out for 1 h to block the active site. The nanoflowers were then resuspended by centrifugation in 500 μL of PBS containing 1% BSA and finally stored in a brown flask at 4 °C.

[0080] VII) Preparation of the sandwich complex

[0081] 200 μL of immunogenic MNPs was mixed with 500 μL of MNPs containing different concentrations (5 × 10⁻⁶). 3 -5×10 9 The sample of Shewanella (cfu / mL) was incubated with 100 μL of CoOOH NFs at 15 rpm for 30 min to form an MNP-bacteria-CoOOH sandwich complex, thus obtaining the initial solution.

[0082] 8) Initial solution enrichment

[0083] The initial solution prepared above was placed on a magnet in a test tube to magnetically separate the complex. The magnetically adsorbed complex was collected and washed 3-5 times with 500 μL PBST to remove impurities, thus obtaining a colorimetric biosensor.

[0084] To better utilize the technical solution provided in this application, the following are the usage instructions for the colorimetric biosensor:

[0085] 1) Catalytic reaction of the sensor under test

[0086] The colorimetric biosensor for detecting Shewanella prepared in Example 2 was injected into 200 μL of TMB, catalyzed for 15 min, and terminated with 100 μL of dilute sulfuric acid (1.5 M).

[0087] 2) Measurement of absorbance

[0088] The solution after the catalytic reaction was transferred to a 96-well plate, and its absorbance was measured using a microplate reader. A standard curve was constructed using the absorbance values ​​at the peak values ​​of a series of Shewanella concentrations, and the concentration of Shewanella in the test solution was calculated.

[0089] To demonstrate the effectiveness of the technical solution provided in this application, the following are the detection data of the colorimetric biosensor for detecting Shewanella provided in this application.

[0090] Five rounds of immunization were first performed on New Zealand rabbits using Shewanella bacteria. For the initial immunization, Freund's complete adjuvant was used to fully emulsify the antigen. The booster immunization procedure was similar to the initial immunization. For the subsequent four immunizations, Freund's incomplete adjuvant was used for full emulsification. Multiple subcutaneous injections were administered at different sites visible on the animal's back or neck. After five immunizations, rabbit polyclonal antiserum was obtained. Antibody titer primarily reflects the physical state of the antibody and its retention time in the body, expressed based on the degree of binding to the antigen. Antibody titer is directly proportional to antibody efficacy. To obtain the prepared polyclonal antibody, an indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the antibody titer. An antibody titer exceeding 64,000 indicates a high antibody titer, suitable for further experiments.

[0091] This embodiment uses SDS-PAGE electrophoresis to determine the purification efficiency of the MR-1 antibody. See attached image. Figure 2 The total molecular weight of pAb was approximately 70 kDa, with the heavy chain having a molecular weight of approximately 55 kDa and the light chain having a molecular weight of 20–25 kDa. The results showed that the pAb purified by the octanoic acid-ammonium sulfate method had no obvious impurity bands, indicating good purification effect and high purity, and the results were satisfactory. MALDI-TOF measurements were performed on the purified antibody. (See attached image) Figure 3 The molecular weight of the Shewanella antibody was determined to be 66464 kDa. The molecular weight obtained by SDS-PAGE and MALDI-TOF assays is roughly consistent. This allows for further use in subsequent experiments.

[0092] This embodiment uses a UV-Vis spectrophotometer to verify the peroxidase-like properties of CoOOH nanoflowers. See attached image. Figure 4(a) Only when CoOOH NFs are mixed with TMB and H2SO4 is the color distinctly yellow, while H2SO4, TMB, and TMB+H2SO4 are almost colorless, and CoOOH is brown. This indicates that CoOOH NFs have good peroxidase-like enzyme activity. In this example, different concentrations (1-500 μg / mL) of CoOOH NFs were used to catalyze TMB substrates, and their absorbance was measured using UV light. See Appendix. Figure 4 (b) The absorbance value increased with the concentration of CoOOH NFs. The absorbance value (A) showed a good linear relationship with the concentration of CoOOH NFs (C), ranging from 1 to 120 μg / mL, indicating the feasibility of CoOOH NFs as a label.

[0093] In this embodiment, the prepared CoOOH nanoflowers were characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). See Appendix. Figure 5 SEM images of the flower-like CoOOH nanosheets show that the CoOOH sheets aggregate to form a flower-like nanostructure. Preparing CoOOH nanosheets into a flower shape increases the surface area and allows for the coupling of more antibodies. The flower-like structure of CoOOH is clearly visible in the TEM images. To further confirm the formation of the sandwich complex, TEM imaging was used to characterize the complex. (See attached image.) Figure 6 The images shown verify their successful formation.

[0094] Testing the sensitivity of biosensors

[0095] A calibration model was established between the absorbance value of the biosensor and the concentration of the target bacteria to determine the concentration of Salmonella in unknown samples. The biosensor was used to perform three parallel detections on pure cultured Shewanella cells at concentrations of 5 × 10⁻⁶. 3 ~5×10 9 CFU / mL. See attached file. Figure 7 As can be seen, the higher the concentration of Shewanella, the greater the color change in the bacterial sample.

[0096] The absorbance value (A) shows 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).

[0097] This embodiment evaluates the specificity of the biosensor by detecting target bacteria (Shewanella MR-1) and non-target bacteria (Shewanella putrefactive bacteria, Geobacterium, Escherichia coli, and Staphylococcus aureus). See Appendix. Figure 8Clearly, the saturation values ​​for non-target bacteria (Escherichia coli 0.1529, Staphylococcus aureus 0.1023, Shewanella putrefactive bacteria 0.2233, and Geobacterium tumefaciens 0.2304) were significantly lower than those for Shewanella (0.7977), validating the good specificity of the biosensor. Three repeated experiments were conducted, and the fluctuations in absorbance and standard errors were small, indicating good reproducibility of the method.

[0098] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A colorimetric biosensor for detecting Shewanella, characterized in that, Including magnetic nanoparticles with streptavidin-modified surfaces, biotin-modified Shewanella MR-1 polyclonal antibody, immune flower-shaped cobalt hydroxyoxide nanozyme, and Shewanella MR-1. The surface is modified with streptavidin-modified magnetic nanoparticles, biotin-modified Shewanella MR-1 polyclonal antibody, and an MNP-Shewanella-CoOOH NFs complex with a sandwich structure formed by an immunoflorescent cobalt hydroxyoxide nanozyme. Carboxylated CoOOH nanoparticles were conjugated with MR-1 antibody to prepare an immunoflorescent cobalt hydroxyoxide nanozyme.

2. The method for preparing the colorimetric biosensor for detecting Shewanella according to claim 1, characterized in that, The steps are as follows: 1) Culture the bacteria and determine the total number of colonies. 2) Antibody preparation With a concentration of 10 9 Shewanella bacteria culture treated with CFU / mL was subcutaneously injected into the back of female New Zealand white rabbits at different sites using emulsified antigen, with 0.2 mL injected at each site. A booster immunization was given 30 days after the initial immunization, followed by booster immunizations every 10 days for a total of 3 times. Freund's incomplete adjuvant was used for booster immunizations. Ten days after the final immunization, blood was collected from the ear vein, incubated at 37°C for 1 hour, then placed at 4°C overnight, centrifuged at 3000 rpm, and the supernatant was collected to obtain the Shewanella antibody solution, which was then purified. 3) Preparation of immunomagnetic nanoparticles 200 μL of streptavidin-modified MNPs and 10 μL of biotinylated polyclonal antibody were added to a centrifuge tube containing 500 μL of PBS and blocked with 1 wt% BSA. The mixture was then incubated at 15 rpm for 45 minutes. After washing with PBST to remove excess polyclonal antibody, immune MNPs were formed and stored in 500 μL of PBS at 4°C. 4) Preparation of immune CoOOH nanoflowers Centrifuge 10 mL of CoOOH NFs at 10,000 rpm for 10 minutes to remove the supernatant, and resuspend in 2 mL of deionized water; take 5 mL each of EDC and NHS diluents, and mix the CoOOH diluent with EDC / NHS to achieve a CoOOH / EDC / NHS volume ratio of 1:1:1; mix at 15 rpm for 1 h; mix the reaction mixture with 250 μL of the antibody prepared in step 2), and incubate at 15 rpm for 1 h at room temperature; finally, obtain immune CoOOH nanoflowers. After successfully immobilizing the antibody on CoOOH NFs, add 200 μL of 10.0% BSA and incubate for 1 h to block the active site; then centrifuge and resuspend in 500 μL of PBS containing 1% BSA, and finally store in a brown flask at 4 °C; 5) Preparation of sandwich complex 200 μL of immunized MNPs, 500 μL of samples containing different concentrations of Shewanella, and 100 μL of immunized CoOOH NFs were incubated at 15 rpm for 30 min to form an MNP-Shewanella-CoOOH NFs complex, which was then used to obtain the initial solution. The initial solution was then enriched to obtain the sensor to be tested.

3. The method for preparing a colorimetric biosensor for detecting Shewanella according to claim 2, characterized in that, The bacterial culture comprises the following steps in sequence: (1) Preparation of liquid culture medium: Add 4.2g LB broth powder and 200mL deionized water to the conical flask, stir and dissolve by ultrasonication, seal the flask, and sterilize it at high temperature in an autoclave to obtain LB liquid culture medium; (2) Bacterial resuscitation: After thawing the frozen bacteria in a 4°C freezer, add them to the LB liquid medium in step (1), mix well, and incubate in a shaker for 24 hours; (3) Prepare solid LB medium: Dissolve 5g NaCl, 2.5g yeast, 5g tryptone and 10g agar in 500mL deionized water and mix well; pour the prepared liquid medium into multiple petri dishes to make LB solid medium.

4. The method for preparing a colorimetric biosensor for detecting Shewanella according to claim 2, characterized in that, The concentrations of the Shewanella samples were 5 × 10⁻⁶. 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.

5. The method for preparing a colorimetric biosensor for detecting Shewanella according to claim 2, characterized in that, The initial solution enrichment method involves placing the initial solution on a magnet through a test tube to magnetically separate the complex, collecting the substances adsorbed by the magnet, and then washing with 500 μL PBST 3–5 times to remove impurities, thereby obtaining a colorimetric biosensor.

6. The method of using the colorimetric biosensor for detecting Shewanella as described in claim 1, characterized in that, 1) Inject 200 μL of TMB into the colorimetric biosensor, catalyze for 15 min, and terminate with 100 μL of 1.5 M dilute sulfuric acid; 2) Transfer the solution after the catalytic reaction to a 96-well plate and measure its absorbance using an ELISA reader; plot a standard curve using the absorbance values ​​at the peak values ​​of a series of Shewanella concentrations, and calculate the concentration of Shewanella in the test solution.

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