Material for detecting viable staphylococcus aureus by colorimetric method as well as preparation method and application of material

Abstract

The invention discloses a material for detecting viable staphylococcus aureus by a colorimetric method as well as a preparation method and application of the material, the material takes CuCo2S4 as a carrier, a virulent phage is fixed on the surface, and the virulent phage is a phage SapYZU01. The CuCo2S4 material prepared by the invention has the characteristics of high detection speed, good specificity, high sensitivity, low equipment dependency, simplicity in operation, low cost and the like; the method can solve the problems that in the prior art, staphylococcus aureus detection is labor-consuming, time-consuming and high in price, and some detection materials and detection means cannot distinguish live and dead staphylococcus aureus bacteria, and meets the requirement for visual rapid detection of staphylococcus aureus bacteria in food samples.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a material for detecting live Staphylococcus aureus by colorimetry, a preparation method and an application thereof. Background Art

[0002] Staphylococcus aureus is an important foodborne pathogenic microorganism, widely distributed in air, water, dust, and human and animal skin and mucous membranes. It can cause local suppurative infection, pneumonia, pericarditis, and even septicemia, sepsis and other acute and chronic infections, seriously threatening human health. Staphylococcus aureus can also produce enterotoxins and cause food poisoning. If there are more than 10 Staphylococcus aureus in food, 5 The detection rate of enterotoxins is significantly increased when the number of CFU / g is higher. Furthermore, at a dose of less than 1 μg, susceptible individuals can develop symptoms within 1-6 hours. Therefore, rapid and accurate detection of this bacterium is crucial for treating infected patients, preventing transmission, and ensuring food safety.

[0003] However, existing detection methods have certain limitations. The main detection methods currently include traditional plate culture method, polymerase chain reaction (PCR) technology and immunological assay, but they have disadvantages such as long time consumption, complicated operation, expensive equipment and low sensitivity. Cutting-edge technologies such as dot blot assay (DBA), nanomaterial-based enzyme-linked immunosorbent assay (ELISA) and lateral flow assay (LFA) are used but are expensive and require professional laboratories and personnel, and most methods cannot distinguish between live and dead bacterial cells. In addition, in on-site detection, existing detection technologies are limited by long detection time. Therefore, it is of great practical significance to develop a detection method that is fast, reliable, simple to operate, highly sensitive and specific, low-cost, visual, can target live bacteria, distinguish between live and dead cells, and is anti-interference. Summary of the Invention

[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a CuCo2S4 material for colorimetric detection of live Staphylococcus aureus. The CuCo2S4 material has the characteristics of fast detection speed, good specificity, high sensitivity, low equipment dependence, simple operation, and low cost. It can solve the problems in the prior art that the detection of Staphylococcus aureus is laborious and time-consuming, expensive, and some detection materials and detection methods cannot distinguish between live and dead Staphylococcus aureus bacteria. The material is suitable for the requirements of visual rapid detection of live Staphylococcus aureus in food samples.

[0005] The present invention also provides a preparation method and application of the CuCo2S4 material for detecting live Staphylococcus aureus by the colorimetric method.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a CuCo2S4 material for detecting live Staphylococcus aureus by colorimetric method. The material uses CuCo2S4 as a carrier and has a virulent phage fixed on its surface, and the virulent phage is phage SapYZU01.

[0007] The phage SapYZU01 fixes the phage head on the CuCo2S4 surface through covalent binding, and the phage tail is exposed.

[0008] The method for preparing the CuCo2S4 material for detecting live Staphylococcus aureus by colorimetric method of the present invention comprises the following steps:

[0009] (1) Pour the SapYZU01 phage solution and Staphylococcus aureus suspension onto the bottom solid culture medium to form a double-layer plate, and culture overnight; pick phage plaques and inoculate them into the Staphylococcus aureus suspension, culture overnight, and then centrifuge to obtain the phage suspension;

[0010] (2) CuCl2·2H2O, CoCl2·6H2O, urea, and thioacetamide are dissolved in ultrapure water, and the resulting solution is continuously stirred. Subsequently, the solution is transferred to a Teflon-lined autoclave and heated. The mixture is cooled to room temperature, dispersed in anhydrous ethanol and H2O, and stirred to obtain a precipitate. The precipitate is dried and dispersed to obtain a CuCo2S4 solution.

[0011] (3) The CuCo2S4 solution and the phage suspension were mixed and cultured to prepare a SapYZU01@CuCo2S4 solution.

[0012] In step (1), the SapYZU01 phage solution and the Staphylococcus aureus ATCC 29213 bacterial suspension are added to a sterile centrifuge tube containing LB semi-solid medium, and the mixture is immediately poured onto the bottom LB solid medium to form a double-layer plate, which is cultured overnight; a phage plaque from a single double-layer plate is picked and inoculated into the Staphylococcus aureus ATCC 29213 bacterial suspension, cultured overnight, and then centrifuged to obtain a phage suspension.

[0013] Preferably, in step (1), 100 μL of phage solution and 100 μL of Staphylococcus aureus ATCC 29213 bacterial suspension are added to a sterile centrifuge tube containing 5 mL of LB semi-solid medium, and the mixture is immediately poured onto the bottom LB solid medium to form a double-layer plate, which is cultured overnight at 37°C; a phage plaque from a single double-layer plate is picked and placed in an LB test tube containing 100 μL of Staphylococcus aureus ACTT 29213 bacterial suspension, and the plate is cultured overnight at 37°C and 120 r / min, and then centrifuged to obtain a phage suspension.

[0014] Preferably, in step (2), 0.5 mmol CuCl2·2H2O, 1 mmol CoCl2·6H2O, 1 mmol urea, and 2 mmol thioacetamide are dissolved in 25 mL ultrapure water, and the resulting solution is stirred continuously for 60 min. Subsequently, the solution is transferred to a Teflon-lined autoclave and heated at 180°C for 24 h. After cooling, the black product is collected by centrifugation, washed three times with ultrapure water and anhydrous ethanol, and dried at 60°C overnight. The precipitate is dispersed in a MES solution to obtain a CuCo2S4 solution.

[0015] In step (3), CuCo2S4 and phage suspension are mixed, stirred, and cultured overnight to prepare a SapYZU01@CuCo2S4 solution.

[0016] In step (3), 40 mg of EDC and 40 mg of NHS were dissolved in 2 mL of MES for standby use, and then 10 mg of CuCo2S4 was dissolved in the MES mixture and mixed with 1 mL of phage solution. The mixture was stirred for 30 minutes, incubated at 37°C overnight, stirred for another 2 hours, and stored at 4°C for standby use to prepare a SapYZU01@CuCo2S4 solution.

[0017] The titer of the phage suspension in step (3) is 7.89×10 8 PFU / mL or above.

[0018] The invention discloses an application of the CuCo2S4 material for colorimetric detection of live Staphylococcus aureus in efficient, specific and visual detection of live Staphylococcus aureus.

[0019] The invention discloses an application of the CuCo2S4 material for detecting live Staphylococcus aureus by a colorimetric method in distinguishing live and dead Staphylococcus aureus.

[0020] The application process includes the following steps:

[0021] 1) Add SapYZU01@CuCo2S4 material solution to the buffer solution;

[0022] 2) Add the sample to be tested, wait for the bacteria and phage in the solution to be fully adsorbed, then add H2O2 solution and TMB solution in sequence, wait for the color reaction to develop, and observe the color with the naked eye or measure the absorbance using a UV spectrophotometer.

[0023] Furthermore, the application process includes the following steps:

[0024] 1) Place HAc-NaAc buffer in the centrifuge tubes of the experimental group and the blank control group, and then add the potent phage-modified peroxidase solution respectively;

[0025] 2) Add the sample to be tested to the centrifuge tube of the experimental group in 1). After the bacteria and phage in the solution are fully adsorbed, add H2O2 solution and TMB solution in sequence. Wait for the color reaction to develop. Observe the color with the naked eye or measure the absorbance using a UV spectrophotometer.

[0026] The concentration of the TMB solution is 0.1-50 mM, the concentration of the H2O2 solution is 10-1000 mM, and the pH range of the HAc-NaAc buffer is 3.0-9.0.

[0027] Preferably, the concentration of the TMB solution is 12 mM, the concentration of the H2O2 solution is 12 mM, the pH of the HAc-NaAc buffer is 4.0, the adsorption time is 2 min, and the color development reaction time is 12 min.

[0028] The present invention fixes the bacteriophage SapYZU01 onto the surface of CuCo2S4 nanoparticles to prepare a detection material, a SapYZU01@CuCo2S4 solution. The test solution and the SapYZU01@CuCo2S4 solution are thoroughly mixed in a buffer solution, followed by the addition of H2O2 and TMB solution. The solution color is detected, and the concentration of Staphylococcus aureus is calculated. The surface amino groups of the CuCo2S4 material prepared by the present invention are covalently bonded to the carboxyl groups on the surface of the bacteriophage, while the tail fibers remain free for bacterial capture. The CuCo2S4 material prepared by the present invention for colorimetric detection of live Staphylococcus aureus has the advantages of rapid detection, good specificity, high sensitivity, low equipment dependence, simple operation, and low cost.

[0029] This invention covalently immobilizes the virulent bacteriophage SapYZU01 on the surface of the nanozyme CuCo2S4. The resulting SapYZU01@CuCo2S4 exhibits peroxidase-like activity, catalyzing a colorimetric reaction of TMB to produce blue oxidized TMB (TMBox). Upon addition of Staphylococcus aureus, the virulent phage SapYZU01 selectively inhibits the peroxidase-like activity of SapYZU01@CuCo2S4 through a specific biological interaction with S. aureus. More importantly, unlike many existing test materials that cannot distinguish between live and dead S. aureus, the SapYZU01@CuCo2S4 prepared in this invention can distinguish between live and dead S. aureus with excellent selectivity and interference resistance. This invention proposes immobilizing virulent phage on the surface of CuCo2S4 nanomaterials for specific detection of S. aureus, resolving the challenges of existing techniques, which are labor-intensive, time-consuming, expensive, and unable to distinguish between live and dead S. aureus bacteria.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] The CuCo2S4 material SapYZU01@CuCo2S4 provided by the present invention has high specificity and good stability for colorimetric detection of live Staphylococcus aureus, and the phage activity therein is good. The detection method of the present invention can rapidly detect Staphylococcus aureus in samples by colorimetric method, with a detection time of <15 minutes and a minimum detection concentration of 78 CFU / mL. In addition, the detection material SapYZU01@CuCo2S4 and the detection method of the present invention can avoid interference from Shigella flexneri, Enterobacter sakazakii, Vibrio fluvii, Salmonella enterica, enteropathogenic Escherichia coli, Yersinia enterocolitica, Bacillus cereus, Staphylococcus epidermidis, Staphylococcus xylosus, Staphylococcus albus, and mixtures thereof. It has strong specificity for Staphylococcus aureus and can distinguish between live and dead Staphylococcus aureus. It has the advantages of fast detection speed, high specificity, high sensitivity, low equipment dependence, simple operation, and low cost, and is suitable for the rapid visual detection of live Staphylococcus aureus in food samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a picture of the plaque morphology of bacteriophage SapYZU01.

[0033] Figure 2 TEM image of SapYZU01.

[0034] Figure 3 This is the EDS image of CuCo2S4.

[0035] Figure 4 A is the SEM image of CuCo2S4; B is the XRD image of CuCo2S4; C is the XPS image of CuCo2S4; D is the TEM image of SapYZU01@CuCo2S4; E is the laser scanning confocal microscope image of SapYZU01@CuCo2S4, Staphylococcus aureus and SapYZU01@CuCo2S4+Staphylococcus aureus;.

[0036] Figure 5 Spotting of bacteriophage SapYZU01, SapYZU01@CuCo2S4 and CuCo2S4 in a double-layer plate of Staphylococcus aureus.

[0037] Figure 6 Steady-state kinetic diagrams of SapYZU01@CuCo2S4 catalyzed by different concentrations of TMB substrate and fixed concentration of H2O2, and different concentrations of H2O2 substrate and fixed concentration of TMB.

[0038] Figure 7A is the full spectrum of UV-visible spectra of the four reaction systems; B, C and D are the full spectrum of CuCo2S4 in the presence of hydroxyl radicals (·OH), singlet oxygen ( 1 O2) and superoxide anion radicals (O2· - ) in the presence of EPR spectra; E and F are respectively ·OH or 1 EPR spectra of CuCo2S4, Staphylococcus aureus, and SapYZU01@CuCo2S4+Staphylococcus aureus in the presence of O2.

[0039] Figure 8 A is the optimization of buffer pH value in the detection conditions; B is the optimization of temperature in the detection conditions; C is the optimization of H2O2 concentration in the detection conditions; D is the optimization of TMB concentration in the detection conditions; E is the optimization of SapYZU01@CuCo2S4 dosage in the detection conditions; F is the optimization of SapYZU01@CuCo2S4 phage concentration in the detection conditions.

[0040] Figure 9 This is the full UV-Vis spectrum of the prepared SapYZU01CuCo2S4+H2O2+TMB colorimetric system with or without Staphylococcus aureus.

[0041] Figure 10 A shows the effect of incubation time of SapYZU01@CuCo2S4 and Staphylococcus aureus on the color reaction; B shows the change of reaction time on the color system with or without Staphylococcus aureus; C shows the ratio of absorbance at 652 nm to reaction time of the color system with or without Staphylococcus aureus.

[0042] Figure 11 Stability of SapYZU01@CuCo2S4 at room temperature for one month.

[0043] Figure 12 A is the full UV-Vis spectrum of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system under different Staphylococcus aureus concentrations; B is the linear fit of the absorbance of the colorimetric system at 652 nm and the logarithm of the Staphylococcus aureus concentration; C is the selectivity of the SapYZU10@CuCo2S4+H2O2+TMB colorimetric system; D is the anti-interference ability of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system.

[0044] Figure 13 In the picture, A is the coating result of inactivated and well-active Staphylococcus aureus; B is the detection result of inactivated and well-active Staphylococcus aureus by the SapYZU01@CuCo2S4 detection system.

[0045] Figure 14 A, B, and C represent the effects of common food additives, different NaCl concentrations, and pH values on the colorimetric detection of Staphylococcus aureus; D represents the real food used in this study; and E represents the performance of SapYZU01@CuCo2S4+H2O2+TMB in detecting Staphylococcus aureus in real food. DETAILED DESCRIPTION

[0046] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0047] The Staphylococcus aureus strains YZUsa1, YZUsa15, YZUsa48, YZUsa81, and YZUsa84 used in this invention were provided by Yangzhou University. For details, see the article: Novel SCCmec type XII methicillin-resistant Staphylococcus aureus isolates identified from a swine production and processing chain. Other strains were purchased commercially.

[0048] The bacteriophage SapYZU01 used in the present invention is a publicly available phage provided by Yangzhou University. For details, see the master's thesis: Isolation and Identification of the Broad-Spectrum Lytic Staphylococcus aureus Phage SapYZU15, Antibacterial Activity and Application Research, Yangzhou University, 2023.

[0049] Example 1

[0050] Culture of Staphylococcus aureus

[0051] Staphylococcus aureus YZUsa1, YZUsa15, YZUsa48, YZUsa81 and YZUsa84 stored in -20℃ refrigerator; Staphylococcus aureus ATCC 29213, Staphylococcus xylosus ATCC29971, Staphylococcus albus ATCC 8032 purchased from ATCC culture collection; Staphylococcus epidermidis CMCC 26069, Shigella flexneri CICC 21534, Enterobacter sakazakii CICC 21545, Vibrio fluvii CICC 21612, Salmonella enterica CICC21513, enteropathogenic Escherichia coli CICC 10664, Yersinia enterocolitica CICC 21669 and Bacillus cereus CICC purchased from CICC culture collection 21261; After thawing in a 4°C environment, streak with 10 μL of inoculated loop and culture at 37°C for 36 hours, then pick a single colony and inoculate it into 5 mL of LB liquid medium and culture it in a constant temperature shaker at 37°C and 120 r / min for 24 hours; to ensure good bacterial activity, transfer the above-obtained bacterial solution under the same operation and culture it for 24 hours; centrifuge the bacterial solution at 4°C and 8000 r / min for 10 minutes, discard the supernatant to obtain a bacterial pellet; add 1 mL of sterile saline (0.85% NaCl) to resuspend the bacteria, centrifuge at 4°C and 8000 r / min for 10 minutes to wash away the residual culture medium, and repeat this operation twice; the obtained bacterial suspension is gradiently diluted with sterile saline to obtain a concentration of 10 1 -10 9 CFU / mL of Staphylococcus aureus YZUsa1, YZUsa15, YZUsa48, YZUsa81 and YZUsa84 bacterial suspensions and suspensions of other strains.

[0052] Example 2

[0053] Preparation of phage suspension

[0054] The virulent bacteriophage used in the present invention belongs to the Podoviridae family and is named Staphylococcus aureus phage SapYZU01.

[0055] The specific operation is as follows: add 10% of the titer of Staphylococcus aureus ATCC 29213 to a sterile centrifuge tube containing 5 mL of LB semi-solid medium. 8100 μL each of the phage liquid with PFU / mL and the Staphylococcus aureus ATCC29213 bacterial suspension cultured to the logarithmic phase were immediately poured onto the bottom LB solid culture medium to make a double-layer plate, and cultured overnight at 37°C; the phage plaques on the double-layer plate were picked and placed in an LB test tube containing 100 μL of the logarithmic phase Staphylococcus aureus YZUsa1 bacterial suspension, and cultured overnight at 37°C and 120 r / min, and then centrifuged to obtain the phage SapYZU01 suspension.

[0056] The phage plaques formed were counted using the traditional counting method. The results showed that the titer of SapYZU01 suspension against strain YZUsa1 reached about 10 8 PFU / mL or more, and the plaques formed on the plate are bright, clear, and uniform in size (such as Figure 1 shown).

[0057] Example 3

[0058] 1. Preparation and characterization of SapYZU01@CuCo2S4

[0059] The specific steps for preparing SapYZU01@CuCo2S4 are as follows: 0.5 mmol CuCl2·2H2O, 1 mmol CoCl2·6H2O, 1 mmol urea, and 2 mmol thioacetamide are dissolved in 25 mL of ultrapure water. The resulting solution is then stirred continuously for 60 minutes. The solution is then transferred to a Teflon-lined autoclave and heated at 180°C for 24 hours. After cooling, the black product is collected by centrifugation, washed three times with ultrapure water and anhydrous ethanol, and dried at 60°C overnight. The precipitate is dispersed to obtain a CuCo2S4 solution, which is then re-centrifuged before use. 40 mg of EDC and 40 mg of NHS were dissolved in 2 mL of MES for later use. Subsequently, 10 mg of CuCo2S4 was dissolved in the MES mixture and mixed with 1 mL of phage solution (prepared in Example 2). The mixture was stirred for 30 minutes, incubated at 37°C overnight, stirred for another 2 hours, and stored at 4°C for later use to prepare a SapYZU01@CuCo2S4 solution.

[0060] 2. Characterization of CuCo2S4

[0061] The synthesized CuCo2S4 was characterized by TEM, XRD, XPS and EDS. Figure 4 A It can be observed that the TEM image shows that CuCo2S4 presents an irregular three-dimensional nanoparticle structure. Figure 4The XRD pattern of B confirms the crystal structure of the synthesized CuCo2S4 nanoparticles, and the observed diffraction peaks correspond to the (022), (113), (004), (115), (044) and (137) planes with wavelengths of approximately 26.5°, 31.2°, 37.9°, 49.9°, 54.7° and 77.3°, respectively. Figure 4 The XPS spectrum of C shows that the full spectrum of CuCo2S4 is composed of O, S, Co and Cu. Figure 3 The EDS image shows that the four elements (O, S, Co and Cu) are evenly distributed. The two sub-peaks at 932.1eV and 952.1eV belong to Cu 2p 3 / 2 and Cu 2p 1 / 2 orbital, the binding energies of 932.1 and 952.1 eV are attributed to Cu + , the binding energies of 934.7 and 954.5 eV are attributed to Cu 2+ The Co 2p spectrum is divided into six peaks, including two satellite peaks and the peak corresponding to Co 2p 1 / 2 and Co 2p 3 / 2 The S2p spectrum consists of two peaks, namely S2 p 3 / 2 (161.28 eV) and S2 p 1 / 2 (162.58 eV). The above results indicate that the nanozyme CuCo2S4 was successfully synthesized.

[0062] 3. Characterization of SapYZU01@CuCo2S4

[0063] The bacteriophage SapYZU01 was successfully immobilized on the CuCo2S4 nanozyme by covalent bonding and named SapYZU01@CuCo2S4. The microscopic morphology of bacteriophage SapYZU01, CuCo2S4 and SapYZU01@CuCo2S4 was observed by transmission electron microscopy (TEM). Figure 2 As shown, the bacteriophage SapYZU01 has a regular polyhedral head and a short tail, belonging to the Podoviridae family; CuCo2S4 presents an irregular three-dimensional nanoparticle structure with a slightly rough surface and good dispersion; after the bacteriophage SapYZU01CuCo2S4 is incubated, the head of SapYZU01 adheres closely to the surface of CuCo2S4, with the tail facing outward, and maintains a complete biological structure ( Figure 4 D). SapYZU01@CuCo2S4 and Staphylococcus aureus were stained with the highly sensitive DNA fluorescent dye SYBR and blue DAPI fluorescent dye. The specific operation is as follows: the phage concentrate (about 10 8PFU / mL) and 100-fold diluted CuCo2S4 (10 mg CuCo2S4 dissolved in 2 mL of MES mixture in Example 3) were mixed at a volume ratio of 1:2 and incubated at 37°C for 6 h. 200 uL was taken in a 1.5 mL centrifuge tube and 20 uL of 60x SYBR solution (final concentration was 6x) was added under dark conditions. The cells were stained for 20 min and washed 4-8 times with deionized water. After washing, the cells were redissolved in 100 uL of deionized water. An appropriate amount of the solution was added dropwise to a glass slide and observed under a confocal microscope. The results are shown in Figure 2. Figure 4 E shows that the laser scanning confocal microscopy image of SapYZU01@CuCo2S4+Staphylococcus aureus appears blue-green, indicating that they are successfully bound.

[0064] The dot plaque method was used to verify the activity of phages in the SapYZU01@CuCo2S4 solution. The specific operation was as follows: 10 mL of heated LB solid medium was spread on a plate and placed on a sterile operating table to be solidified; 100 μL of Staphylococcus aureus YZUsa1 suspension cultured to the logarithmic phase was thoroughly mixed with 5 mL of LB semi-solid medium, and spread on a solid plate to be solidified; 10 μL of CuCo2S4 solution, SapYZU01@CuCo2S4 solution and 10 μL of LB semi-solid medium were respectively taken. 8 PFU / mL of phage suspension was dropped onto the above double-layer plate, ensuring SapYZU01 or CuCo2S4, and placed in a 37°C constant temperature incubator for overnight culture. Figure 5 As shown, phage plaques appeared in the area where SapYZU01@CuCo2S4 solution and phage SapYZU01 were added, and the transparency was similar, while no phage plaques appeared in the CuCo2S4 solution, indicating that SapYZU01 in SapYZU01@CuCo2S4 still had biological activity.

[0065] The above results indicate that phage SapYZU01 was successfully immobilized on the CuCo2S4 surface and SapYZU01@CuCo2S4 still retained its biological activity.

[0066] Example 4

[0067] Steady-state kinetics and peroxidase-like activity of SapYZU01@CuCo2S4

[0068] The peroxidase-like properties of SapYZU01@CuCo2S4 were further evaluated by steady-state kinetics by varying the concentration of one substrate while keeping the concentration of the other substrate constant. SapYZU01@CuCo2S4 (prepared in Example 3) was catalyzed by adding different concentrations of TMB substrate (6-60mM) and a fixed concentration of H2O2 (12mM), and different concentrations of H2O2 substrate (50-500M) and a fixed concentration of TMB (12mM). Figure 6 As shown, when hydrogen peroxide is used as the substrate, the V max and K m 5.53×10 -8 M / s and 0.96 mM. In contrast, when TMB was used as substrate, the V max and K m They are 17.08×10 -8 M / s and 5.15mM. Lower K m The K values of SapYZU01@CuCo2S4 for TMB and hydrogen peroxide indicate that the affinity between the enzyme and the substrate is stronger. m The V of SapYZU01@CuCo2S4 to TMB and hydrogen peroxide is lower than that of other nanozymes (Ag / rGO, N / graphene-ZnFe2O4, and PVP / PtRu NZs) (Table 1). max The values are higher than those of other nanozymes (Cu-MOF and MoO2; Table 1). Therefore, the steady-state kinetics indicate that the prepared SapYZU01@CuCo2S4 has excellent peroxidase-like activity.

[0069] Table 1. Comparison of steady-state kinetic parameters of SapYZU01@CuCo2S4 and other nanozyme oxidase activities

[0070]

[0071]

[0072] The peroxidase-like activity of SapYZUalpha@MnFeO was investigated using the TMB-HRP-H2O2 colorimetric system. Several 5 mL centrifuge tubes were divided into groups a, b, c, and d (n=3). 2700 μL, 2800 μL, 2800 μL, and 2800 μL of HAc-NaAc buffer solution (pH 4.0, 0.2 M) were added to each tube. 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3 was added to groups a, b, and d in sequence. 100 μL of H2O2 solution (10 mM, dissolved in pure water) was added to each group a, c, and d, and 100 μL of TMB solution (10 mM, dissolved in anhydrous ethanol) was added to each group a, b, and c. The reaction was carried out at room temperature for 10 minutes, and the results were immediately detected using a UV-visible spectrometer. Figure 7 As shown in Figure A, the SapYZU01@CuCo2S4+TMB+H2O2 reaction system has a distinct absorption peak at 652 nm. The SapYZU01@CuCo2S4+TMB system exhibits a slight peak at 652 nm, while the other two systems show no such change. This indicates that SapYZU01@CuCo2S4 has peroxidase-like activity and can oxidize the colorless TMB into the blue product TMBox in the presence of H2O2.

[0073] To further investigate the catalytic mechanism of SapYZU01@CuCo2S4, electron paramagnetic resonance (EPR) analysis was performed to verify the reactive oxygen species (ROS); 5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6-tetramethyl-4-piperidine were used as free radical scavengers. Figure 7 As shown in B and C, the characteristic signal of ·OH shows four peaks with relative signal intensities of 1:2:2:1, while 1 The characteristic signal of O2 shows three peaks of equal intensity, which is consistent with the peaks of ·OH and 1 Typical EPR spectrum of O2 Figure 1 consistent with the results of the previous studies, indicating that they exist in the catalytic system. Figure 7 As shown in D, O2· – The signal of is weaker and the peak is more dispersed, indicating that the O2· – Therefore, the SapYZU01@CuCo2S4+TMB+H2O2 system resulted in ·OH, O2· – and 1 In order to confirm the detection mechanism of SapYZU01@CuCo2S4, Staphylococcus aureus was added to the reaction system and EPR was used to detect ·OH and 1 O2 signal ( Figure 7(E and F) The typical ·OH peak is significantly attenuated, indicating that the host bacteria attenuate the signal, thereby reducing TMBox synthesis. These results suggest that S. aureus binding to SapYZU01@CuCo2S4 blocks its enzyme activation site, reducing its oxidase-like activity and leading to specific recognition by the bacterium and reduced ·OH release upon adsorption.

[0074] Example 5

[0075] Optimization of detection conditions for color development system

[0076] 1. Optimization of pH value of HAC-NaAC buffer

[0077] The specific operation is as follows: several 5 mL centrifuge tubes were divided into 7 groups (n=3), 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3, 100 μL of H2O2 solution (10 mM, dissolved in pure water), and 100 μL of TMB solution (10 mM, dissolved in anhydrous ethanol) were added to 2700 μL of HAC-NaAC buffer (0.2 M, pH 3.0-9.0) in sequence, and the mixture was reacted for 10 min. The results were detected by UV-visible spectrometer. Figure 8 As shown in Figure A, the absorbance first increases and then decreases, reaching a maximum value at pH 4.0. This indicates that the catalytic activity of the SapYZU01@CuCo2S4 system in this experiment is best at pH 4.0. Therefore, the pH value of the buffer was set to 4.0 in subsequent experiments.

[0078] 2. Optimization of detection temperature

[0079] The specific operation is as follows: 5 mL centrifuge tubes were divided into 5 groups (n=3), and 2700 μL of HAc-NaAc buffer (0.2 M, pH 4.0), 100 μL of SapYZU01@CuCo2S4 solution prepared in Example 3, 100 μL of H2O2 solution (10 mM, dissolved in pure water), and 100 μL of TMB solution (10 mM, dissolved in ethanol) were added in sequence at intervals. The reaction was carried out in a constant temperature water bath at 20°C, 30°C, 40°C, 50°C, and 60°C for 10 minutes, and the test was performed. The results showed that ( Figure 8 B), the absorbance of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system at 652 nm first increased and then decreased with the increase of reaction temperature, reaching a maximum value at 40°C. For experimental convenience, room temperature (25°C) was used as the detection temperature in subsequent experiments.

[0080] 3. Optimization of H2O2 concentration

[0081] Take 5 groups of 5mL centrifuge tubes (n=3), use HAc-NaAc buffer (pH 4.0, 0.2M), add 100μL of SapYZU01@CuCo2S4 solution (n=3) prepared in Example 2, 100μL of TMB solution (10mM, dissolved in anhydrous ethanol) in 3mL system, and then add 100μL of H2O2 solution of different concentrations (6mM-18mM, dissolved in pure water), react at room temperature for 10min, and detect. Figure 8 Figure C shows that the absorbance of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system at 652 nm increases with increasing H2O2 concentration. To minimize material usage and reduce detection system cost, the H2O2 concentration was set to 12 mM in the following experiments.

[0082] 4. Optimization of TMB concentration

[0083] Take 5 groups of 5mL centrifuge tubes (n=3), use HAc-NaAc buffer (pH 4.0, 0.2M), add 100μL of SapYZU01@CuCo2S4 solution (n=3) prepared in Example 3, 100μL of H2O2 solution (12mM, dissolved in pure water), and then add 100μL of different concentrations of TMB solution (6mM-18mM, dissolved in anhydrous ethanol) to each 3mL system, react at room temperature for 10min, and detect. Figure 8 Figure D shows that the absorbance at 652 nm of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system increases with increasing TMB concentration. To minimize material usage and reduce detection system cost, the TMB concentration was set to 12 mM in the following experiments.

[0084] 5. Optimization of SapYZU01@CuCo2S4 dosage

[0085] The specific operation is as follows: Take several 5mL centrifuge tubes (n=3), add HAc-NaAc buffer (pH 4.0, 0.2M), 10μL, 50μL, 100μL, 150μL, 200μL, 250μL and 300μL of the SapYZU01@CuCo2S4 solution prepared in Example 3 in sequence, then add 100μL of H2O2 solution (12mM, dissolved in pure water) and 100μL of TMB solution (12mM, dissolved in anhydrous ethanol), respectively, and make up the HAc-NaAc buffer to make the system 3mL. React at room temperature for 10 minutes and record the absorbance value. The results are as follows. Figure 8As shown in Figure E, the absorbance of the SapYZU01@CuCo2S4+H2O2+TMB reaction system at 652 nm increases with the increase in the amount of SapYZU01@CuCo2S4. Due to factors such as test cost and enzyme activity, the amount of SapYZU01@CuCo2S4 solution in subsequent experiments was determined to be 100 μL.

[0086] 6. Optimization of SapYZU01@CuCo2S4 phage concentration

[0087] The specific operation is as follows: the titer is 7.89×10 8 SapYZU01@CuCo2S4 was prepared according to the method of Example 3 at a volume ratio of 3:1, 2:1, 1:1, 1:2 and 1:3. Ten groups of 5 mL centrifuge tubes (n=3) were taken, and 2600 μL (5 experimental groups) and 2700 μL (5 blank control groups) of HAc-NaAc buffer (pH 4.0, 0.2 M) were added respectively. 100 μL of the above-mentioned SapYZU01@CuCo2S4 solution with different ratios was added to each of the 10 groups; then 100 μL of the 10 μL solution with a concentration of 10 μL was added to the experimental group. 8 CFU / mL of Staphylococcus aureus YZUsa1 was incubated at room temperature for 10 minutes. After incubation, 100 μL of H2O2 solution (12 mM, dissolved in pure water) was added first, followed by 100 μL of TMB solution (12 mM, dissolved in ethanol), and the mixture was reacted at room temperature for 10 minutes. The color was then developed and tested. The results showed that ( Figure 8 F), the difference in absorbance at 652 nm of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system between the experimental group and the control group first stabilized and then decreased with the increase of the volume ratio of SapYZU01. When the volume ratio was 2:1 (the phage volume was 66 μL and the titer of phage SapYZU01 was 7.89×10 8 Therefore, the optimal concentration of bacteriophage SapYZU01 in SapYZU01@CuCo2S4 is 2.63×10 8 PFU / mL. Subsequently, the optimal ratio of CuCo2S4 solution and SapYZU01 suspension was 2:1, and the optimal phage concentration was 2.63×10 8 PFU / mL, and SapYZU01@CuCo2S4 was prepared (other preparation steps were the same as in Example 3) for detection.

[0088] Example 6

[0089] Verify the feasibility of SapYZU01@CuCo2S4 colorimetric detection of Staphylococcus aureus

[0090] The specific steps are as follows: 2700 μL and 2600 μL of HAc-NaAc buffer solution (pH 4.0, 0.2 M) were added to 5 mL centrifuge tubes of group a (blank control group) and group b (experimental group) (n=3), respectively. Then 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3 was taken into each of the two groups, and 100 μL of Staphylococcus aureus YZUsa1 (10 8 CFU / mL) in group b and incubate at room temperature for 10 minutes; then, 100 μL of H2O2 solution (12 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in anhydrous ethanol) were added to the two groups in sequence and reacted for 10 minutes. The absorption spectrum of the reaction system was recorded. Figure 9 As shown, group b has no obvious absorption peak at a wavelength of 652 nm, while group a has an obvious absorption peak at a wavelength of 652 nm, indicating that Staphylococcus aureus can inhibit the color change of the SapYZU01@CuCo2S4+H2O2+TMB reaction system, proving that it is feasible to detect Staphylococcus aureus using the SapYZU01@CuCo2S4 system colorimetric method.

[0091] Example 7

[0092] Optimization of detection conditions for Staphylococcus aureus using SapYZU01@CuCo2S4

[0093] 1. Optimization of incubation time of SapYZU01@CuCo2S4 and Staphylococcus aureus

[0094] The specific operation is as follows: several 5 mL centrifuge tubes were taken and divided into 11 groups (n = 3), and 2600 μL of HAc-NaAc buffer solution (pH 4.0, 0.2 M), 100 μL of SapYZU01@CuCo2S4 solution prepared in Example 3 and 100 μL of Staphylococcus aureus YZUsa1 (10 8 CFU / mL) solution, incubate at room temperature for 2 min, 5 min, 8 min, 11 min, 14 min, 17 min, 20 min, and 23 min, then add 100 μL of H2O2 solution (12 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in anhydrous ethanol) in sequence, react for 10 min, and detect the absorbance at 652 nm. Figure 10 The absorbance of the reaction system shown in A gradually increased over time and stabilized at 25 minutes. The lowest absorbance of the reaction system was recorded when the recognition time between SapYZU01@CuCo2S4 and S. aureus was 2 minutes. To reduce detection time, the incubation time between the SapYZU01@CuCo2S4 system and S. aureus was set to 2 minutes in subsequent experiments.

[0095] 2. Optimization of reaction time of color development system

[0096] The specific operation is as follows: 100 μL of 10 9 CFU / mL of Staphylococcus aureus YZUsa1 was added to a centrifuge tube containing 2600 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3. After incubation at room temperature for 2 minutes, 100 μL of H2O2 solution (12 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in ethanol) were added and reacted at room temperature. Detection was performed every time the reaction time was 1, 3, 6, 9, 12, 15, 18, 21, and 24 minutes. The results are shown in FIG. Figure 10 As shown in BC, as the reaction time increases, the absorbance at 652nm of the two Staphylococcus aureus color development systems increases. However, the absorbance ratio at 652nm of the two color development systems decreases within 12 minutes and remains unchanged after 12 minutes. Therefore, the reaction time of the color development system is set to 12 minutes. In summary, the present invention subsequently conducted experiments under the optimal conditions (buffer pH of 4.0, SapYZU01@CuCo2S4 solution dosage of 100μL, incubation time of 12 minutes, and color development reaction time of 12 minutes).

[0097] Example 8

[0098] Stability of SapYZU01@CuCo2S4 solution

[0099] The specific operation is as follows: operate according to the above optimal conditions, perform a test every 3 days, and make 3 parallels. The results are as follows: Figure 11 As shown in the figure, the enzyme activity of the SapYZU01@CuCo2S4 solution measured remained stable within 30 days, indicating that the method has good stability.

[0100] Example 9

[0101] Detection of Staphylococcus aureus standard curve

[0102] The specific operation is as follows: Under the optimal conditions, 8 groups of different Staphylococcus aureus YZUsa1 concentrations (the final concentration in the 3mL system is 10 2 -10 8CFU / mL), 100 μL of each was added to a centrifuge tube containing 2600 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) and 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3. After incubation for 12 minutes, 100 μL of H2O2 solution (10 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in ethanol) were added, reacted for 12 minutes, and detected. The results are shown in FIG. Figure 12 As shown in A, with the addition of Staphylococcus aureus, the adsorption peak at 652nm of the color system gradually decreases. Then the absorbance of the color system at 652nm is fitted with the concentration of Staphylococcus aureus. Figure 12 As shown in Figure B, the absorbance of the color system at 652 nm is linearly related to the logarithm of the Staphylococcus aureus concentration, and the equation is: y = -0.109x + 0.95, and the detection range is 10 2 -10 8 CFU / mL, and the limit of detection (LOD) was calculated as low as 78 CFU / mL.

[0103] Example 10

[0104] Selectivity and interference of Staphylococcus aureus

[0105] The specific operation is as follows: 7 bacterial strains other than Staphylococcus were selected (Shigella flexneri CICC 21534, Enterobacter sakazakii CICC 21545, Vibrio fluvii CICC 21612, Salmonella enterica CICC 21513, enteropathogenic Escherichia coli CICC 10664, Yersinia enterocolitica CICC 21669, Bacillus cereus CICC 21261 purchased from CICC Culture Collection Center) and 3 bacterial strains of Staphylococcus epidermidis CMCC 26069, Staphylococcus xylosus ATCC 29971 and Staphylococcus albus ATCC 8032, 100 μL of each or 100 μL of the mixture of the two in a 1:1 ratio was added to 100 μL of the SapYZU01@CuCo2S4 solution prepared in Example 3, and 2600 μL of HAc-NaAc buffer (pH 4.0, 0.2 M) to form a 3 mL system. After incubation at room temperature for 2 minutes, 100 μL of H2O2 solution (12 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in ethanol) were added, and the reaction was carried out at room temperature for 12 minutes, and then the results were detected. Figure 12 As shown in Figure C, the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system has good selectivity for Staphylococcus aureus. In addition, the anti-interference results of the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system are shown in Figure 4. Figure 12As shown in D, the 10 bacterial strains had no interference with the detection of Staphylococcus aureus. The results showed that the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system can be used for the detection of Staphylococcus aureus under complex conditions.

[0106] Example 11

[0107] Detection of live and dead Staphylococcus aureus

[0108] The active Staphylococcus aureus strains YZUsa1, YZUsa15, YZUsa48, YZUsa81, and YZUsa84 were treated at 90°C for 15 minutes and inactivated. 100 μL of the active or inactivated Staphylococcus aureus strains cultured to the logarithmic phase were added to a 100 μL SapYZU01@CuCo2S4 solution and HAc-NaAc buffer (pH 4.0, 0.2M) to form a 3 mL system. After incubation at room temperature for 2 minutes, 100 μL of H2O2 solution (12 mM, dissolved in pure water) and 100 μL of TMB solution (12 mM, dissolved in ethanol) were added. The reaction was carried out at room temperature for 12 minutes and then tested. The results are shown in FIG. Figure 13 As shown in the figure, the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system cannot detect dead Staphylococcus aureus. The results show that the SapYZU01@CuCo2S4+H2O2+TMB colorimetric system can distinguish between live and dead Staphylococcus aureus.

[0109] Example 12

[0110] Simulating real sample testing and analysis of influencing factors

[0111] The specific operation is as follows: Potato beef stew, curry chicken, fried pork, steamed duck with mushrooms and black pepper beef were purchased from the supermarket as simulation samples. They were first sterilized in boiling water, cooled to room temperature, and then added with a final concentration of 1×10 2 , 1×10 4 and 1×10 6CFU / mL of Staphylococcus aureus YZUsa1 suspension. At the same time, the mixed Staphylococcus aureus suspensions with different concentrations added to the simulated sample were operated using the traditional counting method; the above-mentioned mixed Staphylococcus aureus suspension was detected according to the optimized reaction conditions and the SapYZU01@CuCo2S4 solution prepared under the optimal conditions. Three parallel samples were set for each concentration of the mixed Staphylococcus aureus suspension, and the concentrations were calculated according to the simulated sample standard curve and the traditional plate counting method, and the spiked recovery rate and relative standard deviation were calculated. Five common food additives (potassium sorbate, 0.0075%; sodium pyrophosphate, 0.5%; sodium hexametaphosphate, 0.5%; sodium tripolyphosphate, 0.5%; disodium succinate, 2%) were added to 5 ml of LB medium. Different amounts of NaCl were added to 5 tubes of LB liquid medium, so that the NaCl concentration was 2%, 4%, 6%, 8%, and 10%. Seven tubes of LB liquid medium were adjusted to pH 3-9. Then, a final concentration of 1×10 2 , 1×10 4 and 1×10 6 CFU / mL of Staphylococcus aureus YZUsa1 suspension was used. The SapYZU01@CuCo2S4 colorimetric method and plate count method were used to detect Staphylococcus aureus in the sample. The results are shown in Table 2 below. The spiked recoveries of Staphylococcus aureus in the simulated sample and LB medium ranged from 90.16% to 114.29%, with RSDs between 0.67% and 3.10%, demonstrating that this method can be applied to the detection of actual samples. The effects of food additives, NaCl concentration, and pH value on the detection are shown in Table 2. Figure 14 As shown, it can be ignored.

[0112] Table 2. Determination of Staphylococcus aureus in food samples by SapYZU01@CuCo2S4 colorimetric method

[0113]

Claims

1. A CuCo2S4 material for colorimetric detection of live Staphylococcus aureus, characterized in that: The material uses CuCo2S4 as a carrier and has a virulent phage fixed on its surface, wherein the virulent phage is phage SapYZU01.

2. The CuCo2S4 material for colorimetric detection of live Staphylococcus aureus bacteria according to claim 1, characterized in that The bacteriophage SapYZU01 preferably has its head fixed on the CuCo2S4 surface by covalent bonding, with the tail of the bacteriophage exposed.

3. A method for preparing a CuCo2S4 material for colorimetric detection of live Staphylococcus aureus, characterized in that: The steps include: (1) Pour the SapYZU01 phage solution and Staphylococcus aureus suspension onto the bottom solid culture medium to form a double-layer plate, and culture overnight; pick phage plaques and inoculate them into the Staphylococcus aureus suspension, culture overnight, and then centrifuge to obtain the phage suspension; (2) dissolving CuCl2·2H2O, CoCl2·6H2O, urea, and thioacetamide in ultrapure water, and then continuously stirring the resulting solution. The solution is then heated for reaction, and the mixture is cooled to room temperature, dispersed in anhydrous ethanol and H2O, and stirred to obtain a precipitate; the precipitate is dried and dispersed to obtain a CuCo2S4 solution; (3) The CuCo2S4 solution and the phage suspension were mixed and cultured to prepare a SapYZU01@CuCo2S4 solution.

4. The preparation method according to claim 3, characterized in that In step (1), the SapYZU01 phage solution and the Staphylococcus aureus ATCC 29213 bacterial suspension were added to a sterile centrifuge tube containing LB semi-solid medium, and the mixture was immediately poured onto the bottom LB solid medium to form a double-layer plate, which was cultured overnight; a phage plaque from a single double-layer plate was picked and inoculated into the Staphylococcus aureus ATCC 29213 bacterial suspension, cultured overnight, and then centrifuged to obtain a phage suspension.

5. The preparation method according to claim 3, characterized in that In step (3), CuCo2S4 and phage suspension are mixed and cultured overnight to prepare SapYZU01@CuCo2S4 solution.

6. The preparation method according to claim 5, characterized in that In step (3), CuCo2S4 and the phage suspension are mixed and stirred, cultured overnight at 30-37°C and 100-120 r / min, and stirred to prepare a SapYZU01@CuCo2S4 solution.

7. The preparation method according to claim 5, characterized in that The titer of the phage suspension in step (3) is 7.89×10 8 PFU / mL or above.

8. Use of the CuCo2S4 material for colorimetric detection of live Staphylococcus aureus bacteria according to claim 1 in efficient, specific, and visual detection of live Staphylococcus aureus bacteria.

9. Use of the CuCo2S4 material for detecting live Staphylococcus aureus by the colorimetric method according to claim 1 in distinguishing live from dead Staphylococcus aureus.

10. The use according to claim 8, characterized in that The application process includes the following steps: (1) Adding SapYZU01@CuCo2S4 material solution to the buffer solution; (2) Add the sample to be tested, wait for the bacteria and phage in the solution to be fully adsorbed, then add H2O2 solution and TMB solution in sequence, wait for the color reaction to develop, and observe the color with the naked eye or use a UV spectrophotometer to measure the absorbed light.

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