Phenylboronic acid functionalization-based Cu2-XSe-Au nano-enzyme and application thereof

Abstract

The invention discloses a phenylboronic acid functionalization-based Cu2-XSe-Au nano-enzyme and application thereof.The Cu2-xSe is synthesized firstly, then HAuCl4 is reduced to Au NPs on the surface of the Cu2-xSe to obtain a Cu2-xSe-Au nano-material, and then 4-mercaptophenylboronic acid is modified on the Cu2-xSe-Au nano-material through the action of an Au-S. The material has excellent peroxidase-like activity and photo-thermal performance, and can be used as a photothermal material for photothermal therapy. The fluorescent probe can be combined with a cis-diol structure on the surface of Escherichia coli O157: H7 through covalent bonds and an electrostatic adsorption effect, so that colorimetric and photo-thermal dual-mode detection of E. coli O157: H7 is realized. According to the present invention, the novel nanometer material-bacterium-antibody sandwich mode is adopted to detect E. coli O157: H7, and has advantages of low cost, high stability, good biocompatibility and the like, such that the new direction and the new idea are provided for the detection research of Escherichia coli.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme technology, and in particular to a nanozyme based on 4-mercaptophenylboronic acid (MPBA) functionalized Cu 2- x A label-free colorimetric and photothermal dual-mode immunoassay platform based on Se-Au nanozymes for rapid and sensitive detection of Escherichia coli O157:H7. Background Art

[0002] In recent years, nanozyme material systems have been widely used in biosensing, biotherapy and environmental treatment due to their unique optical and excellent photothermal properties, as well as good stability, easy modification and simple preparation. In particular, copper selenide compounds with non-stoichiometric ratios (Cu 2-x Se), due to its unique crystal defect structure, tunable plasma resonance properties and enzyme mimicking activity, has become an ideal candidate material for constructing nanoprobes. 2-x Se-Au heterogeneous nanocomposites, through synergistic effects between their components, not only possess excellent peroxidase-like activity, enabling the construction of colorimetric probes, but also possess unique photothermal properties, enabling the construction of multifunctional probes. However, current research has largely focused on a single detection mode, and there remains a gap in integrating these multiple properties to construct dual-mode quantitative detection platforms, particularly for the rapid detection of foodborne pathogens.

[0003] Escherichia coli O157:H7 ( E. coli O157:H7) is one of the most dangerous foodborne pathogens, which can cause severe illnesses such as inflammation, abdominal pain, bloody diarrhea, hemolytic uremic syndrome, hemorrhagic colitis, and even death. E. coli The contamination problem of O157:H7 has attracted widespread attention worldwide. In order to reduce disease outbreaks, ensure public health and protect human health, it is crucial to establish a rapid, reliable and sensitive analytical method for detecting Escherichia coli.

[0004] Currently, it has been widely reported E. coli O157:H7 detection strategies, such as enzyme-linked immunosorbent assay, fluorescence, microfluidics, lateral flow immunoassay, surface-enhanced Raman, electrochemical methods, etc. Although these analytical methods have made great contributions to the screening and detection of bacteria, the applicability of these technologies is affected by uncontrollable factors: (1) A complex and expensive coupling process is required between antibodies and signal tag nanomaterials; (2) The biological activity of antibodies is affected by the external environment (such as ion concentration, pH value and temperature); (3) A single signal recognition mode cannot guarantee the sensitivity and accuracy of detection results in complex external environments.

[0005] Therefore, in view of the existing deficiencies, it is crucial to construct a sensing platform that is free of antibody labeling and has multiple signal recognition modes for the detection of Escherichia coli.

[0006] In the already public E. coli In the patent related to O157:H7 detection, CN119470891A discloses a bacterial detection method based on MIL(53)-Fe nanozyme activity and its application. The bacterial detection method combines an aptamer with an iron-based metal organic framework to form a bacterial probe, and uses MIL(53)-Fe to catalyze TMB in the presence of hydrogen peroxide to generate TMB. + performance, while the generated TMB + Acidification to form TMB 2+ , using TMB 2+ The etching of gold nanorods causes the color of the gold nanorods to change, thereby realizing the detection of bacteria. This detection method is simple and fast, and the minimum detection limit for Staphylococcus aureus is 1.16 CFU / mL, and the minimum detection limit for Escherichia coli is 1.02 CFU / mL, which has broad application prospects. In addition, the detection results of the present invention are visible to the naked eye and can be used as a basis for qualitative detection. This method shows high sensitivity and visualization advantages in pure solution systems, but in complex matrix detection scenarios, it may face challenges such as inhibition of nanozyme activity, decreased aptamer specificity, and interference in signal interpretation.

[0007] CN119395116A describes an in-situ preparation method for silicon-gold-aptamer nanowire arrays for E. coli detection. This method uses nanowire array material produced by an in-situ synthesis method on a silicon wafer. Gold nanoparticles and E. coli aptamers are in situ loaded onto the nanowire arrays by chemical reduction. A metal film is then coated on the back of the silicon wafer. This back surface is then connected to a circuit, allowing the electrical signal generated by the silicon-gold-aptamer nanowire array during testing to pass through the silicon wafer's conductive input circuit. The E. coli aptamers specifically adsorb E. coli in the test solution onto the material surface, generating a current response linearly related to the bacterial concentration. The large surface area of ​​the electrodes in the in-situ synthesized nanoarray structure provides the sensor with excellent sensitivity. This high-performance nanobiosensor provides a practical new method for rapid on-site detection of foodborne pathogens. However, this method involves complex material synthesis, is highly instrument-dependent, and the stability of the E. coli aptamers can be affected by various factors, such as temperature, pH, and ionic strength. In practical applications, different test environments may affect the specific binding ability of aptamers to E. coli, thereby affecting the accuracy and reliability of the test results.

[0008] CN118604337A discloses a colorimetric sensor based on bacterial in situ deposition of manganese dioxide nanozymes, as well as its preparation method and application. This patented invention discloses a colorimetric sensor based on bacterial in situ deposition of manganese dioxide nanozymes, as well as its preparation method and application. Antibody-modified immunomagnetic bead probes are used as capture elements to separate and enrich Escherichia coli in a sample. A nucleic acid aptamer with specific recognition properties is modified on the surface of the E. coli. Using E. coli as a template, the affinity of the phosphate groups in the aptamer for manganese ions is utilized to synthesize dense manganese dioxide nanozymes in situ in E. coli under alkaline conditions. The nanozyme effectively catalyzes the oxidation of a TMB substrate to produce color. The color change of the TMB oxidation product is closely related to the bacterial content, making it suitable for quantitative detection of the pathogenic E. coli O157:H7. This invention effectively increases the loading capacity of the nanozyme, achieves colorimetric signal amplification, and improves the sensitivity of the method. The sensor features simple operation, rapid detection, high specificity, and low cost, and has potential application value in the field of real-time bacterial detection. However, the detection conditions are relatively harsh. The in situ synthesis of manganese dioxide nanozymes needs to be carried out under alkaline conditions, and there are strict requirements on the pH value of the reaction system. In addition, there are interference factors. Various interfering substances may exist in actual samples, such as other metal ions, organic matter, or substances with redox activity. These substances may compete with manganese ions for binding to aptamers, affecting the synthesis of manganese dioxide nanozymes, or directly interfere with the oxidative color development reaction of TMB, resulting in deviations in the test results and false positives or false negatives. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the present invention proposes a Cu 2-x Se-Au nanozymes and their applications first synthesize Cu through a two-step reduction reaction. 2-x Se, and then reduced HAuCl4 to Au NPs on its surface by its own redox reaction to obtain Cu 2-x Se-Au nanomaterials; Next, 4-mercaptophenylboronic acid was modified on Cu through Au-S bonding. 2-x On the Se-Au nanomaterial, this material has excellent peroxidase-like activity and photothermal properties, and can combine with the cis-diol structure on the surface of Escherichia coli O157:H7 through covalent bonds and electrostatic adsorption to achieve specific capture of target bacteria.

[0010] The technical solution for achieving the purpose of the present invention is: A Cu-based phenylboronic acid functionalized 2-X The preparation method of Se-Au nanozyme comprises the following steps: Step 1: Add cetyltrimethylammonium bromide (CTAB) solution and deionized water to a round-bottom flask, add SeO2 solution and ascorbic acid solution in sequence under vigorous stirring, and stir to react for a while; Step 2: Add CuSO4·5H2O solution and ascorbic acid solution to the mixed solution in step 1 under stirring, and then vigorously stir the reaction at a certain temperature for a period of time to obtain Cu 2-x Se solution; The solution was purified using a dialysis bag and centrifuged to remove large particles before use; Step 3: Purify the Cu 2-x After the Se solution was diluted, a certain amount of HAuCl4 solution was added and the mixture was allowed to stand at room temperature for a period of time. After standing, the mixture was centrifuged and then redispersed in deionized water to obtain Cu 2-X Se-Au nanomaterials; Step 4: Under vigorous stirring, add 4-mercaptophenylboronic acid (MPBA) ethanol solution to the 2-X The solution of Se-Au nanomaterials was stirred and reacted at room temperature for a period of time, and then centrifuged and washed with ethanol and deionized water several times before redispersion to obtain phenylboronic acid functionalized Cu2-xSe-Au nanozyme (Cu 2-x Se-Au@MPBA) was stored at 4°C until use.

[0011] Furthermore, in step 1, the volume of the CTAB solution is 0.1-10 mL, and the concentration is 1-100 mmol / L; The volume of deionized water is 1-10 mL; The volume of SeO2 solution is 0.1-10mL, and the concentration is 0.01-1 mol / L; The volume of ascorbic acid solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; The stirring time is 1-60 min.

[0012] Furthermore, in step 2, the volume of the CuSO4·5H2O solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; The volume of ascorbic acid solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; Stirring time is 1-3 h; The molecular weight cut-off of the dialysis bag is 10 KDa, and the purification time is 22-25 hours.

[0013] Furthermore, in step 3, Cu 2-x The volume ratio of Se solution to HAuCl4 solution was 2:1; The mixed solution was allowed to stand at room temperature for 2-3 h, the centrifuge speed was 12000 rpm, and the centrifugation time was 10-15 min.

[0014] Furthermore, in step 4, the volume of the MPBA ethanol solution is 1-1000 μL, and the concentration is 0.001-100 μg / mL; Contains Cu 2-X The volume of the Se-Au nanomaterial solution was 1–10 mL; The reaction time is 1-8 h under stirring at room temperature, the centrifuge speed is 12000 rpm, and the centrifugation time is 10-15 min.

[0015] Furthermore, in steps 1, 2 and 4, the stirring speed is 20-2000 rpm / min.

[0016] The above preparation method was used to prepare 4-mercaptophenylboronic acid functionalized Cu 2-X Se–Au nanozymes, prepared Cu 2-x Se has good dispersion and uniform size, appearing as single spherical nanoparticles. Through spontaneous redox reaction, 2-x Cu was obtained by in situ growth of Au NPs on Se surface. 2-x Se-Au, from Cu 2-x TEM and EDS-mapping images of Se-Au show that 2-x Many relatively small nanoparticles can be observed on the surface of Se, which are uniform and dispersed, indicating that Au has successfully grown to Cu 2-x Se surface. Then, 4-mercaptophenylboronic acid was modified onto Cu by Au-S bonding. 2-x Se-Au, with Cu 2-x Compared with Se-Au, the MPBA modified Cu 2-x The morphology, particle size and dispersion of Se-Au@MPBA were almost not affected. Combined with elemental mapping analysis, Cu 2-x Se-Au@MPBA contains five elements: Cu, Se, Au, S, and B, among which S and B elements come from MPBA, indicating that MPBA has been successfully modified onto Au NPs, proving the successful preparation of the material.

[0017] The present invention further provides a Cu based on phenylboronic acid functionalization 2-X Se–Au nanozymes were used for the colorimetric and photothermal dual-mode detection of Escherichia coli O157:H7.

[0018] Specifically, the application of Cu based on phenylboronic acid functionalization 2-X Se–Au nanozyme detects Escherichia coli O157:H7. The detection method includes the following steps: Step A: Cu 2-XSe-Au@MPBA was co-incubated with Escherichia coli O157:H7 to form a complex. Specifically, 50 μL of 2-X Se-Au@MPBA was mixed with 1 mL of E. coli O157:H7 at different gradient concentrations and incubated at 37°C and 1000 rpm for 30 min; Step B: The complex solution is drawn through a syringe and passed through a filter containing E. coli O157:H7 monoclonal antibodies using an extrapolation effect. The antibodies on the filter membrane will capture E. coli, forming a "nanomaterial-bacteria-antibody" sandwich structure. Specifically, the immune filter membrane is rinsed several times with 1 mL of deionized water and Tris-HCl containing 0.05% Tween-20, and then the incubated immune complex solution is drawn with a 1 mL medical syringe for incubation and rinsing for 2 minutes. Finally, it is rinsed with a buffer solution to remove Cu that is not bound to bacteria. 2-X Se-Au@MPBA nanomaterials; Step C: Using Cu 2-X The peroxidase-like activity and photothermal properties of Se-Au@MPBA enable colorimetric and photothermal dual-mode detection of E. coli O157:H7, specifically: Colorimetric detection: Place the treated filter membrane in a centrifuge tube containing Tris-HCl buffer solution, then add TMB and H2O2, incubate at 37°C, 1000 rpm in the dark for 20 min, and measure the OD value at 652 nm using a microplate reader. 652 nm The standard curve was constructed by combining the signal change at and the logarithmic value of different concentrations of E. coli O157:H7; Photothermal detection: The treated filter membrane was irradiated with a laser with a power of 1.59 W / cm 2 After irradiation with an 808 nm laser for 2 min, the temperature change on the filter membrane was recorded in real time using a handheld thermal imager. A standard curve of the logarithmic value of different concentrations of Escherichia coli O157:H7 and the change in filter membrane temperature (ΔT) was established. ΔT was calculated using the formula ΔT=ΔT1-ΔT0, where ΔT1 and ΔT0 refer to the temperature changes of the immune filter membrane before and after laser irradiation in the sample group and the control group, respectively.

[0019] Furthermore, the concentration of the E. coli O157:H7 solution in step A of the detection method was 2.08×10 7 -2.08×10 2 CFU / mL; The volume of Tris-HCl with 0.05% Tween-20 in step B is 1-1000 μL, pH = 7.4; The volume of Tris–HCl in step C was 1–1000 μL; The volume of TMB was 1–1000 μL, and the concentration was 0.0001–10 mmol / L; The volume of H2O2 is 1-1000 μL, and the concentration is 0.0001-1 mol / L.

[0020] The detection platform constructed by this invention does not require the traditional antibody labeling process. Functionalized nanomaterials can directly capture bacteria, avoiding the activity loss and steric hindrance effects during the antibody immobilization process, significantly improving the stability and reproducibility of the detection system. It can be used to detect E. coli O157:H7 in a variety of real-world samples, including water, milk, and orange juice, meeting the needs of pathogen detection in different scenarios such as food safety monitoring and clinical diagnosis. The detection process is relatively simple to operate, and the nanomaterials are simple to prepare and relatively low in cost, showing good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Cu in the embodiment 2-x Schematic diagram of the preparation process of Se-Au@MPBA.

[0022] Figure 2 Cu prepared in this example 2-X Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS-mapping) of Se-Au@MPBA; in, Figure 2 A is Cu 2-X TEM image of Se-Au@MPBA; Figure 2 BG is Cu 2-X EDS-mapping diagram of Se-Au@MPBA.

[0023] Figure 3 Cu prepared in this example 2-x Steady-state kinetic analysis of Se-Au; in, Figure 3 A is Cu 2-x Se-AuCu 2-x Michaelis-Menten curve of Se-Au under H2O2 concentration; Figure 3 B is Cu 2-x Lineweaver-Burk double reciprocal curve of Se-Auu in H2O2; Figure 3 C is Cu 2-x Michaelis-Menten curve of Se-Au at different TMB concentrations; Figure 3 D is Cu 2-xLineweaver-Burk double reciprocal plot of Se-Au in TMB.

[0024] Figure 4 Based on Cu 2-x Se-Au@MPBA constructed immunosensor platform for detection E. coli Schematic diagram of O157:H7; in, Figure 4 A is the process of preparing immune filter membrane; Figure 4 B is colorimetric and photothermal dual mode detection E. coli O157:H7 process.

[0025] Figure 5 This is a graph showing the results of colorimetric and photothermal dual-mode detection; in, Figure 5 A is based on Cu 2-x The immunosensor platform constructed with Se-Au@MPBA nanomaterials detects different E. coli UV-visible absorption spectrum of O157:H7 (the inset shows the color change of the corresponding solution); Figure 5 B is ΔOD 652 nm and E. coli The linear relationship between the logarithmic values ​​of O157:H7 concentration; Figure 5 C is different E. coli Temperature-time curve of O157:H7 concentration; Figure 5 D is ΔT and E. coli The linear relationship between the logarithmic values ​​of O157:H7 concentrations. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited thereto. Example

[0027] Reference Figure 1 , a Cu-based phenylboronic acid functionalized 2-X The preparation method of Se-Au nanozyme comprises the following steps: Step 1: Synthesis of Cu via a two-step reduction reaction 2-x Se, specifically, 1.6 mL of CTAB solution (concentration of 30 mmol / L) and 4.8 mL of deionized water were added to a 50 mL round-bottom flask, and 0.1 mL of SeO2 solution (concentration of 0.2 mol / L) and 0.6 mL of ascorbic acid solution (concentration of 0.2 mol / L) were added in sequence under vigorous stirring at 2000 rpm / min, and stirred for 10 min; Step 2: Under stirring, 0.1 mL of CuSO4·5H2O solution (concentration of 0.4 mol / L) and 0.8 mL of ascorbic acid solution (concentration of 0.2 mol / L) were quickly added to the mixed solution of step 1, and then stirred vigorously at 30°C for 1.5 h to obtain Cu 2-x Se solution; The solution was purified using a 10 KDa dialysis bag for 24 h and centrifuged to remove large particles before use; Step 3: Purify the Cu 2-x Se solution was diluted 50 times, and the Cu 2-x The volume ratio of Se solution to HAuCl4 solution was 2:1. HAuCl4 solution was added and allowed to stand at room temperature for 3 h. After centrifugation at 12000 rpm for 10 min, the solution was redispersed in deionized water to obtain Cu 2-X Se-Au nanomaterials are reserved; Step 4: Under vigorous stirring, quickly add 400 μL of MPBA (50 μg / mL) ethanol solution to the solution containing 2 mL of Cu 2-x The solution of Se-Au nanomaterials was stirred continuously at room temperature for 4 h, centrifuged at 12,000 rpm for 10 min, washed three times with ethanol and deionized water, and then redispersed to obtain phenylboronic acid-functionalized Cu2-xSe-Au nanozyme (Cu2-xSe-Au@MPBA), which was stored at 4°C for future use.

[0028] Reference Figure 2 Cu 2-X Transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS-mapping) of Se-Au@MPBA, such as Figure 2 As shown in AB, 4-mercaptophenylboronic acid was modified to Cu by Au-S bonding. 2-x Se-Au, with Cu 2- x Compared with Se-Au, the MPBA modified Cu 2-x The morphology, particle size and dispersion of Se-Au@MPBA were almost unaffected. Combined with elemental mapping analysis, e.g. Figure 2 As shown in CG, Cu 2-x Se-Au@MPBA contains five elements: Cu, Se, Au, S, and B, among which S and B elements come from MPBA, indicating that MPBA has been successfully modified onto Au NPs, proving the successful preparation of the material.

[0029] Reference Figure 3 Cu 2-xSteady-state kinetic analysis of Se-Au was performed, and the Cu 2-x K of Se-Au to H2O2 and TMB m 0.7416 mmol L -1 and 1.08 mmol L -1 , V max 3.55×10 -7 mol L -1 s -1 and 1.67×10 -6 mol L -1 s -1 ,Depend on Figure 3 It can be seen from AD that Cu 2-x K of Se-Au to H2O2 and TMB m The value is lower than or equal to that of other nanozymes, indicating that Cu 2-x Se-Au has a stronger affinity for H2O2 and TMB, showing excellent peroxidase-like activity. m The value is lower, but Cu 2-x V of Se-Au nanozyme max larger, which means that its catalytic activity is good and suitable for utilizing Cu 2-x The peroxidase-like activity of Se-Au nanomaterials was used to construct a colorimetric sensing platform.

[0030] Reference Figure 4 , using phenylboronic acid-functionalized Cu 2-X Se-Au nanozyme detects Escherichia coli O157:H7, and the detection method includes the following steps: Step A: Cu 2-X Se-Au@MPBA was co-incubated with Escherichia coli O157:H7 to form a complex. Specifically, 50 μL of 2-X Se-Au@MPBA was mixed with 1 mL of different gradient concentrations (2.08×10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 CFU / mL) of Escherichia coli O157:H7 mixed solution was incubated at 37°C and 1000 rpm for 30 min; Step B: The complex solution is drawn through a syringe and passed through a filter containing E. coli O157:H7 monoclonal antibody using extrapolation. The antibody captures E. coli on the filter membrane, forming a "nanomaterial-bacteria-antibody" sandwich structure. Specifically, the immune filter membrane is rinsed several times with 1 mL of deionized water and Tris-HCl (10 mmol / L, pH=7.4) containing 0.05% Tween-20, and then the incubated immune complex solution is drawn with a 1 mL medical syringe for incubation and rinsing for 2 minutes. Finally, it is rinsed with buffer solution to remove Cu that is not bound to bacteria. 2-X Se-Au@MPBA nanomaterials; The preparation process of immune filter membrane is as follows Figure 4 As shown in A; a nitrocellulose filter membrane (NC membrane with a diameter of 13 mm and a pore size of 2 μm) was washed with a Na2CO3-NaHCO3 buffer solution (100 mmol L -1 , pH = 9.6) washed three times, and then added E. coli -Ab (12.5 μg mL -1 ), incubated at 4°C for 12 h; the incubated filter membrane was washed with PBST (10 mmol L -1 , pH = 7.4, PBS buffer solution containing 0.05% Tween-20) for three times, and finally placed in a vacuum dryer at 25 ° C for 12 h. The treated filter membrane was sealed and stored at 4 ° C for further use; Step C: Utilize the peroxidase-like activity and photothermal properties of nanomaterials to realize colorimetric and photothermal dual-mode detection of E. coli O157:H7, refer to Figure 4 B, specifically: Colorimetric detection: Place the treated filter membrane in a centrifuge tube containing 800 μL Tris-HCl buffer solution, then add 100 μL TMB (1 mmol / L) and 100 μL H2O2 (100 mmol / L). Incubate at 37°C, 1000 rpm in the dark for 20 min, and measure the OD value at 652 nm using a microplate reader. 652 nm The standard curve was constructed by combining the signal change at and the logarithmic value of different concentrations of E. coli O157:H7; Photothermal detection: The treated filter membrane was irradiated with an 808 nm laser at a power of 1.59 W / cm² for 2 minutes. The temperature change on the filter membrane was recorded in real time using a handheld thermal imager. A standard curve was established between the logarithmic value of different E. coli O157:H7 concentrations and the change in filter membrane temperature (ΔT). ΔT was calculated using the formula ΔT1-ΔT0, where ΔT1 and ΔT0 refer to the temperature changes of the immune filter membrane before and after laser irradiation in the sample and control groups, respectively.

[0031] The results of colorimetric and photothermal dual-mode detection are shown in the figure below. Figure 5 As shown, Figure 5 A is based on Cu 2-x The immunosensor platform constructed with Se-Au@MPBA nanomaterials detects different E. coli UV-visible absorption spectrum of O157:H7 (the inset shows the color change of the corresponding solution). E. coli With the increase of O157:H7 concentration, the absorbance signal at 652 nm showed a gradient enhancement trend, which was due to the formation of the "nanomaterial-E. coli-antibody" sandwich structure.

[0032] Reference Figure 5 B is ΔOD 652 nm and E. coli The linear relationship between the logarithmic values ​​of O157:H7 concentration. 652 nm Value and E. coli The logarithm of O157:H7 concentration is 10 2 -10 7 CFU mL -1 There is a good linear relationship within the range, and the linear regression equation is y=0.1663 x-0.0787 (R 2 =0.9932, n=3), and the LOD value of this method was calculated to be 26.34 CFU mL -1 (n=11, 3σ / K).

[0033] Reference Figure 5 C and D are photothermal detection analysis, E. coli O157:H7 concentrations in the range 10 1 -10 5 CFU mL -1 The maximum temperature rise (ΔT) within 2 minutes showed a good linear correlation with the logarithm of its concentration (y = 17.3771 x -11.9586, R 2 =0.9933), and the LOD value of this method was calculated to be 1.91 CFU mL -1 , the sensitivity is increased by 13.8 times compared with the colorimetric mode.

[0034] As can be seen from the examples, using Cu 2-x The peroxidase-like activity and photothermal properties of Se-Au nanomaterials enable dual-mode colorimetric and photothermal detection of E. coli O157:H7. This synergistic dual-mode detection not only increases sensitivity by 14-fold (the photothermal detection limit is 1.91 CFU / mL, a 14-fold increase compared to the colorimetric method's 26.34 CFU / mL), but also significantly improves detection reliability through signal cross-validation.

Claims

1. A Cu-based phenylboronic acid functionalized 2-X The preparation method of Se-Au nanozyme is characterized in that: The following steps are involved: Step 1: Add CTAB solution and deionized water to a round-bottom flask, add SeO2 solution and ascorbic acid solution in sequence under vigorous stirring, and stir to react for a while; Step 2: Add CuSO4·5H2O solution and ascorbic acid solution to the mixed solution in step 1 under stirring, and then vigorously stir the reaction at a certain temperature for a period of time to obtain Cu 2-x Se solution; The solution was purified using a dialysis bag and centrifuged to remove large particles before use; Step 3: Purify the Cu 2-x After the Se solution was diluted, a certain amount of HAuCl4 solution was added to the mixture and allowed to stand at room temperature. After standing, the mixture was centrifuged and then redispersed in deionized water to obtain Cu 2-X Se-Au nanomaterials; Step 4: Under vigorous stirring, add the MPBA ethanol solution to the 2-X The solution of Se-Au nanomaterial was stirred continuously at room temperature for a period of time, centrifuged after stirring, and then washed several times with ethanol and deionized water before redispersion to obtain phenylboronic acid functionalized Cu2-xSe-Au nanozyme, which was stored at 4°C for future use.

2. Cu according to claim 1 2-X The preparation method of Se-Au nanozyme is characterized by: In step 1, the volume of CTAB solution is 0.1-10 mL, and the concentration is 1-100 mmol / L; The volume of deionized water is 0.1-10 mL; The volume of SeO2 solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; The volume of ascorbic acid solution is 0.1-100 mL, and the concentration is 0.01-1 mol / L; The stirring time is 1-60 min.

3. Cu according to claim 1 2-X The preparation method of Se-Au nanozyme is characterized by: In step 2, the volume of CuSO4·5H2O solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; The volume of ascorbic acid solution is 0.1-10 mL, and the concentration is 0.01-1 mol / L; Stir vigorously at 25-35°C for 1-3 h; The molecular weight cut-off of the dialysis bag is 10 KDa, and the purification time is 22-25 hours.

4. Cu according to claim 1 2-X The preparation method of Se-Au nanozyme is characterized by: In step 3, Cu 2-x The volume ratio of Se solution to HAuCl4 solution was 2:1; The mixed solution was allowed to stand at room temperature for 2-3 h, the centrifuge speed was 12000 rpm, and the centrifugation time was 10-15 min.

5. Cu according to claim 1 2-X The preparation method of Se-Au nanozyme is characterized by: In step 4, the volume of MPBA ethanol solution is 1-1000 μL, and the concentration is 0.001-100 μg / mL; Contains Cu 2-X The volume of the Se-Au nanomaterial solution was 1–10 mL; The stirring time at room temperature is 1-8 h, the centrifuge speed is 12000 rpm, and the centrifugation time is 10-15 min.

6. Cu according to claim 1 2-X The preparation method of Se-Au nanozyme is characterized by: In steps 1, 2 and 4, the stirring speed is 20-2000 rpm / min.

7. Phenylboronic acid functionalized Cu obtained by the preparation method according to any one of claims 1 to 6 2-X Se–Au nanozymes.

8. Phenylboronic acid functionalized Cu according to claim 7 2-X The application of Se-Au nanozymes is characterized by: Phenylboronic acid-functionalized Cu2-XSe–Au nanozyme was used to detect Escherichia coli O157:H7. The detection method includes the following steps: Step A: Cu 2-X Se-Au@MPBA was co-incubated with Escherichia coli O157:H7 to form a complex. Specifically, 50 μL of 2-X Se-Au@MPBA was mixed with 1 mL of E. coli O157:H7 at different gradient concentrations and incubated at 37°C and 1000 rpm for 30 min; Step B: The complex solution is drawn up through a syringe and passed through a filter containing a monoclonal antibody against E. coli O157:H7 using an extrapolation effect. The antibody captures the E. coli on the filter, forming a "nanomaterial-bacteria-antibody" sandwich structure. Specifically, the immune filter membrane was washed several times with 1 mL of deionized water and Tris-HCl containing 0.05% Tween-20, and then the immune complex solution was drawn up with a 1 mL medical syringe for incubation and washing for 2 minutes. Finally, it was washed with a buffer solution to remove the Cu that was not bound to the bacteria. 2-X Se-Au@MPBA nanomaterials; Step C: Using Cu 2-X The peroxidase-like activity and photothermal properties of Se-Au@MPBA enable colorimetric and photothermal dual-mode detection of E. coli O157:H7, specifically: Colorimetric detection: Place the treated filter membrane in a centrifuge tube containing Tris-HCl buffer solution, then add TMB and H2O2, incubate at 37°C, 1000 rpm in the dark for 20 min, and measure the OD value at 652 nm using a microplate reader. 652 nm The standard curve was constructed by combining the signal change at and the logarithmic value of different concentrations of E. coli O157:H7; Photothermal detection: The treated filter membrane was irradiated with a laser with a power of 1.59 W / cm 2 After irradiation with an 808 nm laser for 2 min, the temperature change on the filter membrane was recorded in real time using a handheld thermal imager. A standard curve of the logarithmic value of different concentrations of Escherichia coli O157:H7 and the change in filter membrane temperature ΔT was established. ΔT was calculated using the formula ΔT=ΔT1-ΔT0, where ΔT1 and ΔT0 refer to the temperature changes of the immune filter membrane before and after laser irradiation in the sample group and the control group, respectively.

9. The use according to claim 8, characterized in that: The concentration of the E. coli O157:H7 solution in step A of the detection method is 2.08×10 7 -2.08×10 2 CFU / mL; The volume of Tris-HCl with 0.05% Tween-20 in step B is 1-1000 μL, pH = 7.4; The volume of Tris–HCl in step C was 1–1000 μL; The volume of TMB was 1–1000 μL, and the concentration was 0.0001–10 mmol / L; The volume of H2O2 is 1-1000 μL, and the concentration is 0.0001-1 mol / L.

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