Detection method of bacterial endotoxin and application thereof

Abstract

The invention belongs to the technical field of chemical analysis and detection, and particularly provides a bacterial endotoxin detection method and application thereof. The detection method comprises the following steps: mixing a to-be-detected sample, Ag-coated AuNPs nano-enzyme, 3, 3 ', 5, 5'-tetramethyl benzidine and hydrogen peroxide, then adding a sodium acetate buffer solution with the pH value of 4.0-4.1, standing and incubating, determining the Raman intensity through a Raman spectrometer, and calculating to obtain the bacterial endotoxin content in the to-be-detected sample. The nano-enzyme shows excellent peroxidase-like activity and surface enhanced Raman scattering activity, colorless TMB can be oxidized into a blue product oxTMB in the presence of H2O2, negatively charged bacterial endotoxin can be combined with the positively charged nano-enzyme through electrostatic interaction, so that the catalytic activity of the nano-enzyme is reduced, the peroxidase-like activity of the nano-enzyme is inhibited, and the surface enhanced Raman scattering activity of the nano-enzyme is inhibited. And the oxTMB blue color generated by the nano-enzyme oxidized TMB and H2O2 system becomes light, so that the detection method with high sensitivity and strong selectivity is established.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a method for detecting bacterial endotoxin and application thereof. Background Art

[0002] Bacterial endotoxins are lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria. They are the main cause of clinical pyrogenic adverse reactions. When bacterial cells are lysed, LPS is released into the environment. LPS molecules carry a high negative charge because they contain phosphate groups and carboxyl groups. In the human body, Gram-negative bacterial infection can lead to the accumulation of chemically stable LPS molecules in the blood, which can easily induce fever, septic shock, sepsis, intestinal inflammation and even multiple organ failure. Pharmacopoeias of various countries have established strict LPS limits of 0.25 EU / mL for water for injection (WFI) (EU = unit of measurement for endotoxin activity), which has a high purity to avoid adverse effects on patient safety. The Chinese Pharmacopoeia stipulates that the maximum acceptable concentration of LPS for injection is 5EU / kg / h, and the maximum acceptable concentration of LPS for intrathecal injection is 0.2EU / kg / h. Therefore, real-time monitoring of LPS content in production is of great significance to ensure the safety of sterilized products. Currently, the detection methods for LPS include the in vivo rabbit pyrogen test (RPT) and the in vitro horseshoe crab test (LAL), both of which use an aqueous extract of horseshoe crab blood cells, namely the LAL reagent or lysate reagent. The LAL test is the most widely used high-sensitivity test because it is a cascade system that amplifies the enzymatic reaction with LPS, and this method requires a long measurement time, taking up to 2 h, and is expensive. In addition, there are also unfavorable differences in the reactivity between batches of LAL reagents. Therefore, an alternative technology for detecting LPS is urgently needed. Summary of the invention

[0003] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art, provide a method for detecting bacterial endotoxins and its application, utilize the POD-like and SERS activities of the core-shell structured AgAu@AuNPs and the inhibitory effect of bacterial endotoxins on their nanozyme activity, and establish a new colorimetric-SERS dual-mode detection method for bacterial endotoxins.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide a method for detecting bacterial endotoxins, wherein a sample to be tested, Ag@AuNPs nanozymes, 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide are mixed, and then a sodium acetate buffer solution with a pH of 4.0-4.1 is added for static incubation, and the Raman intensity is measured by a Raman spectrometer to calculate the bacterial endotoxin content in the sample to be tested; The Ag@AuNPs nanozyme oxidizes colorless 3,3',5,5'-tetramethylbenzidine to blue oxidized 3,3',5,5'-tetramethylbenzidine in the presence of H2O2; The preparation method of the Ag@AuNPs nanozyme comprises the following steps: S1. Dissolve chitosan, citric acid, MnCl2·4H2O, FeCl3·6H2O and p-phenylenediamine in acetic acid solution, mix by ultrasonication, transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor for hydrothermal reaction, cool naturally to room temperature after the reaction is completed, filter and centrifuge, take the supernatant and vacuum dry to obtain iron and copper doped carbon dots CS@Fe,Mn / CDs nanomaterials; S2. Mix HAuCl4 solution and AgNO3 solution, add to deionized water, heat to 90~95 ℃, stir for 10~5 minutes, add sodium citrate, continue heating and stirring for a preset time, add HAuCl4 and CS@Fe,Mn / CDs, continue stirring for a preset time, and immediately cool the resulting solution in an ice bath to obtain Ag@AuNPs nanozyme.

[0005] Furthermore, the concentration of AgAu@AuNPs is 1 mg / mL, the dosage is 10-20 μL, the concentration of TMB is 5 mmol / L, the dosage is 10-20 μL, and the concentration of H2O2 is 50 mmol / L, the dosage is 10-20 μL.

[0006] Furthermore, the static incubation temperature is 35° C. to 37° C., and the incubation time is 10 to 15 minutes.

[0007] Furthermore, the wavelength range of the Raman spectroscopy detection is 650nm ~ 654nm.

[0008] Furthermore, the preparation process of step S1 is specifically as follows: 0.3-0.5 g chitosan, 0.3-0.5 g citric acid, 0.20-0.40 g MnCl2·4H2O, 0.18-0.4 g FeCl3·6H2O and 0.15-0.35 g p-phenylenediamine are dissolved in 30-50 mL of 0.05-0.1% (v / v) acetic acid solution, and ultrasonically treated for 20-30 min. The mixed solution is transferred to a polytetrafluoroethylene high-pressure reactor, placed in a microwave digester 50 W and heated to 180 ° C for reaction for 1-2 h. After the reaction is completed, it is naturally cooled to room temperature, and large particles of impurities are removed with a 0.22 μm filter membrane. Then, the supernatant is vacuum dried after high-speed centrifugation to obtain the product.

[0009] Furthermore, the preparation process of step S2 is specifically as follows: 10 mmol / L HAuCl4 200~300 μL and 2.0 mg / mL AgNO3 200~300 μL are mixed, added to 30~40 mL deionized water, heated to 90~95 ° C, stirred for 10~15 min, and then 1 mg / mL sodium citrate 30~40 μL is added. After continuing to heat and stir for 10~15 min, 10 mmol / L HAuCl4 200~300 μL and 1.6 mg / mL CS@Fe,Mn / CDs 20-30 μL are added, and stirring is continued for 5~10 min. The resulting solution is immediately cooled in an ice bath to obtain.

[0010] Furthermore, the Michaelis constant Km of the Ag@AuNPs nanozyme for the substrate TMB is 0.65 mmol / L; the Michaelis constant Km of the Ag@AuNPs nanozyme for the substrate H2O2 is K m It is 0.43mmol / L.

[0011] The second aspect of the present invention is to provide the application of the above detection method in the quality control of injections.

[0012] Furthermore, the injection includes any one of sodium chloride injection, metronidazole injection, gentamicin sulfate injection or ampicillin injection.

[0013] The third aspect of the present invention is to provide a bacterial endotoxin detection kit, comprising the Ag@AuNPs nanozyme prepared as above.

[0014] The method for detecting bacterial endotoxins using the above kit comprises: Using Ag@AuNPs nanozyme as a catalyst, in the presence of hydrogen peroxide, SERS is used to in situ monitor the Ag@AuNPs nanozyme-catalyzed oxidation of the chromogenic substrate TMB to obtain information on the oxidation of TMB by the colored substance; and in the presence of bacterial endotoxins, the signal of oxidized TMB in the sample to be tested is detected in situ by SERS, and the concentration of bacterial endotoxins in the sample to be tested is detected by comparing the two sets of SERS spectra.

[0015] In some embodiments, this kit can also be used to achieve in situ SERS ultrasensitive monitoring of hydrogen peroxide in the sample to be tested, by adding this nanozyme as a SERS substrate to TMB solutions with different concentrations of hydrogen peroxide, changing the colorless TMB into blue oxidized TMB, and monitoring the change information of the oxidized TMB through Raman spectroscopy to obtain a SERS spectrum, and through the changes in the SERS spectrum, rapid detection of hydrogen peroxide can be obtained.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses chitosan, p-phenylenediamine, and Fe- and Mn-doped carbon dots as reducing agents and stabilizers to prepare AgAu@AuNPs nanomaterials with a core-shell structure. AgAu@AuNPs exhibit excellent peroxidase-like and surface enhanced Raman scattering (SERS) activities. In the presence of H2O2, AgAu@AuNPs can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue product oxTMB. In addition, AgAu@AuNPs with a core-shell structure as a SERS substrate can significantly enhance the Raman signal and peroxidase-like activity of oxTMB. Negatively charged bacterial endotoxins can bind to positively charged AgAu@AuNPs through electrostatic interactions, resulting in a decrease in the catalytic activity of the nanozyme and inhibition of its peroxidase-like activity, making the blue color of oxTMB produced by the AgAu@AuNPs oxidation of TMB and the H2O2 system lighter, thereby establishing a new colorimetric-SERS dual-mode method for bacterial endotoxin detection with high sensitivity and strong selectivity.

[0017] (2) The new colorimetric-SERS dual-mode method for bacterial endotoxin detection established in the present invention has fully demonstrated its reproducibility and ease of operation. The results of actual sample detection showed that the spiked recovery rate reached 86.9%~106.8%, and the detection limits were 0.038 EU / L and 0.041 EU / L, respectively. The sensitivity was nearly 50 times higher than that of the Chinese Pharmacopoeia horseshoe crab reagent detection method, and the detection time only took 15 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM image of CS@Fe,Mn / CDs prepared in Example 1; Figure 2 This is the transmission electron microscopy TEM image of the AgAu@AuNPs nanozyme prepared in Example 1; Figure 3 UV-vis absorption spectrum of AgAu@AuNPs prepared in Example 1; Figure 4 The SERS spectra of AgAu@AuNPs catalyzing the oxidation of peroxidase chromogenic substrates TMB and H2O2 in Example 1; Figure 5 The UV-vis absorption spectra of the AgAu@AuNPs catalyzed oxidation of the peroxidase chromogenic substrate TMB and H2O2 in Example 1; Figure 6 Fitting Michaelis-Menten plot for AgAu@AuNPs catalyzed TMB; Figure 7 Fitting Michaelis-Menten plot for the catalysis of H2O2 by AgAu@AuNPs; Figure 8 Fitting Michaelis-Menten plot for AgAu@AuNPs+LPS catalyzed TMB; Fig. 9 Fitting Michaelis-Menten plot for the catalysis of H2O2 by AgAu@AuNPs+LPS; Fig.10 UV-vis absorption spectra were detected for AgAu@AuNPs; Fig.11 The linear fitting curve corresponding to the UV-vis absorption spectrum of AgAu@AuNPs; Fig.12 SERS spectra of AgAu@AuNPs were detected; Fig.13 The linear fitting curve corresponding to the SERS spectrum of AgAu@AuNPs detection; Fig.14 SERS spectra of the selective results of AgAu@AuNPs for detecting LPS; Fig.15 UV-vis absorption spectra of AgAu@AuNPs for LPS selective detection results. DETAILED DESCRIPTION

[0019] In order to explain the technical scheme and beneficial effects of the present invention more clearly, the present invention is described in detail below in conjunction with the accompanying drawings and Examples. It should be clear that the reference drawings described are only partial embodiments of the present invention, which are only used to explain the present invention and cannot be construed as limitations of the present invention. Unless otherwise specified, the equipment and reagents used in the present invention are conventional commercial products in the art.

[0020] The present invention uses synthesized chitosan, p-phenylenediamine, and Fe- and Mn-doped carbon dots as reducing agents and stabilizers to prepare AgAu@AuNPs nanomaterials with a core-shell structure. AgAu@AuNPs exhibit excellent peroxidase-like and surface enhanced Raman scattering (SERS) activities. AgAu@AuNPs with peroxidase-like activity can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue product oxTMB in the presence of H2O2. In addition, AgAu@AuNPs with a core-shell structure as a SERS substrate can significantly enhance the Raman signal and peroxidase-like activity of oxTMB. The inventors found in the study that bacterial endotoxins can inhibit AgAu@AuNPs peroxidase, reducing the absorbance and SERS signal of oxTMB. Thus, a nanozyme-based colorimetric-SERS dual-mode biosensor was constructed. The absorbance and SERS signal were linearly related to the endotoxin concentration in the range of 0.16~4.33 EU / L (colorimetric) and 0.016~5.83 EU / L (SERS), and the detection limits were 0.038 EU / L and 0.041 EU / L, respectively. This method was applied to the detection of endotoxins in injection samples and biological products, with a recovery rate of 86.9%~106.8% and an RSD of no more than 5.3%. The method has the characteristics of high sensitivity, strong specificity, simple operation and rapidity.

[0021] The following is a detailed description with reference to specific embodiments.

[0022] Example 1 This example provides the determination of bacterial endotoxin (LPS) in injection samples.

[0023] 1. Preparation of CS@Fe,Mn / CDs nanomaterials: 0.3 g chitosan, 0.3 g citric acid, 0.20 g MnCl2·4H2O, 0.18 g FeCl3·6H2O and 0.15 g p-phenylenediamine were dissolved in 30 mL of 0.05-0.1% (v / v) acetic acid solution, and ultrasonically treated for 20 min. The mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester 50 W and heated to 180°C for 1 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles were removed with a 0.22 μm filter membrane. After high-speed centrifugation, the supernatant was vacuum dried to obtain iron and copper doped carbon dots CS@Fe,Mn / CDs nanomaterials; the prepared CS@Fe,Mn / CDs nanomaterials were subjected to transmission electron microscopy (TEM) analysis, as shown in FIG. Figure 1 As shown, the synthesized CS@Fe,Mn / CDs are typical carbon dots with amorphous spherical shapes and uniform distribution.

[0024] 2. Preparation of AgAu@AuNPs nanozyme: Mix 200 μL of 10 mmol / L HAuCl4 and 200 μL of 2.0 mg / mL AgNO3, add to 30 mL of deionized water, heat to 90 °C, stir for 15 min, add 30 μL of 1 mg / mL sodium citrate, continue heating and stirring for 10 min, add 200 μL of 10 mmol / L HAuCl4 and 20 μL of 1.6 mg / mL CS@Fe,Mn / CDs, continue stirring for 5-10 min, and immediately cool the resulting solution in an ice bath to obtain AgAu@AuNPs nanozyme; the prepared AgAu@AuNPs nanozyme was subjected to transmission electron microscopy (TEM) analysis, as shown in FIG. Figure 2 As shown, the synthesized AgAu@AuNPs nanozyme presents a core-shell structure; AgAuNPs are prepared at the same time, namely: 200 μL of 10 mmol / L HAuCl4 and 200 μL of 2.0 mg / mL AgNO3 are mixed, added to 30 mL of deionized water, heated to 90 °C, stirred for 10-15 min, and then 30 μL of 1 mg / mL sodium citrate is added. After continuing heating and stirring for 10-15 min, AgAuNPs are obtained.

[0025] 3. Peroxidase-like and SERS activity of AgAu@AuNPs: TMB was used as the chromogenic substrate to investigate the POD-like activity of AgAu@AuNPs. 50 μL of 1 mg / mL AgAu@AuNPs, 50 μL of 5 mmol / L TMB, 50 μL of 50 mmol / L H2O2, 100 μL of 0.5 EU / L LPS or no LPS were added to a 5 mL stoppered colorimetric tube, and the volume was adjusted to 3 mL with pH 4.0 acetate buffer solution, shaken, and allowed to stand for 5-10 min. The absorbance A was measured at a wavelength of 652 nm. Figure 3 As shown in the figure, TMB showed an obvious blue color. After adding LPS, the absorbance decreased significantly. At the same time, the absorbance of TMB + H2O2 + AgAu@AuNPs system decreased significantly at 1190, 1330, 1402 and 1605 cm -1 The SERS characteristic peaks appeared at Figure 4 This is due to the peroxidase-like oxidation of the Raman-active oxTMB by the AgAu@AuNPs nanozyme; the UV-vis absorption spectrum of AgAu@AuNPs shows an obvious localized surface plasmon resonance (LSPR) peak at 536 nm (as shown in Figure 5As shown in Figure 2, this indicates that AgAu@AuNPs have strong SERS electromagnetic enhancement (EM). The peroxidase activity of AgAuNPs was also investigated, and its peroxidase activity was significantly lower than that of AgAu@AuNPs.

[0026] The Michaelis-Menten catalytic kinetic parameters were also determined (e.g. Figure 8-9 As shown in Table 1), the Michaelis constants of AgAu@AuNPs for substrates TMB and H2O2 K m The reaction rates were 0.65 mmol / L and 0.43 mmol / L, respectively, and the reaction rate constants were 36.6×10 -7 and 1.34×10 -7 mol / L·s, TMB and H2O2 after adding LPS K m were 1.27 mmol / L and 3.91 mmol / L, and the reaction rate constants were 2.03×10 -7 and 0.98×10 -7 mol / L·s, indicating that the addition of LPS greatly reduced the affinity and reaction rate of AgAu@AuNPs nanozymes with substrates TMB and H2O2.

[0027] Table 1. Results of determination of Michaelis-Menten catalytic kinetic parameters.

[0028]

[0029] 4. LPS working curve production (1) Preparation of colorimetric detection curve of LPS: 10 μL of 1 mg / mL AgAu@AuNPs, 10 μL of 5 mmol / L TMB, 10 μL of 50 mmol / L H2O2, 10 μL of bacterial endotoxin LPS of different concentrations, and 50 μL of pH 4.0 acetate buffer solution were dropped into a microplate, mixed, and allowed to stand for 5-10 min. The absorbance A was measured at a wavelength of 652 nm. The standard curve was plotted with LPS concentration as the horizontal axis and A as the vertical axis to obtain the regression equation, as shown in the figure. Figure 10-11 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0030] (2) Preparation of SERS detection curve for LPS: 10 μL of 1 mg / mL AgAu@AuNPs, 10 μL of 5 mmol / L TMB, 10 μL of 50 mmol / L H2O2, 10 μL of bacterial endotoxin LPS of different concentrations, and 50 μL of pH 4.0 acetate buffer solution were dropped into a microplate, mixed, and allowed to stand for 5-10 min. The solution was scanned for 10 s at 785 nm excitation light and 500 mW laser power. A portable Raman instrument was used to perform Raman spectroscopy on the test solution, with LPS concentration as the horizontal axis and 1605 cm -1 The Raman intensity at the position is taken as the ordinate, and the standard curve is drawn to obtain the regression equation, see Table 2. Figure 12-13 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0031] Table 2. Linear equation, correlation coefficient, relative standard deviation, linear range

[0032] 5. Method specificity investigation: Figure 14-15 To treat β-D glucose, dextran, Cu 2+ 、Zn 2+ Mg 2+ 、Al 3+ , Ca 2+ The effect on the detection system, LPS concentration is 10 EU / L, and the concentration of all interfering substances is 100 times the LPS concentration. The results show that only LPS has a significant inhibitory effect on AgAu@AuNPs+H2O2+TMB, and other substances have almost no effect, and the method has good selection specificity.

[0033] 6. Determination of LPS in various injection samples: Under the same test conditions as the working curve in step 4, the AgAu@AuNPs nanozyme colorimetry / SERS was applied to the determination of various clinical injections, and the samples included NaCl injection, metronidazole injection, gentamicin sulfate injection, and ampicillin sodium injection. 2, 20, and 100 EU / L LPS standard solutions were added to various injection samples, and the synthetic samples were subjected to spike recovery tests. The results are shown in Table 3. The results showed that the LPS spike recovery measured in the synthetic samples was between 86.9% and 105.8%, and the RSD was less than 6% (n=6). The results showed that the method established by the present invention can be used for the detection of LPS in injections.

[0034] Table 3. Sample spike recovery and RSD of the method of the present invention (n = 6).

[0035]

[0036] Example 2: Determination of LPS in a disposable infusion set 1. Preparation of CS@Fe,Mn / CDs nanomaterials: 0.5 g chitosan, 0.5 g citric acid, 0.40 g MnCl2·4H2O, 0.4 g FeCl3·6H2O and 0.35 g p-phenylenediamine were dissolved in 50 mL of 0.1% (v / v) acetic acid solution, and ultrasonically treated for 30 min. The mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester heated to 180°C for 2 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles were removed with a 0.22 μm filter membrane. After high-speed centrifugation, the supernatant was vacuum dried to obtain iron and copper doped carbon dot CS@Fe,Mn / CDs nanomaterials.

[0037] 2. Preparation of AgAu@AuNPs nanozyme: Mix 300 μL of 10 mM HAuCl4 and 300 μL of 2.0 mg / mL AgNO3, add to 40 mL of deionized water, heat to 95°C, stir for 10 min, add 40 μL of 1 mg / mL sodium citrate, continue heating and stirring for 15 min, add 300 μL of 10 mM HAuCl4 and 30 μL of 1.6 mg / mL CS@Fe,Mn / CDs, continue stirring for 5-10 min, and immediately cool the resulting solution in an ice bath to obtain AgAu@AuNPs nanozyme.

[0038] 3. Preparation of LPS working curve: Same as Example 1.

[0039] 4. Determination of LPS in disposable infusion set samples (1) Preparation of test solution: Under sterile conditions, inject 10 mL of 10% sodium chloride injection into the lumen of each infusion set and 15 mL of 10% sodium chloride injection into the lumen of each blood transfusion set. After repeated rinsing for 5 times, seal both ends and keep warm in a (37±1)℃ constant temperature box for 2 h. After taking out, collect the test solution into a sterile pyrogen-free glass container with a stopper to obtain the test solution. At the same time, use 10% sodium chloride injection containing 10 EU / mL bacterial endotoxin as the extraction medium and the same extraction conditions to obtain the positive control solution. (2) Determination of LPS: 10µL of 1mg / mL AgAu@AuNPs, 10μL of 5mmol / L TMB, 10μL of 50mmol / L H2O2, and 10μL of the test solution and positive control solution were added to the ELISA plate. 50μL of pH 4.0 acetate buffer solution was dropped into the microtiter plate and mixed. The plate was allowed to stand for 5-10 min. The absorbance was measured at 652nm and scanned for 10 s at 785nm excitation light and a laser power of 500 mW. A portable Raman instrument was used to perform Raman spectroscopy on the test solution. The Raman intensity was measured and substituted into the regression equation. The results were not detected, 9.5±0.5 EU / mL, and 10.1±0.3 EU / mL, respectively.

[0040] For matters not mentioned above, the prior art applies.

[0041] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting bacterial endotoxins, characterized in that: The sample to be tested, Ag@AuNPs nanozyme, 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide were mixed, and then a sodium acetate buffer solution with pH 4.0-4.1 was added for static incubation. The Raman intensity was measured by a Raman spectrometer to calculate the bacterial endotoxin content in the sample to be tested. The Ag@AuNPs nanozyme oxidizes colorless 3,3',5,5'-tetramethylbenzidine to blue oxidized 3,3',5,5'-tetramethylbenzidine in the presence of H2O2; The preparation method of the Ag@AuNPs nanozyme comprises the following steps: S1. Dissolve chitosan, citric acid, MnCl2·4H2O, FeCl3·6H2O and p-phenylenediamine in acetic acid solution, mix by ultrasonication, transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor for hydrothermal reaction, cool naturally to room temperature after the reaction is completed, filter and centrifuge, take the supernatant and vacuum dry to obtain iron and copper doped carbon dots CS@Fe,Mn / CDs nanomaterials; S2. Mix HAuCl4 solution and AgNO3 solution, add to deionized water, heat to 90~95 ℃, stir for 10~5 min, add sodium citrate, continue heating and stirring for a preset time, add HAuCl4 and CS@Fe,Mn / CDs, continue stirring for a preset time, and immediately cool the resulting solution in an ice bath to obtain Ag@AuNPs nanozyme.

2. The detection method according to claim 1, characterized in that The concentration of the AgAu@AuNPs is 1 mg / mL, and the dosage is 10-20 μL. The concentration of TMB is 5 mmol / L, and the dosage is 10-20 μL. The concentration of H2O2 is 50 mmol / L, and the dosage is 10-20 μL.

3. The detection method according to claim 2, characterized in that The temperature of the static incubation is 35° C. to 37° C., and the incubation time is 10 to 15 minutes.

4. The detection method according to claim 3, characterized in that The wavelength range of the Raman spectrum detection is 650nm~654nm.

5. The detection method according to claim 1, characterized in that The preparation process of step S1 is specifically as follows: 0.3-0.5 g of chitosan, 0.3-0.5 g of citric acid, 0.20-0.40 g of MnCl2·4H2O, 0.18-0.4 g of FeCl3·6H2O and 0.15-0.35 g of p-phenylenediamine are dissolved in 30-50 mL of 0.05-0.1% (v / v) acetic acid solution, and ultrasonic treatment is performed for 20-30 min. The mixed solution is transferred to a polytetrafluoroethylene high-pressure reactor, placed in a microwave digester 50 W and heated to 180° C. for reaction for 1-2 h. After the reaction is completed, it is naturally cooled to room temperature, and large particles of impurities are removed with a 0.22 μm filter membrane. The mixture is then centrifuged at high speed, and the supernatant is vacuum dried to obtain the product.

6. The detection method according to claim 5, characterized in that The preparation process of step S2 is specifically as follows: 10 mmol / L HAuCl4 200~300 μL and 2.0 mg / mL AgNO3 200~300 μL are mixed, added to 30~40 mL deionized water, heated to 90~95 ° C, stirred for 10~15 min, and then 1 mg / mL sodium citrate 30~40 μL is added. After continuing to heat and stir for 10~15 min, 10 mmol / L HAuCl4 200~300 μL and 1.6 mg / mL CS@Fe,Mn / CDs 20~30 μL are added, and stirring is continued for 5-10 min. The resulting solution is immediately cooled in an ice bath to obtain the obtained solution.

7. The detection method according to claim 6, characterized in that The Michaelis constant of the Ag@AuNPs nanozyme for the substrate TMB K m is 0.65 mmol / L; the Michaelis constant of the Ag@AuNPs nanozyme for the substrate H2O2 K m It is 0.43mmol / L.

8. Use of the detection method as described in any one of claims 1 to 7 in the quality control of injections.

9. The use according to claim 8, characterized in that The injection includes any one of sodium chloride injection, metronidazole injection, gentamicin sulfate injection or ampicillin injection.

10. A bacterial endotoxin detection kit, characterized in that: The invention comprises the Ag@AuNPs nanozyme prepared according to any one of claims 1 to 7.

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