A method for detecting bacterial endotoxin and its application

Abstract

The present invention belongs to the technical field of chemical analysis and detection, and specifically provides a method for detecting bacterial endotoxin and its application. The detection method is to mix a sample to be tested, Ag@AuNPs nanozyme, 3,3',5,5'-tetramethylbenzidine, and hydrogen peroxide, and then add a sodium acetate buffer solution with a pH of 4.0 to 4.1, and let it stand for incubation. The Raman intensity is measured by a Raman spectrometer, and the content of bacterial endotoxin in the sample to be tested is calculated. The nanozyme exhibits excellent peroxidase-like and surface-enhanced Raman scattering activities. In the presence of H2O2, it can oxidize colorless TMB into a blue product oxTMB. Negatively charged bacterial endotoxin can bind to the positively charged nanozyme through electrostatic interaction, resulting in a decrease in the catalytic activity of the nanozyme, inhibiting its peroxidase-like activity, and making the blue color of oxTMB produced by the nanozyme oxidizing TMB and H2O2 system become lighter, thereby establishing a highly sensitive and selective detection method.

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 its application. Background Art

[0002] Bacterial endotoxin is lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria and is the main cause of clinical pyrogenic adverse reactions. When bacterial cells lyse, LPS is released into the environment. The LPS molecule carries a relatively high negative charge due to the presence of phosphate groups and carboxyl groups. In the human body, Gram-negative bacterial infections can lead to the accumulation of LPS molecules with stable chemical properties in the blood, which can easily induce fever, septic shock, sepsis, intestinal inflammation, and even multiple organ failure. Pharmacopoeias in various countries have set strict LPS limits of 0.25 EU / mL for water for injection (WFI) (EU = measurement unit of endotoxin activity). Its purity is relatively high to avoid adverse effects on the safety of patients. The Chinese Pharmacopoeia stipulates that the maximum acceptable concentration of LPS for injection is 5 EU / kg / h, and the maximum acceptable concentration of intrathecal injection of LPS is 0.2 EU / kg / h. Therefore, real-time monitoring of LPS content during production is of great significance for ensuring the safety of sterilized products. Currently, the detection methods for LPS include in vivo rabbit pyrogen test (RPT) and in vitro limulus reagent test (LAL), both of which use aqueous extracts of horseshoe crab blood cells, namely LAL reagent or lysate reagent. The LAL test is the most widely used highly sensitive test because it is a cascade system that amplifies the enzymatic reaction with LPS. And this method requires a long measurement time, taking 2 h, and is expensive. In addition, there are also adverse differences in the reaction activities between batches of LAL reagents. Therefore, there is an urgent need for an alternative technology for detecting LPS. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for detecting bacterial endotoxin and its application in view of the above deficiencies of the prior art. By using the peroxidase-like (POD) and surface-enhanced Raman scattering (SERS) activities of the core-shell structure AgAu@AuNPs and the inhibitory effect of bacterial endotoxin on its nanozyme activity, a new colorimetric-SERS dual-mode detection method for bacterial endotoxin is established.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] In the first aspect of the present invention, a method for detecting bacterial endotoxin is provided. A sample to be tested, Ag@AuNPs nanozyme, 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 and left to stand and incubate. The Raman intensity is measured by a Raman spectrometer, and the content of bacterial endotoxin in the sample to be tested is calculated.

[0006] The Ag@AuNPs nanozyme oxidizes colorless 3,3',5,5'-tetramethylbenzidine to blue oxidized 3,3',5,5'-tetramethylbenzidine in the presence of 2 O 2 ;

[0007] The preparation method of the Ag@AuNPs nanozyme comprises the following steps:

[0008] S1. Dissolve chitosan, citric acid, MnCl 2 ·4H 2 O, FeCl 3 ·6H 2 O and p-phenylenediamine in an acetic acid solution, transfer the mixed solution to a polytetrafluoroethylene autoclave for hydrothermal reaction after ultrasonic mixing, naturally cool to room temperature after the reaction is completed, filter and centrifuge, and take the supernatant for vacuum drying to obtain iron and copper-doped carbon dots CS@Fe,Mn / CDs nanomaterials;

[0009] S2. Mix HAuCl 4 solution and AgNO 3 solution, add them to deionized water, heat to 90 - 95 °C, stir for 10 - 5 min, add sodium citrate, continue heating and stirring for a preset time, then add HAuCl 4 and CS@Fe,Mn / CDs, continue stirring for a preset time, and immediately place the resulting solution in an ice bath to cool to obtain the Ag@AuNPs nanozyme.

[0010] Furthermore, the concentration of the 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 2 O 2 is 50 mmol / L, and the dosage is 10 - 20 μL.

[0011] Furthermore, the temperature of the static incubation is 35 °C - 37 °C, and the incubation time is 10 - 15 min.

[0012] Furthermore, the wavelength range of the Raman spectroscopy detection is 650 nm - 654 nm.

[0013] Furthermore, the specific preparation process of step S1 is as follows: take 0.3 - 0.5 g of chitosan, 0.3 - 0.5 g of citric acid, 0.20 - 0.40 g of MnCl 2 ·4H 2 O, 0.18 - 0.4 g of FeCl 3 ·6H 2O 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, ultrasonicated for 20 - 30 min. The mixed solution is transferred to a polytetrafluoroethylene high - pressure reactor and heated to 180 °C in a microwave digestion instrument at 50 W for 1 - 2 h. After the reaction is completed, it is naturally cooled to room temperature. Large - particle impurities are removed with a 0.22 μm filter membrane, and then it is centrifuged at high speed. The supernatant is dried in vacuo to obtain the product.

[0014] Further, the preparation process of step S2 is specifically as follows: Take 10 mmol / L HAuCl 4 200 - 300 μL and 2.0 mg / mL AgNO 3 200 - 300 μL and mix them. Add them to 30 - 40 mL of deionized water, heat to 90 - 95 °C, stir for 10 - 15 min, then add 30 - 40 μL of 1 mg / mL sodium citrate, continue to heat and stir for 10 - 15 min, then add 10 mmol / L HAuCl 4 200 - 300 μL and 20 - 30 μL of 1.6 mg / mL CS@Fe,Mn / CDs, continue to stir for 5 - 10 min, and immediately place the obtained solution in an ice bath to cool to obtain the product.

[0015] Further, the Michaelis constant Km of the Ag@AuNPs nanozyme for the substrate TMB is 0.65 mmol / L; the Michaelis constant 2 O 2 for the substrate H K m is 0.43 mmol / L.

[0016] The second aspect of the present invention is to provide the application of the above - mentioned detection method in the quality control of injections.

[0017] Further, the injection includes any one of sodium chloride injection, metronidazole injection, gentamicin sulfate injection or ampicillin injection.

[0018] The third aspect of the present invention is to provide a bacterial endotoxin detection kit, which includes the Ag@AuNPs nanozyme prepared as above.

[0019] The method for detecting bacterial endotoxin with the above - mentioned kit includes:

[0020] Using Ag@AuNPs nanozyme as a catalyst, in the presence of hydrogen peroxide, the catalytic oxidation of the chromogenic substrate TMB by Ag@AuNPs nanozyme was monitored in situ by SERS to obtain information on the oxidation of TMB by the colored substance; and in the presence of bacterial endotoxin, the signal of oxidized TMB in the sample to be tested was detected in situ by SERS. By comparing two groups of SERS spectra, the concentration of bacterial endotoxin in the sample to be tested was detected.

[0021] In some embodiments, this kind of kit can also be used to achieve in-situ SERS ultrasensitive monitoring of hydrogen peroxide in the sample to be tested. The nanozyme is added as a SERS substrate to the solution of TMB with different concentrations of hydrogen peroxide. The colorless TMB is changed to blue oxidized TMB. By monitoring the change information of oxidized TMB by Raman spectroscopy, an SERS spectrum is obtained. Through the change of the SERS spectrum, rapid detection of hydrogen peroxide is achieved.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) In the present invention, a core-shell structured AgAu@AuNPs nanomaterial is prepared by using chitosan, p-phenylenediamine, and doped Fe and Mn carbon dots as reducing agents and stabilizers. AgAu@AuNPs exhibits excellent peroxidase-like and surface-enhanced Raman scattering (SERS) activities. In the presence of H 2 O 2 AgAu@AuNPs can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue product oxTMB. In addition, the core-shell structured AgAu@AuNPs as a SERS substrate can significantly enhance the Raman signal and peroxidase-like activity of oxTMB. Negatively charged bacterial endotoxin can bind to positively charged AgAu@AuNPs through electrostatic interaction, 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 oxidation of TMB and H 2 O 2 system become lighter. Thus, a new highly sensitive and selective colorimetric-SERS dual-mode method for the detection of bacterial endotoxin is established.

[0024] (2) For the new colorimetric-SERS dual-mode method for the detection of bacterial endotoxin established in the present invention, its reproducibility and operational convenience are fully demonstrated. The results in the detection of actual samples show that the spiked recovery rates reach 86.9% - 106.8%, and the detection limits are 0.038 EU / L and 0.041 EU / L respectively, which is nearly 50 times more sensitive than the limulus reagent detection method in the Chinese Pharmacopoeia, and the detection time only requires 15 minutes. Description of the Drawings

[0025] Figure 1TEM image of CS@Fe,Mn / CDs prepared in Example 1;

[0026] Figure 2 TEM image of AgAu@AuNPs nanozyme prepared in Example 1;

[0027] Figure 3 UV-vis absorption spectrum of AgAu@AuNPs prepared in Example 1;

[0028] Figure 4 SERS spectrum of AgAu@AuNPs catalyzing the oxidation of peroxidase chromogenic substrate TMB and H 2 O 2 ;

[0029] Figure 5 UV-vis absorption spectrum of AgAu@AuNPs catalyzing the oxidation of peroxidase chromogenic substrate TMB and H 2 O 2 ;

[0030] Figure 6 Michaelis-Menten plot for AgAu@AuNPs catalyzing TMB;

[0031] Figure 7 Michaelis-Menten plot for AgAu@AuNPs catalyzing H 2 O 2 ;

[0032] Figure 8 Michaelis-Menten plot for AgAu@AuNPs + LPS catalyzing TMB;

[0033] Figure 9 Michaelis-Menten plot for AgAu@AuNPs + LPS catalyzing H 2 O 2 ;

[0034] Figure 10 UV-vis absorption spectrum for AgAu@AuNPs detection;

[0035] Figure 11 Linear fitting curve corresponding to the UV-vis absorption spectrum for AgAu@AuNPs detection;

[0036] Figure 12 SERS spectrum for AgAu@AuNPs detection;

[0037] Figure 13The linear fitting curve corresponding to the SERS spectrum detected by AgAu@AuNPs;

[0038] Figure 14 The SERS spectrum of the selective result of AgAu@AuNPs for detecting LPS;

[0039] Figure 15 The UV-vis absorption spectrum of the selective result of AgAu@AuNPs for detecting LPS. Detailed implementation manners

[0040] In order to more clearly and comprehensively explain the technical solution and beneficial effects of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be clear that the described reference drawings are only partial embodiments of the present invention, which are only used to explain the present invention and should not be construed as a limitation to the present invention. Unless otherwise specified, the equipment and reagents used in the present invention are conventional commercially available products in the technical field.

[0041] The present invention prepares a core-shell structured AgAu@AuNPs nanomaterial by using synthesized chitosan, p-phenylenediamine, and Fe- and Mn-doped carbon dots as reducing agents and stabilizers. AgAu@AuNPs exhibits excellent peroxidase-like and surface-enhanced Raman scattering (SERS) activities. The peroxidase-like active AgAu@AuNPs can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue product oxTMB in the presence of H 2 O 2 In addition, the core-shell structured AgAu@AuNPs as an SERS substrate can significantly enhance the Raman signal and peroxidase-like activity of oxTMB. The inventors of the present invention found in the research that bacterial endotoxin can inhibit the peroxidase-like activity of AgAu@AuNPs, reducing the absorbance and SERS signal of oxTMB. Thus, a nanozyme-based colorimetric-SERS dual-mode biosensor was constructed. In the range of endotoxin concentration of 0.16 - 4.33 EU / L (colorimetry) and 0.016 - 5.83 EU / L (SERS), the absorbance and SERS signal are linearly related to the endotoxin concentration, and the detection limits are 0.038 EU / L and 0.041 EU / L, respectively. Applying this method to the detection of endotoxin in injection samples and biological products, the recoveries are between 86.9% and 106.8%, and the RSD does not exceed 5.3%. The method has the characteristics of high sensitivity, strong specificity, simple operation, and rapidity.

[0042] The following will be described in detail with specific examples.

[0043] Example 1

[0044] This example provides the determination of bacterial endotoxin (LPS) in injection samples.

[0045] 1. Preparation of CS@Fe,Mn / CDs nanomaterials: 0.3 g of chitosan, 0.3 g of citric acid, 0.20 g of MnCl 2 ·4H 2 O, 0.18 g of FeCl 3 ·6H 2 O and 0.15 g of p-phenylenediamine were dissolved in 30 mL of 0.05 - 0.1% (v / v) acetic acid solution and ultrasonicated for 20 min. The mixed solution was transferred to a polytetrafluoroethylene autoclave and heated to 180 °C at 50 W in a microwave digestion instrument for 1 h. After the reaction was completed, it was naturally cooled to room temperature. Large particle impurities were removed with a 0.22 μm filter membrane, and then it was centrifuged at high speed. The supernatant was dried in vacuo to obtain iron and copper doped carbon dots CS@Fe,Mn / CDs nanomaterials; the prepared CS@Fe,Mn / CDs nanomaterials were analyzed by transmission electron microscopy (TEM). As Figure 1 shown, the synthesized CS@Fe,Mn / CDs showed a typical amorphous spherical uniform distribution of carbon dots.

[0046] 2. Preparation of AgAu@AuNPs nanozyme: 200 μL of 10 mmol / L HAuCl 4 and 200 μL of 2.0 mg / mL AgNO 3 were mixed, added to 30 mL of deionized water, heated to 90 °C, stirred for 15 min, then 30 μL of 1 mg / mL sodium citrate was added, and after continuing to heat and stir for 10 min, 200 μL of 10 mmol / L HAuCl 4 and 20 μL of 1.6 mg / mL CS@Fe,Mn / CDs were added, and stirring was continued for 5 - 10 min. The resulting solution was immediately cooled in an ice bath to obtain AgAu@AuNPs nanozyme; the prepared AgAu@AuNPs nanozyme was analyzed by transmission electron microscopy (TEM). As Figure 2 shown, the synthesized AgAu@AuNPs nanozyme presented a core-shell structure; at the same time, AgAuNPs were prepared, that is: 200 μL of 10 mmol / L HAuCl 4 and 200 μL of 2.0 mg / mL AgNO 3 were mixed, added to 30 mL of deionized water, heated to 90 °C, stirred for 10 - 15 min, then 30 μL of 1 mg / mL sodium citrate was added, and after continuing to heat and stir for 10 - 15 min, AgAuNPs were obtained.

[0047] 3. Peroxidase-like and SERS activities of AgAu@AuNPs: Using TMB as the chromogenic substrate, the POD-like activity of AgAu@AuNPs was investigated. 50 μL of AgAu@AuNPs with a concentration of 1 mg / mL, 50 μL of TMB with a concentration of 5 mmol / L, and 50 μL of H 2 O 2 with a concentration of 50 mmol / L were added to a 5 mL stoppered colorimetric tube. 100 μL of LPS with a concentration of 0.5 EU / L was added or not added, and the volume was made up to 3 mL with pH 4.0 acetate buffer solution. After shaking well and standing for 5 - 10 min, the absorbance A was measured at a wavelength of 652 nm. As Figure 3 shown, TMB showed an obvious blue color. After adding LPS, the absorbance decreased significantly. At the same time, SERS characteristic peaks appeared at 1190, 1330, 1402, and 1605 cm 2 O 2 in the TMB + H -1 +AgAu@AuNPs system (as Figure 4 shown). This is because the peroxidase-like activity of the AgAu@AuNPs nanozyme oxidized the Raman-active oxTMB. The UV-vis absorption spectrum of AgAu@AuNPs showed an obvious local surface plasmon resonance (LSPR) peak at 536 nm (as Figure 5 shown), indicating that AgAu@AuNPs had strong SERS electromagnetic enhancement (EM). At the same time, the peroxidase-like activity of AgAuNPs was investigated, and its peroxidase-like activity was significantly lower than that of AgAu@AuNPs.

[0048] The Michaelis catalytic kinetic parameters were also determined in the experiment (as Figures 8 - 9 and Table 1 shown). The Michaelis constants of AgAu@AuNPs for the substrates TMB and H 2 O 2 were 0.65 mmol / L and 0.43 mmol / L respectively, and the reaction rate constants were 36.6×10 K m and 1.34×10 -7 mol / L·s respectively. After adding LPS, the -7 of TMB and H 2 O 2 were 1.27 mmol / L and 3.91 mmol / L respectively, and the reaction rate constants were 2.03×10 K m and 0.98×10 -7 and 0.98×10 -7mol / L·s, indicating that the addition of LPS significantly reduces the affinity and reaction rate of AgAu@AuNPs nanozyme with substrate TMB and H 2 O 2 2

[0049] Table 1. Determination results of Michaelis catalytic kinetic parameters.

[0050]

[0051] 4. Preparation of LPS working curve

[0052] (1) Preparation of colorimetric detection LPS working curve: Add 10 μL of 1 mg / mL AgAu@AuNPs, 10 μL of 5 mmol / L TMB, 10 μL of 50 mmol / L H 2 O 2 2 Figures 10 - 11 10 μL, and 10 μL of bacterial endotoxin LPS with different concentrations, and 50 μL of pH 4.0 acetate buffer solution into a microplate, mix well, let stand for 5 - 10 min, measure the absorbance A at a wavelength of 652 nm, take the LPS concentration as the abscissa and A as the ordinate to draw a standard curve, and obtain the regression equation, as

[0053] shown; the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2. Figures 12 - 13

[0054] (2) Preparation of SERS detection LPS working curve: Add 10 μL of 1 mg / mL AgAu@AuNPs, 10 μL of 5 mmol / L TMB, 10 μL of 50 mmol / L H 2 O 2 2

[0055] 10 μL, and 10 μL of bacterial endotoxin LPS with different concentrations, and 50 μL of pH 4.0 acetate buffer solution into a microplate, mix well, let stand for 5 - 10 min, scan for 10 s at an excitation light of 785 nm and a laser power of 500 mW, use a portable Raman spectrometer to perform Raman spectroscopy detection on the test solution, take the LPS concentration as the abscissa and the Raman intensity at 1605 cm -1 as the ordinate to draw a standard curve, and obtain the regression equation, as shown in Table 2, as

[0056] shown; the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1. Figures 14 - 15 β-D-glucose, dextran, and Cu that have a large interference on the limulus test method 2+, Zn 2+ , Mg 2+ , Al 3+ , Ca 2+ Effect on the detection system, LPS concentration is 10 EU / L, and the concentration of all interfering substances is the LPS concentration and 100 times. The results show that only LPS has an obvious inhibitory effect on AgAu@AuNPs+H 2 O 2 +TMB, and other substances have almost no effect, indicating that the method has good selectivity.

[0057] 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. The samples to be determined included NaCl injection, metronidazole injection, gentamicin sulfate injection, and ampicillin sodium injection. In various injection samples, LPS standard solutions of 2, 20, and 100 EU / L were added, and a standard addition recovery test was carried out on the synthetic samples. The results are shown in Table 3. The results show that the recovery rate of LPS measured in the synthetic samples is between 86.9% and 105.8%, and the RSD is less than 6% (n = 6). The results indicate that the method established in the present invention can be used for the detection of LPS in injections.

[0058] Table 3. Standard addition recovery rate and RSD of the samples of the method of the present invention (n = 6).

[0059]

[0060] Example 2: Determination of LPS in disposable infusion sets

[0061] 1. Preparation of CS@Fe,Mn / CDs nanomaterials: 0.5 g of chitosan, 0.5 g of citric acid, 0.40 g of MnCl 2 ·4H 2 O, 0.4 g of FeCl 3 ·6H 2 O and 0.35 g of p-phenylenediamine were dissolved in 50 mL of 0.1% (v / v) acetic acid solution, and ultrasonic treatment was carried out for 30 min. The mixed solution was transferred to a polytetrafluoroethylene high-pressure reaction kettle and placed in a microwave digestion instrument at 50 W and heated to 180 °C for 2 h. After the reaction was completed, it was naturally cooled to room temperature, and large-particle impurities were removed with a 0.22 μm filter membrane, and then centrifuged at high speed. The supernatant was vacuum dried to obtain iron and copper-doped carbon dot CS@Fe,Mn / CDs nanomaterials.

[0062] 2. Preparation of AgAu@AuNPs nanozyme: Take 10 mM HAuCl 4300 μL and 2.0 mg / mL AgNO 3 were mixed with 300 μL, added to 40 mL of deionized water, heated to 95 °C, stirred for 10 min, then 40 μL of 1 mg / mL sodium citrate was added, and after continuous heating and stirring for 15 min, 10 mM HAuCl 4 300 μL and 30 μL of 1.6 mg / mL CS@Fe,Mn / CDs were added, and stirring was continued for 5 - 10 min. The resulting solution was immediately cooled in an ice bath to obtain AgAu@AuNPs nanozyme.

[0063] 3. Preparation of the working curve of LPS: The same as in Example 1.

[0064] 4. Determination of LPS in disposable infusion set samples

[0065] (1) Preparation of the test solution: Under aseptic conditions, 10 mL was injected into the inner cavity of each infusion set, and 15 mL of 10% sodium chloride injection was injected into the inner cavity of the blood transfusion set. After repeated rinsing 5 times, both ends were sealed and placed in an incubator at (37 ± 1) °C for 2 h. After taking out, the test solution was collected into a sterile and pyrogen-free stoppered glass container to obtain the test solution; at the same time, 10% sodium chloride injection containing 10 EU / mL of bacterial endotoxin was used as the extraction medium, and under the same extraction conditions, a positive control solution was obtained;

[0066] (2) Determination of LPS: 10 μL of 1 mg / mL AgAu@AuNPs, 10 μL of 5 mmol / L TMB, 10 μL of 50 mmol / L H 2 O 2 10 μL, and 10 μL each of the test solution and the positive control solution were added to the microplate, 50 μL of pH 4.0 acetate buffer solution was added dropwise into the microplate, mixed well, and left standing for 5 - 10 min. The absorbance was measured at 652 nm, scanned at an excitation wavelength of 785 nm and a laser power of 500 mW for 10 s, and the Raman spectrum of the test solution was detected using a portable Raman spectrometer to measure the Raman intensity, and substituted into the regression equation, which were not detected and 9.5 ± 0.5 EU / mL, 10.1 ± 0.3 EU / mL respectively.

[0067] Where not otherwise involved above, it shall apply to the prior art.

[0068] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration purposes and not for limiting the scope of the present invention. Those skilled in the technical field to which the present invention pertains can make modifications or supplements to the described specific embodiments or use similar ways for substitution, but will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above embodiments based on the technical essence of the present invention should all be included within 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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