Method for detecting bacterial endotoxin in nano-enzyme gel colorimetric-fluorescence dual mode

Abstract

The invention discloses a method for detecting bacterial endotoxin in a nano-enzyme gel colorimetric-fluorescence dual-mode, which is characterized in that a synthesized iron and copper doped carbon dot nano-material CS (at) Fe and Cu / CDs is used as a reducing agent to reduce KMnO4 to form CS (at) Fe and Cu / CDS-MnO2 nano-enzyme, CS (at) Fe and Cu / CDS-MnO2 show unique fluorescence and peroxidase-like characteristics, and the fluorescence quantum yield reaches 76%; the preparation method comprises the following steps: preparing CS-coated Fe, Cu / CDs-MnO2 nano-enzyme, mixing CS-coated Fe, Cu / CDs-MnO2 nano-enzyme with an agarose solution and a 3, 3 '-diaminobenzidine tetrahydrochloride solution, dropwise adding the mixture into a cell culture pore plate, and cooling to obtain a nano-enzyme hydrogel microwell plate; the nano-enzyme hydrogel microwell plate is applied to colorimetric-fluorescence dual-mode detection of bacterial endotoxin, and a nano-enzyme gel-based fluorescence and colorimetric dual-mode biosensor is constructed; the method disclosed by the invention has the characteristics of high sensitivity, strong specificity, simplicity in operation, rapidness and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a method and a detection reagent for detecting bacterial endotoxins in a nanoenzyme gel colorimetric-fluorescence dual mode. Background Art

[0002] Endotoxin, also known as lipopolysaccharide (LPS), is a highly pathogenic toxin released from the cell membrane after the death of Gram-negative bacteria. It can induce serious diseases such as sepsis, intestinal inflammation, diabetes, fever, septic shock, and even death. Due to the possibility of severe immune response, LPS detection is an important part of ensuring the safety of sterilized products. 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. The in vitro Limulus amebocyte lysate assay (LAL) and the in vivo rabbit pyrogen test (RPT) test are standard methods for detecting LPS. Enzyme-linked immunosorbent assay (ELISA) and fluorescence technology are also used for LPS detection. However, both methods have certain limitations: ELISA faces the problem of non-specific binding of LPS to microplates, while fluorescence sensing requires the use of special fluorescent groups. To date, the LAL method is regarded as the gold standard method due to its advantages such as simple operation, accurate detection results and high sensitivity. However, the LAL assay is sensitive to the environment, such as pH and temperature, as well as various inhibitors and activators. In addition, the horseshoe crab reagent is extracted from the lysate of amebocytes in the blood of the American horseshoe crab or the eastern horseshoe crab. In recent years, due to overfishing of horseshoe crabs and environmental deterioration, the number of horseshoe crabs in my country has decreased sharply. It has now been listed as a national second-class protected animal, which has brought animal ethical issues and high costs. The traditional horseshoe crab test endotoxin detection method will gradually be replaced. Therefore, there is still an urgent need to develop cost-effective and efficient alternative LAL strategies.

[0003] Dual-signal biosensors that combine colorimetric and fluorescence methods can largely eliminate instrumental and human errors, making the sensor more reliable. Carbon dots (CDs) are widely used in probe detection due to their ultra-small size (below 10 nm), excellent optical and catalytic properties, low toxicity, and easy surface modification. However, the poor electron transfer ability of unmodified CDs reduces its catalytic activity and limits its application. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing a nanozyme hydrogel microplate and detecting bacterial endotoxins in a colorimetric-fluorescence dual-mode. The present invention uses chitosan, o-phenylenediamine doped Fe, and Cu carbon dots as reducing agents to reduce KMnO4 to form CS@Fe,Cu / CDs-MnO2 nanozymes. CS@Fe,Cu / CDs-MnO2 exhibits unique fluorescence and peroxidase-like properties, and the fluorescence quantum yield reaches 76%. On the one hand, the CS@Fe,Cu / CDs-MnO2 nanozyme with peroxidase-like activity and positive charge can oxidize 3,3'-diaminobenzidine tetrahydrochloride (DAB) and H2O2 to produce a brown product (oxDAB), and can bind to negatively charged bacterial endotoxins through electrostatic interactions, resulting in a decrease in the catalytic activity of the nanozyme; on the other hand, oxDAB quenches the fluorescence of the CS@Fe,Cu / CDs-MnO2 nanozyme through the inner filter effect and static quenching. CS@Fe,Cu / CDs-MnO2 nanozyme was loaded on hydrogel, and a nanozyme gel-based fluorescence and colorimetric dual-mode biosensor was constructed with the assistance of a smartphone.

[0005] The method for detecting bacterial endotoxins by the nanoenzyme gel colorimetric-fluorescence dual mode of the present invention is as follows: (1) 0.3-0.5 g chitosan, 0.3-0.5 g citric acid, 0.15-0.35 g CuCl2·2H2O, 0.18-0.4 g FeCl3·6H2O, and 0.15-0.35 g o-phenylenediamine were dissolved in 30-50 mL acetic acid solution with a volume concentration of 0.05-0.1%, and ultrasonically treated for 20-30 min. The mixture was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester. After reacting at 170-190°C for 1-2 h, it was naturally cooled to room temperature. Large particles were removed with a 0.22 μm filter membrane, and then high-speed centrifuged to collect the supernatant and vacuum dried to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs. The high-speed centrifugation is carried out at 8000-10000 r / min for 5-10 min; (2) Dissolve 0.020-0.025 g of potassium permanganate in 30-40 mL of CS@Fe,Cu / CDs solution, stir and mix at room temperature, centrifuge at high speed, collect the supernatant, and vacuum dry to obtain CS@Fe,Cu / CDs-MnO2 nanozyme; The concentration of the CS@Fe,Cu / CDs solution is 1 mg / mL, the dosage is 30-40 mL, and the high-speed centrifugation is performed at 8000-10000 r / min for 5-10 min; (3) Dissolve agarose powder in 20 mL of deionized water, heat in a microwave until completely dissolved, add 100-500 μL of CS@Fe,Cu / CDs-MnO2 nanozyme solution and 100-500 μL of DAB solution, mix well, and then drop the mixture into the cell culture well plate in sequence, cool to room temperature, and prepare a nanozyme hydrogel microplate; The amount of agarose used is 100-200 mg, the concentration of CS@Fe,Cu / CDs-MnO2 nanozyme solution is 1 mg / mL, and the concentration of 3,3'-diaminobenzidine tetrahydrochloride solution is 5 mmol / L; (4) 4) Different concentrations of bacterial endotoxin and H2O2 were dropped into the nanozyme hydrogel microplate, incubated at 30°C for 10-15 min, and then photographed under natural light or fluorescence conditions. The RGB values ​​of the images were read using the image processing software Image J, converted into grayscale values, and the linear relationship between the grayscale value and the bacterial endotoxin concentration was determined. The H2O2 concentration is 50-100mmol / L, and the dosage is 10-20μL; (5) Drop the sample to be tested and H2O2 into the nanozyme hydrogel microplate, incubate at 30°C for 10-15 min, take photos under natural light or fluorescence conditions, read the RGB value of the image using image processing software Image J, and obtain the bacterial endotoxin content in the sample to be tested based on the RGB value; The H2O2 concentration is 50-100 mmol / L, and the dosage is 10-20 μL.

[0006] Another object of the present invention is to provide a detection reagent and a detection kit, comprising the nanoenzyme hydrogel microplate prepared by the above method, H2O2, and a bacterial endotoxin standard.

[0007] The present invention can also use an enzyme reader to perform colorimetry or fluorescence to obtain absorbance values ​​or fluorescence intensities, and establish a linear relationship between the absorbance value or fluorescence intensity and the bacterial endotoxin concentration; after incubating the sample to be tested, the absorbance value or fluorescence intensity is obtained by colorimetry or fluorescence, and the bacterial endotoxin content in the sample to be tested is obtained according to the absorbance value or fluorescence intensity.

[0008] The advantages of the present invention are: 1. The present invention utilizes o-phenylenediamine selected in the preparation of CS@Fe,Cu / CDs to enhance the fluorescence intensity, chitosan to make its surface positively charged, and Fe,Cu doping to improve its POD-like activity, so that the stability and POD-like activity of the prepared CS@Fe,Cu / CDs-MnO2 nanozyme are significantly improved. Since the negatively charged bacterial endotoxin can bind to the positively charged CS@Fe,Cu / CDs-MnO2 nanozyme through electrostatic interaction, the catalytic activity of the nanozyme is reduced, and its peroxidase-like activity is inhibited, so that the brown color of oxDAB produced by the oxidized DAB and H2O2 system of CS@Fe,Cu / CDs-MnO2 nanozyme becomes lighter; and oxDAB quenches the fluorescence of CS@Fe,Cu / CDs-MnO2 nanozyme through the inner filtration effect and static quenching effect, thereby establishing a highly sensitive and selective bacterial endotoxin colorimetric-fluorescence dual-mode detection method; 2. During the nanozyme hydrogel microplate detection process, the colorimetric-fluorescence signal is collected by a smart phone, and its reproducibility and ease of operation are fully reflected. The endotoxin concentration is in the range of 0.125~175 EU / L, the absorbance is linearly related to the endotoxin concentration, and the detection limit is 0.058 EU / L; the fluorescence intensity is linearly related in the range of 0.05~90 EU / L, and the detection limit is 0.036 EU / L. This method is applied to the detection of endotoxin in injection samples and biological products, and the recovery rate is between 85.3% and 103.2%, and the RSD does not exceed 4.5%. The sensitivity is nearly 50 times higher than that of the Chinese Pharmacopoeia horseshoe crab reagent detection method, and the detection time only takes 15 min. The method of the present invention has the characteristics of high sensitivity, strong specificity, simple operation and rapidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the transmission electron microscope TEM image of CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1; Figure 2 The fluorescence spectrum of CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1; Figure 3 The UV-vis absorption spectra of the CS@Fe,Cu / CDs-MnO2 nanozyme catalyzing the oxidation of the peroxidase chromogenic substrate and H2O2 in Example 1; Figure 4 The fluorescence spectra of the substrate DAB and DAB+H2O2 catalyzed by the CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1, where +DAB represents CS@Fe,Cu / CDs-MnO2+DAB, and +oxDAB represents CS@Fe,Cu / CDs-MnO2+DAB+H2O2; Figure 5It is the fitted Michaelis-Menten diagram of TMB catalyzed by CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+LPS in Example 1 of the present invention, wherein Figure a is TMB catalyzed by CS@Fe,Cu / CDs-MnO2, and Figure b is TMB catalyzed by CS@Fe,Cu / CDs-MnO2+LPS; Figure 6 It is the fitted Michaelis-Menten diagram of CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+LPS catalyzing H2O2 in Example 1 of the present invention, wherein Figure a is CS@Fe,Cu / CDs-MnO2 catalyzing H2O2, and Figure b is CS@Fe,Cu / CDs-MnO2+LPS catalyzing H2O2; Figure 7 Zeta potential detection results, from left to right in the figure are CS@Fe, Cu / CDs-MnO2 nanozyme, CS@Fe, Cu / CDs-MnO2+LPS and LPS; Figure 8 The fluorescence spectrum of CS@Fe,Cu / CDs-MnO2 and the UV-vis absorption spectrum of oxDAB in Example 1 of the present invention; Fig. 9 The fluorescence lifetime of CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+oxDAB in Example 1 of the present invention; Fig.10 is a transmission electron microscope (SEM) image of the hydrogel in Example 1; Fig.11 The linear fitting curves of the colorimetric gray value (a) and the fluorescent gray value (b) of the CS@Fe,Cu / CDs-MnO2 nanozyme for detecting LPS in Example 1 of the present invention; Fig.12 The results of CS@Fe,Cu / CDs-MnO2 nanozyme's selectivity for detecting LPS, where Figure a is the colorimetric detection result and Figure b is the fluorescence detection result; Fig.13 These are the standard curves of LPS concentration and absorbance value and the standard curve of LPS concentration and fluorescence intensity in Example 2. DETAILED DESCRIPTION

[0010] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Determination of bacterial endotoxin (LPS) in various injection samples 1. Preparation of iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs 0.3g chitosan, 0.3g citric acid, 0.15g CuCl2·2H2O, 0.18g FeCl3·6H2O, and 0.15g o-phenylenediamine were dissolved in 30mL of 0.05% (v / v) acetic acid solution, and ultrasonically treated for 20min. The mixture was transferred to a polytetrafluoroethylene high-pressure reactor, placed in a microwave digester and heated to 180℃ at 50W for 1h, then naturally cooled to room temperature, and large particles were removed with a 0.22μm filter membrane. The mixture was centrifuged at 10000r / min for 5min, and the supernatant was collected and vacuum dried to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs. 2. CS@Fe,Cu / CDs-MnO2 nanozyme 0.020g potassium permanganate was dissolved in 30mL of 1mg / mL CS@Fe,Cu / CDs solution, stirred at room temperature, centrifuged at 10000r / min for 30min, the supernatant was collected, and vacuum dried to obtain CS@Fe,Cu / CDs-MnO2 nanozyme; CS@Fe,Cu / CDs-MnO2 nanozyme was analyzed by transmission electron microscopy (TEM), and the results are shown in Figure 1 ,The results showed that the synthesized CS@Fe,Cu / CDs-MnO2 nanozymes were uniformly distributed in aggregated amorphous spheres.

[0011] 3. Fluorescence performance test of CS@Fe,Cu / CDs-MnO2 nanozyme (1) Determination of the quantum yield (QY) of CS@Fe,Cu / CDs-MnO2 nanozyme. H2SO4 solution containing 0.1 mol / L quinine sulfate (QY at 360 nm is 54%, η = 1.33) was used as the standard solution, and the value of fluorescence quantum yield was calculated according to the following formula: QY = Q R (I S / I R (A R / A S ) (η 2 S / η 2 R ), where the subscript "S" is the sample, the subscript "R" is quinine sulfate, A is the absorbance at the excitation wavelength, I is the integrated emission intensity, and η is the solvent refractive index; the QY of CS@Fe,Cu / CDs-MnO2 nanozyme was detected to be 76%; (2) Fluorescence properties of CS@Fe,Cu / CDs-MnO2 nanozymes: Figure 2As shown, the fluorescence of CS@Fe,Cu / CDs-MnO2 nanozyme exhibits excitation-dependent fluorescence behavior at 370-430nm, which is consistent with the characteristics of most carbon nanomaterials.

[0012] 4. Detection of peroxidase-like activity of CS@Fe,Cu / CDs-MnO2 nanozyme The POD-like activity of CS@Fe,Cu / CDs-MnO2 nanozyme was investigated using 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and DAB as chromogenic substrates. 100 μL of 1 mg / mL CS@Fe, Cu / CDs-MnO2 nanozyme, 50 mmol / L 100 μL of H2O2 was mixed with 100 μL of 5 mmol / L substrates ABTS, TMB, OPD, and DAB, respectively. After incubation at 30°C for 10 min, the absorbance was measured at 654, 415, and 564 nm using a UV-visible spectrophotometer. The results were as follows: Figure 3 As shown in the figure, the four substrates all showed different degrees of color change. DAB can not only be oxidized to produce a significant characteristic absorption peak, but the oxidation product can also quench the fluorescence intensity of CS@Fe,Cu / CDs-MnO2 nanozyme ( Figure 4 Therefore, DAB was selected as the chromogenic substrate in this study.

[0013] The experiment also carried out the determination of Michaelis-Menten catalytic kinetic parameters. Figure 5 , 6 Table 1. Michaelis constants of CS@Fe,Cu / CDs-MnO2 nanozymes for substrates TMB and H2O2 K m The reaction rates were 0.98 mmol / L and 0.88 mmol / L, respectively, and the reaction rate constants were 2.04×10 -7 and 2.38×10 -7 mol / L·s, TMB and H2O2 after adding LPS K m are 2.04mmol / L and 8.58mmol / L, and the reaction rate constants are 0.25×10 -7 and 0.74×10 -7 mol / L·s, indicating that the addition of LPS greatly reduced the affinity and reaction rate of CS@Fe,Cu / CDs-MnO2 nanozyme with substrates TMB and H2O2.

[0014] Table 1 Michaelis-Menten catalytic kinetic parameters ; 5. LPS detection principle CS@Fe,Cu / CDs-MnO2 nanozyme catalyzes DAB and H2O2 to generate a brown product (oxDAB), which causes fluorescence quenching of CS@Fe,Cu / CDs-MnO2 nanozyme through the inner filter effect (IFE). LPS can inhibit the POD activity of CS@Fe,Cu / CDs-MnO2 nanozyme, reduce the generation of oxDAB, and restore the fluorescence of CS@Fe,Cu / CDs-MnO2 nanozyme.

[0015] Zeta potential plays a key role in evaluating the stability of nanozyme systems. Lower zeta potential, whether positive or negative, shows a higher tendency to aggregate or adsorb. Therefore, the Zeta potential of CS@Fe,Cu / CDs-MnO2 nanozyme before and after adsorption of LPS was measured. The results are shown in Figure 2. Figure 7 As shown, the introduction of LPS causes the zeta potential of the nanozyme to decrease, close to zero. When LPS is not present in the system, the solution is brown and the fluorescence signal is very low. On the contrary, when LPS is present in the system, the solution is very light brown and the fluorescence intensity is high. In addition, the concentration of LPS in the system is positively correlated with the changes in color and fluorescence. This result indicates that the competitive binding of oxDAB and LPS to CS@Fe,Cu / CDs-MnO2 leads to a decrease in POD-like activity. Therefore, a dual-mode detection method for LPS was constructed using oxDAB UV-visible absorption and CS@Fe,Cu / CDs-MnO2 nanozyme fluorescence as detection signals.

[0016] In order to further study the interaction mechanism between CS@Fe,Cu / CDs-MnO2 nanozyme and oxDAB, the UV-visible absorption spectrum and fluorescence spectrum of CS@Fe,Cu / CDs-MnO2 nanozyme were measured. Figure 8 The results show that the emission spectrum of CS@Fe,Cu / CDs-MnO2 nanozyme at 475nm overlaps significantly with the UV-visible absorption spectrum of oxDAB at 466nm, providing the possibility of inner filter effect (IFE) and fluorescence resonance energy transfer (FRET). Generally, fluorescence lifetime and zeta potential are considered to be important means to distinguish IFE from FRET. Fig. 9 As shown in Figure 2, there is no significant difference in fluorescence lifetime when oxDAB is present or absent in the system. In addition, the Zeta potentials of CS@Fe,Cu / CDs-MnO2 and oxDAB are 18.55 m V and 19.07 m V, respectively. Figure 7 ), the same positive charge causes electrostatic repulsion between the two substances, preventing the FRET effect from occurring, so fluorescence quenching is the IFE mechanism.

[0017] 6. Nanozyme gel preparation 200 mg of agarose powder was dissolved in 20 mL of deionized water and heated in a microwave oven until completely dissolved. Then, 500 μL of CS@Fe,Cu / CDs-MnO2 nanozyme solution (1 mg / mL) and 500 μL of DAB (5 mmol / L) were added and mixed. Then, 150 μL of the mixture was quickly dropped into a 96-well microplate and cooled to room temperature to prepare a nanozyme hydrogel microplate. The prepared hydrogel was characterized by SEM, as shown in FIG. Fig.10 As shown, the hydrogel exhibits a hierarchically interconnected porous structure with a high specific surface area.

[0018] 7. LPS working curve production Different concentrations of bacterial endotoxin LPS 10µL, a blank without bacterial endotoxin and 100mmol / L H2O210µL were continuously dripped into the nanozyme hydrogel plate and incubated at 30℃ for 15min. Pictures were collected by smartphone under natural light and fluorescence, respectively. The images were processed using image processing software Adobe photoshop CC 2015.5. The RGB values ​​of the images taken by the mobile phone were read and converted into gray values ​​according to the gray value = 0.299 R + 0.587G + 0.114B. The gray value showed a good linear relationship with the LPS concentration in a certain concentration range. The standard curve was drawn, see Fig.11 a, 11b, and the regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0019] Table 2 Linear equation, correlation coefficient, relative standard deviation, linear range

[0020] 8. Method specificity investigation Select bD glucose, dextran, Cu 2+ 、Zn 2+ Mg 2+ 、Al 3+ , Ca 2+ The specificity of the detection system of the present invention (the method is the same as step 7) was tested, the LPS concentration was 10 EU / mL, and the concentration of other interfering substances was 100 times the LPS concentration; result Fig.12 It shows that only LPS has a significant inhibitory effect on CS@Fe,Cu / CDs-MnO2+H2O2+DAB ( Fig.12 a), while only LPS has a restorative effect on the fluorescence quenching of CS@Fe,Cu / CDs-MnO2 by oxDAB, and other substances have almost no effect ( Fig.12 b) The method has good selection specificity.

[0021] 9. Determination of LPS in various injection samples Using the same test conditions as those used in preparing the working curve in step 7, the CS@Fe,Cu / CDs-MnO2 nanozyme colorimetric / fluorescent probe was applied to the determination of various clinical injection solutions. The samples included NaCl injection, 5% glucose injection, Xuesetong injection, and ampicillin sodium injection.

[0022] 0.2, 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 show that the spike recovery of LPS measured in the synthetic samples was between 85.3% and 103.2%, and the RSD was less than 5% (n=6). The results show that the method established in the present invention can be used for the detection of LPS in injections; Table 3 Sample spike recovery and RSD of the method of the present invention (n = 6) .

[0023] Example 2: Determination of LPS in a single-use infusion set 1. Dissolve 0.5g chitosan, 0.5g citric acid, 0.35g CuCl2·2H2O, 0.4g FeCl3·6H2O and 0.35g o-phenylenediamine in 50 mL of 0.1% (v / v) acetic acid solution, and ultrasonically treat for 30 min. Transfer the mixture to a polytetrafluoroethylene high-pressure reactor, place it in a microwave digester, heat it to 180°C at 50W for 2h, and then cool it naturally to room temperature. Use a 0.22μm filter membrane to remove large particles, centrifuge it at 8000 r / min for 10min, collect the supernatant, and vacuum dry it to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs. 2. Dissolve 0.025 g potassium permanganate in 40 mL of 1 mg / mL CS@Fe,Cu / CDs, stir at room temperature for 40 min, centrifuge at 000 rpm for 10 min, collect the supernatant, and vacuum dry to obtain CS@Fe,Cu / CDs-MnO2 nanozyme; 3. Dissolve 200 mg of agarose powder in 20 mL of deionized water and heat in a microwave oven until completely dissolved. Then add 500 μL of CS@Fe,Cu / CDs-MnO2 nanozyme (1 mg / mL) and 500 μL of DAB (5 mmol / L). After mixing, quickly drop 150 μL of the mixture into a 96-well microplate and cool to room temperature to prepare a nanozyme hydrogel microplate. 4. Preparation of LPS working curve: Different concentrations of bacterial endotoxin LPS 10µL and a blank without bacterial endotoxin and 100mmol / L H2O210µL were continuously dripped into the nanozyme hydrogel plate and incubated at 30℃ for 15min. The absorbance was measured at a wavelength of 458nm. The standard curve was drawn with LPS concentration as the horizontal axis and absorbance A as the vertical axis to obtain the regression equation, see Fig.13 a; Regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 4; Under an excitation wavelength of 410 nm, the fluorescence emission at 475 nm was measured, and a standard curve was drawn with LPS concentration as the abscissa and fluorescence intensity as the ordinate to obtain the regression equation, see Fig.13 b; Regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 4; Table 4 Linear equation, correlation coefficient, relative standard deviation, linear range ; 5. 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 inject 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 hours. 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: Add 20 µL of 50 mmol / L H2O2, 10 µL of the test solution and the positive control solution to the gel ELISA plate, mix well, let stand for 15 min, measure the absorbance at a wavelength of 458 nm, and at the same time, measure the fluorescence emission at 475 nm under an excitation wavelength of 410 nm. Substitute the two into the regression equation. The result is that no LPS is detected in the infusion set and the blood transfusion set, and the LPS content in the positive control is 9.5±0.5 EU / mL.

Claims

1. A method for detecting bacterial endotoxins by nanozyme gel colorimetry-fluorescence dual mode, characterized in that: Here are the steps: (1) 0.3-0.5 g chitosan, 0.3-0.5 g citric acid, 0.15-0.35 g CuCl2·2H2O, 0.18-0.4 g FeCl3·6H2O, and 0.15-0.35 g o-phenylenediamine were dissolved in 30-50 mL acetic acid solution with a volume concentration of 0.05-0.1%. After ultrasonic treatment, the mixture was placed in a microwave oven at 170-190°C for 1-2 h, and then naturally cooled to room temperature. Large particles were removed with a 0.22 μm filter membrane, and then the mixture was centrifuged at high speed, the supernatant was collected, and vacuum dried to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs. (2) Dissolve potassium permanganate in the CS@Fe,Cu / CDs solution, stir and mix at room temperature, centrifuge at high speed, collect the supernatant, and vacuum dry to obtain the CS@Fe,Cu / CDs-MnO2 nanozyme; (3) Dissolve agarose in 20 mL of deionized water, heat until completely dissolved, add 100-500 μL of CS@Fe,Cu / CDs-MnO2 nanozyme solution and 100-500 μL of 3,3'-diaminobenzidine tetrahydrochloride solution, mix well, and quickly drop the mixture into the cell culture well plate, cool to room temperature, and prepare the nanozyme hydrogel microplate; (4) Different concentrations of bacterial endotoxin and H2O2 were dripped into the nanozyme hydrogel microplate, incubated at 30°C for 10-15 min, and then photographed under natural light or fluorescence conditions. The RGB values ​​of the images were read using image processing software, converted into grayscale values, and the linear relationship between the grayscale value and the bacterial endotoxin concentration was determined. (5) Drop the sample to be tested and H2O2 into the nanozyme hydrogel microplate, incubate at 30°C for 10-15 min, take pictures under natural light or fluorescence conditions, use image processing software to read the RGB value of the captured image, and obtain the bacterial endotoxin content in the sample to be tested based on the RGB value.

2. The method for detecting bacterial endotoxins by nanoenzyme gel colorimetry-fluorescence dual mode according to claim 1, characterized in that: The dosage of potassium permanganate is 0.020-0.025g; the concentration of CS@Fe,Cu / CDs solution is 1mg / mL, and the dosage is 30-40mL; the dosage of agarose is 100-200mg, the concentration of CS@Fe,Cu / CDs-MnO2 nanoenzyme solution is 1mg / mL, and the concentration of 3,3'-diaminobenzidine tetrahydrochloride solution is 5mmol / L.

3. The method for detecting bacterial endotoxins by nanoenzyme gel colorimetry-fluorescence dual mode according to claim 1, characterized in that: High-speed centrifugation is performed at 8000-10000 r / min for 5-10 min.

4. The method for detecting bacterial endotoxins by nanoenzyme gel colorimetry-fluorescence dual mode according to claim 1, characterized in that: The H2O2 concentration is 50-100mmol / L and the dosage is 10-20μL.

5. The method for detecting bacterial endotoxins by nanoenzyme gel colorimetry-fluorescence dual mode according to claim 1, characterized in that: After incubation, the absorbance value or fluorescence intensity is obtained by colorimetry or fluorescence method using an enzyme reader, and a linear relationship between the absorbance value or fluorescence intensity and the bacterial endotoxin concentration is established; after incubation of the sample to be tested, the absorbance value or fluorescence intensity is obtained by colorimetry or fluorescence method.

6. A detection reagent or kit, characterized in that: It includes the nanoenzyme hydrogel microporous plate and H2O2 used in the method described in claim 1.

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