A method for detecting bacterial endotoxins using nanoenzyme gel colorimetric-fluorescence dual-mode

Abstract

The present invention discloses a method for detecting bacterial endotoxins by using a nanoenzyme gel in a colorimetric-fluorescence dual-mode. In the present invention, a synthetic iron- and copper-doped carbon dot nanomaterial CS@Fe,Cu / CDs is used as a reducing agent to reduce KMnO4 to form a CS@Fe,Cu / CDs-MnO2 nanoenzyme. The CS@Fe,Cu / CDs-MnO2 exhibits unique fluorescence and peroxidase-like properties, and a fluorescence quantum yield of 76% is achieved. The CS@Fe,Cu / CDs-MnO2 nanoenzyme is then mixed with an agarose solution and a 3,3'-diaminobenzidine tetrahydrochloride solution, and the mixture is dropwise added to a cell culture well plate. The mixture is cooled to obtain a nanoenzyme hydrogel microplate. The nanoenzyme hydrogel microplate is applied to the colorimetric-fluorescence dual-mode detection of bacterial endotoxins, thereby constructing a nanoenzyme gel-based fluorescence and colorimetric dual-mode biosensor. The method of the present invention has the characteristics of high sensitivity, strong specificity, simple operation, rapidity, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and specifically to a method and a detection reagent for detecting bacterial endotoxins using 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 of Gram-negative bacteria upon death. It can induce serious illnesses such as sepsis, intestinal inflammation, diabetes, fever, septic shock, and even death. Due to the potential for severe immune responses, LPS detection is a crucial component in ensuring the safety of sterilized products. The Chinese Pharmacopoeia stipulates that the maximum acceptable concentration of LPS for injection is 5 EU / kg / h, and the maximum acceptable concentration for intrathecal LPS is 0.2 EU / kg / h. Therefore, real-time monitoring of LPS levels during production is crucial for ensuring the safety of sterilized products. The in vitro Limulus Amebocyte Lysate (LAL) assay and the in vivo Rabbit Pyrogen Test (RPT) assay are standard methods for LPS detection. Enzyme-linked immunosorbent assay (ELISA) and fluorescence techniques are also used for LPS detection. However, both methods have limitations: ELISA suffers from nonspecific binding of LPS to microplates, while fluorescence sensing requires the use of specialized fluorophores. To date, the LAL assay has been considered the gold standard due to its simplicity, accuracy, and high sensitivity. However, the LAL assay is sensitive to environmental factors such as pH and temperature, as well as various inhibitors and activators. Furthermore, the Limulus lysate (LAL) is extracted from the lysate of amebocytes in the blood of either Limulus polyphemus or Limulus orientalis. In recent years, due to overfishing and environmental degradation, the number of Limulus lysate in my country has decreased dramatically, and it is now listed as a Class II national protected species. This raises animal ethics concerns and high costs, and the traditional LAL test for endotoxin detection will gradually be replaced. Therefore, there remains 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, resulting in better sensor reliability. 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 ease of surface modification. However, the poor electron transfer ability of unmodified CDs reduces their catalytic activity, limiting their application. Summary of the Invention

[0004] To address the challenges of existing technologies, the present invention provides a nanozyme hydrogel microplate preparation and a method for colorimetric-fluorescence dual-mode detection of bacterial endotoxins. Chitosan, o-phenylenediamine-doped Fe, and Cu carbon dots are used as reducing agents to reduce KMnO4 to form the CS@Fe,Cu / CDs-MnO2 nanozyme. The CS@Fe,Cu / CDs-MnO2 nanozyme exhibits unique fluorescence and peroxidase-like properties, with a fluorescence quantum yield of 76%. The peroxidase-like and positively charged CS@Fe,Cu / CDs-MnO2 nanozyme oxidizes 3,3'-diaminobenzidine tetrahydrochloride (DAB) and H2O2 to produce a brown product (oxDAB). The product binds to negatively charged bacterial endotoxins through electrostatic interactions, resulting in reduced catalytic activity. Furthermore, oxDAB quenches the fluorescence of the CS@Fe,Cu / CDs-MnO2 nanozyme through an inner filtration effect and static quenching. CS@Fe,Cu / CDs-MnO2 nanozyme was loaded on hydrogel, and with the assistance of a smartphone, a nanozyme gel-based fluorescence and colorimetric dual-mode biosensor was constructed.

[0005] The method for detecting bacterial endotoxins using the nanoenzyme gel colorimetric-fluorescence dual mode of the present invention is as follows:

[0006] (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 of 0.05-0.1% acetic acid solution, and ultrasonicated for 20-30 min. The mixture was transferred to a polytetrafluoroethylene autoclave and placed in a microwave digester. The mixture was reacted at 170-190 °C for 1-2 h, and then cooled to room temperature naturally. Large particles were removed with a 0.22 μm filter membrane, and the supernatant was collected by high-speed centrifugation and vacuum dried to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs.

[0007] The high-speed centrifugation is carried out at 8000-10000 r / min for 5-10 min;

[0008] (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;

[0009] 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;

[0010] (3) Dissolve agarose powder in 20 mL of deionized water and microwave-heat until completely dissolved. Then add 100-500 μL of CS@Fe,Cu / CDs-MnO2 nanozyme solution and 100-500 μL of DAB solution. After mixing, quickly add the mixture dropwise to the cell culture well plate and cool to room temperature to prepare the nanozyme hydrogel microplate.

[0011] 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;

[0012] (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 photographed 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;

[0013] The H2O2 concentration is 50-100 mmol / L, and the dosage is 10-20 μL;

[0014] (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, read the RGB values ​​of the captured images using image processing software Image J, and obtain the bacterial endotoxin content in the sample to be tested based on the RGB values;

[0015] The H2O2 concentration is 50-100 mmol / L, and the dosage is 10-20 μL.

[0016] 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.

[0017] The present invention can also use an enzyme reader to perform colorimetry or fluorescence to obtain absorbance values ​​or fluorescence intensity, and establish a linear relationship between the absorbance value or fluorescence intensity and the bacterial endotoxin concentration; after incubation of 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.

[0018] The advantages of the present invention are:

[0019] 1. The present invention utilizes o-phenylenediamine selected in the preparation of CS@Fe,Cu / CDs to enhance fluorescence intensity, chitosan to make its surface positively charged, and Fe,Cu doping to enhance its POD-like activity, thereby significantly improving the stability and POD-like activity of the prepared CS@Fe,Cu / CDs-MnO2 nanozyme. Since negatively charged bacterial endotoxins 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, causing the brown color of oxDAB produced by the oxidized DAB and H2O2 system of CS@Fe,Cu / CDs-MnO2 nanozyme to become lighter; and oxDAB quenches the fluorescence of the CS@Fe,Cu / CDs-MnO2 nanozyme through the inner filtration effect and static quenching, thereby establishing a highly sensitive and selective bacterial endotoxin colorimetric-fluorescence dual-mode detection method;

[0020] 2. During the nanozyme hydrogel microplate detection process, the colorimetric-fluorescence signal is collected by a smartphone, and its reproducibility and ease of operation are fully demonstrated. The absorbance is linearly related to the endotoxin concentration in the range of 0.125~175 EU / L, 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 endotoxins in injection samples and biological products, with a recovery rate between 85.3% and 103.2%, and an RSD of no more than 4.5%. The sensitivity is nearly 50 times higher than that of the Chinese Pharmacopoeia Limulus Amebocyte Lysate (LAL) test method, and the detection time only takes 15 minutes. The method of the present invention has the characteristics of high sensitivity, strong specificity, simple operation, and rapidity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the transmission electron microscopy (TEM) image of the CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1;

[0022] Figure 2 This is the fluorescence spectrum of CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1;

[0023] Figure 3 The UV-vis absorption spectra of the CS@Fe,Cu / CDs-MnO2 nanozyme catalyzing the oxidation of peroxidase chromogenic substrate and H2O2 in Example 1;

[0024] Figure 4 The fluorescence spectra of the CS@Fe,Cu / CDs-MnO2 nanozyme catalyzed by the substrate DAB and DAB+H2O2 in Example 1. In the figure, +DAB represents CS@Fe,Cu / CDs-MnO2+DAB, and +oxDAB represents CS@Fe,Cu / CDs-MnO2+DAB+H2O2;

[0025] Figure 5 The fitted Michaelis-Menten plots of TMB catalyzed by CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+LPS in Example 1 of the present invention are shown, wherein Figure a shows TMB catalyzed by CS@Fe,Cu / CDs-MnO2, and Figure b shows TMB catalyzed by CS@Fe,Cu / CDs-MnO2+LPS;

[0026] Figure 6 The fitted Michaelis-Menten plots of H2O2 catalyzed by CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+LPS in Example 1 of the present invention are shown in Figure a, where Figure a shows H2O2 catalyzed by CS@Fe,Cu / CDs-MnO2 and Figure b shows H2O2 catalyzed by CS@Fe,Cu / CDs-MnO2+LPS.

[0027] Figure 7 Zeta potential detection results. From left to right, the figures are CS@Fe,Cu / CDs-MnO2 nanozyme, CS@Fe,Cu / CDs-MnO2+LPS and LPS.

[0028] 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 are shown;

[0029] Figure 9 The fluorescence lifetime of CS@Fe,Cu / CDs-MnO2 and CS@Fe,Cu / CDs-MnO2+oxDAB in Example 1 of the present invention;

[0030] Figure 10 1 is a transmission electron microscope (SEM) image of the hydrogel in Example 1;

[0031] Figure 11 The linear fitting curves of the colorimetric gray value (Figure a) and the fluorescent gray value (Figure b) of the CS@Fe,Cu / CDs-MnO2 nanozyme in Example 1 of the present invention for detecting LPS;

[0032] Figure 12 The results of CS@Fe,Cu / CDs-MnO2 nanozyme's selective detection of LPS, where Figure a is the colorimetric detection result and Figure b is the fluorescence detection result;

[0033] Figure 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

[0034] The present invention is further illustrated by the following examples, 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.

[0035] Example 1: Determination of bacterial endotoxin (LPS) in various injection samples

[0036] 1. Preparation of Iron- and Copper-doped Carbon Dot Nanomaterials CS@Fe,Cu / CDs

[0037] 0.3 g chitosan, 0.3 g citric acid, 0.15 g CuCl2·2H2O, 0.18 g FeCl3·6H2O, and 0.15 g o-phenylenediamine were dissolved in 30 mL of 0.05% (v / v) acetic acid solution and sonicated for 20 min. The mixture was transferred to a polytetrafluoroethylene autoclave and placed in a microwave digester, heated to 180°C at 50 W for 1 h, and then naturally cooled to room temperature. Large particles were removed with a 0.22 μm filter membrane, and the mixture was centrifuged at 10,000 rpm for 5 min. The supernatant was collected and dried in vacuo to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs.

[0038] 2. CS@Fe,Cu / CDs-MnO2 nanozyme

[0039] 0.020 g potassium permanganate was dissolved in 30 mL of 1 mg / mL CS@Fe,Cu / CDs solution, stirred at room temperature, centrifuged at 10000 rpm for 30 min, and 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). The results are shown in Figure 1 ,The results showed that the synthesized CS@Fe,Cu / CDs-MnO2 nanozymes were aggregated amorphous spheres and evenly distributed.

[0040] 3. Fluorescence performance test of CS@Fe,Cu / CDs-MnO2 nanozyme

[0041] (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 a standard solution, and the fluorescence quantum yield value was calculated according to the following formula: QY = Q R (I S / I R ) (A R / A S ) (η 2S / η 2 R ), where subscript "S" is the sample, subscript "R" is quinine sulfate, A is the absorbance at the excitation wavelength, I is the integrated emission intensity, and η is the refractive index of the solvent; the QY of CS@Fe,Cu / CDs-MnO2 nanozyme was found to be 76%;

[0042] (2) Fluorescence properties of CS@Fe,Cu / CDs-MnO2 nanozymes: Figure 2 As shown, the fluorescence of CS@Fe,Cu / CDs-MnO2 nanozyme exhibits excitation-dependent fluorescence behavior at 370-430 nm, which is consistent with the characteristics of most carbon nanomaterials.

[0043] 4. Detection of peroxidase-like activity of CS@Fe,Cu / CDs-MnO2 nanozyme

[0044] The POD-like activity of CS@Fe,Cu / CDs-MnO2 nanozyme was investigated using 2,2′-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and DAB as chromogenic substrates.

[0045] 100 μL of 1 mg / mL CS@Fe,Cu / CDs-MnO2 nanozyme and 50 mmol / L 100 μL of H2O2 was mixed with 100 μL each of 5 mmol / L substrates ABTS, TMB, OPD, and DAB. 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.

[0046] The Michaelis-Menten catalytic kinetic parameters were also determined. 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 Km 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.

[0047] Table 1 Michaelis-Menten catalytic kinetic parameters

[0048] ;

[0049] 5. LPS detection principle

[0050] The CS@Fe,Cu / CDs-MnO2 nanozyme catalyzes the reaction of DAB and H2O2 to form a brown product (oxDAB). OxDAB quenches the fluorescence of the CS@Fe,Cu / CDs-MnO2 nanozyme through the inner filter effect (IFE). LPS inhibits the POD activity of the CS@Fe,Cu / CDs-MnO2 nanozyme, reduces the formation of oxDAB, and restores the fluorescence of the CS@Fe,Cu / CDs-MnO2 nanozyme.

[0051] Zeta potential plays a key role in evaluating the stability of nanozyme systems. Lower zeta potential, whether positive or negative, indicates 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 in the figure, the introduction of LPS causes the zeta potential of the nanozyme to decrease, approaching 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 the UV-visible absorption of oxDAB and the fluorescence of CS@Fe,Cu / CDs-MnO2 nanozyme as detection signals.

[0052] 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 8The results showed that the emission spectrum of CS@Fe,Cu / CDs-MnO2 nanozyme at 475nm significantly overlapped 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 between IFE and FRET. Figure 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 occurrence of the FRET effect, so fluorescence quenching is the IFE mechanism.

[0053] 6. Nanozyme Gel Preparation

[0054] 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 added dropwise to 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. Figure 10 As shown, the hydrogel exhibits a hierarchically interconnected porous structure with a high specific surface area.

[0055] 7. LPS working curve production

[0056] Different concentrations of bacterial endotoxin LPS 10µL of the blank without bacterial endotoxin and 10µL of 100mmol / L H2O2 were continuously dripped into the nanozyme hydrogel plate and incubated at 30℃ for 15min. Pictures were taken with a smartphone under natural light and fluorescence, and 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 = 0.299R+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 Figure 11 a, 11b, and the obtained regression equation, correlation coefficient, relative standard deviation, and linear range are shown in Table 2.

[0057] Table 2 Linear equation, correlation coefficient, relative standard deviation, linear range

[0058]

[0059] 8. Method specificity investigation

[0060] 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, with an LPS concentration of 10 EU / mL and the concentration of other interfering substances being 100 times the LPS concentration;

[0061] result Figure 12 It shows that only LPS has a significant inhibitory effect on CS@Fe,Cu / CDs-MnO2+H2O2+DAB ( Figure 12 a). Meanwhile, only LPS has a restorative effect on the fluorescence quenching of CS@Fe,Cu / CDs-MnO2 by oxDAB, while other substances have almost no effect ( Figure 12 b) The method has good selection specificity.

[0062] 9. Determination of LPS in various injection samples

[0063] 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 measured samples included NaCl injection, 5% glucose injection, Xuesetong injection, and ampicillin sodium injection.

[0064] LPS standard solutions at concentrations of 0.2, 20, and 100 EU / L were added to various injection samples, and spike recovery tests were conducted on the synthetic samples. The results are shown in Table 3. The results show that the spike recovery of LPS measured in the synthetic samples ranged from 85.3% to 103.2%, with an RSD of less than 5% (n=6). These results indicate that the method established in the present invention can be used for the detection of LPS in injections.

[0065] Table 3 Sample spike recovery and RSD of the method of the present invention (n = 6)

[0066] .

[0067] Example 2: Determination of LPS in a disposable infusion set

[0068] 1. Dissolve 0.5 g chitosan, 0.5 g citric acid, 0.35 g CuCl2·2H2O, 0.4 g FeCl3·6H2O, and 0.35 g o-phenylenediamine in 50 mL of 0.1% (v / v) acetic acid solution and ultrasonicate for 30 min. Transfer the mixture to a polytetrafluoroethylene autoclave and place it in a microwave digester heated to 180°C at 50 W for 2 h. Then cool it naturally to room temperature and remove large particles with a 0.22 μm filter membrane. Centrifuge at 8000 r / min for 10 min, collect the supernatant, and vacuum dry to obtain iron- and copper-doped carbon dot nanomaterials CS@Fe,Cu / CDs.

[0069] 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;

[0070] 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, 150 μL of the mixture was quickly added dropwise to a 96-well microplate and cooled to room temperature to prepare a nanozyme hydrogel microplate.

[0071] 4. Preparation of LPS working curve: Different concentrations of bacterial endotoxin LPS 10µL of the 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. Figure 13 a; Regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 4;

[0072] 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 horizontal axis and fluorescence intensity as the vertical axis to obtain the regression equation. Figure 13 b; Regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 4;

[0073] Table 4 Linear equation, correlation coefficient, relative standard deviation, linear range

[0074] ;

[0075] 5. Determination of LPS in disposable infusion set samples

[0076] (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;

[0077] (2) Determination of LPS: Add 20µL of 50mmol / L H2O2, 10µL of the test solution and the positive control solution to the gel ELISA plate, mix well, let it stand for 15 minutes, and measure the absorbance at a wavelength of 458nm. At the same time, measure the fluorescence emission at 475nm under an excitation wavelength of 410nm. Substitute them into the regression equation respectively. 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 using a nanozyme gel colorimetric-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 of 0.05-0.1% acetic acid solution. 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) Potassium permanganate was dissolved in the CS@Fe,Cu / CDs solution, stirred at room temperature, centrifuged at high speed, and the supernatant was collected and vacuum dried 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 add the mixture dropwise to the cell culture well plate. Cool to room temperature to prepare the nanozyme hydrogel microplate. (4) Different concentrations of bacterial endotoxins and H2O2 were dripped into the nanozyme hydrogel microplate. After incubation at 30°C for 10-15 min, pictures were taken under natural light or fluorescence conditions. The RGB values ​​of the images were read using image processing software and converted into grayscale values ​​to determine the linear relationship between the grayscale value and the bacterial endotoxin concentration. (5) Drop the sample to be tested and H2O2 into the nanozyme hydrogel microplate, incubate at 30°C for 10-15 minutes, 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 nanozyme 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 carried out at 8000-10000 r / min for 5-10 minutes.

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: The invention comprises the nanoenzyme hydrogel microporous plate and H2O2 used in the method according to claim 1.

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