Au (at) Pt nano-enzyme probe as well as application and kit thereof

Abstract

The invention discloses an Au (at) Pt nano-enzyme probe as well as application and a kit thereof. The Au (at) Pt nano enzyme probe is composed of Au (at) Pt nano enzyme and a CD20 antibody; the CD20 antibody is modified on the surface of the Au (at) Pt nano-enzyme. According to the preparation method disclosed by the invention, the Au (at) Pt nano-enzyme with a core-shell structure and a hydrodynamic size stabilized at 25nm is synthesized through specific steps, and the Au (at) Pt nano-enzyme probe modified by the CD20 monoclonal antibody (Rituximab) is prepared, so that the traditional two-step immunohistochemical detection is replaced. A proper DAB buffer solution and a proper reaction solution are prepared, so that clear and accurate chromogenic reaction is initiated, and a solid guarantee is provided for the reliability of an immunohistochemical staining result. Compared with the prior art, the immunohistochemical process is simplified, the cost is reduced, the detection efficiency and accuracy are improved, and an efficient and reliable scheme is provided for clinical diagnosis and scientific research.

Description

Technical Field

[0001] The present invention relates to an Au@Pt nanozyme probe, its application and a kit, belonging to the field of nanozyme probes. Background Art

[0002] Malignant lymphoma is a common malignant tumor globally, and its incidence shows an upward trend. Among them, non-Hodgkin lymphoma (NHL) accounts for a high proportion, and B-cell lymphoma accounts for a relatively large proportion in NHL. CD20 is a transmembrane protein on the surface of B lymphocytes and plays a key role in processes such as B-cell development. Due to its wide expression, it has become an important marker for the diagnosis and treatment of diseases such as NHL.

[0003] Immunohistochemistry (IHC), as a key laboratory technique for detecting and localizing specific antigens in tissue samples, plays an irreplaceable role in the field of medical diagnosis, especially in tumor pathology. It realizes the visualization and localization of target proteins by acting antibodies labeled with enzymes or fluorescent substances on tissue sections. The conventional process of this technique includes steps such as sample fixation and sectioning, antigen retrieval, blocking non-specific binding sites, incubating with specific primary antibodies, incubating with labeled secondary antibodies, and color development or fluorescence imaging. Although IHC has become an indispensable tool in the medical and scientific research fields due to its high specificity and sensitivity, its detection effect depends to a great extent on high-quality antibodies and appropriate antigen retrieval methods. Usually, traditional immunohistochemistry adopts a double-antibody strategy, which requires the use of two antibodies, with a cumbersome operation process and high costs.

[0004] In recent years, nanomaterials have attracted much attention due to their excellent properties in many aspects. Many nanomaterials exhibit catalytic properties similar to natural enzymes. Compared with natural enzymes based on proteins or RNA, nanozymes have better resistance to the environment, especially suitable for reactions under relatively harsh conditions such as high temperature. As a dual-substrate nanozyme, peroxidase-like nanozyme can catalyze the production of a large amount of oxidative free radicals from hydrogen acceptors in the presence of the first substrate hydrogen acceptor, and then rapidly oxidize the second reaction substrate hydrogen donor. Among them, there are various types of hydrogen acceptor substrates, and various enzyme activities such as peroxidase, glutathione peroxidase, and lipid peroxidase can be simulated according to the differences in hydrogen donors. Moreover, the generated free radicals can oxidize various hydrogen donor substrates including small molecule metabolites and biological macromolecules.

[0005] In the pathological diagnosis of malignant lymphoma cells, there is an urgent need for an efficient and accurate detection method. Although the existing technologies are constantly evolving, there are still many problems to be solved, such as the complex operation and high cost of traditional immunohistochemistry methods, and the insufficient substrate selectivity of nanozymes. Therefore, developing a new technology or method that can simplify the immunohistochemistry operation process, improve the detection efficiency and accuracy, and overcome the defects of nanozymes themselves has extremely important practical significance for the diagnosis and treatment of malignant lymphoma. Summary of the Invention

[0006] Object of the Invention: The technical problem to be solved by the present invention is to provide an Au@Pt nanozyme probe and its application and kit.

[0007] Technical Solution: To solve the above technical problem, the present invention provides an Au@Pt nanozyme probe, which is composed of an Au@Pt nanozyme and a CD20 antibody; the CD20 antibody is modified on the surface of the Au@Pt nanozyme. This Au@Pt nanozyme probe can recognize and bind to the CD20 antigen in the sample in a complex sample environment, so as to accurately guide the Au@Pt nanoparticles to stain the target area during the subsequent staining process, improving the targeting and accuracy of staining.

[0008] Among them, the particle size of the gold nanoparticle core in the Au@Pt nanozyme is 5-15 nm, and the thickness of the platinum nanoparticle layer wrapped around the periphery is 5-10 nm, forming a tight core-shell nanostructure. Moreover, this Au@Pt nanozyme, as a catalyst in the immunohistochemical DAB staining process, can accelerate the color development reaction rate of the DAB substrate.

[0009] Among them, the hydrodynamic size of the Au@Pt nanozyme probe is 18 nm - 35 nm, stably dispersed in the buffer solution, and the storage method is to store at 4 °C to maintain the activity and stability of the antibody and extend the shelf life of the kit.

[0010] The present invention also provides the application of the Au@Pt nanozyme probe in the preparation of an immunohistochemical DAB staining reagent or kit. The present invention simplifies the process by introducing nanozymes, improves selectivity by modifying nanozymes, optimizes staining using their activity, and constructs a stable system to improve the diagnostic efficiency and accuracy.

[0011] The present invention also provides an immunohistochemical DAB staining reagent or kit containing the Au@Pt nanozyme probe.

[0012] Among them, it also contains a DAB substrate solution; the DAB substrate solution contains 3,3-diaminobenzidine and hydrochloric acid.

[0013] Among them, it also contains a DAB buffer solution; the DAB buffer solution contains tris(hydroxymethyl)aminomethane and hydrochloric acid.

[0014] Among them, the pH value range of the DAB buffer solution is precisely controlled between 3.0 and 4.0, and the ionic strength is maintained between 0.08 mol / L and 0.15 mol / L. The formula has been verified by optimization experiments and can provide a stable and suitable reaction environment such as acidity, alkalinity, and ionic strength for the immunohistochemical DAB staining reaction targeting the CD20 antigen, ensuring the smooth progress of the staining process, making the staining result clear and accurate within 3 - 10 minutes and not easily fading.

[0015] Among them, the concentration of the Au@Pt nanozyme probe is 0.05 - 0.2 mg / mL, which ensures sufficient catalytic activity in the staining reaction and at the same time avoids adverse effects on the staining effect caused by too high or too low concentration.

[0016] The present invention also provides the application of the Au@Pt nanozyme probe or the immunohistochemical DAB staining reagent in the preparation of a kit for detecting B lymphocytes.

[0017] The present invention also provides a kit for detecting B lymphocytes, which contains the Au@Pt nanozyme probe or the immunohistochemical DAB staining reagent.

[0018] The present invention also provides a method for synthesizing Au@Pt nanozyme particles, comprising the following steps:

[0019] 1. Preparation of gold core:

[0020] (1) Mix ultrapure water and chloroauric acid aqueous solution, and mix them thoroughly with a stirrer to obtain reaction solution A. Among them, the accurate concentration and dosage of chloroauric acid are crucial for the formation and size control of the gold core.

[0021] (2) Sequentially mix ultrapure water, trisodium citrate aqueous solution, potassium carbonate aqueous solution, and tannic acid aqueous solution, and mix them evenly with a stirrer to obtain reaction solution B. Among them, trisodium citrate, potassium carbonate, and tannic acid act as reducing agents and stabilizers in the reaction, and their concentrations and ratios are adjusted to ensure the stable formation of the gold core.

[0022] (3) Put reaction solution A and reaction solution B into a water bath and heat to 60 °C and maintain this temperature. Turn on the mechanical stirring device and quickly pour reaction solution B into reaction solution A at a stable speed to occur a reaction to form the gold core. After reacting for a period of time, let the solution cool naturally at room temperature and then make up the volume with ultrapure water.

[0023] 2. Modification of ultrasmall platinum nanoparticles

[0024] (1) In the three-necked flask with the gold core added, install a double condenser series device, heat the solution to the boiling state through an electric heating mantle, and maintain it for 10 minutes to ensure the uniformity and stability of the reaction system.

[0025] (2) Add aqueous silver nitrate solution drop by drop to the reaction system, with an appropriate time interval between each drop to ensure sufficient reaction. Then, quickly add aqueous sodium citrate solution and aqueous chloroplatinic acid solution, and continue the reaction. During this process, silver nitrate plays a role in seeding induction, and sodium citrate acts as a reducing agent to promote the reduction of chloroplatinic acid to platinum nanoparticles, which are then modified on the surface of the gold core. After the reaction is completed, let the solution cool at room temperature and then make up the volume to the original with ultrapure water again.

[0026] The present invention also provides a preparation method of the Au@Pt nanozyme probe, which includes the following steps:

[0027] 1. Determine the coupling ratio

[0028] (1) Centrifuge the Au@Pt NPs solution, carefully remove the supernatant, and retain the precipitate.

[0029] (2) Resuspend the precipitate with dilute potassium carbonate aqueous solution with pH = 9.5 and then concentrate it. The pH value of the potassium carbonate aqueous solution is crucial for the stability of the subsequent antibody binding reaction.

[0030] (3) Continuously dilute the CD20 antibody with dilute potassium carbonate aqueous solution by a factor of 2 for 10 times to obtain a series of CD20 antibody solutions with different concentrations.

[0031] (4) Take each diluted CD20 antibody solution, add it to the Au@Pt NPs solution, mix well by shaking, and then let it stand at room temperature to allow the antibody to fully contact with the Au@Pt NPs.

[0032] (5) Then add aqueous sodium chloride solution, let it stand at room temperature, and observe the color change of the solution. Take the group before the lowest concentration group whose color has not changed, and increase the antibody concentration of this group by 25% as the final antibody concentration for preparing the probe.

[0033] 2. Prepare the Au@Pt NPs probe

[0034] (1) Adjust the pH value of the Au@Pt NPs to 9.5.

[0035] (2) Accurately add the CD20 antibody according to the ratio determined in the previous step, mix well by shaking, and then let it stand at room temperature to allow the antibody to fully bind to the Au@Pt NPs.

[0036] (3) Add aqueous bovine serum albumin solution for blocking treatment to reduce non-specific binding; then, centrifuge the solution to remove the supernatant, and resuspend the precipitate with pure water.

[0037] Among them, the preparation components of the DAB substrate (solution A) and the buffer (solution B):

[0038] (1) DAB substrate (solution A)

[0039] In the composition system of the DAB substrate (solution A), 3,3-diaminobenzidine (DAB) is used as the core chromogenic substance, and its concentration is precisely set at 1 mg / mL. This concentration ensures the sensitivity and clarity of the chromogenic effect while avoiding excessive background staining or precipitation caused by too high a concentration, and too low a concentration will result in weak chromogenesis that is difficult to observe. Hydrochloric acid plays a key role in adjusting the pH value in solution A. By adding hydrochloric acid, the pH value of solution A is strictly controlled within the range of 3.0 - 4.0. This pH range is crucial for maintaining the chemical stability of DAB and its subsequent efficient reaction with the enzyme-catalyzed products, ensuring that DAB can smoothly participate in the immunohistochemical chromogenic reaction in a suitable acid-base environment.

[0040] (2) DAB buffer solution (solution B)

[0041] In solution B, hydrochloric acid and tris(hydroxymethyl)aminomethane (Tris) work together to regulate the pH value of solution B within the range of 3.0 - 4.0, providing the optimal active environment for horseradish peroxidase and ensuring its efficient catalysis of subsequent reactions. Hydrogen peroxide, as the key substrate for the horseradish peroxidase-catalyzed reaction, has a stable concentration of 1% in solution B. This concentration can ensure that under the action of the enzyme, it can effectively oxidize DAB in solution A, thereby triggering a clear and accurate chromogenic reaction, providing a solid guarantee for the reliability of immunohistochemical staining results.

[0042] Among them, in the present invention, the buffer solution and reaction reagents are all prepared from sterile and endotoxin-free raw materials and are filtered and sterilized through a filter membrane with a pore size of 0.22 μm or smaller to prevent microbial contamination from interfering with the staining reaction.

[0043] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0044] 1. Through the screening of different nanoenzymes, the Au@Pt nanoparticles of the present invention are determined, and the reactant ratio, reaction temperature, and reaction time are precisely regulated to successfully synthesize Au@Pt nanoparticles with a core-shell structure and uniform particle size. Its hydrodynamic size is stably maintained at 25 nm, and this size characteristic is conducive to playing a catalytic role in the immunohistochemical reaction.

[0045] 2. The traditional immunohistochemical process relies on a double-antibody system, which is complex to operate and costly. The present invention innovatively modifies the CD20 antibody on the surface of the nanoenzyme and applies it to immunohistochemical detection. Only a single antibody is required to complete the detection process, greatly simplifying the immunohistochemical operation steps and significantly reducing the experimental cost and technical difficulty.

[0046] 3. Through systematic optimization of different pH ranges and different buffer systems, the present invention effectively improves the efficiency of the immunohistochemistry process. Experimental results show that under optimized conditions, the immunohistochemistry reaction time is significantly shortened, and at the same time, the staining effect is clearer and more stable, saving valuable time for clinical diagnosis and scientific research work and improving the reliability of test results.

[0047] 4. The present invention innovatively introduces nanozymes as carriers and chromogenic catalysts, and only one antibody needs to be surface-modified to complete the detection. Nanozymes not only significantly improve the catalytic performance but also simplify the operation process of immunohistochemistry and reduce costs. Description of the Drawings

[0048] Figure 1 It is a schematic diagram of the antibody conjugation of the Au@Pt NPs probe;

[0049] Figure 2 It is a transmission electron microscope image of Au and Au@Pt nanoparticles; Figure 2 In a, the average size of the Au NPs particles is about 13 nm, Figure 2 In b, the average size of the Au@Pt NPs particles is about 24 nm;

[0050] Figure 3 It is a transmission electron microscope EDS Mapping image of Au@Pt NPs;

[0051] Figure 4 It is a transmission electron microscope EDS Mapping image of the Au@Pt NPs probe;

[0052] Figure 5 It is the Anano value of Au NPs, Pt NPs, Au@Pt NPs particles and the Au@Pt nanozyme probe;

[0053] Figure 6 It is the curve of the absorbance change with time of Au NPs, Pt NPs, Au@Pt NPs particles and the Au@Pt nanozyme probe at different concentrations;

[0054] Figure 7 It is the hydrodynamic size distribution and ζ potential of Au NPs, Au@Pt NPs and the Au@Pt probe;

[0055] Figure 8 It is the staining effect of the kit;

[0056] Figure 9 It is the verification of accuracy and repeatability;

[0057] Figure 10Microscopic imaging photos of Rituximab-Au@Pt probe-labeled malignant lymphoma Raji cells: (a) experimental group, (b) competitive inhibition group, (c) unrelated cell group, (d) unrelated probe group, (e) bare gold group, (f) blank control group. Detailed implementation manners

[0058] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0059] Example 1: Synthesis and characterization of Au@Pt nanozyme particles

[0060] Add 79 ml of ultrapure water and 1 ml of chloroauric acid aqueous solution with a concentration of 10 mg / ml into a three-necked flask, and stir evenly to obtain reaction solution A. Add 16 ml of ultrapure water, 4 ml of trisodium citrate aqueous solution with a concentration of 0.01 g / ml, 0.2 ml of potassium carbonate aqueous solution with a concentration of 0.0138 g / ml, and 0.7 ml of tannic acid aqueous solution with a concentration of 0.01 g / ml into a conical flask in sequence, and mix evenly with a stirrer to obtain reaction solution B. Heat reaction solutions A and B in a water bath to 60 °C and maintain this temperature. Quickly pour reaction solution B into reaction solution A under mechanical stirring. After the reaction is completed, cool to room temperature and make up the volume to 100 ml with ultrapure water to obtain a gold core solution, and store it at 4 °C. The hydrodynamic size distribution and potential of Au NPs and Au@Pt NPs are as Figure 7 shown. The hydrodynamic size of the gold core solution is about 14 nm, and the potential is -28 mV. The hydrodynamic size of Au@Pt particles is 24 nm, and the potential is -46 mV.

[0061] Take the above-prepared gold core solution and place it in a three-necked flask. Connect two condensers in series and heat with an electric heating mantle to boiling and maintain for 10 min to ensure the uniformity and stability of the reaction system. Dilute 0.1 ml of silver nitrate aqueous solution with a concentration of 10 mg / ml to 1 ml with pure water, and then add it dropwise to the reaction system at appropriate intervals for each drop to ensure sufficient reaction. Then add 0.5 ml of trisodium citrate aqueous solution with a concentration of 114.1 mg / ml and 0.25 ml of chloroplatinic acid aqueous solution with a concentration of 10 mg / ml, and continue the reaction for 2 h. During this process, silver nitrate plays a role of seed induction, and trisodium citrate acts as a reducing agent to promote the reduction of chloroplatinic acid to platinum nanoparticles and modify them on the surface of the gold core. After cooling, make up the volume with ultrapure water and store at 4 °C.

[0062] Use high-resolution transmission electron microscopy (TEM) to observe the morphologies of Au@Pt nanoparticles and Au nanoparticles (gold cores), as Figure 2 shown. The Au@Pt nanoparticles exhibit a clear core-shell structure. The size of the gold core is within the expected range of 5 nm to 15 nm, and the thickness of the peripheral platinum nanoparticle layer is within the range of 5 nm to 10 nm. Determine its elemental composition and distribution by energy dispersive spectroscopy (EDS) ( Figure 3) It was confirmed that the existence and proportion of the gold core and platinum shell met the expectations.

[0063] Example 2: Preparation of Au@Pt nanozyme probe

[0064] As Figure 1 shown, the Au@Pt NPs solution with a concentration of 0.05 mg / ml was centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was discarded, and the precipitate was retained. The precipitate was resuspended with a 0.1 M dilute K2CO3 aqueous solution at pH = 9.5 and concentrated to 0.10 mg / ml.

[0065] Rituximab (CD20 antibody, Shanghai Jerryxing Biomedical Technology Co., Ltd., JRX50149) at 10 mg / ml was serially diluted 10 times with a 0.1 M dilute K2CO3 aqueous solution at pH = 9.5 to obtain a series of CD20 antibody solutions with different concentrations. One aliquot of each diluted CD20 antibody solution was added to 1 ml of Au@Pt NPs at a concentration of 0.1 mg / ml, shaken well, and allowed to stand at room temperature for 5 min. Then, a 10 mg / ml NaCl aqueous solution at pH = 9.5 was added, and the mixture was allowed to stand at room temperature for 1 min, and the color change was observed. The group before the lowest concentration group with no color change was taken, and the antibody concentration of this group was increased by 25% as the final antibody concentration (25 μg / ml) for preparing the probe.

[0066] The pH of the Au@Pt NPs solution (0.1 mg / ml, 1 ml) was adjusted to 9.5 with a 0.1 M K2CO3 aqueous solution, 2.5 μl of rituximab (10 mg / ml) was added, shaken well, and allowed to stand at room temperature for 5 min. Then, a 10 mg / ml BSA aqueous solution was added to block for 5 minutes to reduce non-specific binding. Centrifugation was carried out at 12,000 rpm for 30 min at 4 °C, and the precipitate was resuspended with pure water and stored at 4 °C. EDS Mapping images of the Au@Pt NPs probe under transmission electron microscope Figure 4 ) It was clearly seen that the distribution of element S was consistent with that of the nanoparticles, indicating that the antibody was successfully attached to the surface of the nanoparticles. The hydrodynamic size of the Au@Pt nanoprobe was 45 nm, and the potential was -8 mV Figure 7 ).

[0067] Example 3: Study on the catalysis of hydrogen peroxide oxidation of TMB by nanocarriers

[0068] Prepare a 3,3’,5,5’-tetramethylbenzidine (TMB) solution with a concentration of 10 mg / mL using DMSO, and prepare a 0.1 M HAc-NaAc buffer solution with pure water, adjusting the pH to 3.6. At room temperature, add 200 μL of HAc-NaAc buffer solution with pH 3.6, 10 μL of aqueous solutions of nanoparticles or nanozyme probes with concentrations of 16 mg / mL, 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL, and 10 μL of 10 mg / mL TMB solution dissolved in DMSO into a 96-well plate, and pipette evenly to avoid bubbles as much as possible. Add 10 μL of 30% H2O2 solution to the above mixed solution and quickly place it in an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance change at 650 nm within 200 s of the reaction system.

[0069] The results show that ( Figure 5 and Figure 6 ), Au NPs, Pt NPs, and Au@Pt NPs particles all show peroxidase-like activity, but the peroxidase-like activity of Au@Pt NPs is significantly improved, which are 4.9 times and 3.6 times the peroxidase activities of single-element Au and Pt nanozymes, respectively. Among them, Figure 5 The four pictures in Figure 6 respectively represent the Anano values of four materials (Au NPs, Pt NPs, Au@Pt NPs particles, and Au@Pt nanozyme probes), which are used to measure the pod enzyme activity. The larger the value, the higher the activity. Figure 5 The four pictures in

[0070] Example 4: Immunohistochemical staining and effect evaluation

[0071] Prepare the DAB substrate solution: an aqueous solution containing 3,3-diaminobenzidine (DAB) and hydrochloric acid. Among them, DAB is used as the core chromogenic substance, and its final concentration is accurately set to 1 mg / mL. This concentration can ensure the sensitivity and clarity of the chromogenic effect while avoiding excessive background staining or precipitation due to too high a concentration, and too low a concentration will result in weak chromogenicity and difficulty in observation. Hydrochloric acid plays a key role in adjusting the pH value in the DAB substrate solution. By adding hydrochloric acid, the pH value of the DAB substrate solution is strictly controlled within the range of 3.0 - 4.0. This pH range is crucial for maintaining the chemical stability of DAB and its subsequent efficient reaction with the enzyme-catalyzed products, ensuring that DAB can smoothly participate in the immunohistochemical chromogenic reaction in a suitable acid-base environment.

[0072] Prepare DAB buffer: An aqueous solution containing hydrochloric acid, tris(hydroxymethyl)aminomethane (Tris), and hydrogen peroxide. Among them, the final concentration of Tris is 200 mmol / L. Hydrochloric acid and Tris act synergistically to adjust the pH value of the DAB buffer to 3.0 - 4.0, providing an optimal active environment for horseradish peroxidase and ensuring its efficient catalysis of subsequent reactions. Hydrogen peroxide, as the key substrate for the catalytic reaction of horseradish peroxidase, is stably maintained at a concentration of 1% in the DAB buffer. This concentration can ensure that under the action of the enzyme, DAB in the DAB substrate solution can be effectively oxidized, thereby triggering a clear and accurate color reaction, providing a solid guarantee for the reliability of immunohistochemical staining results.

[0073] Take 100 μl of rituximab-Au@Pt probe solutions with different concentrations (0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml), drop them into round droplets on Raji cell smears, place them in a wet box, and incubate in an incubator at 37 °C for 1 hour. After taking them out, wash them 3 times with PBS-T (0.01 M, containing 1% TW-20 solution, pH = 7.4) solution, 5 minutes each time. Mix the DAB substrate solution and the DAB buffer in equal volumes, and drop them onto the incubated cell smears for color development. As Figure 8 The staining effect of the cell smears shows that compared with the traditional immunohistochemical staining method, the staining results are observed under a microscope, and the tumor cells are clearly stained with high contrast. Among them, Figure 8 The probe concentration in the upper left figure in the middle is 0.05 mg / ml, the probe concentration in the upper right figure is 0.1 mg / ml, the probe concentration in the lower left figure is 0.15 mg / ml, and the probe concentration in the lower right figure is 0.2 mg / ml. As Figure 9 shown, through the detection of multiple groups of samples, the detection results of the method of the present invention have good accuracy and repeatability, and reliable results can be stably obtained under different laboratory environments, demonstrating the high efficiency and stability of the immunohistochemical staining method of the present invention.

[0074] Example 5 Study on Specific Adsorption of Probe and Target Antigen

[0075] Experimental group (Rituximab-Au@Pt probe binds to Raji cells to evaluate the specific binding ability of the probe to target cells): Take 100 μl of 0.1 mg / ml rituximab-Au@Pt probe solution, drop it into a round droplet on a Raji cell smear, place it in a wet box, and incubate in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0076] Competitive inhibition group (Rituximab-Au@Pt probe binds to Raji cells with the cell binding sites blocked by 100-fold Rituximab to verify the binding specificity): Drop the rituximab solution (10 mg / ml, 100 μl) onto the Raji cell smear, place it in a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it once with PBS-T solution. Repeat the above steps once. Then take 100 μl of 0.1 mg / ml rituximab-Au@Pt probe solution, drop it into a round droplet on the Raji cell smear, put it into a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0077] Unrelated cell group (Rituximab-Au@Pt probe binds to K562 cells to exclude non-specific binding of the probe to non-target cells): Prepare a K562 cell smear in the same way as preparing the Raji cell smear. Take 100 μl of 0.1 mg / ml rituximab-Au@Pt probe solution, drop it into a round droplet on the K562 cell smear, put it into a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0078] Unrelated probe group (human IgG-Au@Pt probe binds to Raji cells to exclude the influence of non-specific probes): Prepare the human IgG-Au@Pt probe in the same way as coupling the rituximab-Au@Pt probe. Take 100 μl of 0.1 mg / ml human IgG-Au@Pt probe solution, drop it into a round droplet on the Raji cell smear, put it into a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0079] Naked gold group (naked Au@Pt binds to Raji cells to evaluate the non-specific adsorption of the nanoparticles themselves): Take Au@Pt nanoparticles (0.1 mg / ml, 100 μl), drop it into a round droplet on the Raji cell smear, put it into a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0080] Blank control group: Take PBS (0.01 M, pH = 7.4, 100 μl), drop it into a round droplet on the Raji cell smear, put it into a wet box, and incubate it in an incubator at 37 °C for 1 hour. After taking it out, wash it 3 times with PBS-T solution, 5 minutes each time. Observe with a bright-field microscope.

[0081] As Figure 10As shown in Fig. a, in the experimental group, during the co-incubation of Rituximab-Au@Pt probe with Raji cells, the Rituximab monoclonal antibody specifically binds to CD20 on the surface of Raji cells. After removing the excess probe, AuNPs are specifically labeled on the cell surface. Therefore, clear cell outlines can be observed under the microscope, and DAB precipitation shows a brownish-yellow precipitate. Compared with the photos of other groups, due to reasons such as the stability of the microscope and the quality of the glass slides, there is certain system noise in this detection system, manifested as black particles in the background, but there are obvious differences compared with the cell outlines, which does not affect the recognition of tumor cells.

[0082] As Figure 10 shown in Fig. b, the CD20 on the surface of Raji cells in the competitive inhibition group has been blocked by an excessive amount of Rituximab antibody, so it can no longer specifically bind to the Rituximab-Au probe. No brownish-yellow precipitate can be observed at the cell outline in the field of view of this group of experiments, which is similar to the situation of the blank control group ( Figure 10 Fig. f). Therefore, it can be considered that Raji cells are not labeled by nanoparticles, which is consistent with the expectation. The competitive inhibition group can be used to detect the degree of non-specific adsorption. From the results of this group of experiments, it can be seen that there is no irregular scattered brownish-yellow precipitate in the background, which is consistent with the situation of the experimental group.

[0083] As [[ID=ll]] Figure 10 shown in Fig. c, the irrelevant cell group uses K562 cells for the experiment. K562 cells do not express the target CD20 recognized by the Rituximab monoclonal antibody. Therefore, when the Rituximab-Au@Pt probe is co-incubated with K562 cells, the cells cannot bind to the probe, and there is almost no residual Rituximab-Au@Pt probe on the cell surface after washing. It can be seen under the field of view that the K562 cells are in good condition and the appearance is complete, which is similar to the situation of the blank control group ( Figure 10 Fig. f). It can be inferred from this that K562 cells cannot be recognized and labeled by the Rituximab-Au@Pt probe, which is consistent with the expectation. i

[0084] As Figure 10 shown in Fig. d, the irrelevant probe group uses human IgG to replace the Rituximab monoclonal antibody to conjugate with Au@Pt NPs to prepare the probe. Since human IgG cannot specifically recognize the CD20 molecule on the surface of Raji cells, when the human IgG-Au@Pt probe is co-incubated with Raji cells, no binding occurs, and the cells cannot be successfully labeled after washing. Observing the cell smear of this group under the field of view, it is similar to the situation of the blank control group ( Figure 10 Fig. f), which meets the expectation. This further proves that this detection method is specific for malignant lymphoma cells overexpressing CD20.

[0085] As Figure 10As shown in e, Raji cells in the bare gold group were treated with Au@Pt NPs without protein modification at the same concentration. Since there was no antibody conjugated to the Au@Pt NPs, there was no specificity for the cells. At the same time, due to the lack of protection by antibodies and stabilizers, the Au@Pt NPs were extremely prone to aggregation when treated with buffer solution and irregularly deposited on the cell surface. When observing the cell smear of the bare nanoparticle group under the microscope, due to the unevenness of the cells, they were more likely to aggregate at the cell edges. However, since the Au@Pt NPs had no specific recognition and binding ability to the cell surface, different from the experimental group where the cell edges were uniformly stained, in the photos of the bare nanoparticle group, a large amount of precipitate aggregated at the edges of some cells, while there was almost no precipitate on the surface of some cells.

[0086] As Figure 10 As shown in f, Raji cells in the blank control group were incubated with the same volume of PBS (0.01 M, pH = 7.4) for the same length of time, and after washing, they were untreated Raji cell smears. When observed under the microscope, there was no difference in the cell morphology between the blank control group and other groups.

Claims

1. An Au@Pt nanozyme probe, characterized in that, The Au@Pt nanozyme probe is composed of an Au@Pt nanozyme and a CD20 antibody; the CD20 antibody is modified on the surface of the Au@Pt nanozyme.

2. The Au@Pt nanozyme probe according to claim 1, wherein In the Au@Pt nanozyme, the particle size of the gold nanoparticle core is 5-15 nm.

3. The Au@Pt nanozyme probe according to claim 1, wherein In the Au@Pt nanozyme, the thickness of the platinum nanoparticle layer wrapped around the periphery is 5-10 nm.

4. Use of the Au@Pt nanozyme probe according to claim 1 in the preparation of an immunohistochemical DAB staining reagent or kit.

5. An immunohistochemical DAB staining reagent or kit, characterized in that, It contains the Au@Pt nanozyme probe according to any one of claims 1 to 3.

6. The immunohistochemical DAB staining reagent or kit according to claim 5, characterized in that, It also contains a DAB substrate solution; the DAB substrate solution contains 3,3-diaminobenzidine and hydrochloric acid.

7. The immunohistochemical DAB staining reagent or kit according to claim 5, wherein It also contains a DAB buffer solution; the DAB buffer solution contains tris(hydroxymethyl)aminomethane and hydrochloric acid.

8. The immunohistochemical DAB staining reagent or kit according to claim 5, characterized in that, The concentration of the Au@Pt nanozyme probe is 0.05-0.2 mg / mL.

9. Use of the Au@Pt nanozyme probe according to claim 1 or the immunohistochemical DAB staining reagent according to any one of claims 5 to 8 in the preparation of a kit for detecting B lymphocytes.

10. A kit for detecting B lymphocytes, characterized in that, It contains the Au@Pt nanozyme probe according to claim 1 or the immunohistochemical DAB staining reagent according to any one of claims 5 to 8.

Images