A method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes

Abstract

The present invention discloses a preparation method of a dual-target tumor cell detection reagent based on cerium-based nanozymes, which relates to the field of biochemistry technology, comprising: step one, preparing cerium folate-based nanozymes: Ce(NO3)3·6H2O and FA aqueous solution are mixed and stirred, bovine serum albumin is added, and the mixture is reacted under alkaline conditions in a water bath to obtain cerium folate-based nanozymes Ce@BSA-FA; step two, preparing Ki67 cerium folate-based nanozymes coupled with AP-labeled antibodies: EDC·HCl, NHS and Ce@BSA-FA are dissolved in deionized water, activated, and Ki67 antibodies and alkaline phosphatase AP are added to carry out cross-linking reaction; step three, preparing p16 INK4a Cerium folate-based nanozyme coupled with HRP-labeled antibody: EDC·HCl, NHS and Ce@BSA‑FA were dissolved in deionized water, activated and then added with p16 INK4a The antibody and horseradish peroxidase HRP undergo a cross-linking reaction. The dual-target tumor cell detection reagent prepared by the method of the present invention reduces the amount of antibody used, simplifies the operation process, and has better color development efficiency and detection sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes. Background Art

[0002] Immunohistochemistry (IHC) is a technique with strong specificity, high sensitivity, and accurate localization. It can also organically combine morphological and functional studies, making it widely used in many fields of biological and medical research. In the study of tumor pathology diagnosis, the role and significance of IHC is even more critical.

[0003] In order to more accurately determine the co-expression of antigen proteins in tumor cells, many hospitals have introduced immunohistochemical double-staining technology, which can mark two different antigens with red and brown respectively on the same pathological tissue slide. Depending on the expression and characteristics of the antigens, combined testing can be performed as needed.

[0004] Because a pathological tissue slide typically contains multiple different cell types, tumor cells may only account for a small fraction of these cells. Therefore, when the proportion of tumor cells is low, determining whether two protein markers stained on two slides are expressed in the same cell population depends primarily on experience and speculation rather than direct observation. This can lead to difficulties in inference and even misjudgment due to lack of experience.

[0005] At present, the commonly used double-staining kit adopts the primary antibody-secondary antibody immunodetection method, which has a complex process flow and a long operation time. In addition, the current technology has problems such as inaccurate tumor cell positioning, weak signal intensity at low concentrations of double-staining antibodies, and more nonspecific interference at high concentrations. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of inaccurate tumor cell positioning in the prior art, weak signal intensity at low concentrations of double-stained antibodies, and high non-specific interference at high concentrations, and to provide a method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes, wherein the dual-targeted tumor cell detection reagent comprises ki67 folate cerium-based nanozymes coupled with AP-labeled antibodies, p16 INK4aThe preparation method of the dual-target tumor cell detection reagent of cerium folate-based nanozyme coupled with HRP-labeled antibody includes the following steps: Step 1, preparing cerium folate-based nanozyme: mixing Ce(NO3)3·6H2O and FA aqueous solution to obtain a stable complex, wherein FA refers to folic acid, adding bovine serum albumin (BSA) to the obtained stable complex, and placing it in a water bath to react under alkaline conditions, dialyzing the obtained reaction product, ultrafiltration centrifugation and vacuum freeze drying to obtain a black solid powder cerium folate-based nanozyme, namely Ce@BSA-F A; Step 2, prepare Ki67 folate cerium-based nanozyme coupled with AP-labeled antibody: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, i.e., EDC·HCl, N-hydroxysuccinimide, i.e., NHS, and Ce@BSA-FA were dissolved in deionized water and stirred for activation at room temperature. After activation, Ki67 antibody and alkaline phosphatase AP were added for cross-linking reaction, and the uncross-linked Ki67 antibody and AP were removed by centrifugation and dialysis, and the solution was diluted to the working solution concentration using the preservation solution; Step 3, prepare p16 INK4a Conjugating cerium folate-based nanozyme with HRP-labeled antibody: EDC·HCl, NHS and Ce@BSA-FA were dissolved in deionized water and stirred at room temperature for activation. After activation, p16 was added. INK4a The antibody was cross-linked with horseradish peroxidase (HRP), and the uncross-linked p16 was removed by centrifugation and dialysis. INK4a Antibodies and HRP were diluted to working concentration using storage buffer.

[0007] Optionally, step one includes: mixing and stirring 0.1 mol / L Ce(NO3)3·6H2O and 0.1 mol / L FA aqueous solution to form a stable complex; adding bovine serum albumin (BSA) at a concentration of 25 mg / mL and mixing in a constant temperature water bath at 37°C; using 1 mol / L KOH solution, adjusting the pH value of the reaction system to 13, and the color of the solution changes from milky white to black, and continuing to stir at a constant temperature until the reaction is completed; transferring the obtained reaction product solution to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialysis with deionized water for purification; the dialyzed suspension is centrifuged and concentrated at 4000 rpm in a 10 kDa ultrafiltration centrifuge tube, and the collected precipitate is repeatedly washed three times with deionized water to remove residual reagents; and vacuum freeze-drying is performed to obtain a black solid powder Ce@BSA-FA.

[0008] Optionally, step 2 includes: dissolving 57 mg EDC·HCl, 85.5 mg NHS, and 6 mg Ce@BSA-FA in 9 mL deionized water, activating and stirring at room temperature for 8 hours; adding Ki67 antibody and alkaline phosphatase AP, and continuing to stir at room temperature for 24 hours; after the reaction is completed, centrifuging to retain the precipitate, dialyzing for 24 hours to remove the uncross-linked Ki67 antibody and AP, and diluting to the working solution concentration using preservation solution.

[0009] Optionally, the step three comprises: dissolving 57 mg EDC·HCl, 85.5 mg NHS and 6 mg Ce@BSA-FA in 9 mL deionized water, activating and stirring at room temperature for 8 hours; adding p16 INK4a Antibody and horseradish peroxidase HRP were stirred at room temperature for 24 hours. After the reaction, the precipitate was centrifuged and dialyzed for 24 hours to remove the uncrosslinked p16 INK4a Antibodies and HRP were diluted to working concentration using storage buffer.

[0010] The beneficial effects of the present invention are:

[0011] The cerium folate-based nanozyme provided by the present invention can target tumor cells with high expression of folate receptors due to its folic acid ligand, thereby achieving the effect of specifically recognizing tumor cells and reducing nonspecific interference of double-stained antibodies;

[0012] The cerium folate-based nanozyme has multiple binding sites that can couple with AP, HRP, and antibodies to form a coupling complex. The presence of multiple binding sites can not only increase the loading capacity of the enzyme and antibody, but also significantly maintain the stability of the enzyme and antibody.

[0013] The preparation process of the present invention is simple, the product stability is high, the tumor detection operation process is reduced, the operation time is shortened, and the color development efficiency and detection sensitivity are higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the steps of the method of the present invention;

[0015] Figure 2 TEM image of cerium folate-based nanozyme;

[0016] Figure 3 This is the TGA image of cerium folate-based nanozyme;

[0017] Figure 4 This is the UV-visible spectrum of cerium folate-based nanozyme;

[0018] Figure 5 FT-IR spectrum of cerium folate-based nanozyme;

[0019] Figure 6 This is a photo of cerium folate-based nanozymes stored in different media for 7 days;

[0020] Figure 7 Schematic diagram of the structure of Ki 67 cerium folate-based nanozyme coupled with AP-labeled antibody;

[0021] Figure 8 For p16 INK4a Schematic diagram of the structure of cerium folate-based nanozyme coupled with HRP-labeled antibody;

[0022] Figure 9 To evaluate the ·OH scavenging ability of cerium folate-based nanozymes;

[0023] Figure 10 The EPR spectra of ·OH scavenged by cerium folate-based nanozymes after incubation for different times;

[0024] Figure 11 O2 for cerium folate-based nanozymes - Clearance capacity assessment;

[0025] Figure 12 After incubation for different time, the cerium folate-based nanozymes cleared O2 - EPR spectrum of

[0026] Figure 13 To evaluate the hydrogen peroxide scavenging ability of cerium folate-based nanozymes;

[0027] Figure 14 Live-dead staining imaging of L929 cells after 24 hours of treatment with cerium folate-based nanozymes;

[0028] Figure 15 The results of hemolysis experiments of cerium folate-based nanozymes at different concentrations are shown;

[0029] Figure 16 The results are from traditional immunocytochemistry staining experiments;

[0030] Figure 17 These are the results of immunocytochemical staining experiments using the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the embodiments and experimental verification drawings:

[0032] Embodiment 1 of the present invention:

[0033] The present invention provides a method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes, which uses cerium-based nanozymes to carry folic acid to achieve targeted positioning of tumor cell folate (FA) receptors, and has both ki67 and p16 INK4a Detection function, the dual-target tumor cell detection reagent is mainly composed of ki67 folate cerium-based nanozyme coupled with alkaline phosphatase (AP) labeled antibody, p16 INK4a The cerium folate nanozyme is coupled with horseradish peroxidase (HRP) labeled antibody, which mainly involves the preparation of bovine serum albumin-encapsulated cerium folate nanozyme (Ce@BSA-FA) and the use of it as a supporting skeleton to couple antibodies and signal markers to form cerium folate nanozyme-coupled antibodies, referring to Figure 1 , the preparation method comprises:

[0034] Step 1: Preparation of cerium folate-based nanozymes:

[0035] Ce(NO3)3·6H2O and folic acid (FA) aqueous solution were mixed to obtain a stable complex, followed by addition of bovine serum albumin (BSA), and the mixture was reacted in a water bath under alkaline conditions. The obtained reaction product was dialyzed, ultrafiltration centrifuged, and vacuum freeze-dried to obtain a black solid powder cerium folate-based nanozyme Ce@BSA-FA.

[0036] The prepared cerium folate nanozyme, carrying folic acid, can specifically bind to folate receptors on the surface of tumor cells, achieving efficient and precise targeting of tumor cells. This targeting greatly improves the specificity of the test, reduces interference caused by nonspecific binding, and facilitates more accurate identification of tumor cells.

[0037] In this step, cerium nitrate hexahydrate (Ce(NO3)3·6H2O) provides cerium ions (Ce³⁺) as the core component of the nanozyme; folic acid (FA) serves as a targeting ligand, and its γ-carboxyl group can coordinate with Ce³⁺ and specifically bind to the folate receptor (FR) of tumor cells to achieve active targeting; bovine serum albumin (BSA) serves as a stabilizer and coating material to provide biocompatibility.

[0038] Alkaline conditions can be achieved by adjusting the pH value of the reaction system with potassium hydroxide (KOH).

[0039] Step 2: Preparation of Ki67 cerium folate-based nanozyme coupled with AP-labeled antibody:

[0040] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and Ce@BSA-FA were dissolved in deionized water and stirred at room temperature. The carboxyl groups on the surface of the nanozyme were activated by EDC and NHS. After activation, Ki67 antibody and alkaline phosphatase AP were added for cross-linking reaction. The uncross-linked Ki67 antibody and AP were removed by centrifugation and dialysis, and the nanozyme was diluted to a working solution using preservation solution.

[0041] Step 3: Prepare p16 INK4a Cerium folate-based nanozyme coupled with HRP-labeled antibody:

[0042] Step 3 is similar to the preparation method of Ki67 folate cerium-based nanozyme coupled with AP-labeled antibody in step 2, except that the antibody and enzyme are replaced by p16 INK4a Antibodies and horseradish peroxidase (HRP) were prepared by dissolving EDC·HCl, NHS and Ce@BSA-FA in deionized water and stirring them at room temperature for activation. After activation, p16 INK4a The antibody was cross-linked with horseradish peroxidase (HRP), and the uncross-linked p16 was removed by centrifugation and dialysis. INK4a Antibodies and HRP were diluted to working solution using storage solution.

[0043] It should be noted that the preservation solution in the present invention includes: one of PBS, Tris or HEPES buffer, 1% to 2% bovine serum albumin, and 0.01% to 1% Proclin 300. The working solution is usually diluted 10 to 100 times.

[0044] The steps of using the dual-target tumor cell detection reagent based on cerium-based nanozymes of the present invention include: after tissue blocking 1. adding p16-HRP coupled antibody (i.e. p16 INK4a 1. Incubate the mixture of a cerium folate nanozyme-conjugated HRP-labeled antibody and a Ki67-AP-conjugated antibody (i.e., a Ki67 cerium folate nanozyme-conjugated AP-labeled antibody) for 5-10 minutes and wash. 2. Add AP colorimetric solution, incubate for 3-5 minutes, and wash. 3. Add DAB colorimetric solution, incubate for 1-2 minutes, and wash. 4. Counterstain with hematoxylin. The DAB colorimetric solution consists of: Solution A substrate: H₂O₂; Solution B enhancement: NiCl₂; Solution C chromogen: diaminobenzidine. The AP colorimetric solution consists of: Solution A diluent: Tris buffer; Solution B substrate: α-naphthol AS-BI phosphate; Solution C chromogen: Fast Red TR salt.

[0045] The principle of the present invention is:

[0046] Reactive oxygen species (ROS) are highly oxidative molecules generated by cellular metabolism in organisms. Common ROS include superoxide anions (·O2 - ), hydroxyl radicals (·OH), and hydrogen peroxide (H2O2). ROS play a crucial role in the development and progression of many diseases, particularly in the tumor microenvironment, where ROS concentrations are high. Therefore, nanocarriers that can respond to ROS are of great significance. The cerium folate-based nanozyme provided by the present invention has excellent ROS scavenging properties and can passively target ROS-overexpressing tumor cells. The folate receptor (FR) is a glycosylated membrane protein whose primary function is to mediate cellular uptake of folic acid (vitamin B9). Folic acid is a key cofactor in DNA synthesis, repair, and methylation reactions and is crucial for cell proliferation. In normal tissues, FR expression is usually low (such as physiological expression in the kidneys and placenta), but tumor cells, due to their strong metabolic needs, often overexpress FR to obtain more folic acid to support their rapid proliferation. That is, folate receptors are highly expressed on the surface of many tumor cells, while their expression in normal cells is relatively low or even non-existent. Utilizing this characteristic, the folate cerium-based nanozyme prepared by the method of the present invention can target the folate receptors of tumor cells. This targeting mechanism greatly improves the specificity of detection, effectively avoids nonspecific binding with normal cells, reduces interference from background signals, and lays a solid foundation for the accurate identification and detection of tumor cells.

[0047] Furthermore, the cerium folate nanozyme possesses multiple binding sites, allowing it to couple with more enzyme molecules (such as AP and HRP) and antibodies to form coupled complexes. The presence of multiple binding sites not only increases the loading capacity of the enzyme and antibody but also significantly maintains their stability. In complex biological environments, enzymes and antibodies are susceptible to inactivation due to various factors. The multiple binding sites of the cerium folate nanozyme provide stable support and protection for the enzyme and antibody, ensuring their continued activity during the detection process, thereby guaranteeing the accuracy and reliability of the test results.

[0048] The folic acid on the surface of the cerium folate nanozyme undergoes a coupling reaction with the amino group of HRP or AP through its γ-carboxyl group, forming a stable cerium folate nanozyme coupling complex. This not only retains the enzymatic activity of HRP or AP, allowing these enzymes to function normally in subsequent detection reactions, but also inherits the ability of the cerium folate nanozyme to target folate receptors. The coupling complexes, Ce@BSA-FA-HRP or Ce@BSA-FA-AP, have good stability and biocompatibility under physiological conditions, making them suitable for in vivo applications.

[0049] The reagent prepared by the method of the present invention greatly improves the precise positioning of tumor cells, reduces the amount of antibodies used, and has better color development efficiency and detection sensitivity.

[0050] Embodiment 2 of the present invention:

[0051] In an embodiment of the present invention, a method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes includes:

[0052] S1, preparation of cerium folate-based nanozymes:

[0053] 0.1 mol / L Ce(NO3)3·6H2O and 0.1 mol / L FA aqueous solution were mixed and stirred for 12 hours to form a stable complex; then, 25 mg / mL bovine serum albumin (BSA) was added and the mixture was placed in a constant temperature water bath at 37°C and mixed thoroughly. For example, 0.25 mL of Ce(NO3)3·6H2O, 0.25 mL of FA aqueous solution, and 4.5 mL of BSA were taken; 1 mol / L KOH solution was used to adjust the pH value of the reaction system to 13, and the color of the solution changed from milky white to black. The constant temperature stirring was continued until the reaction was completed; the obtained reaction product solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water for 24 hours; the dialyzed suspension was passed through a 10 kDa ultrafiltration centrifuge tube at 4000 The collected precipitate was washed three times with deionized water to remove residual reagents after centrifugation and concentrated at rpm. Black solid powder Ce@BSA-FA was obtained after vacuum freeze-drying for 24 hours.

[0054] S2, preparation of Ki67 cerium folate-based nanozyme coupled with AP-labeled antibody:

[0055] 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA were weighed and dissolved in 9 mL of deionized water. The mixture was activated and stirred at room temperature for 8 hours. Ki67 antibody and alkaline phosphatase (AP) were added, and stirring was continued at room temperature for 24 hours. After the reaction, the precipitate was centrifuged and dialyzed for 24 hours to remove uncross-linked Ki67 antibody and AP. The precipitate was then diluted to a working solution using preservation solution.

[0056] S3, preparation of p16 INK4a Cerium folate-based nanozyme coupled with HRP-labeled antibody:

[0057] 57 mg EDC·HCl, 85.5 mg NHS and 6 mg Ce@BSA-FA were weighed and dissolved in 9 mL deionized water, and activated and stirred at room temperature for 8 hours. After activation, p16 INK4aAntibody and horseradish peroxidase HRP were stirred at room temperature for 24 hours. After the reaction, the precipitate was centrifuged and dialyzed for 24 hours to remove the uncrosslinked p16 INK4a Antibodies and HRP were diluted to working solution using storage solution.

[0058] The difference between Example 2 and Example 1 is that Example 2 clarifies the specific material values ​​and operation values ​​in each step. Those skilled in the art should understand that the purpose of Example 1 is to provide a universal overall preparation process and technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different production scales, product requirements, etc.), and the specific data involved in Example 2 are based on experience or the preferred settings determined under laboratory conditions. It can be understood that under the overall technical framework of the present invention, in actual operation, the existing technology can be flexibly selected or the operating conditions can be adjusted according to the specific circumstances to achieve the same operating effect. For example, the operating condition of stirring time, stirring for 8 hours or 24 hours is not fixed and can be adjusted according to the dosage scale. If the preparation scale is large and the dosage is large, the stirring time is correspondingly extended, and if the dosage is small, the stirring time is appropriately shortened, as long as the expected or the same or similar technical effect as in the embodiment of the present invention can be achieved.

[0059] At 37°C, FA and cerium ions were co-assembled step by step in BSA to form Ce@BSA-FA. Figure 2 This is the TEM image of Ce@BSA-FA, the sample has a chain structure. Figure 3 The thermogravimetric curve of Ce@BSA-FA is shown in Figure 2. The mass loss in the temperature range of 200-300 °C is mainly attributed to the thermal decomposition of FA, while the mass loss in the temperature range of 300-500 °C corresponds to the thermal decomposition of BSA. The UV-Vis spectrum of Ce@BSA-FA is shown in Figure 2. Figure 4 The characteristic peak at 285 nm is attributed to the double bond absorption peak of tryptophan (Trp) and tyrosine (Tyr) residues in BSA; the characteristic peak at 360 nm originates from the absorption of the conjugated double bond in the benzene ring structure of FA. Figure 5 Fourier transform infrared spectroscopy (FT-IR) analysis results of Ce@BSA and Ce@BSA-FA. 1612 cm −1 The characteristic peak at 3670 cm is attributed to the amide I band of BSA, which is produced by the stretching vibration of the C=O group in the peptide bond; −1 The characteristic peak at is related to the stretching vibration shift of the NH and OH groups in FA. To preliminarily evaluate whether the cerium-based nanozyme has the stability in physiological environment, the stability of Ce@BSA-FA in water, phosphate buffered saline (PBS, pH = 7.2-7.4) and DMEM culture medium was observed for 7 days. Figure 6Comparison images of the material before and after seven days of stabilization in different media. Visual observation of the material's state in different solutions revealed no significant differences in the cerium-based nanozyme's initial state after seven days of stabilization in water, PBS, and DMEM, demonstrating the stability of the cerium-based nanozyme under physiologically similar conditions.

[0060] The structure of Ki67 cerium folic acid-based nanozyme coupled with AP-labeled antibody is shown below: Figure 7 Folic acid (yellow) coordinated BSA (blue network) coated with cerium ions (red). Folic acid gives the material better free radical scavenging properties and provides its γ-carboxyl group to couple with the amino group of AP (pink), obtaining Ki67 folate cerium-based nanozyme coupled with alkaline phosphatase (AP) labeled antibody, which has good stability and biocompatibility under physiological conditions and is suitable for in vivo applications.

[0061] p16 INK4a The structure of cerium folate-based nanozyme coupled with HRP-labeled antibody is shown below: Figure 8 Folic acid (yellow) coordinated BSA (blue network) coated cerium ions (red). Folic acid gives the material better free radical scavenging performance and provides its γ-carboxyl group to couple with the amino group of HRP (purple) to obtain p16 INK4a The cerium folate-based nanozyme coupled with horseradish peroxidase (HRP)-labeled antibody has good stability and biocompatibility under physiological conditions and is suitable for in vivo applications.

[0062] Application Example 1:

[0063] The ·OH radical scavenging ability of Ce@BSA-FA was studied using salicylic acid as a chromogenic substrate. Figure 9 ). 1 mL of ferrous sulfate solution (5 mmol / L), 1 mL of 2% H2O2, 1 mL of ethanol-salicylic acid solution (2.5 mmol / L) and 1 mL of deionized water were mixed and reacted in a water bath at 37 °C for 30 minutes, which was recorded as the positive control group. A series of the above mixed solutions were prepared, and 1 mL of different concentrations of Ce@BSA-FA (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL) were added in sequence. The reaction was in a water bath at 37 °C for 30 minutes. Each experiment was repeated three times. The ·OH free radical scavenging ability of Ce@BSA-FA was evaluated by comparing the changes in absorbance. When the concentration was 50 μg / mL, the ·OH scavenging rate of Ce@BSA-FA was 79%.

[0064] Application Example 2:

[0065] The EPR test was performed using DMPO as an indicator to evaluate the ability of Ce@BSA-FA to scavenge ·OH. Figure 10 As shown in the figure, with the extension of reaction time, the characteristic signal peak intensity of ·OH gradually weakened, indicating that ·OH was decreasing. When the reaction time was extended to 30 minutes, the characteristic signal peak of ·OH was almost undetectable, indicating that ·OH in the system had been completely removed by Ce@BSA-FA.

[0066] Application Example 3:

[0067] Evaluation of Ce@BSA-FA O2 scavenging by NBT illumination - Ability ( Figure 11 ). Cerium-based nanozymes 3+ / Ce 4+ Redox cycle can remove O2 - Ce in cerium-based nanozymes 3+ As an active reduced state, it can directly quench O2 - , and Ce 4+ Can be regenerated into Ce by accepting electrons 3+ , forming a dynamic antioxidant mechanism. When the concentration of Ce@BSA-FA increased from 6.25 μg / mL to 50 μg / mL, Ce@BSA-FA had a significant effect on·O2 - Increased the scavenging power from 6% to 49% ( Figure 11 ).

[0068] DMPO was used as a free radical scavenger to dynamically monitor the effect of Ce@BSA-FA on O2 - The results of the clearing process are shown in Figure 12 At the beginning of the reaction (0 minutes), typical O2 - The quartet characteristic signal of Ce@BSA-FA treatment group decreased significantly after 15 minutes of reaction. After 30 minutes of reaction, the peak intensity of O2 - The signal peak of the system completely disappeared, confirming that the O2 - was cleared by Ce@BSA-FA, which was consistent with the NBT light irradiation method. - The clearing results are consistent.

[0069] Application Example 4:

[0070] Figure 13 The results showed that the scavenging ability of Ce@BSA-FA for H2O2 showed a significant concentration gradient dependence. When the concentration was 200 μg / mL, the scavenging rate of Ce@BSA-FA for H2O2 was 60%.

[0071] Application Example 5:

[0072] The cytotoxicity of Ce@BSA-FA was detected by Calcein-AM / PI staining experiment. The L929 cell suspension was transferred to each well of a 96-well culture plate at an appropriate density for inoculation, with 100 μL of cell suspension per well, and cultured until the cells adhered to the wall and grew to an appropriate density, usually about 24 hours. The negative control group was added with DMEM complete medium, while the sample group was added with Ce@BSA and Ce@BSA-FA solutions to concentrations of 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / L, respectively. They were then placed in a CO2 incubator and cultured for 24 hours. After the culture was completed, the 96-well culture plate was removed and the cells were stained according to the operating procedures of the Calcein AM / PI detection kit. The live / dead cells were observed under an inverted fluorescence microscope and the images were processed using Image ( Figure 14 The staining results showed that as the concentration gradient of the cerium-based nanozyme increased, the green fluorescence signal intensity and cell density, representing viable cells, showed a significant increase. This phenomenon directly demonstrated that the cerium-based nanozyme not only did not show obvious cytotoxicity to the L929 cell line, but instead exhibited a concentration-dependent pro-proliferation effect.

[0073] Application Example 6:

[0074] The in vitro biocompatibility of the synergistic therapeutic system was evaluated using a hemolysis assay. One mL of mouse blood was collected using an anticoagulant tube and centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded, and the red blood cells (RBCs) were retained. The RBCs were washed with PBS and resuspended in 10 mL of PBS. Ce@BSA-FA solutions of varying concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, and 600 μg / mL) were prepared as experimental groups. PBS served as a negative control, and ultrapure water served as a positive control. 200 μL of the RBC suspension was mixed with 800 μL of each solution and incubated at room temperature for 4 hours. After incubation, the supernatant was centrifuged, and the UV absorbance of the supernatant was measured to calculate the percentage of RBC hemolysis. Figure 15 The figure shows the hemolysis percentage of Ce@BSA-FA solution after incubation with mouse red blood cells at various concentrations. The hemolysis rate of Ce@BSA-FA was less than 10%, even at the highest concentration of 600 μg / mL studied, demonstrating that Ce@BSA-FA has good blood compatibility and biocompatibility.

[0075] In summary, Ce@BSA-FA has a chain structure and is stable in different solutions. It has a concentration gradient-dependent effect in scavenging free radicals (·OH, ·O2⁻, H2O2), is non-toxic to the L929 cell line, and has a pro-proliferation effect. The hemolysis rate is less than 10%, and it shows good cytocompatibility and blood compatibility in biocompatibility experiments.

[0076] Application Example 7:

[0077] By comparing the traditional immunocytochemical staining with the dual-target tumor cell detection reagent based on cerium-based nanozymes of the present invention (i.e., Ki67 folate cerium-based nanozyme coupled with AP-labeled antibody and p16 INK4a The purpose of this study is to verify the advantages of the detection reagent of the present invention in shortening the detection process, reducing the cost of antibodies and improving the accuracy of results.

[0078] Control group (traditional immunocytochemistry staining):

[0079] Cervical exfoliated cells were used for conventional immunocytochemical staining: 1. Prepare cervical cell smears using the natural sedimentation method and fix and soak in 95% ethanol for 30 minutes. 2. Perform antigen retrieval using EDTA antigen retrieval solution, pH 9.0, in an autoclave. 3. Add peroxidase blocker and incubate at room temperature for 10 minutes, then rinse with TBST rinse buffer. 4. Add Ki-67 and p16 monoclonal antibodies and incubate at 37°C for 45 minutes, then rinse with TBST rinse buffer. 5. Add HRP- and AP-conjugated secondary antibodies and incubate at 37°C for 30 minutes, then rinse with TBST rinse buffer. 6. Add chromogenic reagent (Accelerated Red) and incubate at room temperature for 15 minutes, then rinse with TBST rinse buffer. 7. Add DAB chromogenic solution and incubate at room temperature for 5 minutes, then rinse with tap water. 8. Counterstain with Mayer's hematoxylin solution for 30-60 seconds at room temperature, then rinse with tap water to return the stain to blue. Rapidly dehydrate, transparently seal, and observe under a microscope. Figure 16 As shown in the figure, conventional immune cell staining results show that the cytoplasm of cervical exfoliated cells is stained brown and the nucleus is stained red, indicating a positive result. However, the experimental process is long and there are certain non-specific reactions.

[0080] Experimental group (using the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention):

[0081] The tissue used was cervical exfoliated cells, and the ki67 cerium folic acid-based nanozyme prepared by the method in Example 2 was coupled with AP-labeled antibodies and p16 INK4aRapid immunocytochemical staining with cerium folate nanozyme coupled to HRP-labeled antibody: 1. Prepare cervical cell smears using the natural sedimentation method and fix them in 95% ethanol for 15 minutes. 2. Perform antigen retrieval using EDTA antigen retrieval solution, pH 9.0, in a pressure cooker. 3. Add peroxidase blocker and incubate at room temperature for 3 minutes; rinse with TBST rinse buffer. 4. Add Ki67 cerium folate nanozyme coupled to AP-labeled antibody and p16 INK4a 5. Add the cerium folic acid-based nanoenzyme coupled with the HRP-labeled antibody, incubate at room temperature for 10 minutes; rinse with TBST rinse buffer. 6. Add the accelerated red color development solution, incubate at room temperature for 5 minutes; rinse with TBST rinse buffer. 7. Add the DAB color development solution, incubate at room temperature for 2 minutes; rinse with tap water. 8. Add Mayer's hematoxylin staining solution for counterstaining at room temperature for 30 to 60 seconds, rinse with tap water, and return to blue. Dehydrate quickly, transparently seal, and observe under a microscope. Figure 17 As shown, the results of rapid immunocytological staining showed that the cytoplasm of cervical exfoliated cells was stained brown and the nucleus was stained red, and the result was positive.

[0082] From the above experimental and control groups, it can be seen that the detection reagents of the present invention and traditional detection reagents can both target and locate the relevant antigens, but the traditional detection reagent staining process takes a long time, while the experimental time using the detection reagents of the present invention is significantly shortened, especially in the antibody incubation and color development steps, the time is greatly reduced (this is because the experimental method of the present invention is more stable, more efficient, and more specific, so it can effectively shorten the antibody binding time). In addition, the detection reagents of the present invention omit the use of secondary antibodies, directly saving antibody costs. And from Figure 14 and Figure 15 From the staining results, it can be seen that there are brown backgrounds and blue impurities in the background of traditional immunocytochemistry staining images, indicating that it has certain nonspecific reactions. The staining background of the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention is clean, the cell nucleus and cytoplasm are clearly stained, the staining intensity is higher, there is no nonspecific background, and the staining effect is better, indicating that the detection reagent of the present invention can more accurately locate the target antigen and has higher detection accuracy.

[0083] In summary, the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention can shorten the detection operation time, reduce the amount of antibody used, and has excellent color development efficiency and detection sensitivity.

[0084] In the above embodiments of the present invention, the description of each embodiment has its own emphasis. For parts not described in detail in a particular embodiment, reference can be made to the relevant description of other embodiments. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes, characterized in that: The dual-target tumor cell detection reagent includes ki67 folate cerium-based nanozyme coupled with AP-labeled antibody, p16 INK4a The preparation method of the dual-targeted tumor cell detection reagent of the cerium folate-based nanozyme coupled with the HRP-labeled antibody includes: Step 1: Preparation of cerium folate-based nanozymes: Ce(NO3)3·6H2O and FA aqueous solution were mixed and stirred to obtain a stable complex, wherein FA refers to folic acid. Bovine serum albumin (BSA) was added to the obtained stable complex, and the complex was placed in a water bath to react under alkaline conditions. The obtained reaction product was dialyzed, ultrafiltered and centrifuged, and vacuum freeze-dried to obtain a black solid powder cerium folate-based nanozyme, namely Ce@BSA-FA. Step 2: Preparation of Ki67 cerium folate-based nanozyme coupled with AP-labeled antibody: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and Ce@BSA-FA were dissolved in deionized water and activated by stirring at room temperature. After activation, Ki67 antibody and alkaline phosphatase (AP) were added for cross-linking reaction. Uncross-linked Ki67 antibody and AP were removed by centrifugation and dialysis, and the solution was diluted to the working solution concentration using preservation solution. Step 3: Prepare p16 INK4a Cerium folate-based nanozyme coupled with HRP-labeled antibody: EDC·HCl, NHS and Ce@BSA-FA were dissolved in deionized water and activated by stirring at room temperature. After activation, p16 was added INK4a The antibody was cross-linked with horseradish peroxidase (HRP), and the uncross-linked p16 was removed by centrifugation and dialysis. INK4a Antibodies and HRP were diluted to working concentration using storage buffer.

2. The method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes according to claim 1, characterized in that: The step one comprises: 0.1 mol / L Ce(NO3)3·6H2O and 0.1 mol / L FA aqueous solution were mixed and stirred to form a stable complex; 25 mg / mL bovine serum albumin (BSA) was added and the mixture was placed in a constant temperature water bath at 37°C for mixing; 1 mol / L KOH solution was used to adjust the pH value of the reaction system to 13, and the color of the solution changed from milky white to black. Stirring at constant temperature was continued until the reaction was completed; the obtained reaction product solution was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed with deionized water for purification; the dialyzed suspension was centrifuged at 4000 rpm using a 10 kDa ultrafiltration centrifuge tube, and the collected precipitate was repeatedly washed with deionized water three times to remove residual reagents; and black solid powder Ce@BSA-FA was obtained after vacuum freeze-drying.

3. The method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes according to claim 1, characterized in that: The second step includes: 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA were dissolved in 9 mL of deionized water and activated with stirring at room temperature for 8 hours. After activation, Ki67 antibody and alkaline phosphatase (AP) were added, and stirring was continued at room temperature for 24 hours. After the reaction, the precipitate was centrifuged and dialyzed for 24 hours to remove uncross-linked Ki67 antibody and AP, and then diluted to the working solution concentration with preservation solution.

4. The method for preparing a dual-targeted tumor cell detection reagent based on cerium-based nanozymes according to claim 1, characterized in that: The step three includes: 57 mg EDC·HCl, 85.5 mg NHS and 6 mg Ce@BSA-FA were dissolved in 9 mL deionized water and activated with stirring at room temperature for 8 hours. After activation, p16 INK4a Antibody and horseradish peroxidase HRP were stirred at room temperature for 24 hours. After the reaction, the precipitate was centrifuged and dialyzed for 24 hours to remove the uncrosslinked p16 INK4a Antibodies and HRP were diluted to working concentration using storage buffer.

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