Preparation method of dual-targeting tumor cell detection reagent based on cerium-based nano-enzyme

Abstract

The invention discloses a preparation method of a cerium-based nano-enzyme-based dual-targeting tumor cell detection reagent, and relates to the technical field of biochemistry, and the preparation method comprises the following steps: 1, preparing a cerium folate-based nano-enzyme: mixing Ce (NO3) 3.6 H2O and an FA aqueous solution, stirring, adding bovine serum albumin, and reacting under a water bath alkaline condition to obtain the cerium folate-based nano-enzyme Ce (at) BSA-FA; step 2, preparing a ki67 folic acid cerium-based nano-enzyme coupled AP labeled antibody: dissolving EDC.HCl, NHS and Ce (at) BSA-FA in deionized water, activating, and then adding the ki67 antibody and alkaline phosphatase AP to carry out cross-linking reaction; and step 3, preparing a p16INK4a folic acid cerium-based nano-enzyme coupled HRP labeled antibody: dissolving EDC.HCl, NHS and Ce at BSA-FA in deionized water, activating, and then adding the p16INK4a antibody and horse radish peroxidase HRP to carry out cross-linking reaction. According to the dual-targeting tumor cell detection reagent prepared by the method disclosed by the invention, the dosage of antibodies is reduced, the operation process is simplified, and the reagent has better color development efficiency and detection sensitivity.

Description

Technical Field

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

[0002] Immunohistochemistry (IHC) has strong specificity, high sensitivity, accurate localization, and can organically combine morphological research with functional research. It is widely used in many fields of biological and medical research. In the research of tumor pathology diagnosis, the role and significance of immunohistochemistry technology are more crucial.

[0003] In order to more accurately judge the co-expression of antigen proteins in tumor cells, many hospitals have introduced immunohistochemical double staining technology. This technology can separately label two different antigens with red and brown on the same pathological tissue slide, and can be detected according to the different antigen expressions and characteristics as needed.

[0004] Since there are usually multiple different types of cells on a pathological tissue section, tumor cells may only account for a part, or even a very small part. Therefore, when the proportion of tumor cells is low, whether the two protein indexes stained on two slides are co-expressed in the same group of cells mainly depends on experience and speculation, rather than direct observation, which is prone to difficult inference or even misjudgment due to lack of experience and other reasons.

[0005] Currently, the commonly used double staining kit adopts the immune detection method of primary antibody-secondary antibody, with a complex process flow, long operation time, and problems in the current technology such as inaccurate localization of tumor cells, weak signal intensity at low concentration of double staining antibodies, and more non-specific interferences at high concentration. Summary of the Invention

[0006] The object of the present invention is to solve the problems in the prior art such as inaccurate localization of tumor cells, weak signal intensity of double staining antibodies at low concentration, and more non-specific interferences at high concentration, and to provide a preparation method of a dual-targeted tumor cell detection reagent based on cerium-based nanozymes. The dual-targeted tumor cell detection reagent includes ki67 folic acid cerium-based nanozyme conjugated AP-labeled antibody, p16 INK4aFolic acid cerium-based nanozyme conjugated with HRP-labeled antibody. The preparation method of the dual-target tumor cell detection reagent includes: Step 1, preparing folic acid cerium-based nanozyme: Mix Ce(NO3)3·6H2O and FA aqueous solution and stir to obtain a stable complex. Here, FA refers to folic acid. Add bovine serum albumin BSA to the obtained stable complex and place it in a water bath to react under alkaline conditions. Dialyze, ultrafiltrate by centrifugation, and vacuum freeze-dry the obtained reaction product to obtain a black solid powder of folic acid cerium-based nanozyme, namely Ce@BSA-FA; Step 2, preparing ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody: Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, namely EDC·HCl, N-hydroxysuccinimide, namely NHS, and Ce@BSA-FA in deionized water, stir and activate at room temperature. After activation, add ki67 antibody and alkaline phosphatase AP to carry out a cross-linking reaction. Remove the uncross-linked ki67 antibody and AP by centrifugation and dialysis, and dilute to the working solution concentration with a preservation solution; Step 3, preparing p16 INK4a Folic acid cerium-based nanozyme conjugated with HRP-labeled antibody: Dissolve EDC·HCl, NHS and Ce@BSA-FA in deionized water, stir and activate at room temperature. After activation, add p16 INK4a antibody and horseradish peroxidase HRP to carry out a cross-linking reaction. Remove the uncross-linked p16 INK4a antibody and HRP by centrifugation and dialysis, and dilute to the working solution concentration with a preservation solution.

[0007] Optionally, Step 1 includes: Mix 0.1 mol / L Ce(NO3)3·6H2O and 0.1 mol / L FA aqueous solution and stir to form a stable complex; add bovine serum albumin BSA with a concentration of 25 mg / mL and mix in a 37°C constant temperature water bath; use 1 mol / L KOH solution to adjust the pH value of the reaction system to 13, and the solution color changes from milky white to black. Continue to stir constantly at a constant temperature until the reaction ends; Transfer the obtained reaction product solution to a dialysis bag with a molecular weight cut-off of 1000 Da and dialyze and purify with deionized water; Centrifuge and concentrate the dialyzed suspension through a 10 kDa ultrafiltration centrifugal tube at 4000 rpm, and wash the collected precipitate three times with deionized water to remove residual reagents; Vacuum freeze-dry to obtain a black solid powder of Ce@BSA-FA.

[0008] Optionally, Step 2 includes: dissolving 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA in 9 mL of deionized water, activating and stirring at room temperature for 8 hours; after activation, adding ki67 antibody and alkaline phosphatase AP thereto, and continuing to stir at room temperature for 24 hours; after the reaction ends, centrifuging to collect the precipitate, dialyzing for 24 hours to remove the uncrosslinked ki67 antibody and AP, and diluting to the working solution concentration with the preservation solution.

[0009] Optionally, Step 3 includes: dissolving 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA in 9 mL of deionized water, activating and stirring at room temperature for 8 hours; after activation, adding INK4a p16 antibody and horseradish peroxidase HRP thereto, and continuing to stir at room temperature for 24 hours; after the reaction ends, centrifuging to collect the precipitate, dialyzing for 24 hours to remove the uncrosslinked p16 INK4a antibody and HRP, and diluting to the working solution concentration with the preservation solution.

[0010] The beneficial effects of the present invention are as follows: The folic acid cerium-based nanozyme provided by the present invention can target and localize to tumor cells with high expression of folate receptors due to the presence of folic acid ligands, achieving the effect of specifically recognizing tumor cells and reducing the non-specific interference suffered by the double-stained antibody; The folic acid cerium-based nanozyme has multiple binding sites and can conjugate with AP, HRP, and antibodies to form a conjugate complex. The existence of multiple binding sites can not only increase the loading amounts of the enzyme and the antibody, but also greatly maintain the stability of the enzyme and the antibody; The preparation process of the present invention is simple, the product has high stability, reduces the operation process of tumor detection, shortens the operation time, and has higher chromogenic efficiency and detection sensitivity. Description of the Drawings

[0011] Figure 1 It is a schematic diagram of the method steps of the present invention; Figure 2 It is a TEM image of the folic acid cerium-based nanozyme; Figure 3 It is a TGA image of the folic acid cerium-based nanozyme; Figure 4 It is an ultraviolet-visible light spectrum diagram of the folic acid cerium-based nanozyme; Figure 5 It is an FT-IR spectrum diagram of the folic acid cerium-based nanozyme; Figure 6 It is a physical picture of the folic acid cerium-based nanozyme stored in different media for 7 days; Figure 7 It is a schematic diagram of the structure of the ki 67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody; Figure 8 is p16 INK4a Schematic diagram of the structure of folic acid cerium-based nanozyme coupled with HRP-labeled antibody; Figure 9 Evaluation of the ·OH scavenging ability of folic acid cerium-based nanozyme; Figure 10 EPR spectra of folic acid cerium-based nanozyme scavenging ·OH after incubation for different times; Figure 11 is the ·O2 of folic acid cerium-based nanozyme - Scavenging ability evaluation; Figure 12 EPR spectra of folic acid cerium-based nanozyme scavenging ·O2 after incubation for different times - ; Figure 13 Evaluation of the hydrogen peroxide scavenging ability of folic acid cerium-based nanozyme; Figure 14 Live-dead staining imaging of L929 cells after treatment with folic acid cerium-based nanozyme for 24 h; Figure 15 Results of hemolysis experiments with different concentrations of folic acid cerium-based nanozyme; Figure 16 Results of traditional immunocytochemical staining experiments; Figure 17 Results of immunocytochemical staining experiments using the dual-targeted tumor cell detection reagent based on cerium-based nanozyme of the present invention. Detailed implementation manners

[0012] The present invention will be further described below in conjunction with examples and experimental verification drawings: Example 1 of the present invention:

[0013] The present invention provides a preparation method of a dual-targeted tumor cell detection reagent based on cerium-based nanozyme. The cerium-based nanozyme is used to carry folic acid to achieve targeted localization of folic acid (FA) receptors on tumor cells, and at the same time has the detection functions of ki67 and p16 INK4a This dual-targeted tumor cell detection reagent mainly consists of ki67 folic acid cerium-based nanozyme coupled with alkaline phosphatase (AP)-labeled antibody and p16 INK4a Folic acid cerium-based nanozyme coupled with horseradish peroxidase (HRP)-labeled antibody. It mainly involves the preparation of folic acid cerium-based nanozyme encapsulated with bovine serum albumin (Ce@BSA-FA) and the formation of folic acid cerium-based nanozyme coupled antibody by coupling antibody and signal label using it as a support framework. Referring to Figure 1 , this preparation method includes: Step 1, prepare folic acid cerium-based nanozyme: Mix the aqueous solutions of Ce(NO3)3·6H2O and folic acid (FA) to obtain a stable complex. Subsequently, add bovine serum albumin (BSA) and place it in a water bath to react under alkaline conditions. Dialyze, ultrafiltrate by centrifugation, and vacuum freeze-dry the resulting reaction product to obtain a black solid powder of folic acid cerium-based nanozyme Ce@BSA-FA.

[0014] The prepared folic acid cerium-based nanozyme carries folic acid and can specifically bind to the folic acid receptor on the surface of tumor cells, achieving efficient and precise targeting of tumor cells. This targeting greatly improves the specificity of detection, reduces interference caused by non-specific binding, and helps to more accurately identify tumor cells.

[0015] In this step, cerium nitrate hexahydrate (Ce(NO3)3·6H2O) provides cerium ions (Ce³⁺), which serve as the core component of the nanozyme; folic acid (FA) acts as a targeting ligand, and its γ-carboxyl group can coordinate with Ce³⁺ and specifically bind to the folic acid receptor (FR) of tumor cells to achieve active targeting; bovine serum albumin (BSA) acts as a stabilizer and coating material, providing biocompatibility.

[0016] The alkaline condition can be achieved by adjusting the pH value of the reaction system with potassium hydroxide (KOH).

[0017] Step two, prepare the ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody: Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), and Ce@BSA-FA in deionized water, stir at room temperature, activate the carboxyl groups on the surface of the nanozyme through EDC and NHS. After activation, add ki67 antibody and alkaline phosphatase AP to undergo a cross-linking reaction. Remove the uncross-linked ki67 antibody and AP by centrifugation and dialysis, and dilute to the working solution with the preservation solution; Step three, prepare p16 INK4a Folic acid cerium-based nanozyme conjugated with HRP-labeled antibody: The preparation method in step three is similar to that of preparing the ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody in step two, except that the antibody and enzyme are replaced with p16 INK4a Antibody and horseradish peroxidase (HRP), that is, dissolve EDC·HCl, NHS, and Ce@BSA-FA in deionized water, stir and activate at room temperature. After activation, add p16 INK4a Antibody and horseradish peroxidase HRP undergo a cross-linking reaction. Remove the uncross-linked p16 INK4a Antibody and HRP by centrifugation and dialysis, and dilute to the working solution with the preservation solution.

[0018] It is additionally noted that the storage solution in the present invention includes: one of PBS, Tris or HEPES buffer, and 1% to 2% bovine serum albumin, 0.01% to 1% Proclin 300. The working solution is usually diluted 10 to 100 times.

[0019] The steps of using the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention include: 1. adding p16-HRP coupled antibody (i.e., p16 INK4a 1. Add a mixture of cerium folate nanozyme coupled with HRP labeled antibody) and ki67-AP coupled antibody (i.e. ki67 cerium folate nanozyme coupled with AP labeled antibody), incubate 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. Hematoxylin counterstaining. Among them, the composition of DAB colorimetric solution is: A liquid substrate: H2O2; B liquid enhancement: NiCl2; C liquid chromogen: diaminobenzidine; AP colorimetric solution is: A liquid dilution: Tris buffer; B liquid substrate: α-naphthol AS-BI phosphate; C liquid chromogen: Fast Red TR salt.

[0020] The principle of the present invention is: 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), etc. ROS plays an important role in the occurrence and development of many diseases, especially in the tumor microenvironment, where the ROS concentration is high. Therefore, nanocarriers that can respond to ROS are of great significance. The cerium folate-based nanozyme provided by the present invention has good ROS scavenging properties and can be passively targeted to tumor cells overexpressing ROS; folate receptors (FRs) are a type of glycosylated membrane proteins whose main function is to mediate the cell's uptake of folic acid (vitamin B9). Folic acid is a key cofactor in DNA synthesis, repair and methylation reactions, and is essential for cell proliferation. In normal tissues, the expression of FR 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 cerium folate-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.

[0021] In addition, cerium folate-based nanozymes have multiple binding sites, which can couple more enzyme molecules (such as AP, HRP) and antibodies to form a coupling complex. The presence of multiple binding sites can not only increase the load of enzymes and antibodies, but also greatly maintain the stability of enzymes and antibodies. In a complex biological environment, enzymes and antibodies are easily affected by various factors and inactivated, while the multiple binding sites of cerium folate-based nanozymes provide stable support and protection for enzymes and antibodies, ensuring that they can continue to be active during the detection process, thereby ensuring the accuracy and reliability of the test results.

[0022] 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 to form a stable cerium folate nanozyme coupling complex, which not only retains the enzymatic activity of HRP or AP, allowing these enzymes to play a normal catalytic role in subsequent detection reactions, but also inherits the ability to target folate receptors because the cerium folate nanozyme itself has the ability to target folate receptors. The coupling complex Ce@BSA-FA-HRP or Ce@BSA-FA-AP has good stability and biocompatibility under physiological conditions and is suitable for in vivo applications.

[0023] 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.

[0024] Embodiment 2 of the present invention:

[0025] In an embodiment of the present invention, a method for preparing a dual-targeted tumor cell detection reagent based on a cerium-based nanozyme comprises: S1, preparation of cerium folate-based nanozymes: 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 37°C constant temperature water bath 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. The pH value of the reaction system was adjusted to 13 using 1 mol / L KOH solution, 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 purified by dialyzing with deionized water for 24 hours. The dialyzed suspension was filtered 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.

[0026] S2. Preparation of ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody: Weigh 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA and dissolve them in 9 mL of deionized water. Activate and stir at room temperature for 8 hours; after activation, add ki67 antibody and alkaline phosphatase AP, and continue to stir at room temperature for 24 hours; after the reaction, centrifuge to collect the precipitate, dialyze for 24 hours to remove the uncrosslinked ki67 antibody and AP, and dilute to the working solution with the preservation solution.

[0027] S3. Preparation of p16 INK4a Folic acid cerium-based nanozyme conjugated with HRP-labeled antibody: Weigh 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA and dissolve them in 9 mL of deionized water. Activate and stir at room temperature for 8 hours; after activation, add p16 INK4a antibody and horseradish peroxidase HRP, and continue to stir at room temperature for 24 hours; after the reaction, centrifuge to collect the precipitate, dialyze for 24 hours to remove the uncrosslinked p16 INK4a antibody and HRP, and dilute to the working solution with the preservation solution.

[0028] The difference between Example 2 and Example 1 is that Example 2 specifies the specific material consumption values and operation values in each step. Those skilled in the art should understand that the purpose of Example 1 is to provide a general preparation process and technical principle with universality, and cover as many different applicable scenarios and conditions as possible (such as different production scales, product requirements, etc.). The specific data involved in Example 2 are relatively optimal settings determined based on experience or under laboratory conditions. It can be understood that within the overall technical framework of the present invention, in actual operation, existing technologies can be flexibly selected or operation conditions can be adjusted according to specific circumstances to achieve the same operation effect. For example, for the operation condition of stirring time, stirring for 8 hours and 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; if the dosage is small, the stirring time is appropriately shortened, as long as the expected or the same or similar technical effects as those in the examples of the present invention can be achieved.

[0029] Under the temperature condition of 37°C, FA and cerium ions are co-assembled step by step in BSA to form Ce@BSA-FA. Figure 2 This is the TEM image of Ce@BSA-FA, and the sample is in a chain structure. Figure 3is the thermogravimetric curve of Ce@BSA-FA. 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 4 . The characteristic peak at 285 nm is attributed to the double bond absorption peaks of tryptophan (Trp) and tyrosine (Tyr) residues in BSA; the characteristic peak at 360 nm originates from the absorption of conjugated double bonds in the benzene ring structure of FA. Figure 5 shows the Fourier transform infrared spectroscopy (FT-IR) analysis results of Ce@BSA and Ce@BSA-FA. The characteristic peak at 1612 cm −1 is attributed to the amide I band of BSA, which is generated by the stretching vibration of the C=O group in the peptide bond; the characteristic peak at 3670 cm −1 is related to the stretching vibration shift of N-H and O-H groups in FA. To preliminarily evaluate the stability of cerium-based nanozymes in the physiological environment, a 7-day observation experiment was conducted on the stability of Ce@BSA-FA in water, phosphate buffered saline (PBS, pH = 7.2-7.4), and DMEM medium. Figure 6 is the physical comparison diagram of the material before and after standing in different media for 7 days. By visually observing the state change of the material in different solutions, after standing for seven days, there is no significant difference in the state of the material in water, PBS, and DMEM solutions compared with the initial state by naked eye observation, indicating that folic acid cerium-based nanozymes can maintain stability under conditions similar to the physiological environment.

[0030] The structural schematic of ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody is as shown in Figure 7 . Folic acid (yellow) coordinated BSA (blue network) coats cerium ions (red). Folic acid endows the material with better free radical scavenging performance and provides its γ-carboxyl group to couple with the amino group of AP (pink) to obtain ki67 folic acid cerium-based nanozyme conjugated with alkaline phosphatase (AP) labeled antibody, which has good stability and biocompatibility under physiological conditions and is suitable for in vivo applications.

[0031] p16 INK4a The structural schematic of folic acid cerium-based nanozyme conjugated with HRP-labeled antibody is as shown in Figure 8 . Folic acid (yellow) coordinated BSA (blue network) coats cerium ions (red). Folic acid endows the material with better free radical scavenging performance and provides its γ-carboxyl group to couple with the amino group of HRP (purple) to obtain p16 INK4a folic acid cerium-based nanozyme conjugated with horseradish peroxidase (HRP) labeled antibody, which has good stability and biocompatibility under physiological conditions and is suitable for in vivo applications.

[0032] Application Example 1: 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 group of experiments was carried out in parallel 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%.

[0033] Application Example 2: The EPR test was performed using DMPO as an indicator to evaluate the ability of Ce@BSA-FA to remove ·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.

[0034] Application Example 3: Evaluation of Ce@BSA-FA scavenging O2 using NBT photoirradiation - Capacity ( 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 ).

[0035] 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 inFigure 12 In it, at the beginning of the reaction (0 minutes), a typical quartet characteristic signal of ·O2 - could be detected in the system; at 15 minutes of the reaction, the signal peak intensity of the Ce@BSA-FA treatment group decreased significantly; when the reaction was 30 minutes later, it could be seen that the signal peak of ·O2 - completely disappeared, confirming that ·O2 - in the system was scavenged by Ce@BSA-FA, which was consistent with the scavenging result detected by the NBT photolysis method for ·O2 - .

[0036] Application Example 4: Figure 13 The scavenging ability of Ce@BSA-FA for H2O2 showed an obvious concentration gradient dependence. When the concentration was 200 μg / mL, the scavenging rate of Ce@BSA-FA for H2O2 was 60%.

[0037] Application Example 5: 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, 100 μL of cell suspension per well, and cultured until the cells adhered and grew to an appropriate density, usually about 24 hours. The negative control group was added with DMEM complete medium, while the sample groups were added with Ce@BSA and Ce@BSA-FA solutions respectively, so that their concentrations were 3.125 μg / mL, 6.25 μg / mL, and 12.5 μg / L, and then placed in a CO2 incubator for 24 hours. After the culture was completed, the 96-well culture plate was taken out, and then the cells were stained according to the operation steps of the Calcein AM / PI detection kit, and the live / dead cells were observed under an inverted fluorescence microscope and the pictures were processed with 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 live cells showed a significant increasing trend. This phenomenon intuitively indicated that the cerium-based nanozyme not only did not show obvious cytotoxicity to the L929 cell line, but instead showed a concentration-dependent proliferative effect.

[0038] Application Example 6: The in vitro biocompatibility of the synergistic treatment system was evaluated by hemolysis detection. 1 mL of mouse blood was collected in an anticoagulant tube and centrifuged at 8000 rpm for 10 minutes in a centrifuge. After centrifugation, the supernatant was discarded and the red blood cells were retained. The red blood cells were washed with PBS and resuspended in 10 mL of PBS solution. Ce@BSA-FA solutions with different concentrations (100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL) were prepared as the experimental groups, PBS as the negative control group, and ultrapure water as the positive control group. 200 μL of the red blood cell suspension was mixed with 800 μL of each group of solutions and incubated at room temperature for 4 hours. After incubation, centrifugation was performed, and the ultraviolet absorbance of the supernatant was measured and the hemolysis percentage of the red blood cells was calculated. Figure 15 It is a data graph of the hemolysis percentage after co-incubation of Ce@BSA-FA solutions at various concentrations with mouse red blood cells. The hemolysis rate of Ce@BSA-FA is lower than 10%, even at the highest concentration of 600 μg / mL studied, demonstrating that Ce@BSA-FA has good blood compatibility and biocompatibility.

[0039] In summary, Ce@BSA-FA has a chain-like structure, is stable in different solutions, has a concentration gradient dependence in scavenging free radicals (·OH, ·O2⁻, H2O2), is non-toxic to the L929 cell line and has a proliferative effect, and the hemolysis rate is lower than 10%. It shows good cell compatibility and blood compatibility in the biocompatibility experiment.

[0040] Application Example 7: By comparing the effects of traditional immunocytochemical staining and immunocytochemical staining using the dual-targeted tumor cell detection reagent based on cerium-based nanozymes of the present invention (i.e., ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody and p16 INK4a folic acid cerium-based nanozyme conjugated with HRP-labeled antibody), the aim is to verify the advantages of the detection reagent of the present invention in shortening the detection process, reducing the antibody cost, and improving the result accuracy. Among them, Control group (traditional immunocytochemical staining): The tissue used was cervical exfoliated cells, and traditional immunocytochemical staining was performed: 1. The cervical cell smear was prepared by the natural sedimentation method and fixed in 95% ethanol for 30 minutes. 2. Antigen retrieval was performed in a pressure cooker using EDTA antigen retrieval solution pH 9.0. 3. Peroxidase blocker was added dropwise and incubated at room temperature for 10 minutes; rinsed with TBST washing buffer. 4. Ki-67 monoclonal antibody and p16 monoclonal antibody were added dropwise and incubated at 37 °C for 45 minutes; rinsed with TBST washing buffer. 5. HRP-labeled secondary antibody and AP-labeled secondary antibody were added dropwise and incubated at 37 °C for 30 minutes; rinsed with TBST washing buffer. 6. Fast red chromogenic solution was added dropwise and incubated at room temperature for 15 min; rinsed with TBST washing buffer. 7. DAB chromogenic solution was added dropwise and incubated at room temperature for 5 min; rinsed with tap water. 8. Mayer hematoxylin staining solution was added dropwise for counterstaining and stained at room temperature for 30 - 60 seconds, rinsed thoroughly with tap water, and blued. Rapid dehydration, clearing and mounting were performed, and observed under the microscope. As Figure 16 shown, the results of conventional immunocytochemical staining showed that the cytoplasmic staining was brown and the nuclear staining was red in cervical exfoliated cells, and the results were positive. However, the experimental procedure was long and there was a certain degree of non-specific reaction.

[0041] Experimental group (using the dual-target tumor cell detection reagent based on cerium-based nanozyme of the present invention): The tissue used was cervical exfoliated cells, and the ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody and p16 prepared by the method in Example 2 was used INK4a and the folic acid cerium-based nanozyme conjugated with HRP-labeled antibody were used for rapid immunocytochemical staining: 1. The cervical cell smear was prepared by the natural sedimentation method and fixed in 95% ethanol for 15 minutes. 2. Antigen retrieval was performed in a pressure cooker using EDTA antigen retrieval solution pH 9.0. 3. Peroxidase blocker was added dropwise and incubated at room temperature for 3 minutes; rinsed with TBST washing buffer. 4. The ki67 folic acid cerium-based nanozyme conjugated with AP-labeled antibody and INK4a the folic acid cerium-based nanozyme conjugated with HRP-labeled antibody were added dropwise and incubated at room temperature for 10 minutes; rinsed with TBST washing buffer. 5. Fast red chromogenic solution was added dropwise and incubated at room temperature for 5 min; rinsed with TBST washing buffer. 6. DAB chromogenic solution was added dropwise and incubated at room temperature for 2 min; rinsed with tap water. 7. Mayer hematoxylin staining solution was added dropwise for counterstaining and stained at room temperature for 30 - 60 seconds, rinsed thoroughly with tap water, and blued. Rapid dehydration, clearing and mounting were performed, and observed under the microscope. As Figure 17 shown, the results of rapid immunocytochemical staining showed that the cytoplasmic staining was brown and the nuclear staining was red in cervical exfoliated cells, and the results were positive.

[0042] From the above experimental group and control group, it can be seen that both the detection reagent of the present invention and the traditional detection reagent can target and localize relevant antigens. However, the staining process of the traditional detection reagent takes a long time, while the experimental time using the detection reagent of the present invention is significantly shortened, especially in the antibody incubation and color development steps, with a large reduction in time (this is because the experimental method of the present invention has a more stable combination, higher efficiency, and stronger specificity, so it can effectively shorten the antibody binding time). In addition, the detection reagent of the present invention omits the use of the secondary antibody, directly saving the antibody cost. And from Figure 14 and Figure 15 the staining results, it can be seen that there are brown backgrounds and blue impurities in the background of the traditional immunocytochemical staining image, indicating that it has certain non-specific reactions. The staining background of the double-target tumor cell detection reagent based on cerium-based nanozymes of the present invention is clean, the nuclei and cytoplasm are clearly stained, the staining intensity is higher, there is no non-specific background, and the staining effect is better, indicating that the detection reagent of the present invention can more accurately localize the target antigen and has higher detection accuracy.

[0043] In summary, the double-target tumor cell detection reagent based on cerium-based nanozymes of the present invention can shorten the operation time of detection, reduce the amount of antibody used, and has excellent color development efficiency and detection sensitivity.

[0044] In the above embodiments of the present invention, the descriptions of each embodiment have their own emphases. For the parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments. Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a dual-targeted tumor cell detection reagent based on cerium-based nanozyme, characterized in that, The dual-targeted tumor cell detection reagent includes a ki67 folic acid cerium-based nanozyme conjugated with an AP-labeled antibody and p16 INK4a a folic acid cerium-based nanozyme conjugated with an HRP-labeled antibody. The preparation method of the dual-targeted tumor cell detection reagent includes: Step 1: Prepare folic acid cerium-based nanozyme: Mix Ce(NO3)3·6H2O and aqueous FA solution and stir to obtain a stable complex. Here, FA refers to folic acid. Add bovine serum albumin BSA to the obtained stable complex and place it in a water bath to react under alkaline conditions. Dialyze, ultrafiltrate by centrifugation, and vacuum freeze-dry the obtained reaction product to obtain a black solid powder of folic acid cerium-based nanozyme, namely Ce@BSA-FA; Step 2: Prepare ki67 folic acid cerium-based nanozyme-conjugated AP-labeled antibody: Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, i.e., EDC·HCl, N-hydroxysuccinimide, i.e., NHS, and Ce@BSA-FA in deionized water, stir and activate at room temperature. After activation, add ki67 antibody and alkaline phosphatase AP to carry out a cross-linking reaction. Remove the uncross-linked ki67 antibody and AP by centrifugation and dialysis, and dilute to the working solution concentration with the preservation solution; Step 3, prepare p16 INK4a Folic acid cerium-based nanozyme conjugated with HRP-labeled antibody: EDC·HCl, NHS and Ce@BSA-FA were dissolved in deionized water and stirred for activation at room temperature. After activation, p16 was added. INK4a The antibody and horseradish peroxidase HRP underwent a cross-linking reaction, and the uncross-linked p16 was removed by centrifugation and dialysis. INK4a The antibody and HRP were diluted to the working solution concentration using the preservation solution.

2. The preparation method of the dual-targeted tumor cell detection reagent based on cerium-based nanozyme according to claim 1, wherein, The said Step 1 includes: Mix 0.1 mol / L Ce(NO3)3·6H2O and 0.1 mol / L aqueous FA solution and stir to form a stable complex; add bovine serum albumin BSA with a concentration of 25 mg / mL, and mix in a 37°C constant temperature water bath; use 1 mol / L KOH solution to adjust the pH value of the reaction system to 13, and the solution color changes from milky white to black. Continue to stir at a constant temperature until the reaction ends; transfer the obtained reaction product solution to a dialysis bag with a molecular weight cut-off of 1000 Da, and dialyze and purify with deionized water; centrifuge and concentrate the dialyzed suspension through a 10 kDa ultrafiltration centrifugal tube at 4000 rpm, and repeat washing the collected precipitate three times with deionized water to remove residual reagents; obtain a black solid powder Ce@BSA-FA after vacuum freeze-drying.

3. The preparation method of the dual-targeted tumor cell detection reagent based on cerium-based nanozyme according to claim 1, characterized in that, The said Step 2 includes: Dissolve 57 mg EDC·HCl, 85.5 mg NHS, and 6 mg Ce@BSA-FA in 9 mL deionized water, activate and stir at room temperature for 8 hours; after activation, add ki67 antibody and alkaline phosphatase AP thereto, and continue to stir at room temperature for 24 hours; after the reaction ends, centrifuge to retain the precipitate, dialyze for 24 hours to remove the uncross-linked ki67 antibody and AP, and dilute to the working solution concentration with the preservation solution.

4. The preparation method of the dual-targeted tumor cell detection reagent based on cerium-based nanozyme according to claim 1, wherein, The said Step 3 includes: Dissolve 57 mg of EDC·HCl, 85.5 mg of NHS, and 6 mg of Ce@BSA-FA in 9 mL of deionized water, and activate and stir at room temperature for 8 hours; after activation, add p16 INK4a antibody and horseradish peroxidase HRP, and continue to stir at room temperature for 24 hours; after the reaction, centrifuge to collect the precipitate, and dialyze for 24 hours to remove the uncrosslinked p16 INK4a antibody and HRP, and dilute to the working solution concentration with the preservation solution.

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