A magnetic nano-imprinted material of abasic endonuclease and preparation and application thereof

Abstract

The application discloses a kind of base-removing endonuclease magnetic nano imprinting material and its preparation and application.First, avidin is covalently modified on the surface of magnetic nanoparticles, then biotin is combined to reach saturation state, then template molecule APE1 is combined and fixed, then phenylboronic acid derivative is added for modification, then dopamine is added for in-situ polymerization and surface imprinting, then two different molecular weight mPEG-NH2 are used to double close the surface, finally remove template molecule, obtain the magnetic nano imprinting material of APE1.The affinity and specificity of the magnetic nano imprinting material to APE1 and anti-interference ability have breakthrough progress compared with prior art, high-purity APE1 protein can be obtained from complex biological samples (including but not limited to living cells, cell extracts, exosome extracts, etc.) simply and quickly, in-situ capture and separation and purification of APE1 in living cells are realized, which is helpful for detailed and in-depth study on structural change and functional regulation of APE1 in living cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of material preparation, and relates to preparation of a magnetic nano-affinity adsorption material, characterization of the magnetic nano-affinity adsorption material, and application of the magnetic nano-affinity adsorption material in specific identification, capture, separation and enrichment, and purification of target proteins, in particular to a magnetic nano-imprinted material of an apurinic / apyrimidinic endonuclease 1 and a preparation method and application thereof. BACKGROUND

[0002] Apurinic / apyrimidinic endonuclease 1 / oxidation-reduction effector-1 (APE1 for short) is a multifunctional enzyme, which is essential for maintaining cellular homeostasis. As a redox-dependent regulatory factor, APE1 can regulate multiple transcription factors, including NF-kB, AP-1, HIF-1a and STAT3, and plays a crucial role in regulating cell signaling, cell aging and inflammatory pathways. In the nucleus, APE1 is an apurinic / apyrimidinic endonuclease in the base excision repair pathway, and this repair function also regulates the transcription of cytokine genes such as TNFα. The multifunctionality of APE1 makes it a key regulator in the pathogenesis of various diseases such as cancer and nervous system diseases.

[0003] APE1 contains 318 amino acids and has a theoretical molecular weight of 36 kDa. It is currently believed that the complex subcellular localization and multiple functions of APE1 are regulated by the cleavage of the signal sequence at the N-terminus and various post-translational modifications. By separating and enriching APE1 protein in cell lysates and tissue lysates using immunomagnetic beads, acetylation modification and ubiquitination modification of multiple amino acid residues of APE1 have been identified. However, the research results also raise more challenging questions. For example, in the redox functional region of APE1 sequence located at 34-127 aa, multiple ubiquitination sites such as K52, K58, K79, K85, K98, K103 and K125 have been identified, but only two acetylation sites of K85 and K125 have been identified. Do acetylation occur on other sites, but the function is completed and then deacetylated so quickly that it is not detected? How do different post-translational modifications distribute in the cytoplasm and nucleus? Under what conditions does the change of post-translational modification start? Little is known about these issues. To obtain more comprehensive information about post-translational modification changes, methods need to be developed to capture APE1 protein in different cell states in situ in living cells to reveal the dynamic changes of post-translational modification.

[0004] Current available immunomagnetic beads are generally designed to interact with the surface of target cells or to bind to proteins released after cell lysis [5, 6, 8]. There is no report that antibody-modified materials can effectively enter living cells and function normally in the complex intracellular environment. This is closely related to the fact that antibodies need to be exposed on the surface of magnetic beads to function, but are easily destroyed and degraded in living cells. We found strong binding between APE1 and avidin (AVD) in our early studies

[13] . By fixing APE1 on the surface of avidin-modified magnetic cores, and then using the strong adhesion and room temperature self-polymerization properties of dopamine to molecularly imprint APE1 under mild conditions, the obtained magnetic molecularly imprinted nanoparticles have high affinity and specific recognition ability for APE1, and can separate and enrich APE1 from complex biological samples (such as serum, cell lysate, etc.) [14-15]. However, when these materials are used for in situ capture and purification of APE1 in living cells, they are severely interfered by the intracellular environment, and their adsorption capacity, anti-interference ability and purification effect cannot meet the needs of subsequent post-translational modification research and practical application.

[0005] In view of the above problems, it is urgent to develop a new type of high-efficiency adsorption separation material with strong anti-interference ability, large adsorption capacity, and the ability to enter living cells to capture APE1 in situ and obtain high-purity target protein, which can provide a powerful research tool for fully and systematically revealing the rules of structural changes of APE1 in cells and its regulatory relationship with various functions.

[0006] References:

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[0008] 2)Shah F,Logsdon D,Messmann RA,et al.Exploiting the Ref-1-APE1 node in cancer signaling and other diseases:from bench to clinic.npj Precision Onc.2017,1:19.

[0009] 3) Oliveira TT, Coutinho LG, de Oliveira LOA, Timoteo ARdS, Farias GC, Agnez-Lima LF. APE1 / Ref-1 Role in Inflammation and Immune Response. Frontiers in Immunology. 2022, 13:793096.

[0010] 4) Weaver TM, Hoitsma NM, Spencer JJ, et al. Structural basis for APE1 processing DNA damage in the nucleosome. Nature Communications. 2022, 13:5390.

[0011] 5) Nassour H, Wang Z, Saad A, et al. Peroxiredoxin 1 interacts with and blocks the redox factor APE1 from activating interleukin-8 expression. Scientific Reports. 2016, 6:29389.

[0012] 6) Wu HH, Cheng YW, Chang JT, Wu TC, Liu WS, Chen CY, Lee H. Subcellular localization of apurinic endonuclease 1 promotes lung tumor aggressiveness via NF-kappa B activation. Oncogene. 2010, 29:4330-4340.

[0013] 7) Maynard S, Hejl A.M, Dinh T.S.T, Keijzers G, Hansen A.M, Desler C, et al., Defective Mitochondrial Respiration, Altered Dntp Pools and Reduced APE nuclease 1 Activity in Peripheral Blood Mononuclear Cells of Alzheimer's Disease Patients. Aging 2015, 7: 793-815.

[0014] 8) Lopez DJ, Rodriguez JA, S. Molecular Mechanisms Regulating the DNA Repair Protein APE1 : A Focus on Its Flexible N-Terminal Tail Domain. International Journal of Molecular Sciences. 2021, 22: 6308.

[0015] 9) Chattopadhyay R, Das S, Maiti AK, et al. Regulatory role of human AP-endonuclease (APE1 / Ref-1) in YB-1-mediated activation of the multidrug resistance gene MDR1. Molecular and Cellular Biology. 2008, 28(23): 7066-7080.

[0016] 10) Sengupta S, Mantha AK, Mitra S, Bhakat KK. Human AP endonuclease (APE1 / Ref-1) and its acetylation regulate YB-1-p300 recruitment and RNA polymerase II loading in the drug-induced activation of multidrug resistance gene MDR1. Oncogene. 2011, 30(4):482-493.

[0017] 11) Lirussi L, Antoniali G, Vascotto C, et al. Nucleolar accumulation of APE1 depends on charged lysine residues that undergo acetylation upon genotoxic stress and modulate its BER activity in cells. Molecular Biology of the Cell. 2012, 23(20):4079-4096.

[0018] 12) Bhakat KK, Izumi T, Yang SH, Hazra TK, Mitra S. Role of acetylated human AP-endonuclease (APE1 / Ref-1) in regulation of the parathyroid hormone gene. The EMBO Journal. 2003, 22(23):6299-6309.

[0019] 13) Zhai J, Liu Y, Huang S, Fang S, Zhao M. A specific DNA-nanoprobe for tracking the activities of human apurinic / apyrimidinic endonuclease 1 in living cells. Nucleic Acids Research. 2017, 45(6):e45.

[0020] 14) Zhai J, Zhao M, Cao X, Li M, Zhao M. Metal-ion-responsive bionanocomposite for selective and reversible enzyme inhibition. Journal of the American Chemical Society. 2018, 140: 16925-16928.

[0021] 15) Xie H, Sun Y, Zhang R, Zhang Y, Zhao M. Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein. Biopolymers. 2023, 114: e23537. SUMMARY

[0022] In view of the above problems, the purpose of the present application is to provide a magnetic nano-imprinted material of apurinic / apyrimidinic endonuclease 1 / oxidation-reduction effector-1 (APE1) and its preparation method and application, to obtain an APE1 magnetic nano-imprinted material with strong anti-interference ability, large adsorption capacity and the ability to enter living cells, so as to realize in-situ capture and separation and purification of APE1 in living cells.

[0023] To achieve the above technical purposes, the present application first covalently modifies a large amount of avidin (AVD) on the surface of magnetic nanoparticles, adds biotin to completely convert the four subunits of AVD into biotin binding state, removes the excess biotin molecules, and then adds a template molecule APE1 for binding and fixing. Then, a benzene boronic acid derivative (such as 3-hydroxybenzene boronic acid, B-OH) is added to modify the glycosyl on the surface of AVD and pre-assemble with the amino acid on the surface of APE1. Then, dopamine monomers are added for in-situ polymerization and surface imprinting. After the reaction is completed, a large molecular weight (such as 20k) amino-modified methoxy polyethylene glycol (mPEG-NH2) is first used to modify the surface, and then a small molecular weight (such as 2k) mPEG-NH2 is further used to block the non-specific binding sites. Finally, the template molecule is removed to obtain the APE1 magnetic nano-imprinted material. The magnetic nano-imprinted material can be quickly introduced into living cells under the action of a magnetic field. Compared with the currently reported materials [14, 15], the APE1 magnetic nano-imprinted material of the present application has stronger affinity for APE1, larger adsorption capacity, and stronger anti-interference ability. Similarly, for the extraction and purification of APE1 in cell lysate, the operation steps of the present application are simpler, the required time is shorter, the extracted protein has higher purity, and the ability to detect post-translational modifications is greatly enhanced. In particular, the magnetic nano-imprinted material of the present application realizes the in-situ capture and separation and purification of APE1 in living cells, which is not possessed by the reported materials. These advances provide important method support for further revealing the molecular regulation mechanism of APE1 in cells to exert multiple functions.

[0024] The present application adopts the following technical solutions:

[0025] A dealkylation endonuclease magnetic nano-imprinted material sequentially comprises a magnetic core, a silica layer, an avidin layer, a polydopamine layer and a PEG double blocking layer from inside to outside, characterized in that the avidin layer is composed of avidin saturated with biotin and modified by a benzene boronic acid derivative, the polydopamine layer has an imprint of dealkylation endonuclease, and the PEG double blocking layer is composed of two amino-modified methoxy polyethylene glycols with different molecular weights.

[0026] Further, the magnetic core is preferably a magnetic nanoparticle with a diameter of 13-15 nm, the thickness of the silica layer coated on the magnetic core is 1-2 nm, the thickness of the avidin layer is less than or equal to 5 nm, the thickness of the polydopamine layer is 3-5 nm, and the thickness of the PEG double blocking layer is 20-30 nm.

[0027] Further, the magnetic core comprises a ferromagnetic nanoparticle, preferably a Fe3O4 magnetic nanoparticle or a γ-Fe2O3 magnetic nanoparticle.

[0028] Further, in the avidin layer, the biotin binding pockets on the four subunits of avidin are all filled with biotin molecules, and the protein surface has no extra biotin molecules; the glycosyl of avidin is modified with phenylboronic acid derivatives. The phenylboronic acid derivatives can be 3-hydroxyphenylboronic acid (B-OH) and / or 3-aminophenylboronic acid (B-NH2).

[0029] Further, the PEG double blocking layer is composed of an amino-modified methoxy polyethylene glycol (mPEG-NH2) with a large molecular weight (such as 15-30k) and an amino-modified methoxy polyethylene glycol with a small molecular weight (such as 1.5-3k). In some embodiments of the present application, the PEG double blocking layer is composed of 20k mPEG-NH2 and 2k mPEG-NH2.

[0030] Further, the expression of the above-mentioned abasic endonuclease magnetic nano-imprinted material can be represented as MNP@Bio-AVD*@PDA@PEG-2. Wherein, MNP represents a magnetic nanoparticle with a silica layer, such as Fe3O4@SiO2 or γ-Fe2O3@SiO2, Fe3O4 represents a magnetite core, γ-Fe2O3 represents a γ-ferroferric oxide magnetic core, and SiO2 represents a silica layer; Bio-AVD represents an avidin-biotin complex that has been saturated with biotin; Bio-AVD* represents an avidin layer that has been saturated with biotin and modified with phenylboronic acid derivatives; PDA represents a polydopamine layer; and PEG represents an amino-modified methoxy polyethylene glycol layer composed of two kinds of mPEG-NH2.

[0031] The present application also provides a preparation method of the above-mentioned abasic endonuclease magnetic nano-imprinted material, which comprises: first covalently connecting avidin on the surface of the silica layer, then adding biotin to completely convert the four subunits of avidin into biotin binding state; after removing the excess biotin molecules, adding a template molecule APE1 for binding and fixation; then adding phenylboronic acid derivatives to modify the glycosyl on the surface of avidin and pre-assemble with the amino acids on the surface of APE1; then performing a dopamine polymerization reaction on the outer layer, and then stepwise modifying the surface of the polydopamine with two kinds of amino-modified methoxy polyethylene glycol with different molecular weights; finally eluting the template molecule APE1 to obtain the final abasic endonuclease magnetic nano-imprinted material.

[0032] In some embodiments of the present application, the specific steps of the above-mentioned preparation method are as follows:

[0033] 1) Ultrasonically disperse the magnetic core coated with a carboxylated silica layer in pure water, add a compound for activating the carboxyl group, and shake at room temperature (all below refer to 15-28℃) for a period of time to obtain solution A;

[0034] 2) Add avidin to solution A, shake the reaction at room temperature to covalently link avidin to the surface of the silica layer, magnetically separate and wash to remove unreacted material, then re-disperse the resulting magnetic nanoparticles in Tris solution by sonication as solution B;

[0035] 3) Add biotin to solution B to convert all four subunit biotin-binding pockets of the avidin to biotin-bound state; magnetically separate and wash to remove unbound material, then re-disperse the resulting magnetic nanoparticles in Tris solution by sonication as solution C;

[0036] 4) Add APE1 to solution C, incubate at room temperature for a period of time to allow APE1 to bind to avidin, then add a phenylboronic acid derivative for modification as solution D;

[0037] 5) Add dopamine to solution D for polymerization, magnetically separate and wash to remove unreacted material, then re-disperse the resulting magnetic nanoparticles in Tris solution by sonication as solution E;

[0038] 6) Add larger molecular weight amino-modified methoxypolyethylene glycol (mPEG-NH2) to solution E to modify the surface of the polydopamine, magnetically separate and wash to remove unreacted material, then re-disperse the resulting magnetic nanoparticles in Tris solution by sonication, and add smaller molecular weight mPEG-NH2 for secondary modification of the surface of the magnetic nanoparticles, magnetically separate and wash to remove unreacted material;

[0039] 7) The resulting magnetic nanoparticles are washed with elution buffer to remove the template molecule APE1, dried at room temperature to obtain the magnetic nanoimprinted material of the apurinic endonuclease.

[0040] In the above step 1), the carboxylated silica-coated magnetic core is ultrasonically dispersed in pure water at a final concentration of 0.1-0.5 mg / mL; preferably, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added to activate the carboxyl groups, wherein the concentration of EDC is 1-4 mg / mL and the concentration of NHS is 2-8 mg / mL; the activation time at room temperature is 30-60 minutes. Among them, EDC can be replaced by N,N'-dicyclohexyl carbodiimide (DCC) or N,N'-diisopropyl carbodiimide (DIC).

[0041] In the above step 2), the concentration of avidin is 40-200 nM. The avidin is added to make the ratio of avidin to magnetic nanoparticles in the final concentration of the mixed solution 400 nmol / g, and the reaction is shaken at room temperature for 8-16 hours. When the avidin is covalently connected to the surface of the silica layer, the concentration of the nanoparticles should not be too high in order to fully disperse the nanoparticles, and the concentration is preferably 0.1-0.5 mg / mL. After the reaction is completed, in order to remove the unreacted substances in the system, the system can be washed 1-3 times with deionized water, and then ultrasonically dispersed in a Tris solution, the ultrasonic dispersion time is 5-10 minutes, the concentration of the Tris solution is 10-15 mM, and the pH is 8.0-8.5.

[0042] In the above step 3), the concentration of biotin is 200-400 nM, and the concentration of magnetic nanoparticles is 0.1-0.2 mg / mL, and the binding reaction is carried out at room temperature.

[0043] In the above step 4), the concentration of APE1 is 40-80 nM, and the incubation is carried out at room temperature for 20-40 minutes; the benzene boronic acid derivative is preferably 3-hydroxybenzene boronic acid, and the concentration is 600-800 nM, and the incubation is carried out at room temperature for 30-60 minutes.

[0044] In the above step 5), the concentration of dopamine is 0.08-0.12 mg / mL, and the polymerization reaction can be carried out at room temperature, and the time can be between 80-100 minutes, and preferably 90 minutes.

[0045] In the above step 6), both steps of polyethylene glycol modification are carried out at 37°C, and the preferred methoxy polyethylene glycol is 20k and 2k amino-modified, and the concentration of both is 5-10 mg / mL, and the time of the two steps of reaction is 8-12 hours.

[0046] In the above step 7), preferably, the magnetic nanoparticles are ultrasonically dispersed and washed in an elution buffer for multiple times, and then ultrasonically dispersed and washed with ethanol for multiple times, and each washing time is 10-15 minutes. If not used immediately, it should be stored at 4°C. The elution buffer contains 100 mM Bis-propane-Cl, 100 mM MgCl2, 10 mM DTT, and 0.1% Triton X-100.

[0047] Further, the application also provides the application of the above-mentioned apurinic / apyrimidinic endonuclease magnetic nano-separation material in the identification, capture, separation and purification, and enrichment of target protein molecules APE1 in actual biological samples (including but not limited to living cells, cell lysates, tissue lysates, etc.).

[0048] For example, the method for separating and purifying APE1 from a cell lysate can comprise: adding the abasic endonuclease magnetic nano-separation material to the cell lysate for room temperature adsorption, then magnetic separation, collecting the magnetic nanoparticles, washing to remove impurities, and finally eluting APE1 with an elution buffer.

[0049] For another example, the method for in situ capturing and separating and purifying APE1 in living cells comprises: magnetically transfecting the abasic endonuclease magnetic nano-separation material into living cells, culturing for a period of time, then breaking the cells, magnetic separation to collect the magnetic nanoparticles, and finally eluting APE1 with an elution buffer after washing.

[0050] The present application has the following beneficial effects:

[0051] The affinity between AVD and abasic endonuclease I (APE1) is enhanced by 6 times after AVD is converted into a biotin saturated binding state, which means that the amino acid distribution of the AVD binding interface has changed and the binding to APE1 is more close. After binding to the template molecule APE1, a phenylboronic acid derivative is added for reaction, which on the one hand closes the sugar group exposed on the surface of AVD, and on the other hand non-covalently pre-assembles with the polar amino acids on the surface of APE1. In the subsequent polymerization process of dopamine, a more fine imprint pocket is formed around the template molecule. After the completion of the dopamine polymerization reaction, the outermost layer is modified by first large molecular weight PEG and then small molecular weight PEG, which can completely eliminate the non-specific adsorption of different interferents. In summary, the present application enhances the affinity and specificity of the material to APE1 through the auxiliary action of two small molecules of biotin and phenylboronic acid derivative. The non-specific binding and interference of irrelevant proteins are further eliminated by the insertion type closing of double-layer PEG.

[0052] Compared with the best reported technology

[15] , which is also used for the extraction and purification of APE1 in cell lysate, the operation steps of the present application are simpler, the required time is shorter, the purity of the extracted protein is higher, and the ability to detect post-translational modifications is greatly enhanced. In particular, in-situ extraction and separation and purification in living cells are achieved.

[0053] In summary, the present application discloses an abasic endonuclease magnetic nano-imprinted material, a preparation method and application thereof. The magnetic nano-imprinted material is a nanoparticle with a diameter of 50±5 nm. The affinity and specificity to the target protein and the anti-interference ability of the present application have made a breakthrough progress compared with the existing methods. The present application can simply and quickly obtain high-purity APE1 protein from complex biological samples (including but not limited to living cells, cell extracts, exosome extracts, etc.), which is helpful for more detailed and in-depth research on the structural changes and functional regulation of APE1 in living cells. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The effect of different concentrations of biotin added in Tris buffer on the binding ability of MNP@Bio-AVD to APE1 was compared.

[0055] Figure 2 The effect of avidin binding biotin on the binding constant of APE1 was compared by isothermal titration calorimetry, including (a) the binding of free avidin to APE1, (b) the binding of avidin / biotin complex (binding ratio = 1:4, abbreviated as Bio-AVD) to APE1; binding buffer: 10 mM Tris-HCl, pH = 8.5; AVD or Bio-AVD concentration: 20 μM; APE1 concentration: 200 μM.

[0056] Figure 3 The adsorption binding amount of different surface modified magnetic beads (MNP@COOH, MNP@AVD and MNP@SA) to different proteins was compared.

[0057] Figure 4 The adsorption binding amount of different surface modified magnetic beads (MNP@COOH, MNP@AVD and MNP@Bio-AVD) to different proteins under different buffer conditions was compared.

[0058] Figure 5 The structure of (a) different phenylboronic acid derivatives and (b) the selectivity factor of different phenylboronic acid derivative modified dopamine imprinting materials (MIP0) and non-imprinting materials (NIP0) to APE1 were shown, wherein the control group (pDA) was MIP0 and NIP0 without phenylboronic acid modification.

[0059] Figure 6 It is a schematic diagram of the preparation process of the APE1 magnetic nano imprinting material (MIP-APE1) of the application.

[0060] Figure 7 It is a transmission electron micrograph of magnetic nanoparticles MNP@COOH and magnetic nano-imprinted particles MIP-APE1.

[0061] Figure 8 It is a Scatchard plot of the binding amount of (a) magnetic nano-imprinted particles MIP-APE1 and (b) non-imprinted magnetic nano-particles NIP-APE1 to APE1.

[0062] Figure 9 It is the selectivity of MIP-APE1 and NIP-APE1 to several common nucleases. The concentration of each enzyme in the adsorption experiment was 10 nM.

[0063] Figure 10 It is the APE1 activity determination result of HeLa cell lysate after being adsorbed by MIP-APE1 and desorbed and recovered.

[0064] Figure 11 Figure 1 is the SDS-PAGE determination result of the target protein obtained by selective capturing and desorption of APE1 in live HeLa cells and HeLa cell lysate using MIP-APE1. Lane 1: HeLa cell lysate without any treatment; Lane 2: eluate of MIP-APE1 in live cell extract; Lane 3: eluate of MIP-APE1 in cell lysate extract; Lane 4: APE1 standard.

[0065] Figure 12 Figure 2 is the mass spectrum of K7 site acetylation (K(Acetyl)GAVAEDGDELRTEPEAK) of APE1 extracted in cell lysate.

[0066] Figure 13 Figure 3 is the mass spectrum of K197 site acetylation (FLK(Acetyl)GLASR) of APE1 extracted in cell lysate.

[0067] Figure 14 Figure 4 is the mass spectrum of K228 site acetylation (GNKK(Acetyl)NAGFTPQER) of APE1 extracted in cell lysate.

[0068] Figure 15 Figure 5 is the activity determination result of APE1 extracted after MIP-APE1 was introduced into live cells and the remaining APE1 in the cells.

[0069] Figure 16 Figure 6 is the mass spectrum analysis result of acetylation modification on K58 of APE1 captured in live cells.

[0070] Figure 17 Figure 7 is the mass spectrum analysis result of ubiquitination modification on K63 of APE1 captured in live cells.

[0071] Figure 18 Figure 8 is the mass spectrum analysis result of acetylation modification on K203 and S-nitrosylation modification on C208 of APE1 captured in live cells. DETAILED DESCRIPTION

[0072] The present application will be described in detail below with reference to the accompanying drawings and examples. It should be understood by those skilled in the art that the following examples will help to understand the present application, but do not limit the content of the present application. Various modifications and changes can be made within the spirit and scope of the present application, and the protection scope of the present application should be defined by the appended claims.

[0073] Example 1, Effect of biotin on the interaction between avidin (AVD) and APE1

[0074] Our research group first discovered and reported that avidin has a strong binding interaction with APE1, but the binding interface between them is not clear. It has been reported that after avidin binds to biotin, the loop structure of 36-44 will change in conformation, its ability to bind DNA is enhanced, and it can resist protease degradation better. We explored whether the binding of biotin would have a significant impact on the interaction between avidin and APE1.

[0075] After the surface modified avidin magnetic beads MNP@AVD (200 nmol / g) were incubated with an excess of biotin (AVD: biotin = 1:6.25, molar ratio), the excess biotin was removed by washing with Tris buffer, and then the obtained MNP@Bio-AVD (Bio-AVD represents avidin-biotin complex that has been saturated with biotin) was dispersed in Tris buffer containing 20 nM APE1 and different concentrations of biotin, respectively, to determine its adsorption capacity for APE1, and compared with the adsorption capacity of APE1 by directly using MNP@AVD in Tris buffer.

[0076] From Figure 1 It can be seen that when the AVD on the surface of the magnetic beads is completely converted into a biotin binding state, the adsorption capacity for APE1 in Tris buffer is more than that when directly using MNP@AVD to bind, indicating that the binding of biotin is beneficial to enhance the binding interaction between AVD and APE1. However, if the binding buffer contains an excess of biotin, as the biotin concentration increases, the binding interaction of MNP@Bio-AVD with APE1 will gradually decrease, indicating that free biotin in the solution will significantly interfere with the binding interaction between them. This subtle influence is first discovered in the study.

[0077] We continued to use isothermal titration calorimetry (ITC) to compare the changes in avidin binding to APE1 before and after binding to biotin. From Figure 2 The determination results can be obtained that the binding constant of avidin to APE1 is 1.5 x 10 6 M -1 When avidin binds to biotin molecules to saturation, its affinity for APE1 increases by more than 6 times, and the binding constant reaches 9.2 x 10 6 M -1Simultaneously, the binding ratio changed from 1:4 to 1:2. It is speculated that without biotin binding, the tetramer surface of avidin exhibits both specific binding and non-specific adsorption of APE1. The exothermic interference of non-specific adsorption causes the measured binding ratio (the point of maximum curve slope) to occur earlier. When the biotin-binding pockets of all four avidin subunits are filled with biotin molecules, the charge distribution and hydrophobic regions on the AVD surface change. These changes significantly weaken non-specific adsorption, and the specific binding between avidin and APE1 becomes dominant. Key interaction sites can better match, thereby enhancing the binding force. This provides important evidence for improving the performance of existing APE1 molecularly imprinted materials.

[0078] Example 2: Effect of different phenylboronic acid derivatives on polydopamine (PDA) selectivity on APE1

[0079] To fundamentally improve the selectivity of magnetic nanomaterials for APE1, we first compared the selectivity characteristics of magnetic beads with different surface modifications for APE1. From Figure 3 The results showed that the binding amounts of APE1 (36kDa, pI 8.3), deoxyribonuclease I (DNase I, 30kDa, pI 5.0), and 3' exonuclease (TREX1, 33kDa, pI 8.1) on the carboxyl-modified magnetic bead MNP@COOH surface were relatively similar. When the magnetic bead surface was modified with avidin (AVD), the binding amount of APE1 increased significantly, the binding amount of DNase I decreased significantly, and the binding amount of TREX1 also increased, but the increase was less than that of APE1. When the magnetic bead surface was modified with streptavidin (SA), the binding amount of APE1 decreased significantly, the binding amount of DNase I was greater than that of MNP@AVD, and the binding amount of TREX1 was similar to that of MNP@AVD.

[0080] Based on the results of Example 1, we further compared the adsorption of three proteins by different surface-modified magnetic beads in different buffer solutions. Figure 4 As shown, MNP@Bio-AVD exhibited the highest adsorption capacity for APE1 in Tris buffer, representing the most selective condition compared to the other two proteins. Notably, TREX1 showed high binding capacity under various assay conditions, with the highest binding capacity observed in Tris buffer containing excess biotin. These results indicate that TREX1 exhibits strong non-specific adsorption on the aforementioned magnetic beads with different surface modifications, necessitating specific methods to eliminate its interference with the selective capture and purification of target proteins.

[0081] In this example, we first take DNase I as a control protein to explore the method of improving the selectivity of materials to APE1. Phenylboronic acid can covalently bind to sugar, glycoprotein and other cis-diol containing molecules in alkaline aqueous solution, and can also complex the vicinal diol fragments on the polydopamine. We adsorb APE1 on the carboxyl modified magnetic beads MNP@COOH, add dopamine for imprinting reaction, and then add phenylboronic acid derivatives with different functional groups Figure 5 Further modification of the imprinting site is carried out in (a) to investigate the contribution of different phenylboronic acid derivatives to the selectivity of the imprinting material (MIP0) to APE1. As a control, we also prepared a non-imprinted material (NIP0) without adding APE1 template molecules.

[0082] After removing the template, we measured the adsorption amount Q (nmol / g) of APE1 and DNase I of MIP0 and NIP0 modified by different phenylboronic acid derivatives, respectively. The selectivity factor = Q(APE1) / Q(DNase I) is used as a comparison parameter, and the results are shown in Figure 5 As can be seen from (b), compared with pDA-MIP0 without phenylboronic acid derivative modification, the selectivity factor of each MIP material modified by phenylboronic acid derivative to APE1 is increased, indicating that the modification of phenylboronic acid derivative helps to improve the selectivity of the imprinting material to APE1. By comparing the NIP materials without imprinting but only with phenylboronic acid derivative modification, B-C 12 H 25 The modification of B-Nap has little effect. It can be seen that the phenylboronic acid derivative with polar group is more conducive to the combination of the material to APE1. In Figure 5 (b), among all the materials for comparison, the imprinting material modified by phenylboronic acid with hydroxyl group (B-OH) has the most significant contribution to the selectivity of the material to APE1. It is speculated that B-OH with hydroxyl group can interact with APE1 through hydrogen bond to enhance the affinity to APE1 and reduce the non-specific adsorption of other proteins. According to this result, we select B-OH as the small molecule auxiliary ligand for preparing APE1 magnetic nano-imprinting material in the subsequent experiment.

[0083] Example 3, preparation of magnetic nano-imprinting material (MIP-APE1)

[0084] The process of preparing APE1 magnetic nano-imprinting material according to the present application is shown in Figure 5 , which specifically comprises the following steps:

[0085] A 0.5 mg / mL solution of 1.0 mg Fe3O4@SiO2@COOH was prepared by ultrasonic dispersion in 2.0 mL of pure water at room temperature. The carboxyl groups were activated by adding 4.0 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 10.0 mg of N-hydroxysuccinimide (NHS) and shaking for 30 minutes at room temperature. A final concentration of 200 nM of avidin was added and shaken for 8 hours at room temperature. After the reaction was completed, the unreacted material was removed by magnetic separation and the material was washed three times with deionized water. The resulting material was designated MNP@AVD.

[0086] MNP@AVD was ultrasonically dispersed in a 10 mM Tris (pH 8.5) solution at a final concentration of 0.2 mg / mL. Biotin was added at a final concentration of 350 nM to convert the avidin to a biotin-saturated binding state. The unbound material was removed by magnetic separation and washing, and the resulting magnetic nanoparticle product was ultrasonically redispersed in the Tris solution. APE1 was added at a final concentration of 80 nM and incubated for 30 minutes at room temperature. After 3-hydroxybenzeneboronic acid was added at a final concentration of 800 nM and reacted for 30 minutes, dopamine was added at a final concentration of 0.1 mg / mL and polymerized for 1.5 hours at 37 °C. The unreacted material was removed by magnetic separation and washing, and the resulting magnetic nanoparticle product was ultrasonically redispersed in the Tris solution. 20k amino-modified methoxypolyethylene glycol (mPEG-NH2) was added at a final concentration of 5 mg / mL and reacted for 8 hours. The unreacted material was removed by magnetic separation and washing, and the resulting magnetic nanoparticle product was ultrasonically redispersed in the Tris solution. 2k mPEG-NH2 was added at a final concentration of 5 mg / mL and reacted for 8 hours. The unreacted material was removed by magnetic separation and washing. The resulting product was washed three times with elution buffer (100 mM Bis-propane-Cl, 100 mM MgCl2, 10 mM DTT, 0.1% Triton X-100) and three times with ethanol. The product was dried overnight at room temperature to obtain the magnetic nanoprinted material of the apurinic endonuclease. A non-printed magnetic nanoparticle (NIP-APE1) was synthesized as a control by not adding the template molecule APE1, and the other steps were the same. Figure 7 Transmission electron micrographs of (a) magnetic nanoparticles MNP@COOH and (b) magnetic nanoprinted particles MIP-APE1.

[0087] Example 4, Determination of the affinity and selectivity of the magnetic nanoparticles for APE1

[0088] Adsorption experiments were conducted by adding different concentrations of APE1 to Tris solutions containing 0.2 mg / mL MIP-APE1 and NIP-APE1, with final concentrations of APE1 of 2.5 nM, 4 nM, 5 nM, 7.5 nM, 10 nM, 12.5 nM, 15 nM, and 20 nM, respectively. After adsorption, the concentration of remaining APE1 in the supernatant was detected using a DNA probe. The obtained data were analyzed using the Scatchard equation.

[0089]

[0090] Among them Q and Q max represents the protein adsorption capacity per unit mass of magnetic nanoparticles and the maximum protein adsorption capacity per unit mass of magnetic nanoparticles, respectively; c is the protein concentration in the supernatant after adsorption reaches equilibrium; K d The dissociation constant between the magnetic nanoparticles and APE1 is given. The measurement results are as follows: Figure 8 As shown.

[0091] according to Figure 8 Based on the data, we calculated the dissociation constants of MIP-APE1 and NIP-APE1 to APE1 to be 2.6 nM and 172 nM, respectively. The dissociation constant of the magnetic nanoimprinted particle MIP-APE1 reaches the nM level, indicating that MIP-APE1 has a high affinity for APE1.

[0092] Add MIP-APE1 or NIP-APE1 to 200 μL of 10 mM Tris (pH 8.5) solution to a final concentration of 0.05 mg / mL, disperse evenly, and then add APE1 or other control protein to a final concentration of 10 nM. Incubate at 37 °C for 30 min for adsorption. Perform magnetic separation, and use DNA probes (as shown in Table 1) to detect the activity of the remaining APE1 or other control protein in the supernatant after adsorption. Calculate the amount of protein that can be desorbed per unit of magnetic nanoimprinted particle.

[0093] Table 1. DNA probe sequences and their optimal buffer solutions used in the examples.

[0094]

[0095] Note: * indicates phosphorothioylation modification; ROX is 6-carboxy-X-rhodamine; BHQ2 is Black Hole quencher 2; BHQ1 is Black Hole quencher 1; FAM is 6-carboxyfluorescein; I indicates inosine deoxyribonucleotide.

[0096] Depend on Figure 9The adsorption experiment data shown can see that the binding amount of magnetic nano-imprinted particles MIP-APE1 to APE1 is much greater than that of NIP material, close to the saturated adsorption capacity. The binding amount of other three enzymes on MIP-APE1 is obviously less than that of APE1, comparable to or lower than that on NIP-APE1, indicating that the imprinting process significantly improves the selectivity of the material. Compared with the results of Figure 4 The imprinting process and the double-layer PEG blocking of the material surface together reduce the non-specific adsorption of TREX1.

[0097] Example 5. Extraction and purification of APE1 in cell lysate and living cells by MIP-APE1

[0098] 1. Extraction and purification steps of APE1 in cell lysate:

[0099] HeLa cells were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% inactivated fetal bovine serum, and the culture temperature was 37°C, and the culture gas was 5% CO2 / 95% air. When the cell coverage area was 70%-80% of the T7 cell culture bottle, the cells were washed 3 times with PBS, scraped with a cell scraper and collected. Resuspend the cells with 1 / 10 original volume of 1×PBS, lyse with an ultrasonic wave disrupter, and after the suspension is clarified, centrifuge at 10,000×g for 10 minutes to remove the insoluble components. Collect the cell lysate and store it at -80°C.

[0100] Add 0.25 mg of MIP-APE1 to 500 μL of lysate with a protein concentration of about 0.1 mg / mL, and adsorb at room temperature for 20 minutes. Magnetic separation, collect the magnetic nanoparticles. Add another 0.25 mg of MIP-APE1 to the remaining supernatant, and adsorb at room temperature for 20 minutes before magnetic separation. Combine the two portions of magnetic nanoparticles, first wash with Tris (10 mM, pH 8.5) for 3 times, and discard the washing solution; then wash with 0.04% Triton X-100 for 1 time, and discard the washing solution; then wash with 1×Buffer 1 (10 mM Bis-propane-Cl, 10 mM MgCl2, 1 mM DTT, pH 7.0) for 1 time, and discard the washing solution; finally, elute the protein with 10×Buffer 1 (100 mM Bis-propane-Cl, 100 mM MgCl2, 10 mM DTT, 0.1% Triton X-100, pH 7.0). Collect the eluate and perform subsequent analysis and characterization.

[0101] From Figure 10As can be observed, the first MIP-APE1 has successfully extracted more than 60% of APE1 from HeLa cell lysate, and the second MIP-APE1 has extracted less than 15% of APE1 from the remaining APE1. The two MIPs obtained by magnetic separation were combined and washed thoroughly, and the recovery rate of APE1 in the final eluent was (85±3)%, indicating that MIP-APE1 can quickly, quantitatively and highly active extract APE1 from HeLa cell lysate.

[0102] Compared with the method we have reported

[15] , the above steps are greatly simplified. In the reported method

[15] , the cell lysate needs to be first salted out by (NH4)2SO4 to remove most of the high-abundance proteins, and then the biotin-modified proteins in the remaining supernatant are removed by streptavidin-modified magnetic beads MNP@SA. After completing the first two steps of pretreatment, APE1 in the remaining protein solution is extracted and purified by APE1 molecularly imprinted magnetic nanoparticles prepared in the reported method. In the method of the present application, there is no need to perform the first two steps of pretreatment, and high-purity APE1 can be directly extracted by MIP-APE1, which fully demonstrates that the new magnetic nano-imprinted particles have better selectivity, higher affinity and stronger anti-interference ability for APE1.

[0103] 2. In-situ selective capture and separation and purification of APE1 in living cells

[0104] HeLa cells were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% inactivated fetal bovine serum, and the culture temperature was 37°C, and the culture gas was 5% CO2 / 95% air. When the cell coverage area was 70%-80% of the T7 cell culture bottle, the cells were washed 3 times with PBS, and then 0.2 mg / mL MIP-APE1 was added, and the culture was carried out at 37°C under the condition of 5% CO2 at the bottom of the magnetic field for 30 min. After the magnetic transfection was completed, the magnetic field was removed, and the MIP-APE1 that did not enter the cells was washed away with PBS, and then the normal culture condition was changed to continue the culture for 30 min. Washed 3 times with 1×PBS, and then resuspended in pre-cooled 1×PBS (containing PMSF, DDT and protease inhibitors). The cells were broken by grinding with a glass homogenizer, and the MIP-APE1 magnetic beads were collected after magnetic separation. The magnetic beads were washed thoroughly and the protein was eluted with 10×Buffer 1 (100 mM Bis-propane-Cl, 100 mM MgCl2, 10 mM DTT, 0.1% Triton X-100, pH 7.0). The eluate was collected for subsequent analysis.

[0105] Figure 11is the result of SDS-PAGE assay of the eluate. As a control experiment, the eluate collected during the purification of HeLa cell lysate in Example 1 was also assayed synchronously. It can be seen that the HeLa cell lysate without any treatment almost has no band at the position of the target protein, while there are high-abundance impurity proteins with similar molecular weight below the position. After MIP-APE1 purification, a single target band can be seen in lane 3, which confirms the high selectivity and anti-interference ability of the magnetic nanoimprint material. Further, from lane 2, it can be seen that the MIP-APE1 introduced into the living cells also obtained the target protein with high purity.

[0106] To confirm that the purified protein is APE1, the bands in lanes 2 and 3 were subjected to in-gel digestion, respectively, and then the digests were analyzed by liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) (Orbitrap Fusion Lumos Tribrid LC-MS, Thermo Scientific). The results show that APE1 is the main component in the protein enriched in lane 3 (Table 2), which verifies the high selectivity of MIP-APE1 in extracting proteins from cell lysate.

[0107] Table 2. Mass spectrometry analysis results of proteins enriched in HeLa cell lysate

[0108]

[0109] Among the identified APE1 peptides in the cell lysate, acetylation of K7 (K(Acetyl)GAVAEDGDELRTEPEAK) ( Figure 12 ), K197 (FLK(Acetyl)GLASR) ( Figure 13 ) and K228 (GNKK(Acetyl)NAGFTPQER) ( Figure 14 ) sites was found. Acetylation of the N-terminal K7 residue is positively correlated with the transcriptional regulatory activity of APE1 and cell survival. The function of acetylation at K197 site is not clear, which is speculated to regulate the interaction of APE1 with other proteins and control the BER process. K228 residue is located in the DNA binding loop of APE1, and its acetylation may regulate the DNA repair process by affecting DNA binding. In addition, the DNA binding sites (K227, K228 and K276) of APE1 are also reported as the main interaction region of APE1 with Polβ to form a ternary complex APE1-DNA-Polβ34. Therefore, acetylation of K228 may affect the ability of APE1 to recruit downstream proteins.

[0110] For MIP-APE1 introduced into living cells, we used fluorescent probes (Table 1) to measure the activity of APE1 extracted by MIP-APE1. Figure 15 The activity of APE1 in the eluate of MIP-APE1 collected by magnetic separation after the cell membrane of living cells containing MIP-APE1 was broken by a glass homogenizer was demonstrated. The measurement results of APE1 in the remaining cell release were also given. It can be seen that MIP-APE1 successfully captured and enriched APE1 with high activity, and the activity was even higher than that of the remaining part in the cells. APE1 is usually mainly distributed in the nucleus, and the content in the cytoplasm and mitochondria is relatively low. MIP-APE1 cannot enter the nucleus, Figure 15 The results to some extent reflect the behavior of APE1 migrating from the nucleus to the cytoplasm and being captured by MIP-APE1 magnetic beads.

[0111] For Figure 11 The mass spectrometry analysis results of APE1 captured in living cells enriched in lane 2 in FIG. 6 not only confirmed the high purity of APE1, but also identified multiple post-translational modifications that have not been reported so far. For example, acetylation modification on K58 ( Figure 16 ), ubiquitination modification on K63 ( Figure 17 ), acetylation modification on K203 and S-nitrosylation modification on C208 ( Figure 18 ), etc.

[0112] These post-translational modifications were not detected in APE1 extracted from cell lysates, and there is no literature report so far. The ubiquitination modification on K63 may regulate the migration of APE1 from the nucleus to the cytoplasm together with S-nitrosylation of C208 and acetylation of K203. It is worth noting that APE1 with these post-translational modifications may be de-modified or removed after being transported to the cytoplasm, so that these post-translational modifications are difficult to be detected in traditional cell lysates or tissue extracts. MIP-APE1 can enter living cells and timely capture and retain important information of these dynamic post-translational modifications, which fully proves that the material prepared by the present application is superior to the existing material and has the powerful ability to obtain the complete structure information of proteins and dynamic changes.

Claims

1. A magnetic nanoimprinted material for debasing endonuclease, comprising, from the inside out, a magnetic core, a silica layer, an avidin layer, a polydopamine layer, and a PEG double-blocking layer, characterized in that, The avidin layer is composed of avidin saturated with biotin and modified with a phenylboronic acid derivative. The polydopamine layer contains an imprint of abasic endonuclease. The PEG double-blocking layer is composed of two amino-modified methoxy polyethylene glycols with different molecular weights. This abasic endonuclease magnetic nanoimprint material is prepared by the following method: first, avidin is covalently linked to the surface of a silica layer coating a magnetic core; then, biotin is added to completely convert the four subunits of avidin into a biotin-bound state; after removing excess biotin molecules, template molecule APE1 is added for binding and immobilization; then, a phenylboronic acid derivative is added to modify the glycosyl groups on the surface of avidin and pre-assemble it with the amino acids on the surface of APE1; then, dopamine polymerization is carried out in the outer layer; then, on the surface of polydopamine, a stepwise reaction is carried out on the outermost layer, first with a high molecular weight (15-30 kJ) amino-modified methoxy polyethylene glycol, followed by a low molecular weight (1.5-3 kJ) methoxy polyethylene glycol. The amino group of k was used for blocking modification with methoxy polyethylene glycol; finally, the template molecule APE1 was eluted to obtain the debased endonuclease magnetic nanoimprint material.

2. The magnetic nanoimprinted material for debasing endonuclease as described in claim 1, characterized in that, The magnetic core is a magnetic nanoparticle with a diameter of 3-15 nm. The thickness of the silica layer coating the magnetic core is 1-2 nm. The thickness of the avidin layer is less than or equal to 5 nm. The thickness of the polydopamine layer is 3-5 nm. The thickness of the PEG double-blocking layer is 20-30 nm.

3. The magnetic nanoimprinted material for debasing endonuclease as described in claim 1, characterized in that, The magnetic core is either Fe3O4 magnetic nanoparticles or γ-Fe2O3 magnetic nanoparticles.

4. The magnetic nanoimprinted material for debasing endonuclease as described in claim 1, characterized in that, In the avidin layer, the biotin-binding pockets on all four subunits of avidin are filled with biotin molecules, and there are no excess biotin molecules on the protein surface; the glycosyl group of avidin is modified with a phenylboronic acid derivative, which is 3-hydroxyphenylboronic acid and / or 3-aminophenylboronic acid.

5. The magnetic nanoimprinted material for debasing endonuclease as described in claim 1, characterized in that, The PEG double-blocking layer is composed of an amino-modified methoxy polyethylene glycol with a molecular weight of 15-30 k and an amino-modified methoxy polyethylene glycol with a molecular weight of 1.5-3 k.

6. A method for preparing the magnetic nanoimprinted material of debasing endonuclease according to any one of claims 1 to 5, comprising: First, avidin is covalently linked to the surface of a silica layer coating the magnetic core. Then, biotin is added to completely convert the four subunits of avidin into a biotin-bound state. After removing excess biotin molecules, template molecule APE1 is added for binding and immobilization. Next, a phenylboronic acid derivative is added to modify the glycosyl group on the surface of avidin and pre-assemble it with the amino acids on the surface of APE1. Then, dopamine polymerization is carried out in the outer layer. Then, the surface of polydopamine is blocked and modified stepwise by first high molecular weight 15-30 k amino-modified methoxy polyethylene glycol and then low molecular weight 1.5-3 k amino-modified methoxy polyethylene glycol. Finally, template molecule APE1 is eluted to obtain the debased endonuclease magnetic nanoimprinted material.

7. The preparation method according to claim 6, characterized in that, Includes the following steps: 1) A magnetic core coated with a carboxylated silica layer was ultrasonically dispersed in pure water, and a compound activating the carboxyl groups was added. After shaking at room temperature, the resulting solution was A. 2) Avidin was added to solution A, and after shaking at room temperature, avidin was covalently attached to the silica layer surface. Unreacted substances were removed by magnetic separation and washing. The resulting magnetic nanoparticles were then ultrasonically dispersed in Tris solution, resulting in solution B. 3) Biotin was added to solution B, converting the biotin-binding pockets of all four subunits of avidin into a biotin-bound state. Unbound substances were removed by magnetic separation and washing. The resulting magnetic nanoparticles were then ultrasonically dispersed in Tris solution, resulting in solution C. 4) APE1 was added to solution C, and after incubation at room temperature for a period of time, APE1 and avidin were bound. Then, a phenylboronic acid derivative was added for modification, resulting in solution D. 5) Add dopamine to solution D for polymerization, magnetically separate and wash to remove unreacted substances, and then re-disperse the obtained magnetic nanoparticles in Tris solution as solution E; 6) Add a larger molecular weight amino-modified methoxy polyethylene glycol to solution E to modify the surface of polydopamine, magnetically separate and wash to remove unreacted substances, then re-disperse the obtained magnetic nanoparticles in Tris solution, and then add a smaller molecular weight amino-modified methoxy polyethylene glycol to modify the surface of the magnetic nanoparticles a second time, magnetically separate and wash to remove unreacted substances; 7) Wash the template molecule APE1 away from the magnetic nanoparticles obtained in step 6) with elution buffer, and dry at room temperature to obtain the debased endonuclease magnetic nanoimprint material.

8. The preparation method according to claim 7, characterized in that, In step 1), the magnetic cores coated with carboxylated silica are ultrasonically dispersed in pure water to a final concentration of 0.1-0.5 mg / mL. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N,N'-dicyclohexylcarbodiimide, or N,N'-diisopropylcarbodiimide, and N-hydroxysuccinimide, to a final concentration of 1-4 mg / mL, and N-hydroxysuccinimide, to a final concentration of 2-8 mg / mL, are added to activate the carboxyl groups. In step 2), avidin is added to solution A to a concentration of 40-200 nM, corresponding to a magnetic nanoparticle concentration of 0.1-0.5 mg / mL. In step 3), biotin is added to solution B to a concentration of 200-400 nM, corresponding to a magnetic nanoparticle concentration of 0.1-0.2 mg / mL. In step 4), APE1 is added to solution C to a concentration of 40-80 mg / mL. nM, after incubation at room temperature, the concentration of phenylboronic acid derivative added is 600~800 nM; step 5) the concentration of dopamine added to solution D is 0.08 - 0.12 mg / mL; step 6) the concentration of the two amino-modified methoxy polyethylene glycols added to solution E is 5~10 mg / mL.

9. The preparation method according to claim 7, characterized in that, Step 2) After adding avidin, shake and react at room temperature for 8-16 hours, then wash with deionized water to remove unreacted substances from the system; Step 3) Add biotin to carry out the binding reaction at room temperature; Step 4) Add APE1 and incubate at room temperature for 20-40 minutes, then add phenylboronic acid derivative and incubate at room temperature for 30-60 minutes; Step 5) Add dopamine and polymerize at room temperature for 80-100 minutes; Step 6) Both amino-modified methoxy polyethylene glycol modifications are carried out at 37°C for 8-12 hours; Step 7) First, ultrasonically disperse and wash the magnetic nanoparticles multiple times in the elution buffer, then ultrasonically disperse and wash them multiple times with ethanol.

10. The preparation method according to claim 7, characterized in that, The concentration of the Tris solution is 10-15 mM, and the pH is 8.0-8.5; the elution buffer contains 100 mM Bis-propane-Cl, 100 mM MgCl2, 10 mM DTT, and 0.1% Triton X-100.

11. The application of the debased endonuclease magnetic nanomaterial according to any one of claims 1 to 5 in the identification, capture, separation, purification and / or enrichment of the target protein molecule APE1 in biological samples.

12. The application as described in claim 11, characterized in that, The application involves separating and purifying APE1 from cell lysates, including: adding the debased endonuclease magnetic nanoparticle separation material to the cell lysates for room temperature adsorption, followed by magnetic separation, collecting the magnetic nanoparticles, washing to remove impurities, and finally eluting APE1 with elution buffer.

13. The application as described in claim 11, characterized in that, The application involves capturing and purifying APE1 in situ within living cells, including: magnetically transfecting the debased endonuclease magnetic nanoparticle separation material into living cells, culturing for a period of time, then lysing the cells, magnetically separating and collecting the magnetic nanoparticles, and washing them to elute APE1 with an elution buffer.

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