DNA tetrahedron-based magnetic immobilized enzyme reactor, preparation and application
By combining DNA tetrahedra with magnetic graphene oxide and gold nanoparticles to immobilize trypsin and form a magnetically immobilized enzyme reactor, the shortcomings of traditional magnetic nanoparticle immobilized enzyme reactors are overcome, achieving efficient enzyme digestion and reuse.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NATIONAL INSTITUTE OF METROLOGY CHINA
- Filing Date
- 2022-09-21
- Publication Date
- 2026-06-09
Smart Images
Figure CN115505584B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of functionalized magnetic nanomaterials technology, and in particular to a magnetically immobilized enzyme reactor based on DNA tetrahedrons, its preparation and application. Background Technology
[0002] Efficient and rapid protein enzymatic digestion is a crucial component of bottom-up proteomics strategies, significantly impacting the qualitative and quantitative analysis of proteins. Traditional protein solution-based digestion methods typically require over ten hours, severely limiting high-throughput preprocessing and analysis of complex biological samples. Furthermore, the autolysis of free proteases increases sample complexity. In recent years, immobilized enzymes have rapidly developed due to their significantly reduced digestion time and reusability. Enzymes and materials are primarily linked through covalent bonds, physical adsorption, embedding, or cross-linking. Common immobilization matrices include magnetic nanoparticles, mesoporous materials, and metal-organic frameworks.
[0003] Magnetic nanoparticles possess unique magnetic properties. Compared to traditional solution-based enzyme digestion, magnetically immobilized enzyme reactors can rapidly separate enzymes from the digestion system, enabling enzyme recovery and reuse, and providing an economical and efficient experimental approach. However, using magnetic nanoparticles as immobilized enzyme carriers still has some drawbacks. For example, the low specific surface area of magnetic nanoparticles significantly affects the enzyme loading capacity; they are easily oxidized in air or acidic environments; and the tendency of magnetic particles to aggregate affects the activity and dispersion effect of the immobilized enzyme reactor.
[0004] To address the aforementioned shortcomings, new technical approaches are urgently needed to improve enzyme digestion efficiency. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a magnetically immobilized enzyme reactor based on DNA tetrahedra, its preparation and application. The composite magnetically immobilized enzyme reactor uses magnetic nanomaterials as a matrix and has the characteristics of being easy and fast to use and reusable, thus providing certain economic benefits. Moreover, the composite magnetically immobilized enzyme reactor can effectively control the spacing between trypsin molecules by controlling the size of the DNA tetrahedra, thereby effectively avoiding enzyme activity loss caused by enzyme autodigestion, resulting in a significant improvement in enzyme digestion efficiency and a greatly shortened enzyme digestion time.
[0006] The present invention adopts the following technical solution:
[0007] On one hand, the present invention provides a magnetically immobilized enzyme reactor based on DNA tetrahedra, comprising: magnetic graphene oxide, gold nanoparticles immobilized on the magnetic graphene oxide, DNA tetrahedra modified on the gold nanoparticles, and trypsin immobilized on the DNA tetrahedra.
[0008] In addition to any of the possible implementations described above, another implementation is provided, wherein the magnetic graphene oxide is prepared by loading magnetite nanoparticles onto graphene oxide using a solvothermal method, wherein the magnetite nanoparticles have a particle size of 100–500 nm.
[0009] In addition to any of the possible implementations described above, another implementation is provided in which the magnetic nanoparticles can also be metal oxides of Fe, Co, and Ni, preferably Fe3O4 magnetic nanoparticles. Fe3O4 has the highest saturation magnetization among several metal oxides, and Fe3O4 magnetic nanoparticles have the advantages of simple preparation method, wide availability of raw materials, safety and non-toxicity, and good biocompatibility.
[0010] In addition to any of the possible implementations described above, another implementation is provided in which the DNA tetrahedron is synthesized from four DNA single strands via a self-assembly reaction, each DNA single strand containing 22 to 80 deoxyribonucleotide monomers; the 3' or 5' end of the DNA single strand has a functional group, which is a thiol group and a carboxyl group, wherein the thiol group is used for the reaction of the DNA tetrahedron with gold nanoparticles in a magnetically immobilized enzyme reactor, and the carboxyl group is used for the bonding of the DNA tetrahedron with the amino groups in the trypsin structure.
[0011] On the other hand, the present invention also provides a method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedra, comprising: preparing magnetic graphene oxide by a solvothermal method; immobilizing gold nanoparticles on the magnetic graphene oxide; synthesizing DNA tetrahedra with thiol groups at their vertices using a self-assembly reaction; modifying the DNA tetrahedra onto the gold nanoparticles using covalent bonding between Au and S; and immobilizing trypsin on the DNA tetrahedra through covalent bonds formed by carboxyl and amino groups, thereby obtaining the magnetically immobilized enzyme reactor.
[0012] In addition to any of the possible implementations described above, another implementation is provided, wherein the method specifically includes:
[0013] S1. Loading magnetite nanoparticles on the surface of graphene oxide yields product I:Fe3O4@GO;
[0014] S2. Using negatively charged functional groups on the surface of graphene oxide as nucleation centers, gold nanoparticles are grown in situ on the surface of graphene oxide using a reducing metal salt solution (such as HAuCl4) to obtain product II:Fe3O4@GO@AuNPs.
[0015] S3. Using the covalent bond between Au and S, DNA tetrahedra are modified onto the gold nanoparticles in product II to obtain product III: Fe3O4@GO@AuNPs@DNA TET;
[0016] S4. Trypsin is immobilized on the DNA tetrahedron of product III by covalent bonds formed by the carboxyl group of DNA tetrahedron and the amino group of trypsin, to obtain product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin, which is the magnetically immobilized enzyme reactor.
[0017] In addition to any of the possible implementations described above, another implementation is provided in which the preparation method of product I:Fe3O4@GO in step S1 is as follows:
[0018] Graphene oxide and anhydrous sodium acetate were added to an ethylene glycol solution of ferric chloride hexahydrate to obtain a dispersion. The dispersion was sonicated until homogeneous and then transferred to a high-pressure reactor for heating at 180–220°C for 8–12 hours. The reaction product was cooled and washed to obtain product I:Fe3O4@GO.
[0019] In addition to any of the possible implementations described above, another implementation is provided in which the preparation method of product II:Fe3O4@GO@AuNPs in step S2 is as follows:
[0020] Add 30–50 ml of ultrapure water to 60 mg of product I: Fe3O4@GO and mix well to obtain a dispersion. Add 2–4 ml of tetrachloroauric acid solution and 2–4 ml of sodium citrate solution to the dispersion, stir for 10 minutes, then add 2–4 ml of freshly prepared NaBH4 solution under ice bath conditions and continue mechanical stirring for 30–60 minutes. Let the mixed solution stand in the dark for 16–24 hours to obtain product II: Fe3O4@GO@AuNPs.
[0021] In addition to any of the possible implementations described above, another implementation is provided in which the preparation method of product III:Fe3O4@GO@AuNPs@DNA TET in step S3 is as follows:
[0022] Add DNA tetrahedral solution, TE buffer and a certain amount of sodium chloride solution to 15 mg of product II. After incubating the dispersion at 4°C for 12-16 h, product III is obtained. The DNA tetrahedron is prepared by the self-assembly of four 1 μmol / L DNA single strands through complementary base pairing.
[0023] In addition to any of the possible implementations described above, another implementation is provided in which the preparation method of product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin in step S4 is as follows:
[0024] First, EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added to 1 mg of product III to activate the carboxyl groups on the DNA tetrahedra. Then, the activated product III was incubated with the protease solution at 4°C.
[0025] After rotating and incubating for 12 hours, product IV was obtained: Fe3O4@GO@AuNPs@DNA TET@Trypsin.
[0026] In another aspect, the present invention also provides an application of the above-mentioned magnetic immobilized enzyme reactor based on DNA tetrahedrons, wherein the immobilized enzyme reactor is used for rapid protein enzymatic digestion; the rapid enzymatic digestion step is as follows: the immobilized enzyme reactor is mixed and incubated with the denatured protein sample, and after the enzymatic digestion is completed, the magnetic material and the supernatant are separated by an external magnet.
[0027] The beneficial effects of this invention are as follows:
[0028] This invention combines magnetic nanomaterials with DNA tetrahedra, utilizing the ease of separation of magnetic nanomaterials to improve the reusability of immobilized enzyme reactors. Simultaneously, the DNA tetrahedral structure is easy to modify, highly stable, and biocompatible. Furthermore, the size of the DNA tetrahedral material is controllable and possesses a certain degree of rigidity. By controlling the size of the DNA tetrahedron, the spacing between the immobilized enzymes can be effectively controlled, effectively preventing enzyme activity loss due to autodigestion, thereby improving the reusability and enzymatic digestion efficiency of the immobilized enzyme. Attached Figure Description
[0029] Figure 1 The diagram shows a synthetic route of a magnetically immobilized enzyme reactor based on DNA tetrahedrons according to an embodiment of the present invention.
[0030] Figure 2 The images shown are transmission electron microscopy (TEM) images of product I: Fe3O4@GO and product II: Fe3O4@GO@AuNPs (b) during the synthesis process of the example.
[0031] Figure 3 The image shows the four single-stranded DNA sequences that form the synthetic DNA tetrahedron.
[0032] Figure 4The figures shown are hysteresis curves of the products Fe3O4@GO (a), Fe3O4@GO@AuNPs (b), Fe3O4@GO@AuNPs@DNA TET (c) and Fe3O4@GO@AuNPs@DNA TET@Trypsin (d) during the synthesis process of the examples.
[0033] Figure 5 The figure shows a comparison of the amino acid sequence coverage of BSA digested by conventional solution enzyme digestion (a) and by the magnetic immobilized enzyme reactor based on DNA tetrahedrons of the present invention (b). Detailed Implementation
[0034] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered in isolation, but can be combined with each other to achieve better technical effects.
[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0037] An embodiment of the present invention provides a magnetically immobilized enzyme reactor based on DNA tetrahedra, comprising: magnetic graphene oxide, gold nanoparticles immobilized on the magnetic graphene oxide, DNA tetrahedra modified on the gold nanoparticles, and trypsin immobilized on the DNA tetrahedra.
[0038] In one specific embodiment, magnetic iron oxide nanoparticles are loaded onto graphene oxide using a solvothermal method to form magnetic graphene oxide; the particle size of the magnetic iron oxide nanoparticles is 100–500 nm.
[0039] In one specific embodiment, the DNA tetrahedron is synthesized from four DNA single strands through a self-assembly reaction, each DNA single strand containing 22 to 80 deoxyribonucleotide monomers; the 3' or 5' end of the DNA single strand has functional groups, namely thiol and carboxyl groups, wherein the thiol group is used for the reaction of the DNA tetrahedron with gold nanoparticles in the magnetically immobilized enzyme reactor, and the carboxyl group is used for the bonding of the DNA tetrahedron with the amino groups in the trypsin structure.
[0040] like Figure 1As shown in the embodiment of the present invention, a method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedra includes: preparing magnetic graphene oxide by a solvothermal method; immobilizing gold nanoparticles on the magnetic graphene oxide; synthesizing DNA tetrahedra with thiol groups at their vertices using a self-assembly reaction; modifying the DNA tetrahedra onto the gold nanoparticles using covalent bonding between Au and S; and immobilizing trypsin on the DNA tetrahedra through covalent bonds formed by carboxyl and amino groups, thereby obtaining the magnetically immobilized enzyme reactor.
[0041] In one embodiment, the method specifically includes:
[0042] S1. Loading magnetite nanoparticles on the surface of graphene oxide yields product I:Fe3O4@GO;
[0043] S2. Using negatively charged functional groups on the surface of graphene oxide as nucleation centers, gold nanoparticles are grown in situ on the surface of graphene oxide using a reducing metal salt solution (such as HAuCl4) to obtain product II:Fe3O4@GO@AuNPs.
[0044] S3. Using the covalent bond between Au and S, DNA tetrahedra are modified onto the gold nanoparticles in product II to obtain product III: Fe3O4@GO@AuNPs@DNA TET;
[0045] S4. Trypsin is immobilized on the DNA tetrahedron of product III by covalent bonds formed by the carboxyl group of DNA tetrahedron and the amino group of trypsin, to obtain product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin, which is the magnetically immobilized enzyme reactor.
[0046] In one embodiment, in step S1, the preparation method of product I:Fe3O4@GO is as follows: graphene oxide and anhydrous sodium acetate are added to an ethylene glycol solution of ferric chloride hexahydrate to obtain a dispersion; the dispersion is ultrasonicated until homogeneous, transferred to a high-pressure reactor for heating at a temperature of 180–220°C for 8–12 hours; the reaction product is cooled and washed to obtain product I:Fe3O4@GO. In the Fe3O4@GO composite material, the particle size of iron oxide is approximately 100–500 nm. Figure 2 (a) ; and it has strong magnetism, see Figure 4 Curve a.
[0047] In one embodiment, in step S2, the preparation method of product II:Fe3O4@GO@AuNPs is as follows: 40 ml of ultrapure water is added to 60 mg of product I:Fe3O4@GO and mixed thoroughly to obtain a dispersion; 2–4 ml of tetrachloroauric acid solution and 2–4 ml of sodium citrate solution are added to the dispersion successively, and the mixture is stirred for 10 minutes. Then, 2–4 ml of freshly prepared NaBH4 solution is added under ice bath conditions, and mechanical stirring continues for 30–60 minutes; the mixed solution is left to stand in the dark for 16–24 hours to obtain product II:Fe3O4@GO@AuNPs. Transmission electron microscopy characterization of product II:Fe3O4@GO@AuNPs shows that gold particles are abundantly distributed on graphene oxide. Figure 2 (b) In contrast, after magnetic graphene oxide is immobilized with gold nanoparticles, the magnetic properties of the material slightly decrease because some of the gold nanoparticles cover the magnetic particles. Figure 4 Curve b.
[0048] In one embodiment, in step S3, the preparation method of product III: Fe3O4@GO@AuNPs@DNA TET is as follows: DNA tetrahedral solution, TE buffer and a certain amount of sodium chloride solution are added to 15mg of product II, and the dispersion is incubated at 4°C for 12-16h to obtain product III.
[0049] In one embodiment, the DNA tetrahedron consists of four single-stranded DNA strands, each with a concentration of 1 μmol / L (the single-stranded DNA sequence is as follows). Figure 3 Synthesized via self-assembly; the base sequence of the DNA single strand is designed according to the base complementary pairing principle, and each DNA single strand contains 22–80 deoxyribonucleotide monomers; the 3' or 5' end of the DNA single strand has a functional group, which can be a thiol group or a carboxyl group; Figure 4 As shown in curve c, product III:Fe3O4@GO@AuNPs@DNA TET maintained good magnetism during the synthesis process.
[0050] In one embodiment, in step S4, the preparation method of product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin is as follows: First, EDC (i.e., 1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (i.e., N-hydroxysuccinimide) are added to 1 mg of product III to activate the carboxyl groups on the DNA tetrahedron. Then, the activated product III is incubated with a protease solution at 4°C for 12 hours by rotation to obtain product IV. Trypsin and product III:
[0051] After Fe3O4@GO@AuNPs@DNA TET bonding, as Figure 4As shown in curve d, the hysteresis curve of the obtained product IV indicates that the material has good paramagnetic ability and can be rapidly separated using a magnet.
[0052] The present invention will be further illustrated below through comparative experiments and comparisons with specific embodiments:
[0053] Example
[0054] In the following specific embodiments:
[0055] Product I: Fe3O4@GO was prepared by the following steps: 200 mg of ferric chloride hexahydrate was dispersed in 50 ml of anhydrous ethylene glycol and sonicated for 30 min. 50 mg of graphene oxide and 800 mg of anhydrous sodium acetate were added to the dispersion, and sonication was continued for 8 hours until the solution was homogeneous. Subsequently, the homogenized solution was transferred to a high-pressure reactor and heated in an oven at 200 °C for 12 h. After cooling and washing, Product I was obtained.
[0056] Product II: Fe3O4@GO@AuNPs were prepared by the following steps: 60 mg of Fe3O4@GO nanocomposite material was weighed and placed in a round-bottom flask. 40 mL of ultrapure water was added, and the mixture was mechanically stirred to suspend the material uniformly in the ultrapure water. 2 mL of 0.01 mol / L HAuCl4 solution and 2 mL of 0.01 mol / L sodium citrate solution were added to the dispersion successively. After mechanical stirring for 10 min, 2 mL of freshly prepared 0.1 mol / L NaBH4 solution was added under ice bath conditions. Mechanical stirring was continued for 30 min, and the mixture was then allowed to stand in the dark for 16 hours. After the reaction was complete, the precipitate was washed three times each with anhydrous ethanol and ultrapure water to obtain product II.
[0057] Product III: Fe3O4@GO@AuNPs@DNA TET was prepared by the following steps: 15 mg of Fe3O4@GO@AuNPs was weighed, and 100 μL of DNA tetrahedral solution, 500 μL of TE buffer and 10 μL of 50 mmol / L NaCl solution were added. The dispersion was incubated at 4 °C for 12 h by rotation to obtain product III.
[0058] The DNA tetrahedrons used were prepared by the following steps: each single-stranded DNA was prepared into a solution with a concentration of 100 μmol / L. 1 μL of each single strand was added to 96 μL of TE buffer to make the final concentration of each single strand 1 μmol / L; after incubation at 95°C for 20 minutes, the reaction was carried out at 4°C for 30 minutes to allow for self-assembly of DNA tetrahedrons.
[0059] Product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin was prepared through the following steps: A 0.1 mol / L LMES buffer solution (pH=6) was prepared to dissolve EDC and NHS; 1 mg of magnetically immobilized enzyme material was taken, and EDC and NHS solutions in a molar ratio of 2:1 were added to the material. After activating the material for 30 min, the supernatant was discarded, and the material was washed three times with TE buffer. 1 ml of protease solution was added to the material, and the material was then incubated at 4°C for 12 h to obtain the immobilized enzyme reactor.
[0060] Comparative experiment
[0061] Taking the detection of amino acid sequence coverage of bovine serum albumin (BSA) digested with enzymes as an example, the traditional solution digestion method was used as a control to examine the digestion performance of the novel magnetic immobilized enzyme reactor in this invention. 40 μg of BSA was dissolved in 50 mM ammonium bicarbonate, and after denaturation with DTT and IAA, the protein solution was added to the immobilized enzyme reactor and incubated at 37°C for 2 minutes. The magnetic immobilized enzyme reactor and supernatant were separated under an external magnetic field. The supernatant was desalted and used for mass spectrometry analysis. Figure 5 As shown, the amino acid sequence coverage of BSA obtained by conventional solution digestion is 85%. Figure 5 In (a), the amino acid sequence coverage of BSA digested by the novel magnetic immobilized enzyme reactor of this invention is 92%, see [reference]. Figure 5 (b)
[0062] Experimental results show that the novel magnetic immobilized enzyme reactor of this invention has a significantly higher enzyme digestion efficiency than solution enzyme digestion, and reduces the digestion time from 12 hours to 2 minutes, greatly shortening the sample pretreatment time and improving experimental efficiency. At the same time, the immobilized enzyme reactor can be rapidly and effectively separated from the enzyme digestion system using an external magnetic field, allowing the immobilized enzyme material to be reused and generating economic benefits.
[0063] This invention involves a series of functional modifications to magnetic nanoparticles. Using graphene oxide as a carrier for the immobilized enzyme reactor provides a larger specific surface area, thereby increasing the enzyme loading capacity and shortening the digestion time. Gold nanoparticles are easily modified and possess good stability; encapsulating magnetic nanoparticles with gold nanoparticles can improve their tendency to aggregate and their poor stability. The DNA tetrahedron used in this invention is a three-dimensional DNA structure with a tetrahedral shape, formed by the self-assembly of four single-stranded DNA molecules (the base sequence of the single-stranded DNA is designed according to the complementary base pairing principle). DNA tetrahedrons are relatively simple to prepare and have high yields, exhibiting excellent biocompatibility and abundant functional modification sites. Simultaneously, the size of the DNA tetrahedron is controllable, allowing for effective control of the spacing between trypsin molecules, thereby effectively avoiding enzyme activity loss due to autodigestion and further improving the enzymatic digestion efficiency of the immobilized enzyme reactor.
[0064] While several embodiments of the present invention have been provided herein, those skilled in the art should understand that modifications can be made to these embodiments without departing from the spirit of the invention. The above embodiments are merely exemplary and should not be construed as limiting the scope of the invention.
Claims
1. A magnetically immobilized enzyme reactor based on DNA tetrahedra, characterized in that, The magnetically immobilized enzyme reactor comprises: magnetic graphene oxide, gold nanoparticles immobilized on the magnetic graphene oxide, DNA tetrahedra modified on the gold nanoparticles, and trypsin immobilized on the DNA tetrahedra. The magnetic graphene oxide is prepared by loading magnetite nanoparticles onto graphene oxide using a solvothermal method, wherein the magnetite nanoparticles have a particle size of 100–500 nm. The DNA tetrahedron is synthesized from four DNA single strands through a self-assembly reaction. Each DNA single strand contains 22 to 80 deoxyribonucleotide monomers. The 3' or 5' end of the DNA single strand has functional groups, namely thiol and carboxyl groups. The thiol group is used for the reaction of the DNA tetrahedron with gold nanoparticles in the magnetically immobilized enzyme reactor, and the carboxyl group is used for the bonding of the DNA tetrahedron with the amino groups in the trypsin structure. The 5'-3' nucleotide sequences of the four DNA single strands are as follows: F1: SH-TACACAGATCATAGTAGGTAAGTTATCGAAC, F2: SH-ATCTGTGTACAGACGACGAATCCCTATCGGA, F3: SH-TTACAGTCTGCTTCGTCGTCGAGTTCGATAA, F4: COOH-TCAGTCGGTCAGACTGTAAACTACCTACTAGATCCGATAGG.
2. A method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 1, characterized in that, The method includes: preparing magnetic graphene oxide by a solvothermal method; immobilizing gold nanoparticles on the magnetic graphene oxide; synthesizing DNA tetrahedra with thiol and carboxyl groups at their vertices using a self-assembly reaction; modifying the DNA tetrahedra onto the gold nanoparticles using covalent bonding between Au and S; and immobilizing trypsin on the DNA tetrahedra through covalent bonds formed by carboxyl and amino groups to obtain the magnetically immobilized enzyme reactor. The magnetic graphene oxide is prepared by loading magnetite nanoparticles onto graphene oxide using a solvothermal method, wherein the magnetite nanoparticles have a particle size of 100–500 nm. The DNA tetrahedron is synthesized from four DNA single strands through a self-assembly reaction. Each DNA single strand contains 22 to 80 deoxyribonucleotide monomers. The 3' or 5' end of the DNA single strand has functional groups, namely thiol and carboxyl groups. The thiol group is used for the reaction of the DNA tetrahedron with gold nanoparticles in the magnetically immobilized enzyme reactor, and the carboxyl group is used for the bonding of the DNA tetrahedron with the amino groups in the trypsin structure. The 5'-3' nucleotide sequences of the four DNA single strands are as follows: F1: SH-TACACAGATCATAGTAGGTAAGTTATCGAAC, F2: SH-ATCTGTGTACAGACGACGAATCCCTATCGGA, F3: SH-TTACAGTCTGCTTCGTCGTCGAGTTCGATAA, F4: COOH-TCAGTCGGTCAGACTGTAAACTACCTACTAGATCCGATAGG.
3. The method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 2, characterized in that, The method specifically includes: S1. Loading magnetite nanoparticles on the surface of graphene oxide yields product I: Fe3O4@GO; S2. Using the negatively charged functional groups on the surface of graphene oxide as nucleation centers, gold nanoparticles were grown in situ on the surface of graphene oxide using the reducing metal salt solution HAuCl4 to obtain product II: Fe3O4@GO@AuNPs. S3. Using the covalent bond between Au and S, DNA tetrahedra are modified onto the gold nanoparticles in product II to obtain product III: Fe3O4@GO@AuNPs@DNA TET; S4. Trypsin is immobilized on the DNA tetrahedron of product III by covalent bonds formed by the carboxyl group of DNA tetrahedron and the amino group of trypsin, to obtain product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin, which is the magnetically immobilized enzyme reactor.
4. The method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 3, characterized in that, In step S1, the preparation method of product I: Fe3O4@GO is as follows: Graphene oxide and anhydrous sodium acetate were added to an ethylene glycol solution of ferric chloride hexahydrate to obtain a dispersion. The dispersion was sonicated until homogeneous and then transferred to a high-pressure reactor for heating at 180–220°C for 8–12 hours. The reaction product was cooled and washed to obtain product I: Fe3O4@GO.
5. The method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 3, characterized in that, In step S2, the preparation method of product II: Fe3O4@GO@AuNPs is as follows: Add 30–50 mL of ultrapure water to 60 mg of product I: Fe3O4@GO and mix well to obtain a dispersion. Add 2–4 mL of tetrachloroauric acid solution and 2–4 mL of sodium citrate solution to the dispersion, stir for 10 minutes, then add 2–4 mL of NaBH4 solution under ice bath conditions and continue mechanical stirring for 30–60 minutes. Let the mixed solution stand in the dark for 16–24 hours to obtain product II: Fe3O4@GO@AuNPs.
6. The method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 3, characterized in that, In step S3, the preparation method of product III: Fe3O4@GO@AuNPs@DNA TET is as follows: Add DNA tetrahedral solution, TE buffer and sodium chloride solution to 15 mg of product II. After incubating the dispersion at 4°C for 12-16 h by rotation, product III is obtained. The DNA tetrahedron is prepared by self-assembly of four 1 μmol / L DNA single strands through complementary base pairing.
7. The method for preparing a magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 3, characterized in that, In step S4, the preparation method of product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin is as follows: First, EDC (i.e., 1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (i.e., N-hydroxysuccinimide) were added to 1 mg of product III to activate the carboxyl groups on the DNA tetrahedron. Then, the activated product III was incubated with trypsin solution at 4°C for 12-16 hours by rotation to obtain product IV: Fe3O4@GO@AuNPs@DNA TET@Trypsin.
8. An application of the magnetically immobilized enzyme reactor based on DNA tetrahedrons as described in claim 1, characterized in that, The immobilized enzyme reactor is used for rapid protein digestion. The rapid digestion steps are as follows: the immobilized enzyme reactor is mixed and incubated with the denatured protein sample. After the digestion is completed, the magnetic material and the supernatant are separated by an external magnet.