Magnetic nano material based on DNA tetrahedron as well as preparation method and application of magnetic nano material

Abstract

The invention relates to the field of nano materials, in particular to a DNA tetrahedron-based magnetic nano material as well as a preparation method and application thereof. According to the magnetic nano material Fe3O4 (at) DOPA / PEI (at) Au (at) DNA TET-Trypsin based on the DNA tetrahedron, regular distribution of trypsin on the surface of the material is achieved by utilizing the structural characteristics of the DNA tetrahedron, and glycopeptide is enriched by utilizing rich hydrophilic groups on the surface; the preparation method comprises the following steps: introducing PEI on the surface of magnetic Fe3O4, and synthesizing a polymer Fe3O4 (at) DOPA / PEI with rich amino groups on the surface through Schiff base reaction; then, gold nanoparticles are deposited on the polymer through an AuNPs solution, and DNA tetrahedrons with-SH at the tops are successfully attached to the gold nanoparticles through Au-S bonds; and finally, synthesizing the magnetic nano material through amination reaction between amido and carboxyl. Compared with a traditional enrichment material, the magnetic nano material disclosed by the invention has excellent digestion and enrichment performance, and not only can realize rapid digestion of protein but also can perform specific enrichment on glycopeptide within a very short reaction time.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to a magnetic nanomaterial based on DNA tetrahedron, and a preparation method and application thereof. Background Art

[0002] Protein glycosylation, as one of the most common and important post-translational modifications of proteins, plays a vital role in many biological processes. Abnormal protein glycosylation is closely related to the development of many diseases, including chronic respiratory diseases, Alzheimer's disease, glioma, neurological diseases and heart failure. Therefore, further research on protein glycosylation has practical significance for the treatment of diseases and understanding of disease pathology. To date, mass spectrometry-based research strategies are commonly used in glycoproteomics because mass spectrometers can provide highly accurate molecular weight data and rapid analytical detection speeds. Bottom-up strategies have become the main strategy for mass spectrometry-based identification of glycoproteins due to their high selectivity and high throughput.

[0003] When using a bottom-up strategy to identify glycoproteins, the workflow includes three key steps: digestion of the protein into a peptide mixture, enrichment of modified peptides, and mass spectrometry analysis. Traditionally, conventional glycoprotein identification methods based on bottom-up strategies take approximately 19 hours. The disadvantages of this experimental procedure are excessive experimental time and insufficient digestion of low-concentration proteins. These two factors can directly affect the accuracy of qualitative and quantitative analysis in subsequent key experiments. Therefore, the development of new methods for efficient protein digestion is crucial for large-scale glycoproteomics analysis.

[0004] To date, functionalized nanomaterials have attracted widespread attention due to their good protein digestion ability. However, the irregular distribution of trypsin on the surface of the material may lead to the overlap of its active domains, resulting in a decrease in digestion ability. DNA tetrahedron is a three-dimensional DNA nanomaterial composed of four single-stranded DNAs based on the principle of base complementary pairing. The unique structural characteristics and excellent biocompatibility of DNA tetrahedron provide an effective solution to the problem of overlapping active domains. However, there are few reports on the enrichment of glycopeptides by functionalized nanomaterials based on DNA tetrahedrons. Therefore, the development of a functionalized nanomaterial based on DNA tetrahedrons for rapid protein digestion and selectivity of glycopeptides is a new demand. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art and to provide a magnetic nanomaterial based on DNA tetrahedrons and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention is to provide a magnetic nanomaterial based on DNA tetrahedron. The magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin includes: magnetic Fe3O4, polyethyleneimine layer, gold nanolayer and DNA tetrahedron arranged in sequence from inside to outside.

[0008] The second aspect of the present invention is to provide a method for preparing the above-mentioned magnetic nanomaterial, comprising the steps of:

[0009] S1. After uniformly mixing magnetic Fe3O4, Tris-HCl, and L-dopamine, polyethyleneimine, CuSO4, and H2O2 were added in sequence. After ultrasonic mixing, mechanical stirring was performed to react. After the reaction, the product was separated with a magnet, washed, and dried to obtain the polymer Fe3O4@DOPA / PEI.

[0010] S2. placing the polymer Fe3O4@DOPA / PEI in an AuNPs solution, ultrasonically treating it and then mechanically stirring it to deposit the gold nanoparticles on the polymer Fe3O4@DOPA / PEI; separating the product with a magnet, washing it, and drying it to obtain Fe3O4@DOPA / PEI@Au;

[0011] S3, mixing and incubating Fe3O4@DOPA / PEI@Au, TE buffer, DNA tetrahedron solution and NaCl solution to obtain Fe3O4@DOPA / PEI@Au@DNA TET;

[0012] S4. The washed Fe3O4@DOPA / PEI@Au@DNA TET is dispersed in a mixed solution of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for incubation. After the incubation is completed, trypsin solution is added for reaction. After the reaction is completed, the magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin is obtained by washing and drying.

[0013] Preferably, in step S1, the preparation of magnetic Fe3O4 includes: mixing FeCl3·6H2O, diethylene glycol and ethylene glycol and performing ultrasonic dispersion, and then adding NaAc and sodium acrylate to obtain solid magnetic Fe3O4.

[0014] Preferably, in step S2, the preparation of the AuNPs solution includes: adding HAuCl4·4H2O to deionized water, condensing and refluxing under continuous heating, adding sodium citrate dihydrate when heated to 181°C, continuing heating for 15 minutes, and cooling to room temperature to obtain the AuNPs solution.

[0015] Preferably, in step S3, the preparation of the DNA tetrahedron comprises: adding the four DNA single strand solutions into TE buffer for reaction, and obtaining the DNA tetrahedron after the reaction is completed.

[0016] More preferably, the reaction comprises: after 10 min of reaction at 95℃, transferring to 4℃ for 30 min of reaction.

[0017] Preferably, in step S4, the volume ratio of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is 2:1.

[0018] The third aspect of the present application provides application of the above-mentioned magnetic nanomaterial or the magnetic nanomaterial prepared by the above-mentioned preparation method in glycopeptide mass spectrometry detection, and the steps comprise: adding the magnetic nanomaterial to enrich the to-be-detected substance after pretreatment of the to-be-detected substance, washing the magnetic nanomaterial with a washing liquid after the enrichment is completed, eluting the magnetic nanomaterial with an elution buffer, and analyzing the elution liquid by using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer.

[0019] Preferably, the washing liquid is NH4HCO3 buffer.

[0020] Preferably, the elution buffer comprises: acetonitrile, H2O and trifluoroacetic acid; wherein the volume ratio of acetonitrile, H2O and trifluoroacetic acid is 30:69.9:0.1.

[0021] Compared with the prior art, the above technical scheme of the present application has the following technical effects:

[0022] The DNA tetrahedron-based magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin of the present application realizes regular distribution of trypsin on the surface of the material by using the structural characteristics of the DNA tetrahedron, so as to avoid overlapping of the active domains, and enriches glycopeptides by using the rich hydrophilic groups on the surface based on the HILIC strategy; the present application synthesizes magnetic Fe3O4 by using a solvothermal method, introduces PEI on the surface of the magnetic Fe3O4, so that the PEI is wrapped on the magnetic sphere, synthesizes the polymer Fe3O4@DOPA / PEI with rich amine groups on the surface by using a Schiff base reaction; then gold nanoparticles are deposited on the polymer by using an AuNPs solution, and the DNA tetrahedron with -SH on the top is successfully attached to the gold nanoparticles by using an Au-S bond, and can provide a carboxyl reaction site; finally, the magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin is synthesized by an amidation reaction between the amine group and the carboxyl group; compared with traditional enrichment materials, the digestion and enrichment performance of the magnetic nanomaterial of the present application is excellent, and in a very short reaction time, not only can the protein be quickly digested, but also the glycopeptides can be specifically enriched. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Transmission electron micrograph of Fe3O4@DOPA / PEI@Au.

[0024] Figure 2 Infrared spectrogram of Fe3O4@DOPA / PEI, Fe3O4@DOPA / PEI@Au, Fe3O4@DOPA / PEI@Au@DNA TET and Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin.

[0025] Figure 3 Thermogravimetric analysis diagram of Fe3O4@DOPA / PEI, Fe3O4@DOPA / PEI@Au and Fe3O4@DOPA / PEI@Au@DNA TET.

[0026] Figure 4 Atomic force microscope diagram of DNA TET.

[0027] Figure 5 X-ray photoelectron spectrogram of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin.

[0028] Figure 6 is a diagram of the volume ratio of EDC to NHS to the performance of the material in the experiment of preparing Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin; wherein, Figure 6a . EDC: NHS = 3: 1; Figure 6b . EDC: NHS = 2: 1; Figure 6c . EDC: NHS = 1: 1; Figure 6d . EDC: NHS = 1: 2; Figure 6e . EDC: NHS = 1: 3.

[0029] Figure 7 is a diagram of the optimization analysis of the use amount of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin to the selective enrichment effect of glycopeptides; wherein, Figure 7a . 0.1 mg; Figure 7b . 0.5 mg: Figure 7c . 1.0 mg; Figure 7d . 1.5 mg; Figure 7e . 2.0 mg.

[0030] Figure 8 is a diagram of the optimization analysis of the selective enrichment effect of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin to glycopeptides under different elution times under the condition of a certain enrichment time (1 h); wherein, Figure 8a . 1 h; Figure 8b.30min; Figure 8c .10min; Figure 8d .5min.

[0031] Figure 9 is an analysis diagram of the selective enrichment effect of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin on glycopeptides under different enrichment times under the condition of a certain elution time (10min); wherein, Figure 9a .1h; Figure 9b .30min; Figure 9c .10min Figure 9d .5min.

[0032] Figure 10 is a mass spectrum diagram of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin after being digested and enriched in different concentrations of HRP solution; wherein, Figure 10a .0amol·μL -1 ; Figure 10b .1amol·μL -1 ; Figure 10c .0.01amol·μL -1 ; Figure 10d .0.001amol·μL -1 .

[0033] Figure 11 is a mass spectrum diagram of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin after being digested and enriched in different proportions of mixed protein solution; wherein, Figure 11a BSA: HRP = 500:1; Figure 11b BSA: HRP = 1000:1; Figure 11c BSA: HRP = 2000:1; Figure 11d BSA: HRP = 5000:1. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0035] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0036] The present application will be further described below with reference to the drawings and specific embodiments, but not as a limitation of the present application.

[0037] Embodiment 1

[0038] The embodiment provides a preparation method of a DNA tetrahedron-based magnetic nanomaterial, and steps include:

[0039] (1) A certain amount of FeCl3·6H2O, diethylene glycol and ethylene glycol are added into a beaker (100 mL), and ultrasonic treatment is performed until uniform dispersion is achieved, and then a certain amount of NaAc and sodium acrylate is added; the obtained solid solution is washed with deionized water for three times, and the washed product is dried under vacuum, and finally magnetic Fe3O4 is obtained.

[0040] (2) A certain amount of Fe3O4, Tris-HCl (10 mM) and L-DOPA is added into a beaker (500 mL) and ultrasonic treatment is performed until uniform dispersion is achieved, and then a certain amount of PEI, CuSO4 and H2O2 is sequentially added, and ultrasonic treatment is performed until the solution becomes a uniform dark brown solution, and then the mixed solution is mechanically stirred at room temperature for 12 h; after the reaction is completed, the product is separated by a magnet, washed with deionized water for three times, and the washed product is dried under vacuum. Finally, polymer Fe3O4@DOPA / PEI is obtained.

[0041] (3) A certain amount of HAuCl4·4H2O (20 mg·mL -1 ) is added into deionized water, and condensation reflux is performed under continuous heating, and heating is waited until 181 ℃, and then a certain amount of sodium citrate dihydrate (38.8 mM) is rapidly added, and the color of the solution rapidly changes from light yellow to deep purple. After 15 min of continuous heating at 181 ℃, the solution is cooled to room temperature and placed aside, and finally an AuNPs solution is obtained.

[0042] (4) A certain amount of Fe3O4@DOPA / PEI is added into the AuNPs solution and ultrasonic treatment is performed for 30 min, and then the mixed solution is mechanically stirred at room temperature for 1 h after the reaction, and the product is separated by a magnet, washed with anhydrous ethanol for three times, and the washed product is dried under vacuum, and finally Fe3O4@DOPA / PEI@Au is obtained.

[0043] (5) Four DNA single strands are designed according to the principle of base complementary pairing, and are used for preparing a DNA tetrahedron. First, each DNA single strand is dissolved and diluted to a concentration of 100 μmol·L -1 . Then, each DNA single strand solution is added into TE buffer according to a certain amount and mixed uniformly. The final concentration of each DNA single strand solution is 1 μmol·L -1 . The obtained mixed solution is reacted at 95 ℃ for 10 min, and then reacted at 4 ℃ for 30 min. Finally, the product DNA tetrahedron is obtained.

[0044] In the formula, the sequence of each single-stranded DNA is shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] (6) A certain amount of Fe3O4@DOPA / PEI@Au was placed in a centrifuge tube (0.5 mL), and a certain amount of TE buffer and DNA tetrahedron solution (1 μmol·L -1 ) and NaCl solution (10mmol·L -1 ); Then, a certain amount of NaCl solution (10 mmol·L -1 ), twice in total, and incubated with shaking at 4 °C overnight. After the reaction, the product Fe3O4@DOPA / PEI@Au@DNA TET was obtained and stored in a refrigerator at 4 °C.

[0049] (7) Fe3O4@DOPA / PEI@Au@DNA TET was washed with PBS to remove impurities on the surface of the material, and then evenly dispersed in a mixed solution of NHS and EDC with a total volume of 0.3 mL. Subsequently, it was incubated at 37°C with shaking for 30 min. After the reaction, it was washed with PBS to remove surface impurities, and then a certain amount of trypsin solution (1 mg mL -1 ) was evenly mixed with the obtained mixed solution and incubated with shaking at 4°C overnight. Finally, the product was washed with PBS to remove surface impurities and freeze-dried to obtain the target product, Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin.

[0050] The transmission electron microscopy images of the obtained Fe3O4@DOPA / PEI@Au are shown in Figure 2. Figure 1 As shown in Figure 2, the layered structure of the material can be clearly seen. The Fourier transform infrared spectra of Fe3O4@DOPA / PEI, Fe3O4@DOPA / PEI@Au, Fe3O4@DOPA / PEI@Au@DNA TET and Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin are shown in Figure 2. Figure 2 As shown in Figure 2, the changes in functional groups between different materials can be judged. Thermogravimetric analysis of Fe3O4@DOPA / PEI, Fe3O4@DOPA / PEI@Au and Fe3O4@DOPA / PEI@Au@DNA TET are shown in Figure 2. Figure 3 As shown in Figure 2, the thermal stability of the target material and the precursor material can be determined. Figure 4As shown in Figure 2, the three-dimensional structure of DNA tetrahedron can be clearly seen, indicating its objective existence. The X-ray photoelectron spectrum of Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin is shown in Figure 2. Figure 5 As shown, it shows that the target material was successfully prepared.

[0051] Example 2

[0052] Differences in the selective enrichment of glycopeptides by DNA tetrahedron-based magnetic nanomaterials prepared under different EDC to NHS volume ratios:

[0053] (1) Sample preparation: HRP (50 mg) was dispersed in NH4HCO3 (50 mM, 2.5 mL), heated at 100°C for 10 min to denature it, and then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3 (50 mM, 2.25 mL) was added. The mixture was shaken at 56°C for 1 h. Then, a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3 (50 mM, 4.5 mL) was added in the dark and alkylated at 37°C for 1 h to obtain the HRP pretreatment solution.

[0054] (2) Digestion and enrichment: Five 1.0 mg portions of magnetic nanomaterials prepared with different EDC and NHS volume ratios (EDC:NHS=3:1; EDC:NHS=2:1; EDC:NHS=1:1; EDC:NHS=1:2; EDC:NHS=1:3) were dispersed into five centrifuge tubes containing 100 μL of HRP pretreatment solution, enriched at 37°C for 1 h, and then washed three times with NH4HCO3 (50 mM) buffer; then eluted with 10 μL of elution buffer (ACN:H2O:TFA=30.0%:69.9%:0.1%, volume ratio) for 1 h.

[0055] (3) Mass spectrometry analysis: 1 μL of the eluate was taken to target, and the matrix used was DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA = 20%:79.9%:0.1%, volume ratio), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed ( Figure 6 ).

[0056] Example 3

[0057] Different amounts of DNA tetrahedron-based magnetic nanomaterials have different effects on the selective enrichment of glycopeptides.

[0058] (1) Sample preparation: HRP (50 mg) was denatured by heating at 100 °C for 10 min in NH4HCO3(50 mM, 2.5 mL). Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added and shaken at 56 °C for 1 h. Then a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) was added in dark condition and alkylated at 37 °C for 1 h. Finally, HRP pretreatment solution was obtained.

[0059] (2) Digestion and enrichment: Five different amounts (0.1 mg; 0.5 mg; 1.0 mg; 1.5 mg; 2.0 mg) of DNA tetrahedron-based magnetic nanomaterial were dispersed into five centrifuge tubes containing 100 μL of HRP pretreatment solution, respectively, and enriched at 37 °C for 1 h. After that, the materials were washed with NH4HCO3(50 mM) buffer solution for three times. Then 10 μL of elution buffer (ACN:H2O:TFA = 30.0%:69.9%:0.1% by volume) was used to elute for 1 h.

[0060] (3) Mass spectrometry analysis: 1 μL of eluent was spotted on the target. The matrix used was DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA = 20%:79.9%:0.1% by volume). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed (Figure 7).

[0061] Example 4

[0062] The difference of DNA tetrahedron-based magnetic nanomaterials in selective enrichment of glycopeptides at different elution times under the condition of a certain enrichment time.

[0063] (1) Sample preparation: HRP (50 mg) was denatured by heating at 100 °C for 10 min in NH4HCO3(50 mM, 2.5 mL). Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added and shaken at 56 °C for 1 h. Then a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) was added in dark condition and alkylated at 37 °C for 1 h. Finally, HRP pretreatment solution was obtained.

[0064] (2) Digestion and enrichment: Four 1.0 mg of magnetic nanomaterials were dispersed into four centrifuge tubes containing 100 μL of HRP pretreated solution, respectively, and enriched at 37 °C for 1 h, followed by washing the materials with NH4HCO3(50 mM) buffer for three times; then eluted with 10 μL of elution buffer (ACN:H2O:TFA = 30.0%:69.9%:0.1%, volume ratio) for different time (1 h, 30 min, 10 min, 5 min).

[0065] (3) Mass spectrometry analysis: 1 μL of eluate was spotted on the target, and the matrix used was DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA = 20%:79.9%:0.1%, volume ratio) for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (Figure 8).

[0066] Example 5

[0067] The difference of DNA tetrahedron-based magnetic nanomaterials in selective enrichment of glycopeptides under the condition of a certain elution time and different enrichment times.

[0068] (1) Preparation of sample: HRP (50 mg) was dispersed in NH4HCO3(50 mM, 2.5 mL) and denatured by heating at 100 °C for 10 min. Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added, and the mixture was shaken at 56 °C for 1 h, followed by adding a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) under dark conditions, and alkylated at 37 °C for 1 h. Finally, the HRP pretreated solution was obtained.

[0069] (2) Digestion and enrichment: Four 1.0 mg of magnetic nanomaterials were dispersed into four centrifuge tubes containing 100 μL of HRP pretreated solution, respectively, and enriched at 37 °C for different time (1 h, 30 min, 10 min, 5 min), followed by washing the materials with NH4HCO3(50 mM) buffer for three times; then eluted with 10 μL of elution buffer (ACN:H2O:TFA = 30.0%:69.9%:0.1%, volume ratio) for 10 min.

[0070] (3) Mass spectrometry analysis: 1 μL of eluate was spotted on the target, and the matrix used was DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA = 20%:79.9%:0.1%, volume ratio) for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (Figure 9).

[0071] Example 6

[0072] Application of DNA tetrahedron-based magnetic nanomaterials in enriching glycopeptides from HRP solution with different concentrations.

[0073] (1) Preparation of sample: HRP (50 mg) was dispersed in NH4HCO3(50 mM, 2.5 mL) and denatured by heating at 100 °C for 10 min. Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added, and the mixture was shaken at 56 °C for 1 h. Then a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) was added under dark conditions, and the mixture was alkylated at 37 °C for 1 h. The final HRP pretreatment solution was diluted to 10 amol·μL -1 ; 1 amol·μL -1 ; 0.01 amol·μL -1 ; 0.001 amol·μL -1 .

[0074] (2) Digestion and enrichment: Four 1.0 mg of magnetic nanomaterials were dispersed in four centrifuge tubes containing 100 μL of HRP pretreatment solution, respectively, and enriched at 37 °C for 10 min. Then the materials were washed three times with a buffer of NH4HCO3(50 mM). Then 10 μL of elution buffer (ACN:H2O:TFA = 30.0%:69.9%:0.1% by volume) was used to elute the materials for 10 min.

[0075] (3) Mass spectrometry analysis: 1 μL of the eluate was spotted on the target, and the matrix used was DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA = 20%:79.9%:0.1% by volume). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was performed (Figure 10).

[0076] Example 7

[0077] Application of DNA tetrahedron-based magnetic nanomaterials in enriching glycopeptides from mixed protein solution.

[0078] (1) Preparation of sample: HRP (50 mg) was dispersed in NH4HCO3(50 mM, 2.5 mL) and denatured by heating at 100°C for 10 min. Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added, and the mixture was shaken at 56°C for 1 h. Then a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) was added in the dark, and the mixture was alkylated at 37°C for 1 h. Finally, a HRP pretreatment solution was obtained.

[0079] BSA (50 mg) was dispersed in NH4HCO3(50 mM, 2.5 mL) and denatured by boiling at 100°C for 10 min. Then a mixed solution of DTT (5 mM, 0.25 mL) and NH4HCO3(50 mM, 2.25 mL) was added, and the mixture was shaken at 56°C for 1 h. Then a mixed solution of IAA (14 mM, 0.5 mL) and NH4HCO3(50 mM, 4.5 mL) was added in the dark, and the mixture was alkylated at 37°C for 1 h. Finally, a BSA pretreatment solution was obtained. And mixed protein solutions were prepared in different molar ratios (BSA:HRP=500:1; BSA:HRP=1000:1; BSA:HRP=2000:1; BSA:HRP=5000:1).

[0080] (2) Digestion and enrichment: Four 1.0 mg magnetic nanomaterials were dispersed in four centrifuge tubes containing 100 μL of mixed protein solutions with different molar ratios, respectively, and enriched at 37°C for 10 min. Then the materials were washed with a buffer of NH4HCO3(50 mM) for three times. Then 10 μL of elution buffer (ACN:H2O:TFA=30.0%:69.9%:0.1% by volume) was used to elute the materials for 10 min.

[0081] (3) Mass spectrometry analysis: 1 μL of the eluate was spotted on a target, and a matrix of DHB (20 mg / mL) dissolved in a mixed solution (ACN:H2O:TFA=20%:79.9%:0.1% by volume) was used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis (Fig. 11).

[0082] In summary, the magnetic nanomaterials of the present application have excellent digestion and enrichment performance, and can not only realize rapid digestion of proteins but also specific enrichment of glycopeptides in a very short reaction time.

[0083] The above merely describes preferred embodiments of the present application, and is not intended to limit the implementation and protection scope of the present application. Those skilled in the art should be able to understand that any equivalent substitutions and obvious changes made according to the present application description and drawings should be included in the protection scope of the present application.

Claims

1. A magnetic nanomaterial based on DNA tetrahedron, characterized in that: The magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin includes: magnetic Fe3O4, polyethyleneimine layer, gold nanolayer and DNA tetrahedron arranged from the inside to the outside.

2. A method for preparing a magnetic nanomaterial according to claim 1, characterized in that the steps include: S1. After uniformly mixing magnetic Fe3O4, Tris-HCl, and L-dopamine, polyethyleneimine, CuSO4, and H2O2 were added in sequence. After ultrasonic mixing, mechanical stirring was performed to react. After the reaction, the product was separated with a magnet, washed, and dried to obtain the polymer Fe3O4@DOPA / PEI. S2. placing the polymer Fe3O4@DOPA / PEI in an AuNPs solution, ultrasonically treating it and then mechanically stirring it to deposit the gold nanoparticles on the polymer Fe3O4@DOPA / PEI; separating the product with a magnet, washing it, and drying it to obtain Fe3O4@DOPA / PEI@Au; S3, mixing and incubating Fe3O4@DOPA / PEI@Au, TE buffer, DNA tetrahedron solution and NaCl solution to obtain Fe3O4@DOPA / PEI@Au@DNA TET; S4. The washed Fe3O4@DOPA / PEI@Au@DNA TET is dispersed in a mixed solution of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride for incubation. After the incubation is completed, trypsin solution is added for reaction. After the reaction is completed, the magnetic nanomaterial Fe3O4@DOPA / PEI@Au@DNA TET-Trypsin is obtained by washing and drying.

3. The preparation method according to claim 2, characterized in that In step S1, the preparation of magnetic Fe3O4 includes: mixing FeCl3·6H2O, diethylene glycol and ethylene glycol and performing ultrasonic dispersion, and then adding NaAc and sodium acrylate to obtain solid magnetic Fe3O4.

4. The preparation method according to claim 2, characterized in that In step S2, the preparation of the AuNPs solution includes: adding HAuCl4·4H2O to deionized water, condensing and refluxing under continuous heating, adding sodium citrate dihydrate when heated to 181°C, continuing heating for 15 minutes, and cooling to room temperature to obtain the AuNPs solution.

5. The preparation method according to claim 2, characterized in that In step S3, the preparation of DNA tetrahedrons includes: adding four single-stranded DNA solutions to TE buffer for reaction, and obtaining DNA tetrahedrons after the reaction is completed.

6. The preparation method according to claim 5, characterized in that Reactions include: After reacting at 95°C for 10 min, the reaction was transferred to 4°C for 30 min.

7. The preparation method according to claim 2, characterized in that In step S4, the volume ratio of N-hydroxysuccinimide to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 2:

1.

8. Use of the magnetic nanomaterial according to claim 1 or the magnetic nanomaterial prepared by the preparation method according to any one of claims 2 to 7 in glycopeptide mass spectrometry detection, characterized in that: The steps include: After pretreatment of the test substance, magnetic nanomaterials are added for enrichment. After enrichment, the magnetic nanomaterials are washed with a washing solution, and then eluted with an elution buffer. The eluate is analyzed using a matrix-assisted laser desorption ionization time-of-flight mass spectrometer.

9. The use according to claim 8, characterized in that The washing solution was NH4HCO3 buffer.

10. The use according to claim 8, characterized in that The elution buffer comprises acetonitrile, H2O and trifluoroacetic acid; wherein the volume ratio of acetonitrile, H2O and trifluoroacetic acid is 30:69.9:0.1.

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