CN112516972B - Nanometer material capable of selectively enriching and separating sulfur-containing compounds, and preparation method and application thereof - Google Patents
Nanometer material capable of selectively enriching and separating sulfur-containing compounds, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nanomaterial capable of selectively enriching and separating sulfur-containing compounds, which is a core-shell type ferroferric oxide/polydopamine/gold (Fe 3O 4/PDA/Au) composite magnetic nanomaterial. The preparation method of the material comprises the following steps: firstly, preparing amino modified Fe3O4/PDA, and reducing chloroauric acid (HAuCl 4) by using tetrakis (hydroxymethyl) phosphonium chloride (THPC) to obtain gold nanoparticles with small particle size, wherein the gold nanoparticles are adsorbed on the surface of the Fe3O4/PDA through electrostatic action and the viscosity of the PDA. And finally, reducing HAuCl4 for many times through hydroxylamine hydrochloride to form a complete gold nano layer by taking gold nano particles adsorbed on the surface of Fe3O4/PDA as crystal nuclei. The core-shell type Fe3O4/PDA/Au composite nano-material prepared by the method has good dispersibility, stability and biocompatibility, and can be used for selectively enriching and separating sulfur-containing compounds from a sample solution.
Description
Technical Field
The invention relates to a core-shell type magnetic nano material capable of selectively enriching and separating sulfur-containing compounds, a preparation method and application thereof, and Fe of the invention 3 O 4 The PDA/Au composite material can be used for enriching and separating sulfur-containing compounds in the fields of biology, chemistry, medicine and the like.
Background
Sulfur-containing compounds, such as cysteine, glutathione, homocysteine, are widely present in nature and in living bodies, participate in metabolism in the body, and have important physiological functions. The level of sulfhydryl compound in organism is closely related to clinical diseases, such as liver injury, skin diseases and the like caused by lack of cysteine in vivo, and the higher homocysteine possibly causes neurological diseases, alzheimer disease, cardiovascular diseases and the like, and glutathione is taken as the sulfhydryl compound with the largest intracellular content, is an important antioxidant in vivo, can remove excessive free radicals in vivo, and plays an important role in maintaining normal life activities of organism. Therefore, the method has important significance for monitoring the sulfur-containing compounds. However, the substrate environment in which the sulfur-containing compounds exist is complex, the interference is more, and the content of a plurality of sulfur-containing compounds in the substrate is very low, so that the effective enrichment of the sulfur-containing compounds is realized, the interference can be reduced, and the detection sensitivity and accuracy are improved.
The neuropeptide component in animals and the polypeptide toxin component in some animals, wherein the polypeptide containing disulfide bonds generally shows strong activity or strong toxicity due to the stable space structure, is one of important sources for finding active molecules, however, no good separation, detection and identification method exists at present due to the low content and poor stability of the neuropeptide or polypeptide toxin component containing disulfide bonds, and a method for rapidly enriching and detecting the neuropeptides and toxins containing disulfide bonds is urgently needed.
The existing enrichment method of sulfur-containing compounds mainly comprises the following steps: covalent chromatography, immobilized metal ion chromatography, molecularly imprinted polymers, gold nanomaterials, and the like. However, the method generally has the problems of complicated operation and long time consumption; some methods can only enrich one sulfur-containing compound, and have poor universality and small application range.
Disclosure of Invention
The invention aims to: the invention aims to overcome the defects of the prior art and provides a core-shell Fe capable of selectively enriching and separating sulfur-containing compounds 3 O 4 PDA/Au composite magnetic nano material and a preparation method thereof are provided, so that the rapid enrichment and separation of sulfur-containing compounds from complex sample matrixes are realized.
The invention solves the technical problems that: the process of selectively separating the sulfur-containing compounds from the gold nanoparticles mainly comprises the steps of adsorbing the sulfur-containing compounds, washing to remove non-adsorbed non-sulfur compounds, and eluting the sulfur-containing compounds adsorbed on the surfaces of the gold nanoparticles. Because the gold nanometer particle size is small, the gold nanometer particles which adsorb the sulfhydryl compound are required to be separated from the sample solution through high-speed centrifugal operation, the whole enrichment and separation process involves multiple centrifugal separation operations, the operation is complex and time-consuming, and the used gold nanometer cannot be recycled. Fe (Fe) 3 O 4 The nano particles can be rapidly separated from the solution under the external magnetic field due to magnetism, and have the characteristics of simple and rapid operation. At present, most of researches on preparing gold-magnetic composite nano particlesThe gold nanoparticles are directly coated on the surfaces of the magnetic nanoparticles, so that the effect is not ideal. Poly-dopamine (PDA has good hydrophilicity, biocompatibility and electrochemical characteristics, so that the poly-dopamine has important roles in the fields of biomedicine, biosensing and the like 3 O 4 The modification has the advantages of simple, convenient, quick and green method; at present, fe is prepared 3 O 4 The PDA/Au composite nano material is prepared by in situ reduction of HAuCl by PDA 4 (chloroauric acid), at Fe 3 O 4 The gold nano-particles are generated on the surface of the PDA, and are distributed in star points on the surface of the PDA, so that a complete gold nano-layer cannot be formed by the method, the PDA which is not coated by the gold nano-particles is easy to react with amino groups, sulfhydryl groups and other groups in target molecules to form covalent bonds, and enrichment and separation of sulfur-containing compounds are not facilitated. The invention adopts seed growth method, uses NH 2 Repeated reduction of HAuCl with OH HCl (hydroxylamine hydrochloride) as reducing agent 4 (chloroauric acid) forming a gold nano layer on the surface of the PDA, and finally preparing the core-shell Fe 3 O 4 The PDA/Au material has good hydrophilicity, dispersibility and biocompatibility, and can be applied to the fields of biology, chemistry, medicine and the like for selectively enriching and separating sulfur-containing compounds.
The technical scheme is as follows: the invention relates to a core-shell Fe capable of selectively enriching and separating sulfur-containing compounds 3 O 4 PDA/Au magnetic nano material, which uses magnetic Fe 3 O 4 Is taken as an inner core, oxidized and self-polymerized with dopamine on the surface of the inner core under alkaline condition to form a PDA layer, and reduced with THPC to HAuCl 4 The gold nano particles with small particle size are adsorbed on Fe 3 O 4 PDA surface and uses NH as crystal nucleus 2 Reduction of HAuCl by OH HCl 4 Forming a complete gold nano layer and finally preparing the core-shell Fe 3 O 4 PDA/Au composite magnetic nano material.
Specifically, core-shell Fe 3 O 4 The preparation method of the PDA/Au magnetic nano-material comprises the following steps:
PDA coated Fe 3 O 4 Preparation of nanoparticles:
taking Fe of the same mass 3 O 4 (100nm) nanoparticles and dopamine hydrochloride, adding Tris-HCl buffer solution with pH of 8.5, controlling the concentration of the dopamine to be 2mg/mL, stirring for 4 hours at room temperature, separating under an external magnetic field, washing with pure water for multiple times, and obtaining the PDA coated Fe 3 O 4 The nanoparticles are dispersed in pure water;
b. preparing a gold nano seed solution: dissolving 24 μL of 80% THPC (tetrakis (hydroxymethyl) phosphonium chloride) in 2mL of pure water, adding 90mL of pure water and 1mL of 1mol/L sodium hydroxide solution, stirring for 5min, and adding 1% HAuCl 4 After stirring for 5min, the reaction was terminated.
c. Fe loaded with gold nano seeds 3 O 4 Preparation of PDA: taking Fe in the above a 3 O 4 And (3) adding the gold nano seed solution in the step (b) into the PDA suspension, regulating the pH of the reaction solution to 2-3 by using a citric acid solution with the volume ratio of 1% to less than or equal to 1:5, stirring for 0.5-1 h at room temperature, magnetically separating, and washing by using pure water. Gold nano seeds with electronegativity are adsorbed on Fe through electrostatic action 3 O 4 Surface of PDA to obtain Fe 3 O 4 The PDA-Au seeds were dispersed in pure water.
d. Preparation of three-layer core-shell structure Fe 3 O 4 PDA/Au nanomaterial: (1) Taking 10mLFE 3 O 4 PDA-Au seed dispersion, 50mL of pure water was added and stirred for 1min; (2) 0.2mL of 1% HAuCl was added 4 Stirring is continued for 1min; (3) 0.37ml of 0.2mol/L NH was added 2 OH-HCl, reacting for 10min; (4) To the reaction mixture was added 0.25mL of 1% HAuCl 4 0.125mL of 0.2mol/L NH 2 OH HCl, repeating the step (4) for 6-9 times. Magnetic separation and washing to finally obtain core-shell Fe 3 O 4 PDA/Au composite magnetic nano material.
The specific adsorption steps are as follows: addition of Fe to sample solution of Sulfur-containing Compound 3 O 4 PDA/Au magnetic nanomaterial to make Fe 3 O 4 PDA/Au magnetic nano material is fully contacted with sample solution, after a certain period of light shaking, under the external magnetic field, removing supernatant liquor free of sulfur-containing compound, adding dithiothreitol, mercaptoethanol or mercaptoacetic acid to make adsorption on Fe 3 O 4 PDA/Au nano material meterThe sulfur-containing compounds of the surface are eluted.
The invention discovers that Chinese patent 201110190350.5 utilizes PDA to reduce HAuCl in situ through a great deal of experimental study 4 Gold nano-particles are formed on the surface of the PDA, the gold nano-particles are distributed on the surface of the PDA in star points, a finished gold nano-layer cannot be formed, and as a large amount of PDA is not coated with gold nano-particles, the PDA is easy to be irreversibly combined with mercapto groups, amino groups and other groups in target molecules, and cannot be applied to enrichment and separation of sulfur-containing compounds. Chinese patent 201710343036.3 by SiO 2 Coating Fe 3 O 4 By modification of Fe 3 O 4 /SiO 2 The amino and sulfhydryl groups are combined with gold nano-particles, the required reagents are various, the preparation process is complex, the time is long (16-24 h), and a complete gold nano-layer cannot be formed, so that the subsequent enrichment of sulfur-containing compounds is not facilitated. Chinese patent 201210236102.4 by SiO 2 Coating Fe 3 O 4 Preparing gold nano seed solution by taking THPC as a reducing agent, and reducing HAuCl for multiple times by taking formaldehyde as the reducing agent 4 Forming a gold nano layer, although shortening SiO 2 Coating Fe 3 O 4 But uses formaldehyde to reduce HAuCl 4 When the gold nano layer is formed, three-step reduction conditions are complex and time-consuming (the gold nano coating time is as long as 47h in the whole process), the material preparation process is complex, and compared with PDA, siO 2 No better biocompatibility exists. The invention firstly adsorbs gold nano particles with small particle size on the surface of PDA as seeds for gold growth, uses NH 2 OH & HCl is used as a reducing agent to perform multiple reduction on the surface of the nano-gold material to obtain a complete nano-gold layer, and finally the Fe3O4/PDA/Au magnetic nano-material with a core-shell structure is prepared. Therefore, compared with the above comparison document, the invention has the following advantages:
1. preparing a gold nano seed solution by using THPC (tetrakis (hydroxymethyl) phosphonium chloride) as a reducing agent, wherein the diameter of gold nano particles is about 2nm, so that the gold nano particles are distributed more uniformly on the surface of the PDA;
2. using NH 2 Repeated reduction of HAuCl by OH HCl 4 Gold nano thin layers can be formed on the surface of the PDA, and the prepared material is more uniform and is more beneficial to the adsorption of sulfur-containing compounds, thereby realizing the adsorption of the sulfur-containing compoundsQuick enrichment and separation;
3. the preparation time is short, and the preparation of the material can be completed within 4-6 hours.
Firstly, preparing amino modified core-shell structure Fe 3 O 4 PDA nanoparticles; at the same time reduce HAuCl by THPC 4 To obtain negatively charged gold nanoparticles with smaller particle diameter (2 nm), which are easy to adsorb to amino-modified Fe 3 O 4 The PDA surface can be used as a seed for forming a gold nano layer in the next step; the Fe is then described above 3 O 4 Uniformly mixing and stirring PDA and gold nano suspension to obtain assembled gold magnetic composite particles Fe 3 O 4 PDA/Au; finally with Fe 3 O 4 Gold nanoparticles adsorbed on PDA/Au surface and reduced by THPC as crystal nucleus, NH was used 2 The OH-HCl is subjected to multi-step reduction on the surface of the gold shell to obtain a layer of complete gold shell, thereby preparing three-layer core-shell gold magnetic nanoparticle Fe 3 O 4 /PDA/Au。
Gold magnetic nanoparticle Fe with three-layer core-shell structure prepared by the method 3 O 4 PDA/Au with particle size of about 120nm has excellent dispersivity, biocompatibility and magnetic response.
The beneficial effects are that:
(1) Direct in situ reduction of HAuCl using PDA 4 The method has the defect that a complete gold nano layer cannot be formed, and the method prepares core-shell Fe by using a self-assembly technology and a seed growth method 3 O 4 PDA/Au magnetic nano material adopting multiple reduction of HAuCl in preparation of gold nano layer 4 In the mode, the particle size distribution of the prepared nano particles is uniform, and a complete and uniform gold nano layer can be generated on the surface of the nano particles.
(2) Compared with the method for enriching and separating the sulfur-containing compound by the non-magnetic gold nano-particles, the addition of the magnetic nano-material omits repeated centrifugation and filtration, so that the whole preparation process is simple and quick, and the method is particularly suitable for enriching and separating the thiol compound which is easy to oxidize.
Drawings
FIG. 1 is Fe 3 O 4 Particles; fe (Fe) 3 O 4 PDA particleGranulating; core-shell structured gold magnetic nanoparticle Fe 3 O 4 Infrared spectrogram of PDA/Au.
FIG. 2 shows a core-shell gold magnetic nanoparticle Fe 3 O 4 Transmission electron microscopy of PDA/Au and intermediate of the reduction process. FIG. 2a is Fe 3 O 4 A transmission electron microscope image of the particles; FIG. 2b is Fe 3 O 4 Transmission electron microscopy of PDA particles; FIG. 2c is a core-shell particle Fe 3 O 4 Transmission electron microscope image of PDA/Au.
FIG. 3 shows a core-shell gold magnetic nanoparticle Fe 3 O 4 X-ray photoelectron spectroscopy of PDA/Au and intermediate of reduction process. FIG. 3a is Fe 3 O 4 X-ray photoelectron spectroscopy of PDA particles; FIG. 3b is Fe 3 O 4 X-ray photoelectron spectroscopy of PDA/Au particles; FIG. 3c is Fe 3 O 4 X-ray photoelectron spectroscopy of Au in PDA/Au particles.
FIG. 4 shows high performance liquid chromatograms before and after enrichment of a sample solution. FIG. 4a shows that no Fe is added 3 O 4 High performance liquid chromatography of the sample solution of PDA/Au material; FIG. 4b shows the addition of Fe 3 O 4 High performance liquid chromatogram of sample solution after enrichment of PDA/Au material.
FIG. 5 is a graph showing the comparison and detection of the content of natural products of sulforaphane and glucoraphanin before and after enrichment.
Detailed Description
EXAMPLE 1 core-shell Fe 3 O 4 Preparation of PDA/Au magnetic nano material
Core-shell Fe 3 O 4 The preparation process of the PDA/Au magnetic nano-material comprises the following steps:
(1) preparation of PDA coated Fe 3 O 4 Nanoparticles; (2) reduction of HAuCl using THPC 4 Obtaining gold nanoparticles with small particle size; (3) gold nanoparticles are adsorbed on Fe through electrostatic effect and viscosity of PDA 3 O 4 Surface of PDA; (4) by NH 2 Multiple reduction of HAuCl by OH HCl 4 Forming a complete gold nano layer and preparing Fe with a three-layer core-shell structure 3 O 4 PDA/Au nanomaterial.
The specific experimental procedure is as follows:
a. preparation of PDA coated Fe 3 O 4 And (3) nanoparticles: weighing Fe 3 O 4 100mg of (100 nm) nanoparticle and 100mg of dopamine hydrochloride are respectively added with 50ml of Tris-HCl buffer solution with pH of 8.5, stirred for 4 hours at room temperature, separated under an external magnetic field, washed for multiple times by pure water, and the prepared PDA coated Fe is obtained 3 O 4 The nanoparticles were dispersed in 10mL of purified water. 584cm in the IR spectrum of FIG. 1 -1 The absorption peak at this point is Fe 3 O 4 Characteristic absorption peaks of Fe-O bonds on the surface; 3426cm -1 Is the O-H stretching vibration peak affected by hydrogen bond. Fe (Fe) 3 O 4 PDA and Fe 3 O 4 Compared with nano particles, fe 3 O 4 The absorption peak of the Fe-O bond in PDA is red shifted. At 2961 and 2848cm -1 The absorption peak appearing at the position is the expansion vibration of C-H on the PDA; at 1618cm -1 、1509cm -1 、1445cm -1 1261cm -1 The absorption peaks at PDA are respectively C= O, N-H, C=C on the aromatic ring and C-OH vibration absorption on the aromatic ring. The PDA is described as Fe 3 O 4 A PDA layer is formed on the surface, and Fe is added 3 O 4 Coating the glass fiber with the glass fiber.
b. Preparing a gold nano seed solution: dissolving 24 μL of 80% THPC in 2mL of pure water, adding 90mL of pure water and 1mL of 1mol/L sodium hydroxide solution, stirring for 5min, and adding 1% HAuCl 4 After stirring for 5min, the reaction was terminated.
c. Fe loaded with gold nano seeds 3 O 4 Preparation of PDA: 5mL of Fe in the above a was taken 3 O 4 And (3) adding 25mL of the gold nano seed solution in the step (b) into the PDA suspension, regulating the pH of the reaction solution to 2-3 by using a 1% citric acid solution, stirring for 1h at room temperature, magnetically separating, and washing by using pure water. Gold nano seeds with electronegativity are adsorbed on Fe through electrostatic action and adhesion of PDA 3 O 4 PDA surface to obtain Fe 3 O 4 PDA-Au seeds were dispersed in 20mL pure water.
d. Preparation of three-layer core-shell structure Fe 3 O 4 PDA/Au nanomaterial: (1) 10mL of Fe was taken 3 O 4 PDA-Au seedAdding 50mL of pure water into the dispersion, and stirring for 1min; (2) 0.2mL of 1% HAuCl was added 4 Stirring is continued for 1min; (3) 0.37ml of 0.2mol/L NH was added 2 OH-HCl, reacting for 10min; (4) To the reaction mixture was added 0.25mL of 1% HAuCl 4 0.125mL of 0.2mol/L NH 2 OH HCl, the above step (4) is repeated 9 times. Magnetic separation and washing to finally obtain core-shell Fe 3 O 4 PDA/Au composite magnetic nano material. FIGS. 2 a-c are Fe respectively 3 O 4 、Fe 3 O 4 PDA and Fe 3 O 4 Transmission electron microscope image of PDA/Au. PDA is easy to oxidize and self-polymerize in alkaline solution and forms a PDA layer on the surface of the substrate, as shown in a of FIG. 2, fe 3 O 4 Fe formed by self-polymerization of PDA on its surface 3 O 4 The PDA is a sphere-type structure, wherein the PDA is about 20nm thick (FIG. 2 b); NH (NH) 2 Reduction of HAuCl by OH HCl 4 The formed gold particles continuously grow and wrap on the surface of the THPC-Au seed, and finally the gold nano-shell layer is formed (figure 2 c). XPS for Fe 3 O 4 /PDA、Fe 3 O 4 The PDA/Au surface element composition was verified. As shown in FIGS. 3 a-b, the elements C, O, N, au are present in Fe 3 O 4 Surface of PDA/Au. The Fe2p3 binding at 710.20eV was not detected, further demonstrating that Fe in the composite material 3 O 4 The coating is completely carried out by the PDA, and the coating is consistent with the transmission electron microscope image result; as shown in FIG. 3c, there are two peaks at 83.0 and 86.6eV, ascribed to Au 0 The excitation of Au 4f electrons of the nano-gold particles proves that the nano-gold particles are coated on the surface of the PDA. Comprehensive transmission electron microscope, X-ray photoelectron spectroscopy analysis and infrared analysis results show that the experimental scheme of the invention combining the self-assembly technology and the seed growth method can obtain gold magnetic nanoparticle Fe with three-layer structure coated with complete gold shell 3 O 4 /PDA/Au。
e. And (3) performance verification: the specific adsorption steps are as follows: immersing 50mg of the prepared Fe in 10mL of mixed sample solution (containing cysteine, glutathione, tyrosine and tryptophan) 3 O 4 PDA/Au magnetic nanomaterial to make Fe 3 O 4 The PDA/Au magnetic nano material is fully connected with the sample solutionAfter touching and gently shaking for a certain time, removing supernatant without sulfhydryl compound under external magnetic field, adding dithiothreitol to adsorb on Fe 3 O 4 And eluting the sulfhydryl compound on the surface of the PDA/Au nano-material. FIGS. 4a and 4b are, respectively, the addition of Fe 3 O 4 High performance liquid chromatogram of mixed sample before and after PDA/Au enrichment, as shown in figure, cysteine and glutathione of sulfhydryl-containing compound in solution are adsorbed by material, while signal intensity of tyrosine and tryptophan of sulfhydryl-free compound is kept unchanged, which proves Fe 3 O 4 The PDA/Au material has selective adsorption to only sulfhydryl compounds. (chromatographic peak assignment: 1. Tyrosine; 2. Cysteine; 3. Tryptophan; 4. Glutathione).
EXAMPLE 2 enrichment of thiol-rich Polypeptides in proteomic Studies
(1) Extraction and enzymolysis of mouse brain tissue protein
About 5mg of hypothalamic tissue of a mouse is taken, 40. Mu.l of protein extract (50 mM Tris-HCl solution of 4% SDS) is added, ultrasonic extraction is carried out until the sample is dissolved, 10mM DTT is added, incubation is carried out for 30min, 55mM Iodoacetamide (IAA) is added, incubation is carried out for 30min in a dark place, 220. Mu.l of 80% acetone is added after the addition of 10mM DTT, precipitation is carried out at 4 ℃ overnight, centrifugation is carried out at 16000rpm, supernatant is removed, 220. Mu.l of 80% acetone is added again, the supernatant is removed, and the mixture is left at room temperature for 12min until the acetone is completely volatilized. Adding protein lysate (Tris-HCl buffer solution containing 8M urea), fully dissolving, diluting with Tris-HCl buffer solution until the urea concentration is lower than 1M, adding trypsin according to the mass percent of 1%, carrying out enzymolysis for 12 hours at 37 ℃ to obtain enzymolysis solution, then placing the enzymolysis solution on a Seppak C18 column, washing with trifluoroacetic acid, eluting with 80% acetonitrile solution containing 0.2% trifluoroacetic acid, and collecting eluent.
(2) Enrichment of sulphur-containing peptide fragments
Immersing 50mg of Fe in the eluent obtained in the step (1) 3 O 4 PDA/Au magnetic nanomaterial (prepared in example 1 of the present invention) to allow Fe to be used 3 O 4 PDA/Au magnetic nano material is fully contacted with sample solution, after a certain period of light shaking, under the external magnetic field the supernatant liquor free of mercapto compound is removed, and added with two componentsThiothreitol pair adsorbed on Fe 3 O 4 And eluting the sulfhydryl compound on the surface of the PDA/Au nano-material to obtain the sulfur-containing polypeptide part.
(3) Nano LC-MS/MS high-throughput identification peptide fragment sequence
Injecting the sulfur-containing polypeptide obtained in the step (2) into a Dan U3000 NanonRSLC nano liter liquid phase system, wherein a chromatographic column is 5 mu m reprosil C18AQ (75 mu m multiplied by 150 mm), the loading amount is 1-2 mu L, the flow rate is 200-400 nL/min, the mobile phase A (acetonitrile/formic acid/water=2/0.2/98, v/v/v), the mobile phase B (acetonitrile/formic acid/water=80/0.2/20, v/v/v) and the linear gradient elution is carried out for 150min by 2-30% B;
and using a Thermo Q-Exactive Orbitrap mass spectrometer for peptide fragment analysis, wherein the spraying voltage is 2.5kV, and the ion transmission capillary temperature is 200 ℃; the primary full scanning range of the mass spectrum is m/z 300-2000, and the separation width is 3Da; tandem mass spectrometry adopts a secondary mass spectrometry scanning mode based on primary mass spectrometry data, sequentially selects 5 ions with highest ion intensity in the primary mass spectrometry to carry out Collision Induced Dissociation (CID) secondary tandem mass spectrometry, and adopts Xcalibur software to carry out data acquisition;
the secondary mass spectrum data adopts PEAKS 8.5 software to carry out library searching identification analysis, a corresponding protein database is selected, and the retrieval parameters are set as follows: precursor ion error 10ppm; the ion error is 1Da; 2 sites are allowed to be miscut, and the false positive rate is less than or equal to 1%; the enzyme cutting mode selects pancreatin (Trypsin), and the number of unique peptide fragments is more than or equal to 2; other parameters are default parameters, the score obtained under the above search condition has significance, and P <0.05 is considered as a valid identification result; and identifying and determining the amino acid sequences of all peptide fragments in the enzymolysis liquid of each sample.
The result was identified from them that 6623 peptide fragments were obtained, of which the number of peptide fragments containing Cys (cysteine) was 4650, and the concentration of peptide fragments containing thiol reached 70%. Of 6623 peptide fragments, 1107 peptide fragments containing Met (methionine, another sulfur-containing amino acid) were identified to account for about 17%, and thus, fe by the present invention 3 O 4 The PDA/Au magnetic nano-material can well complete enrichment of the sulfur-containing peptide segment, in particular to enrichment of the sulfhydryl-containing peptide segment.
EXAMPLE 3 enrichment of thiol-containing peptide fragments in extracts of animals of Keratin origin
(1) The preparation method comprises the steps of preparing slices of Chinese medicinal cornu Bubali, pulverizing to obtain powder, adding 20 times of water, decocting and extracting for 3 times, each for 4 hours, mixing the extractive solutions, and concentrating to proper concentration to obtain cornu Bubali water extract.
(2) Enrichment of sulphur-containing peptide fragments
Immersing 50mg of Fe in the buffalo horn water extract obtained in the step (1) 3 O 4 PDA/Au magnetic nanomaterial (prepared in example 1 of the present invention) to allow Fe to be used 3 O 4 PDA/Au magnetic nano material is fully contacted with sample solution, after a certain period of light shaking, under the external magnetic field, removing supernatant fluid containing no sulfhydryl compound, adding dithiothreitol or mercaptoethanol to make adsorption on Fe 3 O 4 And eluting the sulfhydryl compound on the surface of the PDA/Au nano-material to obtain the thiol-containing polypeptide part of the buffalo horn.
(3) Nano LC-MS/MS high-throughput identification peptide fragment sequence
Desalting the buffalo horn thiol-containing polypeptide part obtained in the step (2) by adopting Seppakc18, drying, re-dissolving by using an initial mobile phase, injecting into a Daian U3000 NanoRSLC nano liter liquid phase system, performing linear gradient elution on a chromatographic column with 5 mu m Repro il C18AQ (75 mu m multiplied by 150 mm), a loading amount of 1-2 mu L, a flow rate of 200-400 nL/min, a mobile phase A (acetonitrile/formic acid/water=2/0.2/98, v/v/v), a mobile phase B (acetonitrile/formic acid/water=80/0.2/20, v/v/v) and 2-30% B for 150min;
and using a Thermo Q-Exactive Orbitrap mass spectrometer for peptide fragment analysis, wherein the spraying voltage is 2.5kV, and the ion transmission capillary temperature is 200 ℃; the primary full scanning range of the mass spectrum is m/z 300-2000, and the separation width is 3Da; tandem mass spectrometry adopts a secondary mass spectrometry scanning mode based on primary mass spectrometry data, sequentially selects 5 ions with highest ion intensity in the primary mass spectrometry to carry out Collision Induced Dissociation (CID) secondary tandem mass spectrometry, and adopts Xcalibur software to carry out data acquisition;
the secondary mass spectrum data adopts PEAKS 8.5 software to carry out library searching identification analysis, a bovine protein database is selected, and the retrieval parameters are set as follows: precursor ion error 10ppm; the ion error is 1Da; 2 sites are allowed to be miscut, and the false positive rate is less than or equal to 1%; the enzyme cutting mode selects Non enzyme cutting (Non enzyme), and the number of unique peptide fragments is more than or equal to 2; other parameters are default parameters, the score obtained under the above search condition has significance, and P <0.05 is considered as a valid identification result; and identifying and determining the amino acid sequences of all peptide fragments in the enzymolysis liquid of each sample.
The result was identified from the above, 1019 peptide fragments, wherein the number of peptide fragments containing Cys (cysteine) was 737, and the concentration of peptide fragments containing thiol reached 72%. Some of the thiol-containing peptides identified as obtained contain 4-5 Cys, such as TITPCISSPCAPAAPCTPCVPR, AQASCCRPSYCGQSCCR, RPVCCDPCSLQEGCCR, SCQAVVCRPCCW, etc. It can be seen that by Fe 3 O 4 The PDA/Au magnetic nano-material can well complete enrichment of the peptide segment containing the sulfhydryl group in the cutin traditional Chinese medicine extracting solution.
Example 4 enrichment of natural products of raphanin and raphanin
(1) The sulforaphane and the sulforaphane are rich in cruciferous plants and are common antioxidants. The broccoli contains sulforaphane and glucoraphanin, 500g of broccoli is cut into small pieces, 500ml of water is added for decoction and extraction for 30min, then the obtained product is concentrated to a proper amount, and the volume is fixed to 100ml, so that the broccoli extract is obtained.
(2) Enrichment of raphanin and raphanin
Immersing 50mg of Fe in 100ml of broccoli extract obtained in the step (1) 3 O 4 PDA/Au magnetic nanomaterial (prepared in example 1 of the present invention) to allow Fe to be used 3 O 4 PDA/Au magnetic nano material is fully contacted with sample solution, after a certain period of light shaking, under the external magnetic field, removing supernatant liquor containing no sulfhydryl compound, adding cysteine to make adsorption on Fe 3 O 4 And eluting the sulfhydryl compound on the surface of the PDA/Au nano-material to obtain enriched broccoli extract, and fixing the volume to 100ml to obtain broccoli enriched liquid.
(3) Determination of sulforaphane and sulforaphane
Chromatographic conditions: wondaSil C18 column (4.6 mm. Times.250 mm,5 μm); mobile phase: gradient eluting with methanol-water for 0-20 min, and increasing methanol concentration from 1% to 30%; the detection wavelength is 254nm; the flow rate is 1.0ml/min; column temperature is 30 ℃; the theoretical plate number is not less than 5000 based on sulforaphane.
And (3) respectively carrying out HPLC analysis on the broccoli extract and the broccoli enrichment solution, and measuring the contents of the sulforaphane and the sulforaphane in the two samples by adopting a standard curve method. As shown in fig. 5 before and after enrichment, the contents of sulforaphane and sulforaphane in the broccoli extract are measured before enrichment: 58ng/ml and 1806ng/ml, and the content of the sulforaphane and the sulforaphane in the enriched broccoli enrichment liquid is 1289ng/ml and 23 mu g/ml respectively, and the sulforaphane are enriched by 22.4 times and 12.7 times respectively, which indicates that the total Fe is used for preparing the broccoli 3 O 4 The PDA/Au magnetic nano material can well complete enrichment of two sulfur-containing compounds of the sulforaphane and the sulforaphane.
Claims (1)
1. A method for enriching and separating sulfur-containing compounds, comprising the steps of:
adding a sample solution containing a sulfur compound to core-shell Fe 3 O 4 In the PDA/Au composite magnetic nano material, after the PDA/Au composite magnetic nano material is gently shaken for a certain time, under the action of an externally applied magnetic field, the supernatant liquid without sulfur compounds is removed; or slowly flowing a sample solution containing sulfur compounds through Fe with core-shell type 3 O 4 PDA/Au composite magnetic nano material test tube; then adding dithiothreitol, mercaptoethanol, thioglycollic acid or cysteine, and adsorbing Fe on core-shell type 3 O 4 Eluting the sulfur-containing compound on the surface of the PDA/Au composite magnetic nano-material;
the core-shell Fe 3 O 4 The preparation method of the PDA/Au composite magnetic nano-material comprises the following steps:
a. PDA coated Fe 3 O 4 Preparation of nanoparticles:
taking Fe of the same mass 3 O 4 Adding Tris-HCl buffer solution with pH of 8.5 into the nanoparticles and dopamine hydrochloride, controlling the concentration of the dopamine hydrochloride to be 2mg/mL, stirring at room temperature for 4h, separating under an external magnetic field, washing with pure water for multiple times, and preparing the PDA coated Fe 3 O 4 Nanoparticles, and dispersing them in pure water;
b. preparing a gold nano seed solution:
dissolving 24 μL of 80% THPC in 2mL pure water, adding 90mL pure water and 1mL 1mol/L sodium hydroxide solution, stirring for 5min, adding 1% HAuCl 4 After stirring for 5min, ending the reaction;
c. fe loaded with gold nano seeds 3 O 4 Preparation of PDA:
taking the PDA coated Fe prepared in the step a 3 O 4 Adding a prepared gold nano seed solution into the nanoparticle suspension, regulating the pH value of the solution to 2-3 by using a 1% citric acid solution, stirring for 0.5-1 h at room temperature, performing magnetic separation, washing by pure water, and adsorbing the gold nano seeds with electronegativity on Fe by electrostatic action, wherein the volume ratio of the gold nano seed solution to the nanoparticle suspension is less than or equal to 1:5 3 O 4 Surface of PDA to obtain Fe 3 O 4 Dispersing the PDA-Au seeds in pure water;
d. preparation of core-shell structured Fe 3 O 4 PDA/Au nanomaterial:
(1) 10mL of Fe was taken 3 O 4 Adding 50mL pure water into the PDA-Au seed dispersion liquid, and stirring for 1min;
(2) 0.2mL of 1% HAuCl was added 4 Stirring is continued for 1min;
(3) 0.375mL of 0.2mol/L NH was added 2 OH ‧ HCl, reacting for 10min;
(4) To the reaction mixture was added 0.25mL of 1% HAuCL 4 And 0.125mL of 0.2mol/L NH 2 OH‧HCl；
(5) Repeating the step (4) for 6-9 times, magnetically separating, and washing to obtain core-shell Fe 3 O 4 PDA/Au composite magnetic nano material.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893634A (en) * | 2009-05-20 | 2010-11-24 | 中国科学院生物物理研究所 | Specific detection method of protein or polypeptide cysteine sulfydryl modification and application thereof |
| CN102861921A (en) * | 2011-07-08 | 2013-01-09 | 西南民族大学 | Preparation method of core-shell magnetic/gold nano particles |
| CN104162414A (en) * | 2014-08-01 | 2014-11-26 | 曲阜师范大学 | Vacant shell type poly-dopamine adsorbed nanogold composite material as well as preparation and application thereof |
| CN104258909A (en) * | 2014-08-01 | 2015-01-07 | 曲阜师范大学 | Fe3O4-poly-dopamine-Au nano-composite material as well as preparation method and application thereof |
| CN109369912A (en) * | 2018-11-05 | 2019-02-22 | 南方科技大学 | Method for controllably grafting DNA on surface density of polydopamine nano particle |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8784895B2 (en) * | 2011-03-15 | 2014-07-22 | Northwestern University | Multifunctional metal nanoparticles having a polydopamine-based surface and methods of making and using the same |
-
2019
- 2019-09-19 CN CN201910886669.8A patent/CN112516972B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101893634A (en) * | 2009-05-20 | 2010-11-24 | 中国科学院生物物理研究所 | Specific detection method of protein or polypeptide cysteine sulfydryl modification and application thereof |
| CN102861921A (en) * | 2011-07-08 | 2013-01-09 | 西南民族大学 | Preparation method of core-shell magnetic/gold nano particles |
| CN104162414A (en) * | 2014-08-01 | 2014-11-26 | 曲阜师范大学 | Vacant shell type poly-dopamine adsorbed nanogold composite material as well as preparation and application thereof |
| CN104258909A (en) * | 2014-08-01 | 2015-01-07 | 曲阜师范大学 | Fe3O4-poly-dopamine-Au nano-composite material as well as preparation method and application thereof |
| CN109369912A (en) * | 2018-11-05 | 2019-02-22 | 南方科技大学 | Method for controllably grafting DNA on surface density of polydopamine nano particle |
Non-Patent Citations (1)
| Title |
|---|
| 顾觉奋.《离子交换与吸附树脂在制药工业上的应用》.中国医药科技出版社,2008,第313-314页. * |
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