CN108693354B - Flavone glycoside functional magnetic nanometer affinity probe and its preparation method and application and intracellular target proteins catching method - Google Patents
Flavone glycoside functional magnetic nanometer affinity probe and its preparation method and application and intracellular target proteins catching method Download PDFInfo
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- CN108693354B CN108693354B CN201710217509.5A CN201710217509A CN108693354B CN 108693354 B CN108693354 B CN 108693354B CN 201710217509 A CN201710217509 A CN 201710217509A CN 108693354 B CN108693354 B CN 108693354B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The present invention relates to drug targets protein-enriched, separation and detection fields, disclose a kind of flavone glycoside functional magnetic nanometer affinity probe；The present invention also provides the preparation method of the flavone glycoside functional magnetic nanometer affinity probe and its applications in the capture of drug targets albumen and/or identification of cell；And the method that intracellular target proteins are captured by flavone glycoside functional magnetic nanometer affinity probe.Affinity probe of the invention includes magnetic nano-particle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface, wherein, one end of the coupled structures is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound.The affinity probe only needs a small amount of holoprotein extracting solution that high specific capture can be realized.
Description
Technical field
The present invention relates to drug targets protein-enriched, separation and detection fields, and in particular, to a kind of flavone glycoside function
Magnetic Nano affinity probe and preparation method thereof and its answering in the capture of drug targets albumen and/or identification of cell can be changed
With；And the method that intracellular target proteins are captured by flavone glycoside functional magnetic nanometer affinity probe.
Background technique
Flavone compound is a kind of polyphenols being widely present in plant, it mostly from plant secondary generation
It thanks.Its basic parent nucleus is 2- phenyl chromone, is with C6-C3-C6Mode connect.It is distinguished according to link position and chemical property,
Mainly there are flavonoids, flavonols, flavanone, anthocyanidin, flavanonol, isoflavones, your ear ketone etc..Numerous studies display is yellow
Ketone compounds have free radical resisting, and analgesia is antipyretic, antitumor, mitigate cardiac-cerebral ischemia reperfusion injury, diuresis, and liver protection is immunized
It adjusts, the multiple functions such as antiviral, there is great exploitation potential quality.
With the extensive use of proteomics method, the method based on chemical proteomics confirms active ingredient of Chinese herbs
The research of target proteins identification is more and more in recent years.Presently, there are challenge mainly include two aspects that on the one hand have pharmacology
Active molecule is weaker with the interaction itself between protein, and conventional method is difficult to keep the interaction between them
It is not destroyed；Still further aspect is exactly the expression of drug targets albumen much in trace level, common ESI-MS or
MALDI-MS loses the information of many weight target proteins matters due to detection limit problem.And if in drug targets protein enrichment process
The concentration of target proteins can be improved, reduce the loss in preparation of samples, while life is combined using nanoliter Liquid Chromatography-Tandem Mass Spectrometry
It is identified out will to have more target proteins for object informatics technology.
Summary of the invention
The purpose of the present invention is overcoming, the prior art needs a large amount of cell holoprotein extracting solutions and specificity is lower scarce
It falls into, provides a kind of flavone glycoside functional magnetic nanometer affinity probe, which only needs a small amount of holoprotein extracting solution i.e.
High specific capture can be achieved, the present invention also provides the preparation method of the flavone glycoside functional magnetic nanometer affinity probe and its
Application in the capture of drug targets albumen and/or identification of cell.
The present inventor is had found by in-depth study, is fixed on specific flavone glycoside molecule by chemical modification
Magnetic Nano material surface, can use its with target biomolecule interaction directly from sample solution to protein/more
The target molecules such as peptide carry out enrichment and desalination, to eliminate the sample elution step for being easiest to cause damages；Also, pass through
Quick separating may be implemented using additional magnetic field, enormously simplify separation, shorten time for sample pretreatment；Simultaneously by building
The qualitative of the protein of target can be realized using a small amount of cell holoprotein extracting solution in the method for having found nano-LC-MS/MS
With quantitative analysis.
One aspect of the present invention provides a kind of flavone glycoside functional magnetic nanometer affinity probe as a result, wherein spy that this is affine
Needle includes magnetic nano-particle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface,
In, one end of the coupled structures is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound
It connects.
Second aspect of the present invention provides a kind of preparation method of flavone glycoside functional magnetic nanometer affinity probe, wherein
This approach includes the following steps,
1) compound of flavone glycoside compound and structure shown in formula (2) in the presence of an organic, is carried out first to connect
Touching, obtains the intermediate compound that one end of the compound of structure shown in formula (2) is mutually bonded with flavone glycoside compound；
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains knot shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of the compound of structure is connected with magnetic nano-particle；
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that the amino of amino or BOC protection, n are the whole of 2-10
Number.
Third aspect present invention provides a kind of method for capturing intracellular target proteins, wherein this method includes following step
It is rapid:
1) make obtained by flavone glycoside functional magnetic nanometer affinity probe or preparation method of the invention of the invention
Ketoside functional magnetic nanometer affinity probe is contacted with cell holoprotein extracting solution, so that flavone glycoside functionalization magnetic
Property nanometer affinity probe is in conjunction with intracellular target proteins；
2) unbonded albumen is removed, to isolate the albumen of capture；
3) albumen of capture is identified；
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside
The intracellular target proteins of compound specificity identification.
Fourth aspect present invention provides flavone glycoside functional magnetic nanometer affinity probe or system of the invention of the invention
Ketoside functional magnetic nanometer affinity probe obtained by Preparation Method is in the capture of drug targets albumen and/or identification of cell
Application.
Through the above technical solutions, flavone glycoside functional magnetic nanometer affinity probe of the invention only needs a small amount of cell
The capture of intracellular target proteins can be realized in holoprotein extracting solution, and albumen capture specificity is high.In addition, by of the invention
Flavone glycoside functional magnetic nanometer affinity probe is for passing through simple externally-applied magnetic field step when capturing intracellular target proteins
Target proteins that magnetic Nano affinity probe captures and other Protein Separations can be simplified operation.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the ESI-MS map of radix scutellariae glycoside derivates Qz-1 in embodiment 1.
Fig. 2 is the infrared absorpting light spectra of radix scutellariae glycoside derivates Qz-1 in embodiment 1.
Fig. 3 is according to the four of the scutelloside functional magnetic nanometer affinity probe and alkynyl that are prepared in embodiment 2
The schematic diagram of Fe 3 O magnetic nano-particle.
Fig. 4 is the transmission electron microscope photo of the alkynyl ferriferrous oxide nano-particle in embodiment 2.
Fig. 5 is the TOF-SIMS mass spectrogram that the upper figure in embodiment 2 is MNPs@Qz-1 in embodiment 2, and the following figure is MNPs's
TOF-SIMS mass spectrogram.
Fig. 6 is that scutelloside, the Qz-1 of various concentration in embodiment 6 are active to people's hepatomicrosome enzyme HLM fluorescence probe CDE
It influences.
It is special that the upper figure of Fig. 7 is that the positive control probe in embodiment 7 is captured from HEK293T cell holoprotein extracting solution
Property target proteins matter GCYB1 one peptide fragment of trypsin digestion second order ms figure, the following figure is peptide fragment difference in the upper figure
By the first mass spectrometric figure after weight acetylation isotope reagent label.
Specific embodiment
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
Flavone glycoside functional magnetic nanometer affinity probe provided by the invention is characterized in that the affinity probe includes magnetic
Property nanoparticle, coupled structures and the flavone glycoside compound for being supported on the magnetic nano-particle surface, wherein the idol
The one end for being coupled structure is connected with the magnetic nano-particle surface, and the other end is connected with flavone glycoside compound.
According to the present invention, the magnetic nano-particle refers to the magnetic nanoparticle of tool, such as can be four oxidations three
Fe nanometer particles.
In addition, the magnetic nano-particle may be core-shell structure, for example, its kernel is magnetic particle, outer layer covers
SiO2.It for example can be with as such magnetic nano-particle are as follows: its kernel is ferriferrous oxide nano-particle, outer layer covers SiO2
Magnetic nano-particle.
In accordance with the present invention it is preferred that the average grain diameter of the magnetic nano-particle is 100-400nm, preferably 100-
12nm。
Preferably, the group key that the magnetic nano-particle passes through the group of surface modification and one end of the coupled structures
It closes.
Preferably, the group of the magnetic nano-particle surface modification is alkynyl, the idol being mutually bonded with the alkynyl
The group for being coupled one end of structure is azido, alternatively, the group of the magnetic nano-particle surface modification is azido, and it is described
The group of one end of the coupled structures of nitrine base phase bonding is alkynyl, alternatively, the magnetic nano-particle surface modification
Group is n-hydroxysuccinimide, and one end group for the coupled structures being mutually bonded with the n-hydroxysuccinimide is ammonia
Base.
It is further preferred that compound of the flavone glycoside compound for structure shown in following formula (1), and the formula
(1) compound of structure shown in passes through the carboxyl of 7 connected uronic acids of O of flavone glycoside and the base of the coupled structures other end
Group Xiang Jianhe.
It is further preferred that the coupled structures that the compound of the structure shown in the formula (1) combines is another
The group at end is amino.
In accordance with the present invention it is preferred that the coupled structures are provided by following formula (2) compound represented,
Wherein, R1Indicate azido, R2Indicate that amino, n indicate the integer of 2-10, preferably 2,3,4 or 5.
In the present invention, the atom number of compound shown in formula (1) is as follows:
In a particularly preferred embodiment of the present, in formula (1), R1Indicate azido, R2Indicate amino, n 3；
And the group of the magnetic nano-particle surface modification is alkynyl；The compound of structure shown in formula (2) by azido with it is described
The alkynyl of magnetic nano-particle surface modification is mutually bonded, and passes through 7 companies, O institute of compound of structure shown in amino and the formula (1)
The carboxyl for connecing uronic acid is mutually bonded, also structure as shown in Fig. 2.
Above-mentioned alkynyl is preferably propargyl.
In the present invention, surface modification has the magnetic nano-particle of alkynyl that can obtain by synthesis, can also lead to
Cross it is commercially available, such as can commercially available surface modification have the magnetic nano-particle of propargyl.
Flavone glycoside functional magnetic nanometer affinity probe provided by the invention, it is preferable that relative to 1 milligram of the magnetic
Property nanoparticle, the load capacity of the flavone glycoside compound is 30-45nmol；It is highly preferred that relative to 1 milligram of the magnetic
Property nanoparticle, the load capacity of the flavone glycoside compound is 32-41nmol.
The present invention also provides the preparation method of above-mentioned flavone glycoside functional magnetic nanometer affinity probe, this method include with
Lower step,
1) compound of flavone glycoside compound and structure shown in formula (2) in the presence of an organic, is carried out first to connect
Touching, obtains the intermediate compound that one end of the compound of structure shown in formula (2) is mutually bonded with flavone glycoside compound；
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains knot shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of the compound of structure is connected with magnetic nano-particle；
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that the amino of amino or BOC protection, n are the whole of 2-10
Number.
Preferably, in formula (2), R1Indicate azido, R2Indicate amino, n 2,3,4 or 5；It is highly preferred that in formula (2), R1
Indicate azido, R2Indicate amino, n 3.
According to the method for the present invention, the flavone glycoside compound is the compound of structure shown in following formula (1)；
According to the method for the present invention, in step 1), in the presence of an organic, by flavone glycoside compound and formula (2) institute
Show that the compound of structure carries out the first contact, obtains one end and the flavone glycoside compound phase of the compound of structure shown in formula (2)
The intermediate compound (also flavone glycoside derivative below) of bonding.
According to the method for the present invention, in step 1), the compound of structure shown in the flavone glycoside compound and formula (2)
Molar ratio is 1:0.9-1.3；Preferably 1:1-1.1.
Method in accordance with the invention it is preferred that first contact carries out in the presence of the first catalyst, the flavone sugar
The molar ratio of glycoside compound and first catalyst is 1:1-1.5, preferably 1:1.2-1.3.
As long as being capable of catalytic step 1 as first catalyst) connection reaction, it is preferable that described first urges
Agent is EDCI (1- ethyl -3- (3- dimethylamine propyl) carbodiimide hydrochloride) and HOBt (1- hydroxy benzo triazole).
According to the method for the present invention, in step 1), the organic solvent can for n,N-Dimethylformamide (DMF),
One of methanol and dimethyl sulfoxide are a variety of, preferably n,N-Dimethylformamide.Dosage as organic solvent can be with
For the conventional amount used of this field, for example, relative to 1 mole of flavone glycoside compound, the organic solvent is preferably 0.05-5
Mole, more preferably 0.1-1 moles.
Preferably, it is 15-30 DEG C that the condition of first contact, which includes: the temperature of contact, and the time of contact is that 5-50 is small
When.It is highly preferred that the temperature that the condition of first contact includes: contact is 20-30 DEG C, the time of contact is 20-30 hours.
According to the method for the present invention, in step 2), the intermediate compound and magnetic nano-particle that step 1) is obtained are carried out
Second contact, obtains the flavone glycoside function that the other end of the compound of structure shown in formula (2) is connected with magnetic nano-particle
Change magnetic Nano affinity probe；
According to the method for the present invention, the magnetic nano-particle refers to the magnetic nanoparticle of tool, such as can be four
Fe 3 O nanoparticle.
In addition, the magnetic nano-particle may be core-shell structure, for example, its kernel is magnetic particle, outer layer covers
SiO2.It for example can be with as such magnetic nano-particle are as follows: its kernel is ferriferrous oxide nano-particle, outer layer covers SiO2
Magnetic nano-particle.
According to the method for the present invention, have can be with the compound of structure shown in formula (2) for the magnetic nano-particle surface modification
One end the group that is mutually bonded of group.As the group can the structure according to formula (2) compound one end base
Group is to select, such as can be alkynyl, azido or amino.The group R of one end of the compound of the structure shown in formula (2)1Table
Show azido, the magnetic nano-particle surface is preferably modified with alkynyl, is more preferably modified with propargyl.
Method in accordance with the invention it is preferred that the average grain diameter of the magnetic nano-particle is 100-400nm, preferably
100-120nm。
According to the method for the present invention, in step 2), relative to magnetic nano-particle described in 1mg, the intermediate compound
Dosage is 40-80nmol.
Preferably, second contact carries out in the presence of the second catalyst, second catalyst and the flavone sugar
The molar ratio of glycoside compound is 1:8-15, preferably 1:9-11.
As long as being capable of catalytic step 2 as second catalyst) connection reaction, such as in R1Indicate nitrine
Base, when the magnetic nano-particle surface is preferably modified with alkynyl, second catalyst can be copper sulphate and vitamin C
Sodium.
Furthermore it is preferred that the second contact carries out in the presence of solvent, the solvent for example can be water and/or phosphate-buffered salt
Solution.The dosage of the solvent does not require particularly, can be conventional amount used.
Preferably, it is 15-30 DEG C that the condition of second contact, which includes: the temperature of contact, and the time of contact is 0.2-24
Hour.It is highly preferred that the temperature that the condition of second contact includes: contact is 20-30 DEG C, the time of contact is 1-5 hours.
The present invention also provides a kind of methods for capturing intracellular target proteins, wherein method includes the following steps:
1) make flavone glycoside functional magnetic nanometer affinity probe of the invention or preparation method through the invention made
The flavone glycoside functional magnetic nanometer affinity probe obtained is contacted with cell holoprotein extracting solution, so that flavone glycoside function
Magnetic Nano affinity probe can be changed in conjunction with intracellular target proteins；
2) unbonded albumen is removed, to isolate the albumen of capture；
3) albumen of capture is identified；
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside
The intracellular target proteins of compound specificity identification.
The method of the intracellular target proteins of capture according to the present invention, in step 1), it is preferable that the flavone glycoside function
Changing the dosage that magnetic Nano affinity probe is guided and supported with cell holoprotein can change in a big way, it is preferable that the Huang
Ketoside functional magnetic nanometer affinity probe (with the poidometer of surface group magnetic nano-particle) and cell holoprotein
The weight ratio for guiding and supporting middle total protein is 1:0.01-0.035, preferably 1:0.01-0.031.
As above-mentioned steps 1) in contact conditions may include: the temperature of contact be 3-5 DEG C, the time of contact can be
1-8 hours, preferably 1-2 hours.
The method of the intracellular target proteins of capture according to the present invention in step (2), can abandon supernatant by using centrifugation
The mode of liquid removes unbonded albumen, and such as 14000-16000g is centrifuged 10-15min, and uses phosphate buffered saline solution
(PBS), water washing magnetic bead removes non-specific adsorption protein on magnetic bead three times.
The method of the intracellular target proteins of capture according to the present invention in step (3), can use trypsin digestion egg
It is white, to pass through the albumen of nano-LC-MS/MS identification capture.Further, it is also possible to the method analyzed by Western Blot
Identify the albumen of capture, concrete operations are known to those skilled in the art, and details are not described herein.
The method of the intracellular target proteins of capture according to the present invention, can be to the intracellular target of capture by step (4)
It marks albumen and carries out specific analysis, so that it is determined that flavone glycoside compound specificity identifies intracellular target proteins.As step
4), for example, can the quantitative proteomics method based on stable isotope labeling quantitative comparison, row are carried out to the albumen of capture
Unless binding proteins specific, to obtain the intracellular target proteins of specific recognition.The concrete operation step of this method is this
Well known to the technical staff of field, details are not described herein.
In addition, in order to accurately exclude nonspecific proteins, it is preferable that the magnetism by setting surface group is received
Rice corpuscles is control group, to exclude nonspecific magnetic nano-particle binding protein, with the flavone glycoside functional magnetic
Nanometer affinity probe is compared, and the surface group magnetic nano-particle is not bound with the target egg of flavone glycoside compound effects
White matter.
Preparation the present invention also provides flavone glycoside functional magnetic nanometer affinity probe of the invention or through the invention
Flavone glycoside functional magnetic nanometer affinity probe obtained by method is in the capture of drug targets albumen and/or identification of cell
Application.
The present invention will be described in detail by way of examples below.
In following embodiment, normal embryonic kidney cells (HEK293T) cell is purchased from Chinese Concord Hospital, deuterated-N- acetyl group
Succinimide is purchased from Sigma company (Sigma-Aldrich) Aldrich)；Mass spectrum grade trypsase is purchased from Promege company；
Dithiothreitol (DTT) (DTT) is purchased from Pierce company；Alkynyl ferroferric oxide magnetic nano magnetic bead is limited purchased from the city Ying Rui science and technology
Company；Baicalin is purchased from Han Wei Science and Technology Ltd., 95% or more purity；Memebrane protein, holoprotein extracts kit are purchased from shellfish
Rich biotech firm；Grade trypsase is sequenced and is purchased from Hyclone company；ZiptipC18 is purchased from Minipore company；BCA albumen is fixed
It measures kit and is purchased from Tianjin biochemical technology Co., Ltd.DMEM culture medium (is purchased from GIBCO company), trypsase, fetal calf serum
(being purchased from Sigma company), secondary deionized water is prepared through Millpore system.Trifluoroacetic acid aqueous solution is purchased from Tedia company.DMEM
(high sugar) culture medium (Invitrogen, USA), colourless DMEM culture medium (Invitrogen, USA), pancreatin 0.25%
Trypsin-EDTA (Invitrogen, USA), fetal calf serum FBS (Invitrogen, USA)；Sodium liter electron spray nozzle needle is purchased green
Continuous Science and Technology Ltd.'s (New Objective product).
Embodiment 1
The present embodiment 1 is used to illustrate the preparation and representation of radix scutellariae glycoside derivates Qz-1 of the invention.
Scutelloside (446.2mg, 1mmol, Baicalin, same as below shown in for example above-mentioned formula (1) of structure) is weighed,
HOBt (175.6mg, 1.3mmol) is set in a round bottom flask, and 6ml anhydrous DMF is added, and stirs 30min.N is taken in refrigerator3-
PEG3-NH2(218.3mg, 1mmol (- 11 nitrine -3,6 of 1 amino, 9- trioxaundecane) are dissolved in 2mlDMF, are uniformly mixed, by
It is added dropwise to flask, EDCI (249.2mg, 1.3mmol) is dissolved in 2ml anhydrous DMF, is added dropwise, and reaction a period of time is added excessive
TEA (303.3mg, 3mmol), is stirred to react 72h at normal temperature, adds water stopping reaction three times, CHCl3Extraction, anhydrous magnesium sulfate
It is dried overnight, silica gel column chromatography separating purification, baking oven drying, obtaining pale yellow powder, (radix scutellariae glycoside derivates Qz-1 is (also simple below
Referred to as Qz-1), shown in result such as formula (3)), yield 32.5%.
ESI-MS(m/z):647.2189([M+H+]),29H34N4O13.1H-NMR(DMSO-d6,400MHz)δH(ppm):
8.71 (s, 1H), 8.10 (d, J=8,2H), 8.04 (s, 1H), 7.61 (d, J=8,2H), 7.03 (d, 1H) 5.54 (d, 1H)
5.24 (d, J=8,1H), 5.10 (d, J=8,1H), 3.97 (d, J=8,1H), 3.45 (s, 4H), 3.42 (m, J=8,10H),
3.41(s,6H),1.23(s,1H)；13C-NMR(150MHz,DMSO-d6) (ppm): 183.13,168.62,164.09,
151.89,1489.77,147.29,132.65,131.41,131,2,129.71,126.98,106.77,106.31,101.11,
94.54,76.16,76.03,73.32,71.54,70.27,70.14,69.77,69.32,50.52,29.58。
The radix scutellariae glycoside derivates Qz-1 of preparation characterizes molecular weight by ESI-MS, and infrared absorption spectrum, nuclear-magnetism is respectively adopted
The methods of hydrogen spectrum, the carbon spectrum that resonate characterization molecular structure, can determine the knot of radix scutellariae glycoside derivates Qz-1 from the above spectrum analysis
Structure is the compound of structure shown in formula (3).Characterization result is as depicted in figs. 1 and 2, and wherein Fig. 1 is radix scutellariae glycoside derivates Qz-1's
ESI-MS figure, wherein 647.2189 be the molecular ion peak of (M+1).Fig. 2 is the infrared absorption spectrum of radix scutellariae glycoside derivates Qz-1
Figure, wherein 2100-2120cm-1Position, 2959cm are absorbed for the azido of feature on Qz-1-1、2925cm-1It vibrates and inhales for-CH
It receives, 3430cm-1For-OH absorption of vibrations.
Embodiment 2
The present embodiment 2 be used to illustrate radix scutellariae glycoside derivates-ferroferric oxide magnetic nano affinity probe of the invention (namely
Flavone glycoside functional magnetic nanometer affinity probe of the invention) preparation and representation.
The magnetic nano-particle concentration of alkynyl functionalization is 30mg/ml, and preservation liquid is 50% ethanol water, using preceding super
Sound makes it be uniformly dispersed.Radix scutellariae glycoside derivates Qz-1 is configured to 5mM using DMSO, is diluted to PBS containing 1.25 weights using preceding
Measure %DMSO.Anhydrous cupric sulfate is configured to 5mM using water, and vitamine C sodium is configured to 50mM with water three times using preceding.Scutelloside spreads out
Biology-ferroso-ferric oxide is affine nano-probe (MNPs@Qz-1) construction method are as follows: take clean 1.5ml centrifuge tube, be added in pipe
100 μ L (3mg) magnetic nano-particles, Magnetic Isolation discards preservation liquid, while using water washing 3 times three times, passes through magnetic after washing
Property separation discard water.Then radix scutellariae glycoside derivates Qz-1 1.5m Μ 1ml, ultrasonic 1min are added in centrifuge tube makes magnetic bead again
Dispersion in the solution, sequentially adds 2 μ L 50mM vitamine C sodiums and 2 μ L 5mM anhydrous cupric sulfates, at room temperature, shakes and is incubated for 1-
For 24 hours, Magnetic Isolation, abandoning supernatant is MNPs@Qz-1.
The building of radix scutellariae glycoside derivates-ferriferrous oxide nano probe (MNPs Qz-1) is with four oxygen of alkynyl functionalization
Three Fe nanometer particles (MNPs) are control, and the structural schematic diagram of two kinds of probes is as shown in Figure 3.The wherein original oxidation of alkynyl four three
Fe nanometer particles are a kind of core-shell structure, and kernel is ferroso-ferric oxide, outer layer covers SiO2, surface bond has propargyl.
It is carried out by ferroferric oxide magnetic nano-particles (MNPs) pattern and size of the transmission electron microscope to alkynyl
Characterization, as a result as shown in Figure 4, wherein Fig. 4 is transmission electron microscope photo, and transmission electron microscope results show the ferroso-ferric oxide of alkynyl
The average grain diameter (nm) of magnetic nano-particle is 100-120nm；It is characterized, is tied by Secondary Ion Mass Spectrometry MNPs@Qz-1
Fruit is as shown in figure 5, the TOF-SIMS mass spectrogram that the upper figure in Fig. 5 is MNPs Qz-1, and middle probe MNPs Qz-1 is because occur
Click chemistry reacts to form triazole ring, has C in arrow pointed location2N3 -66.043 (m/z) characterising mass spectrometry peaks, quality error
For the TOF-SIMS mass spectrogram that the following figure in -44.7, Fig. 5 is MNPs, in arrow in the TOF-SIMS mass spectrogram of negative control MNPs
Head pointed location is without the C2N3 -Characterising mass spectrometry peak shows successfully to construct scutelloside functional magnetic nanometer affinity probe.
Embodiment 3
This example 3 is used to the Qz-1 content for illustrating to load in scutelloside functional magnetic nanometer affinity probe of the invention.
Clean 1.5ml centrifuge tube is taken, 100 μ L (3mg) alkynyl ferroferric oxide magnetic nano-particles are added in pipe, so
The volume for being separately added into 25 μ Μ afterwards is the radix scutellariae glycoside derivates Qz-1 of 200ul, 400ul, 600ul, 800ul, 1ml, 1.2ml, is surpassed
Sound 1min is dispersed again in magnetic bead in solution, 2 μ L50mM vitamine C sodiums and 2 μ L5mM anhydrous cupric sulfates is sequentially added, in room
It under temperature, shakes and is incubated for 2h, UV, visible light is passed through by Magnetic Isolation probe and the reaction solution containing Qz-1 respectively after reaction
The variation of Qz-1 absorption value may know that total in 3mg alkynyl ferroferric oxide magnetic nano-particles in spectrographic determination supernatant
The amount of the Qz-1 of valence modification.By can be calculated, 32.6- is covalently bonded in the alkynyl ferriferrous oxide nano-particle of 1mg
The radix scutellariae glycoside derivates of 40.8nmol.
Embodiment 4
The present embodiment 4 is used to illustrate scutelloside functional magnetic nanometer affinity probe capture scutelloside of the invention in cell
The method and condition optimizing of interior target proteins.
(1) culture of HEK293 cell
Cell culture chooses HEK293 cell as experimental subjects.In 5%CO2 and 37 degree Celsius of insulating box culture,
Culture medium are as follows: 1%PS, 90% DMEM, 10% FBS.Contain antibiotic simultaneously.Embryonic kidney cells HEK293 is being added to 10%
Cultivated in DMEM (high sugar) culture medium of fetal calf serum and 1% Pen .- Strep, in 37 DEG C of incubators containing 5% dioxy
Change carbon.Culture to cell covers with culture dish in 100mm culture dish, removes culture medium, collects cell, in HEK293 cell
Holoprotein extract.
(2) with the extraction of holoprotein
At 4 DEG C, 2500g pelleted by centrifugation 5min carefully sucks culture medium, receives as far as possible the cell collected in above-mentioned (1)
Collect cell.Cell is washed twice with cold PBS, blots supernatant as far as possible after washing twice.Every 5 × 106--1×107A cell is (big
About 50mg, 50ul cell volume) in the cold total protein extracting solution of 500ul be added (extract reagent from BestBio holoprotein
Box), after piping and druming mixes, shaken 20 minutes under the conditions of 4 DEG C, until cell sufficiently cracks, without obvious cell precipitation.At 4 DEG C,
It is centrifuged 15min under the conditions of 14000g, supernatant is quickly sucked to the clean centrifuge tube of another pre-cooling, total protein can be obtained.It will be upper
- 80 DEG C of refrigerators of packing save backup or are directly used in capture scutelloside specificity target egg after stating the extraction BCA standard measure of albumen
White matter.Its concrete operations is carried out referring to BestBio holoprotein extracts kit.
(3) scutelloside specificity target proteins are captured
By positive control probe (the scutelloside functional magnetic nanometer affinity probe MNPs@that embodiment 2 obtains of 10mg preparation
Qz-1 it) is separately added into PBS buffer solution with equivalent control probe (ferroferric oxide magnetic nano-particles of alkynyl) and is diluted to
0.4mL, the two are mixed with the cell holoprotein extracting solution of equivalent respectively, are incubated for 2h at 4 DEG C.Magnetic Isolation, centrifugation
(14000g, 10min) removes the protein solution being not bound with, with PBS buffer solution and water washing capture protein nano spy three times
Needle for several times, removes non-specific adsorption protein, measures the protein content combined on MNPs@Qz-1 probe by BCA method.
It can be calculated by BCA working curve, about 11.33 μ g/mg of control probe non-specific adsorption HEK293 protein content, positive control probe
(100nm) captures about 30.90 μ g/mg of albumen quality.It is covalently bonded with according to the alkynyl ferriferrous oxide nano-particle of 1mg
The radix scutellariae glycoside derivates of 32.6-40.8n mol, it is possible to calculate the binding capacity about 1.67- of MNPs Qz-1 and protein
2.04nmol Qz-1/ μ g albumen.
Embodiment 5
The present embodiment 5 is used to illustrate the scutelloside functional magnetic nanometer affinity probe of different-grain diameter of the invention to cell
The load efficiency of middle holoprotein.
According to the method for embodiment 2, by the commercialization alkynyl ferroso-ferric oxide functional magnetic nanoparticle of different-grain diameter
Son is reacted with Qz-1, to prepare the positive control probe of different-grain diameter.Wherein, dispersibility is selected preferably, partial size 100nm, 400nm alkynes
Base ferroferric oxide magnetic nano-particles are prepared for the scutelloside functional magnetic nanometer affinity probe of two kinds of different-grain diameters,
Confirmed by identical characterizing method in embodiment 2.The method after probe according to embodiment 4 has been prepared, albumen is carried out
Then the capture of matter removes the protein solution being not bound with and non-specific adsorption protein, measured by BCA method in difference
The protein content combined on partial size probe is control with the ferroferric oxide magnetic nano-particles (MNPs) of alkynyl.As a result
It has been shown that, partial size are that the albumen quality that the MNPs@Qz-1 of 100nm is captured under the same conditions is more.
Embodiment 6
The present embodiment 6 is used to illustrate that radix scutellariae glycoside derivates Qz-1 does not make the bioactivity of radix scutellariae active constituent scutelloside
It reduces in other words without its physiological activity of large effect.It is intended to the affine spy of functional magnetic nanometer by scutelloside derivatization
Needle captures the target proteins that intracellular target proteins matter illustrates scutelloside.Modification front and back compound pair is measured by 96 well plate methods
It is evaluated in the activity influence of people's hepatomicrosome HLM fluorescence probe DME.
The inhibitor of series of concentrations, specially 125nM, 250nM, 500nM are configured using the PBS containing 1.25%DMSO,
1.25 μM, 2.5 μM, 5 μM, 12.5 μM, 25 μM, 125 μM, 250 μM and 1Mm.It is sequentially added in 96 orifice plates by enzyme source and series
The inhibitor (scutelloside, Qz-1, MNPs@Qz-1) of concentration, enzyme and inhibitor are respectively 20 μ L, are mixed, then in 37 DEG C of conditions
10 μ L DME (making final concentration of 3 μM) starting reaction is then added, so that each enzymatic hydrolysis reaction exists in lower preincubate 30min
In PBS (100mM, pH=6.5) buffer system of 50 μ L systems, under the conditions of 37 DEG C after pre-reaction 30min, 50 μ L are added later
The detection reagent that shines stops reaction, in 37 DEG C of conditioned response 20min, detects bioluminescence with microplate reader.Depression effect is not by
Luminous intensity with inhibitor concentration and control group is converted into the percentage of enzyme inhibition and indicates, is fitted IC50。
As a result as shown in fig. 6, it can be seen from the figure that in the low concentration range, when such as c < 100 μM, scutelloside, Qz-1 pairs
People's hepatomicrosome enzymatic activity is suitable, and when compound concentration is higher, radix scutellariae glycoside derivates Qz-1 is more better compared to scutelloside.
Embodiment 7
The present embodiment 7 is used to illustrate scutelloside functional magnetic nanometer affinity probe capture scutelloside of the invention in cell
The identification method of the nano-LC-MS/MS of interior target proteins.
(1) scutelloside specificity target proteins are captured
By the positive control probe (the scutelloside functional magnetic nanometer affinity probe that embodiment 2 obtains) of 3mg preparation and control
Probe (ferroferric oxide magnetic nano-particles of alkynyl) is separately added into PBS buffer solution and is diluted to 0.4mL, the two respectively with etc.
Cell holoprotein extracting solution (containing 200 μ g total proteins) mixing of amount, is incubated for 2h at 4 DEG C.Centrifugation (14000g, 10min) is gone
Except the protein solution being not bound with, PBS buffer solution is washed 3 times, washes 3 removal non-specific adsorption protein three times
Obtain the specific protein of capture.
(2) protein pre-treatment is captured
8M urea 200 μ L, 95 DEG C of denaturation 5min are added in the albumen of capture, and excess 100Mm DTT is added after cooling at 37 DEG C
Insulating box reacts 4h, and appropriate IAA is added later, reacts 2h in 37 DEG C of insulating boxs, uses molecular cut off for 3K super filter tube later
20mM NH is added in centrifugation (14000g, 30min) desalination 3 times4HCO3Weight is molten, and trypsase is added according to 40:1, digests at 37 DEG C
Overnight.
(3) albumen of identification capture
The supernatant freeze-drying that the enzymolysis liquid centrifugation (14000g, 5min) that step (2) are obtained obtains, using containing 0.1%FA
After 0.22um filter membrane after H2O (mass spectrum is pure) dissolution, final 5 μ L carries out Nano-LC-MS/MS analysis.Chromatographic column used are as follows: C18
Column (75 μ m 15cm, 3 μm, LC Packings).Liquid phase is Dionex Ultramate3000, mobile phase A: 95%H2O, 5%
MeCN, 0.1%FA, Mobile phase B: 95%MeCN, 5%H2O, 0.1%FA, flow velocity are as follows: 300nL/min, gradient: 3%B-45%
B, 120min.Tryptic digestion peptide fragment first passes around trap column desalination 5min, subsequently into through C18After post separation, into EFI
Mist mass spectrum (Xevo G2Q-TOF) detection.Spray voltage+3.03kV, cone voltage+35V.Survey scan:350-1700,
MS/MS scan:50-2000.The parent ion charge number of selection is+2 ,+3 ,+4.
MASCOT v2.6.0 search engine is used to identify the albumen of capture.Quality error setting are as follows: parent ion 30ppm, son
Ion 0.8Da.Methionine is oxidized to variable modification.Trypsase leakage enzyme site maximum is set as 1, Expect threshold value and sets
It is set to 0.05, only with the peptide fragment (each most matched result of second order ms figure) to rank the first in qualification result, only confidence
Albumen (p < 0.05) within degree threshold is considered as identifying the albumen come.
According to the method for embodiment 6, in three parallel laboratory tests, the scutelloside function compared with negative control probe captures protein
It is obviously more that magnetic Nano affinity probe capture protein can be changed, by protein functional assays, it is found that many protein participate in
Important metabolic process in cell, specially substance and energetic supersession (ATPB, CYC, GPR75 etc.), calcium adjust (CALL5),
The functions such as anti-oxidant (PRDX1), heat shock (HSP (30/60/70/90)) and anticancer activity (Caspase14, RFA1) are related,
Action target albumen is mainly thanked to intracellular calcium ion, heat shock inhibition, Inflammatory Pathway and regulation energy related.
The amount of the most of target proteins matter captured as described in Example 4 is conventional enough in nanogram level range
NanoLC-MS/MS, which is analyzed and obtained, firmly believes reliable qualification result.
These results confirm that scutelloside functional magnetic nanometer affinity probe of the invention has stronger capture scutelloside
The ability of target proteins.
(4) specificity verification
The enzymolysis liquid centrifugation (17000g, 5min) that step (2) are obtained obtains supernatant, and positive control probe and control probe obtain
The supernatant arrived reacts 5 hours respectively with 100 times of excessive acetylation labelled reagents under 25 DEG C of stirring conditions, and positive control probe is caught
Peptide fragment of the albumen obtained through trypsin digestion is marked by heavy isotope (deuterated acetic acid-n-succinimidyl ester), control probe
Peptide fragment of the albumen of capture through trypsin digestion is marked by light isotope (acetic acid-n-succinimidyl ester).After label,
Isometric two parts of reaction solutions mixing, is added excessive azanol and reacts 20 minutes under conditions of pH 10-11 to consume
The labelled reagent of amount.The peptide fragment mixed liquor of isotope labelling is lyophilized, and with Ziptip C18Desalination.Obtained peptide fragment solution into
Row Nano-LC-MS/MS analysis.Mass Spectrometry Conditions and the same step of parameter setting (3).
Quantitative analysis uses Mascot Distiller 2.6.0 software, and the same peptide fragment of weight isotope labelling owns
Mass spectrogram is stacked to produce a total mass spectrogram, the peptide fragment and light isotope label peptide fragment of heavy label in total mass spectrogram
Ratio be by calculate isotopic peak peak height ratios obtain.The quantitative ratio of albumen is by calculating the same albumen
The intensity rate average value of all matching peptide fragments obtains.
The results show that 7 scutelloside functional magnetic nanometer affinity probe of the present embodiment has filtered out specific recognition target
Protein G CYB:Guanylate cyclase is guanylate cyclase, and the existing enzymatic activity of the albumen is also a kind of membrane receptor, is participated in
Intracellular biological chemical process, is mainly played a role by signal transduction process.Therefore the protein-specific combination scutelloside.
And negative probes binding capacity is not enriched to this protein, the quantitative result of representative trypsin digestion peptide fragment is as schemed
Shown in 7, illustrate that catching method of the invention can specifically capture scutelloside target proteins in the cell.
From above-described embodiment as can be seen that scutelloside functional magnetic nanometer affinity probe can be just made from the method for the present invention
Intracellular target proteins really and are specifically captured, and the cell holoprotein extracting solution needed is less.
The preferred embodiment of the present invention has been described above in detail, and still, the present invention is not limited thereto.In skill of the invention
In art conception range, can with various simple variants of the technical solution of the present invention are made, including each technical characteristic with it is any its
Its suitable method is combined, and it should also be regarded as the disclosure of the present invention for these simple variants and combination, is belonged to
Protection scope of the present invention.
Claims (50)
1. a kind of method for capturing intracellular target proteins, which is characterized in that method includes the following steps:
1) contact flavone glycoside functional magnetic nanometer affinity probe with cell holoprotein extracting solution, so that flavone sugar
Glycosides functional magnetic nanometer affinity probe is in conjunction with intracellular target proteins；
2) unbonded albumen is removed, to isolate the albumen of capture；
3) albumen of capture is identified；
4) quantitative comparison is carried out to the protein of capture, and excludes non-binding proteins specific, to obtain flavone glycoside chemical combination
The intracellular target proteins of object specific recognition,
Wherein, the flavone glycoside functional magnetic nanometer affinity probe includes magnetic nano-particle, coupled structures and load
Flavone glycoside compound on the magnetic nano-particle surface, wherein one end of the coupled structures and the magnetic Nano
Particle surface is connected, and the other end is connected with flavone glycoside compound.
2. kernel is four oxidations according to the method described in claim 1, wherein, the magnetic nano-particle is core-shell structure
Three Fe nanometer particles, outer layer covers SiO2。
3. according to the method described in claim 2, wherein, the average grain diameter of the magnetic nano-particle is 100-400nm.
4. according to the method described in claim 3, wherein, group and the idol of the magnetic nano-particle by surface modification
It is coupled the group bonding of one end of structure.
5. according to the method described in claim 4, wherein, the group of the magnetic nano-particle surface modification is alkynyl, with institute
The group for stating the one end for the coupled structures that alkynyl is mutually bonded is azido, alternatively, the magnetic nano-particle surface modification
Group be azido, the group of the one end for coupled structures be bonded with the nitrine base phase is alkynyl, alternatively, the magnetic
Property nanoparticle surface modified group be n-hydroxysuccinimide, the coupling being mutually bonded with the n-hydroxysuccinimide
One end group of structure is amino.
6. method described in any one of -5 according to claim 1, wherein the flavone glycoside compound is following formula (1)
The compound of shown structure, and the compound of structure shown in the formula (1) passes through the carboxylic of 7 connected uronic acids of O of flavone glycoside
Base is mutually bonded with the group of the coupled structures other end,
7. according to the method described in claim 6, wherein, the idol that the compound of the structure shown in the formula (1) combines
The group for being coupled the other end of structure is amino.
8. method described in any one of -5 according to claim 1, wherein the coupled structures are as shown in following formula (2)
Compound provides,
Wherein, R1Indicate azido, R2Indicate that amino, n indicate the integer of 2-10.
9. according to the method described in claim 8, wherein, R1Indicate azido, R2Indicate amino, n 2,3,4 or 5.
10. according to the method described in claim 9, wherein, R1Indicate azido, R2Indicate amino, n 3, and the magnetism is received
The group of rice corpuscles surface modification is alkynyl, and the compound of structure shown in formula (2) passes through azido and the magnetic nano-particle
The alkynyl of surface modification is mutually bonded, and the compound of structure shown in the formula (1) passes through 7 connected uronic acids of O of flavone glycoside
Carboxyl is mutually bonded with amino.
11. method described in any one of -5 according to claim 1, wherein relative to 1 milligram of the magnetic nano particle
Son, the load capacity of the flavone glycoside compound are 30-45nmol.
12. according to the method for claim 11, wherein relative to 1 milligram of the magnetic nano-particle, the flavone sugar
The load capacity of glycoside compound is 32-41nmol.
13. according to the method described in claim 8, wherein, the preparation of the flavone glycoside functional magnetic nanometer affinity probe
Method includes the following steps,
1) flavone glycoside compound in the presence of an organic, is carried out first with the compound of structure shown in formula (2) to contact, is obtained
The intermediate compound being mutually bonded to one end of the compound of structure shown in formula (2) with flavone glycoside compound；
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains structure shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of compound is connected with magnetic nano-particle；
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that the amino of amino or BOC protection, n are the integer of 2-10.
14. according to the method for claim 13, wherein n 2,3,4 or 5.
15. method described in 3 or 14 according to claim 1, wherein in step 1), the flavone glycoside compound and formula (2) institute
The molar ratio for showing the compound of structure is 1:0.9-1.3.
16. according to the method for claim 15, wherein in step 1), knot shown in the flavone glycoside compound and formula (2)
The molar ratio of the compound of structure is 1:1-1.1.
17. according to the method for claim 16, wherein first contact carries out in the presence of the first catalyst, described
The molar ratio of flavone glycoside compound and first catalyst is 1:1-1.5.
18. according to the method for claim 17, wherein mole of the flavone glycoside compound and first catalyst
Than for 1:1.2-1.3.
19. according to the method for claim 18, wherein first catalyst is 1- ethyl -3- (3- dimethylamine propyl)
Carbodiimide hydrochloride and 1- hydroxy benzo triazole.
20. according to the method for claim 19, wherein the condition of first contact includes: that the temperature of contact is 20-30
DEG C, the time of contact is 5-50 hours.
21. method described in 3 or 14 according to claim 1, wherein in step 2), relative to magnetic nano-particle described in 1mg,
The dosage of the intermediate compound is 40-80nmol.
22. according to the method for claim 21, wherein second contact carries out in the presence of the second catalyst, described
The molar ratio of second catalyst and the flavone glycoside compound is 1:8-15.
23. according to the method for claim 22, wherein mole of second catalyst and the flavone glycoside compound
Than for 1:9-11.
24. according to the method for claim 23, wherein second catalyst is copper sulphate and vitamine C sodium.
25. according to the method for claim 24, wherein the condition of second contact includes: that the temperature of contact is 20-30
DEG C, the time of contact is 0.2-24 hours.
26. a kind of drug targets albumen capture of flavone glycoside functional magnetic nanometer affinity probe in scutelloside in the cell
And/or the application in identification, wherein the flavone glycoside functional magnetic nanometer affinity probe includes magnetic nano-particle, idol
It is coupled structure and is supported on the flavone glycoside compound on the magnetic nano-particle surface, wherein one end of the coupled structures
It is connected with the magnetic nano-particle surface, the other end is connected with flavone glycoside compound.
27. application according to claim 26, wherein the magnetic nano-particle is core-shell structure, and kernel is four oxygen
Change three Fe nanometer particles, outer layer covers SiO2。
28. application according to claim 27, wherein the average grain diameter of the magnetic nano-particle is 100-400nm.
29. application according to claim 28, wherein the magnetic nano-particle by the group of surface modification with it is described
The group bonding of one end of coupled structures.
30. application according to claim 29, wherein the group of the magnetic nano-particle surface modification is alkynyl, with
The group of the one end for the coupled structures that the alkynyl is mutually bonded is azido, alternatively, the magnetic nano-particle surface is repaired
The group of decorations is azido, and the group of the one end for the coupled structures being bonded with the nitrine base phase is alkynyl, alternatively, described
The group of magnetic nano-particle surface modification is n-hydroxysuccinimide, the idol being mutually bonded with the n-hydroxysuccinimide
One end group for being coupled structure is amino.
31. the application according to any one of claim 26-30, wherein the flavone glycoside compound is following formula
(1) compound of structure shown in, and the compound of structure shown in the formula (1) passes through 7 connected uronic acids of O of flavone glycoside
Carboxyl is mutually bonded with the group of the coupled structures other end,
32. application according to claim 31, wherein the compound of the structure shown in the formula (1) combines described
The group of the other end of coupled structures is amino.
33. the application according to any one of claim 26-30, wherein the coupled structures are by shown in following formula (2)
Compound provide,
Wherein, R1Indicate azido, R2Indicate that amino, n indicate the integer of 2-10.
34. application according to claim 33, wherein R1Indicate azido, R2Indicate amino, n 2,3,4 or 5.
35. application according to claim 34, wherein R1Indicate azido, R2Indicate amino, n 3, and the magnetism is received
The group of rice corpuscles surface modification is alkynyl, and the compound of structure shown in formula (2) passes through azido and the magnetic nano-particle
The alkynyl of surface modification is mutually bonded, and the compound of structure shown in the formula (1) passes through 7 connected uronic acids of O of flavone glycoside
Carboxyl is mutually bonded with amino.
36. the application according to any one of claim 26-30, wherein relative to 1 milligram of the magnetic nano particle
Son, the load capacity of the flavone glycoside compound are 30-45nmol.
37. application according to claim 36, wherein relative to 1 milligram of the magnetic nano-particle, the flavone sugar
The load capacity of glycoside compound is 32-41nmol.
38. application according to claim 33, wherein the preparation of the flavone glycoside functional magnetic nanometer affinity probe
Method includes the following steps,
1) flavone glycoside compound in the presence of an organic, is carried out first with the compound of structure shown in formula (2) to contact, is obtained
The intermediate compound being mutually bonded to one end of the compound of structure shown in formula (2) with flavone glycoside compound；
2) intermediate compound that step 1) obtains is carried out second with magnetic nano-particle to contact, obtains structure shown in formula (2)
The flavone glycoside functional magnetic nanometer affinity probe that the other end of compound is connected with magnetic nano-particle；
In formula (2), R1Indicate azido, alkynyl or amino, R2Indicate that the amino of amino or BOC protection, n are the integer of 2-10.
39. the application according to claim 38, wherein n 2,3,4 or 5.
40. the application according to claim 38 or 39, wherein in step 1), the flavone glycoside compound and formula (2) institute
The molar ratio for showing the compound of structure is 1:0.9-1.3.
41. application according to claim 40, wherein in step 1), knot shown in the flavone glycoside compound and formula (2)
The molar ratio of the compound of structure is 1:1-1.1.
42. application according to claim 41, wherein first contact carries out in the presence of the first catalyst, described
The molar ratio of flavone glycoside compound and first catalyst is 1:1-1.5.
43. application according to claim 42, wherein mole of the flavone glycoside compound and first catalyst
Than for 1:1.2-1.3.
44. application according to claim 43, wherein first catalyst is 1- ethyl -3- (3- dimethylamine propyl)
Carbodiimide hydrochloride and 1- hydroxy benzo triazole.
45. application according to claim 44, wherein the condition of first contact includes: that the temperature of contact is 20-30
DEG C, the time of contact is 5-50 hours.
46. the application according to claim 38 or 39, wherein in step 2), relative to magnetic nano-particle described in 1mg,
The dosage of the intermediate compound is 40-80nmol.
47. application according to claim 46, wherein second contact carries out in the presence of the second catalyst, described
The molar ratio of second catalyst and the flavone glycoside compound is 1:8-15.
48. application according to claim 47, wherein mole of second catalyst and the flavone glycoside compound
Than for 1:9-11.
49. application according to claim 48, wherein second catalyst is copper sulphate and vitamine C sodium.
50. application according to claim 49, wherein the condition of second contact includes: that the temperature of contact is 20-30
DEG C, the time of contact is 0.2-24 hours.
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