CN106124783A - Modified coated nanometer magnetic bead of cuorin and preparation method thereof - Google Patents
Modified coated nanometer magnetic bead of cuorin and preparation method thereof Download PDFInfo
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- CN106124783A CN106124783A CN201610512711.6A CN201610512711A CN106124783A CN 106124783 A CN106124783 A CN 106124783A CN 201610512711 A CN201610512711 A CN 201610512711A CN 106124783 A CN106124783 A CN 106124783A
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- cuorin
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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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
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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
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The invention discloses a kind of modified coated nanometer magnetic bead of cuorin and preparation method thereof, the modified coated nanometer magnetic bead of cuorin includes: modified cuorin, biotin derivative and Streptavidin MagneSphere；Together with modified cuorin and biotin derivative are thus connected to by CO NH structure；Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin, and biotin derivative and Streptavidin connect.Modification cuorin is directly firmly firmly connected with biotin derivative by this modified coated nanometer magnetic bead of cuorin by chemical bond, and the Streptavidin on biotin derivative and Streptavidin MagneSphere surface connects.This modified coated nanometer magnetic bead of cuorin is used directly for the detection of anti-phospholipid antibody, and the detection product prepared relative to the method for conventional physical absorption cuorin, has higher stability.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of modified coated nanometer magnetic bead of cuorin and preparation side thereof
Method.
Background technology
Anticardiolipin antibody is a kind of antibody that can react with the multiple antigenic substance containing structure of phospholipid, and antigen is
Participate in the electronegative phospholipid composition of various kinds of cell film composition.Be primarily present in clinically antiphospholipid syndrome, various self
Immune disease and syphilis are in the patient.
Anti-phospholipid syndrome (APS) includes self property immune disease multiple, is more common in youngster, and men and women's incidence rate is about
For 2:8.Patient may occur in which that one or more show, and can involve multiple system, organ, mainly have: vein and artery thrombosis,
Thrombocytopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infraction, pulmonary hypertension.Pernicious APS can behave as short
The extensive thrombosis of Progressive symmetric erythrokeratodermia in phase, causes multiple organ dysfunction syndrome the most dead.Can be secondary to systemic lupus erythematosus (sle) or
Other autoimmune disease, but (primary antiphospholipid syndrome) also can individually occur.
Also can produce anti-phospholipid antibody in syphilitic's body, syphilis is the property caused by spirochete bacteria Tyreponema pallidum
Spread disease.It is reported, have more than 5 million people's syphilizations every year, including by the 30000 of heredo infection babies.
Syphilis in hiding in the patient and can hide many years, and may result in various clinical manifestation.Wherein it is in syphilis to hide
The patient of phase there is no clinical symptoms, and the secondary stage does not treats even lifelong in patient about 2/3rds.The infected is at latent phase not
There is infectiousness, but be in child's PI congenital syphilis of the Mothers of latent phase.
Anti-phospholipid antibody detection test is widely used in the non-treponema test of phospholipid syndrome diagnosis and syphilis.
This detection has advantage cheap, that quickly and conveniently perform great amount of samples.Additionally, due to the concentration of anti-phospholipid antibody can be along with
The treatment of syphilis is successful and is gradually lowered, and the specific antibody treponema antibody of syphilis can last for several years the most lifelong
The highest.Therefore anti-phospholipid antibody detection test is considered as the finer selection of monitoring syphilis treatment.
The main method of traditional detection anti-phospholipid antibody is the mode of cuorin physical absorption to be spread upon specifically
Above solid such as ELISA Plate, then utilize and be attached to the cuorin above ELISA Plate and the anti-phospholipid antibody knot in sample to be detected
Close, it is achieved the capture to the anti-phospholipid antibody in sample.By the anti-phospholipid antibody of capture being developed the color and measuring luminous concentration, just
Can indirectly read the anti-phospholipid antibody concentration in sample.
Due to the cuorin fixing with physical adsorption way under the influence of the external condition such as solvent, heating easily from solid panel
On dissociate and come off, the less stable of the inspection product therefore prepared by the method.
Summary of the invention
Based on this, it is necessary to provide the preferably modified cuorin of a kind of stability for detecting anticardiolipin antibody to be coated
Nanometer magnetic bead and preparation method thereof.
A kind of modified coated nanometer magnetic bead of cuorin, including: modified cuorin, biotin derivative and Streptavidin
Magnetic bead；
Described modified cuorin is that the hydrophobic fat acid side chain of cuorin is oxidized to be obtained, and described modified cuorin contains
The structural formula of described biotin derivative is:Wherein, described-R-is containing 4
Individual～the saturated alkyl chain of 20 carbon atoms or the polyglycol chain containing 2～10 carbon atoms, in described modified cuorin
'sWith-the NH in described biotin derivative2Generate-CO-NH-structure thus by described modified cuorin and described
Biotin derivative connects together；
Described Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin, described biotin derivative and described
Streptavidin connects.
In one embodiment, described-R-is the saturated alkyl chain containing 4～10 carbon atoms.
In one embodiment, described modified cuorin contains one
In one embodiment, the structural formula of described modified cuorin is as follows:
The preparation method of a kind of above-mentioned coated nanometer magnetic bead of modified cuorin, comprises the steps:
Cuorin and peroxide are fully reacted under conditions of the first solvent exists, obtains modified cuorin, wherein,
The hydrophobic fat acid side chain of described modified cuorin is oxidized, and described modified cuorin contains
Described modified cuorin and biotin derivative are fully reacted under conditions of the second solvent exists, obtains biology
The modified cuorin that element derivant combines, wherein, the structural formula of described biotin derivative is:Its
In, described-R-is the saturated alkyl chain containing 4～20 carbon atoms or the polyglycol chain containing 2～10 carbon atoms,
In described modified cuorinWith-the NH in described biotin derivative2Generate-CO-NH-structure thus by described
Modified cuorin connects together with described biotin derivative；And
The modified cuorin combined by described biotin derivative and Streptavidin MagneSphere mix and fully react, and obtain
The modified coated nanometer magnetic bead of cuorin, wherein, described Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin,
Described biotin derivative and described Streptavidin connect.
In one embodiment, the described abundant reaction under conditions of the first solvent exists by cuorin and peroxide
In operation, described peroxide is benzoyl hydroperoxide, metachloroperbenzoic acid, peracetic acid or Perpropionic Acid, described cuorin
It is 1:1～8 with the mol ratio of described peroxide.
In one embodiment, the described abundant reaction under conditions of the first solvent exists by cuorin and peroxide
In operation, described first solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
In one embodiment, the described condition that described modified cuorin and biotin derivative are existed at the second solvent
In the operation of lower fully reaction, described second solvent be DMSO, DMF, oxolane or pH be the phosphate-buffered of 6.5～8.5
Liquid.
In one embodiment, the described condition that described modified cuorin and biotin derivative are existed at the second solvent
In the operation of lower fully reaction, described modified cuorin is 1:1.5～20 with the mol ratio of described biotin derivative.
In one embodiment, the described modified cuorin that described biotin derivative is combined and Streptavidin MagneSphere
In the operation mixed and fully react, the concentration of the modified cuorin that described biotin derivative combines is 0.1mg/mL～1mg/
ML, the concentration of described Streptavidin MagneSphere is 5mg/mL～15mg/mL.
Modification cuorin is directly firmly spread out with biotin by this modified coated nanometer magnetic bead of cuorin by chemical bond
Biological firm connection, the Streptavidin on biotin derivative and Streptavidin MagneSphere surface connects, and inhales relative to physics
Attached mode, more easily controls the modified cuorin amount of magnetic bead surfaces, and can be by the length of regulation-R-, it is achieved change
Property cuorin amount and nanometer magnetic bead between the control of distance, preferably remain modified cuorin amount and be combined with anti-phospholipid antibody
Space.This modified coated nanometer magnetic bead of cuorin is used directly for the detection of anti-phospholipid antibody, and relative to biography
Detection product prepared by the method for system physical absorption cuorin, has higher stability.
Accompanying drawing explanation
Fig. 1 is the flow chart of the preparation method of the coated nanometer magnetic bead of modified cuorin of an embodiment.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
The coated nanometer magnetic bead of modified cuorin of one embodiment, including: modified cuorin, biotin derivative and chain
Mould Avidin magnetic bead.
Cuorin is the Ester being made up of 3 glycerol, 2 phosphoric acid and 4 long-chain unsaturated alkyls, and this structure contains
There are 2 hydrophilic center and 4 hydrophobic side chains.The structural formula of cuorin is as follows:
Modified cuorin is that the hydrophobic fat acid side chain of cuorin is oxidized to be obtained, and modified cuorin contains
Owing to cuorin has 4 side chains, during cuorin modification, 4 side chains can be the most oxidized, therefore this modification heart phosphorus
Fat may comprise multipleSpecifically, this modification cuorin can comprise 1～8
Preferably, modified cuorin contains one
Particularly preferred, the structural formula of modified cuorin is as follows:
The structural formula of biotin derivative is:
Wherein ,-R-is the saturated alkyl chain containing 4～20 carbon atoms or the poly-second containing 2～10 carbon atoms
Glycol chains.
In modified cuorinWith-the NH in biotin derivative2Generate-CO-NH-structure thus by modification
Cuorin connects together with biotin derivative.
Preferably ,-R-is the saturated alkyl chain containing 4～10 carbon atoms.
Particularly preferred ,-R-is-(CH2)6-。
Owing to the size of Cardiolipin molecules is the least, the space wretched insufficiency that can transform, to hydrophilic phosphate ester center
Carry out modification and can reduce the affinity of cuorin and anti-phospholipid antibody, even make antigen active disappear.
This modified coated nanometer magnetic bead of cuorin, by transforming cuorin hydrophobic side chains, remains heart phosphorus
The modified cuorin at fat hydrophilic phosphate ester center.
Modification cuorin is directly firmly spread out with biotin by this modified coated nanometer magnetic bead of cuorin by chemical bond
Biological firm connection, the Streptavidin on biotin derivative and Streptavidin MagneSphere surface connects, and inhales relative to physics
Attached mode, more easily controls the modified cuorin amount of magnetic bead surfaces, and can be by the length of regulation-R-, it is achieved change
Property cuorin amount and nanometer magnetic bead between the control of distance, preferably remain modified cuorin amount and be combined with anti-phospholipid antibody
Space.This modified coated nanometer magnetic bead of cuorin is used directly for the detection of anti-phospholipid antibody, and relative to biography
Detection product prepared by the method for system physical absorption cuorin, has higher stability.
Additionally, due to each Streptavidin can be combined closely with 4 biotin, this modified coated nanometer of cuorin
Magnetic bead passes through biotin-Streptavidin enlarge-effect, makes detected signal be exaggerated 4 times, greatly improves anti-phospholipid and resists
The detection sensitivity of body.
The preparation method of the above-mentioned modified coated nanometer magnetic bead of cuorin as shown in Figure 1, comprises the steps:
S10, cuorin and peroxide are fully reacted under conditions of the first solvent exists, obtain modified cuorin.
By cuorin and peroxide reactions, the fatty acid side chain of cuorin is aoxidized.
Peroxide is benzoyl hydroperoxide, metachloroperbenzoic acid, peracetic acid or Perpropionic Acid.
The mol ratio of cuorin and peroxide is 1:1～8.
First solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
S10 also includes the operation being purified intermediate product, and purification can be through liquid phase system after ethyl acetate extracts
Standby chromatogram purification.The modified cuorin of purity about 80% can be obtained after purification.
In S10, reaction temperature is 60 DEG C～100 DEG C.
Owing to cuorin has 4 side chains, during cuorin modification, 4 side chains can be the most oxidized, therefore this modification heart phosphorus
Fat may comprise multipleSpecifically, modified cuorin can comprise 1～8
Preferably, modified cuorin contains one
Particularly preferred, the structural formula of modified cuorin is as follows:
S20, the modified cuorin obtained by S10 and biotin derivative are the most anti-under conditions of the second solvent exists
Should, obtain the modified cuorin that biotin derivative combines.
The structural formula of biotin derivative is:
Wherein ,-R-is the saturated alkyl chain containing 4～20 carbon atoms or the poly-second containing 2～10 carbon atoms
Glycol chains.
In modified cuorinWith-the NH in biotin derivative2Generate-CO-NH-structure thus by modification
Cuorin connects together with biotin derivative.
Preferably ,-R-is the saturated alkyl chain containing 4～10 carbon atoms.
Particularly preferred ,-R-is-(CH2)6-。
Biotin derivative can directly be commercially available.
In S20, the second solvent be DMSO, DMF, oxolane or pH be the phosphate buffer of 6.5～8.5.
In S20, modified cuorin is 1:1.5～20 with the mol ratio of biotin derivative.
Modified cuorin and Streptavidin MagneSphere mixing that S30, the biotin derivative obtained by S20 combine are the most abundant
Reaction, obtains the modified coated nanometer magnetic bead of cuorin.
Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin, and biotin derivative and Streptavidin are even
Connect.
Streptavidin MagneSphere can be directly to buy, such as, purchased from MagnaBind company (article No. is 21344).
In the modified cuorin that biotin derivative is combined and Streptavidin MagneSphere mixing the operation fully reacted,
The concentration of the modified cuorin that biotin derivative combines is 0.1mg/mL～1mg/mL, and the concentration of Streptavidin MagneSphere is
5mg/mL～15mg/mL.
The preparation method of this modified coated nanometer magnetic bead of cuorin, by chemical bond directly by firm for modification cuorin
Firmly being connected with biotin derivative, the Streptavidin on biotin derivative and Streptavidin MagneSphere surface connects, phase
For the mode of physical absorption, more easily control the modified cuorin amount of magnetic bead surfaces, and can be by the length of regulation-R-
Degree, it is achieved the control of the distance between modified cuorin amount and nanometer magnetic bead, preferably remains modified cuorin amount and anti-phosphorus
The space of fat antibodies.The coated nanometer magnetic bead of modified cuorin prepared is used directly for the detection of anti-phospholipid antibody,
And the detection product prepared relative to the method for conventional physical absorption cuorin, has higher stability.
Additionally, due to each Streptavidin can be combined closely with 4 biotin, prepared modified cuorin is coated to be received
Rice magnetic bead, by biotin-Streptavidin enlarge-effect, makes detected signal be exaggerated 4 times, greatly improves anti-phospholipid
The detection sensitivity of antibody.
It it is below specific embodiment.
Embodiment 1: the preparation of modified cuorin
0.5mmol cuorin is dissolved in 1mL dry toluene, add 1.5mmol metachloroperbenzoic acid, in stirring by
Gradually it is heated to 110 DEG C, and maintains this thermotonus 72 hours.After reactant liquor is cooled to room temperature, pour in 25mL frozen water,
Solution is extracted 3 times again, combined ethyl acetate by 20mL ethyl acetate.By molten for the ethyl acetate molten 10mL saturated sodium-chloride of merging
After liquid washs 1 time, add anhydrous sodium sulfate and be dried 12 hours.Ethyl acetate, the remaining a small amount of first of solid it is evaporated under liquid negative pressure
Alcohol dissolves, and filters out insoluble matter, and solution preparative liquid chromatography is purified, and obtains about 210mg colourless oil liquid and is modification
Cuorin, MS (ESI+,m/z):1479.83131。
Embodiment 2: the preparation of the modified cuorin that biotin derivative combines
Stirring is lower is sequentially added into 200mg ring ethyoxyl modification cuorin, 110mg terminal amino group in 4mL anhydrous DMF solution
The biotin derivative changed, stirring reaction 10 hours under room temperature.Reactant liquor it is evaporated, residual solid 5mL ethyl acetate under decompression
Dissolving, filter insoluble matter, and be evaporated by the acetic acid ethyl dissolution of gained, the i.e. biotin derivative of the white solid obtained combines
Modified cuorin.
In the present embodiment, the structural formula of biotin derivative is:-R-is-(CH2)6-。
The modified cuorin that biotin derivative prepared by the present embodiment combines needs not move through special purification and i.e. can be used for
Subsequent use.
Embodiment 3: be coated with the preparation of the Streptavidin MagneSphere of modified cuorin
Take the phosphate buffer of the modified cuorin of the biotin derivative combination that 100 μ L contain 0.2mg/mL, add
100 μ L contain the Streptavidin MagneSphere of 10mg/mL, mix latter 37 DEG C and hatch 10 minutes.With 1mL 30mM Tris after Magneto separate
Buffer redissolves, and redissolves Magneto separate again after the mixed liquor concussion of gained, and is buffered by the solid of gained 5mL 30mM Tris
Liquid redissolves, and obtains the Streptavidin MagneSphere solution being coated with modified cuorin that concentration is 0.02mg/mL.
Embodiment 4: the Avidin magnetic bead being coated with modified cuorin reacts with anti-phospholipid sample
Take the Avidin magnetic bead solution being coated with modified cuorin of preparation in 200 μ L embodiments 1～3 respectively, add 5 μ L
Anti-phospholipid antibody serum sample also hatches 30 minutes in 37 DEG C.The Streptavidin magnetic of modified cuorin will be coated with after Magneto separate
Pearl is redissolved to 200 μ L, adds the two of horseradish peroxidase-labeled and resists, 37 DEG C hatch 30 minutes after clean successively, add at the bottom of TMB
Thing liquid is also hatched 10 minutes, adds 100 μ L stop buffers, reads OD value in 10 minutes in microplate reader, obtains the luminous letter of sample
Number value.
Take three cuorin positive serum samples and three cuorin negative serum samples respectively, and with traditional physics
Absorption method is comparison, and the OD value that contrast records obtains table 1 below.
Table 1: embodiment 1～3 measures the OD value of different samples with matched group (physisorphtion)
As can be seen from Table 1, what prepared by embodiment 1～3 is coated with Avidin magnetic bead and the physical absorption of modified cuorin
The magnetic bead (matched group) of the method absorption luminous signal when testing anti-phospholipid antibody sample compares, being coated of embodiment 1～3 preparation
There is an Avidin magnetic bead of modified cuorin luminous signal value relatively matched group is the lowest when measuring negative sample and (reduce 1 times～4
Times), the Avidin magnetic bead being coated with modified cuorin luminous signal when measuring positive sample of embodiment 1～3 preparation simultaneously
Relatively matched group has raising (improve 3～10 times) greatly.
Thus illustrating, the Avidin magnetic bead being coated with modified cuorin of embodiment 1～3 preparation is measuring anti-phospholipid sample
Time, relative to the magnetic bead of traditional physisorphtion, detection sensitivity has and is obviously improved.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. the coated nanometer magnetic bead of modified cuorin, it is characterised in that including: modified cuorin, biotin derivative and
Streptavidin MagneSphere；
Described modified cuorin is that the hydrophobic fat acid side chain of cuorin is oxidized to be obtained, and described modified cuorin contains
The structural formula of described biotin derivative is:Wherein, described-R-be containing 4～
The saturated alkyl chain of 20 carbon atoms or the polyglycol chain containing 2～10 carbon atoms, in described modified cuorinWith-the NH in described biotin derivative2Generate-CO-NH-structure thus by described modified cuorin and described life
Thing element derivant connects together；
Described Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin, described biotin derivative and described strepto-
Avidin connects.
The modified coated nanometer magnetic bead of cuorin the most according to claim 1, it is characterised in that described-R-is containing 4
～the saturated alkyl chain of 10 carbon atoms.
The modified coated nanometer magnetic bead of cuorin the most according to claim 1, it is characterised in that described modified cuorin contains
There is one
The modified coated nanometer magnetic bead of cuorin the most according to claim 3, it is characterised in that described modified cuorin
Structural formula is as follows:
5. a preparation method for the coated nanometer magnetic bead of modified cuorin according to any one of Claims 1 to 4, its feature
It is, comprises the steps:
Cuorin and peroxide are fully reacted under conditions of the first solvent exists, obtains modified cuorin, wherein, described
The hydrophobic fat acid side chain of modified cuorin is oxidized, and described modified cuorin contains
Described modified cuorin and biotin derivative are fully reacted under conditions of the second solvent exists, obtains biotin and spread out
The modified cuorin of bioconjugation, wherein, the structural formula of described biotin derivative is:Its
In, described-R-is the saturated alkyl chain containing 4～20 carbon atoms or the polyglycol chain containing 2～10 carbon atoms,
In described modified cuorinWith-the NH in described biotin derivative2Generate-CO-NH-structure thus by described
Modified cuorin connects together with described biotin derivative；And
The modified cuorin combined by described biotin derivative and Streptavidin MagneSphere mix and fully react, and obtain modification
The coated nanometer magnetic bead of cuorin, wherein, described Streptavidin MagneSphere is the nanometer magnetic bead combining Streptavidin, described
Biotin derivative and described Streptavidin connect.
The preparation method of the modified coated nanometer magnetic bead of cuorin the most according to claim 5, it is characterised in that described general
Cuorin and peroxide are under conditions of the first solvent exists in the operation of fully reaction, and described peroxide is peroxide benzene first
Acid, metachloroperbenzoic acid, peracetic acid or Perpropionic Acid, the mol ratio of described cuorin and described peroxide be 1:1～
8。
The preparation method of the modified coated nanometer magnetic bead of cuorin the most according to claim 5, it is characterised in that described general
Cuorin and peroxide are under conditions of the first solvent exists in the operation of fully reaction, and described first solvent is dichloromethane
Alkane, chloroform, chloroform, benzene or toluene.
The preparation method of the modified coated nanometer magnetic bead of cuorin the most according to claim 5, it is characterised in that described general
Described modified cuorin and biotin derivative are under conditions of the second solvent exists in the operation of fully reaction, described second molten
Agent be DMSO, DMF, oxolane or pH be the phosphate buffer of 6.5～8.5.
The preparation method of the modified coated nanometer magnetic bead of cuorin the most according to claim 5, it is characterised in that described general
In the operation that described modified cuorin and biotin derivative fully react under conditions of the second solvent exists, the described modified heart
Phospholipid is 1:1.5～20 with the mol ratio of described biotin derivative.
The preparation method of the modified coated nanometer magnetic bead of cuorin the most according to claim 5, it is characterised in that described
In the modified cuorin that described biotin derivative is combined and Streptavidin MagneSphere mixing the operation fully reacted, described
The concentration of the modified cuorin that biotin derivative combines is 0.1mg/mL～1mg/mL, the concentration of described Streptavidin MagneSphere
For 5mg/mL～15mg/mL.
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| CN201610512711.6A CN106124783B (en) | 2016-06-30 | 2016-06-30 | Coated nanometer magnetic bead of modified cuorin and preparation method thereof |
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| CN201610512711.6A Active CN106124783B (en) | 2016-06-30 | 2016-06-30 | Coated nanometer magnetic bead of modified cuorin and preparation method thereof |
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| CN114195821A (en) * | 2021-12-17 | 2022-03-18 | 郑州安图生物工程股份有限公司 | Novel cardiolipin derivative and preparation method and application thereof |
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| CN114195821B (en) * | 2021-12-17 | 2024-03-12 | 郑州安图生物工程股份有限公司 | Cardiolipin derivative and preparation method and application thereof |
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| CN106124783B (en) | 2018-10-02 |
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