CN106198956A - 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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- CN106198956A CN106198956A CN201610506083.0A CN201610506083A CN106198956A CN 106198956 A CN106198956 A CN 106198956A CN 201610506083 A CN201610506083 A CN 201610506083A CN 106198956 A CN106198956 A CN 106198956A
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- G—PHYSICS
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
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- 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
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- 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
- G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles
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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；Modification cuorin and biotin derivative by NH CO structure thus are connected together by modified cuorin with biotin derivative；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 and amination obtains, the described modified heart
Phospholipid contains-NH2；
The structural formula of described biotin derivative is:Wherein, described-R-is containing 4
～the saturated alkyl chain of 20 carbon atoms or containing the polyglycol chain of 2～10 carbon atoms, in described modified cuorin-
NH2-NH-CO-structure is generated thus by described modified cuorin and described biotin with the-COOH in described biotin derivative
Derivant 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-a NH2。
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 intermediate product；
In described intermediate product, add excessive ammonia and fully react, obtaining modified cuorin, wherein, the described modified heart
The hydrophobic fat acid side chain of phospholipid is oxidized and amination, and described modified cuorin contains amino；
The biotin derivative of described modified cuorin and the activation of N-hydroxysuccinimide is existed at the second solvent
Under the conditions of fully react, obtain biotin derivative combine modified cuorin, wherein, the structural formula of described biotin derivative
For:Wherein, described-R-is the carbochain containing 4～20 carbon atoms or contains 2～10 carbon
The polyglycol chain of atom ,-the NH in described modified cuorin2With-COOH generation-the NH-CO-in described biotin derivative
Structure thus described modified cuorin is connected together with described biotin derivative the described biotin derivative of generation and combines
Modified cuorin；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
Being 1:1～8 with the mol ratio of described peroxide, described first solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
In one embodiment, described add in described intermediate product in excessive ammonia the operation fully reacted, institute
The mass concentration stating ammonia is 10%～30%.
In one embodiment, the described biotin by described modified cuorin and the activation of N-hydroxysuccinimide derives
Thing is under conditions of the second solvent exists in the operation of fully reaction, and described second solvent is DMSO, DMF, oxolane or pH
It it is the phosphate buffer of 6.5～8.5.
In one embodiment, the described biotin by described modified cuorin and the activation of N-hydroxysuccinimide derives
In the operation that thing fully reacts under conditions of the second solvent exists, described modified cuorin and described N-hydroxysuccinimide
The mol ratio of the biotin derivative of activation is 1:1.5～20.
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 and amination obtains, and modified cuorin contains
There is-NH2。
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 multiple-NH2.Specifically, this modification cuorin can comprise 1～8-NH2。
Preferably, modified cuorin contains-a NH2。
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.
-NH in modified cuorin2-NH-CO-structure is generated thus by the modification heart with the-COOH in biotin derivative
Phospholipid 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 intermediate product.
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.
S20, in the intermediate product that S10 obtains, add excessive ammonia fully reacting, obtain modified cuorin.
By peroxide and ammonia so that the hydrophobic fat acid side chain of cuorin is oxidized and amination, so that
The modified cuorin that must prepare contains amino.
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 multiple amino.Specifically, modified cuorin can comprise 1～8-NH2。
Preferably, modified cuorin contains-a NH2。
Particularly preferred, the structural formula of modified cuorin is as follows:
The mass concentration of ammonia is 10%～30%.
In S20, reaction temperature is 60 DEG C～100 DEG C.
The biotin derivative that S30, the modified cuorin obtained by S20 and N-hydroxysuccinimide (NHS) activate exists
Second solvent fully reacts under conditions of existing, and obtains the modified cuorin that biotin derivative combines.
The structural formula of biotin derivative is:
-R-is the carbochain containing 4～20 carbon atoms or the polyglycol chain containing 2～10 carbon atoms, modified heart phosphorus
-NH in fat2Generate-NH-CO-structure with the-COOH in biotin derivative thus modification cuorin and biotin are derived
Thing connects together and generates the modified cuorin that biotin derivative combines.
Preferably ,-R-is the saturated alkyl chain containing 4～10 carbon atoms.
Particularly preferred ,-R-is-(CH2)6-。
The biotin derivative of NHS activation is biotin derivative NHS ester, and biotin derivative NHS ester can be direct
It is commercially available.
In S30, the second solvent be DMSO, DMF, oxolane or pH be the phosphate buffer of 6.5～8.5.
In S30, the mol ratio of modified cuorin and the biotin derivative of N-hydroxysuccinimide activation be 1:1.5～
20。
Modified cuorin and Streptavidin MagneSphere mixing that S40, the biotin derivative obtained by S30 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 xxxx).
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
Under argon shield, 0.5mmol cuorin is dissolved in 1mL dry toluene, adds the m-chloro peroxide benzene first of 1.5mmol
Acid, is gradually heated up being warming up to 110 DEG C in stirring, and maintains this thermotonus 72 hours.After reactant liquor is cooled to room temperature, pour into
In 25mL frozen water, then extract solution 3 times by 20mL ethyl acetate, combined ethyl acetate.By molten for the ethyl acetate of merging 10mL
After saturated nacl aqueous solution washs 1 time, add anhydrous sodium sulfate and be dried 12 hours.Ethyl acetate it is evaporated under liquid negative pressure, remnants'
Solid adds to 8mL strong aqua ammonia, is gradually heated up being warming up to 100 degrees Celsius, and maintains this thermotonus 8 hours under stirring.Negative
Pressure is evaporated reactant liquor, obtains yellow, viscous liquid, after dissolving with a small amount of methanol, falls insoluble matter with 0.2 μm membrane filtration,
Solution preparative liquid chromatography is purified, and obtains about 83mg white solid.S(ESI+,m/z):1495.29113.
Embodiment 2: the preparation of the modified cuorin that biotin derivative combines
Weighing the prepared modified cuorin of 50mg embodiment 1 to be dissolved in 2mL dry DMF, stirring is lower adds 20mg biotin
Derivant NHS ester, at room temperature stirring reaction 2.5 hours.Being evaporated reactant liquor under decompression, residual solid 5mL ethyl acetate is molten
Solving, filter insoluble matter, and be evaporated by the acetic acid ethyl dissolution of gained, what the i.e. biotin derivative of the white solid obtained combined changes
Property 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 that the biotin derivative that 100 μ L concentration are 0.2mg/mL combines, add
Entering 100 μ L concentration is the Streptavidin MagneSphere of 10mg/mL, mixes latter 37 DEG C and hatches 10 minutes.1mL 30mM is used after Magneto separate
Tris buffer redissolves, and redissolves Magneto separate again after the mixed liquor concussion of gained, and by the solid of gained 5mL 30mM Tris
Buffer redissolves, and obtains the Streptavidin MagneSphere solution being coated with modified cuorin of 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 and amination obtains, described modified cuorin
Containing-NH2；
The structural formula of described biotin derivative is:Wherein, described-R-is containing 4～20
The saturated alkyl chain of individual carbon atom or the polyglycol chain containing 2～10 carbon atoms ,-the NH in described modified cuorin2
Generate-NH-CO-structure with the-COOH in described biotin derivative thus described modified cuorin and described biotin are spread out
Biology 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-a NH2。
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 intermediate product；
In described intermediate product, add excessive ammonia and fully react, obtaining modified cuorin, wherein, described modified cuorin
Hydrophobic fat acid side chain is oxidized and amination, described modified cuorin contains amino；
The condition that the biotin derivative of described modified cuorin and the activation of N-hydroxysuccinimide is existed at the second solvent
Lower fully reaction, obtains the modified cuorin that biotin derivative combines, and wherein, the structural formula of described biotin derivative is:Wherein, described-R-is the carbochain containing 4～20 carbon atoms or contains 2～10 carbon atoms
Polyglycol chain ,-the NH in described modified cuorin2-NH-CO-structure is generated with-the COOH in described biotin derivative
Thus described modified cuorin is connected together with described biotin derivative and generates changing of described biotin derivative combination
Property cuorin；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, described first solvent is dichloromethane, 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 to
Adding in excessive ammonia the operation fully reacted in described intermediate product, the mass concentration of described ammonia is 10%～30%.
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
The biotin derivative of described modified cuorin and the activation of N-hydroxysuccinimide is abundant under conditions of the second solvent exists
Reaction operation in, described second solvent 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
The biotin derivative of described modified cuorin and the activation of N-hydroxysuccinimide is abundant under conditions of the second solvent exists
In the operation of reaction, the mol ratio of the biotin derivative that described modified cuorin activates with described N-hydroxysuccinimide is
1:1.5～20.
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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610506083.0A CN106198956B (en) | 2016-06-30 | 2016-06-30 | Coated nanometer magnetic bead of modified cuorin and preparation method thereof |
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| CN201610506083.0A CN106198956B (en) | 2016-06-30 | 2016-06-30 | Coated nanometer magnetic bead of modified cuorin and preparation method thereof |
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| CN106198956A true CN106198956A (en) | 2016-12-07 |
| CN106198956B CN106198956B (en) | 2018-04-20 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829803A (en) * | 2003-07-18 | 2006-09-06 | Qtl生物***有限公司 | Assays for protease enzyme activity |
| CN101360997A (en) * | 2005-11-18 | 2009-02-04 | 美国政府健康及人类服务部，疾病控制和预防中心 | Modified cardiolipin and uses therefor |
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| US20140051070A1 (en) * | 2011-02-15 | 2014-02-20 | Kyowa Medex Co., Ltd. | Streptavidin-coupled magnetic particles and manufacturing method for same |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1829803A (en) * | 2003-07-18 | 2006-09-06 | Qtl生物***有限公司 | Assays for protease enzyme activity |
| CN101360997A (en) * | 2005-11-18 | 2009-02-04 | 美国政府健康及人类服务部，疾病控制和预防中心 | Modified cardiolipin and uses therefor |
| US20140051070A1 (en) * | 2011-02-15 | 2014-02-20 | Kyowa Medex Co., Ltd. | Streptavidin-coupled magnetic particles and manufacturing method for same |
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
Non-Patent Citations (1)
| Title |
|---|
| LISAY.WU: "Chemoaffinity Capture of Pre-Targeted Prostate Cancer Cells With Magnetic Beads", 《THE PROSTATE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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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Address after: 518000 Shenzhen 1 Longgang biological technology plant 1, Baolong two road, Baolong street, Guangdong Patentee after: SHENZHEN YHLO BIOTECH CO., LTD. Address before: 518000 Guangdong, Shenzhen, Nanshan District Nanshan street, Xinghai Road, Li Shan Industrial Zone, 1-4 1-4 floors. Patentee before: SHENZHEN YHLO BIOTECH CO., LTD. |