Coated nanometer magnetic bead of modified cuorin and preparation method thereofTechnical field
The present invention relates to vitro detection field, more particularly to a kind of coated nanometer magnetic bead of modified cuorin and its preparation sideMethod.
Background technology
Anticardiolipin antibodies are a kind of antibody that can be reacted with a variety of antigenic substances containing structure of phospholipid, and antigen isParticipate in the negatively charged phospholipid composition of various kinds of cell film composition.Clinically be primarily present in antiphospholipid syndrome, it is various itselfImmunity disease and syphilis patient's body.
Anti- phospholipid syndrome (APS) includes itself a variety of property immunity disease, is more common in young man, men and women's incidence rate is aboutFor 2：8.Patient may occur in which one or more performances, can involve multiple systems, organ, mainly have：Vein and Arterial thrombosis,Thrombopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infraction, pulmonary hypertension.Pernicious APS can behave as shortThe extensive thrombosis of progressive in phase, causes multiple organ failure even dead.Can secondary to systemic loupus erythematosus orOther autoimmunity diseases, but (primary antiphospholipid syndrome) also can individually occur.
Also anti-phospholipid antibody can be produced in syphilitic's body, syphilis is the property as caused by spirochete bacteria Tyreponema pallidumSpread the disease.It is reported that 5 million people's syphilizations are had more than every year, including 30,000 babies by heredo infection.Syphilis can hide and hide in patient's body many years, and can cause various clinical manifestations.Wherein hidden in syphilisThe patient of phase has no clinical symptoms, and the secondary stage does not treat even lifelong in patient about 2/3rds.The infected in latent phase notWith infectiousness, but child's may be infected congenital syphilis of the Mothers in latent phase.
Anti-phospholipid antibody detection experiment is widely used in the non-treponema experiment of phosphatide syndrome diagnosis and syphilis.The detection has the advantages that cheap, quick and conveniently great amount of samples is performed.Further, since the concentration of anti-phospholipid antibody can be withThe treatment of syphilis is successful and gradually reduces, and the specific antibody treponema antibody of syphilis can last for several years even throughout one's lifeIt is all high.Therefore anti-phospholipid antibody detection experiment is considered as to monitor the finer selection of syphilis treatment.
The main method of traditional detection anti-phospholipid antibody is to be applied to cuorin specifically with the mode of physical absorptionAbove solid such as ELISA Plate, the cuorin being attached to above ELISA Plate and the anti-phospholipid antibody knot in sample to be detected are then utilizedClose, realize the capture to the anti-phospholipid antibody in sample.By developing the color to the anti-phospholipid antibody of capture and measuring luminous concentration, justThe anti-phospholipid antibody concentration in sample can indirectly be read.
Due to the cuorin that physical adsorption way is fixed under the influence of the external conditions such as solvent, heating easily from solid panelIt is upper to dissociate and come off, therefore the stability for passing through inspection product prepared by this method is poor.
The content of the invention
Based on this, it is necessary to provide a kind of preferably modified cuorin coating of stability for being used to detect anticardiolipin antibodiesNanometer magnetic bead and preparation method thereof.
A kind of coated nanometer magnetic bead of modified cuorin, including：Modified cuorin, biotin derivative and StreptavidinMagnetic bead；
The modified cuorin is obtained for the hydrophobic fat acid side chain of cuorin by oxidation and amination, the modified heartPhosphatide contains-NH2；
The structural formula of the biotin derivative is：Wherein ,-the R- is to contain 4The saturated alkyl chain of~20 carbon atoms or the polyglycol chain containing 2~10 carbon atoms, in the modified cuorin-NH2With-COOH generation-NH-CO- the structures in the biotin derivative so as to by the modified cuorin and the biotinDerivative connects together；
The Streptavidin MagneSphere is the nanometer magnetic bead for combining Streptavidin, the biotin derivative and describedStreptavidin connects.
In one embodiment ,-the R- is the saturated alkyl chain containing 4~10 carbon atoms.
In one embodiment, the modified cuorin contains-a NH2。
In one embodiment, the structural formula of the modified cuorin is as follows：
A kind of preparation method of the above-mentioned coated nanometer magnetic bead of modification cuorin, includes the following steps：
Cuorin and peroxide are fully reacted under the conditions of existing for the first solvent, obtain intermediate product；
Excessive ammonia is added into the intermediate product and is fully reacted, obtains modified cuorin, wherein, the modified heartThe hydrophobic fat acid side chain of phosphatide contains amino by oxidation and amination, the modified cuorin；
The biotin derivative that the modified cuorin and N- hydroxysuccinimides are activated is existing for the second solventUnder the conditions of fully react, obtain biotin derivative combination modification cuorin, wherein, the structural formula of the biotin derivativeFor：Wherein ,-the R- is the carbochain containing 4~20 carbon atoms or contains 2~10 carbonThe polyglycol chain of the atom ,-NH in the modified cuorin2With-COOH generations-the NH-CO- in the biotin derivativeStructure combines so as to which the modified cuorin to be connected together to the generation biotin derivative with the biotin derivativeModification cuorin；And
Modification cuorin and Streptavidin MagneSphere that the biotin derivative combines are mixed and fully reacted, is obtainedThe coated nanometer magnetic bead of modified cuorin, wherein, the Streptavidin MagneSphere is the nanometer magnetic bead for combining Streptavidin,The biotin derivative is connected with the Streptavidin.
In one embodiment, it is described fully to react cuorin and peroxide under the conditions of existing for the first solventIn operation, the peroxide is benzoyl hydroperoxide, metachloroperbenzoic acid, Peracetic acid or Perpropionic Acid, the cuorinMolar ratio with the peroxide is 1：1~8, first solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
In one embodiment, in the operation that excessive ammonia is added into the intermediate product and is fully reacted, instituteThe mass concentration for stating ammonium hydroxide is 10%~30%.
In one embodiment, the biotin that the modified cuorin and N- hydroxysuccinimides are activated derivesIn the operation that thing fully reacts under the conditions of existing for the second solvent, second solvent is DMSO, DMF, tetrahydrofuran or pHFor 6.5~8.5 phosphate buffer.
In one embodiment, the biotin that the modified cuorin and N- hydroxysuccinimides are activated derivesIn the operation that thing fully reacts under the conditions of existing for the second solvent, the modified cuorin and the N- hydroxysuccinimidesThe molar ratio of the biotin derivative of activation is 1：1.5~20.
In one embodiment, the modification cuorin and Streptavidin MagneSphere that the biotin derivative is combinedIn the operation for mixing and fully reacting, the concentration for the modification cuorin that the biotin derivative combines is 0.1mg/mL~1mg/ML, the concentration of the Streptavidin MagneSphere is 5mg/mL~15mg/mL.
This coated nanometer magnetic bead of modified cuorin is directly firmly spread out modified cuorin by chemical bond with biotinBiological firm connection, biotin derivative are connected with the Streptavidin on Streptavidin MagneSphere surface, inhaled relative to physicsAttached mode, more easily controls the modification cuorin amount of magnetic bead surfaces, and can be changed by the length of adjusting-R-, realizationThe control of the distance between property cuorin amount and nanometer magnetic bead, preferably remains modified cuorin amount and is combined with anti-phospholipid antibodySpace.This coated nanometer magnetic bead of modified cuorin is used directly for the detection of anti-phospholipid antibody, and relative to biographyDetection product prepared by the method for system physical absorption cuorin, has higher stability.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of the coated nanometer magnetic bead of modification cuorin of an embodiment.
Embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, it is below in conjunction with the accompanying drawings and specific realExample is applied to be described in detail the embodiment of the present invention.Elaborate in the following description many details in order toFully understand the present invention.But the invention can be embodied in many other ways as described herein, art technologyPersonnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is from following public specific implementationLimitation.
The coated nanometer magnetic bead of modification cuorin of one embodiment, including：Modified cuorin, biotin derivative and chainMould avidin magnetic bead.
Cuorin is the Ester being made of 3 glycerine, 2 phosphoric acid and 4 long-chain unsaturated alkyls, which containsThere are 2 hydrophilic centers and 4 hydrophobic side chains.The structural formula of cuorin is as follows：
Modified cuorin is obtained for the hydrophobic fat acid side chain of cuorin by oxidation and amination, and modified cuorin containsThere is-NH2。
Since cuorin has 4 side chains, 4 side chains can be aoxidized at the same time when cuorin is modified, therefore the modification heart phosphorusFat may include multiple-NH2.Specifically, which can include 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 atomsGlycol chains.
- NH in modified cuorin2So that will the modified heart with the-COOH generation-NH-CO- structures in biotin derivativePhosphatide is connected together with biotin derivative.
Preferably ,-R- is the saturated alkyl chain containing 4~10 carbon atoms.
Particularly preferred ,-R- is-(CH2)6-。
Since the size of Cardiolipin molecules is very small, the space wretched insufficiency that can be transformed, to hydrophilic phosphate centerThe compatibility of cuorin and anti-phospholipid antibody can be reduced by carrying out modification, or even antigen active is disappeared.
This coated nanometer magnetic bead of modified cuorin remains heart phosphorus by transforming cuorin hydrophobic side chainsThe modification cuorin at fat hydrophily phosphate center.
This coated nanometer magnetic bead of modified cuorin is directly firmly spread out modified cuorin by chemical bond with biotinBiological firm connection, biotin derivative are connected with the Streptavidin on Streptavidin MagneSphere surface, inhaled relative to physicsAttached mode, more easily controls the modification cuorin amount of magnetic bead surfaces, and can be changed by the length of adjusting-R-, realizationThe control of the distance between property cuorin amount and nanometer magnetic bead, preferably remains modified cuorin amount and is combined with anti-phospholipid antibodySpace.This coated nanometer magnetic bead of modified cuorin is used directly for the detection of anti-phospholipid antibody, and relative to biographyDetection product prepared by the method for system physical absorption cuorin, has higher stability.
Further, since each Streptavidin can combine closely with 4 biotins, this modified coated nanometer of cuorinMagnetic bead is made detected signal be exaggerated 4 times, is greatly improved anti-phosphatide and resisted by biotin-Streptavidin enlarge-effectThe detection sensitivity of body.
The preparation method of the above-mentioned modified coated nanometer magnetic bead of cuorin as shown in Figure 1, includes the following steps：
S10, fully react cuorin and peroxide under the conditions of existing for the first solvent, obtains 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 molar ratio of cuorin and peroxide is 1：1~8.
First solvent is dichloromethane, chloroform, chloroform, benzene or toluene.
S10 further includes the operation purified to intermediate product, and purifying can be by liquid phase system after ethyl acetate extractsStandby chromatogram purification.The modification cuorin of purity about 80% can be obtained after purification.
In S10, reaction temperature is 60 DEG C~100 DEG C.
Excessive ammonia is added in S20, the intermediate product obtained to S10 and is fully reacted, obtains modified cuorin.
Passing through peroxide and ammonium hydroxide so that the hydrophobic fat acid side chain of cuorin is aoxidized and amination, so thatObtain obtained modified cuorin and contain amino.
Since cuorin has 4 side chains, 4 side chains can be aoxidized at the same time when cuorin is modified, therefore the modification heart phosphorusFat may include multiple amino.Specifically, modified cuorin can include 1~8-NH2。
Preferably, modified cuorin contains-a NH2。
Particularly preferred, the structural formula of modified cuorin is as follows：
The mass concentration of ammonium hydroxide is 10%~30%.
In S20, reaction temperature is 60 DEG C~100 DEG C.
S30, exist the biotin derivative that modification cuorin and N- hydroxysuccinimides (NHS) that S20 is obtained activateFully reacted under the conditions of second solvent is existing, obtain the modification cuorin of biotin derivative combination.
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 fat2Derive with-COOH generation-NH-CO- the structures in biotin derivative so as to which cuorin and biotin will be modifiedThing connects together the modification cuorin that generation 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 esters, and biotin derivative NHS esters can be directIt is commercially available.
The phosphate buffer that in S30, the second solvent is DMSO, DMF, tetrahydrofuran or pH are 6.5~8.5.
In S30, the molar ratio of modified cuorin and the biotin derivative of N- hydroxysuccinimides activation is 1：1.5~20。
S40, the modification cuorin for the biotin derivative combination that S30 is obtained and Streptavidin MagneSphere mixing are simultaneously abundantReaction, obtains the coated nanometer magnetic bead of modified cuorin.
Streptavidin MagneSphere is the nanometer magnetic bead for combining Streptavidin, and biotin derivative and Streptavidin connectConnect.
Streptavidin MagneSphere can be directly to buy, for example, being purchased from MagnaBind companies (article No. xxxx).
In the operation that the modification cuorin and Streptavidin MagneSphere that biotin derivative is combined are mixed and fully reacted,The concentration for the modification cuorin that biotin derivative combines is 0.1mg/mL~1mg/mL, and the concentration of Streptavidin MagneSphere is5mg/mL~15mg/mL.
The preparation method of this coated nanometer magnetic bead of modified cuorin, it is firm will to be directly modified cuorin by chemical bondFirmly it is connected with biotin derivative, biotin derivative is connected with the Streptavidin on Streptavidin MagneSphere surface, phaseFor the mode of physical absorption, the modification cuorin amount of magnetic bead surfaces is more easily controlled, and the length of adjusting-R- can be passed throughDegree, realizes the control of the distance between modified cuorin amount and nanometer magnetic bead, preferably remains modified cuorin amount and anti-phosphorusThe space of fat antibody binding.The obtained coated nanometer magnetic bead of modified 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.
Further, since each Streptavidin can combine closely with 4 biotins, obtained modified cuorin is coated to be receivedRice magnetic bead makes detected signal be exaggerated 4 times, greatly improves anti-phosphatide by biotin-Streptavidin enlarge-effectThe detection sensitivity of antibody.
It is specific embodiment below.
Embodiment 1：The preparation of modified cuorin
Under argon gas protection, 0.5mmol cuorins are dissolved in 1mL dry toluenes, add the m-chloro peroxide benzene first of 1.5mmolAcid, is gradually heated up being warming up to 110 DEG C in stirring, and when maintaining the thermotonus 72 small.After reaction solution is cooled to room temperature, pour intoIn 25mL frozen water, then with 20mL ethyl acetate extraction solution 3 times, combined ethyl acetate.10mL is used by the ethyl acetate of merging is moltenAfter saturated nacl aqueous solution washs 1 time, when addition anhydrous sodium sulfate drying 12 is small.Ethyl acetate is evaporated under liquid negative pressure, it is remainingSolid is added into 8mL concentrated ammonia liquors, is gradually heated up being warming up to 100 degrees Celsius under stirring, and when maintaining the thermotonus 8 small.It is negativePressure is evaporated reaction solution, obtains yellow, viscous liquid, after being dissolved with a small amount of methanol, falls insoluble matter with 0.2 μm of membrane filtration,Solution is purified with preparative liquid chromatography, obtains about 83mg white solids.S(ESI+,m/z):1495.29113.
Embodiment 2：The preparation for the modification cuorin that biotin derivative combines
Weigh modified cuorin made from 50mg embodiments 1 to be dissolved in 2mL anhydrous DMFs, stir lower addition 20mg biotinsDerivative NHS esters, when stirring reaction 2.5 is small at room temperature.Reaction solution is evaporated under decompression, residual solid is molten with 5mL ethyl acetateSolution, filters insoluble matter, and the ethyl acetate dissolving of gained is evaporated, and what obtained white solid i.e. biotin derivative combined changesProperty cuorin.
In the present embodiment, the structural formula of biotin derivative is：- R- is-(CH2)6-。
The modification cuorin that biotin derivative manufactured in the present embodiment combines needs not move through especially purifying and can be used toSubsequent use.
Embodiment 3：It is coated with the preparation of the Streptavidin MagneSphere of modified cuorin
The phosphate buffer of the modification cuorin for the biotin derivative combination that 100 μ L concentration are 0.2mg/mL is taken, is addedEnter the Streptavidin MagneSphere that 100 μ L concentration are 10mg/mL, be incubated 10 minutes for 37 DEG C after mixing.1mL 30mM are used after Magneto separateTris buffer solutions redissolve, and redissolve Magneto separate again after the mixed liquor concussion of gained, and by the solid of gained 5mL 30mM TrisBuffer solution redissolves, and obtains the Streptavidin MagneSphere solution for being coated with modified cuorin of 0.02mg/mL.
Embodiment 4：The avidin magnetic bead and anti-phosphatide sample for being coated with modified cuorin are reacted
The avidin magnetic bead solution for being coated with modified cuorin prepared in 200 μ L embodiments 1~3 is taken respectively, adds 5 μ LAnti-phospholipid antibody serum sample is simultaneously incubated 30 minutes in 37 DEG C.The Streptavidin magnetic of modified cuorin will be coated with after Magneto separatePearl redissolves the secondary antibody for 200 μ L, adding horseradish peroxidase-labeled, 37 DEG C be incubated 30 minutes after clean successively, add TMB bottomsThing liquid is simultaneously incubated 10 minutes, is added 100 μ L terminate liquids, is read OD values in 10 minutes in microplate reader, obtain the luminous letter of sampleNumber value.
Three cuorin positive serum samples and three cuorin negative serum samples are taken respectively, and with traditional physicsAbsorption method is control, contrasts the OD values measured, obtains table 1 below.
Table 1：Embodiment 1~3 and control group (physisorphtion) measure the OD values of different samples
As can be seen from Table 1, the avidin magnetic bead and the physical absorption that are coated with modified cuorin that prepared by embodiment 1~3Luminous signal of the magnetic bead (control group) of method absorption when testing anti-phospholipid antibody sample compares, coating prepared by embodiment 1~3There is the avidin magnetic bead of the modified cuorin luminous signal value when measuring negative sample is significantly low compared with control group (to reduce 1 times~4Times), while the avidin magnetic bead for being coated with modified cuorin of the preparation of embodiment 1~3 luminous signal when measuring positive sampleHave compared with control group and greatly improve (improving 3~10 times).
Thus illustrate, prepared by embodiment 1~3 is coated with the avidin magnetic bead of modified cuorin in the anti-phosphatide sample of measurementWhen, relative to the magnetic bead of traditional physisorphtion, detection sensitivity, which has, to be obviously improved.
Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneouslyTherefore the limitation to the scope of the claims of the present invention cannot be interpreted as.It should be pointed out that for those of ordinary skill in the artFor, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present inventionProtect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.