CN104914243A - Magnetic nanoparticle biological probe, preparation method and application thereof - Google Patents
Magnetic nanoparticle biological probe, preparation method and application thereof Download PDFInfo
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- CN104914243A CN104914243A CN201510269714.7A CN201510269714A CN104914243A CN 104914243 A CN104914243 A CN 104914243A CN 201510269714 A CN201510269714 A CN 201510269714A CN 104914243 A CN104914243 A CN 104914243A
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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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
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- 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
Abstract
The invention discloses a magnetic nanoparticle biological probe, a preparation method and application thereof, in particular to a magnetic nanoparticle biological probe for detecting HBV preS1 antigen, a magnetic immunochromatography test paper strip, a preparation method and a use method thereof. According to the invention, magnetic nanoparticles with carboxyl modified surfaces are adopted as the carrier, the antibody of HBV preS1 is connected to the magnetic nanoparticles' surfaces through EDC and HBV to establish the preparation method of the magnetic nanoparticle biological probe for detecting the HBV preS1 antigen, and the biological probe is adopted as the marker and the antibody of the targeted marker is taken as the capture antibody to establish a sandwich type immunochromatography detection system. The magnetic nanoparticle biological probe has strong specificity, and has good signal sensitivity and stability. The magnetic immunochromatography test paper strip provided by the invetnion on the one hand can realize rapid qualitative detection of the HBV preS1 antigen through visual inspection, and on the other hand can realize quantitative detection of the HBV preS1 antigen through the quantitative magnetic signal of the captured probe.
Description
Technical field
The invention belongs to biotechnology and technical field of nano material, being specifically related to a kind of magnetic nanoparticle bioprobe, magnetic immuno-chromatographic test paper strip and preparation method thereof for detecting HBV preS1 antigen and using method.
Background technology
Hepatitis B (HBV) is the height morbidity of China, and in population of China, infection rate reaches 60%, and in ascendant trend year by year, HBsAg carrier has more than 1.3 hundred million, and more than 10% transfers chronic hepatitis to, and part develops into cirrhosis, and then causes liver cancer.Therefore, the accurate measurements after whether hepatitis B being had in vivo to the accurate judgement and antiviral therapy copied just seems particular importance.Apply the dynamic change of five indexes of hepatitis b serological index and pattern thereof clinically, the state of an illness of monitoring, monitoring HBV infection person, judge the infectiousness of the infected and evaluate the result for the treatment of of antiviral drugs.Phase late 1980s, along with appearance and the maturation of round pcr, HBV-DNA has quantitatively become the goldstandard of diagnosis of hepatitis b, but round pcr is due to the high standard of its requirement of experiment and high cost, makes its utilization and extention greatly limited.
Research in recent years both at home and abroad shows, before HBV, S1 (preS1) antigen is the another new mark that virus exists and copies, and Pre-S1Ag appears at the very early time of acute HBV infection, and with HbcAg (HBeAg) and HBV DNA significant correlation.Pre S 1 antigen and HBV-DNA recall rate meet, and pre S 1 antigen only detects in HBV-DNA positive serum.Pre-s1 protein disappears with HBeAg and disappears, and turns the time with the moon and be proportionate, and like this, pre S 1 antigen can be used as the index that virus sweep is turned out cloudy with virus.The hepatitis B patient propagation hepatitis type B virus of pre S 1 antigen positive danger that is more negative than pre S 1 antigen and asymptomatic HbsAg carrier is larger, thus illustrates that pre S 1 antigen can reflect hepatitis B replication and communicable index.If pre S 1 antigen lasting masculin, AHB is to chronic transformation in instruction.Compare pre-s1 protein in the patients serum of acute hepatitis B, chronic hepatitis B and the HBsAg positive, pre S 1 antigen the moon turns more early, and the course for the treatment of of AHB patient is shorter, and prognosis also better.Illustrate that the detection of pre S 1 antigen and antibody thereof is the clinical diagnosis of acute hepatitis B, observation of curative effect and the good index sentencing data prognosis.
All the time, in China detection HBV employing is that traditional ELISA detects, and because ELISA testing cost is low, charge low, patient's burden is relatively little.So be difficult to the hospital being comprehensively generalized to each department for employing chemiluminescence and electrochemiluminescence detect, relative to traditional ELISA method detection HBV although chemiluminescence and electrochemiluminescence detect there is obvious detection sensitivity and detect linear advantage, for competition chemiluminescence and the time-resolved fluorescence of same item, also there is equipment more stable, the advantage that result is more stable, but operation steps and detection time do not simplify too many compared with conventional ELISA method, testing cost is expensive, also higher to the technical requirement of clinical examination doctor, limit it to promote the use of clinical.
Magnetic Nano sonde method immuno-chromatographic assay technology is the ultimate principle using for reference immune colloidal gold chromatography technology ripe at present, colloid gold particle is replaced to utilize antibody with magnetic Nano material, the sandwich reaction of antigen in test strips, realize the detection technique of magnetic Nano probe mark hepatitis B pre S 1 antigen, both current immune colloidal gold chromatography technology difficult quantitation had been overcome, the deficiency that detection sensitivity is low, have again fast, sensitive, high specific, and quantitative detection can be realized, it is a kind of Fast Detection Technique being suitable for Site Detection, there is good development trend and application prospect.
Magnetic Nano material is the ferriferous oxide (mostly being tri-iron tetroxide) with superparamagnetism is kernel, take monox as shell, when this iron oxide particle volume is reduced to a certain numerical value, thermal perturbation can by suitable with total magnetocrystalline anisotropy energy, like this, intragranular magnetic moment direction just may As time goes on, and entirety repeatedly changes in parallel between a direction of easy axis and another direction of easy axis.From single domain particle aggregate, just there is superparamagnetism.Occur in the temperature range of superparamagnetism at single domain particle aggregate, measure its magnetization curve at different temperature respectively, these magnetization curves must coincide together, and there will not be magnetic hysteresis, and namely the remanent magnetism of aggregate and coercive force are all zero.This superparamagnetism is also the basic guarantee of the biomedical applications of magnetic Nano material.
Can accomplish that Viral Quantification detects because magnetic Nano sonde method detects HBV preS1 antigen, therefore clinician can according to the PD of the mutation analysis patient of the amount of HBV virus, thus better know the rational use of medicines of clinician, save patient to the expense for the treatment of hepatitis B, especially long-term monitoring effect is had more to chronic hepatitis B.The sensitivity of certain Yin Qigao, magnetic Nano sonde method is made to detect HBV preS1 antigen, the verification and measurement ratio of hepatitis B can be improved, make the window phase of detection in advance, prevent the false negative of ELISA method and undetected, so magnetic Nano sonde method detects the detection that HBV preS1 antigen can also be widely used in health check-up.
Summary of the invention
The existence of hepatitis B well can not be reflected in order to overcome " HBV"liang dui ban" " reagent in prior art, quantitatively can not detect HBV, and HBV-DNA quantitative PCR detection experimental cost is high, the defect of length consuming time, the invention provides a kind of magnetic nanoparticle bioprobe for detecting HBV preS1 antigen, magnetic immuno-chromatographic test paper strip and preparation method thereof and using method, its magnetic nanoparticle bioprobe has high specificity, sensitive and the feature of good stability of signal, its magnetic immuno-chromatographic test paper strip can realize detecting with quantitative the fast qualitative of the clear middle HBV preS1 antigen of human peripheral.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A preparation method for magnetic nanoparticle bioprobe, specifically comprises the following steps:
Step 1) carries out surface amination process to the magnetic nanoparticle that particle diameter is 100-200nm, obtains the magnetic nanoparticle of surface amination; Again carboxylated process is carried out to the magnetic nanoparticle of described surface amination, obtain the magnetic nanoparticle of surface carboxyl groups; Step 2) in damping fluid by the magnetic nanoparticle of described surface carboxyl groups, carbodiimides (EDC, also known as carbodiimide) and N-hydroxy-succinamide (NHS) hybrid reaction, reaction after washing, obtain reactant A;
Step 3) by the antibody hybrid reaction of described reactant A and HBV preS1, obtains reactant B in coupling buffer;
Step 4), by described reactant B and the solution hybrid reaction containing amino compound, reacts rear washing, i.e. obtained magnetic nanoparticle bioprobe.
Further, in step 1), described magnetic nanoparticle can be magnetic nanoparticle conventional in field of nanometer material technology; Described magnetic nanoparticle has superparamagnetism; The specific saturation magnetization of described magnetic nanoparticle is preferably at 25-45emu/g; Described magnetic nanoparticle is preferably for having the magnetic particle/silica composite material of nucleocapsid structure, that is: the kernel of described magnetic nanoparticle is magnetic particle, and the shell of described magnetic nanoparticle is monox; Described magnetic particle is preferably ferriferous oxide, as tri-iron tetroxide.The thickness of the shell of described magnetic particle/silica composite material is preferably 5-50nm.
Wherein, described magnetic particle can be prepared by existing document, such as: Magnetite Nanocrystal Clusters with Ultra-High Sensitivity in Magnetic Resonance Imagin(Fangjie Xu, Changming Cheng, Du-Xing Chen, Hongchen Gu, Chemphyschem, 2012,13,336-341).Described magnetic particle/silica composite material can be prepared by existing document, such as: Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry(Xiuqing Xu, Chunhui Deng, Mingxia Gao, Wenjia Yu, Pengyuan Yang, and Xiangmin Zhang, Advanced Materials, 2006, 18, 3289-3293.).
Further, described magnetic nanoparticle preferably adopts following methods to obtain:
(1) magnetic particle is prepared with solvent-thermal method:
Inorganic molysite, stabilizing agent and ethylene glycol are mixed, at 180-220 DEG C, react 8-24 hour, washing, obtains magnetic particle; Described stabilizing agent is sodium citrate or polyacrylic acid (PAA);
Wherein, described washing is preferably washs repeatedly at assist lower ethanol and/or the deionized water of Magneto separate; Wherein, described solvent-thermal method is the method preparing magnetic particle of this area routine; By this area general knowledge, described reaction is carried out under weak basic condition, when described stabilizing agent is polyacrylic acid, preferably also adds urea to control the pH value of reaction system when described mixing;
(2) monox bag is by magnetic particle:
By the mixing of the strong acid solution of above-mentioned obtained magnetic particle and 0.8-1.2mol/L, ultrasonic 10-40 minute, Magneto separate auxiliary under spend deionized water 5-7 time, obtain the magnetic particle after activation; By the magnetic particle after described activation and alcohol-water mixture mixing, alkali and tetraalkyl orthosilicate is added under stirring, the alkali added makes the pH value mixing rear system at 8.0-10.5, reaction 8-24 hour, Magneto separate auxiliary under with alcohol washing 3-5 time, namely obtain described magnetic nanoparticle;
Wherein, the consumption of described tetraalkyl orthosilicate is magnetic particle described in 20-500 μ L/100mg; Alcohol water ratio in described alcohol-water mixture is 60:40-90:10; Described strong acid is the acid that this area routine uses, and is generally hydrochloric acid; Wherein, described alkali is the alkali that this area routine uses, and is preferably NH3H2O; Alcohol in described alcohol-water mixture can be any alcohol that this area routine uses; Described alcohol can be the alcohol that this area routine uses, and is preferably ethanol.By this area general knowledge, described tetraalkyl orthosilicate can divide many batches of interpolations, such as, can add part in initial reaction stage, after reaction a period of time, add remainder.
Further, in step 1), the method for described surface amination process and condition are method and the condition of this area routine, and described surface amination process preferably adopts silane coupling agent to carry out.
Described surface amination process is preferably carried out according to following operation:
Mixed with alcohol-water mixture by described magnetic nanoparticle, adjust ph, to 8.0-11.0, obtains mixed liquor C; Again described mixed liquor C is mixed with aminopropyl triethoxysilane (APTES), the consumption of described aminopropyl triethoxysilane is magnetic nanoparticle described in 15-50 μ L/15mg, stir after 1-3 hour, continue to stir 2-4h at 60-85 DEG C, after washing, namely obtain the magnetic nanoparticle of surface amination.
Wherein, described alcohol-water mixture can be the alcohol-water mixture that this area routine uses, and the alcohol content in described alcohol-water mixture is preferably 60-90v%.Alcohol in described alcohol-water mixture is preferably ethanol.Described adjust ph preferably adopts ammoniacal liquor to carry out.
Further, in step 1), the method for described surface carboxyl groups process and condition are method and the condition of this area routine, and described surface carboxyl groups process preferably adopts acid anhydrides to carry out.
Described surface carboxyl groups process is preferably carried out according to following operation:
The magnetic nanoparticle of described surface amination, succinic anhydride and solvent are mixed, the magnetic nanoparticle that the consumption of described succinic anhydride is surface amination described in 20-60mg/100mg, described solvent is N, dinethylformamide and/or dimethyl sulfoxide, reaction 12-36 hour, after reaction, washing, obtains the magnetic nanoparticle of surface carboxyl groups.
Wherein, the carboxyl-content in the magnetic nanoparticle of described surface carboxyl groups is preferably more than 0.35mmol/g, is more preferably 0.35-5.0mmol/g.
Further, step 2) in, preferably can also with buffer solution 2-3 time before carrying out the mixing of the magnetic nanoparticle of above-mentioned described surface amination, succinic anhydride and solvent, make the magnetic nanoparticle of described surface carboxyl groups resuspended with damping fluid again, the pH value of described resuspended rear system is 4.5-5.5; Described resuspended method and condition are method and the condition of this area routine, are generally to make particle form colloidal solution in a liquid.Described damping fluid can be the damping fluid that this area routine uses, and the pH value of described damping fluid is preferably 4.5-5.5, and described damping fluid is preferably the 2-(N-morpholino containing 0.05-1.5v% tween) ethyl sulfonic acid solution (being called for short MEST solution).
Further, step 2) in, the consumption of described carbodiimides (EDC) is preferably the magnetic nanoparticle of surface carboxyl groups described in 1.2-1.4 μm of ol/1mg, is more preferably the magnetic nanoparticle of surface carboxyl groups described in 1.25 μm of ol/1mg.The consumption of described N-hydroxy-succinamide (NHS) is preferably the magnetic nanoparticle of surface carboxyl groups described in 1.2-1.4 μm of ol/1mg, is more preferably the magnetic nanoparticle of surface carboxyl groups described in 1.25 μm of ol/1mg.The mol ratio of described EDC and described NHS is preferably 1:0.9-0.9:1.
Further, step 2) in, described reaction is as the criterion to react completely.The time of described reaction is preferably 30-60min.
Further, step 2) in, after the reaction, the method of described washing and condition are method and the condition of this area routine, described washing is preferably undertaken by following operation: the 2-(N-morpholino of Tween-20 with containing 0.05-1.5v%) after ethyl sulfonic acid solution (MEST) washs 2 times, then use borate tween solution (being called for short BST solution) to wash 2 times.Wherein, the pH value of described MEST solution is preferably 4.5-5.5.
Further, in step 3), described antibody can be monoclonal antibody (abbreviation monoclonal antibody) and/or polyclonal antibody (being called for short how anti-).According to this area general knowledge, the antibody consumption of described HBV preS1 is saturated or excessive relative to described reactant A.
Further, in step 3), described coupling buffer can be the coupling buffer that this area routine uses.The pH value of described coupling buffer is preferably 8.8-9.2, is more preferably 9.0.Described coupling buffer is preferably borate tween solution (BST solution).
Further, in step 3), described reaction is as the criterion to react completely, and the time of described reaction is preferably 2-4 hour.
Further, in step 4), the described solution containing amino compound can be the conventional solution containing amino material used in field of biology, is preferably one or more in bovine serum albumin solution (BSA solution), Tris damping fluid, glycine solution and polypeptide solution.
Further, in step 4), described reaction is as the criterion to react completely, and the time of described reaction is preferably 30-60min.
Further, in step 4), the method for described washing and condition are method and the condition of this area routine, and described washing is preferably undertaken by following operation: wash 2-4 time with borate tween molten (BST solution).
Present invention also offers a kind of magnetic nanoparticle bioprobe for detecting HBV preS1 antigen obtained by above-mentioned preparation method, it is the magnetic nanoparticle that a kind of surface is connected with HBV preS1 antibody.
Wherein, described magnetic nanoparticle bioprobe needs resuspended in conserving liquid, stores at 2-8 DEG C.Described conserving liquid generally can adopt the cleansing solution used in described step 4).
Present invention also offers a kind of application of described magnetic nanoparticle bioprobe, it is applied in the magnetic immuno-chromatographic test paper strip for detecting HBV preS1 antigen, described magnetic immuno-chromatographic test paper strip comprises a sample pad, one pad, one chromatographic film, one adsorptive pads and a base plate, described pad is coated with described magnetic nanoparticle bioprobe, described chromatographic film there are a detection line (being called for short T line) and a nature controlling line (being called for short C line), described sample pad, described pad, described chromatographic film and described adsorptive pads are connected successively on described base plate, described T line is positioned at the side near described pad, described C line is positioned at the side near described adsorptive pads, described T line contains the antibody of HBV surface antigen antibody or preS1, described C line contains IgG bis-and resists, the antibody of the HBV surface antigen that the HBV preS1 antibody on described magnetic nanoparticle bioprobe and described T line contain or preS1 be different antibody.
Wherein, the content of described magnetic nanoparticle bioprobe is preferably 4-12 μ g.
Wherein, described chromatographic film can be the chromatographic film that this area routine uses, and is preferably nitrocellulose filter.
Wherein, the antibody of described HBV preS1 can be the antibody of the HBV preS1 antigen that field of biology routine uses.The antibody of described HBV preS1 can be monoclonal antibody and/or resist more; According to this area general knowledge, when the antibody of the HBV surface antigen that the HBV preS1 antibody on described magnetic nanoparticle bioprobe and described T line contain or preS1 is monoclonal antibody, these two kinds of monoclonal antibodies are not identical, and it should be the monoclonal antibody of the different epitopes having specific reaction for HBV.
Wherein, described IgG bis-resists the IgG bis-used for this area routine to resist, and is preferably that sheep anti-mouse igg two is anti-or rabbit anti-mouse igg two is anti-.
Wherein, described linking is the linking on the conventional meaning of this area, by this area general knowledge, and described sample pad and described pad, described pad and described chromatographic film, and the joining place of described chromatographic film and described adsorptive pads generally has, and about 1mm's is superimposed.
By this area general knowledge, at regular intervals between described T line and described C line, described spacing range is the spacing range of this area routine, generally at about 0.8cm.
The specification of the magnetic immuno-chromatographic test paper strip for detecting HBV preS1 antigen of the present invention can be specification conventional in magnetic immuno-chromatographic field, can be applicable to magnetic analytical meter (MAR) and carry out Card Reader and analyze.The width of the described magnetic immuno-chromatographic test paper strip for HBV detection is generally at about 0.5cm.
Present invention also offers a kind of preparation method of described magnetic immuno-chromatographic test paper strip, it comprises the following steps: be sprayed on described pad by described magnetic nanoparticle bioprobe, the antibody of described HBV surface antigen antibody or preS1 is fixed on described T line, be fixed on described C line by anti-for described IgG bis-, by described sample pad, described pad, described chromatographic film and described adsorptive pads are engaged on described base plate successively, and make described T line be positioned at the side of close described pad, described C line is made to be positioned at the side of close described adsorptive pads, obtain described magnetic immuno-chromatographic test paper strip.
Wherein, the consumption of described magnetic nanoparticle bioprobe is preferably 4-12 μ g.
Wherein, described magnetic nanoparticle bioprobe preferably also carries out resuspended step before spraying.The described resuspended chromatography buffer that preferably adopts carries out.Described chromatography buffer can be the chromatography buffer that this area routine uses, preferably for containing the phosphate buffer of the sucrose of Tween-20 and 0.02-0.2wt% of the borate buffer solution of the sucrose of Tween-20 and 0.02-0.2wt% of BSA, 0.05-0.5v% of 0.1-1wt% or BSA, the 0.05-0.5v% containing 0.1-1wt%.The pH value of described chromatography buffer is preferably 6.5-9.0.
Wherein, the method for described specking and condition can be method and the condition of this area routine.
Wherein, described fixing method and condition can be method and the condition of this area routine, and general available point film instrument carries out.Described HBV preS1 antibody or the fixing of surface antigen antibody are preferably undertaken by following operation: the solution specking of the described antibody being 0.5-2mg/mL with the speed of 0.75-0.85 μ L/cm by concentration with some film instrument is on described T line.Anti-fixing of described IgG bis-is preferably undertaken by following operation: with some film instrument with the anti-solution specking of the speed of the 0.75-0.85 μ L/cm described IgG bis-that is 1-2mg/mL by concentration on described C line.
Wherein, described linking is the linking on the conventional meaning of this area, by this area general knowledge, and described sample pad and described pad, described pad and described chromatographic film, and the joining place of described chromatographic film and described adsorptive pads generally has, and about 1mm's is superimposed.
In the present invention, obtained magnetic immuno-chromatographic test paper strip according to actual conditions, can carry out cutting when follow-up use, carries out Card Reader analysis to be applicable to magnetic analytical meter (MAR).
Present invention also offers a kind of using method of described magnetic immuno-chromatographic test paper strip, it comprises the following steps: added in described sample pad place by sample, after leaving standstill 5-15min, observes the color at described T line and described C line place; After standing 35min, detect the magnetic signal value of described magnetic immuno-chromatographic test paper strip at described T line and described C line place with magnetic analytical meter.
Wherein, described magnetic analytical meter (MAR) can be the magnetic analytical meter that this area routine uses.
By this area general knowledge, the change of the color at described T line and described C line place and criterion as follows: with visual inspection, if T line and C line place have the band of two obvious bands or T line to be shallower than C lines band, then sample is the positive; If only a band appears in C line place, T line place is without band, then sample is negative.If C line place is without band during detection, then test strips system has problem, and it is invalid that its testing result is considered as.
In the present invention, the quantitative magnetic signal of the sample recorded at described T line 3 place and a negative sample are compared at the quantitative magnetic signal at described T line 3 place, also can evaluate testing result, its result of sample of cloudy value >=2.1 of T sample/T is considered as the positive, and its result of sample of the cloudy value < 2.1 of T sample/T is considered as feminine gender.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Compared with prior art, the invention has the beneficial effects as follows:
The present invention has the magnetic nanoparticle of carboxyl for carrier with finishing, by EDC and NHS, the antibody of HBV preS1 is connected to the surperficial magnetic nanoparticle bioprobe preparation method established for detecting HBV preS1 antigen of magnetic nanoparticle.And with this bioprobe for label, with the antibody of target label for capture antibody, establish sandwich formula immunochromatography detection system.Magnetic nanoparticle bioprobe high specificity of the present invention, the sensitive and good stability of signal.The fast qualitative that magnetic immuno-chromatographic test paper strip of the present invention realizes HBV preS1 antigen by visual inspection on the one hand detects, and is realized the quantitative detection of HBV preS1 antigen on the other hand by the quantitative magnetic signal of institute's capture probe.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of instructions, coordinates accompanying drawing to describe in detail below with preferred embodiment of the present invention.The specific embodiment of the present invention is provided in detail by following examples and accompanying drawing thereof.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, and form a application's part, schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 represents the TEM photo of magnetic nanoparticle of the present invention;
Fig. 2 represents the specific saturation magnetization curve map of magnetic nanoparticle of the present invention;
Fig. 3 represents the structural representation of magnetic immuno-chromatographic test paper strip of the present invention.
Embodiment
Below with reference to the accompanying drawings and in conjunction with the embodiments, describe the present invention in detail.
In following embodiment, the particle diameter of magnetic nanoparticle that is obtained or that adopt is all in the scope of 100-200nm.
embodiment 1magnetic nanoparticle
The preparation method of magnetic nanoparticle, it comprises the steps:
(1) magnetic particle is prepared with solvent-thermal method: be dissolved in 40ml ethylene glycol respectively by 0.85g FeCl36H2O and 0.4033g PAA, add urea 1.2g again to dissolve, add in the stainless steel cauldron of band tetrafluoroethene inner bag after mixing, screw reactor, add thermal response 16h at 220 DEG C after, under Magneto separate is auxiliary, respectively washs 3 times with ethanol and deionized water, obtain the magnetic particle that mean grain size is 90nm;
(2) monox bag is by magnetic particle: mixed by the hydrochloric acid solution of 90mg magnetic particle and 1.0mol/L, ultrasonic 30 minutes, under Magneto separate is auxiliary, spend deionized water 5 times, the alcohol-water mixture 125mL being then 70:30 by magnetic particle and volume ratio of alcohol to water mixes, and under agitation adds ethyl orthosilicate 200 μ L and ammoniacal liquor 2.1mL, react 18 hours, Magneto separate auxiliary under wash 3 times, vacuum drying with ethanol, to obtain final product.
Those magnetic nanoparticles are the magnetic particle/silica composite material with nucleocapsid structure, and its kernel is magnetic particle, and its shell is monox.Shown in Figure 1, Fig. 1 is the TEM photo of magnetic nanoparticle, and its mean grain size is 100nm, shown in Figure 2, and the specific saturation magnetization of magnetic nanoparticle is 29.2emu/g.
embodiment 2magnetic nanoparticle
The preparation method of magnetic nanoparticle, it comprises the steps:
(1) magnetic particle is prepared with solvent-thermal method: be dissolved in 30ml ethylene glycol respectively by 0.81g FeCl36H2O and 2.66g sodium citrate, add in the stainless steel cauldron of band tetrafluoroethene inner bag after mixing, screw reactor, add thermal response 24h at 200 DEG C after, under Magneto separate is auxiliary, respectively wash 3 times with ethanol and deionized water, obtain the magnetic particle that mean grain size is 250nm;
(2) monox bag is by magnetic particle: mixed by the hydrochloric acid solution of 110mg magnetic particle and 1.0mol/L, ultrasonic 15 minutes, under Magneto separate is auxiliary, spend deionized water 7 times, then the alcohol-water mixture 125mL being 90:10 by magnetic particle and volume ratio of alcohol to water mixes, under agitation add ethyl orthosilicate 100 μ L and ammoniacal liquor 2.1mL, react 12 hours, and then add the ethyl orthosilicate of 80 μ L, continue reaction 12 hours, Magneto separate auxiliary under wash 3 times, vacuum drying with ethanol, to obtain final product.
These magnetic nanoparticles are the magnetic particle/silica composite material with nucleocapsid structure, and its kernel is magnetic particle, and its shell is monox.The mean grain size of those magnetic nanoparticles is 280nm, and specific saturation magnetization is 42emu/g, and the average thickness of monox shell is 15nm.
embodiment 3magnetic nanoparticle bioprobe
The preparation method of magnetic nanoparticle bioprobe, it comprises the steps:
Step 1) adds the magnetic nanoparticle obtained by 15mg embodiment 1 or 2 in the 50g volume ratio of alcohol to water ethanol water that is 70:30, add 50 μ L ammoniacal liquor and 35 μ L aminopropyl triethoxysilanes, 2h is stirred under normal temperature, continue to stir 3h under 70 DEG C of water bath condition, with ethanol washing, obtain the magnetic nanoparticle of surface amination; The magnetic nanoparticle of surface amination is mixed with dimethyl sulfoxide (DMSO), add 5.5mg succinic anhydride reaction 24h, 3 times are washed with DMSO under Magneto separate is auxiliary, spend deionized water again 3 times, obtain the magnetic nanoparticle of surface carboxyl groups, the carboxyl-content of the magnetic nanoparticle of those surface carboxyl groups is 0.9-1.5mmol/g;
Step 2) (in this MEST solution, contain the Tween-20 of 0.25v% with the MEST solution of 500 μ L, pH is 5.0-5.5) wash the magnetic nanoparticle of 2mg surface carboxyl groups, this washing is in particular: the magnetic nanoparticle of this MEST solution and surface carboxyl groups is placed in 2mL centrifuge tube, vortex oscillator mixes, then Magneto separate; After washing 2 times, the NHS solution 10 μ L of EDC solution 5 μ L and 0.25mol/L of the magnetic nanoparticle of surface carboxyl groups, 0.5mol/L is mixed, reaction 30min, with MEST solution (component is the same) washing 2 times, wash 2 times with the BST solution (containing the Tween-20 of 0.07v% in this BST solution) that pH value is 9.0 again, obtain reactant A;
Step 3), in BST solution (component is the same), by the mixing of the specific monoclonal antibody of the HBV pre S 1 antigen of reactant A and 150 μ g, is reacted 3h under room temperature, is obtained reactant B;
Step 4), by BSA solution capping 30min under room temperature of the 0.5 ~ 1mg/mL of reactant B and 500 μ L, with BST solution (component is the same) washing 3 times after reaction, to obtain final product.
Obtained magnetic nanoparticle bioprobe conserving liquid is resuspended, stores for future use at 4 DEG C.
embodiment 4magnetic immuno-chromatographic test paper strip
Shown in Figure 3, Fig. 3 is the structural representation of magnetic immuno-chromatographic test paper strip of the present invention, this magnetic immuno-chromatographic test paper strip comprises sample pad 1, pad 2, chromatographic film 7, adsorptive pads 6 and a base plate 5, pad 2 is coated with the magnetic nanoparticle bioprobe obtained by embodiment 3, chromatographic film 7 there is T line 3 and a C line 4, sample pad 1, pad 2, chromatographic film 7 and adsorptive pads 6 are connected successively on base plate 5, T line 3 is positioned at the side near pad 2, C line 4 is positioned at the side near adsorptive pads 6, and T line 3 and C line 4 are at a distance of 0.8cm; The specific monoclonal antibody of T line 3 containing HBsAg, C line 4 is containing goat anti-rabbit IgG antibody, and chromatographic film 7 is nitrocellulose filter.
The preparation method of described magnetic immuno-chromatographic test paper strip, it comprises the steps: the chromatography buffer of the magnetic nanoparticle bioprobe conserving liquid 2 times of volumes obtained by embodiment 3 to carry out resuspended, this chromatography buffer is the borate buffer solution of the sucrose of Tween-20 and 0.02-0.2wt% of BSA, 0.05-0.5wt% containing 0.1-1wt%, and the pH value of this chromatography buffer is 6.5-9.0; Get 5 μ L resuspended after suspending liquid specking on pad 2, the content of the magnetic nanoparticle bioprobe in this suspending liquid is 5 μ g; With the speed of 0.8 μ L/cm, the specific monoclonal antibody of the HBsAg of 1mg/mL is fixed on the T line 3 of chromatographic film 7 with a film instrument, with a film instrument, the goat anti-rabbit IgG antibody of 2mg/mL is fixed on the C line 4 of chromatographic film 7, this chromatographic film 7 is nitrocellulose filter, and T line 3 and C line 4 are at a distance of 0.8cm; Sample pad 1, pad 2, chromatographic film 7 and adsorptive pads 6 are connected successively on base plate 5, sample pad 1 and pad 2, pad 2 and chromatographic film 7, the joining place of chromatographic film 7 and adsorptive pads 6 has the superimposed T line 3 of about 1mm to be positioned at the side of close pad 2, C line 4 is positioned at the side near adsorptive pads 6, to obtain final product.
This magnetic immuno-chromatographic test paper strip according to actual conditions, can carry out cutting in use, as wide in cut out to 0.5cm, carries out Card Reader analysis to be applicable to magnetic analytical meter (MAR).
The using method of this magnetic immuno-chromatographic test paper strip, it comprises the steps: sample to add in sample pad 1 place, after leaving standstill 5-15min, observes the color at T line 3 and C line 4 place; After standing 35min, detect the magnetic signal value of described magnetic immuno-chromatographic test paper strip at T line 3 and C line 4 place with magnetic analytical meter.
T line 3 and C line 4 place color change and criterion as follows: with visual inspection, if T line and C line place have the band of two obvious bands or T line to be shallower than C lines band, then sample be the positive; If only a band appears in C line place, T line place is without band, then sample is negative.If C line place is without band during detection, then test strips system has problem, and it is invalid that its testing result is considered as.
In addition, the quantitative magnetic signal of the sample recorded at T line 3 place and a negative sample are compared at the quantitative magnetic signal at T line 3 place, also can evaluate testing result, its result of sample of cloudy value >=2.1 of T sample/T is considered as the positive, and its result of sample of the cloudy value < 2.1 of T sample/T is considered as feminine gender.
Adopt this magnetic immuno-chromatographic test paper strip to detect a serum sample X, recording it at the magnetic signal at T line place is 234; Detect another serum sample Y, recording it at the magnetic signal at T line place is 89.Detect a hepatitis B negative serum sample, recording it at the magnetic signal at T line place is 47.Calculate according to quantitative detected value, then T sample X/T the moon value=4.97, this value is greater than 2.1, and thus this serum sample X is positive; And T sample Y/T the moon value=1.89, this value is less than 2.1, and thus this serum sample Y is negative.
embodiment 5magnetic nanoparticle bioprobe
Magnetic particle in this embodiment presses document, as document 1.: Magnetite Nanocrystal Clusters with Ultra-High Sensitivity in Magnetic Resonance Imagin(Fangjie Xu, Changming Cheng, Du-Xing Chen, Hongchen Gu, Chemphyschem, 2012,13,336-341) be prepared.Magnetic nanoparticle in this embodiment presses document, as document 2.: Synthesis of Magnetic Microspheres with Immobilized Metal Ions for Enrichment and Direct Determination of Phosphopeptides by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry(Xiuqing Xu, Chunhui Deng, Mingxia Gao, Wenjia Yu, Pengyuan Yang, and Xiangmin Zhang, Advanced Materials, 2006, 18, 3289-3293.) be prepared.The particle diameter of obtained magnetic nanoparticle is all in the scope of 50-300nm.Those magnetic nanoparticles are the magnetic particle/silica composite material with nucleocapsid structure, and its kernel is magnetic particle, and shell is monox.
The preparation method of magnetic nanoparticle bioprobe, it comprises the steps:
Step 1) 50g volume ratio of alcohol to water be add in the alcohol-water mixture of 70:30 magnetic nanoparticle that 15mg mean grain size is 150nm (preparation method see document 1. with document 2., the average thickness of the monox shell of those magnetic nanoparticles is 10nm, specific saturation magnetization is 36emu/g), add 50 μ L ammoniacal liquor and 35 μ L aminopropyl triethoxysilanes, 1h is stirred under normal temperature, continue to stir 2h under 70 DEG C of water bath condition, with ethanol washing, obtain the magnetic nanoparticle of surface amination; By the magnetic nanoparticle of surface amination and N, dinethylformamide (DMF) mixes, add 7.0mg succinic anhydride reaction 24h, 3 times are washed with DMF under Magneto separate is auxiliary, spend deionized water again 3 times, obtain the magnetic nanoparticle of surface carboxyl groups, the carboxyl-content of the magnetic nanoparticle of this surface carboxyl groups is about 1.5mmol/g;
Step 2) (in this MEST solution, contain the Tween-20 of 0.05v% with the MEST solution of 500 μ L, pH is 5.0-5.5) wash the magnetic nanoparticle of 2mg surface carboxyl groups, this washing is in particular: the magnetic nanoparticle of this MEST solution and surface carboxyl groups is placed in 2mL centrifuge tube, vortex oscillator mixes, then Magneto separate; After washing 2 times, the NHS solution 10 μ L of EDC solution 5 μ L and 0.25mol/L of the magnetic nanoparticle of surface carboxyl groups, 0.5mol/L is mixed, reaction 30min, with MEST solution (component is the same) washing 2 times, wash 2 times with the BST solution (containing the Tween-20 of 0.1v% in this BST solution) that pH value is 9.0 again, obtain reactant A;
Step 3), in BST solution (component is the same), by the mixing of the specific monoclonal antibody of the HBV preS1 of reactant A and 150 μ g, is reacted 3h under room temperature, is obtained reactant B;
Step 4), by BSA solution capping 30min under room temperature of the 1mg/mL% of reactant B and 500 μ L, with BST solution (component is the same) washing 3 times after reaction, to obtain final product.
Obtained magnetic nanoparticle bioprobe conserving liquid is resuspended, stores for future use at 4 DEG C.
embodiment 6magnetic immuno-chromatographic kit
Shown in Figure 3, Fig. 3 represents the structural representation of this magnetic immuno-chromatographic test paper strip, and the structure of this magnetic immuno-chromatographic test paper strip is with embodiment 4, and difference is magnetic nanoparticle bioprobe pad 2 is coated with obtained by embodiment 5.
The preparation method of this magnetic immuno-chromatographic test paper strip is with embodiment 4, and difference is to use the magnetic nanoparticle bioprobe obtained by embodiment 5 to be sprayed on pad 2.
This magnetic immuno-chromatographic test paper strip according to actual conditions, can carry out cutting in use, as wide in cut out to 0.5cm, carries out Card Reader analysis to be applicable to magnetic analytical meter (MAR).
The using method of this magnetic immuno-chromatographic test paper strip and determination methods are with embodiment 4.
effect example 1
The magnetic immuno-chromatographic test paper strip of embodiment 4 and embodiment 6 is adopted to detect testing sample.Sample to be tested is added in sample pad 1 place, after leaving standstill 5-15min, observes the color at T line 3 and C line 4 place; After standing 35min, the magnetic signal value of magnetic immuno-chromatographic test paper strip at T line 3 place for detecting HBV preS1 antigen described in detecting with magnetic analytical meter, is the magnetic signal value in table 1.Meanwhile, tested sample is the OD value in table 1 with the light absorption value after conventional ELISA method detects.To detect negative sample used be known is hepatitis B negative patient serum, and positive serum is the patients serum having confirmed as the hepatitis B positive.Usually, the quantitative detection gained magnetic signal of sample positive and to be detected and the ratio of negative sample and S/N value, be T
sample/ T
cloudy value, therefore the sample of S/N value > 2.1 is positive.
Ginseng is shown in Table 1, and table 1 is adopt magnetic immuno-chromatographic test paper strip of the present invention and ELISA method to the testing result of different sample.As can be seen from Table 1, adopt magnetic immuno-chromatographic test paper strip of the present invention for detection HBV preS1 antigen, the coefficient of variation of its measurement result is low, and error is little.
Table 1 adopts magnetic immuno-chromatographic test paper strip of the present invention and ELISA method to the testing result of different sample
effect example 2
Detection method is with effect example 1.
Ginseng is shown in Table 2, and table 2 is adopt magnetic immuno-chromatographic test paper strip of the present invention and ELISA method to the graded series testing result of hepatitis B positive serum standard items.Wherein, positive serum standard items 1-8 is respectively concentration hepatitis B positive serum standard items from high to low.As can be seen from Table 2, the measurement result of its measurement result and ELISA method matches, its R2=0.9834, and linear relationship is good, illustrates that the magnetic immuno-chromatographic test paper strip detection accuracy for detecting HBV preS1 antigen of the present invention is high, high specificity.According to table 2 data, adopt magnetic immuno-chromatographic test paper strip of the present invention to the S/N value > 2.1 of the quantitative testing result of HBV preS1 antigen positive standard items 8.According to the foregoing T of the present invention
sample/ T
cloudy valuedecision method, this testing result is positive.
From the quantitative testing result of table 2, although visual inspection is less than detection line, its quantitative testing result is still effective.As can be seen here, magnetic immuno-chromatographic test paper strip of the present invention is adopted to detect HBV preS1 antigen, highly sensitive, and effectively can avoid the false negative phenomenon that occurs because of simple visual inspection.
Table 2 magnetic immuno-chromatographic test paper strip and ELISA method
To the testing result of different gradient HBV positive serum standard model
effect example 3
Detection method is with effect example 1.
Table 3 is for detecting the detection stability of the magnetic immuno-chromatographic test paper strip of HBV preS1 antigen
Ginseng is shown in Table 3, and table 3 is adopt the estimation of stability experimental result of magnetic immuno-chromatographic test paper strip to human peripheral blood serum sample for detecting HBV preS1 antigen of the present invention.Positive serum standard items 1-3 in table 3 is respectively concentration HBV preS1 antigen positive serum standard panel from high to low, the magnetic signal mean value at test strips T line place when the average 1-3 of T is respectively detecting for the 1st day, the 2nd day and the 3rd day of the sample adopted in the magnetic immuno-chromatographic test paper strip his-and-hers watches for detecting HBV preS1 antigen of the present invention.As shown in Table 3, the maximum rate of change of the magnetic signal detected for 3 times is 11.9%, being less than 15%, illustrating that the detection stability of the magnetic immuno-chromatographic test paper strip for detecting HBV preS1 antigen of the present invention is better.
Above-described embodiment, just in order to technical conceive of the present invention and feature are described, its objective is and is one of ordinary skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.The change of every equivalence done by the essence of content of the present invention or modification, all should be encompassed in protection scope of the present invention.
Claims (10)
1. a preparation method for magnetic nanoparticle bioprobe, is characterized in that, comprises the steps:
Step 1) carries out surface amination process to the magnetic nanoparticle that particle diameter is 100-200nm, obtains the magnetic nanoparticle of surface amination; Again carboxylated process is carried out to the magnetic nanoparticle of described surface amination, obtain the magnetic nanoparticle of surface carboxyl groups;
Step 2) in damping fluid by the magnetic nanoparticle of described surface carboxyl groups, carbodiimides and N-hydroxy-succinamide hybrid reaction, reaction after washing, obtain reactant A;
Step 3) by the antibody hybrid reaction of described reactant A and HBV preS1, obtains reactant B in coupling buffer;
Step 4), by described reactant B and the solution hybrid reaction containing amino compound, reacts rear washing, i.e. obtained magnetic nanoparticle bioprobe.
2. the preparation method of magnetic nanoparticle bioprobe according to claim 1, is characterized in that,
In step 1), the specific saturation magnetization of described magnetic nanoparticle is at 25-45emu/g;
And/or in step 1), described magnetic nanoparticle is the magnetic particle/silica composite material with nucleocapsid structure, and the kernel of described magnetic nanoparticle is magnetic particle, and the shell of described magnetic nanoparticle is monox;
And/or in step 1), the carboxyl-content in the magnetic nanoparticle of described surface carboxyl groups is more than 0.35mmol/g;
And/or in step 1), described magnetic nanoparticle adopts following methods to obtain:
Magnetic particle is prepared with solvent-thermal method:
Inorganic molysite, urea, stabilizing agent and ethylene glycol are mixed, at 180-220 DEG C, react 8-24 hour, washing, obtains magnetic particle; Described stabilizing agent is sodium citrate or polyacrylic acid;
Monox bag is by magnetic particle:
By the mixing of the strong acid solution of above-mentioned obtained magnetic particle and 0.8-1.2mol/L, ultrasonic 10-40 minute, Magneto separate auxiliary under spend deionized water 5-7 time, obtain the magnetic particle after activation; By the magnetic particle after described activation and alcohol-water mixture mixing, alkali and tetraalkyl orthosilicate is added under stirring, the alkali added makes the pH value mixing rear system at 8.0-10.5, reaction 8-24 hour, Magneto separate auxiliary under with alcohol washing 3-5 time, namely obtain described magnetic nanoparticle; Wherein, the consumption of described tetraalkyl orthosilicate is magnetic particle described in 20-500 μ L/100mg, and the alcohol water ratio in described alcohol-water mixture is 60:40-90:10.
3. the preparation method of magnetic nanoparticle bioprobe according to claim 2, is characterized in that,
In step 1), described magnetic particle is ferriferous oxide; The thickness of the shell of described magnetic particle/silica composite material is 5-50nm;
And/or in step 1), the carboxyl-content in the magnetic nanoparticle of described surface carboxyl groups is 0.35-5.0mmol/g.
4. the preparation method of magnetic nanoparticle bioprobe according to claim 1, is characterized in that,
In step 1), described surface amination process adopts silane coupling agent to carry out;
And/or in step 1), described surface carboxyl groups process adopts acid anhydrides to carry out;
And/or, step 2) in, before described mixing, also use buffer solution 2-3 time, then make the magnetic nanoparticle of described surface carboxyl groups resuspended with damping fluid;
And/or, step 2) in, the pH value of described damping fluid is 4.5-5.5;
And/or, step 2) in, described damping fluid is the 2-(N-morpholino containing 0.05-1.5v% tween) ethyl sulfonic acid solution;
And/or, step 2) in, the magnetic nanoparticle that the consumption of described carbodiimides is surface carboxyl groups described in 1.2-1.4 μm of ol/1mg, the magnetic nanoparticle that the consumption of described N-hydroxy-succinamide is surface carboxyl groups described in 1.2-1.4 μm of ol/1mg;
And/or, step 2) in, the time of described reaction is 30-60min;
And/or, step 2) in, after the reaction, described washing is undertaken by following operation: the 2-(N-morpholino of Tween-20 with containing 0.05-1.5v%) after ethyl sulfonic acid solution washing 2 times, then wash 2 times with borate tween solution;
And/or in step 3), described antibody is monoclonal antibody and/or resists more;
And/or in step 3), the pH value of described coupling buffer is 8.8-9.2;
And/or in step 3), described coupling buffer is borate tween solution;
And/or in step 3), the time of described reaction is 2-4 hour;
And/or in step 4), the described solution containing amino compound is one or more in bovine serum albumin solution, Tris damping fluid, glycine solution and polypeptide solution;
And/or in step 4), the time of described reaction is 30-60min;
And/or in step 4), described washing is undertaken by following operation: with borate tween solution washing 2-4 time.
5. the preparation method of magnetic nanoparticle bioprobe according to claim 4, is characterized in that,
In step 1), described surface amination process is carried out according to following operation:
Mixed with alcohol-water mixture by described magnetic nanoparticle, adjust ph, to 8.0-11.0, obtains mixed liquor C; Again described mixed liquor C is mixed with aminopropyl triethoxysilane, the consumption of described aminopropyl triethoxysilane is magnetic nanoparticle described in 15-50 μ L/15mg, stir after 1-3 hour, continue to stir 2-4h at 60-85 DEG C, after washing, namely obtain the magnetic nanoparticle of surface amination;
And/or in step 1), described surface carboxyl groups process is carried out according to following operation:
The magnetic nanoparticle of described surface amination, succinic anhydride and solvent are mixed, the magnetic nanoparticle that the consumption of described succinic anhydride is surface amination described in 20-60mg/100mg, described solvent is N, dinethylformamide and/or dimethyl sulfoxide, reaction 12-36 hour, after reaction, washing, obtains the magnetic nanoparticle of surface carboxyl groups;
And/or, step 2) in, the mol ratio of described carbodiimides and described N-hydroxy-succinamide is 1:0.9-0.9:1;
And/or, step 2) in, before described mixing, described damping fluid during described washing is the 2-(N-morpholino containing 0.05-1.5v% tween) ethyl sulfonic acid solution, the pH value of described resuspended rear system is 4.5-5.5;
And/or, step 2) in, after described reaction, the 2-(N-morpholino of the described Tween-20 containing 0.05-1.5v% during described washing) pH value of ethyl sulfonic acid solution is 4.5-5.5.
6. the magnetic nanoparticle bioprobe obtained by the preparation method in claim 1-5 described in any one.
7. the application of a magnetic nanoparticle bioprobe as claimed in claim 6 in the magnetic immuno-chromatographic test paper strip for detecting HBV preS1 antigen, described magnetic immuno-chromatographic test paper strip comprises a sample pad (1), one pad (2), one chromatographic film (7), one adsorptive pads (6) and a base plate (5), described pad (2) is coated with described magnetic nanoparticle bioprobe, described chromatographic film (7) there are a detection line (3) and a nature controlling line (4), described sample pad (1), described pad (2), described chromatographic film (7) and described adsorptive pads (6) are connected successively on described base plate (5), described detection line (3) is positioned at the side near described pad (2), described nature controlling line (4) is positioned at the side near described adsorptive pads (6), described detection line (3) is containing HBV preS1 antibody or surface antigen antibody, described nature controlling line (4) resists containing IgG bis-, the HBV preS1 antibody that the antibody of the HBV preS1 on described magnetic nanoparticle bioprobe and described detection line (3) contain or surface antigen antibody are different monoclonal antibodies.
8. magnetic immuno-chromatographic test paper strip according to claim 7, is characterized in that, the content of described magnetic nanoparticle bioprobe is 4-12 μ g;
And/or described chromatographic film (7) is nitrocellulose filter;
Described HBV preS1 antibody or surface antigen antibody are monoclonal antibodies and/or resist more.
9. a preparation method for magnetic immuno-chromatographic test paper strip as claimed in claim 7 or 8, is characterized in that, comprises the following steps:
Described magnetic nanoparticle bioprobe is sprayed on described pad (2), the antibody of described HBV label is fixed on described detection line (3), be fixed on described nature controlling line (4) by anti-for described IgG bis-, by described sample pad (1), described pad (2), described chromatographic film (7) and described adsorptive pads (6) are engaged on described base plate (2) successively, and make described detection line (3) be positioned at the side of close described pad (2), described nature controlling line (4) is made to be positioned at the side of close described adsorptive pads (6), obtain described magnetic immuno-chromatographic test paper strip.
10. a using method for magnetic immuno-chromatographic test paper strip as claimed in claim 7 or 8, is characterized in that, comprises the following steps:
Sample is added in described sample pad place, after leaving standstill 5-15min, observes the color at described detection line and described nature controlling line place, can qualitative detection be carried out; After standing 35min, detect the magnetic signal value of described magnetic immuno-chromatographic test paper strip at described detection line and described nature controlling line place with magnetic analytical meter, can quantitatively detect.
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