CN103361064B - Preparation method of signal amplifying type quantum dot immune fluorescent probe and application of signal amplifying quantum dot immune fluorescent probe - Google Patents
Preparation method of signal amplifying type quantum dot immune fluorescent probe and application of signal amplifying quantum dot immune fluorescent probe Download PDFInfo
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Abstract
The invention belongs to technical field of immunoassay, and particularly relates to a preparation method of a signal amplifying type quantum dot immune fluorescent probe and an application of the signal amplifying type quantum dot immune fluorescent probe. At first, According to the method, a microemulsion solvent evaporation method is adopted for preparing a quantum dot-polymer composite nanosphere with controllable grain size and good dispersibility by utilizing quantum dot and polymer solution; and an antibody is covalently coupled on the surface of the composite nanosphere to obtain the signal amplifying type quantum dot immune fluorescent probe. The invention provides an application of the quantum dot immune fluorescent probe in a fluorescence immunoassay method or a fluorescence immunoassay detection kit. Compared with traditional quantum dot immune fluorescent probe, the quantum dot immune fluorescent probe prepared by the method provided by the invention has stronger signals and good detection sensitivity.
Description
Technical field
The invention belongs to immuno analytical method field, be specifically related to a kind of preparation method and application thereof of signal scale-up version quantum dot immune fluorescent probe.
Background technology
Immunoassay technology is that the principle based on specific reaction between antibody and antigen is carried out the detection method of qualitative and quantitative analysis to determinand, the immunodetection of lower concentration antigen is still the major issue that clinical diagnosis and inspection and quarantine field face, accurate, the reliable detection of lower concentration antigen can and be treated important scientific basis is provided in time for the early diagnosis of disease, and one of method that improves the immunodetection limit is the high-sensitive immunological probe of preparation.Fluoroimmunoassay technology is to utilize fluorescent substance traget antibody or antigen molecule, by detecting the Strength Changes of fluorescent signal after the specific binding with analysans, realizes the quantitative and qualitative analysis of target analytes is detected.
Quantum dot (quantum dot, be called for short QDs) nanocrystalline as a kind of novel fluorescence, there are some unique fluorescence properties, as controlled in fluorescent emission wavelength, the narrow symmetry of emission peak, excitation wavelength range is wide, quantum yield is high, good light stability etc., the above-mentioned fluorescent characteristic of quantum dot provides good selection for preparing highly sensitive immune fluorescent probe, at present about preparing quantum dot immune fluorescent probe, there is more report, but quantum dot immune fluorescent probe is when the material to very micro-detects, also exist signal low, the problems such as noise jamming is large are (referring to document: J.Lei, H.Ju, Signal amplification using functional nanomaterials for biosensing, Chem.Soc.Rev.2012, 41, 2122-2134).Therefore current quantum dot immune fluorescent probe preparation method is improved, improving the sensitivity detecting is the urgent demand of present analysis detection field.
Summary of the invention
The object of the invention is to overcome the problems such as current quantum dot immune fluorescent probe signal is low, detection sensitivity is low, a kind of preparation method of signal scale-up version quantum dot immune fluorescent probe is provided, and another object of the present invention is to provide quantum dot immune fluorescent probe prepared by aforesaid method and detects the application in (test kit) at fluorescence immunoassay.
For solving the problems of the technologies described above, technical scheme of the present invention is: first the present invention adopts microemulsion solvent volatilization method, with quantum dot and polymers soln, prepare quantum dot-composite nano-polymers ball, obtain the composite Nano ball of uniform particle diameter, good dispersity, then antibody covalent coupling is surperficial at composite Nano ball, obtain signal scale-up version quantum dot immune fluorescent probe.
Owing to comprising a plurality of quantum dots in quantum dot-composite nano-polymers ball, use its antagonist to carry out mark and be equivalent to a plurality of quantum dots with tense marker a part antibody, improved the ratio between quantum dot and antibody in immune fluorescent probe.Use this signal scale-up version immune fluorescent probe to carry out immunofluorescence detection, when micro-analysans is carried out to immunodetection, strengthened the fluorescent signal that immunofluorescence detects, the sensitivity that has improved immunodetection.
A first aspect of the present invention, is to provide a kind of preparation method of signal scale-up version quantum dot immune fluorescent probe, and the method comprises the following steps:
I. utilize microemulsion solvent to wave legal system for quantum dot-composite nano-polymers ball
The microemulsion solvent method of waving is ordinary method, (K.Landfester, Miniemulsion polymerization and the Structure of Polymer and Hybrid Nanoparticles, Angew.Chem.Int.Ed.2009,48,2-22; )
Described quantum dot is oil-soluble, particle diameter 1～15 nanometer, includes but not limited to: ZnSe, CdSe, CdTe, CdS, InP, CuInSe, CuInS, ZnSe/ZnS, CdSe/ZnS, CdS/ZnS, CdTe/ZnS, InP/ZnS, CdSe/ZnSe, CdSe/ZnSe, CdS/ZnSe, CdTe/ZnSe, InP/ZnSe, CdSe/CdS, CdTe/CdS, InP/CdS, CuInSe/ZnS, CuInS/ZnS, ZnSe/Zn
xcd
1-Xs, CdSe/Zn
xcd
1-Xs, CdS/Zn
xcd
1-Xs, CdTe/Zn
xcd
1-Xs, InP/ZnSe
xs
1-X, ZnSe/ZnSe
xs
1-X, CdSe/ZnSe
xs
1-X, CdS/ZnSe
xs
1-X, CdTe/ZnSe
xs
1-X, InP/ZnSe
xs
1-Xin at least one, 0<X<1 wherein;
Described polymkeric substance is the body material of composite Nano ball, contain simultaneously carboxyl functional group can with antibody coupling, include but not limited to: at least one in polystyrene-acrylic copolymer, polystyrene-maleic anhydride multipolymer, polymethylmethacrylate-Sipacril 2739OF;
Preferably, the oil phase component of preparing quantum dot-composite nano-polymers ball is:
Quantum dot, concentration is 0.1～5 μ mol/L;
Polymkeric substance, concentration is 0.05～5.0mg/mL;
Chloroformic solution, appropriate.
Preferably, the water component of preparing quantum dot-composite nano-polymers ball is: the aqueous solution of polyvinyl alcohol and sodium laurylsulfonate, wherein
Polyvinyl alcohol, concentration is 0.5～2.5%;
Sodium laurylsulfonate, concentration is 0.1～0.5%.
Preparing quantum dot-composite nano-polymers ball is W/O microemulsion.
II. antibody covalent coupling is surperficial at quantum dot-composite nano-polymers ball
Covalent coupling is ordinary method, (Y.Xing, S.Nie, et al, Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry, Nat.Protoc.2007,2,1152)
Described antibody, is for a structural domain of different albumen or albumen or the monoclonal antibody of one section of polypeptide, or polyclonal antibody.
The preparation method of signal scale-up version quantum dot immune fluorescent probe of the present invention, comprises following concrete steps:
I. utilize microemulsion solvent to wave legal system for quantum dot-composite nano-polymers ball
A, oil phase component: by quantum dot and polymer dispersed, in chloroformic solution, by quantum dot and polymer dispersed chloroformic solution, the concentration of quantum dot is 0.1～5 μ mol/L, and the concentration of polymkeric substance is 0.05～5.0mg/mL;
B, water component: polyvinyl alcohol and sodium laurylsulfonate are configured to the aqueous solution, and wherein the concentration of polyvinyl alcohol is 0.5～2.5%, and the concentration of sodium laurylsulfonate is 0.1～0.5%;
In C, 4 ℃ of water-baths, under magnetic agitation, oil-phase solution is added in aqueous phase solution, continue to stir 10～60 minutes, then use Ultrasonic Cell Disruptor that solution is carried out to supersound process 1～10 minute, obtain uniform and stable microemulsion.
Under D, room temperature, microemulsion is placed in to open container, under magnetic agitation, solvent is volatilized gradually, the volatilization time is 6～48 hours, centrifuge washing, the dry mixture nanometer ball that obtains 30～200nm;
II. antibody covalent coupling is surperficial at quantum dot-composite nano-polymers ball
A, composite Nano ball are dispersed in the phosphate buffered saline buffer of pH4.0～6.0, nanometer ball concentration 0.5～5mg/mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyl sulfo-succinimide (sulfo-NHS), concentration 0.1～1.0mmol/L, the two mol ratio is 1/1, hatches 30 minutes under room temperature;
B, in the nanometer ball solution after activation, add antibody, the concentration of antibody is 50～500 μ g/mL, then under room temperature, hatches after 1 hour, adds bovine serum albumin, and concentration is 1～10mg/mL, continues to hatch 2h under room temperature;
C, the reaction product obtaining, turn under the rotating speed of per minute 5000～20000, and centrifugal 0.5～2 hour, discard after centrifugal supernatant, precipitation is dispersed in phosphate buffered saline buffer, obtain signal scale-up version quantum dot immune fluorescent probe.
A second aspect of the present invention, is to provide quantum dot immune fluorescent probe prepared by the aforesaid method application in fluorescence immunoassay detection method or fluorescence immunoassay detection kit.
Utilize signal scale-up version quantum dot immune fluorescent probe prepared by the present invention to carry out fluorescence immunoassay while detecting, first the determined antigen of a series of concentration known is fixed in polyvinylidene difluoride (PVDF) or cellulose acetate membrane, the composite Nano ball of antibody and the antigen on film carry out specific binding to have made mark, then measure the fluorescence intensity of quantum dot on film, set up the standard corresponding relation between quantum dot fluorescence intensity and antigen concentration.
Relation on the film that above process records between the fluorescence intensity of quantum dot and antigen amount is the important foundation data in the present invention, can determine the immunodetection limit of probe by the method, and compares with traditional quantum dot immune fluorescent probe.
In above-mentioned application, film immune response detectable antigens comprises the following steps:
On A, the film after infiltrating, put upper antigenic solution spot, dry under room temperature, then film is infiltrated in 1～5% skim-milk solution, seal 1～6 hour;
B, the fluorescent probe that the present invention is prepared are dispersed in 1～5% skim-milk solution, concentration and probe concentration 1～100 μ g/mL, and the film after sealing is placed in one, and hatches 1～6h;
C, hatch after, by film rinsing 3 times in washing lotion, washing lotion, for the phosphate buffered saline buffer (pH7.4) containing 0.5～0.2% tween 20, is then developed film under UV-irradiation, takes pictures;
D, change antigenic solution concentration, above-mentioned B, C step, measure the fluorescence intensity of the corresponding quantum dot of a series of antigen concentrations, sets up the relation of fluorescence intensity and antigen amount.
Afterwards, then the fluorescence intensity of the quantum dot obtaining while detecting with actual sample therewith standard corresponding relation contrast the antigen concentration that can determine in actual sample.
Compared with prior art, the present invention has the following advantages:
1, the present invention adopts solvent evaporation method to prepare quantum dot-composite nano-polymers ball, equipment and process is simple, the composite Nano ball uniform particle diameter, the condition that prepare are controlled, reproducible, and in nanometer ball, quantum dot concentration is easy to control, simultaneously less on the fluorescence property impact of quantum dot.
2, the present invention uses the immune response of spot film to verify the immunocompetence of immune fluorescent probe, and the method is easy fast, and repeatability is high, the convenient quality control to immune fluorescent probe.
3, the composite Nano ball fluorescent probe that the present invention prepares is stronger than traditional quantum dot immune fluorescent probe signal, noise jamming is little, detection sensitivity is high.
Accompanying drawing explanation
Fig. 1 is the projection electromicroscopic photograph of quantum dot-composite nano-polymers ball of preparing;
Fig. 2 is the immunoreactive spot figure of spot film,
The quantum dot immune fluorescent probe that wherein (a) is prior art, (b) is signal scale-up version quantum dot immune fluorescent probe, carries out respectively the immunoreactive spot figure of spot film, the amount of albumen on numeral spot on figure, and unit is " nanogram ".
Embodiment
Now in conjunction with the embodiments and accompanying drawing, the invention will be further described, but enforcement of the present invention is not limited in this.
Following embodiment if no special instructions method therefor is ordinary method.
Reagent and the instrument in embodiment, used are as follows: CdSe, CdSe/ZnS quantum dot is purchased from Sigma-Aldrich company, CuInS quantum dot is purchased from Ocean Nanotech company, polystyrene-acrylic copolymer, polystyrene-maleic anhydride multipolymer and polymethylmethacrylate-Sipacril 2739OF are Sigma-Aldrich company, polyvinyl alcohol is traditional Chinese medicines reagent company, sodium laurylsulfonate is traditional Chinese medicines reagent company, EDC and sulfo-NHS Wei Sai Mo Feishier company, hepatitis b surface antigen antibody Wei Ye people biotech firm, bovine serum albumin and the sheep anti-mouse igg work biotech firm of making a living, Q700 Ultrasonic Cell Disruptor is Qsonica company.
Embodiment 1: the preparation of quantum dot-composite nano-polymers ball
The preparation of CdSe/ZnS quantum dot-composite nano-polymers ball, comprises the following steps:
A, oil phase component: CdSe/ZnS quantum dot and polystyrene-maleic anhydride multipolymer are dispersed in chloroformic solution, and the concentration of quantum dot is 0.1 μ mol/L, and the concentration of polymkeric substance is 0.05mg/mL;
B, water component: polyvinyl alcohol and sodium laurylsulfonate are configured to the aqueous solution, and wherein the concentration of polyvinyl alcohol is 2.5%, and the concentration of sodium laurylsulfonate is 0.5%;
In C, 4 ℃ of water-baths, under magnetic agitation, oil-phase solution is added in aqueous phase solution, continue to stir 60 minutes, then use Ultrasonic Cell Disruptor to carry out supersound process 10 minutes to solution, obtain uniform and stable microemulsion.
Under D, room temperature, microemulsion is placed in to open container, under magnetic agitation, solvent is volatilized gradually, the volatilization time is 6 hours, and centrifuge washing obtains composite Nano ball after being dried;
(2) antibody and composite Nano ball surface covalent coupling comprise the following steps:
A, composite Nano ball are dispersed in the phosphate buffered saline buffer of pH4.0, nanometer ball concentration 0.5mg/mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyl sulfo-succinimide (sulfo-NHS), concentration 0.1mmol/L, the two mol ratio is 1/1, hatches 30 minutes under room temperature.
B, in the nanometer ball solution after activation, add anti-hepatitis b surface antigen antibody, the concentration of antibody is 5 μ g/mL, then under room temperature, hatches after 1 hour, adds bovine serum albumin, and concentration is 50mg/mL, continues to hatch 2h under room temperature.
C, the reaction product obtaining, turn under the rotating speed of per minute 20000, and centrifugal 2 hours, discard after centrifugal supernatant, precipitation is dispersed in phosphate buffered saline buffer, obtain signal scale-up version quantum dot immune fluorescent probe.
The projection electromicroscopic photograph (* 25000 times) of the quantum dot-composite nano-polymers ball preparing, as shown in Figure 1, the quantum dot-composite nano-polymers ball uniform particle diameter (average 74.2 nanometers), the polydispersity coefficient 0.096 that obtain.
Embodiment 2: the preparation of quantum dot-composite nano-polymers ball
A, oil phase component: CdSe quantum dot and polystyrene-acrylic copolymer are dispersed in chloroformic solution, and the concentration of quantum dot is 2.5 μ mol/L, and the concentration of polymkeric substance is 1.0mg/mL;
B, water component: polyvinyl alcohol and sodium laurylsulfonate are configured to the aqueous solution, and wherein the concentration of polyvinyl alcohol is 0.5%, and the concentration of sodium laurylsulfonate is 0.25%;
In C, 4 ℃ of water-baths, under magnetic agitation, oil-phase solution is added in aqueous phase solution, continue to stir 10 minutes, then use Ultrasonic Cell Disruptor to carry out supersound process 3 minutes to solution, obtain uniform and stable microemulsion.
Under D, room temperature, microemulsion is placed in to open container, under magnetic agitation, solvent is volatilized gradually, the volatilization time is 24 hours, and centrifuge washing obtains mixture nanometer ball after being dried;
(2) antibody and composite Nano ball surface covalent coupling comprise the following steps:
A, composite Nano ball are dispersed in the phosphate buffered saline buffer of pH6.0, nanometer ball concentration 2.5mg/mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyl sulfo-succinimide (sulfo-NHS), concentration 0.5mmol/L, the two mol ratio is 1/1, hatches 30 minutes under room temperature.
B, in the nanometer ball solution after activation, add sheep anti-mouse igg, the concentration of antibody is 25 μ g/mL, then under room temperature, hatches after 1 hour, adds bovine serum albumin, and concentration is 1mg/mL, continues to hatch 2h under room temperature.
C, the reaction product obtaining, turn under the rotating speed of per minute 10000, and centrifugal 1 hour, discard after centrifugal supernatant, precipitation is dispersed in phosphate buffered saline buffer, obtain signal scale-up version quantum dot immune fluorescent probe.
Embodiment 3: the preparation of quantum dot-composite nano-polymers ball
A, oil phase component: CuInS quantum dot and polymethylmethacrylate-Sipacril 2739OF are dispersed in chloroformic solution, and the concentration of quantum dot is 0.1 μ mol/L, and the concentration of polymkeric substance is 0.05mg/mL;
B, water component: polyvinyl alcohol and sodium laurylsulfonate are configured to the aqueous solution, and wherein the concentration of polyvinyl alcohol is 2.5%, and the concentration of sodium laurylsulfonate is 0.5%;
In C, 4 ℃ of water-baths, under magnetic agitation, oil-phase solution is added in aqueous phase solution, continue to stir 60 minutes, then use Ultrasonic Cell Disruptor to carry out supersound process 10 minutes to solution, obtain uniform and stable microemulsion.
Under D, room temperature, microemulsion is placed in to open container, under magnetic agitation, solvent is volatilized gradually, the volatilization time is 6 hours, and centrifuge washing obtains mixture nanometer ball after being dried;
(2) antibody and composite Nano ball surface covalent coupling comprise the following steps:
A, composite Nano ball are dispersed in the phosphate buffered saline buffer of pH4.0, nanometer ball concentration 0.5mg/mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyl sulfo-succinimide (sulfo-NHS), concentration 0.1mmol/L, the two mol ratio is 1/1, hatches 30 minutes under room temperature.
B, in the nanometer ball solution after activation, add anti-hepatitis b surface antigen antibody, the concentration of antibody is 5 μ g/mL, then under room temperature, hatches after 1 hour, adds bovine serum albumin, and concentration is 50mg/mL, continues to hatch 2h under room temperature.
C, the reaction product obtaining, turn under the rotating speed of per minute 20000, and centrifugal 2 hours, discard after centrifugal supernatant, precipitation is dispersed in phosphate buffered saline buffer, obtain signal scale-up version quantum dot immune fluorescent probe.
Embodiment 4: the preparation of quantum dot-composite nano-polymers ball
All the other are with embodiment 1, A, oil phase component: quantum dot and polystyrene-acrylic copolymer are dispersed in chloroformic solution, and the concentration of quantum dot is 5 μ mol/L, and the concentration of polymkeric substance is 2.5mg/mL.
Embodiment 5: the preparation of quantum dot-composite nano-polymers ball
All the other are with embodiment 2, A, oil phase component: quantum dot and polymethylmethacrylate-Sipacril 2739OF are dispersed in chloroformic solution, and the concentration of quantum dot is 1 μ mol/L, and the concentration of polymkeric substance is 3mg/mL.
Embodiment 6: the application of signal scale-up version quantum dot immune fluorescent probe in spot film immunodetection
The signal scale-up version quantum dot immune fluorescent probe preparing with embodiment 1, is applied in spot film immunodetection, and concrete steps are as follows:
On A, the inclined to one side fluorine polyethylene film after infiltrating, put upper hepatitis B surface antigen solution spot, under room temperature, dry 1h, then infiltrates film in 5% skim-milk solution, seals 6 hours.
B, fluorescent probe is dispersed in 5% skim-milk solution, concentration and probe concentration 1 μ g/mL, the film after sealing is placed in probe dispersion liquid, hatches 6h.
C, hatch after, by film rinsing 3 times in washing lotion, washing lotion, for the phosphate buffered saline buffer (pH7.4) containing 0.2% tween 20, is then developed film under UV-irradiation, takes pictures.
D, change antigenic solution concentration, measure the fluorescence intensity of the corresponding quantum dot of a series of antigen concentrations, light intensity can be used Quantity One computed in software, sets up the relation of fluorescence intensity and antigen amount.
Embodiment 7: the spot film immunodetection comparison of different immune fluorescent probes
On A, the inclined to one side fluorine polyethylene film after infiltrating, put upper hepatitis B surface antigen solution spot, under room temperature, dry 1h, then infiltrates film in 5% skim-milk solution, seals 6 hours.
B, fluorescent probe is dispersed in 5% skim-milk solution, concentration and probe concentration 1 μ g/mL, the film after sealing is placed in probe dispersion liquid, hatches 6h.
C, hatch after, by film rinsing 3 times in washing lotion, washing lotion, for the phosphate buffered saline buffer (pH7.4) containing 0.2% tween 20, is then developed film under UV-irradiation, takes pictures.
D, change antigenic solution concentration, measure the fluorescence intensity of the corresponding quantum dot of a series of antigen concentrations, light intensity can be used Quantity One computed in software, sets up the relation (1.25～0.039ng) of fluorescence intensity and antigen amount.
The hepatitis B surface antibody substitution signal scale-up version quantum dot immune fluorescent probe of water-soluble CdSe/ZnS quantum dot (purchased from Invitrogen company) mark of E, use surface band carboxyl, repeat B, C, D, the two result more as shown in Figure 2, the quantum dot immune fluorescent probe of prior art can only detect the protein spots (as shown in Fig. 2 (a)) of 0.156ng, and the signal scale-up version quantum dot immune fluorescent probe preparing by the inventive method can detect the protein spots (as shown in Fig. 2 (b)) of 0.078ng.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (5)
1. a preparation method for signal scale-up version quantum dot immune fluorescent probe, the method comprises the following steps:
I. utilize microemulsion solvent volatilization method to prepare quantum dot-composite nano-polymers ball;
A, oil phase component: by quantum dot and polymer dispersed, in chloroformic solution, the concentration of quantum dot is 0.1～5 μ mol/L, and the concentration of polymkeric substance is 0.05～5.0mg/mL;
B, water component: polyvinyl alcohol and sodium laurylsulfonate are configured to the aqueous solution, and wherein the concentration of polyvinyl alcohol is 0.5～2.5%, and the concentration of sodium laurylsulfonate is 0.1～0.5%;
In C, 4 ℃ of water-baths, under magnetic agitation, oil-phase solution is added in aqueous phase solution, continue to stir 10～60 minutes, then use Ultrasonic Cell Disruptor that mixed solution is carried out to supersound process 1～10 minute, obtain uniform and stable microemulsion;
Under D, room temperature, microemulsion is placed in to open container, under magnetic agitation, solvent is volatilized gradually, the volatilization time is 6～48 hours, centrifuge washing, the dry mixture nanometer ball that obtains 30～200nm; II. antibody covalent coupling is surperficial at quantum dot-composite nano-polymers ball;
Described quantum dot is oil-soluble, particle diameter 1～15 nanometer, comprising: ZnSe, CdSe, CdTe, CdS, InP, CuInSe, CuInS, ZnSe/ZnS, CdSe/ZnS, CdS/ZnS, CdTe/ZnS, InP/ZnS, CdSe/ZnSe, CdSe/ZnSe, CdS/ZnSe, CdTe/ZnSe, InP/ZnSe, CdSe/CdS, CdTe/CdS, InP/CdS, CuInSe/ZnS, CuInS/ZnS, ZnSe/Zn
xcd
1-Xs, CdSe/Zn
xcd
1-Xs, CdS/Zn
xcd
1-Xs, CdTe/Zn
xcd
1-Xs, InP/ZnSe
xs
1-X, ZnSe/ZnSe
xs
1-X, CdSe/ZnSe
xs
1-X, CdS/ZnSe
xs
1-X, CdTe/ZnSe
xs
1-X, InP/ZnSe
xs
1-Xin at least one, 0<X<1 wherein;
Described polymkeric substance comprises: at least one in polystyrene-acrylic copolymer, polystyrene-maleic anhydride multipolymer, polymethylmethacrylate-Sipacril 2739OF.
2. the preparation method of a kind of signal scale-up version quantum dot immune fluorescent probe according to claim 1, is characterized in that,
II. antibody covalent coupling is surperficial at quantum dot-composite nano-polymers ball
A, composite Nano ball are dispersed in the phosphate buffered saline buffer of pH4.0～6.0, nanometer ball concentration 0.5～5mg/mL, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyl sulfo-succinimide (sulfo-NHS), concentration 0.1～1.0mmol/L, the two mol ratio is 1/1, hatches 30 minutes under room temperature;
B, in the nanometer ball solution after activation, add antibody, the concentration of antibody is 50～500 μ g/mL, then under room temperature, hatches after 1 hour, adds bovine serum albumin, and concentration is 1～10mg/mL, continues to hatch 2h under room temperature;
C, the reaction product obtaining, turn under the rotating speed of per minute 5000～20000, and centrifugal 0.5～2 hour, discard after centrifugal supernatant, precipitation is dispersed in phosphate buffered saline buffer, obtain signal scale-up version quantum dot immune fluorescent probe.
3. the preparation method of a kind of signal scale-up version quantum dot immune fluorescent probe according to claim 1 and 2, is characterized in that, described antibody is monoclonal antibody or polyclonal antibody.
4. serve as a mark in fluorescence immunoassay detection method or the fluorescence immunoassay detection kit application of probe of the quantum dot immune fluorescent probe that preparation method as claimed in claim 1 prepares.
5. application according to claim 4, it is characterized in that, this application is that the signal scale-up version quantum dot immune fluorescent probe that utilizes described preparation method as arbitrary in claim 1 to prepare is while carrying out fluorescence immunoassay detection, first the determined antigen of a series of concentration known is fixed in polyvinylidene difluoride (PVDF) or cellulose acetate membrane, the composite Nano ball of antibody and the antigen on film carry out specific binding to have made mark, then measure the fluorescence intensity of quantum dot on film, set up the standard corresponding relation between quantum dot fluorescence intensity and antigen concentration; Afterwards, then the fluorescence intensity of the quantum dot obtaining while detecting with actual sample therewith standard corresponding relation contrast the antigen concentration that can determine in actual sample.
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| CN114891508B (en) * | 2022-05-07 | 2023-03-03 | 河南大学 | Water-soluble InP core-shell quantum dot with high brightness and stability and synthesis method and application thereof |
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