CN106526192A - Quantum dot-antibody fluorescent probe, preparation method, probe and test paper strip - Google Patents
Quantum dot-antibody fluorescent probe, preparation method, probe and test paper strip Download PDFInfo
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- G—PHYSICS
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
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Abstract
The invention provides a quantum dot-antibody fluorescent probe preparation method, a quantum dot-antibody fluorescent probe, and an immunochromatography test paper strip and a preparation method thereof. The quantum dot-antibody fluorescent probe preparation method comprises: (1) adding quantum dots to a borate buffer solution to prepare a quantum dot solution; (2) adding an activator to the quantum dot solution, and activating, wherein the activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a hydrochloride thereof and N-hydroxysuccinimide or a sulfide thereof, a molar ratio of the activator to the quantum dots is 5-9:1, and a molar ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or the hydrochloride thereof to the N-hydroxysuccinimide or the sulfide thereof is 2-3:4-5; and 3) adding antibody to the activated quantum dot solution, and coupling to obtain the quantum dot-antibody fluorescent probe, wherein a molar ratio of the antibody to the quantum dots is 4-2:1.
Description
Technical field
The present invention relates to detection field, and in particular to a kind of preparation method of quantum dot-antibody fluorescence probe, quantum dot-
Antibody fluorescence probe, and the immuno-chromatographic test paper strip containing the probe.
Background technology
Be otherwise known as quantum dot (quantum dots, QDs) semiconductor nano, generally by II-VI group and iii-v unit
Element composition, with size adjustable, exciting light spectrum width, emission spectrum is narrow, stoke shift is larger the features such as.Quantum dot molar absorptivity
Coefficient and fluorescence quantum yield are higher, can produce stronger fluorescence signal, improve the sensitivity of detection.Meanwhile, quantum dot
Photochemical stability is good, fast light bleachability is good, improves the stability of testing result.In sum, quantum dot is a kind of reason
The fluorescence probe material thought, has unique advantage in the detection field based on immunochromatography technique and is widely applied prospect.
At this stage, the quantum dot as fluorescent probe mainly has monokaryon quantum dot (CdSe, CdTe, CdS) and nucleocapsid formula weight
Sub- point (CdSe/ZnS, CdSe/ZnSe), can be based between Organic substance and inorganic metal compound or organo-metallic compound
Pintsch process reacts and prepares in non-polar organic solvent.Synthesize in organic system the quantum dot for obtaining have it is almost ideal
Crystal structure, good monodispersity, stronger light stability and higher fluorescent yield, but used in conventional synthesis process
Tributylphosphine, hypertoxic, inflammable, the unstable organic phosphine compound such as tri octyl phosphine, explosive, expensive, and to operation
Environment has higher requirement, limits the large-scale production and application of quantum dot product.Additionally, preparing quantum dot fluorescence probe
During, quantum dot and the coupled product of corresponding antibody molecule have that homogeneity and dispersibility are poor, be easy to occur to reunite,
The problems such as luminous efficiency is relatively low, emission spectrum half-peak breadth is wide, have impact on the detection performance of Related product.
The content of the invention
In view of the foregoing, the invention provides a kind of being capable of precise control raw material dosage, the quantum of reduction production cost
The preparation method of point-antibody fluorescence probe, quantum dot-antibody fluorescence probe, and the immuno-chromatographic test paper strip containing the probe.
One aspect of the present invention provides a kind of preparation method of quantum dot-antibody fluorescence probe, comprises the steps：
(1) quantum dot is added into borate buffer solution, quantum dot solution is obtained；
(2) activator is added in quantum dot solution and quantum dot is activated, the activator is 1- (3- diformazan ammonia
Base propyl group) -3- ethyl carbodiimides or its hydrochlorate and N-hydroxy-succinamide or its thio thing, the activator and quantum
The mol ratio of point is 5～9:1, wherein, 1- (3- the dimethylamino-propyls) -3- ethyl carbodiimides or its hydrochlorate and N- hydroxyls
The mol ratio of base butanimide or its thio thing is 2～3:4～5；
(3) antibody is added in the quantum dot solution after activation and is coupled, the mol ratio of the antibody and quantum dot
For 4～2:1, obtain quantum dot-antibody fluorescence probe.
Another aspect of the present invention also provide using said method prepare quantum dot-antibody fluorescence probe, the antibody with
The mol ratio of quantum dot is 4～2:1.
Another aspect of the invention also provides a kind of immuno-chromatographic test paper strip, including analyte binding regions and detection line, the sample
Product land is coated with the quantum dot-antibody fluorescence probe prepared using said method.
Further aspect of the present invention also provides the preparation method of above-mentioned immuno-chromatographic test paper strip, comprises the steps：
(1) quantum dot prepared using said method-antibody fluorescence probe is dissolved in into probe dilution liquid, prepared quantum dot-
Antibody fluorescence probe solution；
(2) by the quantum dot-antibody fluorescence probe solution even application in glass fibre element film；
(3) the glass fibre element film for being sprayed with the quantum dot-antibody fluorescence probe is dried at 5～20 DEG C；
(4) by dried glass fibre element film, the nitrocellulose filter for being provided with nature controlling line and detection line, absorbent paper according to
It is secondary to be assembled on end liner, immuno-chromatographic test paper strip is obtained.
Quantum dot-antibody fluorescence probe preparation method that the present invention is provided, the fluorescence of the fluorescent probe for preparing are reclaimed
Rate is high, the response rate >=80%；Accuracy of detection is high, can detect that the detectable substance of pg/mL ranks.Immunochromatography provided by the present invention
Reagent paper, detection sensitivity are high, and sensitivity is up to 0.01ng/ml, easy to operate, and detection time is short, and detection range is wide.
Description of the drawings
Fig. 1 is immuno-chromatographic test paper strip prepared by the embodiment of the present invention.
Main element symbol description
| Immuno-chromatographic test paper strip | 100 |
| End liner | 1 |
| Absorbent paper | 2 |
| Nitrocellulose filter | 3 |
| Glass fibre element film | 4 |
| Detection line | 5 |
| Nature controlling line | 6 |
| Quantum dot-antibody fluorescence probe | 7 |
Following specific embodiment will further illustrate the present invention with reference to above-mentioned accompanying drawing.
Specific embodiment
One aspect of the present invention provides a kind of preparation method of quantum dot-antibody fluorescence probe, comprises the steps：
(1) quantum dot is added into borate buffer solution, quantum dot solution is obtained；
(2) activator is added in quantum dot solution and quantum dot is activated, the activator is 1- (3- diformazan ammonia
Base propyl group) -3- ethyl carbodiimides or its hydrochlorate and N-hydroxy-succinamide or its thio thing, the activator and quantum
The mol ratio of point is 5～9:1, wherein, 1- (3- the dimethylamino-propyls) -3- ethyl carbodiimides or its hydrochlorate and N- hydroxyls
The mol ratio of base butanimide or its thio thing is 2～3:4～5；
(3) antibody is added in the quantum dot solution after activation and is coupled, the mol ratio of the antibody and quantum dot
For 4～2:1, obtain quantum dot-antibody fluorescence probe.
Quantum dot-antibody fluorescence probe prepared by this method, the fluorescence response rate are high, the response rate >=80%；Accuracy of detection is high,
Can detect that the detectable substance of pg/mL ranks.
According to one embodiment of present invention, the preparation method of above-mentioned quantum dot-antibody fluorescence probe further include as
Lower step：
(1) quantum dot is added into the borate buffer solution that pH is 7～8, is mixed, prepared concentration is 0.5～1mg/mL's
Quantum dot solution；
(2) activator is added in quantum dot solution and is activated, 0～5 DEG C of 5～10min of ultrasonic activation, the activation
Agent is 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) or its hydrochlorate (EDC HCl) and N- hydroxysuccinimidyl acyls
Imines (NHS) or its thio thing (Sulfo-NHS), the activator are 5～9 with the mol ratio of quantum dot:1, wherein, the 1-
(3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) or its hydrochlorate (EDC HCl) and N-hydroxy-succinamide
(NHS) or its thio thing (Sulfo-NHS) mol ratio be 2～3:4～5；
(3) antibody is added in the quantum dot solution after activation and is coupled, it is anti-under 35～40 DEG C of temperature conditionss
Answer 1～3 hour, the antibody is 4～2 with the mol ratio of quantum dot:1, obtain quantum dot-antibody fluorescence probe.
In an embodiment of the invention, the quantum dot is the quantum dot that soluble surface has carboxyl, can be
In water phase, then synthesis or oil phase synthesis are entered water-filling and are mutually modified, including CdSe/ZnS or CdSe/ZnSe core-shell quanta dots.
Preferably, the quantum dot is then to enter water-filling by oil phase synthesis to be mutually modified.
In an embodiment of the invention, the quantum dot hydration radius is 25-55nm, the quantum dot and water
Proportion is 4～7.
The hydration radius refers to the radius that quantum dot is determined using particle size analyzer after being dissolved in water.The proportion amount of referring to
Ratio between son point and water.
In an embodiment of the invention, the method in step (2) using ice-bath ultrasonic carries out the activation.
In an embodiment of the invention, 1- (3- the dimethylamino-propyls) -3- ethyl carbodiimides or its salt
The mol ratio of hydrochlorate, N-hydroxy-succinamide or its thio thing and quantum dot is 2:5:1.
Inventor's discovery, the quantum dot-antibody fluorescence probe prepared under mole specific concentration of this activator with quantum dot
Sensitivity is higher, and the detection linear relationship that the immuno-chromatographic test paper strip containing the quantum dot-antibody fluorescence probe is obtained is more preferable.Example
It is such as shown in table 1 below,
The immune layer of quantum dot-antibody fluorescence probe that activator of the table 1 containing variable concentrations proportioning is prepared with quantum dot
The linear coefficient table of analysis test strips
It will be appreciated by those skilled in the art that, 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides and N- hydroxyl ambers
The priority of amber acid imide or its thio thing addition is not intended to limit in proper order, it is preferred that first can add N-hydroxy-succinamide or
Its thio thing, then add 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides.
In an embodiment of the invention, the antibody be for different albumen or albumen a domain,
Or the monoclonal antibody of one section of polypeptide, or polyclonal antibody.
The antibody being preferably added is 4 with the mol ratio of quantum dot:1.
Inventor has found that through many experiments the quantum dot prepared under this mole specific concentration-antibody fluorescence probe is sensitive
Du Genggao, the detection linear relationship that the immuno-chromatographic test paper strip containing the quantum dot-antibody fluorescence probe is obtained are more preferable.Such as following table
Shown in 2：
The immune layer of quantum dot-antibody fluorescence probe that quantum dot of the table 2 containing different mol ratio is prepared with antibody response
The linear coefficient table of analysis test strips
In an embodiment of the invention, the antibody is Procalcitonin monoclonal antibody.
According to one embodiment of present invention, the preparation method of described quantum dot-antibody fluorescence probe, also includes：Will
Sealer is added to the quantum dot-antibody fluorescence probe so as to which quantum dot-antibody is closed, and the sealer is second
Hydramine and bovine serum albumin, the content of the ethanolamine and bovine serum albumin in quantum dot solution are 1-3%；By capping
Solution centrifugal after end, takes precipitation and is dissolved in borate buffer solution.
According to one embodiment of present invention, the capping is further included：Sealer is added to into the quantum
In point-antibody fluorescence probe, 20～40min is reacted under 35～40 DEG C of temperature conditionss, the sealer is in quantum dot solution
In content be 1-3%；Solution centrifugal after capping is terminated, abandons supernatant, is repeated twice, and merges precipitation, will precipitation
It is dissolved in the borate buffer solution that pH is 7～8.
The present invention is closed to the quantum dot after coupling using sealer, and the sealer is the excellent life containing amino
The vacant site of quantum dot surface, after quantum dot with antibody covalent coupling, is used blocking agent by thing biocompatiblity molecules, is protected
The free space conformation of the antibody molecule for being coupled at quantum dot surface has been held, the group of granule after quantum point coupling antibody has been overcome
It is poly-, it is easy to precipitate, immune detection is easy to the problems such as false positive occur.
In an embodiment of the invention, the speed of the centrifugation is 22000rpm, and the time is 15min.
In an embodiment of the invention, by the borate buffer solution (BS solution), add Ox blood serum egg
In vain (BSA), mass percent of the bovine serum albumin in borate buffer solution is than 0.1～0.2%.
The present invention adds bovine serum albumin in borate buffer solution, can protect antibody activity and maintain probe steady
Property.Under the protection of this kind of protection liquid, probe can be up to 3 wheat harvesting periods with cryopreservation.
The present invention adopts ethanolamine and bovine serum albumin to combine and is closed, and finds that ethanolamine is made by experiment repeatedly
For organic molecule, reactivity is higher, effectively can be reacted with the activated sites of quantum dot, reach the effect in terminating reaction site
Really, and bovine serum albumin can close quantum dot and antibody causes nonspecific site, particularly, using ethanolamine and Sanguis Bovis seu Bubali
The quantum dot of albumin joint closing, prepared immunity test strip remolding sensitivity use merely ethanolamine or bovine serum albumin to imitate
Fruit is more preferable.
In an embodiment of the invention, it is of the present invention mix preferably to be vortexed mix, those skilled in the art can be with
Understand, being vortexed to mix includes stirring, vortex vortex mixer etc..
Another aspect of the present invention also provides quantum dot-antibody fluorescence probe prepared by a kind of employing said method, described anti-
Body is 4～2 with the mol ratio of quantum dot:1.
According to one embodiment of present invention, ratio of the quantum dot hydration radius for 25-55nm, the quantum dot and water
Weight is 4～7.
Quantum dot provided by the present invention-antibody fluorescence probe, quantum dot are returned than great, easily centrifugation recovery, response rate height
Yield can reach more than 80%, and rigidity is good, is hardly damaged.
Another aspect of the present invention also provides a kind of immuno-chromatographic test paper strip, including analyte binding regions and detection line, the sample
Product land is coated with above-mentioned quantum dot-antibody fluorescence probe.
Referring to Fig. 1, the immuno-chromatographic test paper strip 100 includes end liner 1, absorbent paper 2, nitrocellulose filter 3 and glass fibers
The plain film 4 of dimension is pasted on end liner 1 successively.The end liner 1 is PVC base plates.One end of the absorbent paper 2 and the one of the end liner 1
End alignment, the other end of the absorbent paper 2 are connected with 3 one end of the nitrocellulose filter, are arranged on the nitrocellulose filter 3
There are banded detection line 5 and nature controlling line 6, the detection line 5 and nature controlling line 6 parallel to the immuno-chromatographic test paper strip 100
Two minor faces.The detection line 5 is coated with antigen/hapten-carrier protein conjugate, and the detection line 5 is located at cellulose nitrate
One end of absorbent paper 2 is away from plain film 3.The nature controlling line 6 is located at the centre of detection line 5 and absorbent paper 2, the nature controlling line bag
There is antibody.The other end of the nitrocellulose filter 3 is connected with one end of glass fibre element film 4, the glass fibre
The other end of plain film 4 is alignd with the other end of the end liner 1.Above-mentioned quantum dot-antibody is coated with the glass fibre element film 4
Fluorescent probe 7.
Another aspect of the present invention also provides the preparation method of above-mentioned immuno-chromatographic test paper strip, comprises the steps：
(1) quantum dot prepared using said method-antibody fluorescence probe 7 is dissolved in into probe dilution liquid, prepared quantum dot-
Antibody fluorescence probe solution, the concentration of the quantum dot-antibody fluorescence probe solution is 0.04-0.06mg/mL；
(2) by 7 solution even application of the quantum dot-antibody fluorescence probe in glass fibre element film 4；
(3) the glass fibre element film 4 for being sprayed with the quantum dot-antibody fluorescence probe 7 is dried at 5～20 DEG C；
(4) by dried glass fibre element film 4, the nitrocellulose filter 3 for being provided with nature controlling line 6 and detection line 5, water suction
Paper 2 is assembled on end liner 1 successively, and immuno-chromatographic test paper strip 100 is obtained.
Inventor is had found by experiment repeatedly, is dried using in a low temperature of 5～20 DEG C, can effectively improve the spirit of test strips
Sensitivity, reaches 0.01mg/ml, reduces the precision of test strips, the precision of the immuno-chromatographic test paper strip than being dried under room temperature
Reduce up to 5.2%.It is as shown in table 3 below,
The immuno-chromatographic test paper strip precision being dried under 37 DEG C of 3 room temperature of table and low temperature
In an embodiment of the invention, the probe dilution liquid be borate buffer solution, 1% Polyethylene Glycol,
1% bovine serum albumin, 0.5% tween.
The immune chromatography test paper of the present invention, detection sensitivity are high, and sensitivity is up to 0.01ng/ml, easy to operate, during detection
Between it is short, detection range is wide.
Embodiment one
Weigh 20mg CdSe/ZnS QDs to be dissolved in 4ml chloroformic solutions, weigh 20mg maleic anhydrides-ten eight (carbon) alkene common
Polymers is dissolved in 2ml chloroforms, and ultrasound dissolves which respectively, and two kinds of solution are mixed 5～10min of ultrasound, and it is molten that revolving removes chloroform
Agent, quantum dot are in drying regime, add 4ml aqueous solutions, ultrasonic 30min to dissolve which, and solution clarification is bright.
Embodiment two
Carboxyl water-soluble quantum dot (concentration is 1mg/mL) prepared by 150ul embodiments one, add 250ul pH be
7.4th, 20mM borate buffer solutions, vortex vortex mixer are mixed；Add the EDC 50ul of 50mg/ml, vortex vortex mixer to mix, add
The NHS 50ul of 100mg/ml, are vortexed and mix；Ice-bath ultrasonic activates 5min；Antibody 50.3ug, vortex vortex mixer is added to mix；Put
37 DEG C of reaction 2h on the rotary incubator.Ethanolamine 3ul (concentration is 100%) and 20%BSA 50ul is added, is mixed；37 DEG C,
It is placed on rotary incubator and reacts 30min, be centrifuged, speed 22000rpm, 15min is centrifuged, abandons supernatant, is repeated twice；Merge heavy
Shallow lake is dissolved in 150ul pH for 8.0, in 10mM borate buffer solutions, as quantum dot-antibody fluorescence probe.
Embodiment three
Carboxyl water-soluble quantum dot (concentration is 5mg/mL) prepared by 150ul embodiments one, adds 250ul pH to be 8,
10mM borate buffer solutions, stirring and evenly mixing；The EDC 50ul of 60mg/ml are added, is vortexed and is mixed, add the Sulfo- of 80mg/ml
NHS 50ul, stirring and evenly mixing；Ice-bath ultrasonic activates 10min；Antibody 50.3ug is added, is vortexed and is mixed；It is placed on rotary incubator
37 DEG C of reaction 2h.Ethanolamine 3ul (concentration is 100%) and 20%BSA 50ul is added, is mixed；37 DEG C, it is placed in rotary incubator
Upper reaction 30min, centrifugation, speed 22000rpm are centrifuged 15min, abandon supernatant, be repeated twice；Merge precipitation and be dissolved in 150ul pH
For, in 8.0,10mM borate buffer solutions, addition mass fraction is 0.1%BSA, is put into cold preservation in low temperature.
Example IV
Probe after coupling is sprayed on glass fibre element film (concentration is 1.5ul/cm), 5-20 DEG C of cold drying 12-
15h；Detection line antigen conjugates and nature controlling line antibody is coated with nitrocellulose filter, and 37 DEG C are dried 12-15h；Will be obtained
Glass fibre element film, nitrocellulose filter and absorbent paper are assembled on end liner (PVC base plates) successively, and immune chromatography test paper is obtained
Bar.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, embodiment of above is only for explaining claims.So protection scope of the present invention is not limited to description.Appoint
What those familiar with the art in the technical scope of present disclosure, the change that can be readily occurred in or replacement,
It is included within protection scope of the present invention.
Claims (11)
1. the preparation method of a kind of quantum dot-antibody fluorescence probe, it is characterised in that comprise the steps：
(1) quantum dot is added into borate buffer solution, quantum dot solution is obtained；
(2) activator is added in quantum dot solution and quantum dot is activated, the activator is 1- (3- dimethylaminos third
Base) -3- ethyl carbodiimides or its hydrochlorate and N-hydroxy-succinamide or its thio thing, the activator and quantum dot
Mol ratio is 5～9:1, wherein, 1- (3- the dimethylamino-propyls) -3- ethyl carbodiimides or its hydrochlorate and N- hydroxyl ambers
The mol ratio of amber acid imide or its thio thing is 2～3:4～5；
(3) antibody being added in the quantum dot solution after activation and is coupled, the mol ratio of the antibody and quantum dot is 4～
2:1, obtain quantum dot-antibody fluorescence probe.
2. the preparation method of quantum dot as claimed in claim 1-antibody fluorescence probe, it is characterised in that the quantum dot is
Soluble surface has the quantum dot of carboxyl, can synthesize or then oil phase synthesis is entered water-filling and be mutually modified in water phase, including
CdSe/ZnS or CdSe/ZnSe core-shell quanta dots.
3. the preparation method of quantum dot as claimed in claim 1-antibody fluorescence probe, it is characterised in that 1- (the 3- diformazans
Aminopropyl) -3- ethyl carbodiimides or its hydrochlorate, N-hydroxy-succinamide or its thio thing and quantum dot mol ratio
For 5:2:1.
4. the preparation method of quantum dot as claimed in claim 1-antibody fluorescence probe, it is characterised in that the quantum dot water
Conjunction radius is 25-55nm, and the quantum dot is 4～7 with the proportion of water.
5. the preparation method of quantum dot as claimed in claim 1-antibody fluorescence probe, it is characterised in that the antibody is drop
Calcium element original monoclonal antibody.
6. the preparation method of quantum dot as claimed in claim 1-antibody fluorescence probe, it is characterised in that also include：Will closing
Agent is added to the quantum dot-antibody fluorescence probe so as to which quantum dot-antibody is closed, and the sealer is ethanolamine
And bovine serum albumin, the content of the ethanolamine and bovine serum albumin in quantum dot solution is 1-3%；Capping is terminated
Solution centrifugal afterwards, takes precipitation and is dissolved in borate buffer solution.
7. the preparation method of quantum dot as claimed in claim 6-antibody fluorescence probe, it is characterised in that also including will be described
In borate buffer solution, bovine serum albumin is added, content of the bovine serum albumin in borate buffer solution is 0.1-
0.2%.
8. a kind of quantum dot-antibody fluorescence probe that prepared by the method for claim 1, it is characterised in that the antibody
Mol ratio with quantum dot is 4～2:1.
9. quantum dot as claimed in claim 8-antibody fluorescence probe, it is characterised in that the quantum dot hydration radius is 25-
55nm, the quantum dot are 4～7 with the proportion of water.
10. a kind of immuno-chromatographic test paper strip, including analyte binding regions and detection line, the analyte binding regions spraying will just like right
Seek the quantum dot-antibody fluorescence probe described in 8.
11. a kind of preparation methoies of immuno-chromatographic test paper strip, it is characterised in that comprise the steps：
(1) quantum dot as claimed in claim 8-antibody fluorescence probe is dissolved in into probe dilution liquid, quantum dot-antibody is obtained glimmering
Light probe solution；
(2) by the quantum dot-antibody fluorescence probe solution even application in glass fibre element film；
(3) the glass fibre element film for being sprayed with the quantum dot-antibody fluorescence probe is dried at 5～20 DEG C；
(4) by dried glass fibre element film, the nitrocellulose filter for being provided with nature controlling line and detection line, absorbent paper successively group
It is mounted on end liner, immuno-chromatographic test paper strip is obtained.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107102132A (en) * | 2017-06-22 | 2017-08-29 | 佛山职业技术学院 | Quantum dot fluorescence probe and preparation method thereof, immune chromatography test paper |
| CN108918882A (en) * | 2018-05-17 | 2018-11-30 | 西安交通大学苏州研究院 | A kind of preparation method of the hs-CRP immuno-chromatographic test paper strip based on quantum dot |
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| CN109097021A (en) * | 2017-06-20 | 2018-12-28 | Tcl集团股份有限公司 | The method for improving near-infrared water-soluble copper sulphur indium quantum dot quantum efficiency |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023210A (en) * | 2009-09-21 | 2011-04-20 | 北京中德大地食品安全技术开发有限责任公司 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
| CN103063828A (en) * | 2011-10-24 | 2013-04-24 | 韩焕兴 | Method for utilizing covalence coupling to prepare photon point-antibody compound |
| CN103487578A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | CRP (C-reactive protein)/PCT (procalcitonin) combined diagnosis test paper and preparation method thereof |
-
2016
- 2016-09-13 CN CN201610823178.5A patent/CN106526192A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023210A (en) * | 2009-09-21 | 2011-04-20 | 北京中德大地食品安全技术开发有限责任公司 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
| CN103063828A (en) * | 2011-10-24 | 2013-04-24 | 韩焕兴 | Method for utilizing covalence coupling to prepare photon point-antibody compound |
| CN103487578A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | CRP (C-reactive protein)/PCT (procalcitonin) combined diagnosis test paper and preparation method thereof |
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| CN109097021A (en) * | 2017-06-20 | 2018-12-28 | Tcl集团股份有限公司 | The method for improving near-infrared water-soluble copper sulphur indium quantum dot quantum efficiency |
| CN107102132A (en) * | 2017-06-22 | 2017-08-29 | 佛山职业技术学院 | Quantum dot fluorescence probe and preparation method thereof, immune chromatography test paper |
| CN110343527A (en) * | 2018-04-08 | 2019-10-18 | 华中师范大学 | Quantum dot fluorescence probe and its preparation method and application and the method and kit for detecting elastase activity |
| CN110343527B (en) * | 2018-04-08 | 2022-04-12 | 华中师范大学 | Quantum dot fluorescent probe, preparation method and application thereof, and method and kit for detecting activity of elastase |
| CN108918882A (en) * | 2018-05-17 | 2018-11-30 | 西安交通大学苏州研究院 | A kind of preparation method of the hs-CRP immuno-chromatographic test paper strip based on quantum dot |
| CN108918858A (en) * | 2018-05-17 | 2018-11-30 | 西安交通大学苏州研究院 | A kind of preparation method of quantum dot-antibody immune complex |
| CN108918858B (en) * | 2018-05-17 | 2021-06-11 | 西安交通大学苏州研究院 | Preparation method of quantum dot-antibody immune complex |
| CN108918882B (en) * | 2018-05-17 | 2021-08-06 | 西安交通大学苏州研究院 | Preparation method of hypersensitive C-reactive protein immunochromatographic test strip based on quantum dots |
| CN109187960A (en) * | 2018-09-10 | 2019-01-11 | 浙江理工大学 | A method of the detection Ancient Silk Textile based on immunochromatography technique |
| CN110082523A (en) * | 2019-04-12 | 2019-08-02 | 西安交通大学苏州研究院 | A kind of quantum dot-preparation method of myoglobins antibody immune complex and the preparation method of test strips |
| CN110231476A (en) * | 2019-05-08 | 2019-09-13 | 桂林理工大学 | Fluorescein mixes immobilized processing method with protein on a kind of solid-phase matrix |
| CN111500769A (en) * | 2020-03-11 | 2020-08-07 | 南京农业大学 | Fluorescence immunochromatography method for detecting SARS-CoV-2 nucleic acid |
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