CN103760363A - Detection method for core-shell quantum dot-based flexible coupled marker immunochromatography test strip - Google Patents
Detection method for core-shell quantum dot-based flexible coupled marker immunochromatography test strip Download PDFInfo
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- CN103760363A CN103760363A CN201410008389.4A CN201410008389A CN103760363A CN 103760363 A CN103760363 A CN 103760363A CN 201410008389 A CN201410008389 A CN 201410008389A CN 103760363 A CN103760363 A CN 103760363A
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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
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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
- 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
- G01N33/58—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
Abstract
The invention provides a detection method for a core-shell quantum dot-based flexible coupled marker immunochromatography test strip, belonging to the technical field of an immunodetection method. According to the method, based on the fact that the core-shell quantum dot has the characteristics of being wide in exciting line, narrow in emission spectrum line, high in quantum size effect and fluorescent quantum efficiency and strong in photochemical stability, quantum dots and protein G or protein A molecules are subjected to covalent linkage by adopting an EDC condensation mode, the flexible coupling of the quantum dots and antibody molecules can be realized by utilizing protein G or protein A molecules as bridges and by means of the specificity of Fc terminal of an antibody, antigenic determinant part of the antibody is not occupied, the antigen immunity activity of the antibody can be greatly improved, and the sensitivity and specificity when the marker product is utilized for immunodetection can be greatly enhanced.
Description
Technical field
The invention belongs to immunologic detection method technical field, be specifically related to the detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip.
Background technology
Utilize at present the test strip of nm of gold colour developing to be widely applied, its ultimate principle is to take microporous barrier as solid phase carrier, antigen or the antibody of bag detected material, add after sample to be tested, through the capillary siphoning effect of microporous barrier, make to detect thing coated antigen or antibody on film in sample and be combined, by the color of nano gold mark thing self, judge testing result.But for the lower sample of some content, the very slight color of nm of gold, is difficult to the naked eye judged result, and sensitivity is low, can only qualitative detection, can not quantitative test.Due to quantum dot, have the characteristics such as excitation spectrum live width, the spectral line of emission are narrow, quantum size effect, high fluorescence efficiency, high light chemical stability, can substitute colloidal nano gold carries out biomarker, replaces absorbing colour developing detect with high-sensitive fluorescence signal.Quantum dot test strips both can be carried out qualitative analysis, can quantitatively detect again.
Through the patent retrieval of prior art is found, the patent of Chinese Patent Application No. 200610024086.7, utilize the characteristic of quantum dot, invented a kind of detection method of quantum dot mark fast immune chromatographic test paper bar, thing to be detected is carried out to qualitative or half-quantitative detection, this invention is by changing particle diameter or the kind of quantum dot, obtain the fluorescence of different wave length, the quantum dot-labeled different antibody with dissimilar, carries out multi-component detection by multicolor fluorescence signal, method is simple and quick, and cost is low.Yet quantum dot is a kind of nano material, there is great surface effect, it is existing that to utilize test strips that quantum spot check is surveyed be all by electrostatic adsorption by quantum dot and antibody molecule, directly the mode such as covalently bound is carried out biomarker, in the process of labelled antibody, be easy to mark to the Fab end with antigenic determinant position of antibody, antibody after mark can not carry out immune response with antigen molecule, because of but a kind of invalid mark, this direct interconnection technique is for the sensitivity of final immune detection, especially specificity has impact significantly, reduce and detect effect, quantitatively detection sensitivity and specificity all cannot reach satisfactory result.And existing quantum dot test strips patent is all to utilize bare nucleus quantum dot to carry out immune detection, because bare nucleus quantum dot has very large weakening compared with the fluorescent characteristic of core-shell quanta dots, thereby applying it to test strips immune detection, effect is necessarily weaker than the result of carrying out immune detection with core-shell quanta dots.
Summary of the invention
The object of the invention is for a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip is provided, this detection method has higher sensitivity and specificity.
The invention provides a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip, comprise as follows:
Step 1: prepare core-shell quanta dots;
Step 2: the core-shell quanta dots that step 1 is obtained and Protein G or albumin A are undertaken covalently bound by the mode of EDC condensation, obtain covalently bound product;
Step 3: the covalently bound product that step 2 is obtained mixes with an antibody molecule of antigen molecule to be detected, obtains the marked product of the flexible coupling of quantum dot antibody;
Step 4: the marked product of the flexible coupling of quantum dot antibody that step 3 is obtained is coated on glass fibre element film, obtain scribbling the glass fibre element film of marked product, another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter are formed to calibration tape and control band, obtain the nitrocellulose filter of parallel coated antibody;
Step 5: on one side polyester or plastic plate, paste successively upper glass cellulose filter membrane, scribble glass fibre element film, the water-absorption fiber element film of marked product, the nitrocellulose filter of parallel coated antibody, the polyester after pasting or plastic plate are made and are obtained immuno-chromatographic test paper strip;
Step 6: the pad end of the immuno-chromatographic test paper strip that step 5 is obtained inserts in testing sample solution, takes out test strips after immunochromatography, determines testing result.
Preferably, described core-shell quanta dots is CdTe/CdS, CdSe/CdS, CdSe/ZnSe or CdSe/ZnS core-shell quanta dots.
Preferably, the mol ratio of an antibody molecule of described covalently bound product and antigen molecule to be detected is 1:1.
Preferably, in described step 4, calibration tape and control band be arranged in parallel, calibration tape and the 8-12mm that is spaced apart that controls band.
Preferably, described forms another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter after calibration tape and control band, room temperature was dried after 20-30 minute, put into again the phosphate buffer that contains bovine serum albumin(BSA) and seal 60-120 minute, envelope after dry, places 4 ℃ and saves backup.
Preferably, described testing sample solution is blood, urine or saliva.
Preferably, in described step 6, after immunochromatography, take out after test strips, be specially: by test strips visual inspection result under uviol lamp, or obtain and detect the curve of spectrum under fluorescence spectrophotometer, by contrasting with typical curve, determine testing result.
Beneficial effect of the present invention
1, the invention provides a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip, the method utilizes core-shell quanta dots to have excitation spectrum live width, the spectral line of emission is narrow, quantum size effect, fluorescence quantum efficiency is high, on the basis of the characteristic that photochemical stability is strong, the mode that adopts EDC condensation is undertaken covalently bound by quantum dot and Protein G or albumin A molecule, utilize Protein G or albumin A molecule to do bridge, rely on itself and most mammal (to comprise people, sheep, rabbit, mouse, horse, pig, the specificity of antibody Fc end monkey etc.), realize the flexible coupling of quantum dot and antibody molecule, do not occupy the antigenic determinant position of antibody, the antigen immune that greatly improves antibody is active, sensitivity and the specificity of utilizing this marked product to carry out immune detection are all greatly improved,
2, the present invention is coated in the marked product of the flexible coupling of the quantum dot antibody obtaining on glass fibre element film, another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter are formed to calibration tape and control band, utilize double antibody sandwich method principle to detect in sample whether contain target antigen molecule in conjunction with flexible marker technology and the photoluminescent property thereof of quantum dot.By calibration tape and the fluorescence spectrum of controlling band on test immuno-chromatographic test paper strip, qualitative or quantitatively judge testing result;
3, the present invention is by the quantum dot-labeled different antibody molecule by different-grain diameter size, just can obtain the fluorescence of different wave length, the different antibody molecule of the flexible coupling of quantum dot by different size size, can produce multicolor fluorescence, realize multi-component detection, calibration tape only needs one, does not need many calibration tapes, experimental implementation simple and fast;
4, the present invention combines the highly sensitive high specific of flexible coupling labelling technique, immunoreactive high specific with the high-efficiency fluorescence characteristic three of quantum dot, this detection method is easy, sensitive, be applicable to blood sample, urine sample, saliva equal samples, can be applicable to the various fields such as food security, environmental monitoring, medical diagnosis, health and epidemic prevention.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes Protein G to realize the schematic diagram of the flexible coupling of core-shell quanta dots and antibody molecule;
Fig. 2 is quantum dot ELISA test strip principle schematic of the present invention.
Embodiment
The preparation method of the core-shell quanta dots described in step 1 of the present invention is prior art, and the nucleocapsid that the method utilizes continuous ionic layer adsorption reaction technology (SILAR) to realize quantum dot is coated.Take one kettle way principle, in same reaction unit, add nuclear fuel material, by controlling the time of nucleus growth, obtain the bare nucleus quantum dot of different-grain diameter size, and then dropwise add husk as raw material, utilize SILAR technology to carry out shell and be coated, last centrifugal purification processing sample obtains core-shell quanta dots, and concrete grammar is referring to article J.Phys.Chem.C2008,112,8587 – 8593.
Core-shell quanta dots of the present invention is preferably the core-shell quanta dots such as CdSe/CdS, CdSe/ZnSe, CdSe/ZnS, by changing the particle size of quantum dot, can realize the tuning of fluorescence color, more preferably adopt green light quantum point (fluorescent emission wave band is at 500-560nm), 3-4nm(fluorescent emission wave band that bare nucleus size is less than 3nm at 560-590nm) gold-tinted quantum dot, be greater than 4nm(fluorescent emission wave band at 590-650nm) red light quantum point carry out the immune detection of test strips.Take CdTe/CdS core-shell quanta dots as example, and concrete grammar is:
1, in distilled water, add tellurium powder and sodium borohydride, obtain the solution of sodium hydrogen telluride, described distilled water is the distilled water with the abundant deoxygenation of nitrogen, and the mol ratio of tellurium powder and sodium borohydride is 1:5;
2, take six perchloric acid hydrate cadmiums, join in the three-necked bottle that contains oxygen-free water, then add mercaptopropionic acid, with 1mol/L sodium hydroxide solution, regulate pH value between 10-11; Six described perchloric acid hydrate cadmiums and the mol ratio of mercaptopropionic acid are 1:2.4;
3, the solution of 1 sodium hydrogen telluride obtaining is added in the three-necked bottle of step 2, be warming up to 100 ℃, synthetic CdTe bare nucleus quantum dot, the time length of reacting by change obtains bare nucleus size and is less than the gold-tinted quantum dot yellow solution that contains green light quantum point green solution, 3-4nm of 3nm, the red light quantum point red solution that is greater than 4nm;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, prepare the husk as raw material of sulphur, according to article J.Phys.Chem.C2008, the experimental procedure of 112,8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, preparation CdTe/CdS core-shell quanta dots.
The core-shell quanta dots that step 1 is obtained described in step 2 of the present invention and Protein G or albumin A are by EDC(1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) mode of condensation carry out covalently bound, be specially: core-shell quanta dots is mixed with EDC condensation agent, the carboxyl group on activation quantum dot surface, stirring at room 20-40 minute, preferably 30 minutes, then add Protein G or albumin A, stirring at room reaction 90-150min, preferred 120min, quantum dot with carboxyl group forms amido link with Protein G or albumin A molecule with amido group under the effect of EDC condensation agent, realization is covalently bound with the quantum dot of carboxyl and protein with amido, with the separation and purification of NAP-5 pillar, obtain covalently bound product, the mol ratio of described core-shell quanta dots, EDC condensation agent, Protein G or albumin A is 1:(100-200): 1.
The covalently bound product obtaining is mixed with an antibody molecule of antigen molecule to be detected described in step 3 of the present invention, obtain the marked product of the flexible coupling of quantum dot antibody, the mol ratio of described covalently bound product and an antibody molecule of antigen molecule to be detected is preferably 1:1, a described antibody molecule treating detectable antigens molecule is not particularly limited, as long as can with the antibody that Protein G or albumin A are connected, be preferably hepatitis B surface antibody (HBsAb), described mixing temperature is preferably room temperature, and incorporation time is preferably 30-60min.The present invention utilize Protein G realize core-shell quanta dots and antibody molecule flexible coupling schematic diagram as shown in Figure 1.
The marked product by the flexible coupling of the quantum dot antibody obtaining described in step 4 of the present invention is coated on glass fibre element film, if what detect is multi-component antigen molecule, after the marked product equal proportion of the flexible coupling of several quantum dot antibody obtaining being mixed, be coated in again on glass fibre element film, obtain scribbling the glass fibre element film of marked product; Another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter are formed after calibration tape and control band, room temperature was dried after 20-30 minute, obtain the nitrocellulose filter of parallel coated antibody, put into again the phosphate buffer that contains bovine serum albumin(BSA) and seal 60-120 minute, envelope after dry, places 4 ℃ and saves backup.Described calibration tape and control band be arranged in parallel, calibration tape and the 8-12mm that is spaced apart that controls band, bandwidth is 2-5mm left and right, another antibody of described antigen molecule to be detected is not particularly limited, can be identical with an antibody molecule of antigen molecule to be detected, also can be different, be preferably hepatitis B surface antibody (HBsAb); Two described anti-being not particularly limited, are preferably goat anti-human igg, rabbit anti-human igg or mouse-anti human IgG.
Described in step 5 of the present invention on one side polyester or plastic plate, paste successively upper glass cellulose filter membrane, scribble glass fibre element film, the water-absorption fiber element film of marked product, the nitrocellulose filter of parallel coated antibody, finally stick thieving paper, unnecessary solution after absorption chromatography, between described film and film, all to closely be connected, the polyester pasting or plastic plate are cut into the wide test strips of 3-6mm, obtain immuno-chromatographic test paper strip, dry encapsulation, 4-25 ℃ keeps in Dark Place.
The pad end of immuno-chromatographic test paper strip sample (absorption pad one end) is inserted in testing sample solution described in step 6 of the present invention, in testing sample, contain target antigen and divide the period of the day from 11 p.m. to 1 a.m, target antigen molecule is first combined with the antibody of quantum dot flexible marker, because chromatography action-reaction bond moves forward along coated nitrocellulose filter, when running into coated antibody, form the antibody complex of the flexible coupling mark of antibody-antigen (object)-quantum dot and be enriched on calibration tape, unnecessary quantum dot antibody labeling product continues to move to control is with position, antibody generation immune response due to two anti-and the flexible couplings of quantum dot, be enriched in again to control and be with, quantum dot ELISA test strip principle schematic of the present invention as shown in Figure 2, then after chromatography effect 5-20 minute, take out test strips, by test strips visual inspection result under uviol lamp, or obtain and detect the curve of spectrum under fluorescence spectrophotometer, by contrasting with typical curve, determine testing result.If calibration tape has fluorescence with control band place, positive result, illustrates in testing sample and contains target antigen molecule, be analyzed with Duplicate Samples, the fluorescence on calibration tape is stronger, shows that the content of object antigen molecule is higher; If only had, control with fluorescence, negative result, illustrates and in testing sample, there is no target antigen molecule; If calibration tape does not all have fluorescence with control band place, illustrate that detection is invalid.Described ultra violet lamp refers to that wavelength is at the common uv lamp of 300-400nm.Fluoroscopic examination spectrometer is the ocean color instrument that can obtain easily in the market.Described testing sample solution is the body fluid such as serum solution, urine, saliva preferably.
Below in conjunction with specific embodiment, the present invention is done to further detailed description.
Embodiment 1
1, take 0.051 gram, tellurium powder (0.4mmol), 0.08 gram of sodium borohydride (2mmol), adds containing in the 10ml distilled water of the abundant deoxygenation of useful nitrogen, obtains the solution of sodium hydrogen telluride;
2, take 0.6714 gram of six perchloric acid hydrate cadmium (1.6mmol), join in the three-necked bottle that contains 100ml oxygen-free water, then add mercaptopropionic acid 342 microlitres (3.84mmol), with 1mol/L sodium hydroxide solution, regulate pH value between 10-11;
3, get in the three-necked bottle in the solution implantation step 2 of sodium hydrogen telluride of 1 step synthesized, be warming up to 100 ℃, synthetic CdTe bare nucleus quantum dot, what the time of reacting by change obtained within 15-30 minute that bare nucleus size is less than 3nm contains green light quantum point green solution;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, prepare the husk as raw material of sulphur, according to article J.Phys.Chem.C2008, the experimental procedure of 112,8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, preparation CdTe/CdS core-shell quanta dots;
5, by 1 μ molCdTe/CdS core-shell quanta dots with at 100 μ molEDC condensation agents, mix, the carboxyl group on activation quantum dot surface, stirring at room 30 minutes, then add 1 μ mol Protein G, stirring at room is reacted 2 hours, obtains the covalently bound product of quantum dot-Protein G with the separation and purification of NAP-5 pillar;
6, the covalently bound product of quantum dot-Protein G is mixed with hepatitis B surface antibody (HBsAb) equal proportion, stirring at room 1 hour, obtains the flexible coupling marked product of quantum dot-Protein G-HBsAb;
7, the flexible coupling marked product of quantum dot-Protein G-HBsAb is sprayed on equably on glass fibre element film, obtains scribbling the glass fibre element film of marked product, dry rear envelope, places 4 ℃ and saves backup; On nitrocellulose filter, with machine, draw two parallel bands, bandwidth is controlled at 2mm left and right, and two band interval 8mm wherein spray HBsAb and form calibration tape on a band; On another band, spray goat anti-human igg's formation control band, room temperature was dried after 20 minutes, obtained the nitrocellulose filter of parallel coated antibody, then put into the phosphate buffer that contains bovine serum albumin(BSA) and seal 60 minutes, envelope after dry, places 4 ℃ and saves backup;
8, on one side plastic plate, paste successively upper glass cellulose membrane, scribble glass fibre element film, the water adsorption glass cellulose membrane of marked product, the nitrocellulose filter of parallel coated antibody, finally stick long 30 centimetres, the thieving paper of wide 2 centimetres, absorb unnecessary solution after chromatography, between film and film, all will closely be connected, make the large plate of immune chromatography test paper, with cutting cutter, the plastic plate pasting is longitudinally cut into the wide immunity test strip of 4mm, dry lower envelope, 4-25 ℃ keeps in Dark Place;
9, the wide immunity test strip sample pad one end of 4mm is inserted in test serum, after chromatography effect 15 minutes, take out test strips observations under uviol lamp, or under fluorescence spectrophotometer, test the fluorescence spectrum of quantum dot, measure 5 minutes report results of HBsAg strong positive sample, measure 20 minutes report results of weak sun and ' negative ' specimens.If the calibration tape on film has green fluorescence with control band place, two fluorescence, illustrate in testing sample and contain HBsAg, positive result, and stronger at the fluorescence of calibration tape, the content of HBsAg is higher; If only had, control with green fluorescence, i.e. a fluorescence, illustrates and in testing sample, there is no HBsAg; If calibration tape does not all have fluorescence with control band, illustrate that detection is invalid.This detection method method sensitivity is 1ng/ml, and specificity is more than 90%, in batch with criticize between repeatability be less than 20%.
Embodiment 2
1, take 0.051 gram of selenium powder (0.4mmol), 0.08 gram of sodium borohydride (2mmol), adds containing in the 10ml distilled water of the abundant deoxygenation of useful nitrogen, obtains the solution of sodium hydrogen telluride;
2, take 0.6714 gram of six perchloric acid hydrate cadmium (1.6mmol), join in the three-necked bottle that contains 100ml oxygen-free water, then add mercaptopropionic acid 342 microlitres (3.84mmol), with 1mol/L sodium hydroxide solution, regulate pH value between 10-11;
3, get in the three-necked bottle in the solution implantation step 2 of sodium hydrogen selenide of 1 step synthesized, be warming up to 100 ℃, synthetic CdSe bare nucleus quantum dot, what the time of reacting by change obtained within 15-30 minute that bare nucleus size is less than 3nm contains green light quantum point green solution;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, prepare the husk as raw material of sulphur, according to article J.Phys.Chem.C2008, the experimental procedure of 112,8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, preparation CdSe/CdS core-shell quanta dots;
5, by 1 μ molCdSe/CdS core-shell quanta dots with at 100 μ molEDC condensation agents, mix, the carboxyl group on activation quantum dot surface, stirring at room 30 minutes, then add 1 μ mol Protein G, stirring at room is reacted 2 hours, obtains the covalently bound product of quantum dot-Protein G with the separation and purification of NAP-5 pillar;
6, the covalently bound product of quantum dot-Protein G is mixed with hepatitis B surface antibody (HBsAb) equal proportion, stirring at room 1 hour, obtains the flexible coupling marked product of quantum dot-Protein G-HBsAb;
7, the flexible coupling marked product of quantum dot-Protein G-HBsAb is sprayed on equably on glass fibre element film, obtains scribbling the glass fibre element film of marked product, dry rear envelope, places 4 ℃ and saves backup; On nitrocellulose filter, with machine, draw two parallel bands, bandwidth is controlled at 2mm left and right, and two band interval 8mm wherein spray HBsAb and form calibration tape on a band; On another band, spray rabbit anti-human igg's formation control band, room temperature was dried after 30 minutes, obtained the nitrocellulose filter of parallel coated antibody, then put into the phosphate buffer that contains bovine serum albumin(BSA) and seal 60 minutes, envelope after dry, places 4 ℃ and saves backup;
8, on one side plastic plate, paste successively upper glass cellulose membrane, scribble glass fibre element film, the water adsorption glass cellulose membrane of marked product, the nitrocellulose filter of parallel coated antibody, finally stick long 30 centimetres, the thieving paper of wide 2 centimetres, absorb unnecessary solution after chromatography, between film and film, all will closely be connected, make the large plate of immune chromatography test paper, with cutting cutter, the plastic plate pasting is longitudinally cut into the wide immunity test strip of 4mm, dry lower envelope, 4-25 ℃ keeps in Dark Place;
9, the wide immunity test strip sample pad one end of 4mm is inserted in test serum, after chromatography effect 10 minutes, take out test strips observations under uviol lamp, or under fluorescence spectrophotometer, test the fluorescence spectrum of quantum dot, measure 5 minutes report results of HBsAg strong positive sample, measure 20 minutes report results of weak sun and ' negative ' specimens.If the calibration tape on film has green fluorescence with control band place, two fluorescence, illustrate in testing sample and contain HBsAg, positive result, and stronger at the fluorescence of calibration tape, the content of HBsAg is higher; If only had, control with green fluorescence, i.e. a fluorescence, illustrates and in testing sample, there is no HBsAg; If calibration tape does not all have fluorescence with control band, illustrate that detection is invalid.This detection method method sensitivity is 1ng/ml, and specificity is more than 90%, in batch with criticize between repeatability be less than 20%.
Embodiment 3
1, take 0.051 gram, tellurium powder (0.4mmol), 0.08 gram of sodium borohydride (2mmol), adds containing in the 10ml distilled water of the abundant deoxygenation of useful nitrogen, obtains the solution of sodium hydrogen telluride;
2, take 0.6714 gram of six perchloric acid hydrate cadmium (1.6mmol), join in the three-necked bottle that contains 100ml oxygen-free water, then add mercaptopropionic acid 342 microlitres (3.84mmol), with 1mol/L sodium hydroxide solution, regulate pH value between 10-11;
3, get in the three-necked bottle in the solution implantation step 2 of sodium hydrogen telluride of 1 step synthesized, be warming up to 100 ℃, synthetic CdTe bare nucleus quantum dot, what the time of reacting by change obtained within 15-30 minute that bare nucleus size is less than 3nm contains green light quantum point green solution;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, prepare the husk as raw material of sulphur, according to article J.Phys.Chem.C2008, the experimental procedure of 112,8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, preparation CdTe/CdS core-shell quanta dots;
5, by 1 μ molCdTe/CdS core-shell quanta dots with in the carboxyl group on 100 μ molEDC condensation agent admixture activation quantum dot surfaces, stirring at room 30 minutes, then add 1 μ mol albumin A, stirring at room is reacted 2 hours, obtains the covalently bound product of quantum dot-albumin A with the separation and purification of NAP-5 pillar;
6, the covalently bound product of quantum dot-albumin A is mixed with hepatitis B surface antibody (HBsAb) equal proportion, stirring at room 1 hour, obtains the flexible coupling marked product of quantum dot-albumin A-HBsAb;
7, the flexible coupling marked product of quantum dot-albumin A-HBsAb is sprayed on equably on glass fibre element film, obtains scribbling the glass fibre element film of marked product, dry rear envelope, places 4 ℃ and saves backup; On nitrocellulose filter, with machine, draw two parallel bands, bandwidth is controlled at 2mm left and right, and two band interval 8mm wherein spray HBsAb and form calibration tape on a band; On another band, spray mouse-anti human IgG formation control band; room temperature was dried after 20 minutes, obtained the nitrocellulose filter of parallel coated antibody, then put into the phosphate buffer that contains bovine serum albumin(BSA) and seal 60 minutes; envelope after dry, places 4 ℃ and saves backup;
8, on one side plastic plate, paste successively upper glass cellulose membrane, scribble glass fibre element film, the water adsorption glass cellulose membrane of marked product, the nitrocellulose filter of parallel coated antibody, finally stick long 30 centimetres, the thieving paper of wide 2 centimetres, absorb unnecessary solution after chromatography, between film and film, all will closely be connected, make the large plate of immune chromatography test paper, with cutting cutter, the plastic plate pasting is longitudinally cut into the wide immunity test strip of 4mm, dry lower envelope, 4-25 ℃ keeps in Dark Place;
9, the wide immunity test strip sample pad one end of 4mm is inserted in test serum, after chromatography effect 5 minutes, take out test strips observations under uviol lamp, or under fluorescence spectrophotometer, test the fluorescence spectrum of quantum dot, measure 5 minutes report results of HBsAg strong positive sample, measure 20 minutes report results of weak sun and ' negative ' specimens.If the calibration tape on film has green fluorescence with control band place, two fluorescence, illustrate in testing sample and contain HBsAg, positive result, and stronger at the fluorescence of calibration tape, the content of HBsAg is higher; If only had, control with green fluorescence, i.e. a fluorescence, illustrates and in testing sample, there is no HBsAg; If calibration tape does not all have fluorescence with control band, illustrate that detection is invalid.This detection method method sensitivity is 1ng/ml, and specificity is more than 90%, in batch with criticize between repeatability be less than 20%.
Embodiment 4
1, take 0.051 gram, tellurium powder (0.4mmol), 0.08 gram of sodium borohydride (2mmol), adds containing in the 10ml distilled water of the abundant deoxygenation of useful nitrogen, obtains the solution of sodium hydrogen telluride;
2, take 0.6714 gram of six perchloric acid hydrate cadmium (1.6mmol), join in the three-necked bottle that contains 100ml oxygen-free water, then add mercaptopropionic acid 342 microlitres (3.84mmol), with 1mol/L sodium hydroxide solution, regulate pH value between 10-11;
3, get in the three-necked bottle in the solution implantation step 2 of sodium hydrogen telluride of 1 step synthesized, be warming up to 100 ℃, synthetic CdTe bare nucleus quantum dot, what the time of reacting by change obtained within 1-10 hour that bare nucleus size is greater than 4nm contains red light quantum point red tan solution;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, prepare the husk as raw material of sulphur, according to article J.Phys.Chem.C2008, the experimental procedure of 112,8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, preparation CdTe/CdS core-shell quanta dots;
5, by 1 μ molCdTe/CdS core-shell quanta dots with at 100 μ molEDC condensation agents, mix, the carboxyl group on activation quantum dot surface, stirring at room 30 minutes, then add 1 μ mol albumin A, stirring at room is reacted 2 hours, obtains the covalently bound product of quantum dot-albumin A with the separation and purification of NAP-5 pillar;
6, the covalently bound product of quantum dot-albumin A is mixed with hepatitis B surface antibody (HBsAb) equal proportion, stirring at room 1 hour, obtains the flexible coupling marked product of quantum dot-albumin A-HBsAb;
7, the flexible coupling marked product of quantum dot-albumin A-HBsAb is sprayed on equably on glass fibre element film, obtains scribbling the glass fibre element film of marked product, dry rear envelope, places 4 ℃ and saves backup; On nitrocellulose filter, with machine, draw two parallel bands, bandwidth is controlled at 2mm left and right, and two band interval 8mm wherein spray HBsAb and form calibration tape on a band; On another band, spray goat anti-human igg's formation control band, room temperature was dried after 30 minutes, obtained the nitrocellulose filter of parallel coated antibody, then put into the phosphate buffer that contains bovine serum albumin(BSA) and seal 60 minutes, envelope after dry, places 4 ℃ and saves backup.
8, on one side plastic plate, paste successively upper glass cellulose membrane, scribble glass fibre element film, the water adsorption glass cellulose membrane of marked product, the nitrocellulose filter of parallel coated antibody, finally stick long 30 centimetres, the thieving paper of wide 2 centimetres, absorb unnecessary solution after chromatography, between film and film, all will closely be connected, make the large plate of immune chromatography test paper, with cutting cutter, the plastic plate pasting is longitudinally cut into the wide immunity test strip of 4mm, dry lower envelope, 4-25 ℃ keeps in Dark Place;
9, the wide immunity test strip sample pad one end of 4mm is inserted in test serum, after chromatography effect 20 minutes, take out test strips observations under uviol lamp, or under fluorescence spectrophotometer, test the fluorescence spectrum of quantum dot, measure 5 minutes report results of HBsAg strong positive sample, measure 20 minutes report results of weak sun and ' negative ' specimens.If the calibration tape on film has red fluorescence with control band place, two fluorescence, illustrate in testing sample and contain HBsAg, positive result, and stronger at the fluorescence of calibration tape, the content of HBsAg is higher; If only had, control with red fluorescence, i.e. a fluorescence, illustrates and in testing sample, there is no HBsAg; If calibration tape does not all have fluorescence with control band, illustrate that detection is invalid.This detection method method sensitivity is 1ng/ml, and specificity is more than 90%, in batch with criticize between repeatability be less than 20%.
Embodiment 5
1, take 0.051 gram, tellurium powder (0.4mmol), 0.08 gram of sodium borohydride (2mmol), adds containing in the 10ml distilled water of the abundant deoxygenation of useful nitrogen, obtains the solution of sodium hydrogen telluride;
2, take 0.6714 gram of six perchloric acid hydrate cadmium (1.6mmol), join in the three-necked bottle that contains 100ml oxygen-free water, then add mercaptopropionic acid 342 microlitres (3.84mmol), with 1mol/L sodium hydroxide solution, regulate pH value between 10-11;
3, get in the three-necked bottle in the solution implantation step 2 of sodium hydrogen telluride of 1 step synthesized, be warming up to 100 ℃, synthetic CdTe bare nucleus quantum dot, the time of reacting by change obtained the quantum dot of different size size within 15-600 minute, according to the difference of quantum point grain diameter size, obtain bare nucleus size and be less than the green glow core-shell quanta dots of 3nm and the ruddiness light core-shell quanta dots that bare nucleus size is greater than 4nm;
4, repeating step 2, prepare the husk as raw material of cadmium; Nine hydrated sodium sulfide powder are dissolved in oxygen-free water, the husk as raw material of preparing sulphur, according to article J.Phys.Chem.C2008,112, the experimental procedure of 8587 – 8593 is utilized continuous ionic layer adsorption reaction technology to carry out shell and is coated, and prepares bare nucleus size and is less than the green glow CdTe/CdS core-shell quanta dots of 3nm and the ruddiness CdTe/CdS core-shell quanta dots that bare nucleus size is greater than 4nm;
5, respectively 1 μ mol bare nucleus size is less than to the green glow core-shell quanta dots of 3nm and ruddiness core-shell quanta dots that 1 μ mol bare nucleus size is greater than 4nm and mixes at 100 μ molEDC condensation agents, the carboxyl group on activation quantum dot surface, stirring at room 30 minutes, then add respectively 1 μ mol Protein G, stirring at room reaction 2 hours, obtains the covalently bound product of green light quantum point-Protein G and the covalently bound product of red light quantum point-Protein G with the separation and purification of NAP-5 pillar;
6, the covalently bound product of green light quantum point-Protein G is mixed with hepatitis B surface antibody (HBsAb) equal proportion, the covalently bound product of red light quantum point-Protein G mixes with hepatitis B e antibody (HBeAb) equal proportion, stirring at room 1 hour, obtains the flexible coupling marked product of green light quantum point-Protein G-HBsAb and the flexible coupling marked product of green light quantum point-Protein G-HBsAb;
7, after the flexible coupling marked product equal proportion of the flexible coupling marked product of green light quantum point-Protein G-HBsAb and green light quantum point-Protein G-HBsAb is mixed, be sprayed on equably on glass fibre element film, obtain scribbling the glass fibre element film of green glow and red light quantum point marked product, envelope after dry, places 4 ℃ and saves backup; On nitrocellulose filter, with machine, draw two parallel bands, bandwidth is controlled at 2mm left and right, and two band interval 8mm wherein spray equivalent HBsAb and HBeAb and form calibration tape on a band; On another band, spray goat anti-human igg's formation control band, room temperature was dried after 30 minutes, obtained the nitrocellulose filter of parallel coated antibody, then put into the phosphate buffer that contains bovine serum albumin(BSA) and seal 60 minutes, envelope after dry, places 4 ℃ and saves backup;
8, on one side plastic plate, paste successively upper glass cellulose membrane, scribble glass fibre element film, the water adsorption glass cellulose membrane of green glow and red light quantum point marked product, the nitrocellulose filter of parallel coated antibody, finally stick long 30 centimetres, the thieving paper of wide 2 centimetres, unnecessary solution after absorption chromatography, between film and film, all to closely be connected, make the large plate of immune chromatography test paper, with cutting cutter, the plastic plate pasting is longitudinally cut into the wide immunity test strip of 4mm, dry lower envelope, 4-25 ℃ keeps in Dark Place;
9, the wide immunity test strip sample pad one end of 4mm is inserted in test serum, after chromatography effect 15 minutes, take out test strips and under fluorescence spectrophotometer, test the fluorescence spectrum of quantum dot, measure 5 minutes report results of HBsAg and HBeAg strong positive sample, measure 20 minutes report results of weak sun and ' negative ' specimens.If the calibration tape on film has green and red fluorescence with control band place, two fluorescence, illustrate in testing sample and contain HBsAg and HBeAg, positive result, and stronger at the fluorescence of calibration tape, the content of HBsAg and HBeAg is higher; If the calibration tape on film has fluorescence with control band place, i.e. two fluorescence, on calibration tape, only has green glow, illustrate in testing sample and contain HBsAg, positive result, only has ruddiness on calibration tape, illustrate and in testing sample, contain HBeAg, positive result, stronger at the fluorescence of calibration tape, the content of HBsAg and (or) HBeAg is higher; If only had, control with fluorescence, i.e. a fluorescence, illustrates and in testing sample, there is no HBsAg and HBeAg; If calibration tape does not all have fluorescence with control band, illustrate that detection is invalid.This detection method method sensitivity is 1ng/ml, and specificity is more than 90%, in batch with criticize between repeatability be less than 20%.
Claims (7)
1. the detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip, is characterized in that, comprises as follows:
Step 1: prepare core-shell quanta dots;
Step 2: the core-shell quanta dots that step 1 is obtained and Protein G or albumin A are undertaken covalently bound by the mode of EDC condensation, obtain covalently bound product;
Step 3: the covalently bound product that step 2 is obtained mixes with an antibody molecule of antigen molecule to be detected, obtains the marked product of the flexible coupling of quantum dot antibody;
Step 4: the marked product of the flexible coupling of quantum dot antibody that step 3 is obtained is coated on glass fibre element film, obtain scribbling the glass fibre element film of marked product, another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter are formed to calibration tape and control band, obtain the nitrocellulose filter of parallel coated antibody;
Step 5: on one side polyester or plastic plate, paste successively upper glass cellulose filter membrane, scribble glass fibre element film, the water-absorption fiber element film of marked product, the nitrocellulose filter of parallel coated antibody, the polyester after pasting or plastic plate are made and are obtained immuno-chromatographic test paper strip;
Step 6: the pad end of the immuno-chromatographic test paper strip that step 5 is obtained inserts in testing sample solution, takes out test strips after immunochromatography, determines testing result.
2. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, is characterized in that, described core-shell quanta dots is CdTe/CdS, CdSe/CdS, CdSe/ZnSe or CdSe/ZnS core-shell quanta dots.
3. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, is characterized in that, the mol ratio of described covalently bound product and an antibody molecule of antigen molecule to be detected is 1:1.
4. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, is characterized in that, in described step 4, calibration tape and control band be arranged in parallel, calibration tape and the 8-12mm that is spaced apart that controls band.
5. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, it is characterized in that, described forms another antibody of antigen molecule to be detected and two anti-being sprayed on respectively on nitrocellulose filter after calibration tape and control band, room temperature was dried after 20-30 minute, put into again the phosphate buffer that contains bovine serum albumin(BSA) and seal 60-120 minute, envelope after dry, places 4 ℃ and saves backup.
6. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, is characterized in that, described testing sample solution is blood, urine or saliva.
7. a kind of detection method based on the flexible coupling label of core-shell quanta dots immuno-chromatographic test paper strip according to claim 1, it is characterized in that, in described step 6, after immunochromatography, take out after test strips, be specially: by test strips visual inspection result under uviol lamp, or obtain and detect the curve of spectrum under fluorescence spectrophotometer, by contrasting with typical curve, determine testing result.
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