CN102269758A - Method for visual detection of antigen-antibody reaction, kit and application thereof - Google Patents
Method for visual detection of antigen-antibody reaction, kit and application thereof Download PDFInfo
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- G01—MEASURING; TESTING
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- 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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The invention relates to a method for visual detection of an antigen-antibody reaction. The method is specifically characterized in that: copper oxide nanoparticle-labeled antibodies react with a sample requiring detection; surface functionalized gold nanoparticles are adopted to detect the copper oxide nanoparticles labeled on the antibodies so as to provide information related to the antigen-antibody reaction, and provide a new tool for the disease detection and the disease diagnosis based on the immunological reaction. With the method provided by the present invention, the detection can be completed by the naked eye without requiring any equipment, and the method is applicable for field work. The invention further provides an application of the method. The invention further provides a kit for the visual detection of the antigen-antibody reaction, and an application thereof.
Description
Technical field
The invention belongs to immunoassay and diagnostic techniques field.Particularly, the present invention relates to the method that the antagonist marking signal amplifies, particularly adopt a kind of surface-functionalized golden nanometer particle, can directly with the naked eye judge antigen-antibody reaction by detecting the antibody labeling thing.
Background technology
When some antigen or specific proteins are detected, can be applied to immunolabelling technique usually.Immunolabelling technique is that not only easily mensuration but also material mark with hypersensitivity were on specific antigen or antibody molecule with some, and the enhancing amplification by these labels shows the character and the content of antigen in the reaction system or antibody.Label commonly used at present comprises fluorescein, enzyme and radioactive nuclide etc.But in actual applications, all there are different defectives in this three big immunolabelling technique.For example in fluorescein-labelled technology, there is the problem of fluorescence lifetime and fluorescence efficiency in fluorescein, and the method complexity of mark on antigen or the antibody; In the enzyme labeling technology, the easy inactivation of enzyme, thus influence the detection limit of determinand; In the isotope labelling technique, nucleic has radioactivity, has strong environmental pollution and health hazard.In addition, adopt above three kinds of methods when some antigen or specific proteins are detected, on playback mode, need just can carry out by each quasi-instrument.Wherein, fluorescein-labelled Technology Need fluorescent microscope, enzyme mark Technology Need microplate reader, detection instruments such as radioactive nuclide Technology Need auto-counter have determined a lot of detections not fall behind and the under-developed area be carried out in condition to the dependence of instrument.Therefore, exist now to simple to operate, reaction system is stablized, be convenient to carry out, environmentally safe and the demand of human body not being had the immune labeled and detection technique of harm.
At present, nanometer technology is current popular research field, and wherein golden nanometer particle is widely used in visual detection owing to its surface plasma resonance effect shows unique color.Mirkin group reported first with the method for surface-functionalized golden nanometer particle by colorimetric detect DNA (list of references: Mirkin, C.A., Letsinger, R.L., Mucic, R.C., Storhoff, J.J.Nature, 1996,382,607-609).Jiang Xing space group reported by the complex functionality golden nanometer particle, utilizes click to react to detect the copper ion (list of references: Yang Zhou, Shixing Wang in the solution, Ke Zhang, XingyuJiang*.Angew.Chem.Int.Ed.2008,47,7454-7456).
Summary of the invention
An object of the present invention is to provide a kind of method of visual detection antigen-antibody reaction, this method adopts surface-functionalized golden nanometer particle, realize the antagonist label, especially to the detection of label copper oxide nanometer particle, and then realize detection to antigen-antibody reaction.Another object of the present invention provides the application of described method.Another purpose of the present invention provides a kind of kit that is used for visual detection antigen-antibody reaction.A further object of the present invention provides the application of described kit.
Be used to realize that the technical scheme of above-mentioned purpose is as follows:
On the one hand, the invention provides a kind of method of visual detection antigen-antibody reaction, this method may further comprise the steps:
1) antigen-antibody reaction: the antibody of copper oxide nanometer particle mark is joined in the detected sample, and fully the antibody of unreacted unnecessary copper oxide nanometer particle mark is removed in the reaction back;
2) visual detection: in the step 1) reaction system, add acid, with the Cu in the copper oxide nanometer particle
2+Discharge; After the reaction, add reductive agent, the golden nanometer particle of surperficial alkynyl functionalization and the golden nanometer particle of surperficial azido functionalization, fully whether the reaction back is according to reacting in the reaction system change in color judgement sample.
Preferably, in described method, before step 1), also comprise the step that detected sample is handled, the purpose of processing be for the antibody that makes mark can with the abundant and stable reaction of sample.
Preferably, before step 1), also comprise the step that detected sample is sealed; Preferably, described sealer is selected from hyclone, lowlenthal serum, horse serum; Further preferably, described sealer is a hyclone.Wherein, preferably, in step 2) in, the capping time is 30～120 minutes, closure temperature is 25～40 ℃; Further preferably, the capping time is 60 minutes, and closure temperature is 37 ℃.
Preferably, in step 1), the antibody of copper oxide nanometer particle mark is one anti-;
Preferably, in step 1), the antibody of copper oxide nanometer particle mark is two anti-;
Preferably, in step 1), the incubation time of antigen-antibody reaction is 30～120 minutes, and incubation temperature is 25～40 ℃; Further preferably, incubation time is 60 minutes, and incubation temperature is 37 ℃.
Preferably, in step 2) in, the acid of adding is selected from HCl, HNO
3, H
2SO
4, HCl more preferably.
Preferably, in step 2) in, the concentration of the acid of adding in reaction system is 0.1～1mM, the acid reaction time is 5～20 minutes; Further preferably, the concentration 1mM of described acid in reaction system, the acid reaction time is 10 minutes.
Preferably, in step 2) in, the reductive agent of adding is selected from sodium ascorbate, sodium citrate, sodium borohydride, more preferably sodium ascorbate.
Preferably, in step 2) in, the concentration of the reductive agent of adding in reaction system is 0.01～0.1M, further preferably, the concentration of described reductive agent in reaction system is 0.05M.
Preferably, in step 2) in, the alkynyl of the golden nanometer particle of the surperficial alkynyl functionalization of adding is the compound that comprises Formula I:
The azido of the golden nanometer particle of the surperficial azido functionalization that adds is the compound that comprises Formulae II:
Preferably, in step 2) in, the mol ratio of the golden nanometer particle of the surperficial alkynyl functionalization of adding and the golden nanometer particle of surperficial azido functionalization is 1: 0.5～1: 2, further preferably, mol ratio is 1: 1.
Preferably, in step 2) in, reductive agent and two kinds of functionalization golden nanometer particles provide simultaneously with the form of detection architecture.Preferably, the concentration of reductive agent in detection architecture is 0.01～0.1mM, more preferably 0.05mM.
Preferably, in step 2) in, the change color of described reaction system and deposited phenomenon occurred in the clock time at 5～30 minutes, further preferably occurred in the clock time at 10～15 minutes.
Preferably, in described method, detected sample comprises the clinical detection sample, and further preferably, detected sample is from suffering from or the patient who suffers from certain disease under a cloud.More preferably, described disease is an immune correlated disease, the immune correlated disease that causes as virus.
On the other hand, the invention provides of the application of described method in fields such as biology, biomedicine, medical tests.
Preferably, the invention provides described method in the detection of immune correlated disease and the application in the diagnosis, further preferably, the immune correlated disease that described immune correlated disease causes for virus.
Preferably, the invention provides described method is used for the reagent of disease detection and diagnosis in preparation application.Further preferably, described disease is an immune correlated disease; More preferably, described immune correlated disease is the viral immune correlated disease that causes.
Another aspect the invention provides a kind of kit that is used for visual detection antigen-antibody reaction, and this kit comprises: the golden nanometer particle of reductive agent, surperficial alkynyl functionalization and the golden nanometer particle of surperficial azido functionalization.
Preferably, the antibody that also comprises the copper oxide nanometer particle mark in the described kit.
Preferably, also comprise copper oxide nanometer particle in the described kit.
Preferably, also comprise antibody in the described kit.Further preferably, described antibody is one anti-or two anti-.
Preferably, the reductive agent in the described kit is selected from sodium ascorbate, sodium citrate, sodium borohydride, more preferably sodium ascorbate.
Preferably, also comprise sealer in the described kit; Further preferably, described sealer is selected from hyclone, lowlenthal serum, horse serum; More preferably, described sealer is a hyclone.
Preferably, also comprise one or more of following reagent in the described kit: positive reference substance, negative control product, sample diluting liquid, damping fluid etc.
On the one hand, the invention provides of the application of described kit again in fields such as biology, biomedicine, medical tests.
Preferably, the invention provides the application of described kit in disease detection and diagnosis.Further preferably, described kit is applied in the detection and diagnosis of immune correlated disease, more preferably, and the immune correlated disease that described immune correlated disease causes for virus.
The principle of described visual detection antigen-antibody reaction is: when existing as antigen that detects target or antibody in the detected sample, mark the antibody of nano cupric oxide particle combine with antigen or antibody in the detected sample, the copper oxide nanometer particle of mark on the antibody discharges Cu because of the adding of acid solution
2+, add reductive agent with Cu
2+Be reduced into Cu (I), Cu (I) makes terminal alkynyl and azido generation annulation (Fig. 1) on the functionalization golden nanometer particle at normal temperatures and pressures as catalyzer, assemble and precipitation thereby golden nanometer particle is reacted and produce, and then become bluish violet from redness by visual inspection detection reaction system, further when golden nanometer particle is seriously assembled, have the appearance of precipitation, thereby realize detection antigen-antibody reaction in the detected sample.In addition, this paper also provides the preparation method of copper oxide nanometer particle labelled antibody, and hereinafter detailed Description Of The Invention is partly specifically described.
Compared with prior art, the present invention has the following advantages at least:
1, the form of reading of the inventive method need be by means of any instrument, and directly naked eyes read testing result, has broken away from present usually three big immunoassay technologies of employing to the dependence of surveying instrument.
2, in detection reaction, part to the functionalization gold nano grain improves, and makes the gold nano grain detection architecture more stable, and is lower to the detection limit that is marked at the copper oxide nanometer particle on the antibody, 1 μ M can be reached, within 10 minutes, just detection can be finished.
3, method of the present invention and kit do not need expensive instrument, and testing process is simple, and cost is low, and good stability is convenient to carry out, and are expected to improve the testing conditions of backward under-developed areas such as Africa.
Below be detailed description of the present invention:
The objective of the invention is to set up a kind of new method of visual detection antigen-antibody reaction, by make mark earlier the antibody and the detected sample of copper oxide nanometer particle react, adopt surface-functionalized golden nanometer particle detection architecture then, the nano cupric oxide particle that is marked on the antibody is detected, thereby provide information, for providing a kind of new tool based on immunoreactive disease detection and diagnosis about antigen-antibody reaction.
The antibody of employed copper oxide nanometer particle mark can provide by the following method among the present invention:
Take by weighing the commercially available copper oxide nanometer particle of 1～5mg and (purchase company in Sigma, particle diameter is less than 50nm), add in 1～5mL PBS damping fluid, ultrasonic dispersion 10～20 minutes, be that antibody diluent 50～500 μ L of 0.1～0.4mg/mL dropwise added in 3 minutes and contain in the solution of copper oxide nanometer particle then with concentration, behind the low-speed oscillation 2～4 hours, centrifugal 5～15 minutes of 8000～11000rpm, remove and contain the supernatant that does not have mark copper oxide nanometer particle antibody, and then the cupric oxide in the centrifuge tube disperseed again with 1～5mL PBS damping fluid, and in solution, add the BSA solution of 20～200 μ L 10%, BSA solution plays function of stabilizer here, stirs centrifugal 5～15 minutes of 5000～8000rpm 30～60 minutes, remove the antibody that precipitation obtains the copper oxide nanometer particle mark, 4 ℃ of preservations are stand-by.
The exemplary technical scheme that the present invention adopts is as follows:
1) antigen-antibody reaction
Adding concentration in 96 orifice plates is the antigen 1 00 μ L of 0.01～0.05mg/mL, 4 ℃ are spent the night, use 200 μ L PBST washing lotions (the PBS damping fluid that contains 0.1% tween) to wash plate three times then, the hyclone that every hole adds 200 μ L 5% seals as sealer, hatches 1～2 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds the antibody 100 μ L through the copper oxide nanometer particle mark, hatches 1～2 hour for 37 ℃.Wash plate three times with 200 μ L deionizations then, finish the antigen-antibody immune response.
2) visual detection
The HCl solution that adds 20 μ L, 0.1～1mM in the hole will be marked at the Cu in the copper oxide nanometer particle on the antibody by the acid-base neutralization reaction
2+Discharge, react after 10 minutes, add surface-functionalized golden nanometer particle detection architecture (the golden nanometer particle surface has alkynyl and azido respectively), the reductive agent in the detection architecture is with Cu
2+Be reduced into Cu (I), Cu (I) makes the terminal alkynyl and the azido generation annulation of golden nanometer particle at normal temperatures and pressures as catalyzer, produce accumulation and deposited phenomenon thereby the golden nanometer particle that makes functionalization reacts, and then can realize Cu in the solution by the variation of visual inspection golden nanometer particle color and deposited phenomenon
2+Detection, the detection by antagonist copper oxide surface nanoparticle label thing realizes the detection to antigen-antibody reaction indirectly.
Beneficial effect of the present invention is:
1, compare with the detection method of existing antibody labeling thing, the present invention need implement not detect by exact instrument, only can finish qualitative detection to the antigen-antibody mark with naked eyes.
2, employed part to the functionalization gold nano grain (alkynyl and azido) is a kind of new Novel Ligands synthetic, that be used for golden nanometer particle in detection reaction, these two kinds of parts make that the gold nano grain detection architecture is more stable, when existing, copper ion is easy to reaction between the part, lower to the detection limit that is marked at the copper oxide nanometer particle on the antibody, 1 μ M can be reached, and within 10 minutes, just detection can be finished.
3, method of the present invention and kit do not need expensive instrument, and testing process is simple, and cost is low, and good stability is convenient to carry out, and are expected to improve the testing conditions of backward under-developed areas such as Africa.
The inventive method is a kind of method of new detection antigen-antibody reaction, can be applied to diagnosis and detection based on immunoreactive major disease, HBV for example, AIDS etc., simultaneously can be made into kit, realize commercialization,, be expected to improve the conditions for diagnostics of backward under-developed areas such as Africa because the cost of whole testing process is low.
Description of drawings
Below, describe embodiments of the invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1: under Cu (I) catalysis, the synoptic diagram of terminal alkynyl on the functionalization golden nanometer particle and azido generation annulation.
Fig. 2: mass spectrum of compound 6 (Fig. 2 A) and infared spectrum (Fig. 2 B).
Fig. 3: mass spectrum of compound 9 (Fig. 3 A) and infared spectrum (Fig. 3 B).
Fig. 4: when adopting method of the present invention that antigen-antibody reaction is detected, the ultra-violet absorption spectrum of detection architecture generation change color.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail.Be to be understood that the embodiment that provides only for to preparation with use ad hoc approach of the present invention to describe, rather than in order to limit the scope of the invention.
Main agents used in the present invention or raw material to be purchased approach as shown in the table:
All the other unlisted common reagent are all purchased in the Beijing Chemical Plant, and specification is AR, do not need to do being further purified processing.
Embodiment 1: compound 1 synthetic
Step:
1, the two mouthfuls of flasks (19#) with 100mL vacuumize, and charge into nitrogen.
2, in this flask, add 50mL toluene under the nitrogen protection; with triphenylchloromethane (4.60g; 16.5mmol) and N; N-diisopropylethylamine (DIEA; 4.20g, 33.0mmol) be dissolved in this toluene the inside, add 11-sulfydryl undecanoic acid (3.00g under the magnetic agitation; 13.8mmol), under the nitrogen protection room temperature reaction 5 hours.
3, reaction is finished, and the solvent decompression is steamed, and adds the 50mL methylene chloride in the product of remnants, and fully after the dissolving, (3 * 100mL) give a baby a bath on the third day after its birth inferiorly, use the anhydrous sodium sulfate drying dichloromethane solution again, standing over night with saturated aqueous common salt.
4, filter to remove the drying agent anhydrous sodium sulfate excessively,, concentrate the crude product that obtains compound 1 the filtrate decompression distillation.After dissolving this crude product with a little methylene chloride, carry out purifying with the method for column chromatography.Eluant, eluent is a sherwood oil: ethyl acetate=10: 1, and obtaining pure product at last is colourless oil liquid, is total to 5.80g (12.6mmol), productive rate is 92%.
Embodiment 2: compound 2 synthetic
Step:
1, in the single port flask (19#) of a 50mL, adds compound 1 (1.50g, 3.3mmol), EDC-HCl (0.69g, 3.6mmol) DMAP of catalytic amount, add the 25mL anhydrous methylene chloride, magnetic agitation makes it dissolving, adds N-hydroxy-succinamide (NHS at last in this mixed liquor, 0.45g, 3.9mmol).
2, this mixed solution stirred one hour at 5 ℃, at room temperature reacted subsequently 24 hours.
3, reaction is finished, and with 25mL methylene chloride dilute reaction solution, (3 * 50mL) give a baby a bath on the third day after its birth time, use the anhydrous sodium sulfate drying organic phase, concentratedly obtain compound 2 (1.80g, 3.23mmol), this compound is the Acibenzolar of compound 1 to use saturated aqueous common salt again.Productive rate is 99%.
Embodiment 3: compound 3 synthetic
Step:
1, with NH
2C
2H
4OC
2H
4OC
2H
4NH
2(8mL 55mmol) is dissolved in the anhydrous methylene chloride, and (after dropwising, at room temperature reaction is spent the night again for 1.80g, dichloromethane solution 10mL 3.23mmol) slowly to drip compound 2 under stirring in this mixed solution.
2, with 50mL methylene chloride dilute reaction solution, (3 * 50mL) wash organic phase three times, use the anhydrous sodium sulfate drying organic phase, and concentrating under reduced pressure obtains the crude product of compound 3 to use saturated aqueous common salt again.
3, with going up sample behind a little methylene chloride dissolving crude product, carry out column chromatography for separation, eluant, eluent is a chloroform: methyl alcohol: ammoniacal liquor=20: 1: 0.05, and obtaining pure product is colourless oil liquid, is total to 1.78g (3.0mmol), productive rate is 94%.
Embodiment 4: compound 4 synthetic
Step:
1, (490.5mg, 5mmol), (690mg, 6mmol), DMAP (100mg) is dissolved among the anhydrous DCM of 30mL NHS, stirs to add EDC (1.15g, 6mmol) 5 ℃ of reaction 1h, stirred overnight at room temperature down with the 4-pentinoic acid.
2, reaction finishes, organic phase with saturated aqueous common salt (3 * 50mL) give a baby a bath on the third day after its birth time, use anhydrous sodium sulfate drying, revolve and desolvate, must yellow oily liquid, productive rate is 99%.
Embodiment 5: compound 5 synthetic
Step:
1, (1g 5mmol) is dissolved among the anhydrous DCM of 30mL, and (3.25g 5.5mmol), stirs under the room temperature and spends the night to add compound 3 under stirring with compound 4.
2, reaction finishes, and concentrates thick product, carries out purifying with the method for column chromatography, and eluant, eluent is that (chloroform: methyl alcohol=50: 1), obtaining pure product is white powder, is total to 3.0g, and productive rate is 79%.
Embodiment 6: compound 6 synthetic
Step:
1, the two mouthfuls of flasks (19#) with 25mL vacuumize, and charge into nitrogen.
2, under the nitrogen protection 0.5mL trifluoroacetic acid (TFA) is added in the 9.5mL anhydrous methylene chloride, obtain the trifluoroacetic acid solution of 5% (v/v).Stir in this solution, add down compound 5 (2.01g, 3mmol), add subsequently triethyl silicane (TESi, 2.3mL, 15mmol), stirred overnight at room temperature.
3, reaction finishes, reaction finishes, and (3 * 5mL) give a baby a bath on the third day after its birth inferiorly, and (2 * 5mL) wash twice, anhydrous sodium sulfate drying to use saturated aqueous common salt then with the sodium hydroxide solution of 0.2M in the back, decompression distillation removes and desolvates, residue is gone up sample with a little methylene chloride dissolving back, carry out column chromatography for separation, eluant, eluent is a chloroform: methyl alcohol=50: 1, obtain white solid, be total to 960mg, productive rate is 77%, and compound 6 is the alkynyl part of functionalization golden nanometer particle.Wherein, the mass spectrogram of compound 6 is seen Fig. 2 A, and infrared spectrum is seen Fig. 2 B.
Embodiment 7: compound 7 synthetic
Step:
1, (1.5g 9.8mmol) joins in the 10mL acetonitrile, and (1.27g, 19.6mmol), 70 ℃ were refluxed 6 hours to add Sodium azide under stirring with the 3-bromo-propionic acid.
2, after reaction finishes, add 30mL DCM dilution, the HCl that adds 50mL 0.1M washes, and uses chloroform extraction then, and the organic phase anhydrous sodium sulfate drying revolves to steam to remove and desolvates, and obtains brown liquid.Productive rate is 99%.
Embodiment 8: compound 8 synthetic
Step:
1, with compound 7 (654mg, 5.7mmol) and compound 3 (2.8g 4.75mmol) is dissolved among the anhydrous DCM of 40mL, adds EDC (1.09g, 5.7mmol) and DMAP (100mg) then, stirred overnight at room temperature.
2, reaction finishes, with saturated common salt wash organic phase (2 * 15mL) twice, use the chloroform extraction water then, merge organic phase, anhydrous sodium sulfate drying, thick product carries out column chromatography for separation, and eluant, eluent is a chloroform: methyl alcohol=20: 1, obtain white solid, be total to 2.82g, productive rate is 86%.
Embodiment 9: compound 9 synthetic
Step:
1, the two mouthfuls of flasks (19#) with 25mL vacuumize, and charge into nitrogen.
2, under the nitrogen protection 0.5mL trifluoroacetic acid (TFA) is added in the 9.5mL anhydrous methylene chloride, obtain the trifluoroacetic acid solution of 5% (v/v).Stir in this solution, add down compound 8 (1.93g, 2.8mmol), add subsequently triethyl silicane (TESi, 2.3mL, 15mmol), stirred overnight at room temperature.
3, reaction finish the back with the sodium hydroxide solution of 0.2M (3 * 5mL) give a baby a bath on the third day after its birth inferior, (2 * 5mL) wash twice to use saturated aqueous common salt then, anhydrous sodium sulfate drying, decompression distillation removes and desolvates, and residue is gone up sample with a little methylene chloride dissolving back, carry out column chromatography for separation, eluant, eluent is a chloroform: methyl alcohol=50: 1 obtains white solid, altogether 830mg, productive rate is 68%, and compound 9 is the azido part of functionalization golden nanometer particle.Wherein, the mass spectrogram of compound 9 is seen Fig. 3 A, and infrared spectrum is seen Fig. 3 B.
Embodiment 10: golden nanometer particle synthetic
41.2mg gold chloride (99.95%) is dissolved in the 100mL water, be heated to boiling under the stirring, the 114mg sodium citrate is dissolved in 10mL water, join in the chlorauric acid solution of boiling then rapidly, solution becomes colorless from yellow, arrives claret again to purple, continues heated and stirred 15 minutes, naturally cool to stirring at room 2 hours, and got the golden nanometer particle of the stable claret of sodium citrate.
Embodiment 11: alkynyl functionalization golden nanometer particle synthetic
The stable golden nanometer particle 1.5mL of sodium citrate will be made, being diluted to 10mL solution with deionized water, dripping the sodium hydroxide solution that concentration is 0.5M then, is 9 until regulating the pH value, under the stirring, the mercaptan part (compound 6,0.5 μ mol) that adds co-stabilizer (11-mercaptoalkyl polyglycol (3), 3 μ mol) and terminal alkynyl functionalization simultaneously, stirred 24 hours, centrifuging 20 minutes through washing three times, obtains the golden nanometer particle of surperficial alkynyl functionalization again after the centrifuging.
Embodiment 12: azido functionalization golden nanometer particle synthetic
The stable golden nanometer particle 1.5mL of sodium citrate will be made, be diluted to 10mL solution with deionized water, drip the sodium hydroxide solution that concentration is 0.5M then, until regulating the pH value is 9, under the stirring, add co-stabilizer (11-mercaptoalkyl polyglycol (3) simultaneously, 3 μ mol) and the mercaptan part (compound 9 of terminal azido functionalization, 0.5 μ mol), stirred 24 hours, centrifuging 20 minutes through washing three times, obtains the golden nanometer particle of surperficial azido functionalization again after the centrifuging.
Embodiment 13: the preparation of functionalization golden nanometer particle detection architecture
The alkynyl functionalization solution of gold nanoparticles for preparing and azido functionalization solution of gold nanoparticles are respectively got 1mL to join in the centrifuge tube, adding concentration in solution is the reductive agent sodium ascorbate 20 μ L of 0.05M, sonic oscillation 5 minutes promptly obtains the detection architecture of functionalization golden nanometer particle.
Embodiment 14: functionalization golden nanometer particle detection architecture is to the detection of copper ion
Get 7 vials, in each bottle, add 1mL functionalization golden nanometer particle detection architecture solution, adding concentration then in bottle successively is the copper-bath 10 μ L of 10mM, 1mM, 100 μ M, 10 μ M, 1 μ M, 100nM and 10nM, after 10 minutes in the bottle copper ion concentration change color has all taken place greater than the detection architecture of 1 μ M (comprising 1 μ M), become bluish violet by redness, detect and be limited to 1 μ M.
Embodiment 15: the anti-OVA of usefulness copper oxide nanometer particle mark rabbit (chicken ovalbumin, ovalbumin,
OVA) antibody
Take by weighing the commercially available copper oxide nanometer particle of 1mg and (purchase company in Sigma, particle diameter is less than 50nm), add in the 1mL PBS damping fluid, ultrasonic dispersion 10 minutes, the anti-OVA antibody of rabbit that with concentration is 0.2mg/mL then (is purchased the company in Bo Aosen, product article No. bs-0283R) dilution 500 μ L dropwise added in 3 minutes and contain in the solution of copper oxide nanometer particle, behind the low-speed oscillation 3 hours, centrifugal 10 minutes of 10000rpm, remove and contain the supernatant that does not have mark copper oxide nanometer particle antibody, and then the cupric oxide in the centrifuge tube disperseed again with 1.5mL PBS damping fluid, and in solution, add the BSA solution of 200 μ L10%, BSA solution plays function of stabilizer here, stirred 30 minutes, centrifugal 10 minutes of 5000rpm removes the excessive oxidation copper that is not marked on the antibody, obtain the anti-OVA antibody of the rabbit complex of copper oxide nanometer particle mark, 4 ℃ of preservations are stand-by.
The anti-OVA antibody response of the rabbit of embodiment 16:OVA antigen and cupric oxide mark
The OVA antigen that adds concentration and be 0.02mg/mL in 96 orifice plates (is purchased the company in Sigma, article No. is A 5503) 100 μ L, 4 ℃ are spent the night, use 200 μ L PBST washing lotions (the PBS damping fluid that contains 0.1% tween) to wash plate three times then, the hyclone that every hole adds 200 μ L 5% seals as sealer, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds the anti-OVA antibody 100 μ L of rabbit through the copper oxide nanometer particle mark, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L deionizations then, remove the anti-OVA antibody of rabbit of unreacted unnecessary cupric oxide mark, finish the antigen-antibody immune response.Simultaneously, credible in order to ensure follow-up testing result, in finishing the process of antigen-antibody reaction, also added rabbit igg onboard in other holes as negative control, BSA is as irrelevant albumen contrast, and blank.The preparation method is with the anti-OVA antibody response of the rabbit of OVA antigen and cupric oxide mark.The concentration that adds 10 μ L in another hole is that 10 μ M copper ions are as positive control.
Embodiment 17: the visual detection of the anti-OVA antibody of mark copper oxide nanometer particle rabbit
The HCl solution that adds 20 μ L 1mmol/L in the every hole of orifice plate will be marked at the Cu in the copper oxide nanometer particle on the antibody by the acid-base neutralization reaction
2+Discharge, react after 10 minutes, add surface-functionalized golden nanometer particle detection architecture 200 μ L, the reductive agent sodium ascorbate in the detection architecture can be with Cu
2+Be reduced into Cu (I), Cu (I) makes terminal alkynyl and azido generation annulation at normal temperatures and pressures as catalyzer, assembles and deposited phenomenon thereby the golden nanometer particle of functionalization is reacted and produce.React after 10 minutes, by being observed visually, treat that the detection architecture in the gaging hole becomes purple from redness, detection architecture in the positive control hole has become blueness by redness, change color does not take place in the detection architecture in negative control, irrelevant albumen contrast and the blank hole, by the ultra-violet absorption spectrum of microplate reader, can see significantly that red shift (Fig. 4) has taken place the ultraviolet absorption peak for the treatment of gaging hole and positive control hole to each hole.Therefore, realized Cu in the solution
2+Detection, thereby indirect realization to the detection of copper oxide nanometer particle labelled antibody, and this detection can be judged by naked eyes.
Embodiment 18: with copper oxide nanometer particle mark goat anti-rabbit igg
The preparation method of reference example 15 is changed to goat anti-rabbit igg (purchase the company in Bo Aosen, article No. is bs-0295G) with the anti-OVA antibody of rabbit wherein.
Embodiment 19:OVA antigen, the anti-OVA antibody of rabbit with the goat anti-rabbit igg of cupric oxide mark
Reaction
Adding concentration in 96 orifice plates is the OVA antigen 1 00 μ L of 0.02mg/mL, 4 ℃ are spent the night, use 200 μ L PBST washing lotions (the PBS damping fluid that contains 0.1% tween) to wash plate three times then, the hyclone that every hole adds 200 μ L 5% seals as sealer, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds the anti-OVA antibody 100 μ L of rabbit, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds the goat anti-rabbit igg 100 μ L through the copper oxide nanometer particle mark, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L deionizations then, remove the goat anti-rabbit igg of unreacted unnecessary cupric oxide mark, finish antigen, anti-, two anti-immune responses.Simultaneously, credible in order to ensure follow-up testing result, in the processes of finishing antigen, one anti-, two anti-reactions, also added rabbit igg onboard in other holes as negative control, BSA is as irrelevant albumen contrast, and blank.The preparation method is with foregoing antigen, anti-, two preparation methods that resist among this embodiment.The concentration that adds 10 μ L in another hole is that 10 μ M copper ions are as positive control.
Embodiment 20: the visual detection of mark copper oxide nanometer particle goat anti-rabbit igg
The HCl solution that adds 20 μ L 1mmol/L in the every hole of orifice plate will be marked at the Cu in the copper oxide nanometer particle on the goat anti-rabbit igg by the acid-base neutralization reaction
2+Discharge, react after 10 minutes, add surface-functionalized golden nanometer particle detection architecture 200 μ L, the reductive agent sodium ascorbate in the detection architecture can be with Cu
2+Be reduced into Cu (I), Cu (I) makes terminal alkynyl and azido generation annulation at normal temperatures and pressures as catalyzer, assembles and deposited phenomenon thereby the golden nanometer particle of functionalization is reacted and produce.React after 10 minutes, by being observed visually, treat that the detection architecture in the gaging hole becomes purple from redness, detection architecture in the positive control hole has become blueness by redness, change color does not take place in the detection architecture in negative control, irrelevant albumen contrast and the blank hole, by the ultra-violet absorption spectrum of microplate reader, can see significantly that red shift has taken place the ultraviolet absorption peak for the treatment of gaging hole and positive control hole to each hole.Therefore, realized Cu in the solution
2+Detection, thereby indirect realization to the detection of copper oxide nanometer particle mark goat anti-rabbit igg.
Embodiment 21: with copper oxide nanometer particle mark rabbit anti-human igg
The preparation method of reference example 15 is changed to rabbit anti-human igg's (purchase the company in Bo Aosen, article No. is bs-0297R) with the anti-OVA antibody of rabbit wherein.
Rabbit anti-human igg's reaction of embodiment 22:HIV-1 gp41 antigen, human serum and cupric oxide mark
The HIV-1 gp41 antigen that adds concentration and be 0.02mg/mL in 96 orifice plates (is purchased the company in Bo Aosen, article No. is bs-0239P) 100 μ L, 4 ℃ are spent the night, use 200 μ L PBST washing lotions (the PBS damping fluid that contains 0.1% tween) to wash plate three times then, the hyclone that every hole adds 200 μ L5% seals as sealer, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds 10 times of dilutions of HIV positive serum, 100 μ L, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L PBST washing lotions then, every hole adds the rabbit anti-human igg 100 μ L through the copper oxide nanometer particle mark, hatches 1 hour for 37 ℃.Wash plate three times with 200 μ L deionizations then, the rabbit anti-human igg who removes unreacted unnecessary cupric oxide mark finishes antigen, anti-, two anti-immune responses.Simultaneously, credible in order to ensure follow-up testing result, in the processes of finishing antigen, one anti-, two anti-reactions, also added negative serum onboard in other holes as negative control, BSA is as irrelevant albumen contrast, and blank.The preparation method is with foregoing antigen, anti-, two preparation methods that resist among this embodiment.The concentration that adds 10 μ L in another hole is that 10 μ M copper ions are as positive control.
Embodiment 23: mark copper oxide nanometer particle rabbit anti-human igg's visual detection
The HCl solution that adds 20 μ L 1mmol/L in the every hole of 96 orifice plates reacts the Cu that will be marked in the last copper oxide nanometer particle of rabbit anti-human igg by acid-base neutralization
2+Discharge, react after 10 minutes, add surface-functionalized golden nanometer particle detection architecture 200 μ L, the reductive agent sodium ascorbate in the detection architecture can be with Cu
2+Be reduced into Cu (I), Cu (I) makes terminal alkynyl and azido generation annulation at normal temperatures and pressures as catalyzer, assembles and deposited phenomenon thereby the golden nanometer particle of functionalization is reacted and produce.React after 10 minutes, by being observed visually, treat that the detection architecture in the gaging hole becomes purple from redness, the detection architecture in the positive control hole has become blueness by redness, and change color does not take place the detection architecture in negative control, irrelevant albumen contrast and the blank hole.Therefore, realized Cu in the solution
2+Detection, thereby indirect realization to copper oxide nanometer particle mark rabbit anti-human igg's detection.Simultaneously, this embodiment proves that also this detection method can be used for detecting qualitatively the HIV blood serum sample, for the disease detection based on immunity provides a kind of new detection method.
Claims (13)
1. the method for a visual detection antigen-antibody reaction said method comprising the steps of:
1) antigen-antibody reaction: the antibody of copper oxide nanometer particle mark is joined in the detected sample, and fully the antibody of unreacted unnecessary copper oxide nanometer particle mark is removed in the reaction back;
2) visual detection: in the step 1) reaction system, add acid; After the reaction, add reductive agent, the golden nanometer particle of surperficial alkynyl functionalization and the golden nanometer particle of surperficial azido functionalization, fully whether the reaction back is according to taking place in the reaction system change in color judgement sample;
Preferably, before step 1), also comprise the step that detected sample is handled;
Preferably, before step 1), also comprise the step that detected sample is sealed; Further preferably, described sealer is selected from hyclone, lowlenthal serum, horse serum; More preferably, described sealer is a hyclone; Preferably, the capping time is 30～120 minutes, and closure temperature is 25～40 ℃; Further preferably, the capping time is 60 minutes, and closure temperature is 37 ℃.
2. method as claimed in claim 1 is characterized by, at described method step 1) in, the antibody of copper oxide nanometer particle mark is one anti-;
Preferably, in step 1), the antibody of copper oxide nanometer particle mark is two anti-.
3. as the method for claim 1 or 2, it is characterized by, at described method step 1) in, the incubation time of antigen-antibody reaction is 30～120 minutes, incubation temperature is 25～40 ℃; Further preferably, incubation time is 60 minutes, and incubation temperature is 37 ℃.
4. as each method among the claim 1-3, it is characterized by, at described method step 2) in, the acid of adding is selected from HCl, HNO
3, H
2SO
4, HCl more preferably;
Preferably, the concentration of the acid of adding in reaction system for O.1～1mM, the acid reaction time is 5～20 minutes; Further preferably, the concentration 1mM of described acid in reaction system, the acid reaction time is 1O minute.
5. as each method among the claim 1-4, it is characterized by, at described method step 2) in, the reductive agent of adding is selected from sodium ascorbate, sodium citrate, sodium borohydride, more preferably sodium ascorbate;
Preferably, the concentration of the reductive agent of adding in reaction system is 0.01～0.1M, and further preferably, the concentration of described reductive agent in reaction system is 0.05M.
6. as each method among the claim 1-5, it is characterized by, at described method step 2) in, the alkynyl of the golden nanometer particle of the surperficial alkynyl functionalization of described adding is the compound that comprises Formula I:
The azido of the golden nanometer particle of the surperficial azido functionalization that adds is the compound that comprises Formulae II:
Preferably, the mol ratio of the golden nanometer particle of the golden nanometer particle of the surperficial alkynyl functionalization of adding and surperficial azido functionalization is 1: 1.
7. as each method among the claim 1-6, it is characterized by, at described method step 2) in, described reductive agent and two kinds of functionalization golden nanometer particles provide simultaneously with the form of detection architecture;
Preferably, the concentration of reductive agent in detection architecture is 0.01～0.1mM, and further preferably, described reductive agent is 0.05mM in the concentration of detection architecture.
8. as each method among the claim 1-7, it is characterized by described method step 2) in, the change color of described reaction system and deposited phenomenon occurred in the clock time at 5～30 minutes, further preferably occurred in the clock time at 10～15 minutes.
9. as each method among the claim 1-8, it is characterized by, described detected sample comprises the clinical detection sample; Preferably, detected sample is from suffering from or the patient who suffers from certain disease under a cloud; Further preferably, described disease is an immune correlated disease; More preferably, described disease is the viral immune correlated disease that causes.
As each method among the claim 1-9 in Application for Field such as biology, biomedicine, medical tests;
Preferably, the invention provides described method in the detection of immune correlated disease and the application in the diagnosis, further preferably, the immune correlated disease that described immune correlated disease causes for virus;
Preferably, the invention provides described method is used for the reagent of disease detection and diagnosis in preparation application; Further preferably, described disease is an immune correlated disease; More preferably, described immune correlated disease is the viral immune correlated disease that causes.
11. a kit that is used for visual detection antigen-antibody reaction, described kit comprises: the golden nanometer particle of reductive agent, surperficial alkynyl functionalization and the golden nanometer particle of surperficial azido functionalization;
Preferably, the antibody that also comprises the copper oxide nanometer particle mark in the described kit;
Preferably, also comprise copper oxide nanometer particle in the described kit;
Preferably, also comprise antibody in the described kit; Further preferably, described antibody is one anti-or two anti-.
12. the kit as claim 11 is characterized by, the reductive agent in the described kit is selected from sodium ascorbate, sodium citrate, sodium borohydride, more preferably sodium ascorbate;
Preferably, also comprise sealer in the described kit; Further preferably, described sealer is selected from hyclone, lowlenthal serum, horse serum; More preferably, described sealer is a hyclone;
Preferably, also comprise one or more of following reagent in the described kit: positive reference substance, negative control product, sample diluting liquid, damping fluid etc.
13. as the application of the kit of claim 11-12 in fields such as biology, biomedicine, medical tests;
Preferably, the invention provides the application of described kit in disease detection and diagnosis; Further preferably, described kit is applied in the detection and diagnosis of immune correlated disease, more preferably, and the immune correlated disease that described immune correlated disease causes for virus.
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| PCT/CN2010/001852 WO2011150541A1 (en) | 2010-06-04 | 2010-11-18 | Visual detection method for antigen/antibody reaction, kit and applications thereof |
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| CN102269758B (en) | 2013-11-20 |
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