CN104251902B - The quantitative detecting method of a kind of 50nm silver particles - Google Patents
The quantitative detecting method of a kind of 50nm silver particles Download PDFInfo
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- CN104251902B CN104251902B CN201410513013.9A CN201410513013A CN104251902B CN 104251902 B CN104251902 B CN 104251902B CN 201410513013 A CN201410513013 A CN 201410513013A CN 104251902 B CN104251902 B CN 104251902B
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- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 59
- 229910052709 silver Inorganic materials 0.000 title claims abstract description 55
- 239000004332 silver Substances 0.000 title claims abstract description 55
- 239000002245 particle Substances 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 53
- 102000036639 antigens Human genes 0.000 claims abstract description 51
- 108091007433 antigens Proteins 0.000 claims abstract description 51
- 108090000790 Enzymes Proteins 0.000 claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 33
- 238000002360 preparation method Methods 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000002835 absorbance Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 65
- 230000003053 immunization Effects 0.000 claims description 52
- 239000002671 adjuvant Substances 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 33
- 238000002649 immunization Methods 0.000 claims description 28
- 238000005406 washing Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 16
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 14
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- 239000003085 diluting agent Substances 0.000 claims description 11
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 9
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- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 15
- 108010058846 Ovalbumin Proteins 0.000 description 13
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- 241000287828 Gallus gallus Species 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
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- 229910052737 gold Inorganic materials 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 3
- 229940038773 trisodium citrate Drugs 0.000 description 3
- 238000004506 ultrasonic cleaning Methods 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 240000007711 Peperomia pellucida Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000705 flame atomic absorption spectrometry Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
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- 238000002156 mixing Methods 0.000 description 2
- 238000011587 new zealand white rabbit Methods 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
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- 241000283073 Equus caballus Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- CMNWUCGLNTVCSI-UHFFFAOYSA-J [Na+].[Na+].[Na+].[Na+].[Cl-].[O-]P([O-])([O-])=O Chemical compound [Na+].[Na+].[Na+].[Na+].[Cl-].[O-]P([O-])([O-])=O CMNWUCGLNTVCSI-UHFFFAOYSA-J 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
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- 238000012875 competitive assay Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
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- 238000004445 quantitative analysis Methods 0.000 description 1
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- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to the quantitative detecting method of a kind of 50nm silver particles. Detection method step comprises: prepares immunogen and envelope antigen, the preparation 50nm silver particles antibody with high specificity of 50nm silver particles, prepare enzyme labelled antibody, measure 50nm silver particles content with absorbance method. Detection method specificity height, highly sensitive, operate relatively simple.
Description
Technical field
The invention belongs to nano material detection technique field, in particular to the quantitative detecting method of a kind of 50nm silver particles.
Background technology
Since multiple century, silver is widely used by everybody as argent usually, such as Silver Jewelry and silverware tool. Nowadays, along with the development of nanotechnology, take nanotechnology as the theme that the new science and technology revolution of core has become current era. During the form of silver Chang Zuowei nanoparticle penetrates into our daily life and produces. Organically antimicrobial comparing with other, nanometer silver has germ resistance and hypotoxicity, and therefore nanometer silver is widely used in antibacterials and foodstuff production. Nanometer silver is environmentally safe and human health contribution greatly, development prospect is extremely wide, and in today of life day by day convenientization, nanometer silver adds the essential product that goods have become consumers in general.
It is known that nanometer silver can enter environment and human body by approach such as directly or indirectly contacts, under the development prospect that nanometer silver is so wide, the biological effect that nanometer silver may be produced by we and the research of potential toxicity are abundant not enough. The report of Bai Chunli academician in 293 Fragrance Hill meetings is pointed out: " while development nanometer; synchronously carry out its safety research; make nanotechnology become the mankind first before it may produce negative effect; just through conscientious research; cause extensive attention, and the technology that finally can promote the well-being of mankind safely. " simultaneously, 2003, Science and Nature be consecutive publications article also, inquire into nano material biological effect, on the impact of environment and health. Therefore, to the research of nano material and important.
At present, size, the aspect and structure form of nanometer silver can be done certain research by transmission type microscope (TEM), atomic force microscope (AFM) and scanning electronic microscope (SEM) etc. by us, but the detection by quantitative for nanometer silver is an extremely challenging job. At present, the quantitative detecting method of nanoparticle is mainly contained inductively coupled plasma mass spectrometry, spectrophotometry, flame atomic absorption spectrometry and electrochemical process etc. by us, but all there is certain defect in aforesaid method, such as inductively coupled plasma mass spectrometry is only suitable for the nanoparticle of specific dimensions, and the nanoparticle for little size is not suitable for; Spectrophotometry and flame atomic absorption spectrometry interfering factors are more, to the higher sample tests result of content still can, but the detectivity of low levels sample is not strong; And electrochemical analysis quantitative analysis stability is poor, sensitivity is low, is not suitable for theoretical application. So, develop a kind of trace analysis methods simple, quick, that be applicable to on-site supervision extremely urgent.
Summary of the invention
For the deficiencies in the prior art, it is an object of the invention to provide the quantitative detecting method of a kind of 50nm silver particles, detection method is to the detection method specificity height of trace 50nm silver particles, highly sensitive, operates relatively simple.
The technical solution used in the present invention is:
A quantitative detecting method for 50nm silver particles, step comprises:
The immunogen of a, preparation 50nm silver particles and envelope antigen;
B, by immunogen injection in animal body, obtained 50nm silver particles antibody with high specificity;
C, prepare enzyme labelled antibody;
D, with absorbance method measure 50nm silver particles content, utilize Direct cELISA by standard substance and the reaction treating test sample competition antibody, the enzyme of enzyme labelled antibody can impel substrate solution luminous, thus records absorbance, namely carrys out detection by quantitative 50nm silver particles content by typical curve.
The preparation method of described 50nm silver particles is:
Take the AgNO of 27.8mg3It is dissolved in 100mL distilled water and dissolves, then transfer in Erlenmeyer flask, slowly stir lower oil bath and be heated to boiling, add the citric acid three sodium solution of 3mL1% fast, continue boiling 35 minutes, collect with brown bottle after cooling,Depositing for subsequent use, gained solution is the solution containing 50nm silver particles.
In described step a, the preparation method of the immunogen of 50nm silver particles is:
Under agitation Thiovanic acid (TGA) aqueous solution is joined in 50nm silver solution, after stirring 15-20min, add the aqueous solution of 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride (EDAC) and N-hydroxy-succinamide (NHS) respectively, 3-4h is stirred under room temperature lucifuge, and then regulate pH9��10, add bovine serum albumin solution (BSA, sigma company)Stir 5-6h, gained solution is loaded in dialysis tubing, with distill water dialysis more than 12 hours, lucifuge cryopreservation.
Described lucifuge cryopreservation temperature is
Described Thiovanic acid aqueous solution mass percent concentration is 9.9%, 50nm silver solution concentration 100 �� g/mL, and the amount of substance of EDAC strength of solution is 10mg/mL, NHS strength of solution to be 10mg/mL, BSA concentration be 10g/L, TGA, EDAC and NHS compares 1:2:4; The volume ratio of nano silver solution, mercaptoacetic acid solution and bovine serum albumin solution is 400:1:10;
The preparation method of described envelope antigen is substantially with the preparation method of immunogen, and difference is in replacing 10g/L bovine serum albumin by mass percent 1% ovalbumin solution (OVA);
In described step b, the preparation method of antibody with high specificity is:
By miscible with volume ratio 1:1 to immunizing antigen and freund's adjuvant, animal is carried out repeatedly immunity, obtained 50nm silver particles antibody with high specificity.
Described repeatedly immunization method is: first time, animal immune was by miscible to immunizing antigen and Freund's complete adjuvant, immunizing dose 0.5mg��1mg/kg/ time, immunization, after immune three weeks, carry out six booster immunizations, booster immunization is by miscible to immunizing antigen and Freund's incomplete adjuvant, carries out immunization, immunizing dose 0.5mg��1mg/kg/ time, six times booster immunization is spaced apart 2-3 week, after six booster immunizations, from animal venous blood collection, serum room temperatureSet to 0 .5-1h, at refrigeratorThe serum of upper strata clarification is drawn, obtained antibody with high specificity after putting 2h.
Described freund's adjuvant preparation method is: by whiteruss and lanolin by ultrasonic 3 hours of 2:1 volume ratio ultrasonic cleaning machine, makes it to mix completely even, obtained incomplete Freund's adjuvant; Adding bacille Calmette-Guerin vaccine in incomplete Freund's adjuvant before use and be complete Freund's adjuvant, the volume ratio of incomplete Freund's adjuvant and bacille Calmette-Guerin vaccine is 6:1.
Described animal comprises Mammals, bird, and Mammals comprises rabbit, horse, sheep, mouse, pig and monkey, it is preferable that rabbit and mouse, and bird comprises chicken, duck and goose, it is preferable that chicken; Described animal is animal of the right age, healthy and strong, nothing infection, it is preferable that buck, and the age of animal should be between twenty and fifty, and rabbit selects 2��5 months monthly ages in age, the adult male rabbit of body weight 2��3Kg, the preferred laying hen of chicken.
The position of described immunization comprises that intraperitoneal, intravenously, intramuscular, lymphoglandula be interior, subcutaneous and intradermal injection, these methods can be used alone or repeatedly combinationally use, it is preferable that in the subcutaneous multi-point injection in back, skin, multi-point injection and leg muscle inject independent or multiple combination injection.
Described adjuvant can also be the inorganic adjuvants such as organic adjuvants such as teichoic acid dipeptides, polysaccharose substance, or alum.
The preparation method of described step c enzyme labelled antibody is:
Getting horseradish peroxidase 10mg and be dissolved in 0.4mL0.05mol/LpH9.6 sodium carbonate buffer and CB damping fluid, add 25% glutaraldehyde 0.1mL after to be dissolved, then 37 DEG C of temperature educate 2 hours; Adding 0-4 DEG C of dehydrated alcohol 2mL afterwards, centrifugal 15 minutes of 2500r/min, incline supernatant liquor; Getting to precipitate and hang with 80% ethanol 4mL is mixed, the same centrifugal, incline ethanol, is inverted by pipe, ethanol is fully flowed out; Precipitate and dissolve with 1mL0.05mol/LpH9.6CB damping fluid, add 0.5-1mL50nm silver particles antibody, be placed on after spending the night in refrigerator, with a small amount of NaH2PO4It is adjusted to neutrality. Finally being purified with saturated ammonium sulphate solution deposit, antibody purification is in 4 DEG C or-20 DEG C of preservations.
Described steps d is specially:
1) bag quilt: make diluent after envelope antigen and water being diluted with 1:50��1:200, preferred 1:100, with diluent bag by 96 hole enzyme plates, every hole adds diluent 80��150 �� L, it is preferable that 100 �� L, places 4 DEG C and spends the night, taking-up washings PBST washs 2��6 times, each 2��6 minutes, it is preferable that wash 3��5 times, each 3��5 minutes.
2) close: adding mass percent concentration is 1%��5%OVA solution, it is preferable that the OVA of 1%; Every hole 100��200 �� L, preferably 150��200 �� L, close the unnecessary part not having envelope antigen, after 30 DEG C��40 DEG C temperature educate 0.5��2.0 hour, taking-up washings PBST washs 2��6 times, each 2��6 minutes, it is preferable that after 37 DEG C of temperature educate 0.5��1.0 hour, taking-up washings PBST washs 3��5 times, each 3��5 minutes.
3) compete: add solution and sample solution that 50 �� l/ hole different concns contain 50nm silver particles, then add and add 50 �� L enzyme labelled antibodies respectively, make it competing reaction. At 30 DEG C��40 DEG C, temperature educates 1��5 hour, it is preferable that at 37 DEG C, temperature educates 3 hours, makes test substance and solid phase antigen compete the binding site on enzyme labelled antibody simultaneously, taking-up washings PBST washs 2��6 times, each 2��6 minutes, it is preferable that wash 3��5 times, each 3��5 minutes.
4) detect: add substrate solution colour developing, every hole 80��120 �� L, it is preferable that every hole 100 �� L, after temperature educates 30 minutes, with the H of 2mol/L2SO4Stop buffer stopped reaction. The photon absorbing intensity of each hole when 490nm is measured by multi-functional microplate reader.
5), according to photon absorbing intensity and the strength of solution drawing curve containing 50nm silver particles, the content of 50nm silver particles in testing sample solution is tried to achieve according to working curve.
The present invention has the following advantages and positively effect:
(1) antibody is practical: the present invention has prepared that specificity is good, much higher clonal antibody of tiring.
(2) provide a kind of quantitative detecting method to nano material: by the polyclonal antibody of the nanometer silver of preparation, enable us to measure trace 50nmAgNPs.
(3) this immune analysis method specificity height, highly sensitive, operates relatively simple.
Embodiment
Experimental drug and instrument:
(1) experiment reagent
Bovine serum albumin (BSA), sheep blood serum albumen (OVA), horseradish peroxidase (HRP), whiteruss, lanolin, anhydrous diethyl ether, glutaraldehyde (25%), liquid bacille Calmette-Guerin vaccine, 75% medical alcohol, sodium hydroxide, urea, anhydrous sodium carbonate, sodium bicarbonate, Sodium phosphate dibasic, ammoniacal liquor, Silver Nitrate, Thiovanic acid (99%, C2H4O2S), N-hydroxy-succinamide (98%, C4H5NO3), trisodium citrate, phosphoric acid, sulfuric acid, ethanol, citric acid, O-Phenylene Diamine, hydrochloric acid, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, ammonium sulfate, sodium tetraborate, Tween-20, hydrogen peroxide, experimental water is ultrapure water.
(2) laboratory apparatus
UV-4100 type ultraviolet-visible spectrophotometer, collecting and distributing type constant-temperature heating magnetic stirring apparatus, magnetic stirring apparatus, dialysis tubing, disposable syringe, multi-functional microplate reader, SCQ50 ultrasonic cleaner, TGL-16G high speed tabletop centrifuge, 96 hole enzyme plates.
(3) preparation of solution
1) 1% trisodium citrate: 50mg trisodium citrate is dissolved in 4.95mL distilled water
2) physiological saline: 0.85gNaCl distilled water holds 100mL surely
3) coating buffer damping fluid (0.05mol/L, pH=9.6 sodium carbonate buffer, CB): NaCO30.0795g, NaHCO30.147g is dissolved in distilled water, and fixed appearance arrives 50mL
4) diluent (0.01mol/L, pH=7.4 phosphate buffered saline buffer, PBS): Na2HPO4��12H2O2.96g, NaH2PO4��2H2O0.2964g, NaCl8.0g, KCl0.2g are dissolved in distilled water, and fixed appearance arrives 1000mL
5) washings (0.01mol/L, pH=7.4, PBST): make by adding volume fraction 0.05% tween in diluent
6) confining liquid: mass percent is ovalbumin (OVA) solution of 1%, and solvent is PBS
7) substrate solution: citric acid 0.5106gNa2HPO4��12H2O1.8408g is dissolved in distilled water, and fixed appearance arrives 50mL, takes 4mg O-Phenylene Diamine and is dissolved in the above-mentioned solution of 10mL, adds the H of 15 �� L30% before use2O2
8) stop buffer: 2mol/L sulphuric acid soln.
Embodiment 1
(1) 50nmAgNPs and 50nm silver particles holoantigen
1) preparation of 50nmAgNPs
Take the AgNO of 27.8mg3It is dissolved in 100mL distilled water and dissolves, then transfer in Erlenmeyer flask, slowly stir lower oil bath and be heated to boiling, add the citric acid three sodium solution of 3mL1% fast, continue boiling 35 minutes, collect with brown bottle after cooling,Depositing for subsequent use, gained solution is 50nmAgNPs.
2) preparation of immunizing antigen and envelope antigen
Get the freshly prepd AgNPs solution of 8mL, the Thiovanic acid (TGA) diluting 10 times of pH7.0 with 20 �� L is modified, stir after 15 minutes, dropwise add the N-hydroxy-succinamide (NHS) of 40 �� L10mg/mL and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) solution of 80 �� L10mg/mL under magnetic stirring, under room temperature, lucifuge stirs 3 hours, adjusting pH with the NaOH of 2mol/L is 9 to 10, add the BSA solution of 200 �� l1%, stir 5 hours at 4 DEG C, gained solution is loaded in dialysis tubing, with distill water dialysis 12 hours, with brown lucifuge reagent bottle 4 DEG C preservation, obtained immunizing antigen.
Envelope antigen preparation is substantially with immunizing antigen preparation, and difference is in the bovine serum albumin solution using 1% ovalbumin solution (OVA) to replace 1%, and the bovine serum albumin solution compound method of 1% is with 1% ovalbumin solution.
(2) preparation of 50nmAgNPs antibody
1) preparation method of antibody
By miscible with volume ratio 1:1 to immunizing antigen and freund's adjuvant, the new zealand white rabbit selected is carried out repeatedly immune by subcutaneous injection, the Freund's complete adjuvant that our injecting immune antigen of animal immune mixes with bacille Calmette-Guerin vaccine and adjuvant for the first time, repeatedly booster immunization afterwards injects after miscible to immunizing antigen and Freund's incomplete adjuvant, after six booster immunizations, the obtained anti-50nmAgNPs antibody of rabbit.
2) preparation of freund's adjuvant
Whiteruss and lanolin are pressed 2:1 volume ratio ultrasonic cleaning machine ultrasonic 3 hours several times, ultrasonic 20 minutes every time, make it to mix completely even, namely drip in emulsifying agent to frozen water mixed solution indiffusion within half a minute and could calculate mixing completely. This water-in-oil emulsifier is referred to as Freund's incomplete adjuvant. Incomplete adjuvant low temperature (0-4 DEG C) is stored, and adds bacille Calmette-Guerin vaccine before use and is Freund's complete adjuvant, and the volume ratio of incomplete Freund's adjuvant and bacille Calmette-Guerin vaccine is 6:1.
3) immunity step
7 about 3 months monthly ages are selected in this experiment, and body weight is that the healthy new zealand male rabbit of about 1.5-2kg is as immunization. Wherein 1 to No. 6 rabbit is experimental group, and No. 7 rabbits are blank group. Injection carries out fundamental immunity containing the antigen of complete Freund's adjuvant first, afterwards with the antigen booster immunization containing incomplete Freund's adjuvant. Being cut totally by the hair at rabbit back with scissors, then with medical alcohol sterilization, adopt the method for the subcutaneous multi-point injection in back, immunizing dose is 1mg/kg/ time, each back subcutaneous inoculation 10 point, each some injection 0.1mL. Starting first time booster immunization after 3 weeks, later every 2 weeks booster immunizations again, after 6 booster immunizations, take a blood sample from rabbit ear vein, every rabbit blood sampling 1mL, serum room temperature (20-25 DEG C) leaves standstill 1h, draws the serum of upper strata clarification after static 2 hours at refrigerator (4 DEG C).
4) preparation of enzyme labelled antibody
Getting horseradish peroxidase 10mg and be dissolved in 0.4mL0.05mol/LpH9.6CB damping fluid, add 25% glutaraldehyde 0.1mL after to be dissolved, then 37 DEG C of temperature educate 2 hours. Adding 4 DEG C of dehydrated alcohol 2mL afterwards, centrifugal 15 minutes of 2500r/min, incline supernatant liquor. Getting to precipitate and hang so that 80% ethanol 4mL is mixed, the same centrifugal, incline ethanol, is inverted by pipe, ethanol is fully flowed out. Precipitate and dissolve with 1mL0.05mol/LpH9.6CB damping fluid, add 1mL40nmAgNPs antibody, be placed on after spending the night in refrigerator, with a small amount of NaH2PO4It is adjusted to neutrality. Finally being purified with saturated ammonium sulphate solution deposit, antibody purification is in 4 DEG C of preservations.
(3) foundation of competitive ELISA analytical method detection 50nmAgNPs
1) bag quilt: by concentration be the envelope antigen OVA-AgNPs bag of 1:100 by 96 hole enzyme plates, every hole 100 �� L, places 4 DEG C and spends the night, and taking-up washings PBST washs 3 times, each 3 minutes.
2) close: add 1%OVA solution, every hole 150 �� L, close the unnecessary part not having envelope antigen, avoid the non-specific of antibody to be adsorbed on enzyme plate. After 37 DEG C of temperature educate 1 hour, take out the same washing 3 times.
3) compete: every hole adds the 50nmAgNPs antibody that the 50 homemade 50nmAgNPs standardized solution of �� L or sample solution and 50 �� LHRP mark, make it competing reaction. After temperature educates 3 hours at 37 DEG C, make test substance and solid phase antigen compete the binding site on enzyme labelled antibody simultaneously. The bottom that added thing should be added in when adding sample plate hole, avoids being added in hole wall top, and notes spattering, and can not produce bubble. Then the same washing.
Add 50nmAgNPs standardized solution and sample solution that 50 �� l/ hole concentration are respectively 0.001,0.01,0.1,1,10,100,1000, then add 50 �� L enzyme labelled antibodies respectively, make it competing reaction. After temperature educates 3 hours at 37 DEG C, making test substance and solid phase antigen compete the binding site on enzyme labelled antibody, taking-up washings PBST washs 3 times simultaneously, each 3 minutes.
4) detect: adding substrate solution colour developing, every hole 100 �� L, after temperature educates 30 minutes, uses stop buffer stopped reaction. The photon absorbing intensity of each hole when 490nm is measured by multi-functional microplate reader.
(4) drafting of typical curve
Linear equation is drawn according to absorbancy and concentration of standard solution:
A=0.5621-0.052logC
R2=0.99091, linearity range is 10-31000ng/mL,
Lowest detectable limit (3 ��) is 0.0093ng/mL.
Finally recording sample solution 50nmAgNPs content is 1.34ng/mL.
Embodiment 2
(1) 50nmAgNPs and 50nm silver particles holoantigen
1) preparation of 50nmAgNPs
Take the AgNO of 27.8mg3It is dissolved in 100mL distilled water and dissolves, then transfer in Erlenmeyer flask, slowly stir lower oil bath and be heated to boiling, add the citric acid three sodium solution of 3mL1% fast, continue boiling 35 minutes, collect with brown bottle after cooling,Depositing for subsequent use, gained solution is 50nmAgNPs.
2) preparation of immunizing antigen and envelope antigen
Get the freshly prepd AgNPs solution of 8mL, the Thiovanic acid (TGA) diluting 10 times of pH7.0 with 20 �� L is modified, stir after 15 minutes, dropwise add the N-hydroxy-succinamide (NHS) of 40 �� L10mg/mL and 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) solution of 80 �� L10mg/mL under magnetic stirring, under room temperature, lucifuge stirs 3 hours, adjusting pH with the NaOH of 2mol/L is 9 to 10, add the BSA (immunizing antigen) or OVA (envelope antigen) of 200 �� l1%, stir 5 hours at 4 DEG C, gained solution is loaded in dialysis tubing, with distill water dialysis 12 hours, with brown lucifuge reagent bottle 4 DEG C preservation.
(2) preparation of 50nmAgNPs antibody
1) preparation method of antibody
By miscible with volume ratio 1:1 to immunizing antigen and freund's adjuvant, the new zealand white rabbit selected is carried out repeatedly immune by subcutaneous injection, the Freund's complete adjuvant that our injecting immune antigen of animal immune mixes with bacille Calmette-Guerin vaccine and adjuvant for the first time, repeatedly booster immunization afterwards injects after miscible to immunizing antigen and Freund's incomplete adjuvant, after six booster immunizations, the obtained anti-50nmAgNPs antibody of rabbit.
2) preparation of freund's adjuvant
Whiteruss and lanolin are pressed 2:1 volume ratio ultrasonic cleaning machine ultrasonic 3 hours several times, ultrasonic 20 minutes every time, make it to mix completely even, namely drip in emulsifying agent to frozen water mixed solution indiffusion within half a minute and could calculate mixing completely. This water-in-oil emulsifier is referred to as Freund's incomplete adjuvant. Incomplete adjuvant low temperature (0-4 DEG C) is stored, and adds bacille Calmette-Guerin vaccine before use and is Freund's complete adjuvant.
3) immunity step
7 about 3 months monthly ages are selected in this experiment, and body weight is that the healthy new zealand male rabbit of about 1.5-2kg is as immunization. Wherein 1 to No. 6 rabbit is experimental group, and No. 7 rabbits are blank group. Injection carries out fundamental immunity containing the antigen of complete Freund's adjuvant first, afterwards with the antigen booster immunization containing incomplete Freund's adjuvant. Being cut totally by the hair at rabbit back with scissors, then with medical alcohol sterilization, adopt the method for the subcutaneous multi-point injection in back, immunizing dose is 1mg/kg/ time, each back subcutaneous inoculation 10 point, each some injection 0.1mL. Starting first time booster immunization after 3 weeks, later every 2 weeks booster immunizations again, after 6 booster immunizations, take a blood sample from rabbit ear vein, every rabbit blood sampling 1mL, serum room temperature (20-25 DEG C) leaves standstill 1h, draws the serum of upper strata clarification after static 2 hours at refrigerator (4 DEG C).
4) preparation of enzyme labelled antibody
Getting horseradish peroxidase 10mg and be dissolved in 0.4mL0.05mol/LpH9.6CB damping fluid, add 25% glutaraldehyde 0.1mL after to be dissolved, then 37 DEG C of temperature educate 2 hours. Adding 4 DEG C of dehydrated alcohol 2mL afterwards, centrifugal 15 minutes of 2500r/min, incline supernatant liquor. Getting to precipitate and hang so that 80% ethanol 4mL is mixed, the same centrifugal, incline ethanol, is inverted by pipe, ethanol is fully flowed out. Precipitate and dissolve with 1mL0.05mol/LpH9.6CB damping fluid, add 1mL40nmAgNPs antibody, be placed on after spending the night in refrigerator, with a small amount of NaH2PO4It is adjusted to neutrality. Finally being purified with saturated ammonium sulphate solution deposit, antibody purification is in 4 DEG C of preservations.
(3) foundation of competitive ELISA analytical method detection 50nmAgNPs
1) bag quilt: by concentration be the envelope antigen OVA-AgNPs bag of 1:100 by 96 hole enzyme plates, every hole 100 �� L, places 4 DEG C and spends the night, and taking-up washings PBST washs 3 times, each 3 minutes.
2) close: add 1%OVA solution, every hole 150 �� L, close the unnecessary part not having envelope antigen, avoid the non-specific of antibody to be adsorbed on enzyme plate. After 37 DEG C of temperature educate 1 hour, take out the same washing 3 times.
3) compete: every hole adds the 50nmAgNPs antibody that the 50 homemade 50nmAgNPs standardized solution of �� L or sample solution and 50 �� LHRP mark, make it competing reaction. After temperature educates 3 hours at 37 DEG C, make test substance and solid phase antigen compete the binding site on enzyme labelled antibody simultaneously. The bottom that added thing should be added in when adding sample plate hole, avoids being added in hole wall top, and notes spattering, and can not produce bubble. Then the same washing.
Add 50nmAgNPs standardized solution and sample solution that 50 �� l/ hole concentration are respectively 0.0001,0.001,0.01,0.1,1,10,100, then add 50 �� L enzyme labelled antibodies respectively, make it competing reaction. After temperature educates 3 hours at 37 DEG C, making test substance and solid phase antigen compete the binding site on enzyme labelled antibody, taking-up washings PBST washs 3 times simultaneously, each 3 minutes.
4) detect: adding substrate solution colour developing, every hole 100 �� L, after temperature educates 30 minutes, uses stop buffer stopped reaction. The photon absorbing intensity of each hole when 490nm is measured by multi-functional microplate reader.
(4) drafting of typical curve
Linear equation is drawn according to absorbancy and concentration of standard solution:
A=0.52152-0.05339logC
R2=0.98342, linearity range is 10--4100ng/mL,
Lowest detectable limit (3 ��) is 0.0076ng/mL.
Finally recording sample solution 50nmAgNPs content is 5.83ng/mL.
With the cross reaction of the golden nanometer particle of enzyme linked immunological direct competitive assay different size and Nano silver grain. Calculate the IC of analogue50Concentration, cross reacting rate=(IC of antigen50The IC of concentration/approximate antigen50Concentration) �� 100%. Result is as follows,
| Different size nano material | Cross reacting rate (%) |
| 50nm spherical silver nanoparticles | 100 |
| 80nm spherical silver nanoparticles | 9.58 |
| 16nm color of spherical gold | <0.01 |
| 40nm color of spherical gold | <0.01 |
| 80nm color of spherical gold | <0.01 |
Can find out that the cross reacting rate with other antigens is all less than 10%, illustrate that the specificity of this 50nmAgNPs antibody is higher.
Claims (6)
1. a quantitative detecting method for 50nm silver particles, step comprises:
The immunizing antigen of a, preparation 50nm silver particles and envelope antigen;
B, immunizing antigen is expelled in animal body, obtained 50nm silver particles antibody with high specificity;
C, prepare enzyme labelled antibody;
D, with absorbance method measure 50nm silver particles content, utilize Direct cELISA, test substance and solid phase antigen in standard solution and testing sample solution compete the binding site on enzyme labelled antibody simultaneously, the enzymatic of enzyme labelled antibody makes substrate solution luminous, thus record absorbance, namely carry out detection by quantitative 50nm silver particles content by typical curve;
In described step a, the preparation method of the immunizing antigen of 50nm silver particles is:
Under agitation mercaptoacetic acid solution is joined in 50nm silver solution, after stirring 15-20min, add 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride solution and N-hydroxy-succinamide solution respectively, stir 3-4h under room temperature lucifuge, and then regulate pH9��10, add bovine serum albumin solution, 5-6h is stirred at 4 DEG C, gained solution is loaded in dialysis tubing, with distill water dialysis more than 12 hours, lucifuge cryopreservation;
Described mercaptoacetic acid solution mass percent concentration is 9.9%, 50nm silver solution concentration is 100 �� g/mL, 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride strength of solution is 10mg/mL, N-hydroxy-succinamide strength of solution is 10mg/mL, bovine serum albumin solution concentration is 10g/L, and the amount of substance of Thiovanic acid, 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimide hydrochloride and N-hydroxy-succinamide compares 1:2:4; The volume ratio of 50nm nano silver solution, mercaptoacetic acid solution and bovine serum albumin solution is 400:1:10.
2. detection method as claimed in claim 1, it is characterised in that: in described step b, the preparation method of antibody with high specificity is:
By miscible with volume ratio 1:1 to immunizing antigen and freund's adjuvant, animal is carried out repeatedly immunity, obtained 50nm silver particles antibody with high specificity.
3. detection method as claimed in claim 2, it is characterized in that: the method for described repeatedly immunity is: first time, animal immune was by miscible to immunizing antigen and Freund's complete adjuvant, immunizing dose 0.5mg��1mg/kg/ time, immunization, immunity is after three weeks, carry out six booster immunizations, booster immunization is by miscible to immunizing antigen and Freund's incomplete adjuvant, carry out immunization, immunizing dose 0.5mg��1mg/kg/ time, six times booster immunization is spaced apart 2-3 week, after six booster immunizations, from animal venous blood collection, the standing 0.5-1h of serum room temperature 20-25 DEG C, the serum of upper strata clarification is drawn after refrigerator 4 DEG C of standing 2h, obtained antibody with high specificity.
4. detection method as claimed in claim 1, it is characterised in that: the preparation method of described step c enzyme labelled antibody is:
Getting horseradish peroxidase 10mg and be dissolved in 0.4mL0.05mol/LpH9.6 sodium carbonate buffer, add 25% glutaraldehyde 0.1mL after to be dissolved, then 37 DEG C of temperature educate 2 hours; Adding 0-4 DEG C of dehydrated alcohol 2mL afterwards, centrifugal 15 minutes of 2500r/min, incline supernatant liquor; Getting to precipitate and hang so that 80% ethanol 4mL is mixed, the same centrifugal, incline ethanol, is inverted by pipe, ethanol is fully flowed out; Precipitate and dissolve with 1mL0.05mol/LpH9.6 sodium carbonate buffer, add 0.5-1mL50nm silver particles antibody with high specificity, be placed on after spending the night in refrigerator, with a small amount of NaH2PO4It is adjusted to neutrality; Finally being purified by enzyme labelled antibody with saturated ammonium sulphate solution deposit, the antibody after purifying is in 4 DEG C or-20 DEG C of preservations.
5. detection method as claimed in claim 1, it is characterised in that: described steps d is specially:
1) bag quilt: making diluent after envelope antigen and water being diluted with 1:50��1:200, with diluent bag by 96 hole enzyme plates, every hole adds diluent 80��150 �� L, places 4 DEG C and spends the night, and takes out, and washs 2��6 times with washings PBST, each 2��6 minutes;
2) close: adding mass percent concentration is 1%��5%OVA solution, every hole 100��200 �� L, close the unnecessary part not having envelope antigen, after 30 DEG C��40 DEG C temperature educate 0.5��2.0 hour, take out, wash 2��6 times with washings PBST, each 2��6 minutes;
3) compete: add standard solution and testing sample solution that 50 �� l/ hole different concns contain 50nm silver particles, then 50 �� L enzyme labelled antibodies are added respectively, make it competing reaction, at 30 DEG C��40 DEG C, temperature educates 1��5 hour, make test substance and solid phase antigen compete the binding site on enzyme labelled antibody simultaneously, take out, wash 2��6 times with washings PBST, each 2��6 minutes;
4) detect: adding substrate solution colour developing, every hole 80��120 �� L, after temperature educates 30 minutes, with the H of 2mol/L2SO4Stop buffer stopped reaction, measures the photon absorbing intensity of each hole when 490nm by multi-functional microplate reader;
5) according to photon absorbing intensity and the standard solution concentration drawing curve containing 50nm silver particles, the content of 50nm silver particles in testing sample solution is tried to achieve according to working curve.
6. detection method as claimed in claim 5, it is characterised in that: described steps d is specially:
1) bag quilt: making diluent after envelope antigen and water being diluted with 1:100, with diluent bag by 96 hole enzyme plates, every hole adds diluent 100 �� L, places 4 DEG C and spends the night, and takes out, and washs 3��5 times with washings PBST, each 3��5 minutes;
2) close: adding mass percent concentration is 1%OVA solution, every hole 150��200 �� L, close the unnecessary part not having envelope antigen, after 37 DEG C of temperature educate 0.5��1.0 hour, take out, wash 3��5 times with washings PBST, each 3��5 minutes;
3) compete: add standard solution and testing sample solution that 50 �� l/ hole different concns contain 50nm silver particles, then 50 �� L enzyme labelled antibodies are added respectively, make it competing reaction, at 37 DEG C, temperature educates 3 hours, make test substance and solid phase antigen compete the binding site on enzyme labelled antibody simultaneously, take out, wash 3��5 times with washings PBST, each 3��5 minutes;
4) detect: adding substrate solution colour developing, every hole 100 �� L, after temperature educates 30 minutes, with the H of 2mol/L2SO4Stop buffer stopped reaction, measures the photon absorbing intensity of each hole when 490nm by multi-functional microplate reader;
5) according to photon absorbing intensity and the standard solution concentration drawing curve containing 50nm silver particles, the content of 50nm silver particles in testing sample solution is tried to achieve according to working curve.
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