CN103323607A - Method for simultaneously measuring two-component plant hormones - Google Patents
Method for simultaneously measuring two-component plant hormones Download PDFInfo
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- 239000003375 plant hormone Substances 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 36
- 239000000523 sample Substances 0.000 claims abstract description 16
- 239000011324 bead Substances 0.000 claims abstract description 15
- 239000002105 nanoparticle Substances 0.000 claims abstract description 15
- 238000005576 amination reaction Methods 0.000 claims abstract description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000010931 gold Substances 0.000 claims abstract description 3
- 229910052737 gold Inorganic materials 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 32
- 238000002372 labelling Methods 0.000 claims description 24
- 239000002245 particle Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 17
- 239000008055 phosphate buffer solution Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000011734 sodium Substances 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 6
- 238000001690 micro-dialysis Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 4
- 239000006148 magnetic separator Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000011065 in-situ storage Methods 0.000 claims description 3
- 238000009830 intercalation Methods 0.000 claims description 3
- 230000002687 intercalation Effects 0.000 claims description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 238000009835 boiling Methods 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000013507 mapping Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 238000001139 pH measurement Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims 1
- 239000008363 phosphate buffer Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000007885 magnetic separation Methods 0.000 abstract 2
- 239000000969 carrier Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 14
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 12
- 241000196324 Embryophyta Species 0.000 description 8
- 238000003556 assay Methods 0.000 description 6
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 6
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 5
- 238000005259 measurement Methods 0.000 description 3
- VMVZMFMKKYTBEF-UHFFFAOYSA-N 2-aminoethanol tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.NCCO VMVZMFMKKYTBEF-UHFFFAOYSA-N 0.000 description 2
- LEOJISUPFSWNMA-UHFFFAOYSA-N ABEI Chemical compound O=C1NNC(=O)C=2C1=CC(N(CCCCN)CC)=CC=2 LEOJISUPFSWNMA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- YYQRGCZGSFRBAM-UHFFFAOYSA-N Triclofos Chemical compound OP(O)(=O)OCC(Cl)(Cl)Cl YYQRGCZGSFRBAM-UHFFFAOYSA-N 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 2
- 239000003617 indole-3-acetic acid Substances 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 229960001147 triclofos Drugs 0.000 description 2
- 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 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 229930192334 Auxin Natural products 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 238000013433 optimization analysis Methods 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for simultaneously measuring two-component plant hormones. The method comprises the following steps: capturing two plant hormones by taking gold nanoparticles and amination magnetic beads as carriers and by taking ABEI-BSA at AuNPs as a nanoparticle label; by taking antibody-labeled ABEI-BSA at AuNPs nanoparticles as a chemiluminiscence probe, separating the captured plant hormones through magnetic separation and centrifugal methods; and measuring chemiluminiscence signals of a magnetic separation solution and a centrifugal separation solution, so that the two plant hormones are detected. The plant hormones IAA and GA can be simultaneously measured through the magnetic beads, the nanoparticles and the simple chemiluminiscence technology only, and the method has the advantages of high simplicity, low cost and high sensitivity.
Description
Technical field
The invention belongs to the chemiluminescence field, relate to the simultaneously method for measuring of a kind of pair of component plant hormone, assay method when being specifically related to plant hormone heteroauxin (IAA) and the two component of gibberellin (GA).
Background technology
Plant hormone is some chemical signal molecules that plant life is concerned, and is synthetic in plant, and causes physiological effect with the concentration of denier, affect and controlling plant grow and to the adaptability of environment.The content of plant hormone in plant is very low.The plant hormone assay method mainly contains in recent years: liquid chromatography-mass spectrography (Lu Qiaomei, Zhang Lan, Chen Tianwen, Lu Minghua, Chen Guonan. liquid chromatography-tandem mass spectrometry is analyzed the content of Plants under Salt Stress hormone. and Chinese science B collects: chemistry 2009,39:785); Gas chromatography-mass spectrography (Barkawi L S, Tam Y Y, Tillman J A, Pederson B, Calio J, Al-Amier H, Emerick M, Normanly J, Cohen J D. A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue. Anal Biochem, 2008,372:177); Immunosensor method (Xiao L. T, Wang R Z. Immunosensor assay:a novel method to analyze phytohormones. PGRSA Quarterly. 2006,33:51) and chemoluminescence method (Hun X, Mei Z H, Wang Z P, He Y H. Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescence probe coupling the DNA signal amplification. Spectrochimica Acta Part A, 2012,95:114) etc.
But, these methods its shortcoming respectively arranged, the present invention utilizes golden nanometer particle and magnetic bead to be carrier, take ABEI-BSA@AuNPs as nanoparticle tags, realized that the two components of plant hormone heteroauxin (IAA) and gibberellin (GA) measure, had advantages of that method is simple, cost is low, highly sensitive.
Summary of the invention
The object of the invention provides a kind of method of measuring simultaneously two component plant hormones, utilize golden nanometer particle and magnetic bead to be carrier, take ABEI-BSA@AuNPs as nanoparticle tags, realize that two components of plant hormone heteroauxin (IAA) and gibberellin (GA) are measured simultaneously.
Technical scheme
A kind of pair of simultaneously method for measuring of component plant hormone, plant hormone is two components of heteroauxin (IAA) and gibberellin (GA), it is characterized in that with golden nanometer particle and amination magnetic bead be carrier, take ABEI-BSA@AuNPs as nanoparticle tags, two components of realization plant hormone heteroauxin (IAA) and gibberellin (GA) are measured simultaneously, and determination step is as follows:
(1) preparation of golden nanometer particle (AuNPs), preparation, the used glass container (volumetric flask of storage solution of gold nanoparticles, brown wide-necked bottle, round-bottomed flask etc.) washed 30 min(minutes with chloroazotic acid (the acid of hydrochloric acid and nitric acid ratio is 1:3) bubble), then rinse dry for standby well with redistilled water; Gold chloride (the HAuCl that in 250 mL round-bottomed flasks, adds 100 mL mass concentrations 0.01%
4), be heated with stirring to boiling, then add fast the trisodium citrate (Na of 1.8 mL mass concentrations 1%
3C
6H
5O
7), continue heating 10 min, stir 15 min, be cooled to room temperature.Transfer in the brown bottle and preserve in the place, cool place;
(2) preparation of anti-IAA antibody labeling golden nanometer particle (AuNPs), get the sample hose of 2 mL, add successively 500 mM(mM/ls of 2 μ L) three (methylol) aminomethane hydrochloride (Tris-HCl) (pH is 8.2), the trichloroethyl phosphate (TCEP) of 6 μ L, 10 mM, 6 μ L 10
-4The mercaptoethylmaine of M, room temperature add the AuNPs that 1 mL step (1) prepares after placing 30 min, and reaction is 12 h(hours under the room temperature), obtain mercaptoethylmaine and modify golden nanometer particle; The mercaptoethylmaine of getting above-mentioned gained modify golden nanometer particle be dispersed in contain 5(wt) in the phosphate buffer solution of % glutaraldehyde, continue to stir 2 h, with phosphate buffer solution eccentric cleaning 3 times, again with it in phosphate buffer solution, add and contain 10 μ g anti-IAA antibody-solutions, 12 h are hatched in 4 ℃ of vibrations, obtain anti-IAA antibody labeling golden nanometer particle;
(3) preparation of anti-GA antibody labeling magnetic bead, in the sample hose of 2 mL, add and contain 10 μ g anti-GA antibody-solutions, add again 1000 μ L and contain 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) of 0.1 M and the N-hydroxy-succinamide (NHS) of 0.2 M, activate 30 min, other gets the small beaker of 10 mL, adds 50 μ L amination magnetic beads (2～3 μ m), 2000 μ L imidazole buffer (0.1 M, pH is 6.8), activate 30 min; With above-mentioned two solution hybrid reactions, 12 h, magnetic resolution is abandoned supernatant, washes three times with phosphate buffer solution again, is settled to 500 μ L with phosphate buffer solution, obtains anti-GA antibody labeling magnetic bead;
(4) preparation of antibody labeling ABEI-BSA@AuNPs
A. the preparation of BSA@AuNPs nano particle is with the HAuCl of 5 mL through constant temperature
4Solution joins in 5 mL concentration, 50 mg/mL bovine serum albumin(BSA) (BSA) solution, and then stirring reaction 2 min add 0.5 mL 1M NaOH, and at 37 ℃ of lower reaction 1 h that continue, obtain BSA@AuNPs nano particle, particle diameter is 2-3 nm, preserves under the room temperature;
B. the preparation of ABEI-BSA@AuNPs, add 1 mL BSA@AuNPs at PBS buffer solution, add again 10 mg NHS, 20 mg EDC, this mixed liquor is stirred 1 h under 37 ℃, then add 1 mL N-(4-ammonia the butyl)-different luminol of N-ethyl (ABEI) solution, at room temperature stir 12-18 h, obtain ABEI-BSA@AuNPs compound;
C. 1 mL ABEI-BSA@AuNPs solution is got in the preparation of antibody labeling ABEI-BSA@AuNPs, adds NHS 3.6 mg, EDC 7.2 mg, at 37 ℃ of lower activation 1 h, adds the ABEI-BSA@AuNPs of 100 μ L, reaction time 12-18 h again; Be divided into two parts, contain 10 μ g anti-IAA antibody-solutions a the adding again, and 12 h are hatched in 4 ℃ of vibrations, namely get IAA antibody labeling ABEI-BSA@AuNPs; Other a 10 μ g anti-GA antibody-solutions, 12 h are hatched in 4 ℃ of vibrations, obtain GA antibody labeling ABEI-BSA@AuNPs;
(5) detection of bi-component plant hormone, object IAA and the GA standard solution of difference amount are mixed respectively, add anti-GA antibody labeling magnetic bead and anti-IAA antibody labeling golden nanometer particle, hatch 1 h for 37 ℃, clean 3 times with PBST solution after the centrifuging (centrifuge speed is 14000 rpm), add again GA antibody labeling ABEI-BSA@AuNPs and IAA antibody labeling ABEI-BSA@AuNPs mixed solution, hatch 1 h for 37 ℃; Subsequently, with the WB cleansing solution washing that includes 2% BSA 3 times, be divided into 2 equal portions, a suction under magnetic separator abandoned supernatant, and then lower floor's solution carry out chemical luminescent detecting with phosphate buffer solution washing 3 times, according to chemiluminescence signal GA carried out quantitatively; A in addition, under magnetic separator, draw supernatant, the gained supernatant is centrifugal under 13000 rpm rotating speeds, collecting precipitation, and with phosphate buffer solution washing 3 times, then carry out chemical luminescent detecting, according to chemiluminescence signal IAA is carried out quantitatively, according to concentration of standard solution and the signal relation typical curve of mapping to get;
(6) sample analysis, with micro/nano level microdialysis probe intercalation model inside plants, plant hormone is carried out microdialysis non-damaged in situ, real time sample, test by step (5) method in passage downstream component to be measured, can obtain plant hormone IAA and GA content according to chemiluminescence signal and step (5) gained typical curve.
Chemical reagent Optimization Analysis pure reagent of the present invention, all solution all configure with redistilled water.
Phosphate buffer solution of the present invention is 0.2 M(pH7.4), compound method: take by weighing 0.2 g KH
2PO
4, 2.9 g Na
2HPO
412H
2O is dissolved in the 1 L water.
PBS buffer solution of the present invention is 0.2 M(pH7.4), compound method: take by weighing 0.2 g KH
2PO
4, 8.0 g NaCl, 2.9 g Na
2HPO
412H
2O and 0.2 g KCl are dissolved in the 1 L water.
The preparation of PBST solution of the present invention: at 0.2 M(pH7.4) add the Tween-20 of 1 mL in the 1 L PBS buffer solution, the volumetric concentration of Tween-20 is 0.1%.
WB cleansing solution of the present invention: take by weighing 2.429 g three (methylol) aminomethanes (Tris), 9.959 g sodium chloride, 0.509 g Tween-20 is after the dissolving of 500 mL ultrapure waters, transfer PH to 8.0 with 0.1 M hydrochloric acid, be diluted to 1000 mL with ultrapure water at last and get final product.
Chemical luminescent detecting of the present invention is selected MPI-E type chemiluminescence analysis system (Xi'an Rui Mai Analytical Instrument Co., Ltd).
Oscillation incubation of the present invention is selected THZ-82A gas bath constant temperature oscillator (Quan Tan city Medical Instruments factory).
Hydro-extractor of the present invention selects Anke-TGL-16C to fly father-in-law's board supercentrifuge (Shanghai City An Ting scientific instrument factory).
PHS-3D type acidometer (Shanghai thunder magnetic instrument plant) is selected in pH measurement of the present invention.
Remarkable result of the present invention
The present invention has studied the relation between variable concentrations IAA and GA and the luminous intensity, has obtained detecting typical curve, the range of linearity and the linear equation of IAA and GA.
When the concentration of IAA was between 0.02 ng/mL-20 ng/mL, along with the variation of IAA concentration, chemiluminescence intensity had significant change.Calculating the nonlinear equation that detects IAA is I
CL=-647.65*exp (x/0.61)-and 5857.63*exp (x/7.16)+6466.90(I
CLIt is the chemiluminescence intensity of system; X is the concentration of IAA, ng/mL; N=12, n represents same concentration mensuration number of times, R
2=0.9997).The concentration of IAA is certain linear relationship with chemiluminescence intensity in 0.02 ng/mL-0.3 ng/mL scope, its equation of linear regression is I
CL=1273.1x-2.44, linearly dependent coefficient R=0.9991, detectability are 0.01 ng/mL(3 σ).
When the concentration of GA was between 0.02 ng/mL-20 ng/mL, along with the variation of GA concentration, chemiluminescence intensity had significant change.Calculating the nonlinear equation that detects GA is I
CL=-6049.94*exp (x/20.42)-and 3765.90*exp (x/3.18)+9815.16(I
CLIt is the chemiluminescence intensity of system; X is the concentration of GA, ng/mL; N=12, R
2=0.9989).The concentration of GA is certain linear relationship with chemiluminescence intensity in 0.02 ng/mL-0.3 ng/mL scope, its equation of linear regression is I
CL=1308.9x+6.322, linearly dependent coefficient R=0.9995, detectability are 0.008 ng/mL(3 σ).
The precision of this assay method is by being that the IAA of 0.3 ng/mL and GA carry out 11 replicate determinations and calculate to concentration, and relative standard deviation is respectively 3.7% and 3.9%, shows that assay method of the present invention has preferably reappearance.
Description of drawings
Fig. 1. measuring principle figure in the time of the two component plant hormone of ABEI-BSA AuNPs nanoparticle tags
Fig. 2. IAA(A) and GA(B) typical curve, wherein A is the typical curve of IAA, horizontal ordinate is IAA concentration, unit is ng/mL, ordinate I
CLIt is the chemiluminescence intensity of system; B is the typical curve of GA, and horizontal ordinate is GA concentration, and unit is ng/mL, ordinate I
CLIt is the chemiluminescence intensity of system.
Embodiment
Step (1) to (5) according to technical scheme obtains IAA(A) and GA(B) typical curve see Fig. 2, wherein mercaptoethylmaine, N-(4-ammonia butyl)-different luminol of N-ethyl (ABEI) are all available from Shanghai Aladdin reagent; Bovine serum albumin(BSA) (BSA), N-hydroxy-succinamide (NHS), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) is available from Acros(New Jersey, USA), three (methylol) aminomethane hydrochloride (Tris-HCl), Chemical Reagent Co., Ltd., Sinopharm Group; Gold chloride (HAuCl
4), trisodium citrate (Na
3C
6H
5O
7) all be purchased from the rich Dihua worker company limited in Tianjin; The amination magnetic bead is doubly thought happy chromatographic technique development centre available from Tianjin.
Method according to invention is measured IAA and GA content, and adopt standard addition method that method is estimated, the sample determination recovery is 96.0 – 102.2%, and measurement result sees Table 1, and method of the present invention has the high characteristics of precision in IAA and GA detection.With micro/nano level microdialysis probe intercalation model plant corn neck, plant hormone is carried out microdialysis non-damaged in situ, real time sample during mensuration.
Table 1. sample analysis measurement result
a7 measurement results
bUnit: ng/mL.
Claims (10)
1. simultaneously method for measuring of two component plant hormones, plant hormone is two components of IAA and GA, it is characterized in that with golden nanometer particle and amination magnetic bead be carrier, take ABEI-BSA@AuNPs as nanoparticle tags, two components of realization plant hormone IAA and GA are measured simultaneously, and determination step is as follows:
(1) preparation of golden nanometer particle AuNPs, preparation, the used glass container of storage solution of gold nanoparticles are washed 30 min with the chloroazotic acid bubble, then rinse dry for standby well with redistilled water; The HAuCl that in 250 mL round-bottomed flasks, adds 100 mL mass concentrations 0.01%
4, be heated with stirring to boiling, then add fast the Na of 1.8 mL mass concentrations 1%
3C
6H
5O
7, continue heating 10 min, stir 15 min, be cooled to room temperature, transfer in the brown bottle and preserve in the place, cool place;
(2) sample hose of 2 mL is got in the preparation of anti-IAA antibody labeling golden nanometer particle, adds successively pH and be the Tris-HCl of 8.2 2 μ L, 500 mM, the TCEP of 6 μ L10 mM, 6 μ L 10
-4The mercaptoethylmaine of M, room temperature add the AuNPs that 1 mL step (1) prepares after placing 30 min, and reaction 12 h under the room temperature get mercaptoethylmaine and modify golden nanometer particle; The mercaptoethylmaine of getting above-mentioned gained modify golden nanometer particle be dispersed in contain 5(wt) in the phosphate buffer solution of % glutaraldehyde, continue to stir 2 h, with phosphate buffer solution eccentric cleaning 3 times, again with it in phosphate buffer solution, add and contain 10 μ g anti-IAA antibody-solutions, 12 h are hatched in 4 ℃ of vibrations, obtain anti-IAA antibody labeling golden nanometer particle;
(3) preparation of anti-GA antibody labeling magnetic bead, in the sample hose of 2 mL, add and contain 10 μ g anti-GA antibody-solutions, add again 1000 μ L and contain the EDC of 0.1 M and the NHS of 0.2 M, activate 30 min, other gets the small beaker of 10 mL, and adding 50 μ L particle diameters is 2～3 μ m amination magnetic beads, 2000 μ L 0.1M, pH is 6.8 imidazole buffers, activates 30 min; With above-mentioned two solution hybrid reactions, 12 h, magnetic resolution is abandoned supernatant, washes three times with phosphate buffer solution again, is settled to 500 μ L with phosphate buffer solution, obtains anti-GA antibody labeling magnetic bead;
(4) preparation of antibody labeling ABEI-BSA@AuNPs
A. the preparation of BSA@AuNPs nano particle is with the HAuCl of 5 mL through constant temperature
4Solution joins in the BSA solution that 5 mL concentration are 50 mg/mL, and then stirring reaction 2 min add 0.5 mL, 1 M NaOH, and reacts 1 hour 37 ℃ of lower continuation, gets BSA@AuNPs nano particle, and particle diameter is 2-3 nm, preserves under the room temperature;
B. the preparation of ABEI-BSA@AuNPs, be that 7.4 PBS buffer solution adds 1 mL BSA@AuNPs at pH, add again NHS 10 mg, EDC 20mg, this mixed liquor was stirred 1 hour under 37 ℃, then add 1 mL N-(4-ammonia butyl)-different luminol solution of N-ethyl, at room temperature stirred 12-18 hour, and obtained ABEI-BSA@AuNPs compound;
C. 1 mL ABEI-BSA@AuNPs solution is got in the preparation of antibody labeling ABEI-BSA@AuNPs, adds NHS 3.6 mg, EDC 7.2 mg, 37 ℃ of lower activation 1 hour, adds the ABEI-BSA@AuNPs of 100 μ L, reaction time 12-18 hour again; Be divided into two parts, contain 10 μ g anti-IAA antibody-solutions a the adding again, and 1 h is hatched in 4 ℃ of vibrations, obtains IAA antibody labeling ABEI-BSA@AuNPs; Other a 10 μ g anti-GA antibody-solutions, 12 h are hatched in 4 ℃ of vibrations, obtain GA antibody labeling ABEI-BSA@AuNPs;
(5) detection of bi-component plant hormone, object IAA and the GA standard solution of difference amount are mixed respectively, add anti-GA antibody labeling magnetic bead and anti-IAA antibody labeling golden nanometer particle, hatch 1 h for 37 ℃, centrifuging, centrifuge speed are 14000 rpm, clean 3 times with PBST solution after separating, add again GA antibody labeling ABEI-BSA@AuNPs and IAA antibody labeling ABEI-BSA@AuNPs mixed solution, hatch 1 h for 37 ℃; Subsequently, with the WB cleansing solution washing that includes 2% BSA 3 times, be divided into 2 equal portions, a suction under magnetic separator abandoned supernatant, and then lower floor's solution carry out chemical luminescent detecting with phosphate buffer solution washing 3 times, according to chemiluminescence signal GA carried out quantitatively; A in addition, under magnetic separator, draw supernatant, the gained supernatant is centrifugal under 13000 rpm rotating speeds, collecting precipitation, and with phosphate buffer solution washing 3 times, then carry out chemical luminescent detecting, according to chemiluminescence signal IAA is carried out quantitatively, according to concentration of standard solution and the signal relation typical curve of mapping to get;
(6) sample analysis, with micro/nano level microdialysis probe intercalation model inside plants, plant hormone is carried out microdialysis non-damaged in situ, real time sample, test by step (5) method in passage downstream component to be measured, can obtain plant hormone IAA and GA content according to chemiluminescence signal and step (5) gained typical curve.
2. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described chemical reagent is analytical reagent, and all solution all configure with redistilled water.
3. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described phosphate buffer is 0.2M, pH=7.4, compound method: take by weighing 0.2 g KH
2PO
4, 2.9 g Na
2HPO
412H
2O is dissolved in the 1L water.
4. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described PBS buffer solution is 0.2 M, pH=7.4, and its compound method is to take by weighing 0.2 g KH
2PO
4, 8.0 g NaCl, 2.9 g Na
2HPO
412H
2O and 0.2 g KCl are dissolved in the 1L water.
5. according to claim 1 simultaneously methods for measuring of two component plant hormones, the compound method that it is characterized in that described PBST solution are to be 0.2M in concentration, add the Tween-20 of 1 mL in the 1 L PBS buffer solution of pH=7.4.
6. according to claim 1 simultaneously methods for measuring of two component plant hormones, it is characterized in that described WB cleansing solution compound method is to take by weighing 2.429g three (methylol) aminomethane, 9.959 g sodium chloride, 0.509 g Tween-20, after the dissolving of 500 mL ultrapure waters, transfer PH to 8.0 with 0.1 M hydrochloric acid, be diluted to 1000 mL with ultrapure water at last.
7. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described chemical luminescent detecting selects MPI-E type chemiluminescence analysis system.
8. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described oscillation incubation selects THZ-82A gas bath constant temperature oscillator.
9. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described hydro-extractor selects Anke-TGL-16C to fly father-in-law's board supercentrifuge.
10. according to claim 1 simultaneously methods for measuring of two component plant hormones is characterized in that described pH measurement selects PHS-3D type acidometer.
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