CN103323607B - 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
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- 239000002105 nanoparticle Substances 0.000 claims abstract description 15
- 239000000523 sample 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
- 238000012360 testing method Methods 0.000 claims description 19
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- 239000000126 substance Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 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
- 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
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- 230000004913 activation Effects 0.000 claims description 4
- 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
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- 229910021529 ammonia Inorganic materials 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
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 238000013019 agitation Methods 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
- 238000001514 detection method Methods 0.000 claims description 2
- 239000006260 foam Substances 0.000 claims description 2
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- 238000010438 heat treatment Methods 0.000 claims description 2
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- 238000012986 modification Methods 0.000 claims description 2
- 230000004048 modification Effects 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
- 238000001139 pH measurement Methods 0.000 claims description 2
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- 239000008363 phosphate buffer Substances 0.000 claims 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
- 229910004042 HAuCl4 Inorganic materials 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
- -1 adds NHS 3.6mg Substances 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
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- 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 13
- 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
- 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
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 5
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- 230000008859 change Effects 0.000 description 4
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- 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
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- 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
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- 240000008042 Zea mays Species 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
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 chemiluminescence field, relate to the method for a kind of pair of component plant hormone Simultaneously test, be specifically related to the Simultaneously test method of plant hormone heteroauxin (IAA) and gibberellin (GA) two component.
Background technology
Plant hormone is the chemical signal molecule that some concern to plant life, synthesizes in plant, and causes physiological effect with the concentration of denier, affects and controls growing and adaptability to environment of plant.The content of plant hormone in plant is very low.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 analyzes the content of Plants under Salt Stress hormone. 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 quantitativeanalysis ofindole-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 analyzephytohormones.PGRSA Quarterly.2006,33:51) with chemoluminescence method (Hun X, Mei Z H, Wang Z P, HeY H.Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescenceprobe coupling the DNA signal amplification.Spectrochimica Acta Part A, 2012,95:114) etc.
But, these methods respectively have its shortcoming, the present invention utilizes golden nanometer particle and magnetic bead to be carrier, with ABEI-BSA@AuNPs for nanoparticle tags, the two components achieving plant hormone heteroauxin (IAA) and gibberellin (GA) measure, and have the advantage that method is simple, cost is low, highly sensitive.
Summary of the invention
The object of the invention is to provide the method for the two component plant hormone of a kind of Simultaneously test, golden nanometer particle and magnetic bead is utilized to be carrier, with ABEI-BSA@AuNPs for nanoparticle tags, realize two component Simultaneously test of plant hormone heteroauxin (IAA) and gibberellin (GA).
Technical scheme
The method of a kind of pair of component plant hormone Simultaneously test, 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, with ABEI-BSA@AuNPs for nanoparticle tags, realize two component Simultaneously test of plant hormone heteroauxin (IAA) and gibberellin (GA), determination step is as follows:
(1) preparation of golden nanometer particle (AuNPs), prepare, store solution of gold nanoparticles glass container (volumetric flask used, brown, wide-mouth bottle, round-bottomed flask etc.) with chloroazotic acid (acid of hydrochloric acid and nitric acid ratio is 1:3) foam washing 30min (minute), then rinse well with redistilled water, dry for standby; Gold chloride (the HAuCl of 100mL mass concentration 0.01% is added in 250mL round-bottomed flask
4), be heated with stirring to boiling, then add the trisodium citrate (Na of 1.8mL mass concentration 1% fast
3c
6h
5o
7), continue heating 10min, stir 15min, be cooled to room temperature.Transfer in brown bottle and preserve in cool place place;
(2) preparation of anti-IAA antibody labeling golden nanometer particle (AuNPs), get the sample hose of 2mL, add three (methylol) aminomethane hydrochloride (Tris-HCl) (pH is 8.2) of 2 μ L500mM (mM/l) successively, the trichloroethyl phosphate (TCEP) of 6 μ L 10mM, 6 μ L 10
-4the mercaptoethylmaine of M, room temperature adds AuNPs prepared by 1mL step (1) after placing 30min, react 12h (hour) under room temperature, obtains mercaptoethylmaine and modifies golden nanometer particle; The mercaptoethylmaine modification golden nanometer particle getting above-mentioned gained is dispersed in the phosphate buffer solution containing 5wt% glutaraldehyde, Keep agitation 2h, by phosphate buffer solution eccentric cleaning 3 times, again by it in phosphate buffer solution, add containing 10 μ g anti-IAA antibody-solutions, 12h is hatched in 4 DEG C of vibrations, obtains anti-IAA antibody labeling golden nanometer particle;
(3) preparation of anti-GA antibody labeling magnetic bead, add containing 10 μ g anti-GA antibody-solutions in the sample hose of 2mL, add 1000 μ L again containing 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) of 0.1M and the N-hydroxy-succinamide (NHS) of 0.2M, activation 30min, separately get the small beaker of a 10mL, add 50 μ L amination magnetic beads (2 ~ 3 μm), 2000 μ L imidazole buffer (0.1M, pH is 6.8), activation 30min; By above-mentioned two solution hybrid reaction 12h, magnetic resolution, abandons supernatant, then washes three times with phosphate buffer solution, 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
The preparation of a.BSA@AuNPs nano particle, by the HAuCl of 5mL through constant temperature
4solution joins in 5mL concentration 50mg/mL bovine serum albumin(BSA) (BSA) solution, and then stirring reaction 2min adds 0.5mL 1M NaOH, and at 37 DEG C, continue reaction 1h, obtain BSA@AuNPs nano particle, particle diameter is 2-3nm, preserves under room temperature;
The preparation of b.ABEI-BSA@AuNPs, 1mL BSA@AuNPs is added at PBS buffer solution, add 10mgNHS, 20mg EDC again, this mixed liquor is stirred 1h at 37 DEG C, then 1mL N-(4-ammonia butyl) the different luminol of-N-ethyl (ABEI) solution is added, at room temperature stir 12-18h, obtain ABEI-BSA@AuNPs compound;
C. the preparation of antibody labeling ABEI-BSA@AuNPs, gets 1mL ABEI-BSA@AuNPs solution, adds NHS 3.6mg, EDC 7.2mg, at 37 DEG C, activate 1h, then add the ABEI-BSA@AuNPs of 100 μ L, reaction time 12-18h; Be divided into two parts again, portion adds containing 10 μ g anti-IAA antibody-solutions, and 12h is hatched in 4 DEG C of vibrations, obtains IAA antibody labeling ABEI-BSA@AuNPs; A 10 μ g anti-GA antibody-solutions in addition, 12h is hatched in 4 DEG C of vibrations, obtains GA antibody labeling ABEI-BSA@AuNPs;
(5) detection of bi-component plant hormone, the object IAA of difference amount and GA standard solution are mixed respectively, add anti-GA antibody labeling magnetic bead and anti-IAA antibody labeling golden nanometer particle, hatch 1h for 37 DEG C, centrifuging (centrifuge speed is 14000rpm) cleans 3 times with PBST solution afterwards, add GA antibody labeling ABEI-BSA@AuNPs and IAA antibody labeling ABEI-BSA@AuNPs mixed solution again, hatch 1h for 37 DEG C; Subsequently, wash 3 times, be divided into 2 equal portions with the WB cleansing solution including 2%BSA, a suction under magnetic separator abandons supernatant, and lower floor's solution phosphate buffer solution washs 3 times, then carries out chemical luminescent detecting, carries out quantitatively according to chemiluminescence signal to GA; A in addition, Aspirate supernatant under magnetic separator, gained supernatant is centrifugal under 13000rpm rotating speed, collecting precipitation, and wash 3 times with phosphate buffer solution, then carry out chemical luminescent detecting, carry out quantitatively according to chemiluminescence signal to IAA, to map to obtain typical curve according to concentration of standard solution and signal relation;
(6) sample analysis, by micro/nano level microdialysis probe intercalation model inside plants, microdialysis non-damaged in situ, real time sample are carried out to plant hormone, test by step (5) method in passages downstream component to be measured, plant hormone IAA and GA content can be obtained according to chemiluminescence signal and step (5) gained typical curve.
Chemical reagent Optimization Analysis pure reagent of the present invention, all solution all configures with redistilled water.
Phosphate buffer solution of the present invention is the phosphate buffer solution of 0.2M (pH=7.4), and its compound method is: take 0.2gKH
2pO
4, 2.9g Na
2hPO
412H
2o is dissolved in 1L water.
PBS buffer solution of the present invention is the PBS buffer solution of 0.2M (pH=7.4), and its compound method is: take 0.2gKH
2pO
4, 8.0g NaCl, 2.9g Na
2hPO
412H
2o and 0.2g KCl is dissolved in 1L water.
The preparation of PBST solution of the present invention: the Tween-20 adding 1mL at the 1L PBS of 0.2M (pH7.4) in buffer solution, the volumetric concentration of Tween-20 is 0.1%.
WB cleansing solution of the present invention: take 2.429g tri-(methylol) aminomethane (Tris), 9.959g sodium chloride, 0.509g Tween-20, after dissolving with 500mL ultrapure water, adjust PH to 8.0 with 0.1M hydrochloric acid, be finally diluted to 1000mL with ultrapure water and get final product.
Chemical luminescent detecting of the present invention selects MPI-E type chemiluminescence analysis system (Xi'an Rui Mai Analytical Instrument Co., Ltd).
Vibration of the present invention is hatched and is selected THZ-82A gas bath constant temperature oscillator (Medical Instruments factory of Quan Tan city).
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 Lei Ci instrument plant) is selected in pH measurement of the present invention.
Remarkable result of the present invention
The present invention have studied the relation between variable concentrations IAA and GA and luminous intensity, obtains the typical curve, the range of linearity and the linear equation that detect IAA and GA.
When the concentration of IAA is between 0.02ng/mL-20ng/mL, along with the change of IAA concentration, chemiluminescence intensity has significant change.Calculating the nonlinear equation detecting IAA is I
cL=-647.65*exp (-x/0.61)-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 represent that same concentration measures number of times, R
2=0.9997).The concentration of IAA is certain linear relationship with chemiluminescence intensity within the scope of 0.02ng/mL-0.3ng/mL, and its equation of linear regression is I
cL=1273.1x-2.44, linearly dependent coefficient R=0.9991, detectability is 0.01ng/mL (3 σ).
When the concentration of GA is between 0.02ng/mL-20ng/mL, along with the change of GA concentration, chemiluminescence intensity has significant change.Calculating the nonlinear equation detecting GA is I
cL=-6049.94*exp (-x/20.42)-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 within the scope of 0.02ng/mL-0.3ng/mL, and its equation of linear regression is I
cL=1308.9x+6.322, linearly dependent coefficient R=0.9995, detectability is 0.008ng/mL (3 σ).
The precision of this assay method is by being that IAA and GA of 0.3ng/mL carries 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 good reappearance.
Accompanying drawing explanation
The Simultaneously test schematic diagram of the two component plant hormone of Fig. 1 .ABEI-BSA@AuNPs nanoparticle tags
Fig. 2 .IAA (A) and GA (B) typical curve, wherein A is the typical curve of IAA, and horizontal ordinate is IAA concentration, and 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
Obtain IAA (A) according to the step (1) to (5) of technical scheme and GA (B) typical curve is shown in Fig. 2, wherein mercaptoethylmaine, N-(4-ammonia butyl) the different luminol of-N-ethyl (ABEI) are all purchased from Shanghai Aladdin reagent; Bovine serum albumin(BSA) (BSA), N-hydroxy-succinamide (NHS), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) is purchased from Acros (NewJersey, USA), three (methylol) aminomethane hydrochloride (Tris-HCl), Chemical Reagent Co., Ltd., Sinopharm Group; Gold chloride (HAuCl
4), trisodium citrate (Na
3c
6h
5o
7) be all purchased from Tianjin Bo Di Chemical Co., Ltd.; Amination magnetic bead doubly thinks happy chromatographic technique development centre purchased from Tianjin.
Method according to invention measures IAA and GA content, and adopt standard addition method to evaluate method, the sample determination recovery is 96.0 – 102.2%, and measurement result is in table 1, and method of the present invention has the high feature of precision in IAA and GA detects.With micro/nano level microdialysis probe intercalation model plant Zea mays neck during mensuration, microdialysis non-damaged in situ, real time sample are carried out to plant hormone.
Table 1. sample analysis measurement result
a7 measurement results
bunit: ng/mL
Claims (10)
1. the method for a two component plant hormone Simultaneously test, plant hormone is two components of IAA and GA, it is characterized in that with golden nanometer particle and amination magnetic bead be carrier, with ABEI-BSA@AuNPs for nanoparticle tags, realize two component Simultaneously test of plant hormone IAA and GA, determination step is as follows:
(1) preparation of golden nanometer particle AuNPs, prepares, stores solution of gold nanoparticles glass container chloroazotic acid foam washing 30min used, then rinse well with redistilled water, dry for standby; The HAuCl of 100mL mass concentration 0.01% is added in 250mL round-bottomed flask
4, be heated with stirring to boiling, then add the Na of 1.8mL mass concentration 1% fast
3c
6h
5o
7, continue heating 10min, stir 15min, be cooled to room temperature, transfer in brown bottle and preserve in cool place place;
(2) preparation of anti-IAA antibody labeling golden nanometer particle, gets the sample hose of 2mL, adds the Tris-HCl that pH is the 2 μ L 500mM of 8.2 successively, the TCEP of 6 μ L10mM, 6 μ L 10
-4the mercaptoethylmaine of M, room temperature adds AuNPs prepared by 1mL step (1) after placing 30min, react 12h under room temperature, obtains mercaptoethylmaine and modifies golden nanometer particle; The mercaptoethylmaine modification golden nanometer particle getting above-mentioned gained is dispersed in the phosphate buffer solution containing 5wt% glutaraldehyde, Keep agitation 2h, by phosphate buffer solution eccentric cleaning 3 times, again by it in phosphate buffer solution, add containing 10 μ g anti-IAA antibody-solutions, 12h is hatched in 4 DEG C of vibrations, obtains anti-IAA antibody labeling golden nanometer particle;
(3) preparation of anti-GA antibody labeling magnetic bead, add containing 10 μ g anti-GA antibody-solutions in the sample hose of 2mL, add the NHS of 1000 μ L containing EDC and 0.2M of 0.1M again, activation 30min, separately get the small beaker of a 10mL, adding 50 μ L particle diameters is 2 ~ 3 μm of amination magnetic beads, 2000 μ L 0.1M, pH is 6.8 imidazole buffers, activation 30min; By above-mentioned two solution hybrid reaction 12h, magnetic resolution, abandons supernatant, then washes three times with phosphate buffer solution, 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
The preparation of a.BSA@AuNPs nano particle, it is in the BSA solution of 50mg/mL that 5mL is joined 5mL concentration through the HAuCl4 solution of constant temperature, stirring reaction 2min, then 0.5mL 1MNaOH is added, and at 37 DEG C, continue reaction 1 hour, obtain BSA@AuNPs nano particle, particle diameter is 2-3nm, preserves under room temperature;
The preparation of b.ABEI-BSA@AuNPs, the PBS buffer solution being 7.4 at pH adds 1mLBSA@AuNPs, add NHS 10mg, EDC 20mg again, this mixed liquor is stirred 1 hour at 37 DEG C, then 1mL N-(4-ammonia butyl) the different luminol solution of-N-ethyl is added, at room temperature stir 12-18 hour, obtain ABEI-BSA@AuNPs compound;
C. the preparation of antibody labeling ABEI-BSA@AuNPs, gets 1mL ABEI-BSA@AuNPs solution, adds NHS 3.6mg, EDC 7.2mg, activates 1 hour, then add the ABEI-BSA@AuNPs of 100 μ L, reaction time 12-18 hour at 37 DEG C; Be divided into two parts again, portion adds containing 10 μ ganti-IAA antibody-solutions, and 1h is hatched in 4 DEG C of vibrations, obtains IAA antibody labeling ABEI-BSA@AuNPs; A 10 μ g anti-GA antibody-solutions in addition, 12h is hatched in 4 DEG C of vibrations, obtains GA antibody labeling ABEI-BSA@AuNPs;
(5) detection of bi-component plant hormone, the object IAA of difference amount and GA standard solution are mixed respectively, add anti-GA antibody labeling magnetic bead and anti-IAA antibody labeling golden nanometer particle, hatch 1h for 37 DEG C, centrifuging, centrifuge speed is 14000rpm, cleans 3 times after being separated with PBST solution, add GA antibody labeling ABEI-BSA@AuNPs and IAA antibody labeling ABEI-BSA@AuNPs mixed solution again, hatch 1h for 37 DEG C; Subsequently, wash 3 times, be divided into 2 equal portions with the WB cleansing solution including 2%BSA, a suction under magnetic separator abandons supernatant, and lower floor's solution phosphate buffer solution washs 3 times, then carries out chemical luminescent detecting, carries out quantitatively according to chemiluminescence signal to GA; A in addition, Aspirate supernatant under magnetic separator, gained supernatant is centrifugal under 13000rpm rotating speed, collecting precipitation, and wash 3 times with phosphate buffer solution, then carry out chemical luminescent detecting, carry out quantitatively according to chemiluminescence signal to IAA, to map to obtain typical curve according to concentration of standard solution and signal relation;
(6) sample analysis, by micro/nano level microdialysis probe intercalation model inside plants, microdialysis non-damaged in situ, real time sample are carried out to plant hormone, test by step (5) method in passages downstream component to be measured, plant hormone IAA and GA content can be obtained according to chemiluminescence signal and step (5) gained typical curve.
2. the method for according to claim 1 pair of component plant hormone Simultaneously test, it is characterized in that described chemical reagent is analytical reagent, all solution all configures with redistilled water.
3. the method for according to claim 1 pair of component plant hormone Simultaneously test, it is characterized in that described phosphate buffer is the phosphate buffer of 0.2M, pH=7.4, its compound method is: take 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
2o is dissolved in 1L water.
4. the method for according to claim 1 pair of component plant hormone Simultaneously test, it is characterized in that described PBS buffer solution is the PBS buffer solution of 0.2M, pH=7.4, its compound method is: take 0.2g KH
2pO
4, 8.0g NaCl, 2.9g Na
2hPO
412H
2o and 0.2g KCl is dissolved in 1L water.
5. the method for according to claim 1 pair of component plant hormone Simultaneously test, is characterized in that the compound method of described PBST solution is the 1L PBS being 0.2M, pH=7.4 in concentration adds 1mL Tween-20 in buffer solution.
6. the method for according to claim 1 pair of component plant hormone Simultaneously test, it is characterized in that described WB cleansing solution compound method takes 2.429g tri-(methylol) aminomethane, 9.959g sodium chloride, 0.509g Tween-20, after dissolving with 500mL ultrapure water, adjust PH to 8.0 with 0.1M hydrochloric acid, be finally diluted to 1000mL with ultrapure water.
7. the method for according to claim 1 pair of component plant hormone Simultaneously test, is characterized in that described chemical luminescent detecting selects MPI-E type chemiluminescence analysis system.
8. the method for according to claim 1 pair of component plant hormone Simultaneously test, is characterized in that described vibration is hatched and selects THZ-82A gas bath constant temperature oscillator.
9. the method for according to claim 1 pair of component plant hormone Simultaneously test, is characterized in that described hydro-extractor selects Anke-TGL-16C to fly father-in-law's board supercentrifuge.
10. the method for according to claim 1 pair of component plant hormone Simultaneously test, is characterized in that PHS-3D type acidometer is selected in described pH measurement.
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