CN106404865A - Microelectrode biosensor for online detection of IAA (auxin) in living plant and application of microelectrode biosensor - Google Patents
Microelectrode biosensor for online detection of IAA (auxin) in living plant and application of microelectrode biosensor Download PDFInfo
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Abstract
The invention relates to a microelectrode biosensing technology, and particularly discloses a microelectrode biosensor for online detection of IAA (auxin) in a living plant. The microelectrode biosensor is characterized in that the specific recognition on the IAA is performed through an IAA-modified antibody on a gold (or platinum) working electrode, and the highly sensitive detection of the IAA is performed through AuNPs (gold nanoparticles) and modified IAA-AuNPs compounds deposited on the electrode. The microelectrode biosensor disclosed by the invention has the advantages that dynamic change information of the IAA in a plant body is mastered in situ and in real time by means of continuously monitoring the IAA in the living plant online, the metabolic process of the IAA is understood, and a theoretical basis is provided for understanding the participation of the IAA in the regulation mechanism of a plant living system; the IAA in the living plant can be continuously monitored online by utilization of the microelectrode biosensor disclosed by the invention, and a to-be-detected specimen is prevented from being essentially damaged; an obtained data result can dynamically reflect the IAA content change in the plant body in real time, and the actual application operation is simple and convenient and is easy to master.
Description
Technical field
The present invention relates to microelectrode biosensing technology, specifically, it is related to a kind of live body on-line checking auximone
Microelectrode biosensor.
Background technology
As a kind of small organic molecule in plant body, auxin (IAA) is primarily involved in the growth of plant and developed
Journey.Auxin generally has positive and negative duality to the induction of the bud that plant embryos bud scale is most advanced and sophisticated, children is tender, leaf etc., depends primarily on it
Concentration and the physiological situation of plant.Under normal circumstances, excessive auxin can promote the generation of ethylene in plant body, upsets plant
Internal physiological equilibrium, thus suppress the growth of plant.On the other hand, the growth of fruit be unable to do without auxin.Seed exists
In growth course, the auxin of generation can change the distribution of its nutrient substance, makes ovary development be fruit.
At present, conventional auxin detection method mainly has enzyme immunity (ELISA) detection method, radioimmunoassay, RIA (RIA)
Method, high performance liquid chromatography (HPLC) method, gas chromatogram (GC) method etc..In addition, in recent years, spectrographic method, electrochemical process etc.
It is applied to the detection of auxin.But current detection method is all the Testing in vitro destroying plant sample, is only capable of reacting a certain
The static concentration of time point or accumulative effect.Traditional detection method need to carry out the pretreatment such as separating-purifying to sample, not only
Complex operation, time-consuming high cost, larger to the destructiveness of sample, and detection can only be sampled in vitro it is impossible to embody IAA in sample
The Real-time and Dynamic change of content.
The content of the auxin in plant body is few and auxin around coexist that matrix composition is extremely complex, its content is easily subject to
The impact of the external conditions such as illumination condition, moisture and temperature, therefore, with going deep into of research, researcher is more desirable to obtain plants
Thing adapts to the Real-time and Dynamic change of auxin during external environment.
Content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of life of live body on-line checking plant
The microelectrode biosensor of long element and its application.
In order to realize the object of the invention, technical scheme is as follows：
In a first aspect, the invention provides a kind of microelectrode biosensor of live body on-line checking auximone, institute
The preparation method stating working electrode in microelectrode biosensor comprises the steps：
S1, metal working electrode or platinum working electrode are immersed HAuCl4In solution, on the working electrode (s through electrochemical deposition
Obtain the working electrode of AuNPs modification；
S2, on the working electrode of S1 gained drop coating mercaptoundecylic acid (MUA), make MUA be assembled into S1 institute by Au S key
On the working electrode obtaining；
S3, on the working electrode of S2 gained, drop coating contains N hydroxysuccinimide (NHS) and 1-3- ethyl carbodiimide
The mixed solution of hydrochlorate (EDC), the carboxyl of activation MUA；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, obtain IAA antibody modification work electricity
Pole；
S5, on the working electrode of S4 gained drop coating K3Fe(CN)6Mediator.
In order to sensitivity and specificity that working electrode to IAA detect is better achieved, the present invention repaiies to working electrode
Panel part is further optimized, specific as follows：
S1, metal working electrode or platinum working electrode are immersed the KNO containing 0.5～1M32-5mM HAuCl4In solution, in work
Make the working electrode that -0.4V electrochemical deposition 200s on electrode obtains AuNPs modification；
S2, on the working electrode of S1 gained drop coating 1～5mM mercaptoundecylic acid (MUA), incubation 1～2h after washed away with water
Unnecessary unreacted MUA, makes MUA be assembled on the working electrode of S1 gained by Au S key；
S3, on the working electrode of S2 gained, drop coating contains 50mM N hydroxysuccinimide and 1-3- ethyl carbodiimide
The mixed solution of hydrochlorate (200mM), after incubation 1-2h, the carboxyl of activation MUA；
In the mixed solution of described N hydroxysuccinimide and 1-3- ethyl-carbodiimide hydrochloride, N hydroxy succinic acid is sub-
Amine is 1 with the molar concentration rate of 1-3- ethyl-carbodiimide hydrochloride:1～1:4；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, incubation 1～4h obtain IAA antibody modification
Working electrode；
S5, on the working electrode of S4 gained drop coating 5～10mM K3Fe(CN)6Mediator.
Preferably, the optimal preparation method of described working electrode includes：
S1, metal working electrode or platinum working electrode are immersed KNO containing 1M33mM HAuCl4In solution, in working electrode
Upper -0.4V electrochemical deposition 200s obtains the working electrode of AuNPs modification；
S2, on the working electrode of S1 gained drop coating 2mM mercaptoundecylic acid (MUA), incubation 1h after with water wash away unnecessary not
The MUA of reaction, makes MUA be assembled on the working electrode of S1 gained by Au S key；
S3, on the working electrode of S2 gained, drop coating contains N hydroxysuccinimide and 1-3- ethyl carbodiimide hydrochloride
The mixed solution of salt, after incubation 1-2h, the carboxyl of activation MUA；
In described mixed solution, the concentration of N hydroxysuccinimide is 50mM, 1-3- ethyl-carbodiimide hydrochloride dense
Spend for 200mM；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, incubation 1h obtain IAA antibody modification
Working electrode；
S5, on the working electrode of S4 gained drop coating 5mM K3Fe(CN)6Mediator.
The present invention deposited golden nanometer particle first on metal working electrode, and research finds, electrode deposits Jenner's grain of rice
After son, the surface area of electrode can be dramatically increased, thus greatly improving the sensitivity of detection.Secondly, the present invention is to HAuCl4Molten
The concentration of liquid is optimized, and finds HAuCl4Concentration effect in 2～5mM is preferable, and the wherein effect of 3mM is best.In this base
On plinth, the present invention is optimized to the condition of electrochemical deposition, AuNPs can be made to deposit in -0.4V electrochemical deposition 200s
More uniform, thus improving the sensitivity of detection.Then, the present invention is spotted with the working electrode that golden nanometer particle is modified
Mercaptoundecylic acid (MUA) is to provide carboxyl, and its concentration is optimized, and finds the MUA effect of 1-5mM preferably, concentration is too
Low be not enough to cover electrode surface, the too high electric conductivity then affecting electrode, and then reduce detection performance, wherein the effect of 2mM is
Good.Further on electrode drop coating NHS and EDC mixed solution, to activate carboxyl.The concentration ratio of NHS and EDC is 1：1-1:4,
Wherein 1:4 effects are best.Then, by anti-for AuNPs- IAA complex drop coating on working electrode, incubation 1-4h obtains IAA antibody and repaiies
The working electrode of decorations.Realize the specific detection to IAA using the antibody of IAA, and AuNPs- anti-IAA complex then can be further
Strengthen detection signal, thus improving the sensitivity of detection.
Further, the preparation method of the anti-IAA of AuNPs- described in S4 complex is：Prepared by reduction of sodium citrate method
Golden nanometer particle, the pH regulator of solution of gold nanoparticles to 7.6 adds 100 μ L to resist in 400 μ L solution of gold nanoparticles
The antibody (1mg/ml) of IAA, after concussion 5min, is incubated 12h in 4 DEG C；Then by mixture in 4 DEG C, 12000rpm centrifugation
30min, to remove the antibody being not connected with；Afterwards precipitation is scattered in the PBS that 1ml contains 1%BSA, pH7.4, is formed
AuNPs- anti-IAA complex.
Further, described microelectrode biosensor also includes Ag/AgCl reference electrode and platinum to electrode.
Further, in order that described microelectrode biosensor is applied to the on-line checking of plant living body, it is to avoid in vitro
Detect the change of sample and loss in the destruction to sample and sampling process, the present invention adopts pin type microelectrode, and microelectrode passes through
The micro electro mechanical processings such as film, etching technique (MEMS) fabrication techniques, prepare above-mentioned working electrode, Ag/AgCl in silicon chip substrate
, to electrode, its outward appearance has the ability penetrating plant tissue for reference electrode and platinum, and length is about 30～50mm.
Second aspect, the invention provides described microelectrode biosensor is in terms of live body on-line checking auximone
Application.
Described plant includes forest, flowers, vegetable, crops etc., and described plant tissue is the stem of plant, leaf, fruit.
Further, the present invention provides a kind of method of live body on-line checking auximone, and methods described includes：
1) described microelectrode biosensor is connected to electrochemical workstation, react with the standard solution of IAA, by electricity
Chemical cycle voltammetry determines that response voltage is 0.2V；Then the standard solution of microelectrode and the IAA of variable concentrations is reacted,
Under 0.2V running voltage, continuous detecting is carried out by chronoamperometry, standard is prepared in the concentration value mapping of peak point current and IAA
Curve；
2) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, in 0.2V running voltage
Under, detect the electric current I at tested position by chronoamperometry, by with step 1) described in standard curve equation contrast, meter
Calculate the real-time IAA concentration obtaining test serum.
In a specific embodiment of the present invention, the stalk selecting Semen Glyciness seedling is material, and In vivo detection IAA contains
Amount.The stalk position that microelectrode biosensor is inserted Semen Glyciness seedling carries out In vivo detection, and is connected with electrochemical workstation
Obtain the curent change that caused with electrode reaction by IAA, go out containing of IAA in biopsy sample juice using the change calculations of this electric current
Amount.
The beneficial effects of the present invention is：
The present invention passes through to modify the specific recognition to IAA for the antibody realization of IAA on the working electrode (s, by electrode
Depositing gold nanoparticles and modify IAA- golden nanometer particle complex and realize the highly sensitive detection of IAA.
The invention provides a kind of live body on-line continuous inspection based on auxin in the plant body of miniature organism sensing technology
Survey method, thus more accurately and real-time understand the dynamic rule of auxin and mechanism of action in plant body.
The present invention, by the on-line checking to IAA in live plant body, grasps auxin in plant body in situ in real time
Dynamic-change information, understands the metabolic process of IAA, the Regulation Mechanism for understanding IAA involved in plant life system provide theoretical according to
According to.Application microbiosensor realizes the on-line checking to IAA in plant living body, and detected sample is not caused with essence wound
Evil；The data result obtaining can in the reflection plant body of Real-time and Dynamic IAA changes of contents, actual operation easy it is easy to
Grasp.
Brief description
Fig. 1 is the simple schematic diagram with detection for the preparation of working electrode of the present invention.
Fig. 2 is the schematic diagram that microelectrode biosensor of the present invention detects plant tissue.
Specific embodiment
Below in conjunction with embodiment, the preferred embodiment of the present invention is described in detail.It will be appreciated that it is following real
Applying providing merely to playing descriptive purpose of example, being not used to the scope of the present invention is limited.The skill of this area
Art personnel, in the case of without departing substantially from spirit of the invention and spirit, can carry out various modifications and replace to the present invention.
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
The preparation of embodiment 1 working electrode
1) golden nanometer particle is prepared by reduction of sodium citrate method.The pH value of solution of gold nanoparticles is adjusted to pH7.6,
Add the antibody (1mg/ml) of 100 anti-IAA in 400 μ L solution of gold nanoparticles, after concussion 5min, be incubated 12h in 4 DEG C.So
Afterwards by mixture in 4 DEG C, 12000rpm centrifugation 30min, to remove the antibody being not connected with.Afterwards precipitation is scattered in 1ml and contains 1%
In the PBS of BSA, pH7.4, form AuNPs- anti-IAA complex.
2), after microelectrode cleaning treatment, immerse KNO containing 1M33mM HAuCl4In solution, -0.4V on metal working electrode
Electrochemical deposition 200s obtains the microelectrode of AuNPs modification.Drop coating 2mM mercaptoundecylic acid on the working electrode after modification
(MUA), wash away unnecessary unreacted MUA with water after incubation 1h, so that MUA is assembled on the gold electrode after modification by Au S key.
3) drop coating 50mM N hydroxysuccinimide (NHS) and 200mM1-3- ethyl carbodiimide salt on the working electrode (s
Anti- for AuNPs- IAA complex, after incubation 1h, is dropped to modification after the carboxyl on activation MUA by the mixed solution of hydrochlorate (EDC)
Gold electrode surfaces, incubation 1h obtains the microelectrode of IAA antibody modification, further drop coating 5mM K on the working electrode (s3Fe(CN)6Matchmaker
Amboceptor.
The application of embodiment 2 microelectrode biosensor
1) microelectrode preparing is connected to electrochemical workstation, react with the standard solution of IAA, followed by electrochemistry
Ring voltammetry determines that response voltage is 0.2V.Microelectrode is reacted with the standard solution of the IAA of variable concentrations, in 0.2V running voltage
Down continuous detecting is carried out by chronoamperometry.By the concentration value mapping of peak point current and IAA, prepare standard curve.
2) do experiment material from culture to the 7th day Semen Glyciness seedling, the tender stem that microelectrode is inserted bean seedlings connects to electrification
Learn work station, on-line continuous measure the concentration change of IAA in 4 days.
3) take the Semen Glyciness seedling tender stem cultivated to the 7th, 8,9,10 days respectively, with traditional liquid chromatography-mass spectrography side of being used in conjunction
Method carries out the detection of IAA to the sample chosen.Liquid chromatography-mass spectrography is used in conjunction result and the contemporaneity that (HPLC-MS) obtains
The result of microelectrode on-line checking is contrasted.
Every group of experiment calculates its meansigma methods in triplicate, obtains result such as table 1：
Table 1 different times Semen Glyciness seedling stem IAA content detection result
| Sample | Microelectrode (ng g-1FW) | HPLC-MS(ng·g-1FW) |
| 7thD | 16.2(±0.24) | 15.7(±0.33) |
| 8thD | 18.6(±0.17) | 18.3(±0.42) |
| 9thD | 19.3(±0.12) | 19.5(±0.39) |
| 10thD | 20.0(±0.04) | 19.8(±0.24) |
As seen from the above table, using microelectrode on-line checking Semen Glyciness seedling stem IAA content and traditional HPLC-MS method measurement
Result data is substantially identical.The method data is reliable, selectivity is high, can achieve to IAA High sensitivity, single-minded identification, is suitable for
In different tissues positions such as the stem of plant, leaf, fruits, in achievable plant body, the live body on-line continuous detection of IAA, contributes to
The regulation rule of solution IAA involved in plant vital movement and mechanism of action.
Embodiment 3
1), after metal working electrode cleaning treatment, immerse KNO containing 0.5M31mM HAuCl4In solution, in metal working electrode
Upper -0.4V electrochemical deposition 200s obtains the microelectrode of AuNPs modification.Drop coating mercaptoundecylic acid (MUA) thereon, after incubation 1h
Wash away unnecessary unreacted MUA with water, make MUA on the gold electrode after Au-S key is assembled into modification.
2) drop coating 50mM N hydroxysuccinimide (NHS) and 200mM1-3- ethyl carbodiimide salt on the working electrode (s
The mixed solution of hydrochlorate (EDC), after incubation 1h, anti-for AuNPs- IAA complex is dropped to the gold electrode surfaces of modification, is incubated 1h
Obtain the microelectrode of IAA antibody modification, further drop coating 5mM K on the working electrode (s3Fe(CN)6Mediator.
3) microelectrode that this is modified is connected to electrochemical workstation, then by the mark of microelectrode and the IAA of variable concentrations
Quasi- solution reaction, carries out continuous detecting by chronoamperometry under running voltage, and the concentration value of peak point current and IAA is made
Figure, prepared standard curve compared with the working electrode containing electro-deposition gold nanoparticles step of preparation under optimal conditionss,
Slope is slightly less than normal, shows the sensitivity decrease of sensor.
4) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 3) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.Its detected value is all less than normal, and larger with the numerical bias that HPLC-MS method detects.
Embodiment 4
1), after microelectrode cleaning treatment, immerse KNO containing 1M33mM HAuCl4In solution, -0.4V on metal working electrode
Electrochemical deposition 200s obtains the microelectrode of AuNPs modification.Drop coating 2mM mercaptoundecylic acid on the working electrode after modification
(MUA), wash away unnecessary unreacted MUA with water after incubation 1h, so that MUA is assembled on the gold electrode after modification by Au S key.
2) drop coating 50mM N hydroxysuccinimide (NHS) and 500mM1-3- ethyl carbodiimide salt on the working electrode (s
The molar concentration rate of NHS and EDC (is adjusted to 1 by the mixed solution of hydrochlorate (EDC):10) carboxylic, after incubation 1h, on activation MUA
After base, anti-for AuNPs- IAA complex is dropped to the gold electrode surfaces of modification, incubation 1h obtains the microelectrode of IAA antibody modification,
Drop coating 5mM K on the working electrode (s further3Fe(CN)6Mediator.
3) microelectrode that this is modified is connected to electrochemical workstation, react with the standard solution of IAA, followed by electrochemistry
Ring voltammetry determines response voltage.Then the standard solution of microelectrode and the IAA of variable concentrations is reacted, logical under running voltage
Cross chronoamperometry and carry out continuous detecting, by concentration value mapping, prepared standard curve and the present invention of peak point current and IAA
The molar concentration rate of NHS and EDC of middle preparation is 1:1-1:Working electrode in the range of 4 is compared, and slope is less, shows sensor
Sensitivity relatively low.
4) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 3) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.Its detected value is all less than normal, and larger with the numerical bias that HPLC-MS method detects.
Comparative example 1
1) after metal working electrode cleaning treatment, drop coating mercaptoundecylic acid (MUA) thereon, (do not carry out deposited Au nanometer
Particle), wash away unnecessary unreacted MUA with water after incubation 1h, so that MUA is assembled on the gold electrode after modification by Au S key.
2) drop coating 50mM N hydroxysuccinimide (NHS) and 200mM1-3- ethyl carbodiimide salt on the working electrode (s
The mixed solution of hydrochlorate (EDC), after incubation 1h, anti-for AuNPs- IAA complex is dropped to the gold electrode surfaces of modification, is incubated 1h
Obtain the microelectrode of IAA antibody modification, further drop coating 5mM K on the working electrode (s3Fe(CN)6Mediator.
3) microelectrode that this is modified is connected to electrochemical workstation, react with the standard solution of IAA, followed by electrochemistry
Ring voltammetry determines response voltage.Then the standard solution of microelectrode and the IAA of variable concentrations is reacted, logical under running voltage
Cross chronoamperometry and carry out continuous detecting, by the concentration value mapping of peak point current and IAA, prepared standard curve is made with us
The standby working electrode containing electro-deposition gold nanoparticles step is compared, and slope is less, shows that the sensitivity of sensor is relatively low.
4) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 3) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.Its detected value is all less than normal, and larger with the numerical bias that HPLC-MS method detects.
Comparative example 2
1) after metal working electrode cleaning treatment, drop coating mercaptoundecylic acid (MUA) thereon, (do not carry out deposited Au nanometer
Particle), wash away unnecessary unreacted MUA with water after incubation 1h, so that MUA is assembled on the gold electrode after modification by Au S key.
2) drop coating 50mM N hydroxysuccinimide (NHS) and 200mM1-3- ethyl carbodiimide salt on the working electrode (s
The mixed solution of hydrochlorate (EDC), after incubation 1h, IAA antibody is dropped to gold electrode surfaces (the not drop coating Jenner grain of rice of modification
Son-anti-IAA complex), incubation 1h obtains the microelectrode of IAA antibody modification, further drop coating 5mM K on the working electrode (s3Fe
(CN)6Mediator.
3) microelectrode that this is modified is connected to electrochemical workstation, react with the standard solution of IAA, followed by electrochemistry
Ring voltammetry determines response voltage.Then the standard solution of microelectrode and the IAA of variable concentrations is reacted, logical under running voltage
Cross chronoamperometry and carry out continuous detecting, by the concentration value mapping of peak point current and IAA, prepared standard curve is made with us
The standby working electrode containing electro-deposition gold nanoparticles step is compared, and slope is less, shows that the sensitivity of sensor is relatively low.
4) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 3) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.Result can only detect that the IAA of the Semen Glyciness seedling cultivated to the 10th day, the IAA of other several days contain
Amount can't detect, and numerical value is less than normal, and larger with the numerical bias that HPLC-MS method detects.
Although, above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. a kind of microelectrode biosensor of live body on-line checking auximone is it is characterised in that described microelectrode is biological
In sensor, the preparation method of working electrode comprises the steps：
S1, metal working electrode or platinum working electrode are immersed HAuCl4The work of AuNPs modification in solution, is obtained through electrochemical deposition
Make electrode；
S2, on the working electrode of S1 gained drop coating MUA, so that MUA is assembled on the working electrode of S1 gained by Au S key；
S3, on the working electrode of S2 gained, drop coating contains N hydroxysuccinimide and 1-3- ethyl-carbodiimide hydrochloride
Mixed solution, the carboxyl of activation MUA；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, obtain the working electrode of IAA antibody modification；
S5, on the working electrode of S4 gained drop coating K3Fe(CN)6Mediator.
2. microelectrode biosensor according to claim 1 is it is characterised in that work in described microelectrode biosensor
The preparation method making electrode comprises the steps：
S1, metal working electrode or platinum working electrode are immersed the KNO containing 0.5～1M32-5mM HAuCl4In solution, in work electricity
Extremely go up the working electrode that -0.4V electrochemical deposition 200s obtains AuNPs modification；
S2, on the working electrode of S1 gained drop coating 1～5mM MUA, incubation 1～2h after wash away unnecessary unreacted MUA with water,
MUA is made to be assembled on the working electrode of S1 gained by Au S key；
S3, on the working electrode of S2 gained, drop coating contains N hydroxysuccinimide and 1-3- ethyl-carbodiimide hydrochloride
Mixed solution, after incubation 1-2h, the carboxyl of activation MUA；
In the mixed solution of described N hydroxysuccinimide and 1-3- ethyl-carbodiimide hydrochloride N hydroxysuccinimide with
The molar concentration rate of 1-3- ethyl-carbodiimide hydrochloride is 1:1～1:4；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, incubation 1～4h obtains the work of IAA antibody modification
Make electrode；
S5, on the working electrode of S4 gained drop coating 5～10mM K3Fe(CN)6Mediator.
3. microelectrode biosensor according to claim 2 is it is characterised in that work in described microelectrode biosensor
The preparation method making electrode comprises the steps：
S1, metal working electrode or platinum working electrode are immersed KNO containing 1M33mM HAuCl4In solution, on the working electrode (s-
0.4V electrochemical deposition 200s obtains the working electrode of AuNPs modification；
S2, on the working electrode of S1 gained drop coating 2mM MUA, incubation 1h after wash away unnecessary unreacted MUA with water, make MUA
It is assembled on the working electrode of S1 gained by Au S key；
S3, on the working electrode of S2 gained, drop coating contains N hydroxysuccinimide and 1-3- ethyl-carbodiimide hydrochloride
Mixed solution, after incubation 1-2h, the carboxyl of activation MUA；
In described mixed solution, the concentration of N hydroxysuccinimide is 50mM, and the concentration of 1-3- ethyl-carbodiimide hydrochloride is
200mM；
S4, on the working electrode of S3 gained drop coating AuNPs- anti-IAA complex, incubation 1h obtains the work of IAA antibody modification
Electrode；
S5, on the working electrode of S4 gained drop coating 5mM K3Fe(CN)6Mediator.
4. the microelectrode biosensor according to any one of claims 1 to 3 is it is characterised in that AuNPs- described in S4
The preparation method of anti-IAA complex is：By the pH regulator of solution of gold nanoparticles to 7.6, add the antibody incubation of anti-IAA, from
It is scattered in the PBS containing 1%BSA, pH7.4 after heart precipitation, form AuNPs- anti-IAA complex.
5. microelectrode biosensor according to claim 4 is it is characterised in that described microelectrode biosensor also wraps
Include Ag/AgCl reference electrode and platinum to electrode.
6. the microelectrode biosensor described in any one of Claims 1 to 5 is in terms of live body on-line checking auximone
Application.
7. application according to claim 6 is it is characterised in that be specially：
1) microelectrode biosensor described in any one of Claims 1 to 5 is connected to electrochemical workstation, with variable concentrations
IAA standard solution reaction, under 0.2V running voltage, continuous detecting is carried out by chronoamperometry, by peak point current and
The concentration value mapping of IAA, prepares standard curve；
2) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under 0.2V running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 1) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.
8. application according to claim 7 is it is characterised in that described plant tissue is the stem of plant, leaf, fruit.
9. a kind of method of live body on-line checking auximone is it is characterised in that methods described includes：
1) microelectrode biosensor described in any one of Claims 1 to 5 is connected to electrochemical workstation, with variable concentrations
IAA standard solution reaction, under 0.2V running voltage, continuous detecting is carried out by chronoamperometry, by peak point current and
The concentration value mapping of IAA, prepares standard curve；
2) microelectrode biosensor is inserted plant tissue to be measured, connect electrochemical workstation, under 0.2V running voltage, lead to
Cross chronoamperometry detect tested position electric current I, by with step 1) described in standard curve equation contrast, be calculated
The instant IAA concentration of test serum.
10. method according to claim 9 is it is characterised in that described plant tissue is the stem of plant, leaf, fruit.
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