CN106404863A - Microelectrode biosensor used for in-vivo online detection of plant zeatin, and application of microelectrode biosensor - Google Patents
Microelectrode biosensor used for in-vivo online detection of plant zeatin, and application of microelectrode biosensor Download PDFInfo
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Abstract
The invention relates to a microelectrode bio-sensing technology, and concretely discloses a microelectrode biosensor used for in-vivo online detection of plant zeatin, and an application of the microelectrode biosensor. The microelectrode biosensor comprises an insulating pedestal and microelectrodes arranged on the insulating substrate, and the microelectrode comprise a work electrode, a reference electrode and a counter electrode, wherein the work electrode is a carbon nanotube-and-polypyrrole-modified platinum black electrode. The microelectrode biosensor adopting the carbon nanotube-and-polypyrrole-modified platinum black electrode to detect the plant zeatin as the work electrode has good electrode stability and catalytic oxidation activity, the detection range can reach 0.5-50 [mu]M, and the detection limit is 0.1 [mu]M. The response signal of the microelectrode biosensor to other interference substances in plants, such as indoleacetic acid, salicylic acid and glucose, is weak, so the microelectrode biosensor has good specificity.
Description
Technical field
The present invention relates to microelectrode biosensing technology, specifically, it is related to a kind of live body on-line checking plant Zea mays element
Microelectrode biosensor.
Background technology
Zeatin is a kind of natural plant soma mitogen, as important plant growth regulating hormone, in plant
The various bioprocess of participation, the division including cell and differentiation in vivo, regeneration after root, the formation of bud and growth, damage etc., also
Photosynthesis and the resistance of plant can be affected.Accordingly, with respect to the physiology function of zeatin, such as synthesis, degraded, metabolism road
The research of footpath and the interaction and other hormones between becomes important topic, but further determines that zeatin is given birth to plant
How in real time, reliably long effect also needs to accurate quantitative analysis method, micro zeatin in plant is carried out qualitative
Quantitative analysis, is an important step during these researchs are carried out.
Content in plant for the hormone is extremely low, and property is again unstable, in addition in cell other compounds interference, therefore measure
Method must be very sensitive and single-minded.With the development of detection method, define biological detection method, Physico-chemical tests method and exempted from
The big detection architecture of epidemic disease detection method three.Although biological detection method is simple but it is susceptible to the interference of cross reaction, and measure the cycle
Long；And used in immunization antibody be not readily available, high cost, sample broke is big；Physico-chemical analysis are again with all kinds of chromatographys
Most commonly seen, but this method is complicated to sample treatment, expensive equipment, and experiment condition is harsh.Especially these methods are to plant sample
Carry out destructive process it is impossible to obtain immediately effectively zeatin concentration it is impossible to realizing in growing process existing in real time
Body is monitored, and therefore needs a kind of sensitivity that plant living body can be carried out with original position dynamic detection electrochemical credit high, easy and simple to handle badly
Analysis method.
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 live body on-line checking plant is beautiful
The microelectrode biosensor of meter Su 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 detection plant Zea mays element, described microelectrode
Biology sensor includes dielectric base and arranges microelectrode on a dielectric base with by MEMS technology, and described microelectrode includes work
Make electrode, reference electrode with to electrode；
Wherein, described working electrode is the platinum black electrode of CNT and polypyrrole modifying.
The present invention is detected to plant Zea mays element using the platinum black electrode of CNT and polypyrrole modifying, because carbon is received
Mitron has catalysed oxidn to zeatin, has big specific surface area simultaneously and is obtained in that larger response current and preferably
Sensitivity；And polypyrrole film has good electric conductivity, and can adsorb and combine CNT, show enhancing catalysis simultaneously
Effect, improves the stability of electrode, obtains the work microelectrode of the detection plant Zea mays element that specificity is good, sensitivity is high, enters
And can be used for plant living body on-line checking zeatin.
Further, the method for modifying of described working electrode is：By clean for surface platinum black electrode immersion containing pyrroles and
In the supporting electrolyte of hydroxyl carbon nano tube, by cyclic voltammetry in -0.2～0.8V, sweep speed is 10～200mV/s
Carry out electropolymerization deposition under conditions of (preferably 50mV/s), obtain the platinum black electrode of CNT and polypyrrole modifying.
Further, in described supporting electrolyte, the concentration of pyrroles is 0.01～5M, and the concentration of hydroxyl carbon nano tube is
0.01～50mg/mL.
Preferably, the concentration of pyrroles is 0.2M in described supporting electrolyte, the concentration of hydroxyl carbon nano tube is 6mg/
mL.Platinum black electrode immersion is lied prostrate using circulation containing in the supporting electrolyte under this pyrrole concentrations and hydroxylating carbon nanotube concentration
Peace method carries out electrochemical polymerization, and described pyrroles's preferred concentration can preferably control pyrroles's voltolisation sum velocity, be allowed to film forming and be difficult
Destroyed, can adsorb with reference to upper CNT does not affect its electric conductivity simultaneously.Described hydroxyl carbon nano tube preferred concentration
Electrode surface area can effectively be increased, strengthen electrode catalyst performance, ensure the uniform of polypyrrole/carbon nano-tube compound film simultaneously
Property and electron transmission efficiency.
Further, described supporting electrolyte is acid solution, and that is, pH value is less than 7, and need to contain the chlorine less than 5M
Ion.Preferably, containing 0.2M potassium chloride and 0.1M sulfuric acid.Potassium chloride is for providing the electrolyte of chlorion, the effect of sulfuric acid
For providing acid electrolyte solution, above-mentioned concentration can make electropolymerization effectively occur.
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 preferably described dielectric base of the present invention has for wearing
The tip portion of thorn plant tissue, realizes the on-line checking of plant living body.Described plant tissue can be the root of plant, stem, Ye Huo
Fruit etc..
Specifically, described dielectric base is in three rib type structures, and three face dielectric base are respectively provided with working electrode, reference
Electrode with to electrode.Described electrode material is arranged on one end of dielectric base, and the other end is provided with conducting element and is used for connecting electricity
Chem workstation.
In order to realize the on-line checking to plant living body, described microorganism biological sensor need to have puncture plant tissue
Ability, therefore, described dielectric base has the tip portion for puncturing plant tissue, and the length of described tip portion can basis
The thickness of required puncture is adjusted.Preferably, the total length of described dielectric base is 1～10cm, tip portion length is 2
～20mm, the width of substrate is 2～20mm, and thickness is 0.1～2mm.In a specific embodiment of the present invention, described exhausted
The specification of edge substrate is：Total length is 5cm, and tip portion length is 5mm, and every face dielectric base width is 3mm, and the insulation of every face is thick
Spend for 0.5mm.
Described dielectric base is selected from silicon chip, stainless steel, lucite etc..
Working electrode after second aspect, the structure based on mentioned microorganism sensor and modification, present invention also offers
Application in terms of detection plant Zea mays element for the aforementioned microelectrode biosensor.
Specifically include following steps：
1) respectively compound concentration be 0,0.1,0.3,0.5,1,3,5,10,30,50 μM of zeatin-phosphoric acid buffer (pH=
6.0) solution, (current potential 0.7～1.1V, current potential increases to carry out differential pulse voltammetry detection using described microelectrode biosensor
Plus 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s), obtain one group of concentration and deduction
The relation curve of the peak current of background current, make working curve, linear equation be i=1.84+0.105c (current unit μ A,
Concentration unit μM), the range of linearity is up to 0.5～50 μM；
2), after cleaning described microelectrode biosensor, three parts of normal concentration (3,15,30 μM) corns are detected first respectively
Plain solution carries out electrochemistry calibration, and evaluation work curve and slope of standard curve deviation are such as within 15% it is believed that electrode can be just
Often work, the working curve after being calibrated；
After correction, testing sample is carried out with differential pulse voltammetry scanning, and (current potential 0.7～1.1V, current potential increases 0.004V, shakes
Width 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s), after activation 20min, after steady testing 5min, obtain
Working curve after correction for the current signal obtaining is calculated the instant concentration of zeatin in testing sample.
Further, described testing sample is root, stem, leaf or the fruit of plant.
The beneficial effects of the present invention is：
The present invention detects plant Zea mays element, table by the use of the platinum black electrode of CNT and polypyrrole modifying as working electrode
Reveal good electrode stability and catalytic oxidation activity, up to 0.5～50 μM, detection is limited to 0.1 μM to detection range.To plant
The response signal of internal other interfering materials such as generation such as heteroauxin, salicylic acid, glucose is weaker, and this electrode shows good
Good specificity.
Further, The invention also achieves in plant zeatin on-line monitoring, really to corn prime information
Synchronous acquisition, obtains more live bodies, the information of dynamic, instant zeatin, is plant vital activity rule, discloses plant life
Life essence of phenomena provides more theoretical foundations.The present invention for traditional bioassay, chemical detection method, the party
Method sample pre-treatments are simple, micro-trauma, and detection method is directly reliably quickly and easily.
Brief description
Fig. 1 is microelectrode biosensor schematic diagram of the present invention.
Fig. 2 is the working electrode schematic diagram of microelectrode biosensor of the present invention.
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.
Embodiment 1 microelectrode biosensor
1st, microelectrode is arranged in the lucite substrate of insulation by MEMS technology, and substrate has tip portion and is used for wearing
Thorn plant tissue, total length is 5cm, and tip portion length is 5mm, and the width of substrate is 3mm, and thickness is 0.5mm；Each substrate
Upper one end is provided with the element for connecting conduction, and the other end is the electrode material of detection, as shown in Figure 1；Wherein reference electricity
Extremely Ag/AgCl, is platinum to electrode, and working electrode is platinum black.
Described dielectric base be in three rib type structures, three face dielectric base are respectively provided with working electrode, reference electrode with right
Electrode.Described electrode material is arranged on one end of dielectric base, and the other end is provided with conducting element and is used for connecting electrochemical operation
Stand.
2nd, use alumina powder in abrasive paper for metallograph polishing electrode, deionized water, EtOH Sonicate clean up respectively.
Subsequently microelectrode is placed in 0.5M dilution heat of sulfuric acid and is circulated voltammetric scan (0～1.5V) and obtains typical cyclic voltammetry spectrum
Figure is it is ensured that electrode surface is clean.
3rd, prepare 0.2M pyrroles/0.2M potassium chloride/6mg/ml hydroxyl carbon nano tube 0.1M sulfuric acid solution, insert microelectrode
Under -0.2～0.8V, 50mV/s, electropolymerization is carried out by cyclic voltammetry and deposits 10min, rear deionized water cleans up,
Obtain the polypyrrole CNT/platinum black electrode modified, as shown in Figure 2.
The application of embodiment 2 microelectrode biosensor
Corn to be measured is " the capital section 968 " of Beijing City Agriculture and Forestry Institute chamber planting, chooses the tender stem of two plants of corn seedlings
For detecting sample.After cleaning, microelectrode is inserted at corn seedling tender stem, starts Electrochemical Detection.
1st, compound concentration is 0,0.1,0.3,0.5,1,3,5,10,30,50 μM of zeatin-phosphate buffer solution (pH respectively
=6.0), using embodiment 1 preparation microelectrode biosensor carry out differential pulse voltammetry detection (current potential 0.7～1.1V,
Current potential increases 0.004V, amplitude 0.05V, pulse width 0.02s, pulse period 0.5s, quiescent time 20s), obtain one group of concentration
With the relation curve of the peak current of background correction electric current, make microelectrode working curve, linear equation is i=1.84+0.105c
(current unit μ A, concentration unit μM), the range of linearity is up to 0.5～50 μM.
2nd, microelectrode after cleaning, detects that three parts of normal concentration (3,15,30 μM) zeatin solution carry out electricity first respectively
Chemistry calibration, evaluation work curve and slope of standard curve deviation are such as within 15% it is believed that electrode can normal work.After correction
Sample is carried out with differential pulse voltammetry scanning, and (current potential 0.7～1.1V, current potential increases 0.004V, amplitude 0.05V, pulse width
0.02s, pulse period 0.5s, quiescent time 20s), after activation 20min, after steady testing 5min, the current signal of acquisition passes through
Working curve after correction is calculated the instant concentration of sample.
Comparative example 1
This comparative example is with the difference of embodiment 1：Pyrroles in embodiment 1 is replaced with aniline and modifies working electrode.
1st, the construction of microelectrode：With embodiment 1.
2nd, working electrode pretreatment：With embodiment 1.
3rd, prepare 0.2M aniline/0.05M potassium chloride/6mg/ml hydroxyl carbon nano tube/0.1M sulfuric acid solution, insert micro- electricity
Pole carries out electropolymerization by cyclic voltammetry under -0.2～0.8V, 50mV/s and deposits 10min, and rear deionized water cleaning is dry
Only, obtain the polyaniline CNT/platinum black electrode modified.
4th, the polyaniline obtaining in step 3 CNT/platinum black microelectrode is placed in 5 μM of zeatin-phosphoric acid buffer (pH=
6.0), in solution, (current potential 0.7～1.1V, current potential increases 0.004V, amplitude 0.05V, pulse to carry out differential pulse voltammetry detection
Width 0.02s, pulse period 0.5s, quiescent time 20s), only observe in the range of current potential is from+0.7 to+1.1V one weak
And wide oxidation peak, peak current be about 0.1826 μ A, less than the electrode detection in embodiment with sample peak current 2.365 μ A,
Sensitivity is worse, illustrates that the microelectrode of comparative example 1 is very poor to the electrocatalytic oxidation activity of zeatin, illustrates that polypyrrole rings in volt-ampere
Work in coordination with answering and enhance electro catalytic activity.
Comparative example 2
This comparative example is with the difference of embodiment 1：The pyrrole concentrations modifying working electrode in embodiment 1 are replaced with
0.8M.
1st, the construction of microelectrode：With embodiment 1.
2nd, working electrode pretreatment：With embodiment 1.
3rd, prepare 0.8M pyrroles/0.05M potassium chloride/6mg/ml hydroxyl carbon nano tube/0.1M sulfuric acid solution, insert micro- electricity
Pole carries out electropolymerization by cyclic voltammetry under -0.2～0.8V, 50mV/s and deposits 10min, and rear deionized water cleaning is dry
Only, obtain the polypyrrole CNT/platinum black electrode modified.
4th, the 5mM potassium ferricyanide/potassium ferrocyanide is placed in the polypyrrole CNT/platinum black microelectrode obtaining in step 3
Carry out in solution finding when AC impedance characterizes, the resistance value that the impedance spectrum of acquisition calculates is more than in embodiment the microelectrode that works
Resistance value, illustrate 0.8M pyrroles polymerization modify obtained a polypyrrole film thicker compared with embodiment, cause the conduction of film
Property decline.
5th, the polypyrrole obtaining in step 3 CNT/platinum black microelectrode is placed in 5 μM of zeatin-phosphoric acid buffer (pH=
6.0), in solution, (current potential 0.7～1.1V, current potential increases 0.004V, amplitude 0.05V, pulse to carry out differential pulse voltammetry detection
Width 0.02s, pulse period 0.5s, quiescent time 20s), only observe in the range of current potential is from+0.7 to+1.1V one weak
Little oxidation peak, peak current be about 0.843 μ A, less than the electrode detection in embodiment with sample peak current 2.365 μ A, sensitive
Property poor, illustrate that the microelectrode of comparative example 2 is poor to the catalytic effect of zeatin, the pyrroles of 0.8M shadow during modifying be described
Ringing the uniformity of the fixation of CNT, having have impact on diffusion on film for the substrate in volt-ampere response thus having influence on electrode inspection
Survey effect.
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 detection plant Zea mays element is it is characterised in that described microelectrode biosensor bag
Include dielectric base and setting microelectrode on a dielectric base, described microelectrode include working electrode, reference electrode with to electrode；
Wherein, described working electrode is the platinum black electrode of CNT and polypyrrole modifying.
2. microelectrode biosensor according to claim 1 is it is characterised in that the method for modifying of described working electrode
For：By in the platinum black electrode supporting electrolyte containing pyrroles and hydroxyl carbon nano tube for the immersion clean for surface, lied prostrate by circulation
Peace method carries out electropolymerization deposition in -0.2～0.8V, sweep speed under conditions of 10-200mV/s, obtains CNT and gathers
The platinum black electrode that pyrroles modifies.
3. microelectrode biosensor according to claim 2 it is characterised in that in described supporting electrolyte pyrroles dense
Spend for 0.01～5M, the concentration of hydroxyl carbon nano tube is 0.01～50mg/mL.
4. microelectrode biosensor according to claim 3 it is characterised in that in described supporting electrolyte pyrroles dense
Spend for 0.2M, the concentration of hydroxyl carbon nano tube is 6mg/mL.
5. the microelectrode biosensor according to claim 3 or 4 is it is characterised in that contain in described supporting electrolyte
0.2M potassium chloride and 0.1M sulfuric acid.
6. microelectrode biosensor according to claim 5 is it is characterised in that contain 0.2M in described supporting electrolyte
Potassium chloride and 0.1M sulfuric acid.
7. the microelectrode biosensor according to any one of Claims 1 to 4 is it is characterised in that described dielectric base has
There is the tip portion for puncturing plant tissue.
8. application in terms of detection plant Zea mays element for the microelectrode biosensor described in any one of claim 1～7.
9. application in terms of live body on-line checking plant Zea mays element for the microelectrode biosensor described in claim 7, it is special
Levy and be, specifically include following steps：
1) prepare a series of zeatin-phosphate buffer solution of variable concentrations respectively, entered using described microelectrode biosensor
Row differential pulse voltammetry detects, obtains the relation curve of one group of concentration and the peak current of background correction electric current, makes work bent
Line；
2) electrochemistry calibration microelectrode biosensor, the working curve after being corrected, carry out differential pulse to testing sample
Voltammetric scan, the current signal of the acquisition working curve after correction is calculated the immediately dense of zeatin in testing sample
Degree.
10. application according to claim 9 is it is characterised in that described testing sample is root, stem, leaf or the fruit of plant.
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| CN114778630A (en) * | 2022-05-06 | 2022-07-22 | 南通大学 | Method for rapidly detecting zeatin in plant body based on flat electrode |
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Cited By (10)
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| CN108303453A (en) * | 2017-12-29 | 2018-07-20 | 北京农业智能装备技术研究中心 | The sensor and its construction method of In vivo detection salicylic acid and heteroauxin simultaneously |
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| CN108717076A (en) * | 2018-05-18 | 2018-10-30 | 山东农业大学 | A kind of electrochemica biological sensor and preparation method thereof of detection zeatin |
| CN109709177A (en) * | 2019-02-27 | 2019-05-03 | 南昌航空大学 | A kind of composite material modified electrode and its preparation method and application |
| CN111505071A (en) * | 2020-04-13 | 2020-08-07 | 商丘师范学院 | Integrated microelectrode sensor for simultaneous detection of pH and AA and preparation method and application thereof |
| CN111505071B (en) * | 2020-04-13 | 2022-06-10 | 商丘师范学院 | Integrated microelectrode sensor for simultaneous detection of pH and AA and preparation method and application thereof |
| CN113984862A (en) * | 2021-09-28 | 2022-01-28 | 杭州电子科技大学 | Built-in plant information micro-nano monitoring device |
| CN113984862B (en) * | 2021-09-28 | 2023-10-24 | 杭州电子科技大学 | Built-in plant information micro-nano monitoring device |
| CN114778630A (en) * | 2022-05-06 | 2022-07-22 | 南通大学 | Method for rapidly detecting zeatin in plant body based on flat electrode |
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| CN106404863B (en) | 2018-11-23 |
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