CN106770570A - Microelectrode biosensor and its application in plant salicylic acid on-line checking - Google Patents
Microelectrode biosensor and its application in plant salicylic acid on-line checking Download PDFInfo
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- CN106770570A CN106770570A CN201611192766.XA CN201611192766A CN106770570A CN 106770570 A CN106770570 A CN 106770570A CN 201611192766 A CN201611192766 A CN 201611192766A CN 106770570 A CN106770570 A CN 106770570A
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Abstract
The present invention provides a kind of microelectrode biosensor, microelectrode 4 8cm long, its tip portion 5 30mm long, width 2mm, thickness 0.5mm；The basal electrode material of the microelectrode is silicon chip, the microelectrode has three-electrode system, comprising Ag/AgCl reference electrodes, the metal working electrode that platinum is modified to electrode and by carbon nano-tube material, three exposed current-carrying parts of electrode are long × wide it is 0.5mm × 0.5mm.Using the microelectrode biosensor, plant salicylic acid content is monitored on-line by live body, record truly and analyze salicylic acid content situation of change in real time, detection method reliability, measurement result is accurate, for the detection of plant salicylic acid provides new method, salicylic acid detection means in plant stress-resistance, disease-resistant research is enriched.
Description
Technical field
The present invention relates to microelectrode biosensing technology, specifically, be related to a kind of microelectrode biosensor and its
Application in plant salicylic acid on-line checking.
Background technology
Salicylic acid (salicylic acid, SA) is the natural activity small molecule phenols thing being widely present in plant tissue
Matter, with important pharmacodynamics effect, involved in plant grow and involved in plant the physiology mistake such as resistance, disease resistance
Journey, has regulating and controlling effect, such as the aging of controllable fruit to plant physiology metabolism.
Detect that salicylic method includes at present：Ultraviolet spectrophotometry, the chromatography of ions, gas chromatography mass spectrometry method, efficient liquid
Phase chromatography, the above method chooses multiple time points to material sampling, by complex process, eventually through Instrumental Analysis water
The content at poplar acid certain time point in plant, by the way of in vitro sampling, constitutes to sample material and destroys, especially precious
Material is likely to result in thoroughly destruction；In addition existing detection plant salicylic acid content technology can not be realized continuously being examined in real time in body
Survey, can only in vitro be sampled by setting time interval in advance, such testing result does not have time continuity, it is impossible to which reflection is planted
Salicylic acid dynamic changes of contents in object.
The content of the invention
It is an object of the invention to provide a kind of new microelectrode biosensor and its in plant salicylic acid on-line checking
In application.
In order to realize the object of the invention, the microelectrode biosensor that the present invention is provided, the microelectrode 4-8cm long, its
Tip portion 5-30mm long, width 2mm, thickness 0.5mm；The basal electrode material of the microelectrode is silicon chip, the microelectrode
With three-electrode system, comprising Ag/AgCl reference electrodes, platinum makees electricity to electrode and by the metal working that carbon nano-tube material is modified
Pole, three exposed current-carrying parts of electrode are long × wide it is 0.5mm × 0.5mm (Fig. 1).
Metal working electrode 2, Ag/AgCl reference electrodes 3, platinum have been sequentially arranged to electrode on the tip of the silicon chip substrate 1
4；The other end of silicon chip substrate is wider, is provided with three wires 5, is connected with above three electrode respectively, wire it is another
Hold for connecting electrochemical workstation.
The microelectrode biosensor can be prepared as follows obtaining：
1) using sputtering technology and etching technique prepared in silicon chip substrate platinum to electrode, Ag/AgCl reference electrodes and
Metal working electrode；
2) carbon nano-tube material solution is prepared：CNT (0.5-1.5 μm of diameter 4-5nm, length) 1mg is dissolved in
In the following mixed liquors of 10mL, 5-10 μ L drop coatings are taken on metal working electrode surface, the 0.05mol/L phosphoric acid after drying in pH6.86 delays
Voltammetric scan is circulated in fliud flushing 10-15 times, is electrolysed 10-15 minutes under 1.3V constant potentials, obtain final product micro- electricity of probe shape
Pole biology sensor.
The mixture formula is：0.05mol/L pyrroles, 0.01mol/L salicylic acids, 0.1mol/L KCl.
The microelectrode biosensor of preparation is in needle probes shape, it is therefore an objective to can be broken through in plant epidermis insertion tissue
Portion.
The present invention also provides application of the microelectrode biosensor in plant salicylic acid on-line checking.
By in microelectrode biosensor insertion plant tissue to be measured, connection electrochemical workstation is circulated volt-ampere
Scanning, realization carries out online salicylic acid detection to plant tissue.
The salicylic acid standard liquid that various concentrations are chosen before detection carries out microelectrode detection, according to Determination of Salicylic Acid and electric current
Relation obtains microelectrode detection salicylic acid working curve；The microelectrode lowest detection is limited to 8.0 × 10-6Mol/L, the range of linearity
It is 2.0 × 10-5-1.2×10-3Mol/L, salicylic acid solution concentration with response peak current linear equation be：I=9 × 10-4C+
11.40, relative coefficient is 0.93；Wherein, I represents electric current, and unit represents concentration for μ A, C, and unit is mol/L.
The root of detection object including plant, stem, leaf or position etc. fruit, insertion depth is 0.5cm or so.Detection target material
Material is the different times of plant growth, for example the fruit in cherry tomato maturity period.
Preferably, under 0.9V operating potentials, salicylic real-time online in plant tissue is carried out using chronoamperometry
Monitoring.And detected under other current potentials, system noise is big, signal is inaccurate, causes testing result inaccurate.
The timing since after electric current is steady, the current data of collection the 10th minute substitutes into linear equation, obtains plant tissue
In salicylic concentration.
The present invention monitors plant salicylic acid content on-line by live body, records truly and analysis salicylic acid content becomes in real time
Change situation, inside plant tissues are pierced into by microelectrode biosensor, using invasive manner, will not cause substantial to plant
Damage or destroy, plant can be with normal growth, the detection method reliability, measurement result is accurate, be that the detection of plant salicylic acid is carried
For new method, salicylic acid detection means in plant stress-resistance, disease-resistant research is enriched.
Brief description of the drawings
Fig. 1 is the structural representation of microelectrode biosensor of the present invention；Wherein, 1- silicon chips substrate, 2- metal working electrodes,
3-Ag/AgCl reference electrodes, 4- platinum is to electrode, 5- wires.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, it is raw materials used to be commercial goods.
Microelectrode biosensor of embodiment 1 and preparation method thereof
The microelectrode biosensor prepared in the present embodiment, 5-30mm, width 2mm, thickness 0.5mm；The microelectrode
Basal electrode material be silicon chip, the microelectrode has three-electrode system, comprising Ag/AgCl reference electrodes, platinum to electrode with
And the metal working electrode modified by carbon nano-tube material, three exposed current-carrying parts of electrode are long × wide it is 0.5mm × 0.5mm
(Fig. 1).
Metal working electrode 2, Ag/AgCl reference electrodes 3, platinum have been sequentially arranged to electrode on the tip of the silicon chip substrate 1
4；The other end of silicon chip substrate is wider, is provided with three wires 5, is connected with above three electrode respectively, wire it is another
Hold for connecting electrochemical workstation.
The microelectrode biosensor of preparation is in needle probes shape, it is therefore an objective to can be broken through in plant epidermis insertion tissue
Portion.
The microelectrode biosensor can be prepared as follows obtaining：
1) using sputtering technology and etching technique prepared in silicon chip substrate platinum to electrode, Ag/AgCl reference electrodes and
Metal working electrode；
2) carbon nano-tube material solution is prepared：The CNT 1mg of diameter 4-5nm, 0.5-1.5 μm of length is dissolved in
In the following mixed liquors of 10mL, 5-10 μ L drop coatings are taken on metal working electrode surface, the 0.05mol/L phosphoric acid after drying in pH6.86 delays
Voltammetric scan is circulated in fliud flushing 10-15 times, is electrolysed 10-15 minutes under 1.3V constant potentials, obtain final product micro- electricity of probe shape
Pole biology sensor.Step 2) mixture formula is：0.05mol/L pyrroles, 0.01mol/L salicylic acids, 0.1mol/L
KCl。
Embodiment 2 carries out real-time online detection using microelectrode biosensor to plant salicylic acid
1st, experimental subjects
The potted plant ripe cherry tomato rice shoot of experiment material selection, is detection with the maturity period fruit on cherry tomato rice shoot
Object, detection time is 10 AM, and random different parts plug in probe on holy girl's fruits, detects salicylic acid content.
2nd, laboratory apparatus and consumptive material
Laboratory apparatus uses electrochemical workstation CHI760E, purchased from Shanghai Chen Hua Instrument Ltd., model C HI76E.
3rd, experimental technique
(1) drafting of standard curve：Microelectrode biosensor connection electrochemical workstation prepared by embodiment 1
CHI760E, the salicylic acid standard liquid for choosing various concentrations carries out microelectrode detection, and operating potential is 0.9V, steady from electric current
After start timing, gather the current data for starting for the 10th minute；Microelectrode inspection is obtained according to Determination of Salicylic Acid and current relationship
Survey salicylic acid working curve.
Determine that for on-line checking salicylic acid probe in detecting concentration range be 2.0 × 10 by cyclic voltammetry-5-1.2×
10-3Mol/L, in this concentration range, response current is presented good linear relationship with concentration, and relational expression is：I (μ A)=9 ×
10-4C (mol/L)+11.40 (coefficient correlation 0.93, n=6), detection is limited to 8.0 × 10-6mol/L。
(2) experimental procedure：
A. potted plant maturity period cherry tomato rice shoot is prepared before testing；
B. the probe that will be prepared is inserted in holy girl's fruits to be measured, inserts carpopodium base portion, fruit side, the fruit of fruit
Bottom, depth is 0.5cm, the salicylic acid content for detecting holy girl's fruits different parts；
C. three same probes are connected to electrochemical workstation CHI760E；
D. salicylic acid is monitored by cyclic voltammetry and redox reaction occurs with pyrroles on the carbon nanotubes, determine work
Current potential is 0.9V；
E. under 0.9V operating potentials, salicylic real time on-line monitoring in cherry tomato is carried out using chronoamperometry；
F. the timing since after electric current is steady, (the 10th minute data before are because being to gather the 10th minute data of beginning
System and environmental disturbances signal it is inaccurate), current data substitute into linear equation, obtain holy girl's fruits reclaimed water poplar acid concentration.
4th, experimental evaluation result
The microelectrode biosensor prepared using Shanghai Chen Hua electrochemical workstation CHI760E and embodiment 1, online inspection
Analysis holy girl's fruits salicylic acid content result is surveyed to show：
Carpopodium base portion：3.1×10-4mol/L
Fruit side：6.2×10-3mol/L
Fruit bottom：2.1×10-3mol/L
Preferably, testing result reliability can realize salicylic acid dynamic on-line monitoring to detection data by electrochemical workstation
Analysis, for live body online study plant salicylic acid content provides new method, realizes the supplement to existing Static Detection means.
5th, microelectrode biosensor testing result analysis of the accuracy
In order to verify the accuracy of microelectrode biosensor testing result, contain with conventional gas-phase chromatography detection salicylic acid
Amount is compared.
(1) extract：Above-mentioned identical holy girl fruits are taken as detection object, holy girl's fruits carpopodium base is cut respectively
Portion, fruit side, each 0.5g of fruit base material, bigcatkin willow is extracted after liquid nitrogen grinding with 1ml acetone and 50ml citric acids extract solution
Acid, is subsequently adding 1ml ethyl acetate (purity 99%), centrifuging and taking supernatant, and gained sample is standby after nitrogen is dried.
(2) esterification：By 100 μ l ether/methyl alcohol (volume ratio 9:1) add with 4 μ l 2mM trimethyl silicone hydride diazomethanes
Enter in sample injection bottle, room temperature places 30min, and adsorption tube one end is deep in sample injection bottle, other end connection suction bottle, sample injection bottle is needed
Sample is collected in water-bath, is eluted with 300 μ l n-hexanes after adsorption tube cooling, preserves -20 DEG C of refrigerators standby.
(3) adsorbent reactivation：After 300 μ l methyl alcohol are added in adsorption tube, then washed 2-3 times with 300 μ l n-hexanes, drying is standby
With.
(4) preparation of gaultherolin hybrid standard product：Prepare 1000 μ g/ml water of n-hexane (purity ＞ 99%) dissolving
Poplar acid methyl esters (Sigma) standard liquid (mother liquor), obtains 400 μ g/ml gaultherolin solution after dilution, be diluted to a series of dense
Degree, makes standard curve.
(5) standard items esterification：The 160 μ g/ml salicylic acids storage μ l of liquid 125 are taken, according to above-mentioned esterification process, is finally used
100 μ l n-hexanes are eluted, and obtain 200 μ g/ml gaultherolin standard items, preserve -20 DEG C of refrigerators standby.
(6) gas-chromatography：Sample is separated using HP-5 quartz capillaries, temperature keeps 1min since 60 DEG C, interval 15
DEG C/min to 250 DEG C, 3min, 280 DEG C of injector temperature, 300 DEG C of detector temperature are kept.It is carrier gas to employ nitrogen as, carrier gas
The detection of flow 1ml/min, FID flame ionic detector, the μ l of sample size 1.
Bigcatkin willow acid recovering rate：0ng, 100ng, 200ng, 400ng gaultherolin are taken, repeat samples are extracted and esterification step
Suddenly, gas Chromatographic Determination content after being eluted with n-hexane.Salicylic acid content is shown in Table 1 in detection cherry tomato fruit sample solution.
The gas Chromatographic Determination salicylic acid content result (unit of table 1：mol/L)
| Content | Carpopodium base portion mol/L | Fruit side | Fruit bottom mol/L |
| Fruit 1 | 2.9×10-4 | 6.1×10-3 | 2.02×10-3 |
| Fruit 2 | 3.4×10-4 | 5.96×10-3 | 2.13×10-3 |
| Fruit 3 | 3.7×10-4 | 6.31×10-3 | 1.96×10-3 |
It can be seen that, using traditional vapor phase method chromatogram testing result and microelectrode biosensor testing result base of the invention
This is consistent, so as to prove the accuracy and reliability of this method testing result.
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, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of microelectrode biosensor, it is characterised in that the microelectrode 4-8cm long, its tip portion 5-30mm long is wide
Degree 2mm, thickness 0.5mm；The basal electrode material of the microelectrode is silicon chip, and the microelectrode has three-electrode system, comprising
Ag/AgCl reference electrodes, the metal working electrode that platinum is modified to electrode and by carbon nano-tube material, three exposed conductive parts of electrode
It is point long × wide to be 0.5mm × 0.5mm.
2. microelectrode biosensor according to claim 1, it is characterised in that on the tip of the silicon chip substrate (1)
Metal working electrode (2), Ag/AgCl reference electrodes (3), platinum have been sequentially arranged to electrode (4)；The other end of silicon chip substrate is wider,
Three wires (5) are provided with, are connected with above three electrode respectively, the other end of wire is used to connect electrochemical operation
Stand.
3. microelectrode biosensor according to claim 1 and 2, it is characterised in that the microelectrode biosensor
Preparation method comprise the following steps：
1) platinum is prepared to electrode, Ag/AgCl reference electrodes and metal working in silicon chip substrate using sputtering technology and etching technique
Make electrode；
2) carbon nano-tube material solution is prepared：The CNT 1mg of diameter 4-5nm, 0.5-1.5 μm of length is dissolved under 10mL
State in mixed liquor, 5-10 μ L drop coatings are taken on metal working electrode surface, after drying in the 0.05mol/L phosphate buffers of pH6.86
It is circulated voltammetric scan 10-15 times, is electrolysed 10-15 minutes under 1.3V constant potentials, the microelectrode for obtaining final product probe shape is biological
Sensor；
Step 2) mixture formula is：0.05mol/L pyrroles, 0.01mol/L salicylic acids, 0.1mol/L KCl.
4. application of the microelectrode biosensor described in any one of claim 1-3 in plant salicylic acid on-line checking.
5. application according to claim 4, it is characterised in that by the biological biography of microelectrode described in claim any one of 1-3
Sensor is inserted in plant tissue to be measured, and connection electrochemical workstation is circulated voltammetric scan, and realization is carried out to plant tissue
Line salicylic acid is detected.
6. application according to claim 5, it is characterised in that the salicylic acid standard liquid that various concentrations are chosen before detection enters
Row microelectrode detects that obtaining microelectrode according to Determination of Salicylic Acid and current relationship detects salicylic acid working curve；The microelectrode
Lowest detection is limited to 8.0 × 10-6Mol/L, the range of linearity is 2.0 × 10-5-1.2×10-3Mol/L, salicylic acid solution concentration with
Respond peak current linear equation be：I=9 × 10-4C+11.40, relative coefficient is 0.93；
Wherein, I represents electric current, and unit represents concentration for μ A, C, and unit is mol/L.
7. the application according to claim any one of 4-6, it is characterised in that detection object includes root, stem, the Ye Huo of plant
Fruit, insertion depth is 0.5cm；It is preferred that holy girl's fruits.
8. application according to claim 7, it is characterised in that under 0.9V operating potentials, is carried out using chronoamperometry
Salicylic real time on-line monitoring in plant tissue.
9. the application according to claim 7 or 8, it is characterised in that the timing since after electric current is steady, gathers the 10th minute
Current data substitute into linear equation, obtain salicylic concentration in plant tissue.
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| CN108398476A (en) * | 2017-12-29 | 2018-08-14 | 北京农业智能装备技术研究中心 | The salicylic biosensor of In vivo detection and its construction method and application |
| CN112694887A (en) * | 2020-12-07 | 2021-04-23 | 黑龙江省农业科学院植物保护研究所 | Light-emitting sensor, construction method thereof and application of light-emitting sensor in detection of salicylic acid content in plants |
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| CN114324518A (en) * | 2021-12-09 | 2022-04-12 | 北京市农林科学院智能装备技术研究中心 | Microelectrode, and rutin and quercetin detection method and application |
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| CN108303453B (en) * | 2017-12-29 | 2020-04-10 | 北京农业智能装备技术研究中心 | Sensor for simultaneously detecting salicylic acid and indoleacetic acid in living body and construction method thereof |
| CN112694887A (en) * | 2020-12-07 | 2021-04-23 | 黑龙江省农业科学院植物保护研究所 | Light-emitting sensor, construction method thereof and application of light-emitting sensor in detection of salicylic acid content in plants |
| CN112694887B (en) * | 2020-12-07 | 2024-04-26 | 黑龙江省农业科学院植物保护研究所 | Luminous sensor, construction method thereof and application thereof in detecting salicylic acid content of plants |
| CN113030230A (en) * | 2021-03-03 | 2021-06-25 | 辽宁大学 | Electrochemical voltammetry for determining salicylic acid content based on p-benzoquinone reduction and application thereof |
| CN113030230B (en) * | 2021-03-03 | 2023-05-30 | 辽宁大学 | Electrochemical voltammetry for measuring salicylic acid content based on p-benzoquinone reduction and application thereof |
| CN114324518A (en) * | 2021-12-09 | 2022-04-12 | 北京市农林科学院智能装备技术研究中心 | Microelectrode, and rutin and quercetin detection method and application |
| CN114324518B (en) * | 2021-12-09 | 2024-02-06 | 北京市农林科学院智能装备技术研究中心 | Microelectrode, detection method of rutin and quercetin and application |
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