CN106841347A - The online test method in situ of reduced glutathione in plant - Google Patents
The online test method in situ of reduced glutathione in plant Download PDFInfo
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Abstract
The present invention provides a kind of online test method in situ of reduced glutathione in plant, it is by microelectrode biosensor of the invention, realize the In vivo detection online in situ of reduced glutathione in plant, using invasive manner, the injury caused to target to be measured is minimum, so that measured target can continued growth, detection method reliability, sensitivity is high, measurement result is accurate, and due to being surveyed in physical examination, the sample process time can be reduced, avoid because of the decomposition of target substance in processing procedure, the error that oxidation etc. is caused, physiological reaction response sensitivity detection efficiency to reduced glutathione is higher than other detection methods.
Description
Technical field
The present invention relates to microelectrode biosensing technology, specifically, it is related to reduced glutathione in a kind of plant
Online test method in situ.
Background technology
Glutathione (Glutathione, GSH) is to be distributed widely in main in plant and microbial cell, content most
The abundant low molecular peptide containing sulfydryl, is a kind of tripeptides (L- glutamy-L- being made up of glutamic acid, cysteine and glycine
Cysteinyl-glycine), molecular formula is C10H17O6SN3.GSH as one of reduced sulfur main in organism, in organism
Played an important role during resisting various stress (damage to plants caused by sudden drop in temperature, arid, heavy metal, fungi etc.), the height of its contents level
It is closely related to the tolerance of various environment-stress with plant.In the last few years, its physiology in higher plant metabolic process
Effect, the especially effect during plant resists active oxygen injury and its with the progress of plant stress-resistance sexual intercourse very
Hurry up.Glutathione is divided into oxidized form (GSSG) and reduced form (GSH), and usually said glutathione is reduced glutathione,
Sulfydryl on its cysteine be its play biological function necessary to.Reduced glutathione is that content is most abundant in plant
The low molecular peptide containing sulfydryl, be the important active substances in plant part, it participates in the shape of disulphide, thioether and thioesters
Into, and the free radical in organism can be removed, it is that intracellular metabolic process and plant are subjected to peroxidating produced during oxidative stress
One of maximally effective scavenger of thing.Oxidized form of glutathione in plant can be converted into reduced glutathione, reduction
Type glutathione and the important indicator that the ratio of oxidized form of glutathione content is reflection plant glutathion inside activity are high
GSH/GSSG ratios are then conducive to the synthesis of protein.There are many researchs to show that GSH is reduced form sulphur in plant in addition
Important storage and types of transportation, with the drought resisting, the winter resistance that improve plant, mitigate the effect such as murder by poisoning of heavy metal.In Genes For Plant Tolerance
The functional activity of many plants is directly or indirectly take part in inverse process.
The method that glutathione content is determined in current plant is a lot, such as fluorescence spectrophotometry, visible spectrophotometry,
Ultraviolet spectrophotometry, iodimetric titration and the HPLC methods that developed in the last few years etc., these methods are carried out by plant tissue
Sampling, detected after milling and extracting, belong to Testing in vitro technology, the information for getting be also a certain moment static concentration or
Cumulative effect, it is impossible to reflect the instantaneous or continuous change of reduced glutathione in plant, therefore suffer from technological means
Limitation, the regulatory mechanism that synthesize in plant for GSH at this stage, degrade, transports and to plant anti-oxidation mechanism of action etc.
Also many aspects are still unclear, in the urgent need to introducing going deep into for new technology and method propulsion research.
The content of the invention
A kind of live body it is an object of the invention to provide plant reduction type glutathione based on biosensor technique is in situ
Online test method, need in the prior art in vitro sampling to overcome, and spatial and temporal resolution is relatively low, it is impossible to realize instantaneous and long-time
The defect of continuous monitoring.
In order to realize the object of the invention, present invention firstly provides a kind of microelectrode biosensor, the base of the microelectrode
Hearth electrode material is silicon chip, and silicon chip is provided with three-electrode system, and comprising Ag/AgCl reference electrodes, platinum is repaiied to electrode and surface
It is decorated with the metal working electrode of glutathione oxidase.
Glutathione oxidase (glutathione oxidase, GSHOx) can be catalyzed glutathione generation glutathione
Disulphide (glutathione disulfide), the real-time sound that enzymatic glutathione changes is recorded by chronoamperometry
Should.
Microelectrode of the invention can be SMD or plug-in type microelectrode.
The microelectrode is SMD microelectrode, surface coated with protective film；The working electrode of the SMD microelectrode is
The metal disk of diameter 5-10mm, reference electrode is the annulus of diameter 8-16mm, and ring width is 2-3mm, and length is 3/5 domain week
It is long, it is the annulus with reference electrode same diameter and width to electrode, length is 1/4 domain girth, reference electrode and to electrode
It is surrounded by half in the outside of the metal disk in a ring respectively, three wires is provided with silicon chip, respectively with above three electrode
It is connected, the other end of wire is used to connect electrochemical workstation.In flake, thickness is less than 2mm, metal electricity to the silicon chip substrate
Pole piece thickness is less than 1mm.
The microelectrode is plug-in type microelectrode；The plug-in type microelectrode 2-8cm long, its tip portion 5-30mm long,
2-5mm wide；Be laid with metal working electrode in the middle of on the tip of the silicon chip substrate, both sides lay respectively Ag/AgCl reference electrodes,
Platinum is to electrode；Exposed metal working electrode diameter 0.5-2mm, Ag/AgCl reference electrode diameter 0.2-0.5mm, platinum is to electrode diameter
0.2-0.5mm；The other end of silicon chip substrate is wider, and three wires are laid with thereon, is connected with above three electrode respectively, wire
The other end be used for connect electrochemical workstation.
The preparation method of the microelectrode biosensor is comprised the following steps：
1) platinum is prepared to electrode, Ag/AgCl reference electrodes and gold in silicon chip substrate using micro-electromechanical technology (MEMS)
Working electrode；
2) microelectrode that will be prepared is placed in 0.5M dilution heat of sulfuric acid, and volt-ampere is circulated under -0.2-1.6V current potentials
Scanning, obtains typical cyclic voltammetric spectrogram, it is ensured that electrode surface is cleaned；
3) Fe is prepared3O4NPs nano particles：It is 20 to take molal weight ratio:1 FeCl3And FeCl2It is dissolved in distilled water,
Adding a certain amount of hydrochloric acid is completely dissolved molysite, and sodium hydroxide solution is then added dropwise over thereto to solution colour by yellowish
Discoloration is brown last until aterrimus；Black precipitate is collected together with magnet, is washed, then rinse one with TMOH
Secondary, precipitation is collected in centrifugation, obtains Oxidation of Fe3O4NPs particles, precipitation nitric acid is washed once, then in 80-90 DEG C of stirring condition
Under, particle is suspended in 0.01-3M nitric acid, until becoming brown；Washing is eventually passed, by Fe3O4NPs nanoparticle suspensions
In the TMOH solution of pH value 11；
4) above-mentioned Fe is taken3O4NPs nano particle TMOH aaerosol solutions, are diluted with 1-100mM sodium citrate solutions, stirring
More than 10min, makes citrate ion uniformly wrap Fe3O4Nano particle；The Fe of citrate is collected with magnet3O4Receive
Rice grain, and being re-dispersed into 1-100mM sodium citrate solutions makes its concentration for 0.1-5mg/ml, adds final concentration of 0.1-
The silver nitrate solution of 100mM, ultrasonic 15min is heated to boiling under stirring, add sodium borohydride solution immediately, sodium borohydride with
The molal weight ratio of silver nitrate is 1:1-1:Stop heating and continuous stirring after 200,10-60min to cooling down, obtain Fe3O4- Ag cores
Shell structural nano particle；
5) by Fe3O4- Ag core-shell structure nanometer particles are deposited on metal working electrode, obtain Fe@Ag/Au electrodes；
6) shitosan and glutathione oxidase mixed liquor are prepared：The 18mg/mL that the PBS of pH value 7.3 is prepared
Glutathione oxidase enzyme liquid mixes in equal volume with 1.0% chitosan solution；Wherein, the preparation of 1.0% chitosan solution
Method is as follows：0.1g shitosans are dissolved in 10mL 0.05M acetums, then the membrane filtration of 0.45 μm of via hole diameter, i.e.,
；
7) by step 6) gained mixing drop-coated in Fe Ag/Au electrode surfaces, dry, obtaining final product surface modification has gluathione
The metal working electrode of peptide oxidizing ferment, so as to complete the preparation of microelectrode biosensor.
The water used in the present invention is distilled water.
The present invention is also provided and carries out original position to reduced glutathione in plant using the microelectrode biosensor
The method of on-line checking.
It is right as detecting using reduced glutathione using plants such as cucumber, tomato, sunflower, cottons as test material
As.Autolab electrochemical workstations, SMD or plug-in type microelectrode are led to using Switzerland ten thousand, by electrochemical method to seedling
Reduced glutathione content in cotyledon, stalk or fruit carries out live body on-line monitoring.
The reduced glutathione standard liquid that various concentrations are chosen before detection carries out microelectrode detection, according to reduced form paddy
The sweet peptide concentration of Guang draws the standard curve of glutathione with current relationship；The microelectrode lowest detection is limited to 0.05mmol/L,
The range of linearity is 0.1-100mmol/L, and reduced glutathione solution concentration is y=with the linear equation of response peak current
0.2668ln (x)+4.8799, relative coefficient is 0.9816.
Under 0.4V operating potentials, the real-time online of reduced glutathione in plant tissue is carried out using chronoamperometry
Monitoring；The timing since after electric current is steady, the current data of collection the 20th minute or so substitutes into linear equation, obtains plant tissue
The concentration of middle reduced glutathione.
The stem of detection object of the invention including plant, leaf or position etc. fruit.For different parts, multi-form is selected
Sensor, SMD sensor be applied to blade, contact pin type sensor be applied to stem and fruit.
In the specific embodiment of the present invention, respectively with cucumber leaves and tamato fruit as detection object.
When being detected to stalk or fruit using plug-in type microelectrode, by microelectrode biosensor insertion
In sample to be tested, insertion depth is 0.2-1cm (preferably 0.5cm), and connection electrochemical workstation is circulated voltammetric scan, realizes
Online reduced glutathione detection is carried out to plant tissue.
When being detected to blade using SMD microelectrode, specific method is as follows：With diameter 1.2-1.5mm (preferably
Pin 1.5mm) pricks a hole on blade, avoids vein position, and a metafiltration scraps of paper are covered on aperture, and filter paper size is not with
It is advisable more than SMD microelectrode, the PBS for drawing pH7.3 is dropped on filter paper, is to soak monoblock filter paper just
Preferably, the metal covering of SMD microelectrode of diaphragm will be removed close on filter paper, with clip by microelectrode, filter paper and leaf
Piece clamper is fixed, and connects electrochemical workstation, carries out the real time on-line monitoring of reduced glutathione in blade.
The present invention can realize the In vivo detection online in situ of reduced glutathione in plant, using invasive manner,
The injury caused to target to be measured is minimum so that measured target can continued growth, detection method reliability, sensitivity is high, determines knot
Fruit is accurate, and due to being surveyed in physical examination, it is possible to reduce the sample process time, it is to avoid because in processing procedure target substance point
The error that solution, oxidation etc. are caused, the physiological reaction response sensitivity detection efficiency to reduced glutathione is detected higher than other
Method.
Brief description of the drawings
Fig. 1 is that the SMD microelectrode biosensor of preparation in the embodiment of the present invention 1 and its metal working electrode surface are repaiied
The enlarged diagram of jewelry.
Fig. 2 is the structural representation of the plug-in type microelectrode biosensor of preparation in the embodiment of the present invention 2.
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.
SMD microelectrode biosensor of embodiment 1 and preparation method thereof
The SMD microelectrode biosensor prepared in the present embodiment, the basal electrode material of the microelectrode is silicon
Piece, silicon chip is provided with three-electrode system, and comprising Ag/AgCl reference electrodes, platinum has glutathione oxygen to electrode and surface modification
Change the metal working electrode of enzyme.
The SMD microelectrode, its surface coated with protective film；The working electrode of the SMD microelectrode is diameter 5mm
Metal disk, reference electrode a diameter of 8mm, ring width 2mm, length are 3/5 domain girth, are 8mm, ring width to electrode diameter
2mm, length is 1/4 domain girth, reference electrode and is surrounded by half in the outside of the metal disk in a ring respectively to electrode, silicon
Three wires are provided with piece, are connected with above three electrode respectively, the other end of wire is used to connect electrochemical workstation.
The microelectrode biosensor can be prepared as follows obtaining：
1) platinum is prepared in silicon chip substrate using micro-electromechanical technology and electricity is made to electrode, Ag/AgCl reference electrodes and metal working
Pole；
2) microelectrode that will be prepared is placed in 0.5M dilution heat of sulfuric acid, and volt-ampere is circulated under -0.2-1.6V current potentials
Scanning, obtains typical cyclic voltammetric spectrogram, it is ensured that electrode surface is cleaned；
3) Fe is prepared3O4NPs nano particles：By 4.595g FeCl3With 1.71g FeCl2It is dissolved in 20mL distilled waters,
2M hydrochloric acid 100mL are added, stirring is completely dissolved up to molysite, and 2M sodium hydroxide solutions to solution face are then added dropwise over thereto
Color is changed into brown finally until aterrimus from faint yellow；Black precipitate is collected together with magnet, is washed, Ran Houyong
0.1MTMOH is rinsed once, centrifugation, collects precipitation, obtains Oxidation of Fe3O4NPs particles, precipitation are washed once with 0.01M nitric acid, so
Afterwards under 80-90 DEG C of stirring condition, particle is set to be suspended in 0.01M nitric acid, until becoming brown；Washing is eventually passed, will
Fe3O4NPs nanoparticle suspensions are in the TMOH solution of 0.1M pH value 11；
4) the above-mentioned Fe of 1mL 0.2mM are taken3O4NPs nano particle TMOH aaerosol solutions, are diluted to 10mM sodium citrate solutions
50mL, stirs 30min so that citrate ion uniformly wraps Fe3O4Nano particle.Citrate is collected with magnet
Fe3O4Nano particle, and being re-dispersed into 40ml 10mM sodium citrate solutions makes its concentration for 0.1mg/ml, adds final concentration
It is the silver nitrate solution of 10mM, ultrasonic 15min is heated to boiling, 1mM 5ml sodium borohydride solutions are added immediately under stirring,
Stop heating and continuous stirring after 15min to cooling down, obtain Fe3O4- Ag core-shell structure nanometer particles；
5) by Fe3O4- Ag core-shell structure nanometer particles are deposited on metal working electrode, obtain Fe@Ag/Au electrodes；
6) shitosan and glutathione oxidase mixed liquor are prepared：Take the 18mg/mL that the PBS of pH value 7.3 is prepared
Glutathione oxidase enzyme liquid 5mL mixes in equal volume with 5mL1.0% chitosan solutions；Wherein, 1.0% chitosan solution
Compound method it is as follows：0.1g shitosans are dissolved in 10mL 0.05M acetums, then the filter membrane of 0.45 μm of via hole diameter
Filtering, obtains final product；
7) by step 6) gained mixing drop-coated in Fe Ag/Au electrode surfaces, dry, obtaining final product surface modification has gluathione
The metal working electrode of peptide oxidizing ferment, so as to complete the preparation of microelectrode biosensor.(Fig. 1)
Plug-in type microelectrode biosensor of embodiment 2 and preparation method thereof
The plug-in type microelectrode biosensor prepared in the present embodiment, the basal electrode material of the microelectrode is silicon
Piece, silicon chip is provided with three-electrode system, and comprising Ag/AgCl reference electrodes, platinum has glutathione oxygen to electrode and surface modification
Change the metal working electrode of enzyme.
The plug-in type microelectrode 3cm long, its tip portion 15mm long, 2mm wide；In the middle of on the tip of the silicon chip substrate
Metal working electrode is laid with, Ag/AgCl reference electrodes, platinum are laid to electrode in both sides respectively；Exposed metal working electrode diameter
1.5mm, Ag/AgCl reference electrode diameter 0.2mm, platinum is to electrode diameter 0.2mm；The other end of silicon chip substrate is wider, thereon cloth
Three wires are provided with, are connected with above three electrode respectively, the other end of wire is used to connect electrochemical workstation.(Fig. 2)
The preparation method of the plug-in type microelectrode biosensor is with embodiment 1.
Embodiment 3 carries out real-time online detection using SMD microelectrode biosensor to plant reduction type glutathione
The SMD microelectrode original position on-line checking cucumber at seedling stage salt stress different time rear blade prepared using embodiment 1
The content of middle reduced glutathione.
1st, the drafting of standard curve
Respectively compound concentration be 0.1,0.5,1,5,10,50, the PBS of 100mM reduced glutathiones (pH=7.0,
10mM) solution, 0.4V is used as operating potential for selection, and the SMD microelectrode prepared using embodiment 1 carries out chronoamperometry inspection
Survey, obtain the relation curve of concentration and electric current, draw the standard curve of glutathione.
The microelectrode lowest detection is limited to 0.05mmol/L, and the range of linearity is 0.1-100mmol/L, reduced form gluathione
Peptide solution concentration is y=0.2668ln (x)+4.8799 with the linear equation of response peak current, and relative coefficient is 0.9816.
2nd, experimental technique
Experiment material is potted plant cucumber (Tianjin spring No. 4) seedling, and using second true leaf as detection object, plant grows to two leaves
After one heart stage, 150mM NaCl treatment is carried out, start within 0,2,4,6 hours detection in treatment respectively, 20min is detected every time.Tool
Body method is：A hole is pricked with the small pin of diameter 1.5mm or so on blade, vein position is avoided, one layer is covered on aperture
Filter paper, filter paper size is advisable with no more than SMD microelectrode, and the PBS (pH7.3) for drawing 100 μ l or so drops in filter paper
On, it is advisable with soaking monoblock filter paper just, by the metal covering of SMD microelectrode close on filter paper, with clip by micro- electricity
Pole, filter paper and blade clamper are fixed, and electrode back segment wiring is connected to electrochemical workstation, and leaf is recorded by chronoamperometry
The real-time change of reduced glutathione concentration in piece.
3rd, experimental result contrast
While real-time detection, it is sampled when NaCl has coerced 0,2,4,6 hours respectively, sample Liquid nitrogen storage, so
The DTNB determination of color GSH for being commonly used using current laboratory afterwards, with reference to Ellman (Ellman G L.Tissue
Sulfhydryl groups.Arch Biochem.Bioph ys, 1959；32:70-77).Weigh the fresh sample (sampling points of 0.5g
It is second true leaf of cucumber seedling), the trichloroacetic acids of 3mL 15% are added, homogenate is worn into, 16000 × g centrifugation 20min take
Clear liquid, is developed the color with 5,5 '-two thiobis (2- nitrobenzoic acids), and OD values are surveyed in 422nm wavelength, while standard curve is made with GSH,
Calculate GSH contents.It is another to be dried the in vitro sample in part, its water content is calculated, leaf water content can be used for on-line checking knot
Unit conversion when fruit and Testing in vitro Comparative result.
The result of Testing in vitro is contrasted with real-time online testing result of the invention, as a result as shown in table 1.As a result
Show the on-line real-time measuremen reliable results of the microelectrode, can be used for scientific research.
The test result of table 1 is contrasted
Embodiment 4 carries out real-time online detection using plug-in type microelectrode biosensor to plant reduction type glutathione
The plug-in type microelectrode original position on-line checking tomato prepared using embodiment 2 is beared fruit phase salt stress different time consequence
The content of reduced glutathione in reality.
1st, the drafting of standard curve
Respectively compound concentration be 0.1,0.5,1,5,10,50, the PBS of 100mM reduced glutathiones (be 10mM with concentration
The PBS of pH7.0 is prepared) solution, 0.4V is used as operating potential for selection, and the SMD microelectrode prepared using embodiment 1 is counted
When current method detection, obtain the relation curve of concentration and electric current, the standard curve of drafting glutathione.
The microelectrode lowest detection is limited to 0.05mmol/L, and the range of linearity is 0.1-100mmol/L, reduced form gluathione
Peptide solution concentration is y=0.2668ln (x)+4.8799 with the linear equation of response peak current, and relative coefficient is 0.9816.
2nd, experimental technique
Experiment material is potted tomato (beauty F1), per 2 plants of basin, to choose the fruit of plant the second fringe diameter about 2cm
As detection object, 150mM NaCl treatment is carried out to plant, respectively in the detection that starts for 0,4,8 hours for the treatment of, every time detection
20min.Specific method is：By in microelectrode biosensor insertion fruit to be measured, insertion depth is that 0.5cm or so (keeps away
Open pericarp part), connection electrochemical workstation is circulated voltammetric scan, realizes the online reduced form gluathione to tamato fruit
Peptide is detected.The timing since after electric current is steady, the current data of collection the 20th minute substitutes into linear equation, in obtaining plant tissue
The concentration of reduced glutathione.
3rd, experimental result contrast
While real-time detection, it is sampled when NaCl has coerced 0,4,8 hours respectively, sample Liquid nitrogen storage, then
The DTNB determination of color GSH commonly used using current laboratory, with reference to Ellman (Ellman G L.Tissue sulfhydryl
Groups.Arch Biochem.Bioph ys, 1959；32:70-77).(sampling point is the second fringe diameter to weigh the fresh samples of 0.5g
The fruit of about 2cm), the trichloroacetic acids of 3mL 15% are added, homogenate is worn into, 16000 × g centrifugation 20min take supernatant, with 5,
5 '-two thiobis (2- nitrobenzoic acids) are developed the color, and OD values are surveyed in 422nm wavelength, while making standard curve with GSH, are calculated GSH and are contained
Amount.Separately part sample is dried, its water content is calculated, fruit water content can be used for on-line checking result and Testing in vitro knot
Unit conversion when fruit contrasts.
The result of Testing in vitro is contrasted with real-time online testing result of the invention, as a result as shown in table 2.As a result
Show the on-line real-time measuremen reliable results of the microelectrode, can be used for scientific research.
The tamato fruit test result of table 2 is contrasted
The method of reductive glutathione in the real-time detection plant in situ that the present invention is provided, wherein, for reproducibility
The enzyme of glutathione detection is glutathione oxidase, is capable of achieving the specific detection to reductive glutathione, is detected sensitive
Degree is high, first detects the glutathione that microelectrode biosensor is used for plant.Detection target material is vegetable material, bag
Crops, flowers, vegetables, forest etc. are included, detection position can be the tissue such as stem, leaf, fruit of plant, and detection time can be
The different times of plant growth.
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 (10)
1. a kind of microelectrode biosensor, it is characterised in that the basal electrode material of the microelectrode is silicon chip, is set on silicon chip
There is three-electrode system, comprising Ag/AgCl reference electrodes, the metal working that platinum has glutathione oxidase to electrode and surface modification is made
Electrode.
2. microelectrode biosensor according to claim 1, it is characterised in that the microelectrode declines electricity for paster
Pole, surface coated with protective film；The working electrode of the SMD microelectrode is the metal disk of diameter 5-10mm, and reference electrode is
The annulus of diameter 8-16mm, ring width is 2-3mm, and length is 3/5 domain girth, is to electrode and reference electrode same diameter and width
The annulus of degree, length is 1/4 domain girth, reference electrode and is surrounded by half in the outer of the metal disk in a ring respectively to electrode
Side, is provided with three wires on silicon chip, be connected with above three electrode respectively, and the other end of wire is used to connect electrochemical operation
Stand；In flake, thickness is less than 2mm to the silicon chip substrate, and metal electrode film thickness is less than 1mm.
3. microelectrode biosensor according to claim 1, it is characterised in that the microelectrode declines electricity for insertion
Pole；The plug-in type microelectrode 2-8cm long, its tip portion 5-30mm long, 2-5mm wide；In on the tip of the silicon chip substrate
Between be laid with metal working electrode, Ag/AgCl reference electrodes, platinum are laid to electrode in both sides respectively；Exposed metal working electrode diameter
0.5-2mm, Ag/AgCl reference electrode diameter 0.2-0.5mm, platinum is to electrode diameter 0.2-0.5mm；The other end of silicon chip substrate compared with
Width, is laid with three wires thereon, is connected with above three electrode respectively, and the other end of wire is used to connect electrochemical operation
Stand.
4. the microelectrode biosensor according to claim any one of 1-3, it is characterised in that the microelectrode is biological to be passed
The preparation method of sensor is comprised the following steps：
1) platinum is prepared to electrode, Ag/AgCl reference electrodes and metal working electrode in silicon chip substrate using micro-electromechanical technology；
2) microelectrode that will be prepared is placed in 0.5M dilution heat of sulfuric acid, and voltammetric scan is circulated under -0.2-1.6V current potentials,
Obtain typical cyclic voltammetric spectrogram, it is ensured that electrode surface is cleaned；
3) Fe is prepared3O4NPs nano particles：It is 20 to take molal weight ratio:1 FeCl3And FeCl2It is dissolved in distilled water, adds
A certain amount of hydrochloric acid is completely dissolved molysite, and sodium hydroxide solution is then added dropwise over thereto to solution colour by yellowish discoloration
For brown is last until aterrimus；Black precipitate is collected together with magnet, is washed, then rinsed once with TMOH, from
The heart, collects precipitation, obtains Oxidation of Fe3O4NPs particles, precipitation nitric acid is washed once, then under 80-90 DEG C of stirring condition, is made
Particle is suspended in 0.01-3M nitric acid, until becoming brown；Washing is eventually passed, by Fe3O4NPs nanoparticle suspensions are in pH value
In 11 TMOH solution；
4) above-mentioned Fe is taken3O4NPs nano particle TMOH aaerosol solutions, with 1-100mM sodium citrate solutions dilute, stirring 10min with
On, citrate ion is uniformly wrapped Fe3O4Nano particle；The Fe of citrate is collected with magnet3O4Nano particle,
And being re-dispersed into 1-100mM sodium citrate solutions makes its concentration for 0.1-5mg/ml, adds final concentration of 0.1-100mM's
Silver nitrate solution, ultrasonic 15min is heated to boiling under stirring, sodium borohydride solution, sodium borohydride and silver nitrate are added immediately
Molal weight ratio is 1:1-1:Stop heating and continuous stirring after 200,10-60min to cooling down, obtain Fe3O4- Ag core shell structures are received
Rice corpuscles；
5) by Fe3O4- Ag core-shell structure nanometer particles are deposited on metal working electrode, obtain Fe@Ag/Au electrodes；
6) shitosan and glutathione oxidase mixed liquor are prepared：The 18mg/mL paddy Guangs that the PBS of pH value 7.3 is prepared
Sweet peptide oxidizing ferment enzyme liquid mixes in equal volume with 1.0% chitosan solution；Wherein, the compound method of 1.0% chitosan solution
It is as follows：0.1g shitosans are dissolved in 10mL 0.05M acetums, then the membrane filtration of 0.45 μm of via hole diameter, obtains final product；
7) by step 6) gained mixing drop-coated in Fe Ag/Au electrode surfaces, dry, obtaining final product surface modification has glutathione oxygen
Change the metal working electrode of enzyme, so as to complete the preparation of microelectrode biosensor.
5. original is carried out to reduced glutathione in plant using microelectrode biosensor described in claim any one of 1-4
The method of position on-line checking.
6. method according to claim 5, it is characterised in that the reduced glutathione mark of various concentrations is chosen before detection
Quasi- solution carries out microelectrode detection, and the standard curve of glutathione is drawn according to reduced glutathione concentration and current relationship；
The microelectrode lowest detection is limited to 0.05mmol/L, and the range of linearity is 0.1-100mmol/L, and reduced glutathione solution is dense
Degree is y=0.2668ln (x)+4.8799 with the linear equation of response peak current, and relative coefficient is 0.9816.
7. method according to claim 6, it is characterised in that under 0.4V operating potentials, carried out using chronoamperometry
The real time on-line monitoring of reduced glutathione in plant tissue；The timing since after electric current is steady, the electricity of collection the 20th minute
Flow data substitutes into linear equation, obtains the concentration of reduced glutathione in plant tissue.
8. the method according to claim any one of 5-7, it is characterised in that detection object includes stem, leaf or the fruit of plant
It is real；For different parts, the sensor of multi-form, SMD sensor is selected to be applied to blade, contact pin type sensor is applicable
In stem and fruit.
9. method according to claim 8, it is characterised in that enter to plant haulm or fruit when using plug-in type microelectrode
During row detection, by microelectrode biosensor insertion sample to be tested, insertion depth is 0.2-1cm, connects electrochemistry work
Voltammetric scan is circulated as station, realization carries out online reduced glutathione detection to plant tissue.
10. method according to claim 8, it is characterised in that cucumber leaves are examined when using SMD microelectrode
During survey, specific method is as follows：A hole is pricked on blade with the pin of diameter 1.2-1.5mm, vein position is avoided, in aperture overlying
The metafiltration scraps of paper of lid one, filter paper size is advisable with no more than SMD microelectrode, and the PBS for drawing pH7.3 drops in filter paper
On piece, it is advisable with soaking monoblock filter paper just, the metal covering of SMD microelectrode of diaphragm will be removed close in filter
On paper, microelectrode, filter paper and blade clamper are fixed with clip, connect electrochemical workstation, carry out reduced form paddy in blade
The real time on-line monitoring of the sweet peptide of Guang.
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