CN105319260B - The online glucose sensing approach of plant and device based on microelectrode biosensing technology - Google Patents
The online glucose sensing approach of plant and device based on microelectrode biosensing technology Download PDFInfo
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Abstract
The present invention provides a kind of online glucose sensing approach of plant based on microelectrode biosensing technology, glucose oxidase microelectrode is inserted in plant tissue to be measured, connect electrochemical workstation and carry out cyclic voltammetry scan, realize and online glucose detection is carried out to plant tissue.The present invention also provides the device for realizing above-mentioned detection method, and described device includes electrochemical workstation and glucose oxidase microelectrode, connected by wire therebetween.The present invention can realize the real-time detection of plant glucose content, plant detection site without it is in vitro, without destructiveness damage, only need clean surface, fixing organization can In vivo detection, obtain continuous result and enter Mobile state analysis.The detection means of the present invention is portable, and testing conditions are simple, can field operation, and realize continuous on-line detection.
Description
Technical field
The present invention relates to electrochemical field, specifically, it is related to a kind of plant based on microelectrode biosensing technology and exists
Line glucose sensing approach and device.
Background technology
Glucose is monose, is sugared containing aldehyde radical.It is widely present in plant, is some carbohydrate (such as sucrose, wheat
Bud sugar, starch, cellulose etc.) main constituents, be the main energy sources material in plant.Glucose is photosynthetic
Product, is the substrate of respiration again, and it is the offer energy that grows of plant, and can strengthen stress resistance of plant.Glucose
Metabolism be whole plant metabolism center, it has linked up the metabolism of protein, lipid and secondary species etc..
At present in plant physiology research, colorimetric method, DNS reagent reducing sugar test methods, liquid phase are used glucose detection more
Chromatography etc..This kind of method need in vitro be sampled to vegetable material, by detecting that concentration of glucose extrapolates plant in leaching liquor
The internal content of strain, higher to sample extraction purity requirement, its sample treatment is complicated and time-consuming, and acquisition is in plant
The concentration at a certain moment, it is impossible to enter Mobile state analysis, long-term follow monitoring.
In order to realize to growth and development of plants and environmental suitability research, live body dynamic detection in situ need to be carried out, at present
The glucose In vivo detection product of in the market is mainly for animal or human body, and because plant has, blade is thin, tissue fluid is few, stem
The features such as stalk lignifying, above-mentioned detection product can not directly apply to plant, therefore urgently Portugal of the exploitation for plant tissue
Grape sugar online test method and device.
The content of the invention
It is an object of the invention to provide a kind of online glucose sensing approach of the plant based on microelectrode biosensing technology
And device.
In order to realize the object of the invention, a kind of online glucose of plant based on microelectrode biosensing technology of the invention
Detection method, this method is to insert glucose oxidase microelectrode in plant tissue to be measured (about 5-20mm), connects electrochemistry
Work station carries out cyclic voltammetry scan, realizes and carries out online glucose detection to plant tissue.
The glucose standards solution that some concentration are chosen before foregoing method, in addition to detection carries out microelectrode detection, root
Microelectrode detection glucose working curve is obtained according to concentration of glucose and current relationship, and utilizes the glucose of known normal concentration
The step of solution is corrected to microelectrode and electrochemical workstation.
The glucose oxidase microelectrode being related in the above method, the long 5-8cm of microelectrode, the long 3- of its tip portion
10mm；The basal electrode material of the microelectrode is silicon chip, and the microelectrode has three-electrode system, includes Ag/AgCl references
Electrode, the metal working electrode that platinum is modified to electrode and by glucose oxidase.
Wherein, metal working electrode 2, Ag/AgCl reference electrodes 3, platinum have been sequentially arranged to electrode on the tip of silicon chip substrate 1
4；The other end of silicon chip substrate is wider, and connecting element 5 is set thereon, above three electrode is corresponded respectively to.(Fig. 1)
The length of silicon chip substrate is 4~6cm, and thickness is 0.5~2mm, and tip portion length is 3~20mm；Response time
Most short is 30s, and lowest detection is limited to 0.06mmol/L, and the range of linearity is 0.01-10mmol/L, glucose concentration and response
The linear correlation property coefficient of peak current is 0.997.
During detection, the connecting element 5 in microelectrode array is connected with electrochemical workstation, is circulated by electrochemical workstation
Voltammetry detects electric current, then current data is scaled into concentration data.
The microelectrode (microelectrode array) is prepared in silicon chip substrate 1 using etching technique and electro-deposition techniques, is prepared
Method comprises the following steps：
1) microelectrode current-carrying part is modified into electrode, Ag/AgCl reference electrodes by platinum plating by electrochemical deposition respectively
And plating metal working electrode；
2) cleaned with abrasive paper for metallograph polishing plating metal working electrode to smooth with deionized water and ultrasound；
3) and then microelectrode is placed in 0.5M sulfuric acid solutions into the progress cyclic voltammetry scan under the conditions of -0.2~1.6V to obtain
To the cyclic voltammetric spectrogram of standard, show that electrode surface is clean；
4) 10mM Cys solution is prepared, drop coating is in gold-plated working electrode surface, the condition of epidemic disaster 100% in room
Lower modification at least 18h, is then washed with deionized water net, the gold electrode modified；
5) particle diameter is prepared for 8~20nm nano gold sol with carbon nano tube dispersion liquid ultrasonic mixing and contains 8-20% nanometers
Gold carbon nano tube dispersion liquid, dispersant liquid drop is applied to step 4) modification gold electrode on, under the conditions of room epidemic disaster 100%
At least 18h is modified, net, the gold electrode modified then is washed with deionized water；
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 50~80%, glucose oxidase is made
Concentration be 0.1~2mol/L solution, by the solution drop coating in step 5) modification gold electrode on, in 2~5 DEG C, humidity
100% time modification at least 24h, is then washed with deionized water only, the metal working electrode modified by glucose oxidase is obtained, in 2
~5 DEG C of storages.
The modification structure schematic diagram of the metal working electrode included in the microelectrode prepared using the above method is shown in Fig. 2.
The present invention further provides for realizing the online glucose inspection of the above-mentioned plant based on microelectrode biosensing technology
The device of survey method, described device includes electrochemical workstation and described glucose oxidase microelectrode, leads to therebetween
Cross wire connection.
The electrochemical workstation being related in the present invention is the abbreviation of electrochemical measurement system, is the conventional survey of electrochemical field
Measure equipment.This measuring system is constituted into a complete machine, fast digital signal generator, high-speed data acquistion system, electricity is included
Position current signal filter, multilevel signal gain, IR drop compensation circuits and potentiostat, galvanostat.It can be directly used for micro-
Steady-state current measurement on electrode.
The present invention can realize the real-time detection of the online glucose of plant, plant detection site without it is in vitro, without destruction
Property damage, only need clean surface, fixing organization can In vivo detection, obtain continuous result and enter Mobile state analysis.The inspection of the present invention
Survey device portable, testing conditions are simple, can field operation, and realize continuous on-line detection, the glucose oxidase of the device is micro-
Electrode has the ability for penetrating plant tissue (including blade, stalk, fruit etc.).
Brief description of the drawings
Fig. 1 is the structural representation of the glucose oxidase microelectrode prepared in the embodiment of the present invention 1；Wherein, 1 is silicon chip
Substrate, 2 be metal working electrode, and 3 be Ag/AgCl reference electrodes, 4 be platinum to electrode, 5 be connecting element.
Fig. 2 is the modification structure schematic diagram of the metal working electrode included in the microelectrode for preparing in the embodiment of the present invention 1.
Fig. 3 is to carry out the working curve result of cyclic voltammetry scan with the microelectrode of preparation in the embodiment of the present invention 1.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The preparation of the glucose oxidase microelectrode of embodiment 1
The long 5cm of glucose oxidase microelectrode prepared in the present embodiment, the long 10mm of its tip portion；The microelectrode
Basal electrode material is silicon chip, and the microelectrode has three-electrode system, comprising Ag/AgCl reference electrodes, platinum to electrode and
The metal working electrode modified by glucose oxidase.(Fig. 1)
Wherein, metal working electrode 2, Ag/AgCl reference electrodes 3, platinum have been sequentially arranged to electrode on the tip of silicon chip substrate 1
4；The other end of silicon chip substrate is wider, and connecting element 5 is set thereon, above three electrode is corresponded respectively to.
The length of silicon chip substrate is 5cm, thickness is 1.5mm, and tip portion length is 4mm；Silicon chip substrate 1 it is sophisticated naked
Dew has been sequentially arranged metal working electrode 2, Ag/AgCl reference electrodes 3, platinum to electrode 4 on part；The other end of silicon chip substrate compared with
Width, sets connecting element 5, corresponds respectively to above three electrode thereon.
It is 30s that the microelectrode response time is most short, and lowest detection is limited to 0.06mmol/L, and the range of linearity is 0.01-
The linear correlation property coefficient of 10mmol/L, glucose concentration and response peak current is 0.997.
During detection, the connecting element 5 in microelectrode array is connected with electrochemical workstation, is circulated by electrochemical workstation
Voltammetry detects electric current, then current data is scaled into concentration data.
The microelectrode (microelectrode array) is prepared in silicon chip substrate 1 using etching technique and electro-deposition techniques, is prepared
Method comprises the following steps：
1) microelectrode current-carrying part is modified into electrode, Ag/AgCl reference electrodes by platinum plating by electrochemical deposition respectively
And plating metal working electrode；
2) cleaned with abrasive paper for metallograph polishing plating metal working electrode to smooth with deionized water and ultrasound；
3) and then microelectrode is placed in 0.5M sulfuric acid solutions into the progress cyclic voltammetry scan under the conditions of -0.2~1.6V to obtain
To cyclic voltammetric spectrogram, show that electrode surface is clean；
4) 10mM Cys solution is prepared, drop coating is in gold-plated working electrode surface, the condition of epidemic disaster 100% in room
Lower modification 18h, is then washed with deionized water net, the gold electrode modified；
5) particle diameter is prepared containing 10% nanogold for 13nm nano gold sol with carbon nano tube dispersion liquid ultrasonic mixing
Carbon nano tube dispersion liquid, step 4 is applied to by dispersant liquid drop) modification gold electrode on, under the conditions of room epidemic disaster 100% modify
18h, is then washed with deionized water net, the gold electrode modified；
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 70%, glucose oxidase concentration is made
For 0.1M solution, by the solution drop coating in step 5) on the gold electrode of modification, modify 24h 100% time in 4 DEG C, humidity, so
After be washed with deionized water net, the metal working electrode modified by glucose oxidase is obtained, in 4 DEG C of storages.
The modification structure schematic diagram of the metal working electrode included in the microelectrode prepared using the above method is shown in Fig. 2.
Respectively compound concentration be 0,0.01,0.05,0.1,0.5,1.0,5.0,10mM glucose-phosphate buffer (pH
=6.8), cyclic voltammetry detection (voltage -1.0~0.6V, sweep speed 50mV/s) is carried out with the microelectrode of preparation, according to Portugal
Grape sugar concentration and the relation of response peak current draw microelectrode working curve (Fig. 3), and linear equation is i=4.8413-
(wherein, c is concentration of glucose, unit μM to 98.58501c；I is electric current, unit nA), the range of linearity is up to 0.01-10mmol/
L.The linear correlation property coefficient of glucose concentration and response peak current is 0.997.
Embodiment 2 detects the glucose content in potato haulm sample based on microelectrode biosensing technology real-time online
After glucose oxidase microelectrode prepared by embodiment 1 is cleaned with deionized water, three parts of standards are first detected respectively
Concentration (0.1mmol/L, 1mmol/L, 5mmol/L) glucose solution, carries out electrochemistry calibration, obtains concentration of glucose and response
After peak current graph of a relation, slope is tried to achieve, when the slope deviation of slope and working curve is within 15%, then it is assumed that enzymatic activity is just
Often, electrode can normal work.
The microelectrode after calibration will be cleaned to insert in potato haulm tissue to be measured, connection electrochemical workstation carries out circulation volt
Peace scanning 5min, the cyclic voltammetry curve stablized, by obtained response peak point current, substitutes into working curve and combined partially
Difference, tries to achieve the glucose content in test serum.
Experimental result is contrasted：
The tissue for cutting microelectrode detection position carries out liquid chromatographic detection, and comparing result is shown in Table 1.As a result this method is shown
Testing result is reliable, and sample process is simple, is easy to field real-time online detection.
The test result of table 1 is contrasted
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 (6)
1. glucose oxidase microelectrode, it is characterised in that the long 5-8cm of microelectrode, the long 3-10mm of its tip portion；It is described
The basal electrode material of microelectrode is silicon chip, and the microelectrode has three-electrode system, includes Ag/AgCl reference electrodes, platinum pair
Electrode and the metal working electrode modified by glucose oxidase；
The preparation method of the microelectrode comprises the following steps：
1) by electrochemical deposition by microelectrode current-carrying part be modified into respectively platinum plating to electrode, Ag/AgCl reference electrodes and
Plate metal working electrode；
2) cleaned with abrasive paper for metallograph polishing plating metal working electrode to smooth with deionized water and ultrasound；
3) and then microelectrode is placed in 0.5M sulfuric acid solutions into the progress cyclic voltammetry scan under the conditions of -0.2~1.6V to be followed
Ring volt-ampere spectrogram, shows that electrode surface is clean；
4) 10mM Cys solution is prepared, drop coating is repaiied in gold-plated working electrode surface under the conditions of room epidemic disaster 100%
At least 18h is adornd, net, the gold electrode modified then is washed with deionized water；
5) particle diameter is prepared into the nanogold containing 8-20% for 8~20nm nano gold sol with carbon nano tube dispersion liquid ultrasonic mixing
Carbon nano tube dispersion liquid, step 4 is applied to by dispersant liquid drop) modification gold electrode on, under the conditions of room epidemic disaster 100% modify
At least 18h, is then washed with deionized water net, the gold electrode modified；
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 50~80%, glucose oxidase concentration is made
For 0.1~2mol/L solution, by the solution drop coating in step 5) on the gold electrode of modification, under 2~5 DEG C, humidity 100%
At least 24h is modified, is then washed with deionized water only, obtains the metal working electrode modified by glucose oxidase, in 2~5 DEG C of storages
Deposit.
2. microelectrode according to claim 1, it is characterised in that be sequentially arranged metal working on the tip of silicon chip substrate (1)
Make electrode (2), Ag/AgCl reference electrodes (3), platinum to electrode (4)；The other end of silicon chip substrate is wider, and connection member is set thereon
Part (5), corresponds respectively to above three electrode.
3. microelectrode according to claim 2, it is characterised in that the length of silicon chip substrate is 4~6cm, thickness is 0.5~
2mm, tip portion length is 3~20mm；It is 30s that the microelectrode response time is most short, and lowest detection is limited to 0.06mmol/L,
The range of linearity is 0.01-10mmol/L, and glucose concentration is 0.997 with the linear correlation property coefficient for responding peak current.
4. the online glucose sensing approach of a kind of plant based on microelectrode biosensing technology, it is characterised in that will by right
The glucose oxidase microelectrode described in any one of 1-3 is asked to insert in plant tissue to be measured, connection electrochemical workstation is followed
Ring voltammetric scan, realizes and carries out online glucose detection to plant tissue.
5. method according to claim 4, it is characterised in that also include the Glucose standards of some concentration of selection before detection
Solution carries out microelectrode detection, and microelectrode detection glucose working curve, and profit are obtained according to concentration of glucose and current relationship
The step of being corrected with the glucose solution of known normal concentration to microelectrode and electrochemical workstation.
6. the online glucometer device of plant based on microelectrode biosensing technology, it is characterised in that described device includes
Glucose oxidase microelectrode described in electrochemical workstation and claim any one of 1-3, is connected therebetween by wire
Connect.
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