CN105319260A - On-line plant glucose detection method and device based on microelectrode biosensing technology - Google Patents
On-line plant glucose detection method and device based on microelectrode biosensing technology Download PDFInfo
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Abstract
The invention provides an on-line plant glucose detection method based on the microelectrode biosensing technology. The method comprises the steps of inserting a glucose oxidase microelectrode into a to-be-detected plant tissue, and connecting an electrochemical workstation for cyclic voltammetry scan, so as to achieve the on-line glucose detection of the plant tissue. The invention also provides a device used for achieving the detection method. The device comprises the electrochemical workstation and the glucose oxidase microelectrode which are connected through a conducting wire. According to the on-line plant glucose detection method and the device, the real-time detection of the plant glucose content can be achieved, the detected part of the plant does not need to be separated from the plant body, and has no destructive damage; the vivo detection can be carried out only through surface cleaning and tissue fixing, and continuous results are obtained for dynamic analysis; the detection device provided by the invention is convenient to carry and simple in detection condition, can be operated in the field, and achieves continuous on-line detection.
Description
Technical field
The present invention relates to electrochemical field, specifically, relate to the online glucose sensing approach of a kind of plant based on microelectrode biosensing technology and device.
Background technology
Glucose is monose, is the sugar containing aldehyde radical.It is extensively present in plant, and being the main constituent of some carbohydrates (as sucrose, maltose, starch, cellulose etc.), is the main energy sources material in plant.Glucose is photosynthetic product, and be again respiratory substrate, growing that it is plant provides energy, and can strengthen stress resistance of plant.The metabolism of glucose is the center of whole plant metabolism, and it has linked up the metabolism of protein, lipid and secondary species etc.
At present in plant physiology research, glucose detection many employings colourimetry, DNS reagent reducing sugar test method, liquid phase chromatography etc.These class methods need sample in vitro to vegetable material, the content in plant body is extrapolated by detecting concentration of glucose in leaching liquor, higher to sample extraction purity requirement, complicated and the length consuming time of its sample preparation, and what obtain is the concentration in a certain moment in plant, cannot carry out performance analysis, long-term follow monitoring.
In order to realize growth and development of plants and environmental adaptation Journal of Sex Research, live body original position need be carried out detect dynamically, glucose In vivo detection product is in the market mainly for animal or human's body, there is due to plant the features such as blade is thin, tissue fluid is few, stem stalk lignification, above-mentioned testing product cannot directly apply to plant, therefore urgently develops the glucose online test method for plant tissue and device.
Summary of the invention
The object of this invention is to provide the online glucose sensing approach of a kind of plant based on microelectrode biosensing technology and device.
In order to realize the object of the invention, the online glucose sensing approach of a kind of plant based on microelectrode biosensing technology of the present invention, the method is that glucose oxidase microelectrode is inserted (about 5-20mm) in plant tissue to be measured, connect electrochemical workstation and carry out cyclic voltammetry scan, realize carrying out online glucose detection to plant tissue.
Aforesaid method, the Glucose standards solution choosing some concentration before also comprising detection carries out microelectrode detection, obtain microelectrode according to concentration of glucose and current relationship and detect glucose working curve, and utilize the step that the glucose solution of known normal concentration corrects microelectrode and electrochemical workstation.
The glucose oxidase microelectrode related in said method, the long 5-8cm of described microelectrode, the long 3-10mm of its tip portion; The basal electrode material of described microelectrode is silicon chip, and described microelectrode has three-electrode system, comprises Ag/AgCl contrast electrode, and platinum is to electrode and the metal working electrode by glucose oxidase enzyme modification.
Wherein, at the bottom of silicon wafer-based 1 tip on be provided with metal working electrode 2, Ag/AgCl contrast electrode 3, platinum in turn to electrode 4; The other end at the bottom of silicon wafer-based is wider, it is arranged Connection Element 5, corresponds respectively to above-mentioned three electrodes.(Fig. 1)
Length at the bottom of silicon wafer-based is 4 ~ 6cm, and thickness is 0.5 ~ 2mm, and tip portion length is 3 ~ 20mm; Response time is the shortest is 30s, and lowest detection is limited to 0.06mmol/L, and the range of linearity is 0.01-10mmol/L, and the linear correlation property coefficient of glucose concentration and response peak electric current is 0.997.
During detection, connect the Connection Element 5 in microelectrode array with electrochemical workstation, detect electric current by electrochemical workstation cyclic voltammetry, then current data is scaled concentration data.
Adopt etching technique and electro-deposition techniques to prepare described microelectrode (microelectrode array) on 1 at the bottom of silicon wafer-based, preparation method comprises the following steps:
1) by electrochemical deposition, microelectrode current-carrying part is modified into platinum plating respectively to electrode, Ag/AgCl contrast electrode and gold-plated working electrode;
2) polish gold-plated working electrode to smooth with abrasive paper for metallograph, ultrasonicly to clean with deionized water;
3) then microelectrode is placed in 0.5M sulfuric acid solution under-0.2 ~ 1.6V condition, carries out the cyclic voltammetric spectrogram that cyclic voltammetry scan obtains standard, show that electrode surface is clean;
4) prepare 10mML-cysteine solution, drip and be applied to gold-plated working electrode surface, under room epidemic disaster 100% condition, modify at least 18h, then clean with deionized water, obtain the gold electrode modified;
5) by particle diameter be the nano gold sol of 8 ~ 20nm and the ultrasonic mixed preparing of the carbon nano tube dispersion liquid carbon nano tube dispersion liquid containing 8-20% nm of gold, dispersant liquid drop is applied to step 4) on the gold electrode modified, at least 18h is modified under room epidemic disaster 100% condition, then clean with deionized water, obtain the gold electrode modified;
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 50 ~ 80%, make the solution that glucose oxidase enzyme concentration is 0.1 ~ 2mol/L, described solution is dripped and is applied to step 5) on the gold electrode modified, 2 ~ 5 DEG C, humidity modifies at least 24h 100% time, then clean with deionized water, obtain by the metal working electrode of glucose oxidase enzyme modification, in 2 ~ 5 DEG C of storages.
Fig. 2 is shown in by the modification structure schematic diagram of the metal working electrode comprised in the microelectrode adopting said method to prepare.
The present invention further provides the device for realizing the online glucose sensing approach of the above-mentioned plant based on microelectrode biosensing technology, described device comprises electrochemical workstation and described glucose oxidase microelectrode, is connected therebetween by wire.
The electrochemical workstation related in the present invention is the abbreviation of electrochemical measurement system, is the measuring equipment that electrochemical field is commonly used.This measuring system is formed a complete machine, include fast digital signal generator, compensating circuit and potentiostat, galvanostat fall in high-speed data acquistion system, glucose current equation traffic filter, multilevel signal gain, IR.The steady-state current that can be directly used on microelectrode is measured.
The present invention can realize the real-time detection of the online glucose of plant, plant detection site without the need in vitro, without destructive damage, only need clean surface, fixing organization get final product In vivo detection, obtain continuous result and carry out performance analysis.Pick-up unit of the present invention is portable, and testing conditions is simple, and can operate in field, and realize continuous on-line detection, the glucose oxidase microelectrode of this device has the ability penetrating plant tissue (comprising blade, stem stalk, fruit etc.).
Accompanying drawing explanation
Fig. 1 is the structural representation of the glucose oxidase microelectrode of preparation in the embodiment of the present invention 1; Wherein, 1 is at the bottom of silicon wafer-based, and 2 is metal working electrode, and 3 is Ag/AgCl contrast electrode, 4 be platinum to electrode, 5 is Connection Element.
Fig. 2 is the modification structure schematic diagram of the metal working electrode comprised in the microelectrode of preparation in the embodiment of the present invention 1.
Fig. 3 is the working curve result of carrying out cyclic voltammetry scan in the embodiment of the present invention 1 with the microelectrode of preparation.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The preparation of embodiment 1 glucose oxidase microelectrode
The long 5cm of glucose oxidase microelectrode prepared in the present embodiment, the long 10mm of its tip portion; The basal electrode material of described microelectrode is silicon chip, and described microelectrode has three-electrode system, comprises Ag/AgCl contrast electrode, and platinum is to electrode and the metal working electrode by glucose oxidase enzyme modification.(Fig. 1)
Wherein, at the bottom of silicon wafer-based 1 tip on be provided with metal working electrode 2, Ag/AgCl contrast electrode 3, platinum in turn to electrode 4; The other end at the bottom of silicon wafer-based is wider, it is arranged Connection Element 5, corresponds respectively to above-mentioned three electrodes.
Length at the bottom of silicon wafer-based is 5cm, thickness is 1.5mm, and tip portion length is 4mm; At the bottom of silicon wafer-based 1 most advanced and sophisticated exposed part on be provided with metal working electrode 2, Ag/AgCl contrast electrode 3, platinum in turn to electrode 4; The other end at the bottom of silicon wafer-based is wider, it is arranged Connection Element 5, corresponds respectively to above-mentioned three electrodes.
The described microelectrode response time is the shortest is 30s, and lowest detection is limited to 0.06mmol/L, and the range of linearity is 0.01-10mmol/L, and the linear correlation property coefficient of glucose concentration and response peak electric current is 0.997.
During detection, connect the Connection Element 5 in microelectrode array with electrochemical workstation, detect electric current by electrochemical workstation cyclic voltammetry, then current data is scaled concentration data.
Adopt etching technique and electro-deposition techniques to prepare described microelectrode (microelectrode array) on 1 at the bottom of silicon wafer-based, preparation method comprises the following steps:
1) by electrochemical deposition, microelectrode current-carrying part is modified into platinum plating respectively to electrode, Ag/AgCl contrast electrode and gold-plated working electrode;
2) polish gold-plated working electrode to smooth with abrasive paper for metallograph, ultrasonicly to clean with deionized water;
3) then microelectrode is placed in 0.5M sulfuric acid solution under-0.2 ~ 1.6V condition, to carry out cyclic voltammetry scan obtain cyclic voltammetric spectrogram, shows that electrode surface is clean;
4) prepare 10mML-cysteine solution, drip and be applied to gold-plated working electrode surface, under room epidemic disaster 100% condition, modify 18h, then clean with deionized water, obtain the gold electrode modified;
5) by particle diameter be the nano gold sol of 13nm and the ultrasonic mixed preparing of the carbon nano tube dispersion liquid carbon nano tube dispersion liquid containing 10% nm of gold, dispersant liquid drop is applied to step 4) on the gold electrode modified, 18h is modified under room epidemic disaster 100% condition, then clean with deionized water, obtain the gold electrode modified;
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 70%, make the solution that glucose oxidase enzyme concentration is 0.1M, described solution is dripped and is applied to step 5) on the gold electrode modified, at 4 DEG C, humidity 100% time modification 24h, then clean with deionized water, obtain by the metal working electrode of glucose oxidase enzyme modification, in 4 DEG C of storages.
Fig. 2 is shown in by the modification structure schematic diagram of the metal working electrode comprised in the microelectrode adopting said method to prepare.
Respectively compound concentration be 0,0.01,0.05,0.1,0.5,1.0,5.0, the glucose-phosphate buffer (pH=6.8) of 10mM, cyclic voltammetry detection (voltage-1.0 ~ 0.6V is carried out with the microelectrode of preparation, sweep velocity 50mV/s), relation according to concentration of glucose and response peak electric current draws microelectrode working curve (Fig. 3), linear equation be i=4.8413-98.58501c (wherein, c is concentration of glucose, unit μM; I is electric current, unit nA), the range of linearity can reach 0.01-10mmol/L.The linear correlation property coefficient of glucose concentration and response peak electric 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 washed with de-ionized water prepared by embodiment 1, first detect three parts of normal concentration (0.1mmol/L, 1mmol/L, 5mmol/L) glucose solutions respectively, carry out galvanochemistry calibration, after obtaining concentration of glucose and response peak current relationship figure, try to achieve slope, when the slope deviation of slope and working curve is within 15%, then think that enzymatic activity is normal, electrode can normally work.
Microelectrode after clean calibration is inserted in potato haulm tissue to be measured, connect electrochemical workstation and carry out cyclic voltammetry scan 5min, obtain stable cyclic voltammetry curve, by the response peak current value obtained, substitute into working curve in conjunction with deviation, try to achieve the glucose content in tissue to be measured.
Experimental result contrasts:
The tissue cutting microelectrode detection position carries out liquid chromatographic detection, and comparing result is in table 1.Result shows that this method testing result is reliable, and sample process is simple, is convenient to field real-time online and detects.
Table 1 test result contrasts
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. glucose oxidase microelectrode, is characterized in that, the long 5-8cm of described microelectrode, the long 3-10mm of its tip portion; The basal electrode material of described microelectrode is silicon chip, and described microelectrode has three-electrode system, comprises Ag/AgCl contrast electrode, and platinum is to electrode and the metal working electrode by glucose oxidase enzyme modification.
2. microelectrode according to claim 1, is characterized in that, the tip of (1) at the bottom of silicon wafer-based is provided with in turn metal working electrode (2), Ag/AgCl contrast electrode (3), platinum to electrode (4); The other end at the bottom of silicon wafer-based is wider, it is arranged Connection Element (5), corresponds respectively to above-mentioned three electrodes.
3. microelectrode according to claim 2, is characterized in that, the length at the bottom of silicon wafer-based is 4 ~ 6cm, and thickness is 0.5 ~ 2mm, and tip portion length is 3 ~ 20mm; The described microelectrode response time is the shortest is 30s, and lowest detection is limited to 0.06mmol/L, and the range of linearity is 0.01-10mmol/L, and the linear correlation property coefficient of glucose concentration and response peak electric current is 0.997.
4. the microelectrode according to any one of claim 1-3, is characterized in that, the preparation method of described microelectrode comprises the following steps:
1) by electrochemical deposition, microelectrode current-carrying part is modified into platinum plating respectively to electrode, Ag/AgCl contrast electrode and gold-plated working electrode;
2) polish gold-plated working electrode to smooth with abrasive paper for metallograph, ultrasonicly to clean with deionized water;
3) then microelectrode is placed in 0.5M sulfuric acid solution under-0.2 ~ 1.6V condition, to carry out cyclic voltammetry scan obtain cyclic voltammetric spectrogram, shows that electrode surface is clean;
4) prepare 10mML-cysteine solution, drip and be applied to gold-plated working electrode surface, under room epidemic disaster 100% condition, modify at least 18h, then clean with deionized water, obtain the gold electrode modified;
5) by particle diameter be the nano gold sol of 8 ~ 20nm and the ultrasonic mixed preparing of the carbon nano tube dispersion liquid carbon nano tube dispersion liquid containing 8-20% nm of gold, dispersant liquid drop is applied to step 4) on the gold electrode modified, at least 18h is modified under room epidemic disaster 100% condition, then clean with deionized water, obtain the gold electrode modified;
6) by glucose oxidase ultrasonic dissolution in the chitosan solution of concentration 50 ~ 80%, make the solution that glucose oxidase enzyme concentration is 0.1 ~ 2mol/L, described solution is dripped and is applied to step 5) on the gold electrode modified, 2 ~ 5 DEG C, humidity modifies at least 24h 100% time, then clean with deionized water, obtain by the metal working electrode of glucose oxidase enzyme modification, in 2 ~ 5 DEG C of storages.
5. the online glucose sensing approach of the plant based on microelectrode biosensing technology, it is characterized in that, glucose oxidase microelectrode described in any one of claim 1-4 is inserted in plant tissue to be measured, connect electrochemical workstation and carry out cyclic voltammetry scan, realize carrying out online glucose detection to plant tissue.
6. method according to claim 5, it is characterized in that, the Glucose standards solution choosing some concentration before also comprising detection carries out microelectrode detection, obtain microelectrode according to concentration of glucose and current relationship and detect glucose working curve, and utilize the step that the glucose solution of known normal concentration corrects microelectrode and electrochemical workstation.
7. based on the online glucometer device of plant of microelectrode biosensing technology, it is characterized in that, described device comprises electrochemical workstation and the glucose oxidase microelectrode described in any one of claim 1-4, is connected therebetween by wire.
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