CN108226241A - The miniature Ratio-type sensor and its construction method of a kind of In vivo detection heteroauxin and application - Google Patents
The miniature Ratio-type sensor and its construction method of a kind of In vivo detection heteroauxin and application Download PDFInfo
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- CN108226241A CN108226241A CN201711421481.3A CN201711421481A CN108226241A CN 108226241 A CN108226241 A CN 108226241A CN 201711421481 A CN201711421481 A CN 201711421481A CN 108226241 A CN108226241 A CN 108226241A
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Abstract
The present invention relates to microelectrode biotechnologies, and in particular to the miniature Ratio-type sensor and its construction method of a kind of In vivo detection heteroauxin and application.The present invention modifies carbon fiber electrode using nafion/cytC/ [Bmim] [BF4], using the oxidation current of cytC as built-in correction signal, dual signal output is formed together with the signal of IAA, so as to build Ratio-type sensor, the stability and reproducibility of IAA sensors are significantly improved, accuracy and reliability when being detected so as to fulfill plant living body IAA.
Description
Technical field
The present invention relates to microelectrode biotechnologies, and in particular to a kind of In vivo detection plant heteroauxin it is miniature
Ratio-type sensor and its construction method and application.
Background technology
Heteroauxin (IAA) is a kind of plant hormone being widely present, and important tune is played in plant various physiological processes
Section act on, such as plant growth, cell division, bloom, apoptosis, gene expression, and to environment-stress respond and adapted to
It is also played an important role in journey, therefore is highly desirable to carry out accurate quantitative analysis to it.IAA in traditional detection plant
Method include high pressure lipuid chromatography (HPLC), gas chromatography-mass spectrography, electrochemical luminescence, Capillary Electrophoresis, Enzyme-linked Immunosorbent Assay,
Radioimmunology etc..These methods need through herborization sample, handle the cumbersome operations such as plant sample, extraction, then
Carry out Testing in vitro.The acquisition of sample would generally cause plant larger damage even to cause Plant death, sample process mistake
Journey can cause the loss of Cucumber, and what Testing in vitro usually reflected is a kind of static and accumulation effect, it is impossible to reflect
Instant messages of the plant hormone in environmental change.Therefore it is badly in need of the biopsy method of IAA in development plant sample.
Electrochemical sensor has the characteristics that high sensitivity, selectivity is good, of low cost, is easily integrated and portable, therefore
It is especially suitable for In vivo detection application.But electrochemical sensor is in application process inevitably by environment and human factor
It influences, reproducibility is poor, seriously restricts its practical application.And the biological sample in viviperception is more complicated, therefore is badly in need of hair
Reproducibility and the preferable miniature electrochemical of reliability are opened up, to be applied to the In vivo detection of plant IAA.
Invention content
The defects of for existing detection technique, the purpose of the present invention is to provide a kind of miniature Ratio-type biographies for detecting IAA
Sensor, so as to preferably be applied to the viviperception of plant.
The solution of the present invention is as follows：
A kind of preparation method for the modified carbon fiber electrode that can be used for detecting IAA, includes the following steps：
1) carbon fiber electrode is ultrasonically treated to (general processing about 3 minutes) in acetone, second alcohol and water successively, then in sulphur
0-1V cyclic voltammetry scans carry out electrochemical pre-treatment in acid solution, until obtaining ideal cyclic voltammogram；
2) with ionic liquid 1-butyl-3-methyl imidazolium tetrafluoroborate ([Bmim] [BF4]) for solvent, preparation contains
The solution of cromoci (cyt C)；By step 1) pretreatment carbon fiber the above-mentioned C containing cyt [Bmim] [BF4]) solution
Middle rotary dipping, it is dry；The carbon fiber of cyt C/ [Bmim] [BF4] modifications is made；
3) carbon fiber made from the step 2) is immersed into certain density nafion (perfluorinated sulfonic acids-polytetrafluorethylene copolymer
Object) in solution, the carbon fiber electrode of nafion/cytC/ [Bmim] [BF4] modifications is made, as can be used for the modification of detection IAA
Carbon fiber electrode.
Further, the step 1) carbon fiber diameter is generally 5-100 μm or so, preferably 10 μm.
Further, a concentration of 0.5-1M of the step 1) sulfuric acid solution, preferably 0.5M.
Further, [Bmim] [BF4] of the C containing cyt described in step 2)) solution concentration be 1-5mg/100ul；It is preferred that
For 2.5mg/100ul.
Further, a concentration of 2-5% (mass fraction) of step 3) the nafion solution, preferably 2%.
Specifically, the above-mentioned modified carbon fiber electrode that can be used for detection IAA, preparation method include the following steps：
1) carbon fiber electrode is ultrasonically treated in acetone, second alcohol and water successively, then in the sulfuric acid of a concentration of 0.5M-1M
0-1V cyclic voltammetry scans carry out electrochemical pre-treatment in solution, until obtaining ideal cyclic voltammogram；
2) [Bmim] [BF4] of C containing cyt described in appropriate (such as 10 μ l) is taken) solution is placed on clean glass plate, and it will
The carbon fiber of step 1) pretreatment is placed in one rotary dipping (general 5min or so) for a period of time, drying at room temperature；Repeat the leaching
Stain drying process 2-5 times；The carbon fiber of cyt C/ [Bmim] [BF4] modifications is made；
3) carbon fiber made from step 2) is immersed in the nafion solution of concentration 2-5% a period of time (general 5min a left sides
It is right), the carbon fiber electrode of nafion/cytC/ [Bmim] [BF4] modifications is made, as can be used for the modified carbon fiber of detection IAA
Electrode.
The invention also includes the modified carbon fiber electrodes that can be used for detection IAA prepared by the above method.
Carbon fiber acts not only as the platform of assembling cyt C molecules, and microelectrode property is also greatly improved cyt C and exists
The electron transport rate of electrode surface.The modification of cyt C also functions to two effects, first, the redox of IAA can be promoted, two
It is that the redox signal of its own can be with the modification situation on detection electrode surface, so as to as built-in correction signal.
The oxidation peak of cyt C is in 0.2V or so, and in 0.7V or so, the oxidation peak of cyt C will not make the oxidation of IAA the oxidation peak of IAA
Into interference.Using the oxidation current of cytC as built-in correction signal, dual signal output is formed together with the signal of IAA, from
And build Ratio-type sensor.The concentration of IAA is reacted with the ratio of cyt C oxidation currents by the oxidation current of IAA, is carried significantly
The high stability of detection.
The present invention also provides a kind of miniature Ratio-type sensor, including be sealed in be connected with each other in capillary glass tube it is above-mentioned
Available for detecting the modified carbon fiber electrode and copper wire of IAA；The copper wire that certain length is exposed in described capillary glass tube one end is led
Line, the other end expose certain length (about 0.5-1mm) described modified carbon fiber and work electrode；The two of the capillary glass tube
End is sealed with insulating materials.
It is fine that miniature Ratio-type sensor of the present invention can both prepare the above-mentioned carbon modified that can be used for detection IAA in advance
Electrode is tieed up, then is assembled into miniature Ratio-type sensor on demand；After can also common carbon fibers be assembled on demand, then carbon is fine
One end of dimension is modified processing as stated above.
The modified carbon fiber electrode of detection IAA or above-mentioned miniature Ratio-type sensor can be used for exist the invention also includes above-mentioned
The application of online In vivo detection plant heteroauxin etc..
The present invention also provides a kind of methods of online In vivo detection plant heteroauxin, include the following steps：
1) it being worked electrode with the above-mentioned modified carbon fiber electrode that can be used for detection IAA, Ag/AgCl are done reference electrode,
Platinum filament is done to electrode, by electrochemical workstation, using related electrochemical method (such as pulse voltammetry, square wave voltammetry, cycle
Voltammetry etc.) a series of IAA containing various concentration of test PBS standard solution, with the electric signal of IAA and the ratio of cyt C electrical signals
The concentration that value corresponds to IAA establishes standard curve；
2) above-mentioned working electrode, Ag/AgCl reference electrodes and platinum filament are inserted into electrode plant tissue to be measured respectively；It is logical
It crosses the method identical to step 1) and carries out related electrochemical method detection, treated in measuring plants according to the acquisition of above-mentioned standard curve
The content of IAA.
Further, treat that measuring plants include crops, flowers, vegetables, forest etc..
Further, detection position includes the tissues such as root, stem, leaf, the fruit of plant.
The raw materials used in the present invention is commercially available to buy.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other each preferably to get the present invention
Example.
The key point of the present invention：
(1) present invention is the method for IAA in In vivo detection plant in situ；
(2) electrode is modified using nafion/cytC/ [Bmim] [BF4], using the oxidation current of cytC as built in
Correction signal, together with IAA signal form dual signal output, so as to build Ratio-type sensor, significantly improve electrification
Learn the reproducibility of sensor；
(3) detection target is plant hormone heteroauxin；
(4) detection target material is vegetable material, including crops, flowers, vegetables, forest etc.；
(5) detection position can be the tissues such as root, stem, leaf, the fruit of plant；
(6) detection target material can be the different times and varying environment of plant growth.
Advantageous effect of the present invention：
The present invention significantly improves the stability and reproducibility of IAA sensors, standard when being detected so as to fulfill plant living body IAA
True property and reliability.
Description of the drawings
Fig. 1 is carbon fiber electrode modification schematic diagram of the present invention.
Fig. 2 represents 30 testing results of non-Ratio-type (A) sensor and Ratio-type (B) sensor to same concentration IAA
Reproducibility compare.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..It is not specified in embodiment specific
Technology or condition person carry out according to the described technology of document in the art or condition or according to product description.It is used
Production firm person is not specified in reagent or instrument, is the conventional products that can be commercially available by regular distributor.
Embodiment 1 builds miniature Ratio-type sensor
(1) a diameter of 10 μm of carbon fiber with copper wire is connect, be sealed in the capillary glass tube drawn, then with exhausted
Edge glue and epoxy resin sealed glass tube, one end exposed portion copper wire make conducting wire, and the other end exposes about 1mm carbon fiber as electrical pole.
By the carbon fiber electrode of preparation successively in acetone, second alcohol and water ultrasound about 3 minutes, then in 0.5M sulfuric acid solutions 0-1V into
Row cyclic voltammetry scan carries out electrochemical pre-treatment, until obtaining ideal cyclic voltammogram.
(2) 5mg cyt C are added in 200ul ionic liquids [Bmim] [BF4], oscillator mixing 5min, Ran Houqu
10 μ l mixtures are placed on clean glass plate, by the carbon fiber electrode rotary dipping in the mixture of step (1) pretreatment
5min, drying at room temperature.This process is repeated 5 times.Then the carbon fiber electrode of cyt C/ [Bmim] [BF4] modifications is immersed certain
The carbon fiber electrode of nafion/cytC/ [Bmim] [BF4] modifications is made in 5min in the nafion solution (2%) of concentration.
The modification schematic diagram of carbon fiber electrode is shown in Fig. 1.
Comparative example 1 builds non-miniature Ratio-type sensor
Construction method differs only in ionic liquid [Bmim] with 1 miniature Ratio-type sensor construction method of embodiment
Cyt C are not added in [BF4], prepare the non-Ratio-type carbon fiber electrically for nafion/ [Bmim] [BF4] modifications for being not added with cyt C
Pole (the only mono signal output of IAA oxidation currents) is as control.
Experimental example 1
3 non-Ratio-type sensors built by the Ratio-type sensors and comparative example 1 of the structure of embodiment 1 are collected respectively,
30 testing results of same concentration IAA are compared, statistical result is shown in Fig. 2.The relative standard of non-Ratio-type sensor is inclined
Poor (RSD) is 16.6%, and the RSD of Ratio-type sensor is 4.3%, and reproducibility is significantly higher than non-Ratio-type sensor.In Fig. 2
A is non-Ratio-type sensor, and B is Ratio-type sensor.
2 online In vivo detection plant heteroauxin of experimental example
The IAA in carbon fiber sensor In vivo detection salt stress processing Sunflower Seedlings after being modified using embodiment 1.
1) IAA standard solution is tested：It is worked electrode with 1 modifying carbon fibers microelectrode of embodiment, Ag/AgCl are done reference
Electrode, platinum filament are done to electrode, the IAA standard solution being configured by the pulse voltammetry test of electrochemical workstation by PBS, with
The concentration of the oxidation current of IAA IAA corresponding with the ratio of cyt C oxidation currents establishes standard curve.
The concentration that control treatment corresponds to IAA with non-Ratio-type sensor prepared by comparative example 1 with the oxidation current of IAA is established
Standard curve.
2) in plant sample IAA In vivo detection：It is punched with puncture needle in axis bar portion, then respectively by work electricity
Pole, Ag/AgCl reference electrodes and platinum filament are inserted into electrode the stem of plant respectively.It is detected by pulse voltammetry, root
The content of the IAA in plant sample is acquired according to standard curve.
Specifically, using Sunflower Seedlings as object to be measured, setting control group (0mm NaCl) and 1 (50mm of processing group
) and processing group 2 (100mm NaCl) NaCl.
By Sunflower Seedlings water planting 15 days, the salt stress processing of various concentration is carried out, at salt stress after 10h, use puncture needle
Sunflower Seedlings stipe part punch, then respectively by after modification Ratio-type carbon fiber electrode, Ag/AgCl reference electrodes and
Platinum filament is inserted into electrode the stem of plant respectively.It is detected by pulse voltammetry, plant sample is acquired according to standard curve
The content of middle IAA.
Experimental result compares：
Cut microelectrode detection position portion of tissue carry out HPLC-MS detections, with sensor detection instant result into
Row comparison, as shown in table 1.The result shows that the variation tendency of the microelectrode sensing detection result is consistent with the trend of HPLC-MS, but
As a result it is more accurate, and the instant IAA concentration in plant living body has been reacted, it is as a result relatively reliable.
IAA contents and the HPLC-MS of the Sunflower Seedlings of 1 Ratio-type IAA sensor In vivo detections salt stress of table processing from
The comparison of body testing result
| Processing group | Sensor (ng/g) | HPLC-MS(ng/g) |
| 0mm | 7.76±0.37 | 7.42±1.49 |
| 50mm | 4.66±0.20 | 4.23±0.85 |
| 100mm | 3.21±0.14 | 3.02±0.58 |
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 preparation method for the modified carbon fiber electrode that can be used for detecting IAA, which is characterized in that include the following steps：
1) carbon fiber electrode is ultrasonically treated in acetone, second alcohol and water successively, then the 0-1V cyclic voltammetrics in sulfuric acid solution
Scanning carries out electrochemical pre-treatment, until obtaining ideal cyclic voltammogram；
2) with ionic liquid [Bmim] [BF4] for solvent, [Bmim] [BF4] solution of the C containing cyt is prepared；Step 1) is pre-processed
Carbon fiber the above-mentioned C containing cyt [Bmim] [BF4]) rotary dipping in solution, it is dry；Cyt C/ [Bmim] [BF4] are made
The carbon fiber of modification；
3) carbon fiber made from the step 2) is immersed in certain density nafion solution, nafion/cytC/ [Bmim] is made
The carbon fiber electrode of [BF4] modification, you can.
2. preparation method according to claim 1, which is characterized in that the step 1) carbon fiber diameter is 5-100 μm, excellent
Select 10 μm.
3. preparation method according to claim 1 or 2, which is characterized in that a concentration of 0.5- of the step 1) sulfuric acid solution
1M, preferably 0.5M.
4. preparation method according to claim 1 or 2, which is characterized in that [Bmim] of the C containing cyt described in step 2)
[BF4]) solution concentration be 1-5mg/100ul；Preferably 2.5mg/100ul；And/or
A concentration of 2-5% of step 3) the nafion solution, preferably 2%.
5. according to claim 1-4 any one of them preparation methods, which is characterized in that include the following steps：
1) carbon fiber electrode is ultrasonically treated in acetone, second alcohol and water successively, then in the sulfuric acid solution of a concentration of 0.5M-1M
Middle 0-1V cyclic voltammetry scans carry out electrochemical pre-treatment, until obtaining ideal cyclic voltammogram；
2) [Bmim] [BF4] of the C containing cyt is taken) solution is placed on clean glass plate, by step 1) pretreatment
Carbon fiber is placed in one rotary dipping for a period of time, drying at room temperature；Repeat the impregnation drying process 2-5 times；Cyt C/ are made
The carbon fiber of [Bmim] [BF4] modification；
3) carbon fiber made from step 2) is immersed to a period of time in the nafion solution of concentration 2-5%, nafion/ is made
The carbon fiber electrode of cytC/ [Bmim] [BF4] modifications, you can.
6. the modified carbon fiber electrode that can be used for detection IAA prepared by any one of claim 1-5 the methods.
7. a kind of miniature Ratio-type sensor, which is characterized in that will including being sealed in the right being connected with each other in capillary glass tube
Ask the 6 modified carbon fiber electrodes and copper wire；The copper wire that certain length is exposed in described capillary glass tube one end does conducting wire, the other end
Modified carbon fiber described in exposing certain length is worked electrode；The both ends of the capillary glass tube are sealed with insulating materials.
8. available for Ratio-type sensing miniature described in the modified carbon fiber electrode or claim 7 of detection IAA described in claim 6
Application of the device in terms of online In vivo detection plant heteroauxin.
A kind of 9. method of online In vivo detection plant heteroauxin, which is characterized in that include the following steps：
1) it is worked and electrode or is sensed with miniature Ratio-type described in claim 7 with modified carbon fiber electrode described in claim 6
The modified carbon fiber electrode of device is worked electrode, and Ag/AgCl are done reference electrode, and platinum filament is done to electrode, passes through electrochemical operation
It stands, the PBS standard solution of IAA containing various concentration a series of is tested using related electrochemical method, with the electric signal and cyt of IAA
The concentration that the ratio of C electrical signal corresponds to IAA establishes standard curve；
2) the step 1) working electrode, Ag/AgCl reference electrodes and platinum filament are inserted into electrode plant tissue to be measured respectively；
It is detected by the related electrochemical method of identical to step 1) method progress, is treated in measuring plants according to the acquisition of above-mentioned standard curve
The content of IAA.
10. according to the method described in claim 9, it is characterized in that, treat that measuring plants include crops, flowers, vegetables, forest；
And/or detection position includes root, stem, leaf, the fruit of plant.
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| CN110265231A (en) * | 2019-06-20 | 2019-09-20 | 河南大学 | A kind of supercapacitor and preparation method thereof based on carbon fiber combination electrode material |
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