CN108254426A - It is prepared for dopamine concentration detection miniature electrochemical in animal brain - Google Patents
It is prepared for dopamine concentration detection miniature electrochemical in animal brain Download PDFInfo
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- CN108254426A CN108254426A CN201711349284.5A CN201711349284A CN108254426A CN 108254426 A CN108254426 A CN 108254426A CN 201711349284 A CN201711349284 A CN 201711349284A CN 108254426 A CN108254426 A CN 108254426A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a kind of preparation methods of miniature dopamine concentration detection electrochemical sensor for being detected in animal brain, using silicon substrate microelectrode array as electrode to be finished, one layer of porous golden Pt nanoparticle is deposited in the electrode site of silicon substrate microelectrode array first with electrodeposition process and chemical etching method, then electropolymerization is recycled to be modified with one layer of perfluorinated sulfonic acid (Nafion) film of deposition in the electrode site of golden Pt nanoparticle above-mentioned, so as to obtain miniature dopamine miniature electrochemical.This being prepared is used for striate nucleus group in the miniature dopamine concentration detected in animal brain detection electrochemical sensor implantation animal brain, and it is connected as working electrode with electrochemical workstation, detection to dopamine concentration in animal brain can be realized by detecting dopamine electrochemical signals, and preparation process is simple, high sensitivity, selectivity are strong, small.
Description
Technical field
The invention belongs to the electrochemical analysis detection technique fields of neurotransmitter dopamine, more particularly to a kind of to be used for animal
Intracerebral dopamine concentration detects the preparation method of miniature electrochemical.
Background technology
Dopamine in nervous system, cardiovascular system and is immunized as important neurotransmitter a kind of in mammal
There is important role in system；In addition, dopamine is as a kind of Catecholamines Neurotransmitters in Blood, mood, spirit with animal,
Body movement, adjusting muscular tone and Endocrine Activity have close contact.And in nervous system dopamine concentration exception
Variation may result in the nervous system diseases such as Parkinson disease, schizophrenia, depression and epilepsy.Therefore, it is neural
The accurate detection of dopamine concentration is of great significance for the study of incident mechanism and prophylactic treatment of above-mentioned disease in system.
The detection method of dopamine mainly has ultraviolet-visible spectrophotometry, fluorescence analysis, high performance liquid chromatography, height
Capillary electrophoresis is imitated, it is expensive although these above-mentioned methods have the advantages that detectable limit low etc., its device complexity,
The shortcomings of sample preparation steps are cumbersome, and temporal resolution is low and is unable to real-time online detection, makes their applications in practice
It is subject to certain restrictions.Compared to the above, electrochemical methods are with price is cheap, high sensitivity, stability are good, the time
High resolution and the advantages that be suitable for on-line real-time measuremen, becomes the hot spot of academia's research.
Chinese patent application (publication number：CN105572200 one kind) is disclosed to detect under the conditions of existing for ascorbic acid
Modification glassy carbon electrode of dopamine and preparation method thereof modifies vitreous carbon with polymeric ionic liquid/polypyrrole/graphene oxide
Electrode can be realized in the presence of ascorbic acid to the detection of dopamine in phosphate buffer in vitro.Chinese patent application is (open
Number：CN106198665 a kind of electrochemical sensor quantitatively detected for levodopa amine and preparation method thereof) is disclosed, is used
NiFe2O4/ reduced graphene modified electrodes realize the quantitative detection of levodopa amine in phosphate buffer in vitro.It is Chinese special
Profit application (publication number：CN106442689) disclose a kind of preparation of the Dopamine Sensor based on nickel oxide-carbon nanotube with
Using, it can be achieved that the dopamine in phosphate buffer detects in vitro.Chinese patent application (publication number：CN102636549 it is) public
The electrochemical sensor and its assay method of dopamine in a kind of human serum of micella identification have been opened, can have been realized in human body blood
The detection of dopamine concentration in final proof product.Several dopamine electrochemical sensors disclosed in above-mentioned patent, are mainly used for more
The isolated measuring of bar amine, is not carried out the detection of dopamine concentration in animal brain.This is mainly due to made in above-mentioned patent
Standby sensor bulk is big, sensitivity is more low.
Following element is needed to have for detecting electrochemical sensor in animal brain：(1) volume of sensor is sufficiently small.
Since live body intracerebral to be implanted into is detected, the volume of sensor cannot be excessive, otherwise brain tissue can be caused excessive
Damage.In general, the size prepared in the electrode of body electrochemical sensor is a μm rank；(2) good sensor selection
Property and sensitivity.It can be by selecting suitable material modified electrode, to enhance the main interference that sensor excludes dopamine detection
The influence of object (such as ascorbic acid), and the sensitivity of sensor can be improved.There are two types of monometallics for porous gold Pt nanoparticle tool
The synergistic effect of nano particle compared with corresponding monometallic nano particle (gold or Pt nanoparticle), has the surface of bigger
Product, faster electron transport rate, numerous advantages such as stronger mechanical stability and higher electro catalytic activity.In addition, perfluor
Sulfonic acid (Nafion) film is widely used amberplex in Dopamine Sensor, can effectively exclude dopamine inspection
The main chaff interferent surveyed, such as the influence of ascorbic acid.It is film modified using porous golden Pt nanoparticle and perfluorinated sulfonic acid (Nafion)
Silicon substrate microelectrode array is expected to prepare the miniature electrochemical that can be used for dopamine concentration detection in animal brain.And it is current,
In the electrochemical analysis detection technique or patent document of the existing dopamine of data-searching carried out according to applicant, do not have still
It prepares and can be used in animal brain using porous golden Pt nanoparticle and the film modified silicon substrate microelectrode array of perfluorinated sulfonic acid (Nafion)
The report of the miniature electrochemical of dopamine concentration detection.
Invention content
Existing defects or deficiency for the above-mentioned prior art, the object of the present invention is to provide one kind in animal brain
Dopamine concentration detects the preparation method of miniature electrochemical, and this method prepares simple, high sensitivity, selective strong, body
Product is small, is suitable for the quick detection of dopamine concentration in animal brain.
In order to realize above-mentioned task, the present invention uses following technical solution：
A kind of preparation method for dopamine concentration detection miniature electrochemical in animal brain, which is characterized in that
It prepares as follows：
Step 1, silicon substrate microelectrode array is immersed in gold chloride, chloroplatinic acid and copper sulphate mixed solution；Using three electricity
Polar body system, the electrode site above silicon substrate microelectrode array to be finished are working electrode, and platinum electrode is to electrode, Ag/
Deposition voltage of the AgCl electrodes for reference electrode, then application -0.2V, sedimentation time 500-600s, so as to obtain golden platinoid
The silicon substrate microelectrode array of three metal alloy nanoparticles modification；Then above-mentioned silicon substrate microelectrode array is immersed copper etching liquid
In, the copper component in alloy nanoparticle is fallen with selective etch, the silicon substrate for finally obtaining porous golden Pt nanoparticle modification is micro-
Electrod-array；
Step 2, the above-mentioned silicon substrate microelectrode array being prepared is immersed in as working electrode in perfluorinated sulfonic acid solution,
The deposition voltage of application+0.5-0.7V, sedimentation time 80-120s so that perfluorinated sulfonic acid electropolymerization to silicon substrate microelectrode array
In corresponding electrode site；After deposition is completed, silicon substrate microelectrode array is toasted in insulating box so that electrode site
The perfluorinated sulfonic acid of upper electropolymerization forms firm film, finally obtains and detects miniature electrochemistry for dopamine concentration in animal brain
Sensor.
According to the present invention, the silicon substrate microelectrode array described in step 1 is 300-500 μm wide, 100-200 μm thick, long 8-10mm,
The electrode site on microelectrode array surface is 20-100 μm of round gold of diameter or platinum site；
Further, the gold chloride described in step 1, chloroplatinic acid and copper sulphate mixed solution use the sulfuric acid of 0.5mol/L
Solution is formulated, and the total concentration of gold chloride, chloroplatinic acid and copper-bath is 1mmol/L, gold chloride, chloroplatinic acid and copper sulphate
Concentration ratio be 1：1：1；The copper etching liquid is 60% salpeter solution, and etch period is 1 hour.
Preferably, the perfluorinated sulfonic acid solution described in step 2 is the perfluor sulphur of a concentration of 5%-10% being configured with alcohol
Acid solution.
Insulating box described in described in step 2, temperature setting are 150 DEG C -200 DEG C, baking time 5min-10min.
It is walked using prepared by the above method for dopamine concentration detection miniature electrochemical, preparation in animal brain
Rapid simple, high sensitivity, selectivity are strong, small, to study such as Parkinson disease, schizophrenia, depression and epilepsy
Etc. the pathogenesis of the nervous system diseases provide a kind of strong tool.
Description of the drawings
Fig. 1 is the structure diagram of silicon substrate microelectrode array used in embodiment 1；
Fig. 2 is the dopamine oxidation current stepped curve that embodiment 2 obtains；
Fig. 3 is the working curve that the dopamine concentration that embodiment 2 obtains detects miniature electrochemical；
Fig. 4 is the anti-interference object test curve that the dopamine concentration that embodiment 3 obtains detects miniature electrochemical；
Fig. 5 is in the thorn of stimulus intensity 300 μ A and 600 μ A (frequency 130Hz, 100 μ s of pulsewidth stimulate 5s) in embodiment 4
Collected dopamine oxidation peak current under swashing, arrow represents that stimulation starts in figure.
The present invention is described in further detail with reference to the accompanying drawings and examples.
Specific embodiment
Embodiment 1：For the preparation of dopamine concentration detection miniature electrochemical in animal brain
Step 1：The structure diagram of silicon substrate microelectrode array used in this example is as shown in Figure 1, including pad area and not
With size electrodes site area, each electrode in different size electrodes sites area is connected to each connection on pad area by contact conductor
On point, the total length of silicon substrate microelectrode array for 15mm (i.e. from the front tip in different size electrodes sites area to pad area distally
The total length at edge is 15mm).
Silicon substrate microelectrode array is 300 μm -500 μm wide, 100-200 μm thick, long 8-9mm, silicon substrate microelectrode array surface
Electrode site is the round carat (measure of the purity of gold) points of 50 μm of diameter, by the use of round carat (measure of the purity of gold) point as electrode site to be finished.By silicon substrate microelectrode battle array
Row are immersed in gold chloride, chloroplatinic acid and copper sulphate mixed solution (gold chloride, chloroplatinic acid and the use of copper sulphate mixed solution
The sulfuric acid solution of 0.5mol/L is formulated, and the total concentration of gold chloride, chloroplatinic acid and copper-bath is 1mmol/L, gold chloride,
The concentration ratio of chloroplatinic acid and copper sulphate is 1：1：1)；Using three-electrode system, 50 μ above silicon substrate microelectrode array to be finished
M circles carat (measure of the purity of gold) point is working electrode, platinum electrode is to electrode, and Ag/AgCl electrodes are reference electrode, then application -0.2V
Deposition voltage, deposition optimal time are 600s, so as to obtain the silicon substrate microelectrode of golden three metal alloy nanoparticle of platinoid modification
Array；Then above-mentioned silicon substrate microelectrode array is immersed in 60% salpeter solution 1 hour, alloy nano is fallen with selective etch
Copper component in particle finally obtains the silicon substrate microelectrode array of porous golden Pt nanoparticle modification.
Step 2：50 μm of electrode sites that the above-mentioned golden Pt nanoparticle being prepared is modified are immersed as working electrode
To the mass concentration being configured with alcohol in 5% perfluorinated sulfonic acid (Nafion) solution, the deposition voltage of application+0.5V deposits
100s so that on perfluorinated sulfonic acid (Nafion) electropolymerization to the corresponding electrode site of silicon substrate microelectrode array；After deposition is completed,
Silicon substrate microelectrode array baking 5min in insulating box (200 DEG C of set temperature) so that the perfluor sulphur of electropolymerization in electrode site
Sour (Nafion) forms firm film, finally obtains and detects miniature electrochemical for dopamine concentration in animal brain.
Embodiment 2：It is drawn for the working curve of dopamine miniature electrochemical in animal brain
Step 1：Dopamine concentration prepared by embodiment 1 is detected into miniature electrochemical as working electrode, platinum
Silk electrode is to electrode, and Ag/AgCl electrodes are reference electrode, are put into the electrolytic cell containing 10mL pH=7.4 phosphate buffers
In, three-electrode system is formed, and be connected on electrochemical workstation；
Step 2：Using constant-voltage power supply voltammetry, the constant voltage of application+0.3V between working electrode and reference electrode, so
0.05 μM, 0.1 μM, 0.2 μM, 0.7 μM, 1 μM, 2 μM, 2 μM, 2 μM, 2 μ are added dropwise successively in the case of electrolytic cell uniform stirring afterwards
M, 2 μM of dopamine standard solution obtains dopamine oxidation current stepped curve as shown in Figure 2.
According to the relationship between obtained dopamine oxidation current value and the dopamine concentration of standard solution added in, draw
This is used for the linear work curve (as shown in Figure 3) of dopamine microsensor in animal brain.
The relationship of dopamine oxidation current and dopamine concentration is as follows：
Ipa(pA)=(73.4 ± 3.1) * CDA(μM)。
Embodiment 3：For the anti-interference object test of dopamine concentration detection miniature electrochemical in animal brain
Step 1：Using prepared by embodiment 1 for dopamine concentration detection miniature electrochemical in animal brain as
Working electrode, platinum electrode are to electrode, and Ag/AgCl electrodes are reference electrode, are put into containing 10mL pH=7.4 phosphoric acid buffers
In the electrolytic cell of liquid, three-electrode system is formed, and be connected on electrochemical workstation；
Step 2：Using constant-voltage power supply voltammetry, the constant voltage of application+0.3V between working electrode and reference electrode, so
1 μM of dopamine, 12 μM of dihydroxyphenyl acetic acids, 12 μM of A ascorbic acid, 0.5 μ is added dropwise successively in the case of electrolytic cell uniform stirring afterwards
M dopamines, 1 μM of dopamine solution, wherein dihydroxyphenyl acetic acid and ascorbic acid are the main chaff interferents of dopamine intracerebral detection, are obtained
To oxidation current stepped curve as shown in Figure 3.It can be seen from the figure that when adding in 1 μM of dopamine solution, dopamine concentration
The miniature electrochemical of detection generates a larger oxidation current, when adding in 12 μM of dihydroxyphenyl acetic acid, oxidation electricity
Stream has almost no change, and illustrates obtained to detect miniature electrochemical to dihydroxyphenyl for dopamine concentration in animal brain
Acetic acid has good inhibiting effect.And when adding in 12 μM of ascorbic acid, oxidation current produces small change, increases
About 3pA, according to the definition of selection ratio, (Selectivity ratio are defined as aoxidizing caused by the substance of comparable sodium
The ratio between size of current), the selection ratio that dopamine Ascorbic Acid is calculated is 290 ± 14 (n=5), although this explanation institute
What is prepared cannot completely inhibit the oxidation of ascorbic acid for dopamine concentration detection miniature electrochemical in animal brain, but
It is the selection ratio that can obtain a more satisfied dopamine Ascorbic Acid.And 0.5 μM then added in and 1 μM more
Bar amine aqueous solution all produces corresponding larger oxidation current, this, which demonstrates added in chaff interferent dopamine will not be detected, produces
Raw apparent interference.
Embodiment 4：It is carried out in rat brain for the miniature electrochemical that dopamine concentration in animal brain detects more
The application of bar amine concentration detection
Step 1：Miniature electrochemical is detected for dopamine concentration in animal brain using prepared by embodiment 1, and
The working curve for the miniature electrochemical of dopamine concentration detection in animal brain is drawn according to embodiment 2；
Step 2：Healthy SD rat is taken, intraperitoneal injection Nembutal sodium solution is anaesthetized, and rat solid is fixed on head
Dopamine concentration detection miniature electrochemical is implanted to rats with left corpus straitum brain core by position indicator as working electrode
Group, and platinum filament is positioned over rat brain offside to electrode and Ag/AgCl reference electrodes and is contacted with cortex；A diameter of 127 μm of tungsten
Silk stimulating electrode implantation left side median forebrain bundle, carries out electro photoluminescence, stimulation parameter is as follows to it：Stimulate 100 μ s of pulsewidth, frequency of stimulation
130Hz, stimulation time 5s, 300 μ A of stimulus intensity and 600 μ A (as shown in Figure 5).Using constant-voltage power supply voltammetry, it is used at this
Dopamine concentration detects the constant voltage of application+0.3V between miniature electrochemical and reference electrode in animal brain, it is possible to
Detect the Dopamine In Striatum concentration variable signal caused by the electro photoluminescence of median forebrain bundle core group, the results are shown in Figure 4.From figure
In it can be seen that, it is collected under the stimulation of stimulus intensity 300 μ A and 600 μ A (frequency 130Hz, 100 μ s of pulsewidth, stimulate 5s)
Dopamine oxidation peak current is 41pA and 200pA, and the working sensor curve drawn using embodiment 2 can calculate to obtain dopamine
Concentration.
Claims (6)
1. a kind of preparation method for dopamine concentration detection miniature electrochemical in animal brain, which is characterized in that press
It is prepared by following steps：
Step 1, silicon substrate microelectrode array is immersed in gold chloride, chloroplatinic acid and copper sulphate mixed solution；Using three electrode bodies
It is that the electrode site above silicon substrate microelectrode array to be finished is working electrode, platinum electrode is to electrode, Ag/AgCl electricity
The deposition voltage of extremely reference electrode, then application -0.2V, sedimentation time 500-600s, so as to obtain golden three metal of platinoid
The silicon substrate microelectrode array of alloy nanoparticle modification；Then above-mentioned silicon substrate microelectrode array is immersed in copper etching liquid, with choosing
Selecting property etches away the copper component in alloy nanoparticle, finally obtains the silicon substrate microelectrode battle array of porous golden Pt nanoparticle modification
Row；
Step 2, the above-mentioned silicon substrate microelectrode array being prepared is immersed in as working electrode in perfluorinated sulfonic acid solution, applied
The deposition voltage of+0.5-0.7V, sedimentation time 80-120s so that perfluorinated sulfonic acid electropolymerization is corresponding to silicon substrate microelectrode array
Electrode site on；After deposition is completed, silicon substrate microelectrode array is toasted in insulating box so that electrode site powers on
The perfluorinated sulfonic acid of polymerization forms firm film, finally obtains and detects miniature electrochemical sensing for dopamine concentration in animal brain
Device.
2. the method as described in claim 1, which is characterized in that the silicon substrate microelectrode array described in step 1 is 300-500 μm wide,
100-200 μm thick, long 8-10mm, the electrode site on microelectrode array surface is 20-100 μm of round gold of diameter or platinum site.
3. the method as described in claim 1, which is characterized in that gold chloride, chloroplatinic acid and copper sulphate mixing described in step 1
Solution is formulated using the sulfuric acid solution of 0.5mol/L, and the total concentration of gold chloride, chloroplatinic acid and copper-bath is 1mmol/
L, the concentration ratio of gold chloride, chloroplatinic acid and copper sulphate is 1：1：1；The copper etching liquid is 60% salpeter solution, during etching
Between be 1 hour.
4. the method as described in claim 1, which is characterized in that the perfluorinated sulfonic acid solution described in step 2 is is configured with alcohol
A concentration of 5%-10% perfluorinated sulfonic acid solution.
5. the method as described in claim 1, which is characterized in that the insulating box described in described in step 2, temperature setting are
150 DEG C -200 DEG C, baking time 5min-10min.
6. prepared by one of them the method for claim 1 to 5 detects miniature electrochemistry for dopamine concentration in animal brain
Sensor.
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Cited By (4)
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| CN110940712A (en) * | 2019-11-14 | 2020-03-31 | 杭州电子科技大学 | Preparation method of miniature electrochemical sensor for detecting dopamine |
| CN111380932A (en) * | 2018-12-29 | 2020-07-07 | 华东师范大学 | Ratio type ion selective microelectrode array and preparation method and application thereof |
| CN111850657A (en) * | 2020-07-30 | 2020-10-30 | 苏州科技大学 | Preparation method and application of polypyrrole/phytic acid/graphene oxide composite anti-corrosion coating |
| CN113325059A (en) * | 2021-05-26 | 2021-08-31 | 辽宁师范大学 | Electrochemical detection device for measuring dopamine by virtue of micro-volume selectivity |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111380932A (en) * | 2018-12-29 | 2020-07-07 | 华东师范大学 | Ratio type ion selective microelectrode array and preparation method and application thereof |
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| CN111850657A (en) * | 2020-07-30 | 2020-10-30 | 苏州科技大学 | Preparation method and application of polypyrrole/phytic acid/graphene oxide composite anti-corrosion coating |
| CN111850657B (en) * | 2020-07-30 | 2022-10-18 | 苏州科技大学 | Preparation method and application of polypyrrole/phytic acid/graphene oxide composite anti-corrosion coating |
| CN113325059A (en) * | 2021-05-26 | 2021-08-31 | 辽宁师范大学 | Electrochemical detection device for measuring dopamine by virtue of micro-volume selectivity |
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