CN106053570B - A kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification - Google Patents
A kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification Download PDFInfo
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Abstract
The present invention relates to a kind of microcysin LR electrochemical detection methods of graphene signal amplification, and this approach includes the following steps：(1) it is (9 11) volume ratio to be added dropwise respectively on the surface of MCH/Au NPs/Au electrodes:(4‑6):(2‑4):Endonuclease enzyme reaction buffer solution, graphene adaptor complex, microcysin LR solution to be measured and the endonuclease enzyme solutions of (1 3)；(2) MCH/Au NPs/Au electrodes are cultivated to 12 hours at 25 35 DEG C, electrode surface is gently eluted with ethyl alcohol and ultra-pure water, removal combines weaker graphene, then dries under a nitrogen, electrochemical method detection is carried out after taking-up, measures the concentration of microcysin LR solution.Highly sensitive electrochemical analysis techniques are utilized for the method that the present invention is established and the aptamer of high specific recognition ability is effectively combined, realize the detection of the hypersensitive, high specificity of Environmental Trace microcysin LR, and instrument is inexpensively portable, method is simple and practicable, can quickly obtain result.
Description
Technical field
The present invention relates to a kind of detection methods of microcapsule algae toxin, more particularly, to a kind of amplification of graphene signal
Microcapsule algae toxin electrochemical detection method.
Background technology
In recent years, environmental problem is particularly acute, and the case where blue algae bloom all occur in many waters.Microcystin
(Microcystins, MCs) is a kind of biotoxin generated in eutrophication water by cyanobacteria, it is by seven amino acid
Biologically active cyclic annular seven peptides constituted.Microcystin has numerous species, the isomery having confirmed that at present
Body is just up to more than 90 kinds.Wherein microcapsule algae toxin (Microcystin-LR, MC-LR) is one such isomers, micro-
There is a kind of the most universal and strongest Microcystin of toxicity in capsule microcystins-LR, to microcystin in water body in water
Element-LR contents, which carry out control and highly sensitive monitoring, has very important Significance for Environment.
Currently, the method for measuring microcapsule algae toxin mainly uses traditional instrument analysis method, such as high-efficient liquid phase color
Spectrometry, liquid-mass chromatography method etc., the sensitivity and accuracy that these methods have had, however analytic process is often extremely complex, takes
Power and take.In recent years, there is research worker to be acted on using the specific recognition of antibody, build the immune of microcapsule algae toxin
Sensor, realize to microcapsule algae toxin it is relatively simple, efficiently detect, and obtain higher sensitivity and selectivity.
However, the cost of microcapsule algae toxin antibody extraction is higher, process is extremely complex, and extracting cycle is longer, therefore is actually answering
It is rarely reported in.
Aptamer is a series of short chains that high specificity combination target substance is provided by SELEX technology screenings
Nucleotide sequence has the specific recognition performance similar to antibody, and have to be combined in vitro, and synthesis cycle is short, is not required to
Want complicated extraction step, and stability well equal many properties better than antibody.Due to its unique performance, it is based on distinct methods
The aptamer sensor of development is with a wide range of applications.Since the aptamer of microcapsule algae toxin is in 2004
After year is screened out, the aptamer sensor for microcapsule algae toxin measurement, which has, largely to be reported, such as fluorescence method, colorimetric
Method etc..Wherein, fluorescence aptamer sensor needs to carry out fluorophor label to aptamers to obtain response signal, therefore marks
Process is complicated, time-consuming, thereby increases and it is possible to which that the compatibility between microcapsule algae toxin and aptamers has certain influence.Colorimetric aptamers pass
Sensor, method is simple, and quickly, but often sensitivity is relatively low.And electrochemical analysis method is since it is with easy to operate, response is fast
Speed, and high sensitivity, it is environmental-friendly, easily realize the advantages that monitoring on-line, of increased attention and application.Therefore,
It is contemplated that by highly sensitive electrochemical analysis method, it is combined, constructs micro- with the aptamer technology with high-affinity
The electrochemical aptamer sensor of capsule microcystins-LR, and then establish for realizing highly sensitive, highly selective detection microcystin
Element-LR model electrochemical analysis methods.It is quantitative quick that Chinese patent 200810242879.5 discloses a kind of microcapsule algae toxin
The preparation and application of detection sensor, but the patent is to select the electrochemical sensor of biological antibody alternatively property mechanism,
Relative to aptamer, the cost of antibody culture and period are high compared with aptamer, and process is complex, is not well suited for
In actually detected middle use.
Invention content
Simple, cost is prepared it is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of
The electrochemical detection method of cheap, highly sensitive highly selective detection microcapsule algae toxin.
The purpose of the present invention can be achieved through the following technical solutions：
It is base electrode that gold nanoparticle (Au NPs), which modifies naked gold electrode (Bare Au), while by graphite outside system
Alkene and aptamers are compounded to form graphene-adaptor complex, are combined with endonuclease, constructed it is a kind of simply, quickly
Electrochemical detection method is, it can be achieved that highly sensitive and highly selective detection MC-LR.
A kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification, this approach includes the following steps：
(1) it is (9-11) volume ratio to be added dropwise successively on the surface of MCH/Au NPs/Au electrodes:(4-6):(2-4):(1-3)
Endonuclease enzyme reaction buffer solution, graphene-adaptor complex, microcapsule algae toxin solution and endonuclease to be measured
Enzyme solutions；
(2) MCH/Au NPs/Au electrodes are cultivated to 1~2 hour at 25-35 DEG C, with ethyl alcohol and ultrapure water wash electricity
Pole surface, removal combine weaker graphene, then dry under a nitrogen, and electrochemical method detection is carried out after taking-up, measure micro-
The concentration of capsule microcystins-LR solution.
The MCH/Au NPs/Au electrodes are prepared using following steps：
(1) using gold electrode as working electrode, the KCl of the 0.05-0.2mol/L containing a concentration of 1~5mmol/L gold chlorides
Solution is electrolyte, and using three-electrode system, cyclic voltammetry scanning is carried out in -1.2-1.0V potential ranges, scans the number of turns
It is enclosed for 10-25, obtains Au NPs/Au electrodes；
(2) obtained Au NPs/Au electrodes are immersed in the ethanol solution of the 6- sulfydryl -1- hexanols containing 30-60mmol/L
It seals, takes out electrode after being cultivated 24 hours under room temperature, use ethyl alcohol and water wash respectively, obtain MCH/Au NPs/Au electrodes,
Drying for standby in nitrogen.
Graphene-the adaptor complex is prepared using following steps：
(1) in the solution of the microcapsule algae toxin aptamer of 10~20 μm of ol/L, addition a concentration of 0.1~
The graphene aqueous solution of 0.3mg/mL, places it in ice-water bath, ultrasound three hours, obtains black dispersion suspension；
(2) after centrifuging obtained black dispersion suspension 5-10 minutes, supernatant is taken, it is multiple to obtain graphene-aptamers
Object is closed, is refrigerated at 4 DEG C spare.
The base sequence of the aptamer of the microcapsule algae toxin is：
5’-GGCGC-CAAAC-AGGAC-CACCA-TGACA-ATTAC-CCATA-CCACC-TCAT T-ATGCC-
CCATC-TCCGC-3’。
The endonuclease enzyme reaction buffer solution is that (50-200mM contains 12- to Tris-HCl buffer solutions reaction buffer
50mM MgCl2,0.5-2mM CaCl2, PH=7.5,25-35 DEG C).
The unit of activity of the enzyme of the endonuclease enzyme solutions is 500-1500U/mL.
The electrochemical method is differential pulse voltammetry, according between peak point current and microcapsule algae toxin concentration
Linear relationship establish working curve.
The electrolyte that the electrochemical method uses is pH=7.0, the ferrocenecarboxylic acid containing 3-10mmol/L and 0.05-
The phosphate buffer solution of the 10-30mmol/L of 0.3mol/L sodium perchlorates.
It is used for the selective enumeration method of MC-LR in the present invention, can specifically use following methods：
The chaff interferent of 100 times of MC-LR solution concentrations is added into the solution containing MC-LR, using differential pulse voltammetry
Measure the peak current response of mixed solution.It is realized to MC-LR's according to the variation that peak current caused by MC-LR and chaff interferent is added
Selectivity analysis；The chaff interferent is paraquat, Acetamiprid, metrifonate, Cupric sulfate, one kind of omethoate or glyphosate.
The present invention is using microcapsule algae toxin aptamer as recognition element, by itself and highly sensitive electricity analytical method phase
In conjunction with further by designing and using endonuclease auxiliary graphene signal amplification, realizing to Microcystin-
Highly sensitive, the specific analysis of LR.It is basal electrode to select the gold electrode of gold nanoparticle deposition modification, can utilize gold nano
Particle provides more electrochemical surface areas and better electric conductivity, to provide more sensitive electrochemical signals.It
MCH monolayers are assembled in electrode surface afterwards, to the electron transmission " channel " of " blocking " electrode surface.Graphene-is suitable simultaneously
Ligand complex is present in system, in the presence of there is no microcapsule algae toxin in system, work of the graphene due to aptamers
With will not self-assemble on electrode.But when in system there are when microcapsule algae toxin, microcapsule algae toxin meeting and aptamers
In conjunction with to make graphene expose, exposed graphene can self-assemble to Au NPs/ by the effect with MCH
Au electrode surfaces so that the current signal being " blocked " is " on " again, and obtains the increase of response current value.Further
Ground, after endonuclease DNase I are added dropwise in system, endonuclease can be by the aptamer combined with MC-LR point
Solution, makes MC-LR expose again, exposed MC-LR can continue to be combined with new aptamers again so that more
Graphene, which exposes, to be assembled on electrode, is so recycled, and realizes the enlarge-effect to current signal, to utilize the electricity of gained
The concentration relationship between signal and MC-LR is flowed, realizes and the high sensitive electrochemical of MC-LR is detected.
The present invention will be easy to operate, and respond rapid electrochemical analysis method has specific recognition to act on to MC-LR
Aptamer technology combines, and in conjunction with the characteristics of the excellent electric conductivity of graphene and bioaffinity, has invented a kind of novelty
MC-LR electrochemical detection methods.Compared with prior art, the present invention has the following advantages：
(1) compared with existing biosensor, the present invention uses gold nanoparticle deposition modified gold electrode for matrix electricity
Pole material utilizes the excellent electric conductivity of graphene and good bio-compatibility.Further by designing and utilizing endonuclease
Enzyme assists graphene signal amplification, realizes highly sensitive, the specific analysis to MC-LR.
(2) present invention is compound with graphene by the aptamer of MC-LR, obtains graphene-adaptor complex.Due to
Aptamer substantially increases selectivity so that the Electrochemical Detection side to the specificity binding ability of test substance MC-LR
Method selective in the similar interfering substance of structure of 100 times of test substance concentration can identify test substance MC-LR.
(3) compared with the method for traditional detection microcapsule algae toxin, the method for the invention established is utilized highly sensitive
Electrochemical analysis techniques and the aptamer of high specific recognition ability be effectively combined, realize Environmental Trace MC-
The detection of the hypersensitive of LR, high specificity, and instrument is inexpensively portable, method is simple and practicable, can quickly obtain result.
Description of the drawings
Fig. 1 is Au NPs nano junction compositions prepared by the present invention；
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
A kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification, wherein involved sensor
Specific manufacturing process is as follows：
(1) gold electrode is pre-processed first, processing mode is as follows：Gold electrode surfaces are placed in the anthropophagy of fresh configuration
Fish (H2SO4/H2O2=7:It 3v/v) impregnates 10-15 minutes in washing lotion, after being cleaned using ultra-pure water after taking-up, is respectively adopted successively
Grain size is 1.0 μm, 0.3 μm, 0.05 μm of alundum (Al2O3) with " 8 " word it is careful be polishing to any surface finish.Ultra-pure water is used later
The alundum (Al2O3) of electrode surface is rinsed well, electrode is respectively placed in ultra-pure water, ethyl alcohol, ultra-pure water ultrasound 5 to 10 successively
Minute.After ultrasound, electrode is taken out immediately, using it as working electrode, platinum electrode is to electrode, and SCE is reference electrode
It carries out cyclic voltammetric (CV) to scan, until obtaining the cyclic voltammogram of stable repetition, then shows that gold electrode has pre-processed
Finish.CV scan setting parameters are as follows：Potential range -0.2-1.55V sweeps fast 50mV/s.By the gold electrode pre-processed with ultrapure
Water wash is clean, takes out, is placed in the gold chloride (containing 0.1mol/L KCl) of 3mmol/L after nitrogen drying, and continuation is with it
Working electrode restores the gold in solution using CV methods, to form gold nanoparticle (Au NPs) in electrode surface, obtains gold
Nano-particle electrode.Parameter setting is as follows：Potential range -1.2~-0.2V, sweeps fast 50mV/s, and the scanning number of turns 15 is enclosed.Obtain Au
NPs electrodes, as shown in Figure 1.
(2) in the solution of the MC-LR aptamers of 10 μm of ol/L, the graphene water of a concentration of 0.15mg/mL is added
Solution places it in ice-water bath, ultrasound three hours, obtains black dispersion suspension.Later after five minutes by suspension centrifugation,
Supernatant is taken, graphene-adaptor complex is obtained.Graphene-the adaptor complex prepared is put into 4 DEG C of refrigerator cold-storages
It is spare.
(3) the Au NPs electrodes prepared in (1) step are immersed in the ethanol solution of MCH of 40mmol/L, after sealing
It is cultivated 24 hours under room temperature, Au NPs electrode surfaces is made to form fine and close MCH molecular layers.Electrode is taken out later, uses second respectively
After the elution of alcohol and water gently, MCH/Au NPs/Au electrodes are obtained, in a nitrogen atmosphere drying for standby.
Embodiment 2
Before to MC-LR measurement of concetrations, the MCH/Au NPs/Au electrodes prepared are taken out, 10 μ are added dropwise in electrode surface
L endonucleases (DNase I) reaction buffer (pH=7.0, the NaCl containing a concentration of 100mmol/L), 5 μ L graphenes-
Adaptor complex, the DNase I solution (1000U/mL) of the MC-LR of the concentration to be measured of 3 μ L, 2 μ L, 30 DEG C are placed in by electrode
It is taken out after cultivating a hour in biochemical cultivation case.Electrode surface is gently eluted with ethyl alcohol and ultra-pure water, it is weaker to remove combination
Graphene.Then electrode is dried in a nitrogen atmosphere, DPV detections are carried out after taking-up.Electrochemical Detection containing
In the phosphate buffer solution (pH=7.0) of the 20mmol/L of the sodium perchlorate of 5mmol/L ferrocenecarboxylic acids and 0.1mol/L into
Row.
The variation of electric current and the concentration of MC-LR are 1.0 × 10-12~1.0 × 10-10It is closed at good linear within the scope of mol/L
System, related coefficient is about that 0.99. lowest detections are limited to 8.0 × 10-13mol/L.This minimum detection limit is enough to detect micro- in water body
The existing MC-LR of amount.
Embodiment 3
The electrochemical aptamer sensor that will be prepared, by 1.0 × 10-11Mol/L MC-LR respectively with a concentration of 1.0 ×
10-9The chaff interferent of mol/L mixes be measured two-by-two.Omethoate, glyphosate, paraquat, Cupric sulfate, Acetamiprid, enemy are investigated
The interference experiment that hundred three kinds of worm environment common contaminants measure MC-LR.Using test condition in embodiment 2, measures electric current and ring
It answers, result of study is shown, other chaff interferents of 100 times of MC-LR concentration influence to be less than caused by the electric current of MC-LR
10.0%.As it can be seen that prepared aptamer sensor has high selectivity to MC-LR.
Embodiment 4
(1) gold electrode is pre-processed by the processing mode of embodiment 1, the gold electrode pre-processed is drenched with ultra-pure water
Wash clean takes out after nitrogen drying, is placed in the gold chloride (containing 0.1mol/L KCl) of 1mmol/L, continues using it as work
Electrode restores the gold in solution using CV methods, to form gold nanoparticle (Au NPs) in electrode surface, obtains gold nano
Granule electrode.Parameter setting is as follows：Potential range -1.2~-0.2V, sweeps fast 50mV/s, and the scanning number of turns 10 is enclosed.Obtain Au NPs
Electrode, as shown in Figure 1.
(2) in the solution of the MC-LR aptamers of 10 μm of ol/L, the graphene that a concentration of 0.1mg/mL is added is water-soluble
Liquid places it in ice-water bath, ultrasound three hours, obtains black dispersion suspension.Later after five minutes by suspension centrifugation, it takes
Supernatant obtains graphene-adaptor complex.It is standby that the graphene-adaptor complex prepared is put into 4 DEG C of refrigerator cold-storages
With.
(3) the Au NPs electrodes prepared in (1) step are immersed in the ethanol solution of MCH of 30mmol/L, after sealing
It is cultivated 24 hours under room temperature, Au NPs electrode surfaces is made to form fine and close MCH molecular layers.Electrode is taken out later, uses second respectively
After the elution of alcohol and water gently, MCH/Au NPs/Au electrodes are obtained, in a nitrogen atmosphere drying for standby.
(4) the MCH/Au NPs/Au electrodes prepared are taken out, 9 μ L endonucleases (DNase is added dropwise in electrode surface
I) reaction buffer (pH=7.0, the NaCl containing a concentration of 100mmol/L), 4 μ L graphenes-adaptor complex, 2 μ L's
The DNase I solution (1000U/mL) of the MC-LR of concentration to be measured, 1 μ L, electrode is placed in 25 DEG C of biochemical cultivation case and cultivates 1
It is taken out after hour.Electrode surface is gently eluted with ethyl alcohol and ultra-pure water, weaker graphene is combined with removal.Then electrode is existed
It is dried under nitrogen atmosphere, DPV detections is carried out after taking-up.Electrochemical Detection in ferrocenecarboxylic acid containing 5mmol/L and
It is carried out in the phosphate buffer solution (pH=7.0) of the 10-30mmol/L of the sodium perchlorate of 0.1mol/L.
The variation of electric current and the concentration of MC-LR can obtain being enough to detect water in a certain range at good linear relationship
The detection limit of micro existing MC-LR in body.
Embodiment 5
(1) gold electrode is pre-processed by the processing mode of embodiment 1, the gold electrode pre-processed is drenched with ultra-pure water
Wash clean takes out after nitrogen drying, is placed in the gold chloride (containing 0.1mol/L KCl) of 5mmol/L, continues using it as work
Electrode restores the gold in solution using CV methods, to form gold nanoparticle (Au NPs) in electrode surface, obtains gold nano
Granule electrode.Parameter setting is as follows：Potential range -1.2~-0.2V, sweeps fast 50mV/s, and the scanning number of turns 25 is enclosed.Obtain Au NPs
Electrode, as shown in Figure 1.
(2) in the solution of the MC-LR aptamers of 20 μm of ol/L, the graphene that a concentration of 0.3mg/mL is added is water-soluble
Liquid places it in ice-water bath, ultrasound three hours, obtains black dispersion suspension.Later after ten minutes by suspension centrifugation, it takes
Supernatant obtains graphene-adaptor complex.It is standby that the graphene-adaptor complex prepared is put into 4 DEG C of refrigerator cold-storages
With.
(3) the Au NPs electrodes prepared in (1) step are immersed in the ethanol solution of MCH of 30mmol/L, after sealing
It is cultivated 24 hours under room temperature, Au NPs electrode surfaces is made to form fine and close MCH molecular layers.Electrode is taken out later, uses second respectively
After the elution of alcohol and water gently, MCH/Au NPs/Au electrodes are obtained, in a nitrogen atmosphere drying for standby.
(4) the MCH/Au NPs/Au electrodes prepared are taken out, 11 μ L endonucleases (DNase is added dropwise in electrode surface
I) reaction buffer (pH=7.0, the NaCl containing a concentration of 100mmol/L), 6 μ L graphenes-adaptor complex, 4 μ L's
The DNase I solution (1000U/mL) of the MC-LR of concentration to be measured, 3 μ L, electrode is placed in 35 DEG C of biochemical cultivation case and cultivates 2
It is taken out after hour.Electrode surface is gently eluted with ethyl alcohol and ultra-pure water, weaker graphene is combined with removal.Then electrode is existed
It is dried under nitrogen atmosphere, DPV detections is carried out after taking-up.Electrochemical Detection in ferrocenecarboxylic acid containing 5mmol/L and
It is carried out in the phosphate buffer solution (pH=7.0) of the 10-30mmol/L of the sodium perchlorate of 0.1mol/L.
The variation of electric current and the concentration of MC-LR can obtain being enough to detect water in a certain range at good linear relationship
The detection limit of micro existing MC-LR in body.
Embodiment 6
(1) gold electrode is pre-processed by the processing mode of embodiment 1, the gold electrode pre-processed is drenched with ultra-pure water
Wash clean takes out after nitrogen drying, is placed in the gold chloride (containing 0.1mol/L KCl) of 3mmol/L, continues using it as work
Electrode restores the gold in solution using CV methods, to form gold nanoparticle (Au NPs) in electrode surface, obtains gold nano
Granule electrode.Parameter setting is as follows：Potential range -1.2~-0.2V, sweeps fast 50mV/s, and the scanning number of turns 20 is enclosed.Obtain Au NPs
Electrode, as shown in Figure 1.
(2) in the solution of the MC-LR aptamers of 15 μm of ol/L, the graphene that a concentration of 0.3mg/mL is added is water-soluble
Liquid places it in ice-water bath, ultrasound three hours, obtains black dispersion suspension.After suspension is centrifuged 7 minutes later, take
Supernatant obtains graphene-adaptor complex.It is standby that the graphene-adaptor complex prepared is put into 4 DEG C of refrigerator cold-storages
With.
(3) the Au NPs electrodes prepared in (1) step are immersed in the ethanol solution of MCH of 20mmol/L, after sealing
It is cultivated 24 hours under room temperature, Au NPs electrode surfaces is made to form fine and close MCH molecular layers.Electrode is taken out later, uses second respectively
After the elution of alcohol and water gently, MCH/Au NPs/Au electrodes are obtained, in a nitrogen atmosphere drying for standby.
(4) the MCH/Au NPs/Au electrodes prepared are taken out, 10 μ L endonucleases (DNase is added dropwise in electrode surface
I) reaction buffer (pH=7.0, the NaCl containing a concentration of 100mmol/L), 5 μ L graphenes-adaptor complex, 3 μ L's
The DNase I solution (1000U/mL) of the MC-LR of concentration to be measured, 2 μ L, electrode is placed in 30 DEG C of biochemical cultivation case and is cultivated
It is taken out after 1.5 hours.Electrode surface is gently eluted with ethyl alcohol and ultra-pure water, weaker graphene is combined with removal.It then will be electric
Pole is dried in a nitrogen atmosphere, and DPV detections are carried out after taking-up.Electrochemical Detection in ferrocenecarboxylic acid containing 5mmol/L and
It is carried out in the phosphate buffer solution (pH=7.0) of the 10-30mmol/L of the sodium perchlorate of 0.1mol/L.
The variation of electric current and the concentration of MC-LR can obtain being enough to detect water in a certain range at good linear relationship
The detection limit of micro existing MC-LR in body.
Above-mentioned is that this can be understood and applied for the ease of those skilled in the art to the description of embodiment
Invention.Person skilled in the art obviously easily can make various modifications to these embodiments, and illustrating herein
General Principle be applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to realities here
Example is applied, those skilled in the art's announcement according to the present invention, the improvement made for the present invention and modification all should be in the present invention
Protection domain within.
Claims (6)
1. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification, which is characterized in that this method include with
Lower step：
(1) it is (9-11) volume ratio to be added dropwise successively on the surface of MCH/Au NPs/Au electrodes:(4-6):(2-4):The core of (1-3)
Sour inscribe enzyme reaction buffer solution, graphene-adaptor complex, microcapsule algae toxin solution to be measured and endonuclease are molten
Liquid；
(2) MCH/Au NPs/Au electrodes are cultivated to 1~2 hour at 25-35 DEG C, with ethyl alcohol and ultrapure water wash electrode table
Face, removal combine weaker graphene, then dry under a nitrogen, and electrochemical method detection is carried out after taking-up, measure Microcystis aeruginosa
The concentration of toxin solution；
Graphene-the adaptor complex is prepared using following steps：
(1) in the solution of the microcapsule algae toxin aptamer of 10~20 μm of ol/L, a concentration of 0.1~0.3mg/ is added
The base sequence of the graphene aqueous solution of mL, the aptamer of the microcapsule algae toxin is：5’-GGCGC-CAAAC-
AGGAC-CACCA-TGACA-ATTAC-CCATA-CCACC-TCATT-ATGCC-CCATC-TC CGC-3 ', place it in ice-water bath
In, ultrasound three hours obtains black dispersion suspension；
(2) after centrifuging obtained black dispersion suspension 5-10 minutes, supernatant is taken, graphene-adaptor complex is obtained,
It is refrigerated at 4 DEG C spare；
The endonuclease is DNase I endonucleases.
2. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification according to claim 1, special
Sign is that the MCH/Au NPs/Au electrodes are prepared using following steps：
(1) using gold electrode as working electrode, the KCl solution of the 0.05-0.2mol/L containing a concentration of 1~5mmol/L gold chlorides
Cyclic voltammetry scanning is carried out in -1.2-1.0V potential ranges using three-electrode system for electrolyte, the scanning number of turns is 10-
25 circles, obtain Au NPs/Au electrodes；
(2) obtained Au NPs/Au electrodes are immersed in the ethanol solution of the 6- sulfydryl -1- hexanols containing 30-60mmol/L and are sealed,
Electrode is taken out after being cultivated 24 hours under room temperature, ethyl alcohol and water wash is used respectively, MCH/Au NPs/Au electrodes is obtained, in nitrogen
Middle drying for standby.
3. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification according to claim 1, special
Sign is that the endonuclease enzyme reaction buffer solution is Tris-HCl buffer solution reaction buffers, and 50-200mM contains 12-
50mM MgCl2,0.5-2mM CaCl2, pH=7.5,25-35 DEG C.
4. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification according to claim 1, special
Sign is that the unit of activity of the enzyme of the endonuclease enzyme solutions is 500-1500U/mL.
5. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification according to claim 1, special
Sign is that the electrochemical method is differential pulse voltammetry, according between peak point current and microcapsule algae toxin concentration
Linear relationship establishes working curve.
6. a kind of microcapsule algae toxin electrochemical detection method of graphene signal amplification according to claim 5, special
Sign is that the electrolyte that the electrochemical method uses is pH=7.0, the ferrocenecarboxylic acid containing 3-10mmol/L and 0.05-
The phosphate buffer solution of the 10-30mmol/L of 0.3mol/L sodium perchlorates.
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| CN112014453B (en) * | 2020-07-24 | 2023-01-31 | 南京师范大学 | Method for detecting microcystin based on annular allosteric DNA electrochemical reaction |
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