CN108535345A - A kind of nontoxic optical electro-chemistry competitive immunoassay method of the zearalenone based on peptide sensor - Google Patents
A kind of nontoxic optical electro-chemistry competitive immunoassay method of the zearalenone based on peptide sensor Download PDFInfo
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Abstract
The present invention discloses a kind of nontoxic optical electro-chemistry competitive immunoassay method of the zearalenone based on peptide sensor.The construction method of the sensing interface is and immobilization zearalenone antibody (Ab) in turn using carbon nanohorn, the gold cone of mercaptoethylmaine functionalization and polyglycine as substrate；With the nano rutile-type TiO of tyrasamine functionalization2The peptide chain for seeing crystal for immobilization particular sequence be situated between as photoelectricity probe；Since peptide chain (peptide) has the function of simulating zearalenone, zearalenone can be with the zearalenone antibody of immobilization on the photoelectricity probe competitive binding sensing interface of label.The photoelectricity probe on sensing interface is attached under the catalysis of horseradish peroxidase, it can be further combined with tyrasamine rutile TiO2It is situated between and sees crystal complex, photosignal is further amplified.The nontoxic photoelectric analysis method for zearalenone can be set up based on the phenomenon.
Description
Technical field
The invention belongs to new function materials and bio-sensing detection technique field, and in particular to one kind being based on peptide sensor
Zearalenone nontoxic optical electro-chemistry competitive immunoassay method.
Background technology
Optical electro-chemistry（PEC）Detection is using light as excitation signal, using photoelectric current as detection signal, by using not
Energy with form makes excitation and detection signal not interfere with each other as excitation signal and detection signal, thus background signal is relatively low,
It can get higher sensitivity.In the building process of optical electro-chemistry sensor, the response of the selection of light-sensitive material for signal
It is most important, in presently used material, TiO2Nano material is because of its unique photocatalytic activity, nontoxicity, excellent chemistry
And physical stability, become the ideal material of photocatalysis and optical electro-chemistry sensor.TiO2The sight crystal that is situated between is that crystal is sub- single
What first ordered arrangement was constituted, compared to traditional TiO2Monocrystalline, TiO2It is situated between and sees crystal with more excellent solar energy conversion and urge
Change performance, PEC performances can be significantly improved.However, TiO2Energy gap is larger, can only be by ultraviolet excitation, therefore in visible light
Area's photoelectric conversion efficiency is relatively low.Tyrasamine makes electronics transfer to TiO as a kind of easy substance aoxidized by autoxidation2
Surface is reacted with photohole, can promote the transmission of electronics, reduces the compound of electron-hole pair, improves photoelectric current.
Mycotoxin is the secondary metabolic product of various fungal species, the crops polluted by mycotoxin, strong to human body
Very big problem is carried out in health and economy-zone.Zearalenone (Zearalenone), also known as F-2 toxin are to be distributed most wide fungi poison
One of element, it is mainly derived from the metabolite of Gibberella zeae bacterium.Zearalenone is a kind of heat safe fungi, extensively
It is distributed in contaminated cereal and agricultural and sideline product, dairy produce, especially in corn and its fabricated product.Zearalenone has
There are Reproductive and developmental toxicity, immunotoxicity and strong Teratogenesis toxicity etc., endocrine can also be impacted, and may induce swollen
Tumor.Therefore develop a kind of nontoxic sensitive detection method of zearalenone and have become the direction that researchers are studied.Exempt from
Epidemic disease determination method has good sensitivity and specificity, is usually used in Quantitative detection toxin.The core master of this method
If antibody and corresponding antigen, combined with detection substance by specific recognition site.In the tradition of zearalenone
In detection method, antigen is mainly the standard solution of zearalenone, and this antigen can generate danger due to its toxicity to human body
Evil.Existing researcher has found that peptide chain can be used as new antigen and replace traditional antigen at present, with traditional zearalenone antigen
It compares, peptide chain has nontoxicity, and is easily combined with other peroxidase or protein.Existing experimental group successfully synthesizes can mould
The peptide chain of label and zearalenone standard solution competitive binding are fixed on electrode surface by the peptide chain of quasi- zearalenone
Antibody, pass through the variation of signal, realize to the Sensitive Detection of zearalenone.
In this experiment, carbon nanohorn is fixed on glassy carbon electrode surface as base material and utilizes its good electric conductivity,
Light induced electron can be made preferably to be transmitted to electrode surface, the introducing of gold cone equally makes electronics faster transmit, reduces electron-hole
It is compound, by modified electrode surface aggregate glycine, increasing specific surface area, more antibody can be fixed.The present invention passes through
A kind of a kind of peptide chain (peptide) that can simulate zearalenone of introducing, the corn of the peptide chain of label and various concentration is red
The mould fixed antibody of ketenes standard solution competitive binding, due to TiO2The photoelectric effect of-ta, photoelectric current are remarkably reinforced, photoelectric current
Signal and zearalenone concentration are linear in a certain range.Electrode after reaction is incubated with HRP- tyrasamine mixed solutions
It educates, measures impedance, the concentration of impedance value and zearalenone is linear in a certain range, by photoelectric current and impedance value,
The highly sensitive detection of zearalenone can be achieved, the successful structure of the sensor carries for the nontoxic detection of zearalenone
Platform is supplied.
Invention content
The object of the present invention is to provide a kind of competitions of the nontoxic optical electro-chemistry of zearalenone based on peptide sensor to exempt from
Epidemic disease analysis method.
To realize that goal of the invention, the present invention adopt the following technical scheme that：
（1）The pretreatment of GCE：GCE mechanical grindings first on the chamois leather for be covered with alumina powder polish, and table is washed away with secondary water
Face residual powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water thoroughly wash；
（2）The preparation of poly (Gly)/Au NCs/CNHs/Ab modified electrodes：The carbon nanohorn of 3 a concentration of 3mg/ml of μ L is added dropwise
Suspension is dried under clean glassy carbon electrode surface, infrared lamp, is cooled to room temperature, mercaptoethylmaine (MEA) solution and gold cone (Au
NCs) solution reaction obtains MEA/Au NCs composite solutions, and the MEA/Au NCs solution drop coatings of 4 μ L are then arrived modified
Electrode surface, Drying and cooling is to room temperature in baking oven；It is 7.0 that the electrode modified, which is immersed in the pH containing 1mM glycine,
In PBS buffer solutions, it is in -0.5-1.8V ranges in potential window, is that 0.1V/s is scanned to get to Poly to sweep speed
(Gly)/Au NCs/CNHs modified electrodes；Finally, the modified electrode obtained is soaked in the Gibberella zeae alkene of 1 mg/mL
Ketone antibody-solutions are incubated 50 min under 4 °C, then remove extra Gibberella zeae alkene using the phosphate buffer solution of pH7.0
Ketone antibody A b,Electrode immerses to 1 h of BSA of a concentration of 1.0 wt.% again, nonspecific activity site on enclosed-electrode surface,
It washes away after surface residual liquid to get to Poly (Gly)/Au NCs/CNHs/Ab modified electrodes；
（3）The preparation of Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes：The TiO of 3 mg/mL2Suspension with
The tyramine solution of 5mg/mL is according to 1:2 volume ratio mixing and absorption 30min, centrifugation, with secondary water washing, removes unadsorbed junket
Amine obtains TiO by obtained solid with secondary water-dispersed2Tyrasamine compound (RMC-ta) solution.Utilize the hydroxyl on tyrasamine
It is reacted with the carboxyl on peptide chain, the peptide chain (peptide) that will simulate zearalenone is connect with RMC-ta compounds, is formed
The peptide chain peptide@ta-RMC of label.Poly (Gly)/Au NCs/CNHs/Ab modified electrodes are immersed to the corn of various concentration
Zeranol（ZEN）Standard solution be incubated under 4 °C in the mixed solution of the labeled peptide that can simulate zearalenone
30min combines the antibody for being fixed on electrode surface using competitive reaction；Electrode surface is rinsed with the phosphate buffer solution of pH7.0
And naturally dry at ambient temperature, obtain Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes；
（4）The detection of zearalenone：It is measured using three-electrode system, with Poly (Gly)/Au NCs/CNHs/Ab/
Peptide modified electrodes are working electrode, and Ag/AgCl is reference electrode, and platinum electrode is to be worked using optical electro-chemistry electrode
Station is detected, and setting voltage is 0.1V, and switch lamp is carried out every 10s, the monochromatic light excitation light source of xenon lamp transmitting using it is preceding by
Monochromator filters；In the PBS buffer solutions of pH 6.5,1 × 10 is detected by optical electro-chemistry work station-6 ng/mL–1
A series of zearalenone standard solution of various concentrations of ng/mL is believed by recording the different electric currents generated before and after switch lamp
Number, drawing curve；Testing sample solution is detected instead of zearalenone standard solution, and the result of detection can pass through
Working curve checks in.
(5) amino acid sequence table of the above-mentioned peptide chain (peptide) that can simulate zearalenone is Asp-Ala-V
Al-Ile-Leu-Leu-Met is bought in the Hangzhou bio tech ltd Dan Gang.
Above-mentioned rutile TiO2It is situated between and sees crystal（RMC）The preparation of material：
0.5 g neopelexes（SDBS）It is dissolved in 25 mL, 2.2 mol/mL HNO3In solution, stir 15 minutes.
Then 0.5 mL isopropyl titanates are added（IV）, 48 h are stirred under 80 °C.Then, products therefrom through centrifuging, with ultra-pure water, ethyl alcohol
After washing 4-5 times, it is dried overnight under 60 °C.Above-mentioned product calcines 1h to remove remaining organic matter in 400 °C of air,
Rutile TiO is made2It is situated between and sees crystal.
The preparation of above-mentioned gold cone (Au NCs)：
Before spin coating polystyrene bead, teflon films are cut into square (1.5x1.5cm) first and with ethyl alcohol and secondary
Water rinses, and is then cleaned 3 minutes with plasma water.Draw the polystyrene bead solution of 1mL a concentration of 2.5 w/v % and by its from
The heart, the volume ratio for being then transferred to ethyl alcohol and methanol is 2:In 1 mixed solution.The table that volume ratio is 0.2 % is added in the solution
The concentration of polystyrene bead is then adjusted to about 5 w/v % by face activating agent (TX100).Then polystyrene bead is spin-coated on
On clean teflon films, and a few minutes are placed at room temperature, make solvent seasoning.Use O2Plasma etching polystyrene bead/
The surfaces teflon certain time.The gold of 50nm is coated on the surface finally by thermal evaporation, obtains golden cone.
A kind of nontoxic optical electro-chemistry of zearalenone based on peptide sensor of the present invention competes immune sensing
Device, including it is reference electrode that working electrode, platinum electrode, which are to electrode and Ag/ AgCl, which is characterized in that the work electricity
Pole uses Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes, 1 be prepared by the method for following step）
The polishing of glass-carbon electrode：Glass-carbon electrode mechanical grinding first on the chamois leather for be covered with alumina powder polishes, and is washed away with secondary water
Remained on surface powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water are thoroughly washed
It washs；2）The preparation of Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes：The carbon of 3 a concentration of 3mg/ml of μ L is added dropwise
Nanometer angle suspension dried under clean glassy carbon electrode surface, infrared lamp, be cooled to room temperature, mercaptoethylmaine (MEA) solution with
Gold cone (Au NCs) solution reaction, obtains MEA/Au NCs composite solutions, then arrives the MEA/Au NCs solution drop coatings of 4 μ L
The electrode surface of modified, Drying and cooling is to room temperature in baking oven；The electrode modified is immersed in the pH containing 1mM glycine
For in 7.0 PBS buffer solutions, be in -0.5-1.8V ranges in potential window, with sweep speed be 0.1V/s be scanned to get
To Poly (Gly)/Au NCs/CNHs modified electrodes；Finally, the modified electrode obtained is soaked in the corn of 1 mg/mL
Zeranol antibody-solutions are incubated 50 min under 4 °C, then remove extra corn using the phosphate buffer solution of pH7.0
Zeranol antibody A b。Electrode immerses to 1 h of BSA of a concentration of 1.0 wt.% again, nonspecific activity on enclosed-electrode surface
Site is washed away after surface residual liquid to get to Poly (Gly)/Au NCs/CNHs/Ab modified electrodes；The TiO of 3 mg/mL2It is outstanding
The tyramine solution of supernatant liquid and 5mg/mL are according to 1:2 volume ratio mixing and absorption 30min, centrifugation, with secondary water washing, removal is not inhaled
Attached tyrasamine obtains TiO by obtained solid with secondary water-dispersed2Tyrasamine compound (RMC-ta) solution.Using on tyrasamine
Hydroxyl reacted with the carboxyl on peptide chain, the peptide chain (peptide) of zearalenone will be simulated and connected with RMC-ta compounds
It connects, forms the peptide chain peptide@ta-RMC of label.Poly (Gly)/Au NCs/CNHs/Ab modified electrodes are immersed different dense
The zearalenone of degree（ZEN）In 4 ° in the mixed solution of standard solution and the labeled peptide that can simulate zearalenone
It is incubated 30min under C, the antibody for being fixed on electrode surface is combined using competitive reaction；Electricity is rinsed with the phosphate buffer solution of pH7.0
Pole surface and at ambient temperature naturally dry obtain Poly (Gly)/Au NCs/CNHs/Ab/ZEN modified electrodes；
It is of the present invention a kind of based on TiO2It is situated between and sees detection side of the optical electro-chemistry sensor for zearalenone of crystal
Method, which is characterized in that steps are as follows：1）It is measured using three-electrode system, with Poly (Gly)/Au NCs/CNHs/Ab/
Peptide modified electrodes are working electrode, and Ag/AgCl is reference electrode, and platinum electrode is auxiliary electrode, utilizes optical electro-chemistry work
Make station to be detected, setting voltage is 0.2V, switch lamp is carried out every 10s, before the monochromatic light excitation light source use of xenon lamp transmitting
It is filtered by monochromator；2）In the PBS buffer solutions of pH7.0,1 × 10 is detected by optical electro-chemistry work station-6 ng/
A series of zearalenone standard solution of different solubility between mL -1 ng/mL, passes through what is generated before and after record switch lamp
Different current signals, drawing curve；Testing sample solution is detected instead of zearalenone standard solution, detection
As a result it can be checked in by working curve.By the electrode after reaction, the reaction was continued with HRP- tyramine solutions, by measuring resistance, record
The variation of impedance value can equally detect the variation of zearalenone concentration.
The present invention remarkable advantage be：
(1) rutile TiO being sensitized with tyrasamine2It is situated between and sees crystal as label substance markers peptide chain, rutile TiO2It is situated between and sees crystal
With higher porosity, the introducing of butyric acid enhances the transmission speed of electronics, with individual conventional Ti O2Monocrystal material is compared,
Effectively improve the sensitivity of photoelectric conversion efficiency and sensor.The peptide chain (peptide) that ZEN will be simulated introduces sensing interface,
The antibody for being fixed on electrode surface is combined using competitive reaction, by the variation of optical signal, realizes the Sensitive Detection to object.
(2) antigen is combined to form space steric effect with antibody specificity, and sensor obtained is realized to Gibberella zeae
The super sensitivity detection of ketenes.
(3) it is used as antigen by introducing to simulate with the peptide chain of zeranol (peptide) and is at war with standard solution
Reaction, platform is provided for the nontoxic detection of zearalenone.
Description of the drawings
Fig. 1 is that the preparation process of the signal-on type optical electro-chemistry sensors of zearalenone of the present invention is shown
It is intended to.
Fig. 2 A are various concentration 1 × 10-6ng/mL–1 ng/mL（a-g）Zearalenone standard solution, sensing electrode
Photocurrent response figure.
Fig. 2 B are the linear relationship chart of the photocurrent response and zearalenone concentration of standard solution of sensing electrode.
Fig. 3 A are the impedance diagram after the electrode after immune response is incubated with HRP- tyramine solutions.
Fig. 3 B are impedance value and relational graph the phenomenon that zearalenone concentration of standard solution.
Specific implementation mode
The present invention is further illustrated the present invention with the following example, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1
A kind of nontoxic optical electro-chemistry competitive immunoassay method (as shown in Figure 1) of the zearalenone based on peptide sensor：
（1）The pretreatment of glass-carbon electrode：Glass-carbon electrode mechanical grinding first on the chamois leather for be covered with alumina powder polishes, with two
Remained on surface powder is removed in secondary washing, then moves into ultrasonic water bath and clean, until clean up, finally sequentially uses ethyl alcohol, diluted acid and
Water thoroughly washs；
（2）A concentration of 3 mg/mL CNHs suspension of 3 μ L is added dropwise to dry under clean glassy carbon electrode surface, infrared lamp, it is cooling
To room temperature；
（3）Mercaptoethylmaine (MEA) solution and gold cone (Au NCs) solution reaction, obtain MEA/Au NCs composite solutions, then by 3 μ L
Au NCs/MEA composite solutions drop coatings are placed in baking oven and dry, then cool down at room temperature to the electrode surface modified；
（4）The electrode modified is immersed in the PBS buffer solutions that the pH containing 1mM glycine is 7.0, in potential window
In -0.5-1.8V ranges, to be scanned to get electric to Poly (Gly)/Au NCs/CNHs modifications for 0.1V/s with sweeping speed
Pole；
（5）Modified electrode is incubated 1h under 4 °C in zearalenone antibody A b (2 mg/mL) solution, then uses pH
7.0 phosphate buffer solution washes away extra Ab, then electrode is immersed to 1 h of BSA of a concentration of 1.0 wt.%, enclosed-electrode table
Nonspecific activity site on face；After washing away surface residual liquid, electrode surface is rinsed simultaneously with the pH phosphate buffer solutions for being 7.0
Poly (Gly)/Au NCs/CNHs/Ab modified electrodes are made in naturally dry at ambient temperature；
（6）The preparation of Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes：The TiO of 3 mg/mL2Suspension with
The tyramine solution of 5mg/mL is according to 1:2 volume ratio mixing and absorption 30min, centrifugation, with secondary water washing, removes unadsorbed junket
Amine obtains TiO by obtained solid with secondary water-dispersed2The compound of tyrasamine（RMC-ta）Solution.Utilize the hydroxyl on tyrasamine
Base is reacted with the carboxyl on peptide chain, and the peptide chain (peptide) that will simulate zearalenone is connect with RMC-ta compounds, shape
At the peptide chain peptide@ta-RMC of label.Poly (Gly)/Au NCs/CNHs/Ab modified electrodes are immersed to the jade of various concentration
Zearlenone（ZEN）In the mixed solution of standard solution and the labeled peptide chain that can simulate zearalenone under 4 °C
It is incubated 30min, the antibody for being fixed on electrode surface is combined using competitive reaction；Electrode is rinsed with the phosphate buffer solution of pH7.0
Surface and at ambient temperature naturally dry obtain Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes.
Embodiment 2
A kind of nontoxic optical electro-chemistry competitive immunoassay method of the zearalenone based on peptide sensor, steps are as follows：
（1）It is measured using three-electrode system, with Poly (Gly)/Au NCs/CNHs/Ab/peptide made from embodiment 1
Modified electrode is working electrode, and Ag/AgCl is reference electrode, and platinum electrode is to be carried out using optical electro-chemistry work station to electrode
Detection, setting voltage are 0.1V, carry out switch lamp every 10s, the monochromatic light excitation light source that xenon lamp emits is using preceding by monochromator
Filtering；
（2）In the PBS buffer solutions of pH 7.0,1 × 10 is detected by optical electro-chemistry work station-6 ng/mL–1 ng/
A series of zearalenone standard solution of various concentrations of mL, by recording the different current signals generated before and after switch lamp,
Drawing curve.Fig. 2 A are various concentration 1 × 10-6ng/mL–1 ng/mL（a-g）Zearalenone standard solution passes
The photocurrent response of sense electrode.Fig. 2 B are the line of the photocurrent response and zearalenone concentration of standard solution of sensing electrode
Sexual intercourse figure.
（3）Testing sample solution is detected instead of zearalenone standard solution, the result of detection can pass through work
It is checked in as curve.
Containing 5 mM Fe (CN)6 3-/4-0.1 M KCl solution in, a series of concentration are carried out by electrochemical workstation
The impedance detection of standard solution, drawing curve.Fig. 3 A are concentration 1 × 10-6Within the scope of ng/mL -0.1 ng/mL
The impedance value that zearalenone standard solution generates.Fig. 3 B are the linear relationship of sensing electrode impedance response and normal concentration.
Claims (4)
1. a kind of nontoxic optical electro-chemistry competitive immunoassay method of zearalenone based on peptide sensor, feature exist
In including the following steps：
（1）Glass-carbon electrode（GCE）Pretreatment：GCE mechanical grindings first on the chamois leather for be covered with alumina powder polish, with two
Remained on surface powder is removed in secondary washing, then moves into ultrasonic water bath and clean, until clean up, finally sequentially uses ethyl alcohol, diluted acid and
Water thoroughly washs；
（2）The preparation of Poly (Gly)/Au NCs/CNHs/Ab modified electrodes：The carbon nanohorn of 3 a concentration of 3mg/ml of μ L is added dropwise
Suspension is dried under clean glassy carbon electrode surface, infrared lamp, is cooled to room temperature, and mercaptoethylmaine (MEA) solution and gold are bored
(Au NCs) solution reaction, obtains MEA/Au NCs composite solutions, then arrives the MEA/Au NCs composite solution drop coatings of 4 μ L
The electrode surface of modified, Drying and cooling is to room temperature in baking oven；The electrode modified is immersed in the pH containing 1mM glycine
It is to be scanned, obtained for 0.1V/s with sweeping speed in -0.5-1.8V ranges in potential window in 7.0 PBS buffer solutions
Poly (Gly)/Au NCs/CNHs modified electrodes；Finally, the corn that the modified electrode obtained is soaked in 1 mg/mL is red
Mould ketenes antibody-solutions, are incubated 50 min under 4 °C, and it is red then to remove extra corn using the phosphate buffer solution of pH7.0
Mould ketenes antibody A b,Electrode immerses to 1 h of BSA of a concentration of 1.0 wt.% again, nonspecific activity position on enclosed-electrode surface
Point washes away after surface residual liquid to get to Poly (Gly)/Au/CNHs/Ab modified electrodes；
（3）The preparation of Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes：The TiO of 3 mg/mL2Suspension with
The tyramine solution of 5mg/mL is according to 1:2 volume ratio mixing and absorption 30min, centrifugation, with secondary water washing, removes unadsorbed junket
Amine obtains TiO by obtained solid with secondary water-dispersed2Tyrasamine compound (RMC-ta) solution；Utilize the hydroxyl on tyrasamine
It is reacted with the carboxyl on peptide chain, the peptide chain (peptide) that will simulate zearalenone is connect with RMC-ta compounds, is formed
The peptide chain peptide@ta-RMC of label；Poly (Gly)/Au NCs/CNHs/Ab modified electrodes are immersed to the corn of various concentration
Zeranol（ZEN）In the mixed solution of standard solution and the labeled peptide chain (peptide) that can simulate zearalenone
It is incubated 30min under 4 °C, the antibody for being fixed on electrode surface is combined using competitive reaction；It is rushed with the phosphate buffer solution of pH7.0
Electrode surface and at ambient temperature naturally dry are washed, Poly (Gly)/Au NCs/CNHs/Ab/peptide modification electricity is obtained
Pole；
（4）The detection of zearalenone：It is measured using three-electrode system, with Poly (Gly)/Au NCs/CNHs/Ab/
Peptide modified electrodes are working electrode, and Ag/AgCl is reference electrode, and platinum electrode is auxiliary electrode, utilizes optical electro-chemistry work
Make station to be detected, setting voltage is 0.2V, switch lamp is carried out every 10s, before the monochromatic light excitation light source use of xenon lamp transmitting
It is filtered by monochromator；In the PBS buffer solutions of pH 7.0,1 × 10 is detected by optical electro-chemistry work station-6 ng/mL–
A series of zearalenone standard solution of various concentrations between 1 ng/mL, by being generated not before and after record switch lamp
Same current signal, drawing curve；Testing sample solution is detected instead of zearalenone standard solution, the knot of detection
Fruit is checked in by working curve.
2. according to the method described in claim 1, it is characterized in that, the rutile TiO2It is situated between and sees crystal（RTM）Material by
Prepared by following methods：0.5 g neopelexes（SDBS）It is dissolved in 25 mL, 2.2 mol/mL HNO3In solution,
Stirring 15 minutes；Then 0.5mL isopropyl titanates are added（IV）, 48 h are stirred under 80 °C；Then, products therefrom is through centrifuging, using
After ultra-pure water, ethyl alcohol wash 4-5 times, it is dried overnight under 60 °C；It is residual to remove that above-mentioned product calcines 1h in 400 °C of air
Rutile TiO is made in the organic matter stayed2It is situated between and sees crystal（RTM）.
3. a kind of nontoxic optical electro-chemistry of zearalenone based on peptide sensor competes immunosensor, including work electricity
It is reference electrode that pole, platinum electrode, which are to electrode and Ag/ AgCl, which is characterized in that the working electrode uses Poly
(Gly)/Au/CNHs/Ab/peptide modified electrodes, the Poly (Gly)/Au/CNHs/Ab/peptide modified electrodes by
What following methods were prepared：1）The polishing of glass-carbon electrode：Glass-carbon electrode is mechanical first on the chamois leather for be covered with alumina powder
Sanding and polishing washes away remained on surface powder with secondary water, then moves into ultrasonic water bath and clean, until cleaning up, sequentially finally
With ethyl alcohol, diluted acid and water thoroughly wash；2）The preparation of Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes：It is added dropwise
The carbon nanohorn suspension of 3 a concentration of 3mg/ml of μ L is dried under clean glassy carbon electrode surface, infrared lamp, is cooled to room temperature,
Mercaptoethylmaine (MEA) solution and gold cone (Au NCs) solution reaction, obtain MEA/Au NCs composite solutions, then by 4 μ L's
MEA/Au NCs solution drop coatings are to the electrode surface of modified, and Drying and cooling is to room temperature in baking oven；The electrode modified is soaked
It steeps in the PBS buffer solutions that the pH containing 1mM glycine is 7.0, is in -0.5-1.8V ranges, to sweep speed in potential window
It is scanned to get to Poly (Gly)/Au NCs/CNHs modified electrodes for 0.1V/s；Finally, the modified electrode that will be obtained
The zearalenone antibody-solutions of 1 mg/mL are soaked in, 50 min are incubated under 4 °C, it is then slow using the phosphoric acid of pH7.0
It rushes solution and removes extra zearalenone antibody A b,Electrode is immersed to 1 h of BSA of a concentration of 1.0 wt.%, closing electricity again
Nonspecific activity site in pole surface is modified after washing away surface residual liquid to get to Poly (Gly)/Au NCs/CNHs/Ab
Electrode；The TiO of 3 mg/mL2The tyramine solution of suspension and 5mg/mL are according to 1:2 volume ratio mixing and absorption 30min, centrifugation,
With secondary water washing, unadsorbed tyrasamine is removed, by obtained solid with secondary water-dispersed, obtains TiO2Tyrasamine compound
(RMC-ta) solution；It is reacted with the carboxyl on peptide chain using the hydroxyl on tyrasamine, the peptide chain of zearalenone will be simulated
(peptide) it is connect with RMC-ta compounds, the peptide chain peptide@ta-RMC of label is formed, by Poly (Gly)/Au NCs/
CNHs/Ab modified electrodes immerse the zearalenone of various concentration（ZEN）Standard solution with labeled to simulate corn red
It is incubated 30min under 4 °C in the mixed solution of the peptide chain (peptide) of mould ketenes, is combined using competitive reaction and is fixed on electrode
The antibody on surface；Electrode surface and at ambient temperature naturally dry are rinsed with the phosphate buffer solution of pH7.0, obtains Poly
(Gly)/Au NCs/CNHs/Ab/peptide modified electrodes.
4. a kind of nontoxic optical electro-chemistry of zearalenone based on peptide sensor described in claim 3 competes immune sensing
Device is used for the detection method of zearalenone, which is characterized in that steps are as follows：1）It is measured using three-electrode system, with
Poly (Gly)/Au NCs/CNHs/Ab/peptide modified electrodes are working electrode, and Ag/AgCl is reference electrode, platinum electrode
It for auxiliary electrode, is detected using optical electro-chemistry work station, setting voltage is 0.2V, and switch lamp, xenon lamp hair are carried out every 10s
The monochromatic light excitation light source penetrated is filtered using preceding by monochromator；2）In the PBS buffer solutions of pH7.0, pass through optical electro-chemistry work
Make station and is detected 1 × 10-6A series of zearalenone standard solution of different solubility between ng/mL -1 ng/mL, leads to
The front and back different current signals generated of overwriting switch lamp, drawing curve；Testing sample solution replaces zearalenone
Standard solution is detected, and the result of detection can be checked in by working curve.By electrode and the HRP- tyramine solutions after reaction after
Continuous reaction records the variation of impedance value, can equally detect the variation of zearalenone concentration by measuring resistance.
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