CN105717180A - Preparation method and application of photoelectrochemical aflatoxin biosensor based on two-dimensional composite nanomaterial - Google Patents
Preparation method and application of photoelectrochemical aflatoxin biosensor based on two-dimensional composite nanomaterial Download PDFInfo
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Abstract
The invention discloses a preparation method of a photoelectrochemical aflatoxin biosensor and belongs to the technical field of novel nano functional materials and biosensors. A novel two-dimensional composite nanomaterial Mn-TiO2/g-C3N4 is prepared firstly; through good bio-compatibility and the large specific area of the material, an aflatoxin antibody is loaded, then alkaline phosphatase is fixed through the interlinking effect of glutaraldehyde, in the detection process, due to the fact that alkaline phosphatase can catalyze L-ascorbic acid-2-trisodium phosphate salt AAP to generate L-ascorbic acid AA in situ, then an electron donor is provided for photoelectric detection, through the influences of specific quantitative combination of the antibody and an antigen on electron transmission capacity, the intensity of photoelectric currents is correspondingly reduced, and finally the photoelectrochemical biosensor which is low in cost, high in sensitivity, good in specificity, rapid in detection and easy to prepare and detects aflatoxin is prepared.
Description
Technical field
The present invention relates to the preparation method of a kind of Optical Electro-Chemistry aflatoxin biosensor.Belong to Nano-function thin films and biosensor technology field.
Background technology
Aflatoxin is the compound that a class chemical constitution is similar, is the derivant of dihydrofuran coumarin.Aflatoxin is present in soil, animals and plants, various nut, the most easily pollutes the grain oil products such as Semen arachidis hypogaeae, Semen Maydis, rice, Semen sojae atricolor, Semen Tritici aestivi, is a mycotoxicosis class mycotoxin maximum, that extremely highlight human health risk.Aflatoxin mainly has Aflatoxins M1, AFB1, aflatoxin B 2, aflatoxin G 1, AFG 2 etc. several.It is 1 class carcinogen that aflatoxin delimited by World Health Organization (WHO), and toxicity big 68 times than arsenicum is only second to meat poisoning mycin, is currently known mycotoxicosis the strongest.The hazardness of aflatoxin is that people and animal livers tissue are had destruction, may result in hepatocarcinoma the most dead time serious, and Homemade fermented food also can detect aflatoxin, and grain and oil and the goods kind recall rate in the most hot and humid area are higher.
At present, the method for detection aflatoxin mainly has chromatography, mass spectrography etc..This type of method instrument is valuable, operation complexity, and laboratory personnel just can detect after needing professional training.Therefore, R&D costs are low, it is fast, highly sensitive to detect, the aflatoxin sensor of high specificity is significant.
Optical Electro-Chemistry sensor, due to the feature such as highly sensitive, testing cost is low, was paid close attention to by increasing researcher in recent years.Optical Electro-Chemistry sensor is to cause electron-hole pair to separate based on additional light source activation Electrophotosensitivmaterial material, under potential condition the most partially, it is achieved the electronics quick transmission on electrode, quasiconductor and trim and analyte, and forms photoelectric current.In optimal conditions, the change of analyte concentration can directly affect the size of photoelectric current, and recycling biological immune combines, it is possible to realize the qualitative and quantitative analysis to analyte according to the change of photoelectric current.
Optical Electro-Chemistry sensor most critical technology is exactly the raising of the performances such as the size to photoelectric current and stability.Titanium dioxide is a kind of photocatalyst and light induced electron host material being most widely used, owing to sheet-like titanium dioxide nanomaterial can expose more high miller index surface, there is higher photocatalytic activity, titanium dioxide nanoplate has ratio nanoparticle preferably application prospect, and the research for titanium dioxide nanoplate also receives much concern.And the photo-generate electron-hole of single titanium dioxide nano material is to the most compound, thus cause weakening of photosignal, and the sensitivity that titanium dioxide poorly conductive also limit the Optical Electro-Chemistry sensor built by single titanium dioxide nano material is the highest, is unfavorable for actual application.But, semiconductor nano material modified or be combined special nano material, the valid density of photo-generated carrier pair can be effectively improved, improving photoelectric transformation efficiency, and be greatly improved detection sensitivity.Therefore, design, prepare efficient, stable titanium dioxide nanoplate and trim is the key technology preparing Optical Electro-Chemistry sensor.
Summary of the invention
It is an object of the invention to provide a kind of prepare simple, highly sensitive, detection quickly, the preparation method of the Optical Electro-Chemistry aflatoxin biosensor of high specificity, prepared sensor, can be used for quick, the Sensitive Detection of aflatoxin.Based on this purpose, a kind of New Two Dimensional nano composite material, i.e. additive Mn titanium dioxide nanoplate In-situ reaction carbonitride two-dimensional nano composite Mn-TiO are the method comprises the steps of firstly, preparing2/g-C3N4, utilize the good biocompatibility of this material and big specific surface area, aflatoxin antibody in load, then by the crosslinked action fixed base acid phosphatase of glutaraldehyde, when detecting, L-AA AA is produced in situ owing to alkali phosphatase can be catalyzed L-AA-2-tricresyl phosphate sodium salt AAP, and and then for Photoelectric Detection provide electron donor, recycling antibody is combined the impact on electron transport ability with the specific quantification of antigen, photo-current intensity is reduced accordingly, finally achieve the structure of the biosensor using unmarked PhotoelectrochemicalMethod Method detection aflatoxin.
The technical solution used in the present invention is as follows:
1. a preparation method for Optical Electro-Chemistry aflatoxin biosensor based on two-dimensional nano composite, described two-dimensional nano composite is additive Mn titanium dioxide nanoplate In-situ reaction carbonitride two-dimensional nano composite Mn-TiO2/g-C3N4, described Optical Electro-Chemistry aflatoxin biosensor is by working electrode, Mn-TiO2/g-C3N4, aflatoxin antibody, bovine serum albumin, glutaraldehyde, alkali phosphatase composition；
It is characterized in that, described preparation method includes following preparation process:
a. Mn-TiO2/g-C3N4Preparation；
B. the preparation of Optical Electro-Chemistry aflatoxin biosensor；
Wherein, step a prepares Mn-TiO2/g-C3N4Concretely comprise the following steps:
First, take 0.8 ~ 1.2 mmol manganese salt and join in 5 mL butyl titanates, in whipping process, be slowly added to 0.5 ~ 0.8
ML Fluohydric acid., reacts at 160 ~ 200 DEG C 18 ~ 24 hours, after being cooled to room temperature in a kettle., after ultra-pure water and dehydrated alcohol centrifuge washing three times, is vacuum dried at 50 DEG C；Secondly, 150 ~ 250 are taken
The dried solid of mg mixes with 400mg tripolycyanamide, and grind into powder；Then, putting in Muffle furnace by the powder of grinding, programming rate is 1 ~ 3 DEG C/min, calcines 0.5 ~ 5 hour at 480 ~ 560 DEG C；Finally, the powder after calcining is cooled to room temperature, i.e. prepares Mn-TiO2/g-C3N4；
Described manganese salt is selected from one of following: manganese sulfate, manganese chloride, manganese nitrate；
Step b prepares concretely comprising the following steps of Optical Electro-Chemistry aflatoxin biosensor:
(1) with ITO electro-conductive glass as working electrode, at the Mn-TiO of electrode surface drop coating 8 ~ 12 uL2/g-C3N4Colloidal sol, dries under room temperature；
(2) the electrode buffer solution PBS that will obtain in step (1), continues the aflatoxin antibody-solutions at electrode surface drop coating 8 ~ 12 L 10 g/mL, preserves and dry in 4 DEG C of refrigerators；
(3) the electrode PBS that will obtain in step (2), continues at the bovine serum albumin solution that electrode surface drop coating 8 ~ 12 L concentration is 100 g/mL, preserves and dry in 4 DEG C of refrigerators；
(4) the electrode PBS that will obtain in step (3), continues the glutaraldehyde solution at electrode surface drop coating 2 ~ 4 L, preserves and dry in 4 DEG C of refrigerators；
(5) the electrode PBS that will obtain in step (4), continues at the alkaline phosphatase enzymatic solution that electrode surface drop coating 6 ~ 10 L concentration is 20 g/mL, preserves and dry in 4 DEG C of refrigerators；
(6) the electrode PBS that will obtain in step (5), preserves in 4 DEG C of refrigerators after drying, i.e. prepares Optical Electro-Chemistry aflatoxin biosensor；
Described Mn-TiO2/g-C3N4Colloidal sol is by the Mn-TiO of 50 mg2/g-C3N4Powder is dissolved in 10 mL ultra-pure waters, and the hydrosol prepared after ultrasonic 30 min；
Described PBS is the phosphate buffered solution of 10mmol/L, and the pH value of described phosphate buffered solution is 7.4；
Described glutaraldehyde solution be volume ratio be the glutaraldehyde water solution of 2.5%.
The preparation method of Optical Electro-Chemistry aflatoxin biosensor based on two-dimensional nano composite the most of the present invention, it is characterised in that described aflatoxin is selected from one of following: Aflatoxins M1, AFB1, aflatoxin B 2, aflatoxin G 1, AFG 2.
The application of the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method the most of the present invention, it is characterised in that include following applying step:
A. standard solution preparation: prepare the aflatoxin standard solution of one group of variable concentrations including blank standard specimen；
B. working electrode is modified: be working electrode by the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, by the drop coating respectively of the aflatoxin standard solution of the variable concentrations of preparation in step b to working electrode surface, 4 DEG C of refrigerators preserve；
C. working curve is drawn: saturated calomel electrode, be connected on Optical Electro-Chemistry detection equipment as reference electrode, platinum electrode as auxiliary electrode, the working electrode composition three-electrode system modified with step b；Successively add Tris HCl buffer solution and the L-AA-2-tricresyl phosphate sodium salt AAP solution of 5 mL 10 mmol/L of 15mL pH=9.6 in a cell；Use i-t means of testing, according to the relation between photocurrent values and the aflatoxin concentration of standard solution of gained, drawing curve；
D. the detection of aflatoxin: replace the aflatoxin standard solution in step a with testing sample, detect according to the method in step b and c, according to intensity level and the working curve of response signal, obtains the content of aflatoxin in testing sample.
The useful achievement of the present invention
(1) Optical Electro-Chemistry aflatoxin biosensor of the present invention preparation is simple, easy to operate, it is achieved that the selective enumeration method quick, sensitive, high to sample, and low cost, can be applicable to portable inspectiont, has market development prospect；
(2) present invention is prepared for novel light-sensitive material Mn-TiO first2/g-C3N4Fully contact with titanium dioxide nanoplate due to manganese growth in situ on titanium dioxide nanoplate, utilize the metal surface plasma body effect of manganese, effectively prevent photo-generate electron-hole to being combined, although it is good to solve titanium dioxide nanoplate photocatalysis effect, but photoelectric current produces less technical problem；Simultaneously because carbonitride g-C3N4Good electric conductivity and titanium dioxide nanoplate thereon fully dispersed, greatly increase the photocatalytic activity of titanium dioxide nanoplate and solve titanium dioxide nanoplate poorly conductive and be unfavorable for preparing the technical problem of Optical Electro-Chemistry sensor, therefore, effective preparation of this material, has important scientific meaning and using value；
(3) present invention is first by Mn-TiO2/g-C3N4It is applied in the preparation of Photoelectrochemistrbiosensor biosensor, significantly improves the valid density of photo-generated carrier, substantially increase the detection sensitivity of Optical Electro-Chemistry sensor so that Photoelectrochemistrbiosensor biosensor achieves the application in real work；The application of this material, is also associated biomolecule sensor, as Electrochemiluminescsensor sensor, electrochemical sensor etc. provide Technical Reference, has the most potential use value.
Detailed description of the invention
Embodiment 1 Mn-TiO2/g-C3N4Preparation
First, take 0.8 mmol manganese salt and join in 5 mL butyl titanates, in whipping process, it is slowly added to 0.5 mL Fluohydric acid., reacts in a kettle. at 160 DEG C 24 hours, after being cooled to room temperature, after ultra-pure water and dehydrated alcohol centrifuge washing three times, it is vacuum dried at 50 DEG C；Secondly, the 150 dried solids of mg and 400 are taken
Mg tripolycyanamide mixes, and grind into powder；Then, putting in Muffle furnace by the powder of grinding, programming rate is 1 DEG C/min, calcines 5 hours at 480 DEG C；Finally, the powder after calcining is cooled to room temperature, i.e. prepares Mn-TiO2/g-C3N4；
Described manganese salt is manganese sulfate.
Embodiment 2 Mn-TiO2/g-C3N4Preparation
First, take 1.0 mmol manganese salt and join in 5 mL butyl titanates, in whipping process, it is slowly added to 0.6 mL Fluohydric acid., reacts in a kettle. at 180 DEG C 21 hours, after being cooled to room temperature, after ultra-pure water and dehydrated alcohol centrifuge washing three times, it is vacuum dried at 50 DEG C；Secondly, the 200 dried solids of mg and 400 are taken
Mg tripolycyanamide mixes, and grind into powder；Then, putting in Muffle furnace by the powder of grinding, programming rate is 2 DEG C/min, calcines 2 hours at 520 DEG C；Finally, the powder after calcining is cooled to room temperature, i.e. prepares Mn-TiO2/g-C3N4；
Described manganese salt is manganese chloride.
Embodiment 3 Mn-TiO2/g-C3N4Preparation
First, take 1.2 mmol manganese salt and join in 5 mL butyl titanates, in whipping process, it is slowly added to 0.8 mL Fluohydric acid., reacts in a kettle. at 200 DEG C 18 hours, after being cooled to room temperature, after ultra-pure water and dehydrated alcohol centrifuge washing three times, it is vacuum dried at 50 DEG C；Secondly, the 250 dried solids of mg and 400 are taken
Mg tripolycyanamide mixes, and grind into powder；Then, putting in Muffle furnace by the powder of grinding, programming rate is 3 DEG C/min, calcines 0.5 hour at 560 DEG C；Finally, the powder after calcining is cooled to room temperature, i.e. prepares Mn-TiO2/g-C3N4；
Described manganese salt is manganese nitrate.
Embodiment 4 The preparation method of Optical Electro-Chemistry aflatoxin biosensor
(1) using a width of 1 cm, a length of 4 cm ITO electro-conductive glass as working electrode, at the Mn-TiO of electrode surface drop coating 8 L2/g-C3N4Colloidal sol, dries under room temperature；
(2) the electrode buffer solution PBS that will obtain in step (1), continues the aflatoxin antibody-solutions at electrode surface drop coating 8 L 10 g/mL, preserves and dry in 4 DEG C of refrigerators；
(3) the electrode PBS that will obtain in step (2), continues at the bovine serum albumin solution that electrode surface drop coating 8 L concentration is 100 g/mL, preserves and dry in 4 DEG C of refrigerators；
(4) the electrode PBS that will obtain in step (3), continues the glutaraldehyde solution at electrode surface drop coating 2 L, preserves and dry in 4 DEG C of refrigerators；
(5) the electrode PBS that will obtain in step (4), continues at the alkaline phosphatase enzymatic solution that electrode surface drop coating 6 L concentration is 20 g/mL, preserves and dry in 4 DEG C of refrigerators；
(6) the electrode PBS that will obtain in step (5), preserves in 4 DEG C of refrigerators after drying, i.e. prepares Optical Electro-Chemistry aflatoxin biosensor；
Described Mn-TiO2/g-C3N4Colloidal sol is by the Mn-TiO of 50 mg2/g-C3N4Powder is dissolved in 10 mL ultra-pure waters, and the hydrosol prepared after ultrasonic 30 min；
Described PBS is the phosphate buffered solution of 10mmol/L, and the pH value of described phosphate buffered solution is 7.4；
Described glutaraldehyde solution be volume ratio be the glutaraldehyde water solution of 2.5%.
Embodiment 5 The preparation method of Optical Electro-Chemistry aflatoxin biosensor
(1) using a width of 1 cm, a length of 4 cm ITO electro-conductive glass as working electrode, at the Mn-TiO of electrode surface drop coating 10 L2/g-C3N4Colloidal sol, dries under room temperature；
(2) the electrode buffer solution PBS that will obtain in step (1), continues the aflatoxin antibody-solutions at electrode surface drop coating 10 L 10 g/mL, preserves and dry in 4 DEG C of refrigerators；
(3) the electrode PBS that will obtain in step (2), continues at the bovine serum albumin solution that electrode surface drop coating 10 L concentration is 100 g/mL, preserves and dry in 4 DEG C of refrigerators；
(4) the electrode PBS that will obtain in step (3), continues the glutaraldehyde solution at electrode surface drop coating 3 L, preserves and dry in 4 DEG C of refrigerators；
(5) the electrode PBS that will obtain in step (4), continues at the alkaline phosphatase enzymatic solution that electrode surface drop coating 8 L concentration is 20 g/mL, preserves and dry in 4 DEG C of refrigerators；
(6) the electrode PBS that will obtain in step (5), preserves in 4 DEG C of refrigerators after drying, i.e. prepares Optical Electro-Chemistry aflatoxin biosensor；
Described Mn-TiO2/g-C3N4Colloidal sol is by the Mn-TiO of 50 mg2/g-C3N4Powder is dissolved in 10 mL ultra-pure waters, and the hydrosol prepared after ultrasonic 30 min；
Described PBS is 10
The phosphate buffered solution of mmol/L, the pH value of described phosphate buffered solution is 7.4；
Described glutaraldehyde solution be volume ratio be the glutaraldehyde water solution of 2.5%.
Embodiment 6 The preparation method of Optical Electro-Chemistry aflatoxin biosensor
(1) using a width of 1 cm, a length of 4 cm ITO electro-conductive glass as working electrode, at the Mn-TiO of electrode surface drop coating 12 L2/g-C3N4Colloidal sol, dries under room temperature；
(2) the electrode buffer solution PBS that will obtain in step (1), continues the aflatoxin antibody-solutions at electrode surface drop coating 12 L 10 g/mL, preserves and dry in 4 DEG C of refrigerators；
(3) the electrode PBS that will obtain in step (2), continues at the bovine serum albumin solution that electrode surface drop coating 12 L concentration is 100 g/mL, preserves and dry in 4 DEG C of refrigerators；
(4) the electrode PBS that will obtain in step (3), continues the glutaraldehyde solution at electrode surface drop coating 4 L, preserves and dry in 4 DEG C of refrigerators；
(5) the electrode PBS that will obtain in step (4), continues at the alkaline phosphatase enzymatic solution that electrode surface drop coating 10 L concentration is 20 g/mL, preserves and dry in 4 DEG C of refrigerators；
(6) the electrode PBS that will obtain in step (5), preserves in 4 DEG C of refrigerators after drying, i.e. prepares Optical Electro-Chemistry aflatoxin biosensor；
Described Mn-TiO2/g-C3N4Colloidal sol is by the Mn-TiO of 50 mg2/g-C3N4Powder is dissolved in 10 mL ultra-pure waters, and the hydrosol prepared after ultrasonic 30 min；
Described PBS is 10
The phosphate buffered solution of mmol/L, the pH value of described phosphate buffered solution is 7.4；
Described glutaraldehyde solution be volume ratio be the glutaraldehyde water solution of 2.5%.
Embodiment 7 The Optical Electro-Chemistry aflatoxin biosensor of embodiment 1 and 3 preparation, is applied to the detection of aflatoxin, and step is as follows:
(1) standard solution preparation: prepare the aflatoxin standard solution of one group of variable concentrations including blank standard specimen；
(2) working electrode is modified: be working electrode by the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, by the drop coating respectively of the aflatoxin standard solution of the variable concentrations of preparation in step (1) to working electrode surface, 4 DEG C of refrigerators preserve；
(3) working curve is drawn: saturated calomel electrode, be connected on Optical Electro-Chemistry detection equipment as reference electrode, platinum electrode as auxiliary electrode, the working electrode composition three-electrode system modified with step (2)；Successively add Tris HCl buffer solution and the L-AA-2-tricresyl phosphate sodium salt AAP solution of 5 mL 10 mmol/L of 15mL pH=9.6 in a cell；Use i-t means of testing, according to the relation between photocurrent values and the aflatoxin concentration of standard solution of gained, drawing curve；The linear detection range of aflatoxin is: 0.002 ~ 200
Ng/mL, detection is limited to: 0.8 pg/mL；
(4) actual sample detection: replace the aflatoxin standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), intensity level according to response signal and working curve, obtain the content of aflatoxin in testing sample；
Described aflatoxin is Aflatoxins M1.
Embodiment 8 The Optical Electro-Chemistry aflatoxin biosensor of embodiment 2 and 4 preparation, is applied to the detection of aflatoxin, and step is as follows:
(1) standard solution preparation: prepare the aflatoxin standard solution of one group of variable concentrations including blank standard specimen；
(2) working electrode is modified: be working electrode by the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, by the drop coating respectively of the aflatoxin standard solution of the variable concentrations of preparation in step (1) to working electrode surface, 4 DEG C of refrigerators preserve；
(3) working curve is drawn: saturated calomel electrode, be connected on Optical Electro-Chemistry detection equipment as reference electrode, platinum electrode as auxiliary electrode, the working electrode composition three-electrode system modified with step (2)；Successively add Tris HCl buffer solution and the L-AA-2-tricresyl phosphate sodium salt AAP solution of 5 mL 10 mmol/L of 15mL pH=9.6 in a cell；Use i-t means of testing, according to the relation between photocurrent values and the aflatoxin concentration of standard solution of gained, drawing curve；The linear detection range of aflatoxin is: 0.002 ~ 200
Ng/mL, detection is limited to: 0.8 pg/mL；
(4) actual sample detection: replace the aflatoxin standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), intensity level according to response signal and working curve, obtain the content of aflatoxin in testing sample；
Described aflatoxin is AFB1.
Embodiment 9 The Optical Electro-Chemistry aflatoxin biosensor of embodiment 3 and 6 preparation, is applied to the detection of aflatoxin, and step is as follows:
(1) standard solution preparation: prepare the aflatoxin standard solution of one group of variable concentrations including blank standard specimen；
(2) working electrode is modified: be working electrode by the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, by the drop coating respectively of the aflatoxin standard solution of the variable concentrations of preparation in step (1) to working electrode surface, 4 DEG C of refrigerators preserve；
(3) working curve is drawn: saturated calomel electrode, be connected on Optical Electro-Chemistry detection equipment as reference electrode, platinum electrode as auxiliary electrode, the working electrode composition three-electrode system modified with step (2)；Successively add Tris HCl buffer solution and the L-AA-2-tricresyl phosphate sodium salt AAP solution of 5 mL 10 mmol/L of 15mL pH=9.6 in a cell；Use i-t means of testing, according to the relation between photocurrent values and the aflatoxin concentration of standard solution of gained, drawing curve；The linear detection range of aflatoxin is: 0.002 ~ 200
Ng/mL, detection is limited to: 0.8 pg/mL；
(4) actual sample detection: replace the aflatoxin standard solution in step (1) with testing sample, detect according to the method in step (2) and (3), intensity level according to response signal and working curve, obtain the content of aflatoxin in testing sample；
Described aflatoxin is aflatoxin G 1.
Claims (3)
1. a preparation method for Optical Electro-Chemistry aflatoxin biosensor based on two-dimensional nano composite, described two-dimensional nano composite is additive Mn titanium dioxide nanoplate In-situ reaction carbonitride two-dimensional nano composite Mn-TiO2/g-C3N4, described Optical Electro-Chemistry aflatoxin biosensor is by working electrode, Mn-TiO2/g-C3N4, aflatoxin antibody, bovine serum albumin, glutaraldehyde, alkali phosphatase composition；
It is characterized in that, described preparation method includes following preparation process:
a. Mn-TiO2/g-C3N4Preparation；
B. the preparation of Optical Electro-Chemistry aflatoxin biosensor；
Wherein, step a prepares Mn-TiO2/g-C3N4Concretely comprise the following steps:
First, take 0.8 ~ 1.2 mmol manganese salt and join in 5 mL butyl titanates, in whipping process, it is slowly added to 0.5 ~ 0.8 mL Fluohydric acid., reacts in a kettle. at 160 ~ 200 DEG C 18 ~ 24 hours, after being cooled to room temperature, after ultra-pure water and dehydrated alcohol centrifuge washing three times, it is vacuum dried at 50 DEG C；Secondly, 150 ~ 250 are taken
The dried solid of mg mixes with 400mg tripolycyanamide, and grind into powder；Then, putting in Muffle furnace by the powder of grinding, programming rate is 1 ~ 3
DEG C/min, calcine 0.5 ~ 5 hour at 480 ~ 560 DEG C；Finally, the powder after calcining is cooled to room temperature, i.e. prepares Mn-TiO2/g-C3N4；
Described manganese salt is selected from one of following: manganese sulfate, manganese chloride, manganese nitrate；
Step b prepares concretely comprising the following steps of Optical Electro-Chemistry aflatoxin biosensor:
(1) with ITO electro-conductive glass as working electrode, at the Mn-TiO of electrode surface drop coating 8 ~ 12 L2/g-C3N4Colloidal sol, dries under room temperature；
(2) the electrode buffer solution PBS that will obtain in step (1), continues in electrode surface drop coating 8 ~ 12
The aflatoxin antibody-solutions of L 10 g/mL, preserves in 4 DEG C of refrigerators and dries；
(3) the electrode PBS that will obtain in step (2), continues in electrode surface drop coating 8 ~ 12
L concentration is the bovine serum albumin solution of 100 g/mL, preserves and dry in 4 DEG C of refrigerators；
(4) the electrode PBS that will obtain in step (3), continues in electrode surface drop coating 2 ~ 4
The glutaraldehyde solution of L, preserves in 4 DEG C of refrigerators and dries；
(5) the electrode PBS that will obtain in step (4), continues in electrode surface drop coating 6 ~ 10
L concentration is the alkaline phosphatase enzymatic solution of 20 g/mL, preserves and dry in 4 DEG C of refrigerators；
(6) the electrode PBS that will obtain in step (5), preserves in 4 DEG C of refrigerators after drying, i.e. prepares Optical Electro-Chemistry aflatoxin biosensor；
Described Mn-TiO2/g-C3N4Colloidal sol is by the Mn-TiO of 50 mg2/g-C3N4Powder is dissolved in 10 mL ultra-pure waters, and the hydrosol prepared after ultrasonic 30 min；
Described PBS is the phosphate buffered solution of 10mmol/L, and the pH value of described phosphate buffered solution is 7.4；
Described glutaraldehyde solution be volume ratio be the glutaraldehyde water solution of 2.5%.
2. the preparation method of Optical Electro-Chemistry aflatoxin biosensor based on two-dimensional nano composite as claimed in claim 1, it is characterised in that described aflatoxin is selected from one of following: Aflatoxins M1, AFB1, aflatoxin B 2, aflatoxin G 1, AFG 2.
3. the application of Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, it is characterised in that include following applying step:
A. standard solution preparation: prepare the aflatoxin standard solution of one group of variable concentrations including blank standard specimen；
B. working electrode is modified: be working electrode by the Optical Electro-Chemistry aflatoxin biosensor prepared by preparation method as claimed in claim 1, by the drop coating respectively of the aflatoxin standard solution of the variable concentrations of preparation in step a to working electrode surface, 4 DEG C of refrigerators preserve；
C. working curve is drawn: saturated calomel electrode, be connected on Optical Electro-Chemistry detection equipment as reference electrode, platinum electrode as auxiliary electrode, the working electrode composition three-electrode system modified with step b；Successively add Tris HCl buffer solution and the L-AA-2-tricresyl phosphate sodium salt AAP solution of 5 mL 10 mmol/L of 15mL pH=9.6 in a cell；Use i-t means of testing, according to the relation between photocurrent values and the aflatoxin concentration of standard solution of gained, drawing curve；
D. the detection of aflatoxin: replace the aflatoxin standard solution in step a with testing sample, detect according to the method in step b and c, according to intensity level and the working curve of response signal, obtains the content of aflatoxin in testing sample.
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