CN106198657B - A kind of preparation method of the parathion sensor based on two-dimension nano materials building - Google Patents

Abstract

The invention discloses a kind of preparation methods of electrogenerated chemiluminescence parathion sensor.Belong to Nano-function thin films and biosensor technology field.The method comprises the steps of firstly, preparing a kind of New Two Dimensional nanocomposites --- titanium dioxide/molybdenum disulfide composite material, i.e. the two-dimensional nano composite material Co-TiO of cobalt doped nano titania square In-situ reaction molybdenum disulfide₂/MoS₂, good biocompatibility and big specific surface area using the material, parathion antibody in load, when being detected, since cobalt doped titanium dioxide can be with catalyzing hydrogen peroxide in-situ preparation O₂, and with the K in the liquid of bottom₂S₂O₈It is electrochemically reacted, generate electrochemiluminescence signal, recycle antibody in conjunction with the specific quantification of antigen to the influence of electron transport ability, so that current strength reduces, to reduce luminous intensity, the building of the Electrochemiluminescsensor sensor using unmarked electrogenerated chemiluminescence method detection parathion is finally realized.

Description

A kind of preparation method of the parathion sensor based on two-dimension nano materials building

Technical field

The present invention relates to a kind of preparation methods of electrogenerated chemiluminescence parathion sensor.Belong to Nano-function thin films With biosensor technology field.

Background technique

Parathion is a kind of broad spectrum activity high poison insecticide, have tag, stomach toxicity, fumigation action, and plant can be infiltrated through It is interior.Parathion acts on quickly insect, can be used for preventing and treating the fruit tree pest insects such as cotton, apple, citrus, pears, peach and wheat red mite etc.. In the soil, parathion can slightly be migrated to deep subsoil by the eluviation of water.Parathion in soil can pass through plant Object root absorption and enter plant in.After people eat this kind of plant or plant containing its residue by mistake, parathion can pass through Alimentary canal, respiratory tract and complete skin and mucous membrane enter human body, it may appear that Nausea and vomiting has a headache, havies loose bowels, whole body weakness nothing The preliminary symptom of the poisonings such as power, long-term consumption or excessive eat will lead to canceration.

Currently, the method for detection parathion mainly has chromatography, mass spectrography etc..Such method instrument is valuable, complicated for operation, Laboratory personnel just can be carried out detection after needing professional training.Therefore, research and development are at low cost, detection is fast, high sensitivity, high specificity Parathion sensor is of great significance.

Electrochemiluminescsensor sensor due to its high sensitivity, specificity it is good, easy to operate the advantages that be widely used in The fields such as clinical diagnosis, Pharmaceutical Analysis, environmental monitoring.The superior Electrochemiluminescsensor sensor of processability, most critical skill Art is exactly the raising of the performances such as effective fixation and the reproducibility of luminous intensity and stability and immune molecule.Titanium dioxide is application A kind of most commonly used photocatalyst material, since sheet-like titanium dioxide nanomaterial can expose more high miller index surfaces, With higher photocatalytic activity, titanium dioxide nanoplate has application prospect preferably than nanoparticle, for titanium dioxide The research of nanometer sheet is also concerned.Meanwhile titanium dioxide poorly conductive is also limited by single titanium dioxide nano material structure The sensitivity for the Electrochemiluminescsensor sensor built is not generally high, is unfavorable for practical application.But on semiconductor nano material Modification or compound special nano material can effectively improve the Resonance energy transfer of semiconductor surface, generate stronger shine Intensity, and greatly improve detection sensitivity.Therefore, designing, preparing efficient, stable titanium dioxide nanoplate and its modifier is Prepare the key technology of Electrochemiluminescsensor sensor.

Molybdenum disulfide (chemical formula MoS₂) nano material, there is two-dimensional layered structure, be most widely used solid profit One of lubrication prescription.Its sheet two-dimension nano materials after removing, is the semiconductor nano material haveing excellent performance, in addition to big ratio Surface area, can be used as the carrier of catalyst and biological antibody, improve load capacity, while also have as co-catalyst excellent Electron transmission performance.

Currently, most of synthesizing mean is all after being separately synthesized, then catalyst and carrier progress is compound, process is numerous Trivial, yield is not high.Therefore, there is the two-dimensional nano composite material of excellent electrogenerated chemiluminescence performance to have In-situ reaction preparation Have wide practical use and important scientific meaning.

Summary of the invention

The purpose of the present invention is to provide it is a kind of prepare simple, high sensitivity, detection quickly, the electroluminescent chemistry of high specificity The preparation method of luminescence sensor, prepared sensor can be used for quick, the Sensitive Detection of parathion.Based on this purpose, originally Invention is prepared for a kind of New Two Dimensional nanocomposite first --- titanium dioxide/molybdenum disulfide composite material, i.e. cobalt doped The two-dimensional nano composite material Co-TiO of nano titania square In-situ reaction molybdenum disulfide₂/MoS₂, utilize the good of the material Good biocompatibility and big specific surface area, parathion antibody in load, when being detected, due to cobalt doped titanium dioxide It can be with catalyzing hydrogen peroxide in-situ preparation O₂, and with the K in the liquid of bottom₂S₂O₈It is electrochemically reacted, generates electrogenerated chemiluminescence letter Number, recycle antibody in conjunction with the specific quantification of antigen to the influence of electron transport ability, so that current strength reduces, thus Luminous intensity is reduced, finally realizes and is passed using the electrogenerated chemiluminescence of unmarked electrogenerated chemiluminescence method detection parathion The building of sensor.

The technical solution adopted by the invention is as follows:

1. a kind of preparation method of the parathion sensor based on two-dimension nano materials building, it is characterised in that described Parathion sensor is made of working electrode, two-dimension nano materials, parathion antibody, bovine serum albumin(BSA)；Two wieners Rice material is the two-dimensional nano composite material Co-TiO of cobalt doped nano titania square In-situ reaction molybdenum disulfide₂/MoS₂；

It is characterized in that, the preparation method includes following preparation step:

A. Co-TiO is prepared₂/MoS₂；

B. parathion sensor is prepared；

Wherein, step a prepares Co-TiO₂/MoS₂Specific steps are as follows:

(1) 0.6 g molybdenum disulfide powder and 0.2 ~ 2.0 mmol cobalt salt is taken to be added to 3 ~ 10 mL n-BuLis jointly molten In liquid, at nitrogen protection and 30 ~ 60 DEG C, stir 12 ~ 48 hours, the solution after being reacted；

(2) using the solution after reaction in nonpolar solvent washing step (1), water-bath is then carried out at 30 ~ 60 DEG C Ultrasonic treatment, after handle, recycle nonpolar solvent carrying out washing treatment after solution, be dried in vacuo, obtain two sulphur of cobalt intercalation Change molybdenum nano material；

(3) the molybdenum disulfide nano material of cobalt intercalation made from 10 ~ 500 mg steps (2) is taken to be added to 5 mL metatitanic acids four In butyl ester, after stirring 1 hour, it is slowly added to 0.5 ~ 0.8 mL hydrofluoric acid while stirring, is then reacting at 160 ~ 180 DEG C It is reacted 18 ~ 20 hours in kettle；

(4) by step (3) resulting reaction product, with ultrapure water and dehydrated alcohol centrifuge washing three times after, it is true at 50 DEG C Sky is dry, obtains Co-TiO₂/MoS₂；

The n-butyllithium solution is the hexane solution of n-BuLi, and concentration is 1.6 mol/L；

The cobalt salt is selected from one of following: cobaltous sulfate, cobalt chloride, cobalt nitrate, organic cobalt compounds；

The nonpolar solvent is selected from one of following: hexane, hexamethylene, carbon tetrachloride, benzene, toluene；

The water bath sonicator processing, processing time are 1 hour；

Step b prepares the specific steps of parathion sensor are as follows:

(1) using ITO electro-conductive glass as working electrode, in the Co-TiO of 8 ~ 12 μ L of electrode surface drop coating₂/MoS₂Colloidal sol, room It is dried under temperature；

(2) electrode obtained in step (1) is cleaned with buffer solution PBS, is continued in 8 ~ 12 μ L of electrode surface drop coating The parathion antibody-solutions of 10 μ g/mL are saved in 4 DEG C of refrigerators and are dried；

(3) electrode obtained in step (2) is cleaned with PBS, continues in 8 ~ 12 μ L concentration of electrode surface drop coating to be 100 The bovine serum albumin solution of μ g/mL is saved in 4 DEG C of refrigerators and is dried；

(4) electrode obtained in step (3) is cleaned with PBS, saves after drying, is obtained to sulphur in 4 DEG C of refrigerators Phosphorus sensor；

The Co-TiO₂/MoS₂Colloidal sol is by the Co-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters, and The hydrosol obtained after 30 min of ultrasound；

The PBS is the phosphate buffer solution of 10 mmol/L, and the pH value of the phosphate buffer solution is 7.4.

2. the application of parathion sensor prepared by preparation method of the present invention, which is characterized in that including as follows Applying step:

A. standard solution is prepared: preparing the parathion standard solution of one group of various concentration including blank standard specimen；

B. working electrode is modified: being work by parathion sensor prepared by preparation method as described in claim 1 The parathion standard solution for the various concentration prepared in step a is distinguished drop coating to working electrode surface, 4 DEG C of refrigerators by electrode Middle preservation；

C. working curve is drawn: using Ag/AgCl electrode as reference electrode, platinum electrode is used as to electrode, with step b The working electrode composition three-electrode system modified, is connected in electrochemiluminescdetection detection equipment；In a cell successively The K of 15 mL is added₂S₂O₈The H of solution and 100 μ L₂O₂Solution；Apply circulation electricity with working electrode of the cyclic voltammetry to assembling Pressure；According to the relationship between the light signal strength of resulting electrogenerated chemiluminescence and parathion antigen concentration of standard solution, draw Working curve；The light signal strength of blank standard specimen is denoted asD ₀, the light signal strength of the parathion standard solution containing various concentration It is denoted asD _i, the difference that response light signal strength reduces is ΔD = D ₀-D _i, ΔDWith the mass concentration of parathion standard solutionCIt Between it is linear, draw ΔD?CWorking curve；

D. the detection of parathion: the parathion standard solution in step a is replaced with sample to be tested, according in step b and c Method detected, according to response light signal strength reduce difference DELTADAnd working curve, obtain parathion in sample to be tested Content；

The K₂S₂O₈Solution is by 1 mol K₂S₂O₈It is dissolved in the buffer solution of pH=6.5 of 10 L and making with 1 mol KCl Standby to obtain, the buffer solution of the pH=6.5 is the phosphate buffer solution that pH value is 6.5；

The H₂O₂Solution is aqueous hydrogen peroxide solution, and the concentration of the aqueous hydrogen peroxide solution is 10%.

Beneficial achievement of the invention

(1) parathion sensor preparation of the present invention is simple, easy to operate, realizes to the quick, clever of parathion Quick, highly selective detection, and it is at low cost, it can be applied to portable inspectiont, there is market development prospect；

(2) present invention is prepared for novel photocatalyst Co-TiO using the method for In-situ reaction for the first time₂/MoS₂, this method master Will be there are three advantage: first is that, due to growth in situ of the cobalt on nano titania square sufficiently with nano titania side Block contact is acted on using the metal surface plasma body of cobalt, effectively increases semiconductor substrate Resonance energy transfer ability, solved Although titanium dioxide nanoplate specific surface area is bigger and mesoporous characteristic is suitable for electrogenerated chemiluminescence host material, electric The technical problem for causing chemiluminescence signal unstable；Second is that due to molybdenum disulfide sheet two-dimension nano materials load characteristic and Nano titania square on it fully dispersed, greatly increases electrogenerated chemiluminescence intensity, solves titanium dioxide Nanoplate difference and electrogenerated chemiluminescence intensity are weak and be unfavorable for the technical issues of preparing Electrochemiluminescsensor sensor；Three It is, since cobalt ions is in this process not only as intercalation material but also as reaction dopant material, finally to use the side of In-situ reaction Method realizes one pot of preparation of the composite material, not only saves time, material loss, and make the two of the cobalt doped prepared TiOx nano square can preferably be evenly spread to above molybdenum disulfide sheet two-dimension nano materials.Therefore, the material Effectively preparation has important scientific meaning and application value；

(3) present invention is for the first time by Co-TiO₂/MoS₂Applied in the preparation of electroluminescent chemiluminescence biosensor, significantly mention The high strength and stability of electrogenerated chemiluminescence, substantially increases the detection sensitivity of Electrochemiluminescsensor sensor, so that Electroluminescent chemiluminescence biosensor realizes application in actual operation；The application of the material is also associated biomolecule sensing Device, such as optical electro-chemistry sensor, electrochemical sensor provide Technical Reference, have extensive potential use value.

Specific embodiment

1 Co-TiO of embodiment₂/MoS₂Preparation

(1) 0.6 g molybdenum disulfide powder and 0.2 mmol cobalt salt is taken to be added in 3mL n-butyllithium solution jointly, in nitrogen At gas shielded and 60 DEG C, stir 12 hours, the solution after being reacted；

(2) using the solution after reaction in nonpolar solvent washing step (1), water bath sonicator is then carried out at 60 DEG C Processing, after handle, recycle nonpolar solvent carrying out washing treatment after solution, be dried in vacuo, obtain the molybdenum disulfide of cobalt intercalation Nano material；

(3) the molybdenum disulfide nano material of cobalt intercalation made from 500 mg steps (2) is taken to be added to 5 mL butyl titanates In, after stirring 1 hour, it is slowly added to 0.5 mL hydrofluoric acid while stirring, then reacts in a kettle 18 hours at 160 DEG C；

(4) by step (3) resulting reaction product, with ultrapure water and dehydrated alcohol centrifuge washing three times after, it is true at 50 DEG C Sky is dry, obtains Co-TiO₂/MoS₂；

The n-butyllithium solution is the hexane solution of n-BuLi, and concentration is 1.6 mol/L；

The cobalt salt is cobaltous sulfate；

The nonpolar solvent is hexane；

The water bath sonicator processing, processing time are 1 hour.

2 Co-TiO of embodiment₂/MoS₂Preparation

(1) 0.6 g molybdenum disulfide powder and 1.0 mmol cobalt salts is taken to be added in 5 mL n-butyllithium solutions jointly, in nitrogen At gas shielded and 30 DEG C, stir 24 hours, the solution after being reacted；

(2) using the solution after reaction in nonpolar solvent washing step (1), water bath sonicator is then carried out at 30 DEG C Processing, after handle, recycle nonpolar solvent carrying out washing treatment after solution, be dried in vacuo, obtain the molybdenum disulfide of cobalt intercalation Nano material；

(3) the molybdenum disulfide nano material of cobalt intercalation made from 200 mg steps (2) is taken to be added to 5 mL butyl titanates In, after stirring 1 hour, it is slowly added to 0.6 mL hydrofluoric acid while stirring, then reacts in a kettle 20 hours at 180 DEG C；

(4) by step (3) resulting reaction product, with ultrapure water and dehydrated alcohol centrifuge washing three times after, it is true at 50 DEG C Sky is dry, obtains Co-TiO₂/MoS₂；

The n-butyllithium solution is the hexane solution of n-BuLi, and concentration is 1.6 mol/L；

The cobalt salt is cobalt chloride；

The nonpolar solvent is carbon tetrachloride；

The water bath sonicator processing, processing time are 1 hour.

The preparation method of 3 parathion sensor of embodiment

(1) using the ITO electro-conductive glass that width is 1 cm, a length of 4 cm as working electrode, 8 μ L's of electrode surface drop coating Co-TiO₂/MoS₂Colloidal sol dries at room temperature；

(2) electrode obtained in step (1) is cleaned with buffer solution PBS, is continued in 8 μ L of electrode surface drop coating, 10 μ The parathion antibody-solutions of g/mL are saved in 4 DEG C of refrigerators and are dried；

(3) electrode obtained in step (2) is cleaned with PBS, continues in 8 μ L concentration of electrode surface drop coating to be 100 μ The bovine serum albumin solution of g/mL is saved in 4 DEG C of refrigerators and is dried；

(4) electrode obtained in step (3) is cleaned with PBS, is saved in 4 DEG C of refrigerators after drying, obtains parathion Sensor；

The Co-TiO₂/MoS₂Colloidal sol is by the Co-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters, and The hydrosol obtained after 30 min of ultrasound；

The PBS is the phosphate buffer solution of 10mmol/L, and the pH value of the phosphate buffer solution is 7.4.

The preparation method of 4 parathion sensor of embodiment

(1) using the ITO electro-conductive glass that width is 1 cm, a length of 4 cm as working electrode, 10 μ L's of electrode surface drop coating Co-TiO₂/MoS₂Colloidal sol dries at room temperature；

(2) electrode obtained in step (1) is cleaned with buffer solution PBS, is continued in 10 μ L 10 of electrode surface drop coating The parathion antibody-solutions of μ g/mL are saved in 4 DEG C of refrigerators and are dried；

(3) electrode obtained in step (2) is cleaned with PBS, continues in 10 μ L concentration of electrode surface drop coating to be 100 μ The bovine serum albumin solution of g/mL is saved in 4 DEG C of refrigerators and is dried；

(4) electrode obtained in step (3) is cleaned with PBS, saves after drying, is obtained to sulphur in 4 DEG C of refrigerators Phosphorus sensor；

The Co-TiO₂/MoS₂Colloidal sol is by the Co-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters, and The hydrosol obtained after 30 min of ultrasound；

The PBS is the phosphate buffer solution of 10 mmol/L, and the pH value of the phosphate buffer solution is 7.4.

The preparation method of 5 parathion sensor of embodiment

(1) using the ITO electro-conductive glass that width is 1 cm, a length of 4 cm as working electrode, 12 μ L's of electrode surface drop coating Co-TiO₂/MoS₂Colloidal sol dries at room temperature；

(2) electrode obtained in step (1) is cleaned with buffer solution PBS, is continued in 12 μ L 10 of electrode surface drop coating The parathion antibody-solutions of μ g/mL are saved in 4 DEG C of refrigerators and are dried；

(3) electrode obtained in step (2) is cleaned with PBS, continues in 12 μ L concentration of electrode surface drop coating to be 100 μ The bovine serum albumin solution of g/mL is saved in 4 DEG C of refrigerators and is dried；

(4) electrode obtained in step (3) is cleaned with PBS, saves after drying, is obtained to sulphur in 4 DEG C of refrigerators Phosphorus sensor；

The Co-TiO₂/MoS₂Colloidal sol is by the Co-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters, and The hydrosol obtained after 30 min of ultrasound；

The PBS is the phosphate buffer solution of 10 mmol/L, and the pH value of the phosphate buffer solution is 7.4.

The parathion sensor of 6 embodiment 1 ~ 5 of embodiment preparation, applied to the detection of parathion, steps are as follows:

(1) standard solution is prepared: preparing the parathion standard solution of one group of various concentration including blank standard specimen；

(2) working electrode is modified: being work by parathion sensor prepared by preparation method as described in claim 1 The parathion standard solution for the various concentration prepared in step (1) is distinguished drop coating to working electrode surface, 4 DEG C of ice by electrode It is saved in case；

(3) working curve is drawn: using saturated calomel electrode as reference electrode, platinum electrode is as auxiliary electrode, with step Suddenly the working electrode that (2) have been modified forms three-electrode system, is connected in electrochemiluminescdetection detection equipment；In a cell The K of 15 mL is successively added₂S₂O₈The H of solution and 100 μ L₂O₂Solution；It is followed with working electrode application of the cyclic voltammetry to assembling Loop voltag；According to the relationship between the light signal strength of resulting electrogenerated chemiluminescence and parathion antigen concentration of standard solution, Draw working curve；The light signal strength of blank standard specimen is denoted asD ₀, the optical signal of the parathion standard solution containing various concentration Intensity is denoted asD _i, the difference that response light signal strength reduces is ΔD = D ₀-D _i, ΔDWith the mass concentration of parathion standard solutionCBetween it is linear, draw ΔD?CWorking curve；The linear detection range of parathion are as follows: 0.003 ~ 100 ng/mL, inspection Rising limit are as follows: 1.1 pg/mL；

(4) detection of parathion: the parathion standard solution in step a is replaced with sample to be tested, according in step b and c Method detected, according to response light signal strength reduce difference DELTADAnd working curve, obtain parathion in sample to be tested Content；

The K₂S₂O₈Solution is by 1 mol K₂S₂O₈It is dissolved in the buffer solution of pH=6.5 of 10 L and making with 1 mol KCl Standby to obtain, the buffer solution of the pH=6.5 is the phosphate buffer solution that pH value is 6.5；

The H₂O₂Solution is aqueous hydrogen peroxide solution, and the concentration of the aqueous hydrogen peroxide solution is 10%.

Claims (1)

1. a kind of preparation method of the parathion sensor based on two-dimension nano materials building, it is characterised in that the parathion Sensor is made of working electrode, two-dimension nano materials, parathion antibody, bovine serum albumin(BSA)；The two-dimension nano materials For the two-dimensional nano composite material Co-TiO of cobalt doped nano titania square In-situ reaction molybdenum disulfide₂/MoS₂；

The Co-TiO₂/MoS₂Specific preparation step are as follows: take 0.6 g molybdenum disulfide powder and 0.2 ~ 2.0 mmol cobalt salt It is added in 3 ~ 10 mL n-butyllithium solutions jointly, at nitrogen protection and 30 ~ 60 DEG C, stirs 12 ~ 48 hours, obtain Solution after reaction；Then water bath sonicator processing is carried out at 30 ~ 60 DEG C, after having handled, recycles nonpolar solvent washing Treated solution, vacuum drying, obtains the molybdenum disulfide nano material of cobalt intercalation；Take cobalt intercalation made from 10 ~ 500 mg Molybdenum disulfide nano material be added in 5 mL butyl titanates, after stirring 1 hour, be slowly added to 0.5 while stirring ~ Then 0.8 mL hydrofluoric acid reacts 18 ~ 20 hours at 160 ~ 180 DEG C in a kettle；Resulting reaction product, use are ultrapure Water and dehydrated alcohol centrifuge washing three times after, be dried in vacuo at 50 DEG C, obtain Co-TiO₂/MoS₂；

The n-butyllithium solution is the hexane solution of n-BuLi, and concentration is 1.6 mol/L；

The cobalt salt is selected from one of following: cobaltous sulfate, cobalt chloride, cobalt nitrate, organic cobalt compounds；

The nonpolar solvent is selected from one of following: hexane, hexamethylene, carbon tetrachloride, benzene, toluene；

The water bath sonicator processing, processing time are 1 hour；

The specific preparation step of the parathion sensor are as follows:

(1) using ITO electro-conductive glass as working electrode, in the Co-TiO of 8 ~ 12 μ L of electrode surface drop coating₂/MoS₂Colloidal sol, at room temperature It dries；

(2) electrode obtained in step (1) is cleaned with buffer solution PBS, is continued in 8 ~ 12 μ L of electrode surface drop coating, 10 μ The parathion antibody-solutions of g/mL are saved in 4 DEG C of refrigerators and are dried；

(3) electrode obtained in step (2) is cleaned with PBS, continues in 8 ~ 12 μ L concentration of electrode surface drop coating to be 100 μ g/ The bovine serum albumin solution of mL is saved in 4 DEG C of refrigerators and is dried；

(4) electrode obtained in step (3) is cleaned with PBS, is saved in 4 DEG C of refrigerators after drying, obtain parathion biography Sensor；

The Co-TiO₂/MoS₂Colloidal sol is by the Co-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters, and ultrasound The hydrosol obtained after 30 min；

The PBS is the phosphate buffer solution of 10 mmol/L, and the pH value of the phosphate buffer solution is 7.4.