CN106053573B - A kind of preparation method of the electrochemical sensor based on load type double-metal co-doped nano composite material - Google Patents

Abstract

The invention discloses a kind of preparation methods of electrochemical sensor based on nano-functional material building.Belong to Nano-function thin films and biosensor technology field.The method comprises the steps of firstly, preparing a kind of New Two Dimensional nano-electrode materials --- load type double-metal co-doped nano composite material FeCo-TiO₂/MoS₂That is the two-dimensional nano composite material of iron, cobalt codoping titanium dioxide nano square In-situ reaction molybdenum disulfide, good biocompatibility and big specific surface area using the material, antibody of clenbuteral in load, when being detected, since iron, cobalt codope titanium dioxide can be with catalyzing hydrogen peroxide in-situ preparation O₂, electrochemical signals are generated, recycle antibody in conjunction with the specific quantification of antigen to the influence of electron transport ability, so that current strength accordingly reduces, finally realize the building of the biosensor using unmarked electrochemical method detection Clenbuterol.

Description

A kind of electrochemical sensor based on load type double-metal co-doped nano composite material Preparation method

Technical field

The present invention relates to a kind of preparation methods of electrochemical sensor based on nano-functional material building.Belong to Performances of Novel Nano-Porous Rice functional material and biosensor technology field.

Background technique

Clenbuterol, i.e. clenobuterol hydrochloride, due to there is significant nutrition " redistribution effects " --- promote animal body egg White matter deposition promotes lipolysis to inhibit fat deposition, can significantly improve lean meat percentage, weight gain and the raising food conversion of trunk Rate.Therefore, it is also referred to as clenbuterol hydrochloride in China, was once used as growth accelerator, the feed addictive of the livestock and poultry such as ox, sheep, fowl, pig. But since Clenbuterol is excessive to human health damage, thus security risk is caused, also thus disabled in the whole world.

Currently, the method for detection Clenbuterol mainly has chromatography, mass spectrography etc..Such method instrument is valuable, operation is multiple Miscellaneous, 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, specificity Strong Clenbuterol sensor is of great significance.

Electrochemica biological sensor due to its high sensitivity, specificity it is good, easy to operate the advantages that be widely used in facing The fields such as bed diagnosis, Pharmaceutical Analysis, environmental monitoring.More, key is wherein studied with unmarked electrochemical immunosensor especially Technology be to improve modified electrode to the fixed amount of antibody and to the signal response speed and size of test bottom liquid.Titanium dioxide is A kind of photocatalyst material being most widely used, simultaneously because good biocompatibility, is also commonly used as electrode matrix material.By More high miller index surfaces can be exposed in sheet-like titanium dioxide nanomaterial, there is higher catalytic activity, titanium dioxide is received Rice piece has application prospect preferably than nanoparticle, and the research of titanium dioxide nanoplate is also concerned.But dioxy The sensitivity that change titanium poorly conductive also limits the electrochemical sensor constructed by single titanium dioxide nano material is not generally high, It is unfavorable for practical application.Modification or compound special nano material, on the one hand increase electrode ratio on semiconductor nano material Surface area enhances electrodes conduct ability, and on the other hand the two can produce concerted catalysis effect, and bigger enhancing is to hydrogen peroxide Solution H₂O₂Catalytic response speed and current responsing signal size, greatly improve detection sensitivity.Therefore, it designs, prepare height Effect, stable titanium dioxide nanoplate and its modifier are the key technologies for preparing electrochemical 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, have for two dimensional nano electrodes material of the In-situ reaction preparation with excellent chemical property extensive Application prospect 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 electrochemical student of high specificity The preparation method of object sensor, prepared sensor can be used for quick, the Sensitive Detection of Clenbuterol.Based on this purpose, originally Invention is prepared for a kind of New Two Dimensional nano-electrode material first --- load type double-metal co-doped nano composite material FeCo- TiO₂/MoS₂, i.e. the two-dimensional nano composite material of iron, cobalt codoping titanium dioxide nano square In-situ reaction molybdenum disulfide utilizes The good biocompatibility of the material and big specific surface area, antibody of clenbuteral in load, when being detected, due to Iron, cobalt codope titanium dioxide can be with catalyzing hydrogen peroxide in-situ preparation O₂, electrochemical signals are generated, antibody and antigen are recycled Specific quantification combine influence to electron transport ability finally to realize so that current strength accordingly reduces using no mark The building of the biosensor of the electrochemical method detection Clenbuterol of note.

The technical solution adopted by the invention is as follows:

1. a kind of preparation method of the electrochemical sensor based on load type double-metal co-doped nano composite material, Electrochemical sensor described in being characterized in that is by working electrode, load type double-metal co-doped nano composite material, Clenbuterol Antibody, bovine serum albumin(BSA) composition；The load type double-metal co-doped nano composite material is iron, cobalt codope titanium dioxide The two-dimensional nano composite material FeCo-TiO of titanium nano square and molybdenum disulfide In-situ reaction₂/MoS₂；

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

A. FeCo-TiO is prepared₂/MoS₂；

B. electrochemical sensor is prepared；

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

(1) 0.6 g molybdenum disulfide powder, 0.2 ~ 2.0 mmol molysite and 0.2 ~ 2.0 mmol cobalt salt is taken to be added jointly Into 3 ~ 10 mL n-butyllithium solutions, at nitrogen protection and 30 ~ 60 DEG C, stir 12 ~ 48 hours, after being reacted Solution；

(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 iron, cobalt is total to intercalation Molybdenum disulfide nano material；

(3) the molybdenum disulfide nano material for taking iron made from 10 ~ 500 mg steps (2), cobalt to be total to intercalation is added to 5 mL In butyl titanate, after stirring 1 hour, it is slowly added to 0.5 ~ 0.8 mL hydrofluoric acid while stirring, then at 160 ~ 180 DEG C It reacts 18 ~ 20 hours in a 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 FeCo-TiO₂/MoS₂；

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

The molysite is selected from one of following: ferric sulfate, iron chloride, ferric nitrate, organoiron compound；

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 electrochemical sensor are as follows:

(1) using ITO electro-conductive glass as working electrode, in the FeCo-TiO of 8 ~ 12 μ L of electrode surface drop coating₂/MoS₂Colloidal sol, It dries at room 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 antibody of clenbuteral solution of 10 μ g/mL is saved in 4 DEG C of refrigerators and is 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, is saved in 4 DEG C of refrigerators after drying, obtains electrification Learn sensor；

The FeCo-TiO₂/MoS₂Colloidal sol is by the FeCo-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters In, 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 electrochemical 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 Clenbuterol standard solution of one group of various concentration including blank standard specimen；

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

C. working curve is drawn: saturated calomel electrode electrode is used as to reference electrode, platinum electrode as to electrode, with The working electrode that step b has been modified forms three-electrode system, connects electrochemical workstation, and 15 are successively added in a cell The H of 5 mol/L of mL PBS and 20 μ L₂O₂；By the working electrode of chronoamperometry detection assembling to H₂O₂Response；Blank The response current of standard specimen is denoted asI ₀, the response current of the Clenbuterol standard solution containing various concentration is denoted asI _i, response current drop Low difference is ΔI = I ₀-I _i, ΔIWith the mass concentration of Clenbuterol standard solutionCBetween it is linear, draw ΔI?CWorking curve；

D. the detection of Clenbuterol: replacing the Clenbuterol standard solution in step a with sample to be tested, according to step b and Method in c is detected, the difference DELTA that electrical signal intensity reduces according to responseIAnd working curve, it obtains in sample to be tested gram The content of Lun Teluo.

Beneficial achievement of the invention

(1) electrochemical sensor of the present invention preparation is simple, easy to operate, realize to sample it is quick, sensitive, 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 New Two Dimensional nano-electrode material FeCo-TiO using the method for In-situ reaction for the first time₂/ MoS₂, this method is mainly there are three advantage: first is that, due to iron, cobalt jointly the growth in situ on nano titania square and It is sufficiently contacted with nano titania square, is acted on, effectively increased semiconductor-based using the metal surface plasma body of iron, cobalt Matter electron transmission ability and catalytic activity, although solving, titanium dioxide nanoplate specific surface area is bigger and mesoporous characteristic is applicable in In electrochemical-based material, but the technical problem that electro-chemical activity is not high and current signal is unstable；Second is that due to curing The load characteristic and nano titania square of molybdenum sheet shape two-dimension nano materials on it fully dispersed, greatly increase electricity Sub- transmission capacity, solves titanium dioxide nanoplate poorly conductive and current responsing signal is weak and be unfavorable for preparing electrochemical sensing The technical issues of device；Third is that since iron, cobalt ions are in this process not only as intercalation material but also as reaction dopant material, most One pot of preparation for realizing the composite material using the method for In-situ reaction afterwards, not only saves time, material loss, and make The nano titania square of the iron, cobalt codope that must prepare can preferably evenly spread to molybdenum disulfide sheet two-dimensional nano Above material.Therefore, effective preparation of the material has important scientific meaning and application value；

(3) present invention is for the first time by FeCo-TiO₂/MoS₂Applied in the preparation of electrochemica biological sensor, significantly improve The strength and stability of current signal substantially increases the detection sensitivity of electrochemical sensor, so that electrochemical biosensor Device realizes application in actual operation；The application of the material is also associated biomolecule sensor, as optical electro-chemistry sensor, Electrochemiluminescsensor sensor etc. provides Technical Reference, has extensive potential use value.

Specific embodiment

1 FeCo-TiO of embodiment₂/MoS₂Preparation

(1) 0.6 g molybdenum disulfide powder, 0.2 mmol molysite and 0.2 mmol cobalt salt is taken to be added to 3mL normal-butyl jointly In lithium solution, at nitrogen protection 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 iron, cobalt is total to two sulphur of intercalation Change molybdenum nano material；

(3) the molybdenum disulfide nano material for taking iron made from 500 mg steps (2), cobalt to be total to intercalation is added to 5 mL metatitanic acids four In butyl ester, after stirring 1 hour, it is slowly added to 0.5 mL hydrofluoric acid while stirring, then reacts 18 in a kettle at 160 DEG C Hour；

(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 FeCo-TiO₂/MoS₂；

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

The molysite is ferric sulfate；

The cobalt salt is cobaltous sulfate；

The nonpolar solvent is hexane；

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

2 FeCo-TiO of embodiment₂/MoS₂Preparation

(1) 0.6 g molybdenum disulfide powder, 1.0 mmol molysite and 1.0 mmol cobalt salts is taken to be added to 5 mL normal-butyls jointly In lithium solution, at nitrogen protection 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 iron, cobalt is total to two sulphur of intercalation Change molybdenum nano material；

(3) the molybdenum disulfide nano material for taking iron made from 200 mg steps (2), cobalt to be total to intercalation is added to 5 mL metatitanic acids four In butyl ester, after stirring 1 hour, it is slowly added to 0.6 mL hydrofluoric acid while stirring, then reacts 20 in a kettle at 180 DEG C Hour；

(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 FeCo-TiO₂/MoS₂；

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

The molysite is iron chloride；

The cobalt salt is cobalt chloride；

The nonpolar solvent is carbon tetrachloride；

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

3 FeCo-TiO of embodiment₂/MoS₂Preparation

(1) 0.6 g molybdenum disulfide powder, 2.0 mmol molysite and 2.0 mmol cobalt salts is taken to be added to the positive fourth of 10 mL jointly In base lithium solution, at nitrogen protection and 50 DEG C, stir 48 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 50 DEG C Processing, after handle, recycle nonpolar solvent carrying out washing treatment after solution, be dried in vacuo, obtain iron, cobalt is total to two sulphur of intercalation Change molybdenum nano material；

(3) the molybdenum disulfide nano material for taking iron made from 10 mg steps (2), cobalt to be total to intercalation is added to 5 mL metatitanic acids four In butyl ester, after stirring 1 hour, it is slowly added to 0.8 mL hydrofluoric acid while stirring, then reacts 24 in a kettle at 200 DEG C Hour；

(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 FeCo-TiO₂/MoS₂；

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

The molysite is ferric acetate；

The cobalt salt is cobalt acetate；

The nonpolar solvent is benzene；

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

The preparation method of 4 electrochemical sensor of embodiment

(1) using glass-carbon electrode as working electrode, in the FeCo-TiO of 8 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 μ L of electrode surface drop coating, 10 μ The antibody of clenbuteral solution of g/mL is saved in 4 DEG C of refrigerators and is 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 electrification Learn sensor；

The FeCo-TiO₂/MoS₂Colloidal sol is by the FeCo-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters In, 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 5 electrochemical sensor of embodiment

(1) using glass-carbon electrode as working electrode, in the FeCo-TiO of 10 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 10 μ L 10 of electrode surface drop coating The antibody of clenbuteral solution of μ g/mL is saved in 4 DEG C of refrigerators and is 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, is saved in 4 DEG C of refrigerators after drying, obtains electrification Learn sensor；

The FeCo-TiO₂/MoS₂Colloidal sol is by the FeCo-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters In, 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 6 electrochemical sensor of embodiment

(1) using glass-carbon electrode as working electrode, in the FeCo-TiO of 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 12 μ L 10 of electrode surface drop coating The antibody of clenbuteral solution of μ g/mL is saved in 4 DEG C of refrigerators and is 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, is saved in 4 DEG C of refrigerators after drying, obtains electrification Learn sensor；

The FeCo-TiO₂/MoS₂Colloidal sol is by the FeCo-TiO of 50 mg₂/MoS₂Powder is dissolved in 10 mL ultrapure waters In, 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 electrochemical sensor of 7 embodiment 1 ~ 6 of embodiment preparation, applied to the detection of Clenbuterol, steps are as follows:

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

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

(3) working curve is drawn: saturated calomel electrode electrode is used as to reference electrode, platinum electrode as to electrode, with The working electrode that step b has been modified forms three-electrode system, connects electrochemical workstation, and 15 are successively added in a cell The H of 5 mol/L of mL PBS and 20 μ L₂O₂；By the working electrode of chronoamperometry detection assembling to H₂O₂Response；Blank The response current of standard specimen is denoted asI ₀, the response current of the Clenbuterol standard solution containing various concentration is denoted asI _i, response current drop Low difference is ΔI = I ₀-I _i, ΔIWith the mass concentration of Clenbuterol standard solutionCBetween it is linear, draw ΔI?CWorking curve；The linear detection range of Clenbuterol are as follows: 0.009 ~ 200 ng/mL, detection limit are as follows: 3.0 pg/mL；

(4) detection of Clenbuterol: the Clenbuterol standard solution in step (1) is replaced with sample to be tested, according to step (2) it is detected with the method in (3), the difference DELTA that electrical signal intensity reduces according to responseIAnd working curve, it obtains to test sample The content of Clenbuterol in product.

Claims (1)

1. a kind of preparation method of the electrochemical sensor based on load type double-metal co-doped nano composite material, feature exist In the electrochemical sensor by working electrode, load type double-metal co-doped nano composite material, antibody of clenbuteral, ox Seralbumin composition；The load type double-metal co-doped nano composite material is iron, cobalt codoping titanium dioxide nano The two-dimensional nano composite material FeCo-TiO of square and molybdenum disulfide In-situ reaction₂/MoS₂；

The FeCo-TiO₂/MoS₂Specific preparation step are as follows: take 0.6 g molybdenum disulfide powder, 0.2 ~ 2.0 mmol iron Salt and 0.2 ~ 2.0 mmol cobalt salt are added to jointly in 3 ~ 10 mL n-butyllithium solutions, in nitrogen protection and 30 ~ 60 DEG C Under, it stirs 12 ~ 48 hours, the solution after being reacted；Using the solution after nonpolar solvent washing reaction, then 30 ~ At 60 DEG C carry out water bath sonicator processing, after handle, recycle nonpolar solvent carrying out washing treatment after solution, be dried in vacuo, obtain The molybdenum disulfide nano material of intercalation is total to iron, cobalt；Iron made from 10 ~ 500 mg, cobalt is taken to be total to the molybdenum disulfide nano of intercalation Material is added in 5 mL butyl titanates, after stirring 1 hour, is slowly added to 0.5 ~ 0.8 mL hydrofluoric acid while stirring, so It reacts 18 ~ 20 hours at 160 ~ 180 DEG C, resulting reaction product, is centrifuged with ultrapure water and dehydrated alcohol in a kettle afterwards After washing three times, it is dried in vacuo at 50 DEG C, obtains FeCo-TiO₂/MoS₂；

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

The molysite is selected from one of following: ferric sulfate, iron chloride, ferric nitrate, organoiron compound；

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 electrochemical sensor are as follows:

(1) using glass-carbon electrode as working electrode, in the FeCo-TiO of 8 ~ 12 μ L of electrode surface drop coating₂/MoS₂Colloidal sol dries in the air at room temperature It is dry；

(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 antibody of clenbuteral solution of g/mL is saved in 4 DEG C of refrigerators and is 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 electrochemistry biography Sensor；

The FeCo-TiO₂/MoS₂Colloidal sol is by the FeCo-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.