CN108375623A - The preparation method and applications of the electrochemical immunosensor of food-borne pathogens are detected based on quick scan anode Stripping Voltammetry technology - Google Patents
The preparation method and applications of the electrochemical immunosensor of food-borne pathogens are detected based on quick scan anode Stripping Voltammetry technology Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention discloses the electrochemical immunosensor preparation method and applications that food-borne pathogens are detected based on quick scan anode Stripping Voltammetry technology, and feature is to include the following steps（1）By amination Fe3O4It is added in nanoparticles solution after being reacted in glutaraldehyde solution, after food-borne pathogens capture antibody response is added after Magneto separate cleans, capturing unit solution is obtained after Magneto separate cleans；（2）By composite nano materials AgNPs@g C3N4Signal element is obtained after being combined with food-borne pathogenic bacteria antibody；（3）Magnetropism glassy carbon electrode surface obtains electrochemical immunosensor after capturing unit, food-borne pathogens sample solution, signal element are added dropwise successively, and application is according to Anodic Stripping peak currenti p With the quantitative relationship between food-borne pathogenic bacteria concentration, food-borne pathogenic bacteria concentration in unknown sample is measured, advantage high sensitivity, high specific, testing result is reliable, step is simple and detection speed is fast.
Description
Technical field
The present invention relates to the detection methods of food-borne pathogens, more particularly, to based on quick scan anode Stripping Voltammetry skill
Art detects the preparation method and applications of the electrochemical immunosensor of food-borne pathogens.
Background technology
Food-borne pathogens refer to that can cause food poisoning or using food as the pathogenic bacteria of communication media.Food exists
The pollution of food-borne pathogens is highly susceptible in the links such as acquisition, processing, transport, the food poisoning and disease therefore risen is quick-fried
Hair event occurs again and again, and the economic loss caused by food-borne pathogens is up to 17,000,000,000 dollars every year in China.Common is food-borne
Pathogenic bacteria have：Vibrio parahemolyticus, Vibrio vulnificus, staphylococcus aureus, Escherichia coli, salmonella etc., traditional detection food
The method of borne pathogen is biochemical culture identification method, this method complex steps, detection cycle is long, time and effort consuming.As molecule is given birth to
Object technology is fast-developing, PCR（PCR）, DNA hybridization and ring mediated isothermal amplification（LAMP）, biochip etc.
Method is also applied to detection food-borne pathogens, obtains preferable accuracy and sensitivity, but also deposit in practical applications
In many problems：False positive probability is higher, expensive equipment, testing cost is high, detecting step is complicated, detection time is grown etc..Therefore,
Sensitive, accurate, easy, quick food-borne pathogens detection method is developed, is active demand.
Anodic stripping voltammetry is a kind of common electrochemical analysis method, is directed to working electrode and applies oxidative scan electricity
Pressure makes the metal on working electrode aoxidize dissolution and generate oxidation current, and quantitative analysis is carried out according to the electric current of oxidation process
Electrochemical methods.Usually before Anodic Stripping, first applies recovery voltage to working electrode, metal ion to be measured is made to work
It is reduced into electrode surface part metal and realizes enrichment concentration, achieve the purpose that improve detection sensitivity.Currently, using functionalization
The report that composite nano materials improve anodic stripping voltammetry detection sensitivity is few.In addition, according to electrochemistry basic theories, sun
Voltage scan rate is bigger when pole dissolves out, and dissolution metal ion quantity is more in the unit interval, and oxidation current is bigger, and detection is sensitive
Degree is also higher, however, since circuit design presence is more highly difficult, such research is rare.
Electrochemical sensing technology is combined by electrochemical immunosensor with immuno analytical method, existing electrochemical sensor
It is sensitive, easy, quick, economical the advantages that, and have the features such as specificity and accuracy of immunoassay.In recent years, various to receive
Rice material especially two-dimension nano materials such as graphene, graphite-phase nitrogen carbide（g-C3N4）Deng using wide in terms of sensor structure
It is general.Currently, disclosing not yet both at home and abroad any about based on quick scan anode Stripping Voltammetry technology detection food-borne pathogens
Electrochemical immunosensor preparation method correlative study report.
Invention content
Technical problem to be solved by the invention is to provide a kind of high sensitivity, high specific, testing results reliably, step
The fast electro-chemistry immunity that food-borne pathogens are detected based on quick scan anode Stripping Voltammetry technology of simple and detection speed
The preparation method and applications of sensor.
Technical solution is used by the present invention solves above-mentioned technical problem：One kind being based on quick scan anode Stripping Voltammetry
Technology detects the electrochemical immunosensor preparation method of food-borne pathogens, includes the following steps：
（1）Capturing unit（cAb@Fe3O4）Preparation
A. by 0.1~0.3 g FeCl2•4H2O and 0.6~0.9 g FeCl3•6H2O is dissolved in the secondary water of 40~50 mL deoxygenations
In, stirred under nitrogen atmosphere makes it be uniformly mixed, and 28 wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, are warming up to
80 DEG C and 2 h of this thermotonus is maintained to be cooled to room temperature under nitrogen protection after reaction, water is cleaned to neutrality, is settled to
50 mL are to get Fe3O4Nanoparticles solution；
B. by 40~50 mL Fe3O4Nanoparticles solution and 0.4~0.6 mL 3- aminopropyl triethoxysilanes（APTES）
It mixes, after 0.5 h of ultrasound, stirs 5~8 h at room temperature, through Magneto separate, cleaning, be dispersed in again in 50 mL water to get amino
Change Fe3O4Nanoparticles solution；
C. in 1~3 mL aminations Fe3O4The glutaraldehyde that 0.1~0.3 mL, 2.5 wt% are added in nanoparticles solution is molten
Liquid reacts 1~3 h at room temperature, and the food-borne pathogens capture of 1~3 mL, 10~50 μ g/mL is added after Magneto separate, cleaning
Antibody（cAb）, after reacting 1~3 h at room temperature, after Magneto separate, cleaning, it is dispersed in the phosphorus of 10~20 pH=7.5~8.2 mL
To get capturing unit in hydrochlorate buffer solution（cAb@Fe3O4）Solution；
（2）Signal element（sAb-AgNPs@g-C3N4）Preparation
A. it weighs 5~10 g melamine powders, calcines 3~5 h in Muffle furnace at 500~600 DEG C, be dried in vacuum overnight
Afterwards, you can obtain nitridation carbon dust, 0.8~1.5 g nitridation carbon dusts is taken to be added to 80~120 mL, 4~6 M HNO3Solution
In, flow back 16~20 h at 120~150 DEG C, and after being cooled to room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by gained
16 h of sediment continuous ultrasound, up to graphite phase carbon nitride after vacuum drying（g-C3N4）；
B. 1~3 mL, 10 mM AgNO are added in 30~60 mL water3Solution is heated to boiling, and adds 1~3 mL
3 mM NaBH of 1wt% citric acid three sodium solutions and 300~500 μ L4Solution is vigorously stirred 1 h under fluidized state, is cooled to room
Wen Hou is centrifuged, and cleaning is distributed in 40~60 mL water to get nano silver particles（AgNPs）Solution；
C. in 10~70 mL nano silver solutions be added 30~70 mg graphite phase carbon nitrides, at room temperature stir 16~
24 h, washing centrifugation, abandon supernatant, are distributed in 40~60 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 120~150 μ L and contains 10~100 mmol/L
1- ethyls-（3- dimethylaminopropyls）Carbodiimide hydrochloride（EDC）With 1~10 mmol/L n-hydroxysuccinimides
（NHS）Mixed solution, 0.8~1.2 M hydrochloric acid tune pH is that 1~2 h is incubated at 4.0~6.0,35 DEG C, is centrifuged, washing, water is fixed
Hold to 200~300 μ L, is 7.0~9.0 with 0.1~0.2 M NaOH solution tune pH, 80~120 μ L, 0.01~1 μ are added
G/mL food-borne pathogenic bacteria antibodies（sAb）It is incubated 3~5 h afterwards, adds 80~100 μ L 2wt% bovine serum albumin(BSA)s（BSA）
Solution is incubated 1~2 h with closing activity site, cleans, and centrifugation is distributed in 5~10 mL water to get signal element sAb-
AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode（MGCE）Successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then successively with 50
The ethanol solution of vt%, the aqueous solution of nitric acid of 50 vt % and distilled water are cleaned by ultrasonic；
B. 5~10 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 5~10 μ L food-borne pathogens samples are then added dropwise
Solution, after being incubated at room temperature 30~50 min, water cleaning adds 5~10 μ L signal units, after being incubated at room temperature 30~50 min,
Water cleans to get electrochemical immunosensor.
The food-borne pathogens include vibrio parahemolyticus, Vibrio vulnificus, staphylococcus aureus, Escherichia coli and
Salmonella.
The method for detecting food-borne pathogens using the electrochemical immunosensor, includes the following steps
Use electrochemical immunosensor described in claim 1 for working electrode, platinum electrode is to electrode, 0.1 M KCl
Solution is supporting electrolyte solution, using the voltage scan rate of 100 V/s, carries out quick scan anode stripping volt ampere analysis,
Measure Anodic Stripping peak currenti p （μA）；Measure a series of corresponding Anodic Stripping peak current of food-borne pathogens of various concentrationsi p Size, establishi p With food-borne pathogenic bacteria concentration（cfu/mL）Between quantitative relationship；It can be measured according to the quantitative relationship
Food-borne pathogenic bacteria concentration in unknown sample.
The principle of the invention：It is intended to, by functionalization Bio-Nano-Materials, electrochemical immunosensor be built, quickly to scan
Anodic stripping voltammetry is detection technique, realizes sensitive, accurate, simple, the quick detection of food-borne pathogens.The first step is caught
Obtain unit（cAb@Fe3O4）Antibody is captured by food-borne pathogens（cAb）It is immobilized in nanometer Fe3O4Nano grain surface forms,
Fe3O4Nano particle has magnetism, is easily adsorbed on magnetic glassy carbon electrode surface, and cAb then can the food-borne cause of specificity capture
Germ.Second step, the captured unit specificity capture of food-borne pathogens.Third walks, signal element（sAb-AgNPs@g-C3N4）
By food-borne pathogenic bacteria antibody（sAb）It is immobilized in g-C simultaneously with nano silver particles3N4Surface forms, and nano silver particles can conduct
The electrochemistry beacon of quick scan anode stripping voltammetry, when carrying out Anodic Stripping, nano silver particles are oxidized to silver ion, production
Raw oxidation current；g-C3N4Surface area is huge, can load a large amount of nano silver particles, greatly improve detection sensitivity；SAb can be with
It identifies food-borne pathogens and is immunized therewith and combine, form the immune compound of capturing unit-food-borne pathogens-signal element
Object is incorporated in electrode surface.4th step detects food-borne pathogens by detection technique of quick scan anode stripping voltammetry,
Food-borne pathogenic bacteria concentration is higher, in conjunction with signal element it is more, the electrochemistry beacon nano silver particles in corresponding signal element
More, oxidation current of the nano silver particles when carrying out Anodic Stripping is also bigger, and principle can carry out food-borne pathogenic accordingly
The quantitative detection of bacterium；Moreover, using fast scanning techniques, voltage scan rate is improved, metal ion number is dissolved out in the unit interval
Amount is more, can significantly improve oxidation current when nano silver particles Anodic Stripping, therefore it is sensitive to further increase detection
Degree.
Compared with the conventional method, the advantage of the invention is that：
（1）It is highly sensitive.The method of the present invention has high sensitivity, and the detection limit of the method for the present invention is about the 100 of existing method
Times or more, the two level amplification principle based on electrochemical signals：When in signal element, g-C3N4It is a kind of two-dimension netted material, tool
There are good thermal stability, chemical stability, biocompatibility and certain electric conductivity, surface area is huge, can load big
Electrochemistry beacon nano silver particles are measured, its electric conductivity is made to greatly enhance, this means that a food-borne pathogens correspond to one
A signal element, but the corresponding a large amount of electrochemistry beacon nano silver particles loaded thereon, realize the level-one of electrochemical signals
Amplification；Second is that using fast scanning techniques, voltage scan rate is improved, when can increase substantially nano silver particles Anodic Stripping
Oxidation current, realize electrochemical signals two level amplification.
（2）High specific.The method of the present invention has high specific, other bacteriums other than object to this detection architecture without
Interference, this is because the immunosensor structure of the present invention is established on the basis of antigen-antibody specificity identification.
（3）Testing result is reliable.The rate of recovery is between 90%~110%.
（4）Step is simple, and detection is quick.After synthetic capturing unit, signal element, it is only necessary to which capturing unit is placed in electrode
Surface plus four food-borne pathogens sample, plus signal unit, Electrochemical Detection steps can be completed；Except antigen-antibody is exempted from
Epidemic disease combined outside institute's required time, and this method is rapidly completed with little need for other times, detection.
（5）The method has universality, need to only change antibody type and inspection to food-borne pathogens not of the same race can be realized
It surveys.
In conclusion the present invention is prepared for one kind detecting food-borne pathogens based on quick scan anode Stripping Voltammetry technology
Electrochemical immunosensor, using composite nano materials AgNPs@g-C3N4Electrochemical immunosensor is built, with g-C3N4Table
A large amount of AgNPs of face load are electrochemistry beacon, and using quick scan anode Stripping Voltammetry technology as detection method, detection is food-borne
Pathogenic bacteria.The result shows that：The method of the present invention high sensitivity, specificity is good, result is accurate and reliable, at low cost, quick, detection process
, there is preferable application prospect in the advantages that simple and quick.
Description of the drawings
Fig. 1 is capturing unit of the present invention (cAb@Fe3O4) preparation flow figure；
Fig. 2 is signal element of the present invention (sAb-AgNPs@g-C3N4) preparation flow figure；
Fig. 3 is the electrochemical immunosensor detection that the quick scan anode Stripping Voltammetry technology of the present invention detects food-borne pathogens
Schematic diagram；
Fig. 4 is the comparison diagram of the present invention quick scan anode leaching and plain scan Anodic stripping；
Fig. 5 is the linear relationship chart of the Anodic Stripping peak point current of various concentration Vibrio vulnificus and the log concentration of Vibrio vulnificus；
Fig. 6 is the linear relationship of the Anodic Stripping peak point current of various concentration vibrio parahemolyticus and the log concentration of Vibrio vulnificus
Figure.
Specific implementation mode
Below in conjunction with attached drawing embodiment, present invention is further described in detail.
Specific embodiment one
A kind of electrochemical immunosensor preparation side for detecting food-borne pathogens based on quick scan anode Stripping Voltammetry technology
Method includes the following steps：
（1）Capturing unit（cAb@Fe3O4）Preparation（Preparation flow is as shown in Figure 1）
A. by 0.1~0.3 g FeCl2•4H2O and 0.6~0.9 g FeCl3•6H2O is dissolved in the secondary water of 40~50 mL deoxygenations
In, stirred under nitrogen atmosphere makes it be uniformly mixed, and 28 wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, are warming up to
80 DEG C and 2 h of this thermotonus is maintained to be cooled to room temperature under nitrogen protection after reaction, water is cleaned to neutrality, is settled to
50 mL are to get Fe3O4Nanoparticles solution；
B. by 40~50 mL Fe3O4Nanoparticles solution and 0.4~0.6 mL 3- aminopropyl triethoxysilanes（APTES）
It mixes, after 0.5 h of ultrasound, stirs 5~8 h at room temperature, through Magneto separate, cleaning, be dispersed in again in 50 mL water to get amino
Change Fe3O4Nanoparticles solution；
C. in 1~3 mL aminations Fe3O4The glutaraldehyde solution of 0.1~0.3 mL 2.5wt% is added in nanoparticles solution,
1~3 h is reacted at room temperature, and the food-borne pathogens that 1~3 mL, 10~50 μ g/mL are added after Magneto separate, cleaning capture antibody
（cAb）, after reacting 1~3 h at room temperature, after Magneto separate, cleaning, it is dispersed in the phosphate of 10~20 pH=7.5~8.2 mL
To get capturing unit in buffer solution（cAb@Fe3O4）Solution；
（2）Signal element（sAb-AgNPs@g-C3N4）Preparation（Preparation flow is as shown in Figure 2）
A. it weighs 5~10 g melamine powders, calcines 3~5 h in Muffle furnace at 500~600 DEG C, be dried in vacuum overnight
Afterwards, you can obtain nitridation carbon dust, 0.8~1.5 g nitridation carbon dusts is taken to be added to 80~120 mL, 4~6 M HNO3Solution
In, flow back 16~20 h at 120~150 DEG C, and after being cooled to room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by gained
16 h of sediment continuous ultrasound, up to graphite phase carbon nitride after vacuum drying（g-C3N4）；
B. 1~3 mL, 10 mM AgNO are added in 30~60 mL water3Solution is heated to boiling, and adds 1~3 mL
3 mM NaBH of 1wt% citric acid three sodium solutions and 300~500 μ L4Solution is vigorously stirred 1 h under fluidized state, is cooled to room
Wen Hou is centrifuged, and cleaning is distributed in 40~60 mL water to get nano silver particles（AgNPs）Solution；
C. in 10~70 mL nano silver solutions be added 30~70 mg graphite phase carbon nitrides, at room temperature stir 16~
24 h, washing centrifugation, abandon supernatant, are distributed in 40~60 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 120~150 μ L and contains 10~100 mmol/L
1- ethyls-（3- dimethylaminopropyls）Carbodiimide hydrochloride（EDC）With 1~10 mmol/L n-hydroxysuccinimides
（NHS）Mixed solution, 0.8~1.2 M hydrochloric acid tune pH is that 1~2 h is incubated at 4.0~6.0,35 DEG C, is centrifuged, washing, water is fixed
Hold to 200~300 μ L, is 7.0~9.0 with 0.1~0.2 M NaOH solution tune pH, 80~120 μ L, 0.01~1 μ are added
G/mL food-borne pathogenic bacteria antibodies（sAb）It is incubated 3~5 h afterwards, adds 80~100 μ L, 2 wt% bovine serum albumin(BSA)s
（BSA）Solution is incubated 1~2 h with closing activity site, cleans, and centrifugation is distributed in 5~10 mL water to get signal element
sAb-AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode（MGCE）Successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then uses 50vt% successively
Ethanol solution, 50 vt % aqueous solution of nitric acid and distilled water be cleaned by ultrasonic；
B. 5~10 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 5~10 μ L food-borne pathogens samples are then added dropwise
Solution, after being incubated at room temperature 30~50 min, water cleaning adds 5~10 μ L signal units, after being incubated at room temperature 30~50 min,
Water cleans to get electrochemical immunosensor.
Above-mentioned food-borne pathogens include vibrio parahemolyticus, Vibrio vulnificus, staphylococcus aureus, Escherichia coli and sand
Door Salmonella.
Specific embodiment two
The method for detecting food-borne pathogens using electrochemical immunosensor prepared by specific embodiment one, principle such as Fig. 3 institutes
Show, includes the following steps
Use electrochemical immunosensor prepared by above-mentioned specific embodiment one for working electrode, platinum electrode is to electrode, and 0.1
M KCl solution is that supporting electrolyte solution carries out quick scan anode Stripping Voltammetry using the voltage scan rate of 100 V/s
Analysis measures Anodic Stripping peak currenti p （μA）；Measure a series of corresponding Anodic Stripping of food-borne pathogens of various concentrations
Peak currenti p Size, establishi p With food-borne pathogenic bacteria concentration（cfu/mL）Between quantitative relationship；It is according to the quantitative relationship
Food-borne pathogenic bacteria concentration in unknown sample can be measured.
As shown in Figure 4, the quick scan anode leaching of the present invention compared with plain scan Anodic stripping it is found that work as other
When condition is identical, the dissolution peak current using quick scan anode leaching be with hundred times of plain scan Anodic stripping with
On.
Specific embodiment three
The preparation and its detection of the electrochemical immunosensor of Vibrio vulnificus are detected based on quick scan anode Stripping Voltammetry technology
Using specifically including following steps：
（1）Capturing unit（cAb@Fe3O4）Preparation（Preparation flow is as shown in Figure 1）
A. by 0.2 g FeCl2•4H2O and 0.8 g FeCl3•6H2O is dissolved in the secondary water of 45 mL deoxygenations, under nitrogen protection
Stirring makes it be uniformly mixed, and 28wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, is warming up to 80 DEG C and maintains this temperature
2 h of degree reaction are cooled to room temperature under nitrogen protection after reaction, and water is cleaned to neutrality, is settled to 50 mL to get Fe3O4It receives
Rice grain solution；
B. by 45 mL Fe3O4Nanoparticles solution and 0.5 mL 3- aminopropyl triethoxysilanes（APTES）Mixing, ultrasound
After 0.5 h, 7 h are stirred at room temperature, through Magneto separate, cleaning, are dispersed in again in 50 mL water to get amination Fe3O4Nanometer
Grain solution；
C. in 2 mL aminations Fe3O4The glutaraldehyde solution of 0.2 mL 2.5wt% is added in nanoparticles solution, at room temperature instead
2 h are answered, the Vibrio vulnificus capture antibody of 2 mL, 10~50 μ g/mL is added after Magneto separate, cleaning（cAb）, 1 is reacted at room temperature
After~3 h, after Magneto separate, cleaning, it is dispersed in the phosphate buffer solution of 15 pH=7.5~8.2 mL single to get capture
Member（cAb@Fe3O4）Solution；
（2）Signal element（sAb-AgNPs@g-C3N4）Preparation（Preparation flow is as shown in Figure 2）
A. it weighs 7 g melamine powders, 4 h is calcined at 550 DEG C in Muffle furnace, after being dried in vacuum overnight, you can obtain nitrogen
Change carbon dust, 1.2 g nitridation carbon dusts is taken to be added to 100 mL, 5 M HNO3In solution, flow back 18 h at 135 DEG C, cooling
To after room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by 16 h of gained sediment continuous ultrasound, after vacuum drying to obtain the final product
Graphite phase carbon nitride（g-C3N4）；
B. 2 mL, 10 mM AgNO are added in 45 mL water3Solution is heated to boiling, and adds 2 mL 1wt% citric acids three
3 mM NaBH of sodium solution and 400 μ L4Solution is vigorously stirred 1 h under fluidized state, after being cooled to room temperature, centrifuges, cleans, point
It is scattered in 50 mL water to get nano silver particles（AgNPs）Solution；
C. 50 mg graphite phase carbon nitrides are added in 40 mL nano silver solutions, stir 20 h at room temperature, wash from
The heart abandons supernatant, is distributed in 50 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 135 μ L and contains 50 mmol/L 1- ethyls-（3-
Dimethylaminopropyl）Carbodiimide hydrochloride（EDC）With 5 mmol/L n-hydroxysuccinimides（NHS）Mixed solution,
0.8~1.2 M hydrochloric acid tune pH is that 1~2 h is incubated at 4.0~6.0,35 DEG C, is centrifuged, and washing, water is settled to 250 μ L, with 0.1
~0.2 M NaOH solution tune pH is 7.0~9.0, and 100 μ L, 0. 1 μ g/mL Vibrio vulnificus antibody is added（sAb）After be incubated 4
H adds 90 μ L 2wt% bovine serum albumin(BSA)s（BSA）Solution is incubated 1~2 h with closing activity site, cleans, centrifuges, point
It is scattered in 7 mL water to get signal element sAb-AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode（MGCE）Successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then uses 50vt% successively
Ethanol solution, 50 vt % aqueous solution of nitric acid and distilled water be cleaned by ultrasonic；
B. 8 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 8 μ L Vibrio vulnificus sample solutions are then added dropwise, and are incubated at room temperature
After 40 min, water cleaning adds 8 μ L signal units, and after being incubated at room temperature 40 min, water cleans to get electrochemistry immuno-sensing
Device；
（4）The quantitative analysis of Vibrio vulnificus
Use electrochemical immunosensor prepared by above-mentioned specific embodiment one for working electrode, platinum electrode is to electrode, and 0.1
M KCl solution is that supporting electrolyte solution carries out quick scan anode Stripping Voltammetry using the voltage scan rate of 100 V/s
Analysis measures Anodic Stripping peak currenti p （μA）；Measure a series of corresponding Anodic Stripping of food-borne pathogens of various concentrations
Peak currenti p Size, establishi p With Vibrio vulnificus concentration（cfu/mL）Between quantitative relationship；It can be surveyed according to the quantitative relationship
Determine Vibrio vulnificus concentration in unknown sample.
As shown in figure 5, Anodic Stripping peak currenti p Linear relationship, line are presented between the logarithm of Vibrio vulnificus solution concentration
Property ranging from 4~104Cfu/mL, linear equation are：Y=- 51.28+132*logx, related coefficientR 2=0.994, detection is limited to 1
cfu/mL.It is linear good, it can be used for unknown sample detection.
Specific embodiment four
Based on quick scan anode Stripping Voltammetry technology detect vibrio parahemolyticus electrochemical immunosensor preparation and its
Detection application, specifically includes following steps：
（1）Capturing unit（cAb@Fe3O4）Preparation
A. by 0.1 g FeCl2•4H2O and 0.6 g FeCl3•6H2O is dissolved in the secondary water of 40 mL deoxygenations, under nitrogen protection
Stirring makes it be uniformly mixed, and 28wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, is warming up to 80 DEG C and maintains this temperature
2 h of degree reaction are cooled to room temperature under nitrogen protection after reaction, and water is cleaned to neutrality, is settled to 50 mL to get Fe3O4It receives
Rice grain solution；
B. by 40 mL Fe3O4Nanoparticles solution and 0.4 mL 3- aminopropyl triethoxysilanes（APTES）Mixing, ultrasound
After 0.5 h, 5 h are stirred at room temperature, through Magneto separate, cleaning, are dispersed in again in 50 mL water to get amination Fe3O4Nanometer
Grain solution；
C. in 1 mL aminations Fe3O4The glutaraldehyde solution of 0.1 mL 2.5wt% is added in nanoparticles solution, at room temperature instead
1 h is answered, the vibrio parahemolyticus capture antibody of 1 mL, 50 μ g/mL is added after Magneto separate, cleaning（cAb）, 1 is reacted at room temperature
After h, after Magneto separate, cleaning, it is dispersed in the phosphate buffer solution of 10 pH=7.5~8.2 mL to get capturing unit
（cAb@Fe3O4）Solution；
（2）Signal element（sAb-AgNPs@g-C3N4）Preparation
A. it weighs 5 g melamine powders, 5 h is calcined at 500 DEG C in Muffle furnace, after being dried in vacuum overnight, you can obtain nitrogen
Change carbon dust, 0.8 g nitridation carbon dusts is taken to be added to 80 mL, 6 M HNO3In solution, flow back 20 h at 120 DEG C, is cooled to
After room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by 16 h of gained sediment continuous ultrasound, up to stone after vacuum drying
Black phase carbon nitride（g-C3N4）；
B. 1 mL, 10 mM AgNO are added in 30 mL water3Solution is heated to boiling, and adds 1 mL 1wt% citric acids three
3 mM NaBH of sodium solution and 300 μ L4Solution is vigorously stirred 1 h under fluidized state, after being cooled to room temperature, centrifuges, cleans, point
It is scattered in 40 mL water to get nano silver particles（AgNPs）Solution；
C. 30 mg graphite phase carbon nitrides are added in 10 mL nano silver solutions, stir 16 h at room temperature, wash from
The heart abandons supernatant, is distributed in 40 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 120 μ L and contains 10 mmol/L 1- ethyls-（3-
Dimethylaminopropyl）Carbodiimide hydrochloride（EDC）With 1 mmol/L n-hydroxysuccinimides（NHS）Mixed solution,
0.8~1.2 M hydrochloric acid tune pH is that 1~2 h is incubated at 4.0~6.0,35 DEG C, is centrifuged, and washing, water is settled to 200 μ L, with 0.1
~0.2 M NaOH solution tune pH is 7.0~9.0, and 80 μ L, 1 μ g/mL vibrio parahemolyticus antibody is added（sAb）After be incubated 3
H adds 80 μ L 2wt% bovine serum albumin(BSA)s（BSA）Solution is incubated 1~2 h with closing activity site, cleans, centrifuges, point
It is scattered in 5 mL water to get signal element sAb-AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode（MGCE）Successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then uses 50vt% successively
Ethanol solution, 50 vt % aqueous solution of nitric acid and distilled water be cleaned by ultrasonic；
B. 5 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 5 μ L vibrio parahemolyticus sample solutions, room temperature is then added dropwise
After being incubated 30~50 min, water cleaning adds 5 μ L signal units, and after being incubated at room temperature 30 min, water cleans to get electrochemistry
Immunosensor；
（4）The quantitative analysis of vibrio parahemolyticus
Use electrochemical immunosensor prepared by above-mentioned specific embodiment one for working electrode, platinum electrode is to electrode, and 0.1
M KCl solution is that supporting electrolyte solution carries out quick scan anode Stripping Voltammetry using the voltage scan rate of 100 V/s
Analysis measures Anodic Stripping peak currenti p （μA）；Measure a series of corresponding Anodic Stripping of food-borne pathogens of various concentrations
Peak currenti p Size, establishi p With vibrio parahemolyticus concentration（cfu/mL）Between quantitative relationship；It is according to the quantitative relationship
Vibrio parahemolyticus concentration in unknown sample can be measured.
As shown in fig. 6, Anodic Stripping peak currenti p Linear close is presented between the logarithm of vibrio parahemolyticus solution concentration
System, the range of linearity are 40~105Cfu/mL, linear equation are：Y=- 192+125*logx, related coefficientR 2=0.992, detection limit
For 10 cfu/mL.It is linear good, it can be used for unknown sample detection.
Specific embodiment five
With above-mentioned specific embodiment three, difference lies in：The food-borne pathogens of detection are staphylococcus aureus, specific steps
It is as follows：
（1）The preparation of capturing unit
A. by 0.3 g FeCl2•4H2O and 0.9 g FeCl3•6H2O is dissolved in the secondary water of 50 mL deoxygenations, under nitrogen protection
Stirring makes it be uniformly mixed, and 28wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, is warming up to 80 DEG C and maintains this temperature
2 h of degree reaction are cooled to room temperature under nitrogen protection after reaction, and water is cleaned to neutrality, is settled to 50 mL to get Fe3O4It receives
Rice grain solution；
B. by 50 mL Fe3O4Nanoparticles solution is mixed with 0.6 mL 3- aminopropyl triethoxysilanes, 0.5 h of ultrasound
Afterwards, 8 h are stirred at room temperature, through Magneto separate, cleaning, are dispersed in again in 50 mL water to get amination Fe3O4Nano particle is molten
Liquid；
C. in 3 mL aminations Fe3O4The glutaraldehyde solution of 0.3 mL 2.5wt% is added in nanoparticles solution, at room temperature instead
3 h are answered, the staphylococcus aureus capture antibody of 3 mL, 10 μ g/mL is added after Magneto separate, cleaning, reacts 3 h at room temperature
Afterwards, it after Magneto separate, cleaning, is dispersed in molten to get capturing unit in the phosphate buffer solution of 20 pH=7.5~8.2 mL
Liquid；
（2）Signal element（sAb-AgNPs@g-C3N4）Preparation
A. it weighs 10 g melamine powders, 3 h is calcined at 600 DEG C in Muffle furnace, after being dried in vacuum overnight, you can obtain nitrogen
Change carbon dust, 1.5 g nitridation carbon dusts is taken to be added to 120 mL, 4 M HNO3In solution, flow back 16 h at 150 DEG C, cooling
To after room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by 16 h of gained sediment continuous ultrasound, after vacuum drying to obtain the final product
Graphite phase carbon nitride（g-C3N4）；
B. 3 mL, 10 mM AgNO are added in 60 mL water3Solution is heated to boiling, and adds 3 mL 1wt% citric acids three
3 mM NaBH of sodium solution and 500 μ L4Solution is vigorously stirred 1 h under fluidized state, after being cooled to room temperature, centrifuges, cleans, point
It is scattered in 60 mL water to get nano silver particles（AgNPs）Solution；
C. 70 mg graphite phase carbon nitrides are added in 70 mL nano silver solutions, stir 24 h at room temperature, wash from
The heart abandons supernatant, is distributed in 60 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 150 μ L and contains 100 mmol/L 1- ethyls-（3-
Dimethylaminopropyl）Carbodiimide hydrochloride（EDC）With the mixed solution of 10 mmol/L n-hydroxysuccinimides, 0.8
~1.2 M hydrochloric acid tune pH are that 1~2 h is incubated at 4.0~6.0,35 DEG C, are centrifuged, and washing, water is settled to 300 μ L, with 0.1~
0.2 M NaOH solution tune pH is 7.0~9.0, and 120 μ L, 0.01 μ g/mL Staphylococcus aureus antibodies are added（sAb）Afterwards
5 h are incubated, 100 μ L 2wt% bovine serum albumin(BSA)s are added（BSA）Solution is incubated 2 h with closing activity site, cleans, from
The heart is distributed in 10 mL water to get signal element sAb-AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode（MGCE）Successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then uses 50vt% successively
Ethanol solution, 50 vt % aqueous solution of nitric acid and distilled water be cleaned by ultrasonic；
B. 10 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 10 μ L staphylococcus aureus sample solutions are then added dropwise,
After being incubated at room temperature 50 min, water cleaning adds 10 μ L signal units, and after being incubated at room temperature 50 min, water cleans to get electrochemistry
Immunosensor；
（4）The quantitative analysis of staphylococcus aureus
Use electrochemical immunosensor prepared by above-mentioned specific embodiment one for working electrode, platinum electrode is to electrode, and 0.1
M KCl solution is that supporting electrolyte solution carries out quick scan anode Stripping Voltammetry using the voltage scan rate of 100 V/s
Analysis measures Anodic Stripping peak currenti p （μA）；Measure a series of corresponding Anodic Stripping of food-borne pathogens of various concentrations
Peak currenti p Size, establishi p With staphylococcus aureus concentration（cfu/mL）Between quantitative relationship；According to the quantitative relationship
Staphylococcus aureus concentration in unknown sample can be measured.
Specific embodiment six
With above-mentioned specific embodiment three, difference lies in：The food-borne pathogens of detection are Escherichia coli.
Specific embodiment seven
With above-mentioned specific embodiment three, difference lies in：The food-borne pathogens of detection are salmonella.
Specific embodiment eight
In order to investigate the accuracy and actual application value of this method, using standard samples recovery, i.e., it is added in tap water certain
The food-borne pathogens of concentration.As shown in Table 1 using the method for the present invention testing result, relative standard deviation（RSD）Less than 5.9%,
The rate of recovery is 93.6~105.4%, as a result satisfactory.Show detection of the present invention for a variety of food-borne pathogens in water sample
As a result accurate and reliable.
The testing result of a variety of food-borne pathogens in 1 tap water of table（n = 5）
It these results suggest that, the present invention successfully constructs a kind of based on the food-borne cause of quick scan anode Stripping Voltammetry technology detection
The electrochemical immunosensor of germ, which highly sensitive can detect food-borne pathogens with high selectivity, easy to operate,
As a result accurate and reliable.By changing the antibody in this immunosensor, you can realize that the high sensitivity of different pathogenic bacteria, height are special
Property detection.
Certainly, above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art
Those of ordinary skill is in the essential scope of the present invention, the variations, modifications, additions or substitutions made, and should also belong to the present invention's
Protection domain.
Claims (3)
1. prepared by a kind of electrochemical immunosensor being detected food-borne pathogens based on quick scan anode Stripping Voltammetry technology
Method, it is characterised in that include the following steps：
（1）The preparation of capturing unit
A. by 0.1~0.3 g FeCl2•4H2O and 0.6~0.9 g FeCl3•6H2O is dissolved in the secondary water of 40~50 mL deoxygenations
In, stirred under nitrogen atmosphere makes it be uniformly mixed, and 28wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, are warming up to 80
DEG C and maintain 2 h of this thermotonus to be cooled to room temperature under nitrogen protection after reaction, water is cleaned to neutrality, is settled to 50
ML is to get Fe3O4Nanoparticles solution；
B. by 40~50 mL Fe3O4Nanoparticles solution is mixed with 0.4~0.6 mL 3- aminopropyl triethoxysilanes, is surpassed
After 0.5 h of sound, 5~8 h are stirred at room temperature, through Magneto separate, cleaning, are dispersed in again in 50 mL water to get amination Fe3O4It receives
Rice grain solution；
C. in 1~3 mL aminations Fe3O4The glutaraldehyde solution of 0.1~0.3 mL 2.5wt% is added in nanoparticles solution,
1~3 h is reacted at room temperature, and the food-borne pathogens capture that 1~3 mL, 10~50 μ g/mL are added after Magneto separate, cleaning is anti-
Body after Magneto separate, cleaning, is dispersed in the phosphate-buffered of 10~20 pH=7.5~8.2 mL after reacting 1~3 h at room temperature
To get capturing unit solution in solution；
（2）The preparation of signal element
A. it weighs 5~10 g melamine powders, calcines 3~5 h in Muffle furnace at 500~600 DEG C, be dried in vacuum overnight
Afterwards, you can obtain nitridation carbon dust, 0.8~1.5 g nitridation carbon dusts is taken to be added to 80~120 mL, 4~6 M HNO3Solution
In, flow back 16~20 h at 120~150 DEG C, and after being cooled to room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by gained
16 h of sediment continuous ultrasound, up to graphite phase carbon nitride after vacuum drying；
B. 1~3 mL, 10 mM AgNO are added in 30~60 mL water3Solution is heated to boiling, and adds 1~3 mL
3 mM NaBH of 1wt% citric acid three sodium solutions and 300~500 μ L4Solution is vigorously stirred 1 h under fluidized state, is cooled to room
Wen Hou is centrifuged, and cleaning is distributed in 40~60 mL water to get nano silver solution；
C. in 10~70 mL nano silver solutions be added 30~70 mg graphite phase carbon nitrides, at room temperature stir 16~
24 h, washing centrifugation, abandon supernatant, are distributed in 40~60 mL water to get composite nano materials AgNPs@g-C3N4Solution；
D. 200 μ L composite nano materials AgNPs@g-C are taken3N4Solution is added 120~150 μ L and contains 10~100 mmol/L 1-
Ethyl-（3- dimethylaminopropyls）The mixing of carbodiimide hydrochloride and 1~10 mmol/L n-hydroxysuccinimides is molten
Liquid, 0.8~1.2 M hydrochloric acid tune pH are that 1~2 h is incubated at 4.0~6.0,35 DEG C, are centrifuged, washing, water is settled to 200~300
μ L are 7.0~9.0 with 0.1~0.2 M NaOH solution tune pH, 80~120 μ L, 0.01~1 food-borne causes of μ g/mL are added
It is incubated 3~5 h after germ antibody, adds 80~100 μ L 2wt% bovine serum albumin solutions and is incubated 1~2 h to close work
Property site, clean, centrifugation, be distributed in 5~10 mL water to get signal element sAb-AgNPs@g-C3N4；
（3）The assembling of electrochemical immunosensor
A. by magnetic glassy carbon electrode successively with 1.0,0.3,0.05 μm of Al2O3It is polished to minute surface, then uses the ethyl alcohol of 50vt% successively
Solution, the aqueous solution of nitric acid of 50 vt % and distilled water are cleaned by ultrasonic；
B. 5~10 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 5~10 μ L food-borne pathogens samples are then added dropwise
Product solution, after being incubated at room temperature 30~50 min, water cleaning adds 5~10 μ L signal units, is incubated at room temperature 30~50 min
Afterwards, water cleaning is to get electrochemical immunosensor.
2. the electrochemistry according to claim 1 for detecting food-borne pathogens based on quick scan anode Stripping Voltammetry technology
Immunosensor preparation method, it is characterised in that：The food-borne pathogens include vibrio parahemolyticus, Vibrio vulnificus, gold
Staphylococcus aureus, Escherichia coli and salmonella.
3. a kind of method detecting food-borne pathogens using electrochemical immunosensor described in claim 1, feature exist
In including the following steps
Use electrochemical immunosensor described in claim 1 for working electrode, platinum electrode is to electrode, 0.1 M KCl
Solution is supporting electrolyte solution, using the voltage scan rate of 100 V/s, carries out quick scan anode stripping volt ampere analysis,
Measure Anodic Stripping peak currenti p ；Measure a series of corresponding Anodic Stripping peak current of food-borne pathogens of various concentrationsi p 's
Size establishes Anodic Stripping peak currenti p With the quantitative relationship between food-borne pathogenic bacteria concentration；According to the quantitative relationship
Measure food-borne pathogenic bacteria concentration in unknown sample.
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