CN104142393A - Biosensor sensitive membrane and application in detection of clenbuterol hydrochloride - Google Patents
Biosensor sensitive membrane and application in detection of clenbuterol hydrochloride Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 239000012528 membrane Substances 0.000 title claims abstract description 39
- 229960001399 clenbuterol hydrochloride Drugs 0.000 title abstract description 5
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 title abstract description 5
- 239000000243 solution Substances 0.000 claims abstract description 81
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 79
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 79
- 239000010931 gold Substances 0.000 claims abstract description 52
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 48
- 229910052737 gold Inorganic materials 0.000 claims abstract description 48
- 230000008878 coupling Effects 0.000 claims abstract description 47
- 238000010168 coupling process Methods 0.000 claims abstract description 47
- 238000005859 coupling reaction Methods 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 45
- 239000000758 substrate Substances 0.000 claims abstract description 32
- 239000011521 glass Substances 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 241000283707 Capra Species 0.000 claims abstract description 27
- QJAOYSPHSNGHNC-UHFFFAOYSA-N octadecane-1-thiol Chemical compound CCCCCCCCCCCCCCCCCCS QJAOYSPHSNGHNC-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000001035 drying Methods 0.000 claims abstract description 11
- 238000011010 flushing procedure Methods 0.000 claims abstract description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 113
- 238000001338 self-assembly Methods 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 36
- 238000005406 washing Methods 0.000 claims description 36
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 33
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- 229910021641 deionized water Inorganic materials 0.000 claims description 22
- 238000001179 sorption measurement Methods 0.000 claims description 16
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 claims description 16
- 238000011068 loading method Methods 0.000 claims description 15
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- 238000000502 dialysis Methods 0.000 claims description 7
- 239000012467 final product Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- 230000009514 concussion Effects 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
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- 229920000151 polyglycol Polymers 0.000 claims description 5
- 239000010695 polyglycol Substances 0.000 claims description 5
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- 230000009467 reduction Effects 0.000 description 4
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- 230000005593 dissociations Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000021393 food security Nutrition 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 3
- 235000015277 pork Nutrition 0.000 description 3
- 238000003380 quartz crystal microbalance Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
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- 239000002184 metal Substances 0.000 description 2
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- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 229940074095 ractopamine Drugs 0.000 description 2
- 229960002052 salbutamol Drugs 0.000 description 2
- 229930195730 Aflatoxin Natural products 0.000 description 1
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 108030001720 Bontoxilysin Proteins 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
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- 125000003368 amide group Chemical group 0.000 description 1
- -1 amino amino, hydroxyl Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001117 clenbuterol Drugs 0.000 description 1
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
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- 239000000470 constituent Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003989 dielectric material Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VZFRNCSOCOPNDB-AJKFJWDBSA-N domoic acid Chemical compound OC(=O)[C@@H](C)\C=C\C=C(/C)[C@H]1CN[C@H](C(O)=O)[C@H]1CC(O)=O VZFRNCSOCOPNDB-AJKFJWDBSA-N 0.000 description 1
- VZFRNCSOCOPNDB-UHFFFAOYSA-N domoic acid Natural products OC(=O)C(C)C=CC=C(C)C1CNC(C(O)=O)C1CC(O)=O VZFRNCSOCOPNDB-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 238000007306 functionalization reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- VZFRNCSOCOPNDB-OXYNIABMSA-N isodomoic acid D Natural products CC(C=C/C=C(/C)C1CNC(C1CC(=O)O)C(=O)O)C(=O)O VZFRNCSOCOPNDB-OXYNIABMSA-N 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 235000020997 lean meat Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
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- 210000004080 milk Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a biosensor sensitive membrane and application in detection of clenbuterol hydrochloride. The sensitive membrane is prepared by the method of: 1) placing a gold membrane loaded on a glass substrate in an ethanol solution of n-Octadecyl mercaptan to perform soaking, and then conducting flushing and drying, thus obtaining a first self-assembled gold membrane; 2) putting the first self-assembled gold membrane in a coupling solution of amino-functionalized graphene or carboxyl-functionalized graphene oxide and goat anti-mouse IgG to perform soaking, then conducting flushing with a phosphate buffer solution and carrying out drying, thus obtaining a second self-assembled gold membrane; and 3) adding an antibody solution dropwise to the surface of the second self-assembled gold membrane to perform antibody assembling so as to make the antibody cover the surface of the second self-assembled gold membrane, conducting standing, then performing flushing with a phosphate buffer solution and carrying out drying. The biosensor sensitive membrane provided by the invention undergoes recognition combination with an antibody through a clenbuterol hydrochloride antigen and its conjugates, and can carry out rapid detection on clenbuterol hydrochloride.
Description
Technical field
The invention belongs to biosensor technology field, be specifically related to a kind of biological sensor sensing film, also relate to the application of this biological sensor sensing film aspect detection clenobuterol hydrochloride simultaneously.
Background technology
In recent years, the frequent generation of the accident causing along with food-safety problem, food-safety problem has caused the extensive concern of all sectors of society.Fast, in sensitive detection food, food cause of disease (salmonella, campylobacter, Escherichia coli etc.) and food contaminant have become the subject matter that researcher faces.
Biology sensor is the sensor as sensitive element with immobilized biological component or biosome itself, is a kind of analysis test apparatus that biochemical reaction can be converted to electric signal.Surface plasmon resonance biosensor is the biology sensor based on surface plasma body resonant vibration (SPR) technology.SPR is very responsive to the electrolytical refractive index in metal surface: different dielectrics, its surface plasma body resonant vibration angle difference; Dielectric of the same race is attached to the amount difference of metal surface, the respective strengths difference of SPR.Based on the biology sensor of this principle, by a kind of molecule with specific recognition attribute, i.e. part, is fixed on metallic film surface conventionally, the analyte in monitoring solution and the cohesive process of this part.In compound formation or dissociation process, the refractive index of metallic film surface solution changes, and can be detected by surface plasmon resonance biosensor in real time.
The advantage that surface plasmon resonance biosensor has becomes a kind of effectively analysis tool in food security research.Ko utilizes Sensitivity enhancement surface plasmon resonance biosensor that anti-salmonella antibody is fixed on to chip surface, utilize the sensor chip after modifying to carry out real-time online detection to salmonella, result shows, the spr sensor after modification can be used for detecting the food-borne pathogens such as salmonella.Leonard etc. utilize surface plasmon resonance biosensor, adopt the method that antibody is fixed on to chip surface to detect Listeria, and Leonard etc. think in the situation that having suitable antibodies to exist, and this research method can be used for the pathogenic bacteria in detection of complex foodstuff samples.Piliarik etc. utilize high flux surface plasmon resonance biosensor, detected the feature nucleotide sequence of brucella, Escherichia coli and Staphylococcus aureus in sensor chip surface fixing DNA probe, this detection can complete in 15 minutes, for the detection of food-borne pathogens provides a kind of fast and convenient detection method.Except pathogenic bacteria, the detection of left drug in food (as: the little molecule objectionable impurities such as melamine, ampicillin, kanamycins, toxin) is also an importance of food security.Nedelkov utilizes surface plasmon resonance biosensor-mass spectrometric hyphenated technique to detect the content of SEB in milk and mushroom, and this research shows, surface plasmon resonance biosensor-mass spectrometric hyphenated technique can be used as the powerful of food analysis.In addition, surface plasmon resonance biosensor is also used to detect the little molecule food contaminants such as botulinum toxin, aflatoxin, vomitoxin, domoic acid, food allergen.Surface plasmon resonance biosensor become food fast, in real time, the important tool of on-line analysis, be widely used in food security field.
The eighties in 20th century, American scientist surprisingly finds that clenobuterol hydrochloride (is commonly called as clenbuterol hydrochloride, a kind of excitant) on raising pork lean meat percentage, there is larger effect, just there is afterwards the method for adding a certain amount of clenobuterol hydrochloride in feed, and promoted the use in feeding live pig as scientific payoffs.But after 1994, a series of poisoning events of clenobuterol hydrochloride that occur because of porker lung, liver are there are, special in Guangdong, there is the larger event of Poisoning Number in Shanghai, the detection of live pig clenobuterol hydrochloride is even more important, but the pork that contains clenobuterol hydrochloride, the color, smell and taste of its meat there is no special difference with the pork of not hydrochloric special human relations Crow, and people cannot debate knowledge by naked eyes, can only rely on coherent detection equipment to detect.
When available technology adopting biology sensor detects the special human relations of hydrochloric acid Crow, due to the special human relations Crow biotinylated molecular weights of hydrochloric acid less (313.7), the method that directly antibody is fixed on to chip surface detection clenobuterol hydrochloride is difficult to realize.
Summary of the invention
The object of this invention is to provide a kind of biological sensor sensing film, the method that solves the chip surface detection clenobuterol hydrochloride that in prior art, antibody is fixed on to biology sensor is difficult to the problem realizing.
Second object of the present invention is to provide the application of a kind of above-mentioned biological sensor sensing film aspect detection clenobuterol hydrochloride.
In order to realize above object, the technical solution adopted in the present invention is: a kind of biological sensor sensing film, prepared by following methods:
1) by loading on golden film on glass substrate and be placed in the ethanolic solution of stearylmercaptan, soak 2～4h and carry out self assembly, after taking-up, successively with ethanol, deionized water rinsing dry, obtain the first self assembly gold film;
2) step 1) gained the first self assembly gold film is placed in to the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg, under 37 DEG C of conditions, soak 2～4h and carry out self assembly, after taking out, rinse and be dried with phosphate buffer (PBS), obtain the second self assembly gold film;
3) antibody-solutions being added drop-wise to step 2) surface of gained the second self assembly gold film carries out antibody assembling, make antibody cover the second self assembly gold film surface, in the environment that 25 DEG C, relative humidity are 40～60%RH, leave standstill after 1～2h, rinse and be dried with phosphate buffer, to obtain final product.
The golden film loading on described in step 1) on glass substrate is prepared by following methods: glass substrate is contained in vacuum evaporation plating machine, vacuumizes with vacuum pump, make the vacuum tightness in plated film chamber reach 1.3 × 10
-2～1.3 × 10
-3pa, heating crucible dissolves highly purified spun gold and flashes to gaseous state gold at the temperature of 1200 DEG C～1400 DEG C; Gaseous state gold particulate, in mobile glass substrate surface deposition, forms one deck continuously and the golden film of light through cooling reduction on glass substrate.The thickness of described golden film is 47～50nm.
Before the described golden film loading on glass substrate uses, through pre-service, described pretreated method is: the golden film loading on glass substrate is put into H
2o
2with in the volume ratio of the concentrated sulphuric acid (mass concentration the is 98%) mixed solution that is 3:7, soak 2～4min, after taking-up, use successively ethanol, deionized water rinsing, drying for standby.
In the ethanolic solution of stearylmercaptan described in step 1), the concentration of stearylmercaptan is 0.28～0.8g/L.
Step 2) described in the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg prepared by following methods:
A. the amino functional functionalized graphene that is 1～5mg/ml by the concentration of 40 parts by volume or carboxyl-functional graphene oxide suspension add in the goat anti-mouse igg suspension that the concentration of 400 parts by volume is 2～10mg/ml, under 18 DEG C, 280rpm condition, 12～24h is cultivated in concussion, obtains potpourri A;
B. by the centrifugal washing twice of step a gained potpourri A, obtain potpourri B;
C. in step b gained potpourri B, add the polyglycol solution that 600 parts by volume mass concentrations are 15%, shaken cultivation 1.5～3h under 15 DEG C, 280rpm condition, obtains mixture C;
D. by the centrifugal washing of step c gained mixture C three times, then add the phosphate buffer of 800 parts by volume, obtain couplings strong solution;
When use, gained couplings strong solution is diluted according to the ratio of 6:100, obtain the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg.
In step b and steps d, the each amount of water of described centrifugal washing is 600 parts by volume, and the time of each centrifugal washing is 10～20min, and centrifugal rotating speed is 12500rpm.
Described amino functional functionalized graphene is prepared by following methods: graphene oxide is distributed in ethylene glycol, and the concentration that makes graphene oxide is 2.5mg/ml, after ultrasonic 2～4h, obtains mixture D; The ratio that is 1:40 according to the volume ratio of ammoniacal liquor and ethylene glycol, adds ammoniacal liquor in mixture D, under 180 DEG C of conditions, reacts after 10～20h, by reaction product vacuum filtration washing, obtains mixture E; Use distill water dialysis 48～96h to remove after foreign particle mixture E, under 60 DEG C of conditions, dry 24～48h, grinds and filters, and to obtain final product.Described filtration is to adopt 500 eye mesh screens to filter.
Described carboxyl-functional graphene oxide is prepared by following methods: graphene oxide is placed in to hydrochloric acid and soaks after 24～48h, and centrifugal and be washed to neutrality, vacuum drying; Dried graphene oxide is mixed with to the suspending liquid that concentration is 1mg/ml; By NaOH and ClCH
2cOONa is according to NaOH, ClCH
2the mass ratio of COONa and graphene oxide is that the ratio of 50:50:1 adds in suspending liquid, and ultrasonic 2～4h, obtains potpourri I; Potpourri I is adjusted to neutrality, and centrifugal and washing, obtains potpourri J; Use distill water dialysis 48～96h to remove after foreign ion potpourri J, under 60 DEG C of conditions, dry 24～48h, grinds and filters, and to obtain final product.Described filtration is to adopt 500 eye mesh screens to filter.
The pH of described phosphate buffer (PBS) is 7.4, is prepared by following methods: by the Na of the KCl of the NaCl of 8.0g, 0.2g, 1.15g
2hPO
4(or the Na of 2.89g
2hPO
412H
2o), the KH of 0.2g
2pO
4be dissolved in 1000ml deionized water, after dissolving completely, at 115 DEG C of high-temperature sterilization 10～15min, under 4 DEG C of conditions, store for future use.
Antibody-solutions described in step 3) is antibody of clenbuteral hydrochloride solution, Anti-ractopamine antibody solution, melamine antibody solution or salbutamol antibody-solutions.Adopt above-mentioned antibody-solutions gained sensitive membrane can be respectively used to detect clenobuterol hydrochloride antigen, Ractopamine antigen, melamine antigen, salbutamol antigen.
The application of above-mentioned biological sensor sensing film aspect detection clenobuterol hydrochloride, described antibody-solutions is antibody of clenbuteral hydrochloride solution, concentration is 4～10 μ g/ml.
Its detection method comprises the following steps:
A. by the couplings of clenobuterol hydrochloride (CLB) and horseradish peroxidase (HRP) according to the dilution proportion of 1:1000 after, mix with the ratio that the clenobuterol hydrochloride of normal concentration is 1:1 according to volume ratio, obtain sample A;
B. described biological sensor sensing film is installed on surface plasmon resonance biosensor, the phosphate buffer that is 7.4 with pH is with the speed on-line rinsing sensitive membrane of 20 μ l/min, until detection curve is basicly stable;
C. sample A is carried out to on-line adsorption detection with the speed of 10 μ l/min, after upon adsorption stablizing, extremely stable with the speed flushing of 20 μ l/min with phosphate buffer, obtain clenobuterol hydrochloride detection curve.
Wherein, the object that adds the couplings of clenobuterol hydrochloride and horseradish peroxidase is to amplify the detected signal of testing sample clenobuterol hydrochloride, improves the detection sensitivity of biological sensor sensing film.
The couplings of described clenobuterol hydrochloride and horseradish peroxidase is prepared by following methods:
I) clenobuterol hydrochloride is dissolved in the deionized water of 0.5 parts by volume, making the concentration of clenobuterol hydrochloride is 0.5～4mg/ml, then adds the hydrochloric acid (1M) of 0.015 parts by volume and the NaNO of 20 parts by volume
2solution (1M), vibration mixes, and obtains potpourri F;
Ii) horseradish peroxidase is dissolved in the deionized water of 1 parts by volume, making the concentration of horseradish peroxidase is 8～11.9mg/ml, then adds the NaOH solution (1M) of 0.02 parts by volume, is cooled to 4 DEG C after mixing, obtains potpourri G;
Iii) potpourri F is added in potpourri G, shaken cultivation 2～4h under 280rpm condition, obtains potpourri H;
Iv) in the phosphate buffer that is 7.4 by potpourri H at pH, dialyse after 1～2 day, add the ethylene glycol of 1 parts by volume, be placed under-20 DEG C of conditions and store for future use.
Biological sensor sensing film of the present invention, taking golden film as substrate, at the little molecule stearylmercaptan of golden film surface self-organization, is fixed on golden film surface by Au-S key by stearylmercaptan; Then the couplings of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg is assembled in its surface, utilize Van der Waals force that amino functional functionalized graphene or carboxyl-functional graphene oxide are fixed on to golden film surface; Finally antibody of clenbuteral hydrochloride is adsorbed on amino functional functionalized graphene or or carboxyl-functional graphene oxide surface on, amino functional functionalized graphene or or carboxyl-functional graphene oxide on amino amino, hydroxyl, the carboxyl condensation that can contain with protein, thereby realize the combination of antibody and Graphene.This sensitive membrane, for biology sensor, is carried out to identity by clenobuterol hydrochloride antigen and conjugates thereof with antibody and is combined, can carry out fast detecting to clenobuterol hydrochloride.When detection, by the sample mixture of clenobuterol hydrochloride and the Clenbuterol couplings sensitive membrane surface of flowing through, the relative response producing by spr sensor, determines the amount of clenobuterol hydrochloride in sample; Or sensitive membrane is placed in the sample mixture of clenobuterol hydrochloride and clenobuterol hydrochloride couplings and cultivates, change by SPR scanning angle, can Quick Measuring test agent in antigen existence whether.
Use biological sensor sensing film of the present invention, can utilize SPR technology to carry out detection in place to clenobuterol hydrochloride.Wherein, the adhesion of goat anti-mouse igg and amino functional functionalized graphene and hydrochloric acid Ke Lunteluo is all relatively good, can play the effect that expands detection signal; When the concentration that detects thing CLB solution is only 10ng/mL, adsorbance is expanded as 140RU from 40RU.The sensitive membrane that relatively prepared by amino functional functionalized graphene and carboxylated functionalization graphene oxide, the CLB solution that concentration is 10ng/mL is respectively 155RU and 140RU all has good detection effect in their lip-deep adsorbances; Sensitive membrane prepared by the carboxyl-functional graphene oxide adopting can reach 0.5ng/mL to the detection bottom line of CLB.Detection method of the present invention, detects the lowest limit lower, have simultaneously simple to operate, save time, be convenient to the advantages such as analysis.
Biological sensor sensing film of the present invention also can be used on QCM (Quartz Crystal Microbalance) (QCM) biology sensor and electrochemica biological sensor.
The present invention adopts electrochemical AC impedance method that the chemical property before and after whole self assembling process and clenobuterol hydrochloride detection is changed and tested, and adopts high flux-surface plasma resonance analyser (HT-SPR) to be combined with antibody and to carry out kinetic measurement in place self assembling process, antigen simultaneously.Result shows, biological sensor sensing film of the present invention is reasonable in design, and Stability Analysis of Structures can be carried out responsive detection fast to CLB.
Brief description of the drawings
Fig. 1 is the preparation flow schematic diagram of the biological sensor sensing film of embodiment 1;
Fig. 2 is the XPS figure of the C1s of amino functional functionalized graphene;
Fig. 3 is the XPS figure of the N1s of amino functional functionalized graphene;
Fig. 4 is the XPS figure of the C1s of carboxyl-functional graphene oxide;
Fig. 5 is the self assembly of embodiment 3 and the curve of adsorption kinetics figure of testing process;
Fig. 6 is the SPR resonance angle variation diagram of different sensitive membrane,
Wherein: a refers to that sensitive membrane is Au;
B refers to that sensitive membrane is Au-C
18sH;
C refers to that sensitive membrane is Au-C
18sH-G-NH
2-IgG;
D refers to that sensitive membrane is Au-C
18sH-G-NH
2-IgG-antibody;
E refers to that sensitive membrane is Au-C
18sH-G-NH
2-IgG-antibody, the simultaneously online clenobuterol hydrochloride that detects;
Fig. 7 is the continuous detecting process kinetics curve map of the clenobuterol hydrochloride solution of embodiment 1 resulting biosensor sensitive membrane to variable concentrations.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
The biological sensor sensing film of the present embodiment, as shown in Figure 1, prepared by following methods:
1) prepare golden film: 2 × 2cm glass substrate is contained in vacuum evaporation plating machine, vacuumizes with vacuum pump, make the vacuum tightness in plated film chamber reach 1.3 × 10
-2pa, heating crucible dissolves highly purified spun gold and flashes to gaseous state gold at the temperature of 1400 DEG C, gaseous state gold particulate, in mobile glass substrate surface deposition, forms one deck continuously and the golden film of light through cooling reduction on glass substrate, and golden film thickness is 47～50nm;
2) the golden film loading on glass substrate is carried out to pre-service: the golden film loading on glass substrate is put into H
2o
2with in the volume ratio of the concentrated sulphuric acid (mass concentration the is 98%) mixed solution that is 3:7, soak 2min, after taking-up, use successively ethanol, deionized water rinsing, drying for standby, is designated as Au;
3) stearylmercaptan that takes 0.0028g is dissolved in the ethanol of 10ml, and ultrasonic 15min makes the ethanolic solution of stearylmercaptan;
4) the pretreated golden film loading on glass substrate is placed in to the ethanolic solution of stearylmercaptan, soak 2h and carry out self assembly, after taking-up, use successively ethanol, deionized water rinsing, and dry up with ear washing bulb, be placed under 4 DEG C of conditions more than 2h, obtain the first self assembly gold film, on golden film, assemble little molecule Stearyl mercaptan, be designated as Au-C
18sH;
5) step 4) gained the first self assembly gold film is placed in to the amino functional functionalized graphene (G-NH of 2ml
2) with the couplings solution of goat anti-mouse igg in, under 37 DEG C of conditions, soak 2h and carry out self assembly, after taking out, rinse and dry up with phosphate buffer (PBS), obtain the second self assembly gold film, be assembled in the first self assembly gold film by the couplings of amino functional functionalized graphene and goat anti-mouse igg, be designated as Au-C
18sH-G-NH
2-IgG;
6) be that the antibody of clenbuteral hydrochloride solution of 6 μ g/ml is added drop-wise to step 5) gained the second self assembly gold film and carries out antibody assembling by 120 μ L, concentration, make antibody of clenbuteral hydrochloride cover the second self assembly gold film surface, in the environment that 25 DEG C, relative humidity are 40%RH, leave standstill after 1h, with phosphate buffer flushing aeration-drying, be placed in again further dry 1h under 37 DEG C of conditions, obtain described biological sensor sensing film, be that antibody of clenbuteral hydrochloride is adsorbed on amino functional functionalized graphene surface, be designated as Au-C
18sH-G-NH
2-IgG-antibody.
Wherein, described amino functional functionalized graphene (G-NH
2) prepared by following methods with the couplings solution of goat anti-mouse igg:
A. the amino functional functionalized graphene suspension that is 1mg/ml by the concentration of 40 μ L adds in the goat anti-mouse igg suspension that the concentration of 400 μ L is 2mg/ml, and under 18 DEG C, 280rpm condition, 12h is cultivated in concussion, obtains potpourri A;
B. by the centrifugal washing twice of step a gained potpourri A, obtain potpourri B; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 10min, and centrifugal rotating speed is 12500rpm;
C. in step b gained potpourri B, add polyglycol (PEG) solution that 600 μ L, mass concentration are 15%, shaken cultivation 1.5h under 15 DEG C, 280rpm condition, obtains mixture C;
D. by the centrifugal washing of step c gained mixture C three times, then add the phosphate buffer of 800 μ L, obtain couplings strong solution; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 10min, and centrifugal rotating speed is 12500rpm;
When use, get 120 μ L couplings strong solutions and be diluted to 2ml, obtain the couplings solution of amino functional functionalized graphene and goat anti-mouse igg.
Wherein, described amino functional functionalized graphene (G-NH
2) prepared by following methods: 100mg graphene oxide is distributed in 40ml ethylene glycol, after ultrasonic 2h, obtains mixture D; 1ml ammoniacal liquor is added in mixture D, reaction system is placed in to the reactor with polytetrafluoroethylsubstrate substrate, under 180 DEG C of conditions, react 10h, by reaction product vacuum filtration washing, obtain mixture E; Use distill water dialysis 48h to remove after foreign particle mixture E, dry 24h under 60 DEG C of conditions, with 500 order stainless steel sift net filtrations, obtains amino functional functionalized graphene after grinding.
The application of the biological sensor sensing film of the present embodiment aspect detection clenobuterol hydrochloride, described antibody-solutions is antibody of clenbuteral hydrochloride solution, concentration is 6 μ g/ml.
Its detection method comprises the following steps:
A. by the couplings of clenobuterol hydrochloride and horseradish peroxidase according to the dilution proportion of 1:1000 after, mix with the ratio that the clenobuterol hydrochloride of normal concentration is 1:1 according to volume ratio, obtain sample A;
B. described biological sensor sensing film is installed on surface plasmon resonance biosensor, the phosphate buffer that is 7.4 with pH is with the speed on-line rinsing sensitive membrane of 20 μ l/min, until detection curve is basicly stable;
C. sample A is carried out to on-line adsorption detection with the speed of 10 μ l/min, after upon adsorption stablizing, extremely stable with the speed flushing of 20 μ l/min with phosphate buffer, obtain clenobuterol hydrochloride detection curve.
Wherein, the object that adds the couplings of clenobuterol hydrochloride and horseradish peroxidase is to amplify the detected signal of testing sample clenobuterol hydrochloride, improves the detection sensitivity of biological sensor sensing film.
The couplings of described clenobuterol hydrochloride and horseradish peroxidase is prepared by following methods:
I) clenobuterol hydrochloride of 0.5mg is dissolved in the deionized water of 0.5ml, then adds the hydrochloric acid (1M) of 15 μ L and the NaNO of 20 μ L
2solution (1M), vibration mixes 15min, obtains potpourri F;
Ii) the horseradish peroxidase of 11.9mg is dissolved in the deionized water of 1ml, then adds the NaOH solution (1M) of 20 μ L, after mixing, be cooled to 4 DEG C, obtain potpourri G;
Iii) potpourri F is added in potpourri G, shaken cultivation 2h under 280rpm condition, obtains potpourri H;
Iv) in the phosphate buffer that is 7.4 by potpourri H at pH, dialyse after 1 day, add the ethylene glycol of 1ml, be placed under-20 DEG C of conditions and store for future use.
Embodiment 2
The biological sensor sensing film of the present embodiment, prepared by following methods:
1) prepare golden film: 2 × 2cm glass substrate is contained in vacuum evaporation plating machine, vacuumizes with vacuum pump, make the vacuum tightness in plated film chamber reach 1.3 × 10
-3pa, heating crucible dissolves highly purified spun gold and flashes to gaseous state gold at the temperature of 1200 DEG C, gaseous state gold particulate, in mobile glass substrate surface deposition, forms one deck continuously and the golden film of light through cooling reduction on glass substrate, and golden film thickness is 47～50nm;
2) the golden film loading on glass substrate is carried out to pre-service: the golden film loading on glass substrate is put into H
2o
2with in the volume ratio of the concentrated sulphuric acid (mass concentration the is 98%) mixed solution that is 3:7, soak 4min, after taking-up, use successively ethanol, deionized water rinsing, drying for standby, is designated as Au;
3) stearylmercaptan that takes 0.0028g is dissolved in the ethanol of 10ml, and ultrasonic 30min makes the ethanolic solution of stearylmercaptan;
4) the pretreated golden film loading on glass substrate is placed in to the ethanolic solution of stearylmercaptan, soak 4h and carry out self assembly, after taking-up, use successively ethanol, deionized water rinsing, and dry up with ear washing bulb, be placed under 4 DEG C of conditions more than 4h, obtain the first self assembly gold film, on golden film, assemble little molecule Stearyl mercaptan, be designated as Au-C
18sH;
5) step 4) gained the first self assembly gold film is placed in to the amino functional functionalized graphene (G-NH of 2ml
2) with the couplings solution of goat anti-mouse igg in, under 37 DEG C of conditions, soak 4h and carry out self assembly, after taking-up, rinse and dry up with phosphate buffer, obtain the second self assembly gold film, be assembled in the first self assembly gold film by the couplings of amino functional functionalized graphene and goat anti-mouse igg, be designated as Au-C
18sH-G-NH
2-IgG;
6) be that the antibody of clenbuteral hydrochloride solution of 8 μ g/ml is added drop-wise to step 5) gained the second self assembly gold film and carries out antibody assembling by 120 μ L, concentration, make antibody of clenbuteral hydrochloride cover the second self assembly gold film surface, in the environment that 25 DEG C, relative humidity are 50%RH, leave standstill after 2h, with phosphate buffer flushing aeration-drying, be placed in again further dry 2h under 37 DEG C of conditions, obtain described biological sensor sensing film, be that antibody of clenbuteral hydrochloride is adsorbed on amino functional functionalized graphene surface, be designated as Au-C
18sH-G-NH
2-IgG-antibody.
Wherein, described amino functional functionalized graphene (G-NH
2) prepared by following methods with the couplings solution of goat anti-mouse igg:
A. the amino functional functionalized graphene suspension that is 1mg/ml by the concentration of 40 μ L adds in the goat anti-mouse igg suspension that the concentration of 400 μ L is 2mg/ml, and under 18 DEG C, 280rpm condition, 24h is cultivated in concussion, obtains potpourri A;
B. by the centrifugal washing twice of step a gained potpourri A, obtain potpourri B; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 10min, and centrifugal rotating speed is 12500rpm;
C. in step b gained potpourri B, add polyglycol (PEG) solution that 600 μ L, mass concentration are 15%, shaken cultivation 3h under 15 DEG C, 280rpm condition, obtains mixture C;
D. by the centrifugal washing of step c gained mixture C three times, then add the phosphate buffer of 800 μ L, obtain couplings strong solution; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 20min, and centrifugal rotating speed is 12500rpm;
When use, get 120 μ L couplings strong solutions and be diluted to 2ml, obtain the couplings solution of amino functional functionalized graphene and goat anti-mouse igg.
Wherein, described amino functional functionalized graphene (G-NH
2) prepared by following methods: 100mg graphene oxide is distributed in 40ml ethylene glycol, after ultrasonic 4h, obtains mixture D; 1ml ammoniacal liquor is added in mixture D, reaction system is placed in to the reactor with polytetrafluoroethylsubstrate substrate, under 180 DEG C of conditions, react 20h, by reaction product vacuum filtration washing, obtain mixture E; Use distill water dialysis 96h to remove after foreign particle mixture E, dry 48h under 60 DEG C of conditions, with 500 order stainless steel sift net filtrations, obtains amino functional functionalized graphene after grinding.
The application of the biological sensor sensing film of the present embodiment aspect detection clenobuterol hydrochloride, described antibody-solutions is antibody of clenbuteral hydrochloride solution, concentration is 8 μ g/ml.
Its detection method comprises the following steps:
A. by the couplings of clenobuterol hydrochloride and horseradish peroxidase according to the dilution proportion of 1:1000 after, mix with the ratio that the clenobuterol hydrochloride of normal concentration is 1:1 according to volume ratio, obtain sample A;
B. described biological sensor sensing film is installed on surface plasmon resonance biosensor, the phosphate buffer that is 7.4 with pH is with the speed on-line rinsing sensitive membrane of 20 μ l/min, until detection curve is basicly stable;
C. sample A is carried out to on-line adsorption detection with the speed of 10 μ l/min, after upon adsorption stablizing, extremely stable with the speed flushing of 20 μ l/min with phosphate buffer, obtain clenobuterol hydrochloride detection curve.
Wherein, the object that adds the couplings of clenobuterol hydrochloride and horseradish peroxidase is to amplify the detected signal of testing sample clenobuterol hydrochloride, improves the detection sensitivity of biological sensor sensing film.
The couplings of described clenobuterol hydrochloride and horseradish peroxidase is prepared by following methods:
I) clenobuterol hydrochloride of 0.5mg is dissolved in the deionized water of 0.5ml, then adds the hydrochloric acid (1M) of 15 μ L and the NaNO of 20 μ L
2solution (1M), vibration mixes 15min, obtains potpourri F;
Ii) the horseradish peroxidase of 11.9mg is dissolved in the deionized water of 1ml, then adds the NaOH solution (1M) of 20 μ L, after mixing, be cooled to 4 DEG C, obtain potpourri G;
Iii) potpourri F is added in potpourri G, shaken cultivation 4h under 280rpm condition, obtains potpourri H;
Iv) in the phosphate buffer that is 7.4 by potpourri H at pH, dialyse after 2 days, add the ethylene glycol of 1ml, be placed under-20 DEG C of conditions and store for future use.
Embodiment 3
The biological sensor sensing film of the present embodiment, prepared by following methods:
1) prepare golden film: 2 × 2cm glass substrate is contained in vacuum evaporation plating machine, vacuumizes with vacuum pump, make the vacuum tightness in plated film chamber reach 1.3 × 10
-2pa, heating crucible dissolves highly purified spun gold and flashes to gaseous state gold at the temperature of 1400 DEG C, gaseous state gold particulate, in mobile glass substrate surface deposition, forms one deck continuously and the golden film of light through cooling reduction on glass substrate, and golden film thickness is 47～50nm;
2) the golden film loading on glass substrate is carried out to pre-service: the golden film loading on glass substrate is put into H
2o
2with in the volume ratio of the concentrated sulphuric acid (mass concentration the is 98%) mixed solution that is 3:7, soak 2min, after taking-up, use successively ethanol, deionized water rinsing, drying for standby, is designated as Au;
3) stearylmercaptan that takes 0.0028g is dissolved in the ethanol of 10ml, and ultrasonic 15min makes the ethanolic solution of stearylmercaptan;
4) the pretreated golden film loading on glass substrate is placed in to the ethanolic solution of stearylmercaptan, soak 2h and carry out self assembly, after taking-up, use successively ethanol, deionized water rinsing, and dry up with ear washing bulb, be placed under 4 DEG C of conditions more than 2h, obtain the first self assembly gold film, on golden film, assemble little molecule Stearyl mercaptan, be designated as Au-C
18sH;
5) step 4) gained the first self assembly gold film is placed in to the carboxyl-functional graphene oxide (GO-COOH) of 2ml and the couplings solution of goat anti-mouse igg, under 37 DEG C of conditions, soak 2h and carry out self assembly, after taking-up, rinse and dry up with phosphate buffer, obtain the second self assembly gold film, be assembled in the first self assembly gold film by the couplings of carboxyl-functional graphene oxide and goat anti-mouse igg, be designated as Au-C
18sH-GO-COOH-IgG;
6) be that the antibody of clenbuteral hydrochloride solution of 4 μ g/ml is added drop-wise to step 5) gained the second self assembly gold film and carries out antibody assembling by 120 μ L, concentration, make antibody of clenbuteral hydrochloride cover the second self assembly gold film surface, in the environment that 25 DEG C, relative humidity are 60%RH, leave standstill after 1h, with phosphate buffer flushing aeration-drying, be placed in again further dry 1h under 37 DEG C of conditions, obtain described biological sensor sensing film, be that antibody of clenbuteral hydrochloride is adsorbed on carboxyl-functional graphene oxide surface, be designated as Au-C
18sH-GO-COOH-IgG-antibody.
Wherein, described carboxyl-functional graphene oxide (GO-COOH) is prepared by following methods with the couplings solution of goat anti-mouse igg:
A. the carboxyl-functional graphene oxide suspension that is 1mg/ml by the concentration of 40 μ L adds in the goat anti-mouse igg suspension that the concentration of 400 μ L is 2mg/ml, and under 18 DEG C, 280rpm condition, 12h is cultivated in concussion, obtains potpourri A;
B. by the centrifugal washing twice of step a gained potpourri A, obtain potpourri B; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 10min, and centrifugal rotating speed is 12500rpm;
C. in step b gained potpourri B, add polyglycol (PEG) solution that 600 μ L, mass concentration are 15%, shaken cultivation 1.5h under 15 DEG C, 280rpm condition, obtains mixture C;
D. by the centrifugal washing of step c gained mixture C three times, then add the phosphate buffer of 800 μ L, obtain couplings strong solution; The each amount of water of described centrifugal washing is 600 μ L, and the time of each centrifugal washing is 10min, and centrifugal rotating speed is 12500rpm;
When use, get 120 μ L couplings strong solutions and be diluted to 2ml, obtain the couplings solution of carboxyl-functional graphene oxide and goat anti-mouse igg.
Wherein, described carboxyl-functional graphene oxide (GO-COOH) is prepared by following methods: graphene oxide is placed in to hydrochloric acid and soaks after 24h, centrifuging under 1250rpm speed conditions, is washed to neutrality, vacuum drying; Dried graphene oxide is mixed with to the suspending liquid that concentration is 1mg/ml; By NaOH and ClCH
2cOONa is according to NaOH, ClCH
2the mass ratio of COONa and graphene oxide is that the ratio of 50:50:1 adds in suspending liquid, and the ultrasonic 2h of water-bath, obtains potpourri I; Potpourri I is adjusted to neutrality with watery hydrochloric acid, centrifugal and wash to product and fully disperse with deionized water, obtain potpourri J; Use distill water dialysis 48h to remove after foreign ion potpourri J, dry 24h under 60 DEG C of conditions, with 500 order stainless steel sift net filtrations, to obtain final product after grinding.
The application of the biological sensor sensing film of the present embodiment aspect detection clenobuterol hydrochloride, described antibody-solutions is antibody of clenbuteral hydrochloride solution, concentration is 4 μ g/ml.
Its detection method comprises the following steps:
A. by the couplings of clenobuterol hydrochloride and horseradish peroxidase according to the dilution proportion of 1:1000 after, mix with the ratio that the clenobuterol hydrochloride of normal concentration is 1:1 according to volume ratio, obtain sample A;
B. described biological sensor sensing film is installed on surface plasmon resonance biosensor, the phosphate buffer that is 7.4 with pH is with the speed on-line rinsing sensitive membrane of 20 μ l/min, until detection curve is basicly stable;
C. sample A is carried out to on-line adsorption detection with the speed of 10 μ l/min, after upon adsorption stablizing, extremely stable with the speed flushing of 20 μ l/min with phosphate buffer, obtain clenobuterol hydrochloride detection curve.
Wherein, the object that adds the couplings of clenobuterol hydrochloride and horseradish peroxidase is to amplify the detected signal of testing sample clenobuterol hydrochloride, improves the detection sensitivity of biological sensor sensing film.
The couplings of described clenobuterol hydrochloride and horseradish peroxidase is prepared by following methods:
I) clenobuterol hydrochloride of 0.5mg is dissolved in the deionized water of 0.5ml, then adds the hydrochloric acid (1M) of 15 μ L and the NaNO of 20 μ L
2solution (1M), vibration mixes 15min, obtains potpourri F;
Ii) the horseradish peroxidase of 11.9mg is dissolved in the deionized water of 1ml, then adds the NaOH solution (1M) of 20 μ L, after mixing, be cooled to 4 DEG C, obtain potpourri G;
Iii) potpourri F is added in potpourri G, shaken cultivation 2h under 280rpm condition, obtains potpourri H;
Iv) in the phosphate buffer that is 7.4 by potpourri H at pH, dialyse after 1 day, add the ethylene glycol of 1ml, be placed under-20 DEG C of conditions and store for future use.
Experimental example 1
This experimental example is to embodiment 1 amino functional functionalized graphene used (G-NH
2) carry out XPS test, by XPSpeak software, the collection of illustrative plates of gained is carried out to swarming, test result is as shown in Figure 2,3.At G-NH
2c1s figure (Fig. 2) in, the peak that contains four, about 284.7eV, 286.3eV, 287.8eV and 285.7eV place, is respectively corresponding C-C/C-H, C-O, O-C=O and tetra-kinds of groups of C-N; G-NH
2in C/O constituent content ratio be respectively 0.55.In addition, at G-NH
2in also there is obvious N1s signal, the content of N element is about 3.1%; By N1s swarming being obtained to two peaks, as shown in Figure 3, be in respectively 399.8eV and 401.3eV place, corresponding to C-N/N-H and N-C=O, illustrate at G-NH
2in not only there is amino group, also there are some amide groups simultaneously.
Experimental example 2
This experimental example carries out XPS test to embodiment 3 carboxyl-functional graphene oxide used (GO-COOH), by XPSpeak software, the collection of illustrative plates of gained is carried out to swarming, and test result as shown in Figure 4.As can be seen from Figure 4, be C-C group at 284.7eV place, be C-O group at 286.3eV place, be C=O group at 287.7eV place, be O-C=O group at 287.8eV place.
Experimental example 3
The curve of adsorption kinetics figure of the self assembling process of embodiment 3 and testing process, as shown in Figure 5.
In the time that self assembly has the golden film surface of stearylmercaptan to pass through G-IgG, CLB antibody, CLB titer, as shown in Figure 5, because adsorption process is attended by de-generation of washing, so curve exists some fluctuations, adsorption curve is more coarse in the variation of curve of adsorption kinetics.What experiment detected employing is online circulation absorption, and the concentration of various solution is all very little, thereby the amount of absorption is very little.GO-COOH was through the absorption of more than 40 minutes, and adsorbance is 100RU, and after being rinsed by phosphate, adsorptive value is reduced to 73RU; The adsorbance of sensitive membrane antagonist is 70RU, becomes 40RU through rinsing adsorbance; When detection, be 130RU to the detected value of clenobuterol hydrochloride.
Experimental example 4
This experimental example detects the SPR resonance angle of different sensitive membrane, and result as shown in Figure 6.
Detect principle according to SPR known, the variation of the tracking detecting sensor chip surface liquid medium refractive index that SPR can realize, in the time that the thickness of golden film surface dielectric changes, its refractive index to light also changes accordingly, causes SPR resonance angle to change.Sensitive membrane that what in Fig. 5, a-d represented respectively is is proof gold film and the resonance angle collection of illustrative plates of the upper stearylmercaptan of assembling, amino functional functionalized graphene and goat anti-mouse igg couplings, antibody of clenbuteral hydrochloride successively thereof, resonance angle collection of illustrative plates when e refers to that sensitive membrane d detects clenobuterol hydrochloride online.As can be seen from Figure 5, compared with proof gold film, when golden film surface is connected to after above material, the corresponding scanning step of SPR resonance angle is down to 28000 from 3800, and this shows that the thickness of golden film increases gradually after self assembly, has also verified the generation of self assembly behavior layer by layer.
Experimental example 5
This experimental example adopts embodiment 1 resulting biosensor sensitive membrane to utilize the method for continuous detecting to detect clenobuterol hydrochloride, and result as shown in Figure 7.
Variable concentrations clenobuterol hydrochloride flows through continuous detecting curve that sensitive membrane surface obtains as shown in Figure 7, comprising 4 continuous detecting processes.The remaining antibody in normal concentration clenobuterol hydrochloride and sensitive membrane surface is in conjunction with 1～4 corresponding rising part on forming curves.Plateau,, correspondence passed into after damping fluid clenobuterol hydrochloride from the process of sensitive membrane surface dissociation.Because antibody is slow from the speed of sensitive membrane surface dissociation, the short time is interior without the dropping signal that significantly dissociates.Along with the increase of clenobuterol hydrochloride concentration and the continuity of detection time, the antibody of biosensor surface and the combination of clenobuterol hydrochloride reach capacity gradually.Its performance on curve of adsorption kinetics is, when 0.5ng/mL curvilinear motion obvious, and along with the increase of concentration afterwards, curvilinear motion is also not obvious.Exactly because this, the antibody limited amount on sensitive membrane surface, along with its variation of carrying out detecting is more and more not obvious.Therefore, embodiment 1 resulting biosensor sensitive membrane to the detection bottom line of clenobuterol hydrochloride at 0.5ng/mL.
In above-described embodiment and experimental example, surface plasmon resonance biosensor used is HT-SPR-2005 type biology sensor.
Claims (10)
1. a biological sensor sensing film, is characterized in that: prepared by following methods:
1) by loading on golden film on glass substrate and be placed in the ethanolic solution of stearylmercaptan, soak 2～4h and carry out self assembly, after taking-up, successively with ethanol, deionized water rinsing dry, obtain the first self assembly gold film;
2) step 1) gained the first self assembly gold film is placed in to the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg, under 37 DEG C of conditions, soak 2～4h and carry out self assembly, after taking-up, rinse and be dried with phosphate buffer, obtain the second self assembly gold film;
3) antibody-solutions being added drop-wise to step 2) surface of gained the second self assembly gold film carries out antibody assembling, make antibody cover the second self assembly gold film surface, in the environment that 25 DEG C, relative humidity are 40～60%RH, leave standstill after 1～2h, rinse and be dried with phosphate buffer, to obtain final product.
2. biological sensor sensing film according to claim 1, is characterized in that: described in load on golden film on glass substrate and use before through pre-service, described pretreated method is: the golden film loading on glass substrate is put into H
2o
2with in the volume ratio of the concentrated sulphuric acid mixed solution that is 3:7, soak 2～4min, after taking-up, use successively ethanol, deionized water rinsing, drying for standby.
3. biological sensor sensing film according to claim 1, is characterized in that: in the ethanolic solution of stearylmercaptan described in step 1), the concentration of stearylmercaptan is 0.28～0.8g/L.
4. biological sensor sensing film according to claim 1, is characterized in that: step 2) described in the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg prepared by following methods:
A. the amino functional functionalized graphene that is 1～5mg/ml by the concentration of 40 parts by volume or carboxyl-functional graphene oxide suspension add in the goat anti-mouse igg suspension that the concentration of 400 parts by volume is 2～10mg/ml, under 18 DEG C, 280rpm condition, 12～24h is cultivated in concussion, obtains potpourri A;
B. by the centrifugal washing twice of step a gained potpourri A, obtain potpourri B;
C. in step b gained potpourri B, add the polyglycol solution that 600 parts by volume mass concentrations are 15%, shaken cultivation 1.5～3h under 15 DEG C, 280rpm condition, obtains mixture C;
D. by the centrifugal washing of step c gained mixture C three times, then add the phosphate buffer of 800 parts by volume, obtain couplings strong solution;
When use, gained couplings strong solution is diluted according to the ratio of 6:100, obtain the couplings solution of amino functional functionalized graphene or carboxyl-functional graphene oxide and goat anti-mouse igg.
5. biological sensor sensing film according to claim 4, is characterized in that: in step b and steps d, the each amount of water of described centrifugal washing is 600 parts by volume, and the time of each centrifugal washing is 10～20min, and centrifugal rotating speed is 12500rpm.
6. according to the biological sensor sensing film described in claim 1,4 or 5, it is characterized in that: described amino functional functionalized graphene is prepared by following methods: graphene oxide is distributed in ethylene glycol, the concentration that makes graphene oxide is 2.5mg/ml, after ultrasonic 2～4h, obtains mixture D; The ratio that is 1:40 according to the volume ratio of ammoniacal liquor and ethylene glycol, adds ammoniacal liquor in mixture D, under 180 DEG C of conditions, reacts after 10～20h, by reaction product vacuum filtration washing, obtains mixture E; Use distill water dialysis 48～96h to remove after foreign particle mixture E, under 60 DEG C of conditions, dry 24～48h, grinds and filters, and to obtain final product.
7. according to the biological sensor sensing film described in claim 1,4 or 5, it is characterized in that: described carboxyl-functional graphene oxide is prepared by following methods: graphene oxide is placed in to hydrochloric acid and soaks after 24～48h, centrifugal and be washed to neutrality, vacuum drying; Dried graphene oxide is mixed with to the suspending liquid that concentration is 1mg/ml; By NaOH and ClCH
2cOONa is according to NaOH, ClCH
2the mass ratio of COONa and graphene oxide is that the ratio of 50:50:1 adds in suspending liquid, and ultrasonic 2～4h, obtains potpourri I; Potpourri I is adjusted to neutrality, and centrifugal and washing, obtains potpourri J; Use distill water dialysis 48～96h to remove after foreign ion potpourri J, under 60 DEG C of conditions, dry 24～48h, grinds and filters, and to obtain final product.
8. the application of biological sensor sensing film as claimed in claim 1 aspect detection clenobuterol hydrochloride, is characterized in that: described antibody-solutions is antibody of clenbuteral hydrochloride solution, concentration is 4～10 μ g/ml.
9. the application of biological sensor sensing film according to claim 8 aspect detection clenobuterol hydrochloride, is characterized in that: its detection method comprises the following steps:
A. by the couplings of clenobuterol hydrochloride and horseradish peroxidase according to the dilution proportion of 1:1000 after, mix with the ratio that the clenobuterol hydrochloride of normal concentration is 1:1 according to volume ratio, obtain sample A;
B. described biological sensor sensing film is installed on surface plasmon resonance biosensor, the phosphate buffer that is 7.4 with pH is with the speed on-line rinsing sensitive membrane of 20 μ l/min, until detection curve is basicly stable;
C. sample A is carried out to on-line adsorption detection with the speed of 10 μ l/min, after upon adsorption stablizing, extremely stable with the speed flushing of 20 μ l/min with phosphate buffer, obtain clenobuterol hydrochloride detection curve.
10. the application of biological sensor sensing film according to claim 8 aspect detection clenobuterol hydrochloride, is characterized in that: the couplings of described clenobuterol hydrochloride and horseradish peroxidase is prepared by following methods:
I) clenobuterol hydrochloride is dissolved in the deionized water of 0.5 parts by volume, making the concentration of clenobuterol hydrochloride is 0.5～4mg/ml, then adds the hydrochloric acid (1M) of 0.015 parts by volume and the NaNO of 20 parts by volume
2solution (1M), vibration mixes, and obtains potpourri F;
Ii) horseradish peroxidase is dissolved in the deionized water of 1 parts by volume, making the concentration of horseradish peroxidase is 8～11.9mg/ml, then adds the NaOH solution (1M) of 0.02 parts by volume, is cooled to 4 DEG C after mixing, obtains potpourri G;
Iii) potpourri F is added in potpourri G, shaken cultivation 2～4h under 280rpm condition, obtains potpourri H;
Iv) in the phosphate buffer that is 7.4 by potpourri H at pH, dialyse after 1～2 day, add the ethylene glycol of 1 parts by volume, be placed under-20 DEG C of conditions and store for future use.
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