CN103792368A - Surface plasma resonance immunosense chip as well as preparation method and application thereof - Google Patents
Surface plasma resonance immunosense chip as well as preparation method and application thereof Download PDFInfo
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- CN103792368A CN103792368A CN201410040559.7A CN201410040559A CN103792368A CN 103792368 A CN103792368 A CN 103792368A CN 201410040559 A CN201410040559 A CN 201410040559A CN 103792368 A CN103792368 A CN 103792368A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
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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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
Abstract
The invention relates to the field of biochips, and particularly relates to a surface plasma resonance immunosense chip as well as a preparation method and application thereof. The surface plasma resonance immunosense chip is prepared by the following steps: fixing a shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or a shrimp tropomyosin monoclonal antibody on a solid phase carrier as a bioprobe; generating surface plasma resonance response by utilizing a gold membrane; modifying sulfydryl on the surface of the gold membrane by utilizing a self-assembly monomolecular layer technique; and fixing the probe on the biochip after the chip is activated. The biochip can be applied to detection of the shrimp tropomyosin monoclonal antibody or the shrimp tropomyosin monoclonal antibody ascites, and detection of the allergen (shrimp tropomyosin), has the advantage of simplicity and convenience in operation, rapidity, high flexibility, no need of being labeled, low cost, simple device, no pollution to the environment and the like, and is hopeful to realize the field real-time detection of a great amount of samples.
Description
Technical field
The present invention relates to biochip field, be specifically related to a kind of surface plasma body resonant vibration immune sensing chip and preparation method thereof and application.
Background technology
Nineteen nineties, the Human Genome Project (Human Genome Project, HGP) and the appearance that develops into biochip, biochip technology and the development of molecular biology related discipline provide advantage.Biochip (biochip) is according to special interactional principle between biomolecule, and biochemical analysis process is bonded to chip surface, thereby realizes the high flux fast detecting to DNA, RNA, polypeptide, protein and other biological composition.Protein-chip (protein chip) is that some non-nucleic acid living matters such as protein or antigen are fixed on miniature base and are obtained, and the probe on chip is configured to protein or chip effective object is that protein person is referred to as protein-chip.
Although biochip technology has been obtained significant progress, obtain attracting attention of common people, but still existing many problems, such as technical costs costliness, complicated operation, poor repeatability, analyst coverage be narrower, conventionally need radioelement mark or fluorescence labeling etc.These problems are mainly manifested in that the preparation, probe of sample is synthetic and fixing, the reading and the aspect such as analyze of the mark of molecule, data.Fluorescence method is to use at present maximum labeling methods, and sensitivity is not high, needs lucifuge, expensive for detection of the laser scanner of result.Isotope-labelling method needs isotope and radioautograph, uses inconvenience, and complicated operation is consuming time, has serious pollution.The present invention does not need mark, and can solve fluorescence labeling and radioelement mark has the problem of pollution to environment, and sample preparation is simple simultaneously, and detection time is short, easy and simple to handle.
The whole world approximately has 30%～40% people to suffer from various anaphylactic diseases.Food irritability reaction is after body is stimulated by same antigen again, the specific immune response of exhibit tissue damage or physiological function disorder, clinical manifestation comprises nettle rash, dermatitis herpetiformis, the irritated syndrome in oral cavity, enteropathy syndrome, asthma and allergic rhinitis etc., even can cause anaphylactic shock, have a strong impact on patient's quality of life.Food hypersenstivity relates to the major issue of food security, and marine product is a wherein important class.Shrimp and goods delicious flavour thereof, nutritious, but there is higher sensitization, and be one of eight large class allergenic foods of FAO's announcement, in irritated patient, approximately there is 20% prawn allergy, children's's incidence of disease, up to 60%, has a strong impact on people's quality of life.All produce shrimp all over the world, output is abundant, along with the globalization of aquatic products trade, the anaphylactogen problem of aquatic products is subject to extensive concern, and corresponding rules have all successively been formulated for the anaphylactogen composition in food in FDA (Food and Drug Adminstration) (FDA), EFSA (EFSA), Japan and Hong-Kong.A lot of countries are more and more stricter to the requirement of imported food anaphylactogen label, and in food, the detection of anaphylactogen has become new food international trade technology barriers.Therefore in food, the detection of anaphylactogen and safety evaluation have become important problem of food security research field.The development of therefore, the detection of shrimp allergen, anaphylactoid research, Claritin is the problem of being badly in need of research.For shrimp allergen in Site Detection food, detect clinically shrimp allergen in serum, study method special, sensitive, quickly significant.
Anaphylactogen (anaphylactogen) is called again allergen, is the antigen that can induce the hypersensitivity of I type; Allergic reaction, claims again allergic reaction, is abnormal, harmful, pathologic immune response.Shrimp allergy belongs to type i allergic reaction, and its mechanism is that anaphylactogen enters after body, brings out the B cell generation specific IgE that can synthesize IgE, and IgE and mast cell, basophilic stippling Cell binding become sensitization target cell.Once IgE is combined with target cell, body will present sensitization.In the time that body contacts same anaphylactogen stimulation again, the IgE of the identical anaphylactogen again entering on the target cell of being combined is in conjunction with specific reaction occurs, cause the allergic symptoms such as oedema, asthma, nettle rash, diarrhoea, be accompanied by patients serum IgE level and raise.The domestic laboratory diagnosis to anaphylactia, mainly adopts anaphylactogen detection and specific IgE, IgG detection method at present, detects the main dependence on import of reagent, and testing cost is higher.Meanwhile, in food, the detection of anaphylactogen and safety evaluation are the important topics in food security field.
At present, the detection of anaphylactogen is mainly enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA), the method is highly sensitive, but testing process complexity, the fluorescent marker contaminated environment of use, instrument is expensive, is restricted in actual applications.Surface plasma body resonant vibration biochip does not need mark, can detect in real time intermolecular interaction, has been widely used in many fields such as Pharmaceutical Analysis, food analysis, environmental monitoring.
Surface plasma body resonant vibration (surface plasmon resonance, SPR) is a kind of physical optics phenomenon based on Maxwell's Electromagnetic theory, and it results from the metal surface with this characteristic of complex permittivity.Applications of surface plasmon resonance (SPR) utilizes P polarized light in the time of glass and metallic film interface experiences total internal reflection, to enter the evanescent wave (evanescent wave) in metallic film, the free electron causing in metal produces surface plasma, in the time that the wave vector of evanescent wave and the wave vector of surface plasma match, the two will resonate, the energy of incident light is absorbed by surface plasma, reflective light intensity sharply declines, SPR phenomenon occurs, and at this moment corresponding incident angle is called resonant angle or resonance angle.If probe or part are fixed on to sensor chip surface, containing the sample of the analysans sensor surface of flowing through, if flow through the material that contains combination with it in the sample of biochip surface, the interaction occurring between them changes the medium refraction index that causes chip surface, causes that SPR resonance angle changes.By the interactional specificity between detection spr signal change detection molecules, concentration, dynamics, affinity, synergy, Interactions Mode etc.In biomolecular interaction analysis process, target material can naturally be present in analyte, analyzes under field conditions (factors), has guaranteed the authenticity of acquired results.Therefore, surface plasmon resonance biosensor have advantages of in real time, exempt from mark, simple to operate, quantitatively detection of biological molecule, detect two or more intermolecular combinations.At present, SPR technology has been widely used in the fields such as food inspection, drug screening, medical diagnosis on disease.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming that overcomes prior art is with not enough, a kind of surface plasma body resonant vibration immune sensing chip is provided, this chip surface is fixed shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody, as probe.
Another object of the present invention is to provide the preparation method of above-mentioned surface plasma body resonant vibration immune sensing chip.
A further object of the present invention is to provide the application of above-mentioned surface plasma body resonant vibration immune sensing chip.
Object of the present invention is achieved through the following technical solutions:
A preparation method for surface plasma body resonant vibration immune sensing chip, comprises following steps:
(1) the golden film that deposit thickness is 40～60nm in substrate of glass is as the solid phase carrier of surface plasma body resonant vibration immune sensing chip;
(2) in double dish, inject and contain HS (CH
2)
10cOOH(sulfydryl undecanoic acid) and HS (CH
2)
6oH(mercaptohexanoic acid) ethanolic solution, chemical modification is carried out in golden film surface; Then inject PBS buffer solution for cleaning, wash off not in conjunction with material; Nitrogen dries up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) in flow cell, pass into PBS buffer solution for cleaning, after baseline stability, add the mixed liquor activation chip of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC); Then pass into PBS buffer solution for cleaning;
(5) in biochip surface fixing shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody, the variation at recording surface plasma resonance angle, the process that monitoring bio probe is fixing, SPR response has significantly rising, probe fixed effect is better, in the time that resonance angle no longer raises, probe fixation procedure finishes; Then pass into PBS buffer solution for cleaning;
(6) add the remaining ester bond of monoethanolamine (Eth) solution sealing deactivation; Then pass into PBS buffer solution for cleaning, obtain surface plasma body resonant vibration immune sensing chip.
Substrate of glass described in step (1) is preferably the circular glass sheet of diameter 20mm, thickness 1mm; Described golden film thickness is preferably 50nm;
HS (CH described in step (2)
2)
10the final concentration of COOH is 0.5～10mM, is preferably 1mM; Described HS (CH
2)
6the final concentration of OH is 4.5～100mM, is preferably 9mM;
The condition of the chemical modification described in step (2) is that 37 ℃ of temperature are bathed, and lucifuge reaction 0.5～48h, is preferably 2h;
The temperature that the described nitrogen of step (2) dries up is 40～60 ℃, is preferably 50 ℃;
The final concentration of the N-hydroxy-succinamide described in step (4) is 0.05mol/L; The final concentration of described N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
The time of the activation chip described in step (4) is 3～60min, is preferably 15min;
The molecular weight of the shrimp tropomyosin described in step (5) is 36kD, and concentration is 0.105mg/mL; Described shrimp tropomyosin monoclonal antibody ascites is to be that 100 times of 15000 shrimp tropomyosin monoclonal antibody ascites dilutions obtain by tiring; The concentration of described shrimp tropomyosin monoclonal antibody is 0.0042mg/mL; The described set time is 10～90min, is preferably 30min;
The concentration of the ethanolamine solutions described in step (6) is 1mol/L, and pH value is 8.5; The time of described sealing deactivation is 1～10min, is preferably 6min;
The number of times of the PBS buffer solution for cleaning described in step (2), (4), (5) and (6) is 1～5 time, and each time of cleaning is 1～5min;
The number of times of the PBS buffer solution for cleaning described in step (2), (4), (5) and (6) is preferably 3 times, and each time of cleaning is preferably 2min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4;
The application of described surface plasma body resonant vibration immune sensing chip, this application comprises the application that utilizes surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detection shrimp tropomyosin monoclonal antibody ascites and the application that utilizes surface plasma body resonant vibration immune sensing chip detection anaphylactogen (shrimp tropomyosin);
The described surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody of utilizing, comprises following steps:
(1) Criterion curve:
The shrimp tropomyosin monoclonal antibody of concentration known or known shrimp tropomyosin monoclonal antibody ascites PBS damping fluid of tiring are configured to the standard solution of variable concentrations or extension rate, take PBS damping fluid as benchmark, each standard solution passes into respectively micro-flow cell of surface plasma body resonant vibration instrument, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve,
(2) detection of unknown sample:
Unknown sample is injected to the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step () obtains, calculates the extension rate of concentration or the unknown shrimp tropomyosin monoclonal antibody ascites of unknown sample Prawn tropomyosin monoclonal antibody;
(3) detection of next sample:
After immune response in step (two) detects and finishes, micro-flow cell first passes into PBS buffer solution for cleaning 1～5 time, and each time of cleaning is 1～5min; Pass into SDS-HCl solution again and clean 1～3 time, each scavenging period is 1～3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 1～5 time, each time of cleaning is 1～3min; Baseline falls back in SPR response, continues to detect next sample.
The probe of the surface plasma body resonant vibration immune sensing chip described in step () is shrimp tropomyosin;
The concentration range of the shrimp tropomyosin monoclonal antibody standard solution described in step () is 1 μ g/mL～4.2mg/mL; Described shrimp tropomyosin monoclonal antibody ascites extension rate scope is 10～150 times;
The detection limit of the standard solution described in step () is preferably 100 μ L; The described immunoreactive reaction time is 3～5min, is preferably 3min; Described surface plasma body resonant vibration sweep limit is 0.5～4 °, is preferably 1～2 °;
The detection limit of the unknown sample described in step (two) is 100 μ L; The described immunoreactive reaction time is 3～5min, is preferably 3min; Described surface plasma body resonant vibration sweep limit is 0.5～4 °, is preferably 1～2 °;
The concentration that in SDS-HCl solution described in step (three), the massfraction of SDS is 1%, HCl is 0.01mol/L.
PBS buffer solution for cleaning number of times described in step (three) is preferably 3 times, and each time of cleaning is preferably 3min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4;
The antigen generation immune response of the antibody in sample and chip surface, forms Ag-Ab bond, and SPR response increases, and shrimp tropomyosin and shrimp tropomyosin monoclonal antibody binding ability are stronger.With the increase of sample Prawn tropomyosin monoclonal anti bulk concentration, dilutability is little, and the same reaction time, more antibody is combined with antigen, and SPR response increases.Method thus, can obtain detecting the typical curve of shrimp tropomyosin monoclonal antibody, and with the increase of extension rate, SPR response declines, and is the curve of falling S.The method is applicable to anaphylactogen antibody test in anaphylactogen antibody screening, patient serum sample.
SPR biochip can the multiple samples of continuous detecting, can carry out the Site Detection of a large amount of samples.At least can 80 samples of continuous detecting, exceed 80 samples, detection sensitivity can reduce, and chip needs regeneration.The method is hopeful to be directly used in clinical, detects the anaphylactogen antibody in patients serum.
The described surface plasma body resonant vibration immune sensing chip detection anaphylactogen (shrimp tropomyosin) that utilizes, comprises following concrete steps:
(I) Criterion curve:
The shrimp tropomyosin of concentration known is configured to the standard solution of variable concentrations with PBS damping fluid, take PBS damping fluid as benchmark, each standard solution passes into respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve,
The detection of (II) unknown sample:
Unknown sample is passed into the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step (I) obtains, calculates the concentration of unknown sample Prawn tropomyosin;
The detection of (III) next sample:
After immune response in step (II) finishes, micro-flow cell first passes into PBS buffer solution for cleaning 1～5 time, and each time of cleaning is 1～5min; Pass into SDS-HCl solution again and clean 1～3 time, each scavenging period is 1～3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 1～5 time, each time of cleaning is 1～3min; Baseline falls back in SPR response, continues to detect next sample.
The probe of the surface plasma body resonant vibration immune sensing chip described in step (1) is shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody;
The concentration range of the standard solution described in step (1) is 1 μ g/mL～2.1mg/mL;
The detection limit of the standard solution described in step (1) is 100 μ L; The described immunoreactive reaction time is 3～5min; The preferred reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 0.5～4 °, is preferably 1～2 °;
The detection limit of the unknown sample described in step (2) is 100 μ L; The described immunoreactive reaction time is 3～5min; The preferred reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 0.5～4 °, is preferably 1～2 °;
The concentration that in SDS-HCl solution described in step (3), the massfraction of SDS is 1%, HCl is 0.01mol/L.
PBS buffer solution for cleaning number of times described in step (3) is preferably 3 times, and each time of cleaning is preferably 3min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4;
Shrimp tropomyosin described in the present invention, shrimp tropomyosin monoclonal antibody ascites and shrimp tropomyosin monoclonal antibody are according to list of references (Huang Jianfang, Wang Caixia, Xiang Junjian, Lv Si, Huang Sheng. the preparation of Environment of Litopenaeus vannamei Low main allergen-tropomyosin monoclonal antibody and allergen epitope [J] thereof. Journal of Immunology, 2012,28(9): 746-749.) prepare;
This method can be carried out detection and the safety evaluation of anaphylactogen in a large amount of food.Use not antibody purification or antibody purification as bioprobe, do not need mark and two anti-, reduced cost, the step that simplifies the operation, shortens detection time.
Principle of the present invention is: utilize self assembled monolayer technology at golden film finishing sulfydryl, activated rear chip can stationary probe.Fix respectively shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites, shrimp tropomyosin monoclonal antibody as bioprobe at solid phase carrier (glass sheet of gold-plated film), utilize golden film to produce surface plasma body resonant vibration (SPR) response.Sample passes into micro-flow cell, with probe generation immune response, by reacting of P polarized light detection biochip probe and sample.When detecting while having in sample with the material of probe matching, resonance angle changes, and SPR detector records testing result, according to typical curve, analyzes experimental result.
The present invention compared with prior art has following outstanding advantage and beneficial effect:
(1) the present invention does not need sample mark, and environmentally safe has overcome the problem of current biochip test method fluorescence labeling or radioelement marking contaminated environment.
(2) the present invention only needs monospecific antibody, and the simple and consumption of sample preparation does not even need purifying less, has simplified operation steps, and experimental implementation is easy, has shortened detection time.
(3) the present invention adopts shrimp tropomyosin or its antibody as probe, and application SPR technology is carried out input, highly sensitive.
(4) present device is simple, does not need expensive detecting instrument, has reduced use cost and the experiment condition of chip, can realize with portable SPR detector the field quick detection of sample.
(5) by the present invention, can be widely used in common lab, be expected in supermarket, market, factory etc. need the food quality place of monitoring in real time, realize the field quick detection of a large amount of samples.
In a word, this technology succeed in developing by really realize biochip technology fast, in real time, feature that can Site Detection, quantitative result, highly sensitive and cost is low, free from environmental pollution rapidly.Surface plasma body resonant vibration biochip test anaphylactogen and monoclonal antibody, for direct method detects, portable devices, experimental implementation is simple, and detection time is short, does not need mark, do not use two to resist, environmentally safe, the Site Detection of a large amount of samples in applicable market, harbour, fairground.Surface plasma body resonant vibration biochip method is the detection, clinical diagnosis, shrimp allergen vaccine design of China's food security field anaphylactogen and formulates China's food allergen tag control and established certain technical foundation, for the identity management of allergen in the detection of food allergen and food provides technical support.
Accompanying drawing explanation
Fig. 1 is biological chips detection system tactic pattern figure of the present invention.
Fig. 2 is chip system mode chart of the present invention.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
N-hydroxy-succinamide (NHS), N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC), monoethanolamine (Eth), lauryl sodium sulfate (SDS), HS (CH
2)
10cOOH(sulfydryl undecanoic acid) and HS (CH
2)
6oH(mercaptohexanoic acid) be purchased from Sigma company of the U.S.; Other reagent is purchased from Beijing chemical reagents corporation.PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the Na that final concentration is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water; The pH value of described PBS damping fluid is 7.4; The concentration that in SDS-HCl solution, the massfraction of SDS is 1%, HCl is 0.01mol/L.
The molecular weight of shrimp tropomyosin is 36kD, tiring of shrimp tropomyosin monoclonal antibody ascites mother liquor is 15000, shrimp tropomyosin monoclonal anti bulk concentration is 4.2mg/mL, according to list of references (Huang Jianfang, Wang Caixia, Xiang Junjian, Lv Si, Huang Sheng. the preparation of Environment of Litopenaeus vannamei Low main allergen-tropomyosin monoclonal antibody and allergen epitope [J] thereof. Journal of Immunology, 2012,28(9): 746-749.) prepare;
The preparation of embodiment 1 surface plasma body resonant vibration immune sensing chip
(1), using the circular glass sheet of diameter 20mm, thickness 1mm as substrate, the golden film that deposit thickness is 50nm in substrate is as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) in double dish, pass into and contain the HS (CH that final concentration is 1mM
2)
10cOOH(sulfydryl undecanoic acid) and 9mM HS (CH
2)
6oH(mercaptohexanoic acid) ethanolic solution 4mL, 37 ℃ temperature bathe, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully clean 3 times, each 2min, washes off not in conjunction with material; 50 ℃ of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) in flow cell, pass into PBS buffer solution for cleaning 3 times, each 2min; After baseline stability, add the mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC), activation chip 15min; Then pass into PBS buffer solution for cleaning 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is that the final concentration of 0.05mol/L, N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
(5) be 36kD in biochip surface fixed member amount, the shrimp tropomyosin 100 μ L that concentration is 0.105mg/mL, as bioprobe, the set time is 30min; Record the variation of SPR resonance angle, the process that monitoring bio probe is fixing; Then pass into PBS buffer solution for cleaning 3 times, each 2min;
(6) add ethanolamine solutions (pH=8.5) the 100 μ L of 1mol/L, the remaining ester bond 6min of sealing deactivation; PBS buffer solution for cleaning 3 times, each scavenging period 2min.
The preparation of embodiment 2 surface plasma body resonant vibration immune sensing chips
(1), using the circular glass sheet of diameter 20mm, thickness 1mm as substrate, the golden film that deposit thickness is 50nm in substrate is as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) in double dish, pass into and contain the HS (CH that final concentration is 1mM
2)
10cOOH(sulfydryl undecanoic acid) and 9mM HS (CH
2)
6oH(mercaptohexanoic acid) ethanolic solution 4mL, 37 ℃ temperature bathe, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully clean 3 times, each 2min, washes off not in conjunction with material; 50 ℃ of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) in flow cell, pass into PBS buffer solution for cleaning 3 times, each 2min; After baseline stability, add the mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC), activation chip 15min; Then pass into PBS buffer solution for cleaning 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is that the final concentration of 0.05mol/L, N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
(5) at the shrimp tropomyosin monoclonal antibody ascites mother liquor 100 μ L of 100 times of biochip surface fixed dilutions, as bioprobe, the time is 30min; Record the variation of SPR resonance angle, the process that monitoring bio probe is fixing; Then pass into PBS buffer solution for cleaning 3 times, each 2min;
(6) add ethanolamine solutions (PH=8.5) the 100 μ L of 1mol/L, the remaining ester bond of sealing deactivation, 6min; PBS buffer solution for cleaning 3 times, each scavenging period 2min.
The preparation method of embodiment 3 surface plasma body resonant vibration immune sensing chips
(1) using the circular glass sheet of diameter 20mm, thickness 1mm as substrate, the golden film that deposit thickness is 50nm in substrate is as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) in double dish, pass into and contain the HS (CH that final concentration is 1mM
2)
10cOOH(sulfydryl undecanoic acid) and 9mM HS (CH
2)
6oH(mercaptohexanoic acid) ethanolic solution 4mL, 37 ℃ temperature bathe, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully clean 3 times, each 2min, washes off not in conjunction with material; 50 ℃ of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) in flow cell, pass into PBS buffer solution for cleaning 3 times, each 2min; After baseline stability, add the mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC), activation chip 15min; Then PBS buffer solution for cleaning 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is that the final concentration of 0.05mol/L, N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
(5) the shrimp tropomyosin monoclonal antibody 100 μ L that are 0.0042mg/mL at biochip surface fixed concentration, as bioprobe, the time is 30min; Record the variation of SPR resonance angle, the process that monitoring bio probe is fixing; Then pass into PBS buffer solution for cleaning 3 times, each 2min;
(6) add monoethanolamine (PH8.5) the 100 μ L of 1mol/L, the remaining ester bond of sealing deactivation, 6min; PBS buffer solution for cleaning 3 times, each scavenging period 2min.
Embodiment 4 utilizes the application of surface plasma body resonant vibration immune sensing chip detection anaphylactogen (shrimp tropomyosin)
(1) Criterion curve:
Be configured to the standard solution of variable concentrations shrimp tropomyosin with PBS damping fluid: the shrimp tropomyosin mother liquor that is 2.1mg/mL by concentration dilutes respectively 10,25,50,75,100 times; Take PBS damping fluid as benchmark, each standard solution is got respectively 100 μ L and passes into successively micro-flow cell of surface plasma resonance instrument, probe generation immune response on the surface plasma body resonant vibration immune sensing chip preparing with embodiment 2, the reaction time is set as 3min; Surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain each standard solution immunoreactive surface plasma resonance kinetic curve occurs, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve;
(2) detection of unknown sample:
The unknown sample of 100 μ L is passed into the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, and the reaction time is set as 3min; Surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain unknown sample immunoreactive surface plasma body resonant vibration kinetic curve occurs, the recurrence typical curve that integrating step (1) obtains, calculates the concentration of unknown sample Prawn tropomyosin;
(3) next sample detection:
After a sample detection finishes, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and each time of cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each 3min, dissociates Ag-Ab bond; Then pass into PBS damping fluid 3 times, each time of cleaning is 3min; Baseline falls back in SPR response, continues to detect next sample.
Concentration is that the shrimp tropomyosin mother liquor of 2.1mg/mL dilutes respectively 10,50,100 times, and sample concentration is respectively: 210 μ g/mL, 42 μ g/mL, 21 μ g/mL, be designated as its actual value; Utilize the content of the above-mentioned sample solution Prawn of surface plasma body resonant vibration immune sensing chip detection tropomyosin: the recurrence typical curve that the data integrating step (1) of measuring by SPR detector obtains, calculates the detected value of sample Prawn tropomyosin concentration simultaneously; Detected value and actual value contrast, as shown in table 1.The absolute deviation of detected value and actual value is all less, and its relative deviation is also all less, illustrates that SPR biological chips detection system has good detectability, and experimental technique is feasible.
Table 1 utilizes the interpretation of result of surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin
Embodiment 5 utilizes surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody ascites
(1) Criterion curve:
Be configured to the shrimp tropomyosin monoclonal antibody ascites standard solution of variable concentrations with PBS damping fluid: be 150,100,80,50,40,20,10 times of 15000 not antibody purification ascites mother liquor dilutions by tiring; Take PBS damping fluid as benchmark, each standard solution respectively 100 μ L passes into micro-flow cell of surface plasma resonance instrument successively, probe generation immune response on the surface plasma body resonant vibration immune sensing chip preparing with embodiment 1, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve;
(2) detection of unknown sample:
The unknown sample of 100 μ L is injected to the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step (1) obtains, calculates the concentration of unknown sample Prawn tropomyosin;
(3) detection of next sample:
After immune response in step (2) finishes, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and each time of cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each scavenging period is 3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 3 times, each time of cleaning is 3min; Baseline falls back in SPR response, continues to detect next sample.
Be that 15000 shrimp tropomyosin monoclonal antibody ascites dilutes respectively 10,50,100,150 by tiring, be designated as its actual value; Utilize the content of the above-mentioned sample solution Prawn of surface plasma body resonant vibration immune sensing chip detection tropomyosin monoclonal antibody ascites: the recurrence typical curve that the data integrating step (1) of measuring by SPR detector obtains, calculates the detected value of sample Prawn tropomyosin concentration simultaneously; Detected value and actual value contrast, as shown in table 2.The absolute deviation of detected value and actual value is all less, and its relative deviation is also all less, illustrates that SPR biological chips detection system has good detectability, and experimental technique is feasible.
Table 2 utilizes the interpretation of result of surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody ascites
Embodiment 6 utilizes the application of surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody
(1) Criterion curve:
Be configured to the shrimp tropomyosin monoclonal antibody standard solution of variable concentrations with PBS damping fluid: the shrimp tropomyosin monoclonal antibody that is 4.2mg/mL by concentration is diluted 300,200,150,75,50 times successively; Take PBS damping fluid as benchmark, each standard solution respectively 100 μ L passes into micro-flow cell of surface plasma resonance instrument successively, probe generation immune response on the surface plasma body resonant vibration immune sensing chip preparing with embodiment 1, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve;
(2) detection of unknown sample:
The unknown sample of 100 μ L is injected to the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step (1) obtains, calculates the concentration of unknown sample Prawn tropomyosin;
(3) continuous detecting of unknown sample:
After immune response in step (2) finishes, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and each time of cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each scavenging period is 3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 3 times, each time of cleaning is 3min; Baseline falls back in SPR response, continues to detect next sample.
The shrimp tropomyosin monoclonal antibody that is 4.2mg/mL by concentration, dilutes respectively 1000,500,100,50 times, and concentration is respectively and is designated as 0.0042,0.0084,0.042,0.084mg/mL, is designated as actual value.The recurrence typical curve that obtains of data integrating step (1) that SPR detector is measured, calculates the predicted value of antibody concentration in sample, and predicted value and actual value contrast, as shown in table 1.The absolute deviation of predicted value and actual value is all less, and its relative deviation is also all less, illustrates that SPR biological chips detection system has good predictive ability, and experimental technique is feasible.
Table 3 utilizes the interpretation of result of surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (9)
1. a preparation method for surface plasma body resonant vibration immune sensing chip, is characterized in that comprising following steps:
(1) the golden film that deposit thickness is 40～60nm in substrate of glass is as the solid phase carrier of surface plasma body resonant vibration immune sensing chip;
(2) in double dish, inject and contain HS (CH
2)
10cOOH and HS (CH
2)
6the ethanolic solution of OH, carries out chemical modification to golden film surface; Then inject PBS buffer solution for cleaning, wash off not in conjunction with material; Nitrogen dries up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) in flow cell, pass into PBS buffer solution for cleaning, after baseline stability, add the mixed liquor activation chip of N-hydroxy-succinamide and N-ethyl-N '-(dimethylamino-propyl) carbodiimide; Then pass into PBS buffer solution for cleaning;
(5) in biochip surface fixing shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody, the variation at recording surface plasma resonance angle, the process that monitoring bio probe is fixing, in the time that resonance angle no longer raises, probe fixation procedure finishes; Then pass into PBS buffer solution for cleaning;
(6) add the remaining ester bond of ethanolamine solutions sealing deactivation; Then pass into PBS buffer solution for cleaning, obtain surface plasma body resonant vibration immune sensing chip.
2. the preparation method of surface plasma body resonant vibration immune sensing chip according to claim 1, is characterized in that:
Substrate of glass described in step (1) is the circular glass sheet of diameter 20mm, thickness 1mm; Described golden film thickness is 50nm;
HS (CH described in step (2)
2)
10the final concentration of COOH is 1mM; Described HS (CH
2)
6the final concentration of OH is 9mM;
The condition of the chemical modification described in step (2) is that 37 ℃ of temperature are bathed, lucifuge reaction 2h;
The temperature that the described nitrogen of step (2) dries up is 50 ℃;
The final concentration of the N-hydroxy-succinamide described in step (4) is 0.05mol/L; The final concentration of described N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
The time of the activation chip described in step (4) is 15min;
The molecular weight of the shrimp tropomyosin described in step (5) is 36kD, and concentration is 0.105mg/mL; Described shrimp tropomyosin monoclonal antibody ascites is to be that 100 times of 15000 shrimp tropomyosin monoclonal antibody ascites dilutions obtain by tiring; The concentration of described shrimp tropomyosin monoclonal antibody is 0.0042mg/mL; The described set time is 30min;
The concentration of the ethanolamine solutions described in step (6) is 1mol/L, and pH value is 8.5; The time of described sealing deactivation is 6min;
The number of times of the PBS buffer solution for cleaning described in step (2), (4), (5) and (6) is 3 times, and each time of cleaning is 2min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4.
3. a surface plasma body resonant vibration immune sensing chip, is characterized in that being prepared by method described in claim 1 or 2.
4. the application of surface plasma body resonant vibration immune sensing chip claimed in claim 3 in biochip field.
5. the application of surface plasma body resonant vibration immune sensing chip according to claim 4 in biochip field, is characterized in that: comprise the application that utilizes surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detection shrimp tropomyosin monoclonal antibody ascites and the application that utilizes surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin.
6. the application of surface plasma body resonant vibration immune sensing chip according to claim 5 in biochip field, is characterized in that: the described surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody of utilizing comprises following steps:
(1) Criterion curve:
The shrimp tropomyosin monoclonal antibody of concentration known or known shrimp tropomyosin monoclonal antibody ascites PBS damping fluid of tiring are configured to the standard solution of variable concentrations or extension rate, take PBS damping fluid as benchmark, each standard solution passes into respectively micro-flow cell of surface plasma body resonant vibration instrument, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve,
(2) detection of unknown sample:
Unknown sample is injected to the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step () obtains, calculates the extension rate of concentration or the unknown shrimp tropomyosin monoclonal antibody ascites of unknown sample Prawn tropomyosin monoclonal antibody;
(3) detection of next sample:
After immune response in step (two) detects and finishes, micro-flow cell first passes into PBS buffer solution for cleaning 1～5 time, and each time of cleaning is 1～5min; Pass into SDS-HCl solution again and clean 1～3 time, each scavenging period is 1～3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 1～5 time, each time of cleaning is 1～3min; Baseline falls back in SPR response, continues to detect next sample.
7. the application of surface plasma body resonant vibration immune sensing chip according to claim 6 in biochip field, is characterized in that:
The probe of the surface plasma body resonant vibration immune sensing chip described in step () is shrimp tropomyosin;
The concentration range of the shrimp tropomyosin monoclonal antibody standard solution described in step () is 1 μ g/mL～4.2mg/mL; Described shrimp tropomyosin monoclonal antibody ascites extension rate scope is 10～150 times;
The detection limit of the standard solution described in step () is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1～2 °;
The detection limit of the unknown sample described in step (two) is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1～2 °;
The concentration that in SDS-HCl solution described in step (three), the massfraction of SDS is 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (three) is 3 times, and each time of cleaning is 3min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4.
8. the application of surface plasma body resonant vibration immune sensing chip according to claim 5 in biochip field, is characterized in that: utilize the application of surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin to comprise following steps:
(I) Criterion curve:
The shrimp tropomyosin of concentration known is configured to the standard solution of variable concentrations with PBS damping fluid, take PBS damping fluid as benchmark, each standard solution passes into respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma resonance kinetic curve of each standard solution, using concentration of standard solution as horizontal ordinate, resonance angle is as ordinate, drawing curve, and carry out polynomial curve fitting, obtain and return typical curve,
The detection of (II) unknown sample:
Unknown sample is passed into the probe generation immune response on micro-flow cell and surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out in chip reaction zone, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of unknown sample, the recurrence typical curve that integrating step (I) obtains, calculates the concentration of unknown sample Prawn tropomyosin;
The detection of (III) next sample:
After immune response in step (II) finishes, micro-flow cell first passes into PBS buffer solution for cleaning 1～5 time, and each time of cleaning is 1～5min; Pass into SDS-HCl solution again and clean 1～3 time, each scavenging period is 1～3min; , Ag-Ab bond is dissociated; Then pass into PBS damping fluid 1～5 time, each time of cleaning is 1～3min; Baseline falls back in SPR response, continues to detect next sample.
9. the application of surface plasma body resonant vibration immune sensing chip according to claim 8 in biochip field, is characterized in that:
The probe of the surface plasma body resonant vibration immune sensing chip described in step (1) is shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody;
The concentration range of the standard solution described in step (1) is 1 μ g/mL～2.1mg/mL;
The detection limit of the standard solution described in step (1) is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1～2 °;
The detection limit of the unknown sample described in step (2) is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1～2 °;
The concentration that in SDS-HCl solution described in step (3), the massfraction of SDS is 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (3) is 3 times, and each time of cleaning is 3min;
Described PBS damping fluid composed as follows: the NaH that final concentration is 2mmol/L
2pO
4, the final concentration Na that is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4.
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