CN103792368B - A kind of surface plasma body resonant vibration immune sensing chip and preparation method thereof and application - Google Patents
A kind of surface plasma body resonant vibration immune sensing chip and preparation method thereof and application Download PDFInfo
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- CN103792368B CN103792368B CN201410040559.7A CN201410040559A CN103792368B CN 103792368 B CN103792368 B CN 103792368B CN 201410040559 A CN201410040559 A CN 201410040559A CN 103792368 B CN103792368 B CN 103792368B
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- 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
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Abstract
The present invention relates to biochip field, be specifically related to surface plasma body resonant vibration immune sensing chip and preparation method thereof and application.Described surface plasma body resonant vibration immune sensing chip is by fixing shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody at solid phase carrier as bioprobe, golden film is utilized to produce surface plasma resonance response, utilize self assembled monolayer technology at golden film finishing sulfydryl, after chip activation, on biochip, stationary probe obtains; This biochip can be applicable to shrimp tropomyosin monoclonal antibody or detects the detection of shrimp tropomyosin monoclonal antibody ascites and the detection of anaphylactogen (shrimp tropomyosin), have easy and simple to handle, quick, highly sensitive, do not need mark, the advantage such as cost is low, equipment is simple, environmentally safe, the scene being expected to realize a large amount of sample is detected in real time.
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, develop into biochip, the appearance of biochip technology and the development of the Human Genome Project (Human Genome Project, HGP) and molecular biology related discipline provide advantage.Biochip (biochip) is according to interactional principle special between biomolecule, and biochemical analysis process is bonded to chip surface, thus realizes detecting fast the high flux of DNA, RNA, polypeptide, protein and other biological composition.Protein-chip (protein chip) is fixed on miniature base by some non-nucleic acid living matters such as protein or antigen to obtain, 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 achieves significant progress, obtain attracting attention of common people, but still there is many problems, such as technical costs costliness, complicated operation, poor repeatability, analyst coverage be narrower, usually need radioelement to mark or fluorescence labeling etc.These problems are mainly manifested in the aspect such as the preparation of sample, probe synthesis and fixing, the mark of molecule, the reading of data and analysis.Fluorescence method uses maximum labeling methods at present, and sensitivity is not high, needs lucifuge, and the laser scanner for testing result is expensive.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 to have pollution problem to environment, sample preparation is simple simultaneously, and detection time is short, easy and simple to handle.
The whole world about has the people of 30% ~ 40% to suffer from various anaphylactic disease.Food irritability reaction is after body is stimulated by same antigen again, the specific immune response of exhibit tissue damage or physiological dysfunction, clinical manifestation comprises nettle rash, dermatitis herpetiformis, oral cavity Hypersensitivity Syndrome, enteropathy syndrome, asthma and allergic rhinitis etc., even can cause anaphylactic shock, have a strong impact on the quality of life of patient.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, be one of eight large class allergenic foods of FAO's announcement, about have 20% prawn irritated in irritated patient, children's's incidence of disease, up to 60%, has a strong impact on the quality of life of people.All produce shrimp all over the world, abundance, along with the globalization of aquatic products trade, the allergen issue of aquatic products is subject to extensive concern, and corresponding regulation has 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 country is 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 the important problem of food security research field one.Therefore, the development of the detection of shrimp allergen, anaphylactoid research, Claritin is the problem being badly in need of research.For shrimp allergen in Site Detection food, detect shrimp allergen in serum clinically, study method special, sensitive, quickly significant.
Anaphylactogen (anaphylactogen) is also called allergen, is the antigen of energy inducing type I hypersensitivity; Allergic reaction, also known as allergic reaction, is abnormal, harmful, pathologic immune response.Shrimp allergy belongs to type i allergic reaction, and its mechanism is after anaphylactogen enters body, and bring out the B cell synthesizing IgE and produce specific IgE, IgE and mast cell, basophilic stippling Cell binding become sensitization target cell.IgE is once be combined with target cell, and body will present sensitization.When body contacts same anaphylactogen stimulation again, the IgE of the identical anaphylactogen again entered on the target cell be combined combines and specific reaction occurs, cause the allergic symptoms such as oedema, asthma, nettle rash, diarrhoea, raise along with patients serum IgE level.The domestic laboratory diagnosis to anaphylactia at present, main employing Allergic skin test and specific IgE, IgG detection method, detect the main dependence on import of reagent, testing cost is higher.Meanwhile, in food, the detection of anaphylactogen and safety evaluation are the important topics of field of food safety.
At present, detection mainly enzyme-linked immunosorbent assay (the Enzyme linkedimmunosorbent assay of anaphylactogen, ELISA), the method is highly sensitive, but testing process is complicated, the fluorescent marker contaminated environment used, instrument price is expensive, is restricted in actual applications.Surface plasma body resonant vibration biochip does not need mark, can detect in real time, be widely used in many fields such as Pharmaceutical Analysis, food analysis, environmental monitoring to intermolecular interaction.
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 to enter evanescent wave (evanescent wave) in metallic film when glass and metallic film interface experiences total internal reflection, the free electron caused in metal produces surface plasma, when 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 sensor chip surface, sample containing analysans flows through sensor surface, if flow through in the sample of biochip surface containing the material combined with it, the interaction occurred between them changes causing the medium refraction index of chip surface, causes SPR resonance angle to change.By detecting the interactional specificity, concentration, dynamics, affinity, synergy, Interactions Mode etc. between spr signal change detection molecules.In biomolecular interaction analysis process, target material can naturally be present in be analyzed in thing, analyzes under field conditions (factors), ensure that the authenticity of acquired results.Therefore, surface plasmon resonance biosensor have in real time, exempt to mark, simple to operate, the advantage that can quantitatively detect biomolecule, detect two or more intermolecular combinations.At present, SPR technique 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 overcoming prior art is with not enough, a kind of surface plasma body resonant vibration immune sensing chip is provided, this chip surface fixes shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody, as probe.
Another object of the present invention is to the preparation method that above-mentioned surface plasma body resonant vibration immune sensing chip is provided.
Another object of the present invention is the application providing 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) deposit thickness is the solid phase carrier of golden film as surface plasma body resonant vibration immune sensing chip of 40 ~ 60nm on the glass substrate;
(2) inject containing HS (CH in double dish
2)
10cOOH (mercaptoundecylic acid) and HS (CH
2)
6the ethanolic solution of OH (sulfydryl hexanol), carries out chemical modification to golden film surface; Then inject PBS buffer solution for cleaning, wash off in conjunction with material; Nitrogen dries up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) pass into PBS buffer solution for cleaning in flow cell, after baseline stability, add the mixed liquor activation chip of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC); Then PBS buffer solution for cleaning is passed into;
(5) shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody is fixed in biochip surface, the change at recording surface plasma resonance angle, the process that monitoring bio probe is fixing, SPR response has and significantly raises, then probe fixed effect is better, when resonance angle no longer raises, probe fixation procedure terminates; Then PBS buffer solution for cleaning is passed into;
(6) add monoethanolamine (Eth) solution and close the remaining ester bond of 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 37 DEG C of temperature baths, and lucifuge reaction 0.5 ~ 48h, is preferably 2h;
The temperature that nitrogen described in step (2) dries up is 40 ~ 60 DEG C, is preferably 50 DEG C;
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 be by tiring be 15000 shrimp tropomyosin monoclonal antibody ascites dilute 100 times and obtain; 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 closed deactivation is 1 ~ 10min, is preferably 6min;
The number of times of step (2), (4), (5) and the PBS buffer solution for cleaning described in (6) is 1 ~ 5 time, and the time of each cleaning is 1 ~ 5min;
The number of times of step (2), (4), (5) and the PBS buffer solution for cleaning described in (6) is preferably 3 times, and the time of each cleaning is preferably 2min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, pH value is 7.4;
The application of described surface plasma body resonant vibration immune sensing chip, this application comprises the application utilizing surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detection shrimp tropomyosin monoclonal antibody ascites and the application utilizing surface plasma body resonant vibration immune sensing chip detection anaphylactogen (shrimp tropomyosin);
The described application utilizing surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detection shrimp tropomyosin monoclonal antibody ascites, 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, with PBS damping fluid for benchmark, each standard solution passes into micro-flow cell of surface plasma body resonant vibration instrument respectively, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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 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 to chip reaction zone, the change of record 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 concentration of unknown sample Prawn tropomyosin monoclonal antibody or the extension rate of unknown shrimp tropomyosin monoclonal antibody ascites;
(3) detection of next sample:
After immune response in step (two) detects and terminates, micro-flow cell first passes into PBS buffer solution for cleaning 1 ~ 5 time, and the time of each cleaning is 1 ~ 5min; Pass into SDS-HCl solution cleaning 1 ~ 3 time again, each scavenging period is 1 ~ 3min; , antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 1 ~ 5 time, the time of each 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 °;
In SDS-HCl solution described in step (three), the massfraction of SDS is the concentration of 1%, HCl is 0.01mol/L.
PBS buffer solution for cleaning number of times described in step (three) is preferably 3 times, and the time of each cleaning is preferably 3min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, pH value is 7.4;
The antigen generation immune response of the antibody in sample and chip surface, formed antibody-antigen conjugates, SPR response increase, shrimp tropomyosin and shrimp tropomyosin monoclonal antibody binding ability stronger.With the increase of sample Prawn tropomyosin MAb concentration, namely dilutability is little, and the same reaction time, more antibody is combined with antigen, and SPR response increases.Method thus, can obtain the typical curve detecting shrimp tropomyosin monoclonal antibody, and with the increase of extension rate, SPR response declines, in falling S curve.The method is applicable to anti-allergen antibodies in anti-allergen antibodies screening, patient serum sample and detects.
SPR biochip can the multiple sample of continuous detecting, can carry out the Site Detection of a large amount of sample.At least can continuous detecting 80 samples, more than 80 samples, detection sensitivity can reduce, and chip needs regeneration.The method is hopeful to be directly used in clinical, detects the anti-allergen antibodies in patients serum.
Described utilizes surface plasma body resonant vibration immune sensing chip detection anaphylactogen (shrimp tropomyosin), comprises following concrete steps:
(I) Criterion curve:
The shrimp tropomyosin PBS damping fluid of concentration known is configured to the standard solution of variable concentrations, with PBS damping fluid for benchmark, each standard solution passes into micro-flow cell of surface plasma resonance instrument respectively, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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,
(II) detection of 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 to chip reaction zone, the change of record 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;
(III) detection of next sample:
After immune response in step (II) terminates, micro-flow cell first passes into PBS buffer solution for cleaning 1 ~ 5 time, and the time of each cleaning is 1 ~ 5min; Pass into SDS-HCl solution cleaning 1 ~ 3 time again, each scavenging period is 1 ~ 3min; , antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 1 ~ 5 time, the time of each 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 (I) is shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody;
The concentration range of the standard solution described in step (I) is 1 μ g/mL ~ 2.1mg/mL;
The detection limit of the standard solution described in step (I) 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 (II) 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 °;
In SDS-HCl solution described in step (III), the massfraction of SDS is the concentration of 1%, HCl is 0.01mol/L.
PBS buffer solution for cleaning number of times described in step (III) is preferably 3 times, and the time of each cleaning is preferably 3min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, 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 carry out detection and the safety evaluation of anaphylactogen in a large amount of food.Use non-antibody purification or antibody purification as bioprobe, do not need mark and two to resist, reduce cost, simplify the operation step, 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 shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites, shrimp tropomyosin monoclonal antibody respectively 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 the reaction of P polarized light detection biochip probe and sample.When detecting in sample the material had with probe matching, resonance angle changes, SPR detector record testing result, according to typical curve, and analysis design mothod 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, overcomes the problem of current biochip test method fluorescence labeling or radioelement marking contaminated environment.
(2) the present invention only needs monospecific antibody, and sample preparation is simple and consumption does not even need purifying less, and simplify operation steps, experimental implementation is easy, shortens detection time.
(3) the present invention adopts shrimp tropomyosin or its antibody as probe, and application SPR technique carries out input, highly sensitive.
(4) present device is simple, does not need expensive detecting instrument, reduces use cost and the experiment condition of chip, can realize the field quick detection of sample with portable SPR detector.
(5) by the present invention, can common lab be widely used in, be expected in supermarket, place that market, factory etc. need food quality to monitor in real time, realize the field quick detection of a large amount of sample.
In a word, this technology succeed in developing by really realize biochip technology quick, real-time, can the feature of Site Detection, can 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, be applicable to the Site Detection of a large amount of sample in market, harbour, fairground.Surface plasma body resonant vibration biochip method is the detection of China's field of food safety anaphylactogen, clinical diagnosis, shrimp allergen vaccine design and formulate China's food allergen tag control and established certain technical foundation, in the detection of food allergen and food, the identity management of allergen 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 thereto.
N-hydroxy-succinamide (NHS), N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC), monoethanolamine (Eth), lauryl sodium sulfate (SDS), HS (CH
2)
10cOOH (mercaptoundecylic acid) and HS (CH
2)
6oH (sulfydryl hexanol) is purchased from Sigma Co., USA; Other reagent is purchased from Beijing chemical reagents corporation.PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L; The pH value of described PBS damping fluid is 7.4; In SDS-HCl solution, the massfraction of SDS is the concentration of 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 MAb 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, in substrate deposit thickness be 50nm golden film as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) passing in double dish containing final concentration is the HS (CH of 1mM
2)
10cOOH (mercaptoundecylic acid) and 9mM HS (CH
2)
6the ethanolic solution 4mL of OH (sulfydryl hexanol), 37 DEG C of temperature baths, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully cleaning 3 times, each 2min, washes off in conjunction with material; 50 DEG C of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) PBS buffer solution for cleaning 3 times are passed in flow cell, each 2min; The mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC) is added, activation chip 15min after baseline stability; Then PBS buffer solution for cleaning is passed into 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is 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, concentration is the shrimp tropomyosin 100 μ L of 0.105mg/mL, and as bioprobe, the set time is 30min; The change of record SPR resonance angle, the process that monitoring bio probe is fixing; Then PBS buffer solution for cleaning is passed into 3 times, each 2min;
(6) add ethanolamine solutions (pH=8.5) the 100 μ L of 1mol/L, close the remaining ester bond 6min of deactivation; PBS buffer solution for cleaning 3 times, each scavenging period 2min.
The preparation of embodiment 2 surface plasma body resonant vibration immune sensing chip
(1) using the circular glass sheet of diameter 20mm, thickness 1mm as substrate, in substrate deposit thickness be 50nm golden film as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) passing in double dish containing final concentration is the HS (CH of 1mM
2)
10cOOH (mercaptoundecylic acid) and 9mM HS (CH
2)
6the ethanolic solution 4mL of OH (sulfydryl hexanol), 37 DEG C of temperature baths, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully cleaning 3 times, each 2min, washes off in conjunction with material; 50 DEG C of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) PBS buffer solution for cleaning 3 times are passed in flow cell, each 2min; The mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC) is added, activation chip 15min after baseline stability; Then PBS buffer solution for cleaning is passed into 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is 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 biochip surface fixed dilution 100 times, as bioprobe, the time is 30min; The change of record SPR resonance angle, the process that monitoring bio probe is fixing; Then PBS buffer solution for cleaning is passed into 3 times, each 2min;
(6) add ethanolamine solutions (PH=8.5) the 100 μ L of 1mol/L, close the remaining ester bond of 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 chip
(1) using the circular glass sheet of diameter 20mm, thickness 1mm as substrate, in substrate, deposit thickness is the solid phase carrier of golden film as surface plasma body resonant vibration biochip of 50nm;
(2) passing in double dish containing final concentration is the HS (CH of 1mM
2)
10cOOH (mercaptoundecylic acid) and 9mM HS (CH
2)
6the ethanolic solution 4mL of OH (sulfydryl hexanol), 37 DEG C of temperature baths, lucifuge, carries out chemical modification 2h to golden film surface; Then pass into PBS buffer solution for cleaning, fully cleaning 3 times, each 2min, washes off in conjunction with material; 50 DEG C of nitrogen dry up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) PBS buffer solution for cleaning 3 times are passed in flow cell, each 2min; The mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC) is added, activation chip 15min after baseline stability; Then PBS buffer solution for cleaning 3 times, each 2min; Wherein, in mixed liquor, the final concentration of N-hydroxy-succinamide is the final concentration of 0.05mol/L, N-ethyl-N '-(dimethylamino-propyl) carbodiimide is 0.05mol/L;
(5) at biochip surface fixed concentration be the shrimp tropomyosin monoclonal antibody 100 μ L of 0.0042mg/mL, as bioprobe, the time is 30min; The change of record SPR resonance angle, the process that monitoring bio probe is fixing; Then PBS buffer solution for cleaning is passed into 3 times, each 2min;
(6) add monoethanolamine (PH8.5) the 100 μ L of 1mol/L, close the remaining ester bond of 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:
The standard solution of variable concentrations shrimp tropomyosin is configured to: be that the shrimp tropomyosin mother liquor of 2.1mg/mL dilutes 10,25,50,75,100 times respectively by concentration with PBS damping fluid; With PBS damping fluid for benchmark, each standard solution gets micro-flow cell that 100 μ L pass into surface plasma resonance instrument successively respectively, probe generation immune response on the surface plasma body resonant vibration immune sensing chip prepared with embodiment 2, the reaction time is set as 3min; Surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record SPR resonance angle, obtain each standard solution and 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, the reaction time is set as 3min; Surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record SPR resonance angle, obtain unknown sample and 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 terminates, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and the time of each cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each 3min, antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 3 times, the time of each 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 10,50,100 times respectively, 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 surface plasma body resonant vibration immune sensing chip detection above-mentioned sample solution Prawn tropomyosin: the recurrence typical curve that the data integrating step (1) measured by SPR detector is obtained simultaneously, calculate the detected value of sample Prawn tropomyosin concentration; 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, and illustrate that SPR biological chips detection system has good detectability, 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:
The shrimp tropomyosin monoclonal antibody ascites standard solution of variable concentrations is configured to: be the non-antibody purification ascites mother liquor dilution 150,100,80,50,40,20,10 times of 15000 by tiring with PBS damping fluid; With PBS damping fluid for 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 prepared with embodiment 1, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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 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 to chip reaction zone, the change of record 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) terminates, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and the time of each cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each scavenging period is 3min; , antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 3 times, the time of each cleaning is 3min; Baseline falls back in SPR response, continues to detect next sample.
By tiring be 15000 shrimp tropomyosin monoclonal antibody ascites dilute 10,50,100,150 respectively, be designated as its actual value; Utilize the content of surface plasma body resonant vibration immune sensing chip detection above-mentioned sample solution Prawn tropomyosin monoclonal antibody ascites: the recurrence typical curve that the data integrating step (1) measured by SPR detector is obtained, 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, and illustrate that SPR biological chips detection system has good detectability, 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:
The shrimp tropomyosin monoclonal antibody standard solution of variable concentrations is configured to: be that the shrimp tropomyosin monoclonal antibody of 4.2mg/mL dilutes 300,200,150,75,50 times successively by concentration with PBS damping fluid; With PBS damping fluid for 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 prepared with embodiment 1, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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 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 to chip reaction zone, the change of record 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) terminates, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, and the time of each cleaning is 3min; Pass into SDS-HCl solution again and clean 3 times, each scavenging period is 3min; , antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 3 times, the time of each cleaning is 3min; Baseline falls back in SPR response, continues to detect next sample.
Be the shrimp tropomyosin monoclonal antibody of 4.2mg/mL by concentration, dilute 1000,500,100,50 times respectively, concentration is respectively and is designated as 0.0042,0.0084,0.042,0.084mg/mL, be designated as actual value.By the recurrence typical curve that the data integrating step (1) that SPR detector is measured obtains, calculate the predicted value of antibody concentration in sample, 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, and illustrate that SPR biological chips detection system has good predictive ability, 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 the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within 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) deposit thickness is the solid phase carrier of golden film as surface plasma body resonant vibration immune sensing chip of 40 ~ 60nm on the glass substrate;
(2) inject containing HS (CH in double dish
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 in conjunction with material; Nitrogen dries up;
(3) above-mentioned solid phase carrier is fixed on SPR detector;
(4) pass into PBS buffer solution for cleaning in flow cell, after baseline stability, add the mixed liquor activation chip of N-hydroxy-succinamide and N-ethyl-N '-(dimethylamino-propyl) carbodiimide; Then PBS buffer solution for cleaning is passed into;
(5) shrimp tropomyosin, shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody is fixed in biochip surface, the change at recording surface plasma resonance angle, the process that monitoring bio probe is fixing, when resonance angle no longer raises, probe fixation procedure terminates; Then PBS buffer solution for cleaning is passed into;
(6) add ethanolamine solutions and close the remaining ester bond of deactivation; Then pass into PBS buffer solution for cleaning, obtain surface plasma body resonant vibration immune sensing chip;
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 concentration of the shrimp tropomyosin described in step (5) is 0.105mg/mL; Described shrimp tropomyosin monoclonal antibody ascites be by tiring be 15000 shrimp tropomyosin monoclonal antibody ascites dilute 100 times and obtain; The concentration of described shrimp tropomyosin monoclonal antibody is 0.0042mg/mL.
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 37 DEG C of temperature baths, lucifuge reaction 2h;
The temperature that nitrogen described in step (2) dries up is 50 DEG C;
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; The described set time is 30 min;
The concentration of the ethanolamine solutions described in step (6) is 1mol/L, and pH value is 8.5; The time of described closed deactivation is 6min;
The number of times of step (2), (4), (5) and the PBS buffer solution for cleaning described in (6) is 3 times, and the time of each cleaning is 2min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, 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 according to 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 and utilize surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detect the application of shrimp tropomyosin monoclonal antibody ascites and utilize the application of 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, it is characterized in that: the described application utilizing surface plasma body resonant vibration immune sensing chip detection shrimp tropomyosin monoclonal antibody or detection shrimp tropomyosin monoclonal antibody ascites, 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, with PBS damping fluid for benchmark, each standard solution passes into micro-flow cell of surface plasma body resonant vibration instrument respectively, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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 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 to chip reaction zone, the change of record 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 concentration of unknown sample Prawn tropomyosin monoclonal antibody or the extension rate of unknown shrimp tropomyosin monoclonal antibody ascites;
(3) detection of next sample:
After immune response in step (two) detects and terminates, micro-flow cell first passes into PBS buffer solution for cleaning 1 ~ 5 time, and the time of each cleaning is 1 ~ 5min; Pass into SDS-HCl solution cleaning 1 ~ 3 time again, each scavenging period is 1 ~ 3min; Antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 1 ~ 5 time, the time of each 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 °;
In SDS-HCl solution described in step (three), the massfraction of SDS is the concentration of 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (three) is 3 times, and the time of each cleaning is 3min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, 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 PBS damping fluid of concentration known is configured to the standard solution of variable concentrations, with PBS damping fluid for benchmark, each standard solution passes into micro-flow cell of surface plasma resonance instrument respectively, with the probe generation immune response on surface plasma body resonant vibration immune sensing chip, surface plasma body resonant vibration scanning is carried out to chip reaction zone, the change of record 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,
(II) detection of 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 to chip reaction zone, the change of record 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;
(III) detection of next sample:
After immune response in step (II) terminates, micro-flow cell first passes into PBS buffer solution for cleaning 1 ~ 5 time, and the time of each cleaning is 1 ~ 5min; Pass into SDS-HCl solution cleaning 1 ~ 3 time again, each scavenging period is 1 ~ 3min; Antibody-antigen conjugates is dissociated; Then pass into PBS damping fluid 1 ~ 5 time, the time of each 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 (I) is shrimp tropomyosin monoclonal antibody ascites or shrimp tropomyosin monoclonal antibody;
The concentration range of the standard solution described in step (I) is 1 μ g/mL ~ 2.1mg/mL;
The detection limit of the standard solution described in step (I) 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 (II) is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1 ~ 2 °;
In SDS-HCl solution described in step (III), the massfraction of SDS is the concentration of 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (III) is 3 times, and the time of each cleaning is 3min;
Described PBS damping fluid composed as follows: final concentration is the NaH of 2mmol/L
2pO
4, final concentration is the Na of 2mmol/L
2hPO
4, final concentration is NaCl and the water of 150mmol/L, pH value is 7.4.
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