CN103792211A - Surface plasma resonance biochip as well as preparation method and application thereof - Google Patents
Surface plasma resonance biochip as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biochips, and particularly relates to a surface plasma resonance biochip as well as a preparation method and application thereof. The surface plasma resonance biochip is prepared by the following steps: fixing a clenbuterol hydrochloride-bovine serum albumin coupling 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 a clenbuterol antibody by utilizing a surface plasma resonance biochip direct method and detection of clenbuterol by utilizing the surface plasma resonance biochip by a competitive inhibition method. The biochip has the characteristics of rapidity, real time and capability of field detection, can rapidly give the quantitative result, is high in flexibility, is low in cost, and does not pollute the environment.
Description
Technical field
The present invention relates to biochip field, be specifically related to a kind of surface plasma body resonant vibration biochip 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.Biochip probe of the present invention is Clenbuterol derivant, is a kind of protein-biochips.
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.
Clenobuterol hydrochloride (Clenbuterol hydrochloride, CLB) is a kind of β
2-receptor stimulating agent (β
2-agonists), because thering is identical pharmacological action with adrenaline, be also referred to as β
2-adrenoceptor agonists (β
2-addrenoeeptor agonists), be mainly used for the treatment of clinically bronchial astehma, spasm, the diseases such as obstructive pneumonia.Early 1980s, a company of the U.S. starts clenobuterol hydrochloride to add in feed, and the effect that it has nutrition reallocation, has improved the lean meat conversion ratio of animal, from then on β greatly
2-receptor stimulating agent is called as " clenbuterol hydrochloride ".The bad reactions such as the people of the long-term edible food that contains clenobuterol hydrochloride can have a headache, uncomfortable in chest, nauseating, nineteen eighty-three there is Food poisoning of clenbuterol event in Spain first, 1998 there is Food poisoning of clenbuterol event in Hong Kong, and the food poisoning causing because of Clenbuterol has in the world exceeded 1000.Clenbuterol and salt thereof, ester be the veterinary drug for banning use of in China and a lot of area, and be defined in animal food and must not detect, but still have some raisers or feed factory illegally to use Clenbuterol, the medicament residue of Clenbuterol detects has become study hotspot.The main method that detects at present clenobuterol hydrochloride is high performance liquid chromatography (High performance liquid chromatography, HPLC) isochromatic spectrum analytic approach, enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, immunological method and the electrochemical method such as ELISA), to normally 2.0 μ g/L of the detectability of Clenbuterol.Said method apparatus expensive, sense cycle is long, operating personnel is required high, is difficult to meet fast, the demand of Site Detection.
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, with not enough, provides a kind of surface plasma body resonant vibration biochip, and this biochip is using clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter (CLB-BSA) as probe.
Another object of the present invention is to provide the preparation method of above-mentioned surface plasma body resonant vibration biochip.
A further object of the present invention is to provide the application of above-mentioned surface plasma body resonant vibration biochip.
Object of the present invention is achieved through the following technical solutions:
A preparation method for surface plasma body resonant vibration biochip, comprises following steps:
(1) the golden film that deposit thickness is 45～55nm in substrate of glass is as the solid phase carrier of surface plasma body resonant vibration biochip;
(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 pass into 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) at the fixing clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter (CLB-BSA) of biochip surface; The variation at recording surface plasma resonance angle, the process that monitoring bio probe is fixing, SPR response has significantly rising, and probe fixed effect is better, and in the time that resonance angle no longer raises, probe fixation procedure finishes; Then inject PBS buffer solution for cleaning;
(6) add the remaining ester bond of monoethanolamine (Eth) solution sealing deactivation; Then inject PBS buffer solution for cleaning; Obtain surface plasma body resonant vibration biochip.
The preferred diameter of substrate of glass described in step (1) is the circular glass sheet that 20mm, thickness are 1mm; The thickness of described golden film 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 4.0～100mM, is preferably 9mM;
The condition of the chemical modification described in step (2) is that 37 ℃ of temperature are bathed, and lucifuge reaction 0.5～6h, 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 10～60min, is preferably 15min;
The concentration of the clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter (CLB-BSA) described in step (5) is 83mg/L; Described regular 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 3～10min, is preferably 6min;
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 3min;
Described 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, pH value is 7.4;
A kind of surface plasma body resonant vibration biochip, obtains by above-mentioned preparation method;
The application of described surface plasma body resonant vibration biochip, comprises and utilizes surface plasma body resonant vibration biochip detect the application of antibody of clenbuteral hydrochloride by direct method and utilize surface plasma body resonant vibration biochip to detect the application of clenobuterol hydrochloride by A competitive inhibition method;
The described surface plasma body resonant vibration biochip that utilizes detects antibody of clenbuteral hydrochloride by direct method, comprises following steps:
(1) Criterion curve:
The antibody of clenbuteral hydrochloride of concentration known is mixed with the standard solution of variable concentrations with PBS damping fluid, take PBS damping fluid as benchmark, each standard solution injects respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration biochip, biochip reaction district is carried out to surface plasma body resonant vibration scanning, 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 biochip, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, 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 concentration of antibody of clenbuteral hydrochloride in unknown sample;
(3) next sample detection:
After immune response in step (two) 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, and 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 preferred concentration range of the antibody of clenbuteral hydrochloride standard solution described in step () is 0.5mg/L～100mg/L;
The detection limit of the standard solution described in step () is preferably 100 μ L; The described immunoreactive reaction time is 3～5min, and 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 (two) is preferably 100 μ L; The described immunoreactive reaction time is 3～5min, and 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 (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 SDS-HCl solution wash number 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 Na that final concentration is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4;
At the fixing clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter (CLB-BSA) of biochip surface, direct-detection antibody of clenbuteral hydrochloride, can carry out antibody screening and research immune response dynamics.Same chip at least can 50 samples of continuous detecting, and a sample detection is complete, passes into PBS damping fluid, then pass into SDS-HCl solution, Ag-Ab bond is dissociated, then pass into PBS damping fluid, baseline falls back in SPR response, can continue to detect next sample.
The described surface plasma body resonant vibration biochip that utilizes detects clenobuterol hydrochloride by competition law, comprises following steps:
(I) Criterion curve:
Clenobuterol hydrochloride standard items are mixed with the standard solution of variable concentrations with PBS damping fluid, the clenobuterol hydrochloride standard solution of variable concentrations mixes with quantitative salt Clenbuterol antibody-solutions respectively, leave standstill, obtain the mixed solution of each standard solution and quantitative antibody of clenbuteral hydrochloride, take PBS damping fluid as benchmark, each standard solution and the quantitatively mixed solution of antibody of clenbuteral hydrochloride inject respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration biochip, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, 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:
The mixed solution of unknown sample extract and quantitative antibody of clenbuteral hydrochloride is injected to micro-flow cell and carry out immune response, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of testing sample, the recurrence typical curve that integrating step (I) obtains, calculates the micromolecular concentration of clenobuterol hydrochloride in unknown sample;
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.
Described in step (I), quantitatively antibody of clenbuteral hydrochloride final concentration in mixed solution is 20mg/L;
The little molecule molecular weight of clenobuterol hydrochloride described in step (I) is 313.7;
The concentration range of the standard solution described in step (I) is 0.5～100 μ g/L;
Time of repose described in step (I) is 1～15min, and preferably the time is 5min;
The detection limit of the mixed solution of the clenobuterol hydrochloride standard items described in step (I) and antibody of clenbuteral hydrochloride is preferably 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 °;
Unknown sample extract described in step (II), comprises following concrete steps:
1. claim 2g pork or pork liver;
2. add the HCl6mL of 0.1M, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
3. get supernatant, add the NaOH2mL of 0.1M, add 6mL ethyl acetate, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
4. getting the supernatant that 3. step obtain dries up in 50 ℃ of nitrogen;
5. add 1mL tri-distilled water to redissolve, obtain unknown sample extract;
Described in step (II), quantitatively antibody of clenbuteral hydrochloride final concentration in mixed solution is 20mg/L; Unknown sample extract is 100 μ L with the mixed solution detection limit of quantitative antibody of clenbuteral hydrochloride; The described immunoreactive reaction time is 3～5min, and 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 (III), the massfraction of SDS is 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (III) 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 Na that final concentration is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4;
Principle of the present invention: fix clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter (CLB-BSA) as bioprobe at solid phase carrier (glass sheet of gold-plated film), utilize golden film to produce surface plasma body resonant vibration (SPR) response, utilize self assembled monolayer technology at golden film finishing sulfydryl, activated rear chip can stationary probe, sample injects micro-flow cell, with probe generation immune response, react (Fig. 1, the Fig. 2) by P polarized light detection biochip probe with sample.When detecting while having in sample with the material of probe matching, resonance angle changes, and SPR detector records testing result, obtains the surface plasma body resonant vibration kinetic curve that detects sample, in conjunction with returning typical curve, and analysis experimental result.The molecular weight (313.7) of clenobuterol hydrochloride, is unsuitable for direct-detection.If the fixing antibody of clenbuteral hydrochloride of biochip surface is as probe, clenobuterol hydrochloride can be combined with antibody, but little near refractive index impact chip surface, the variation of SPR resonance peak is little, and direct-detection effect is bad.In order to detect clenobuterol hydrochloride trace small-molecule substance, the present invention proposes Immunosuppression type biochip method.LB-BSA is as probe for chip surface fixation of C, after mixing with quantitative antibody of clenbuteral hydrochloride, injects the little molecule of CLB flow cell, the CLB-BSA of the little molecule of CLB and chip surface can be combined with antibody of clenbuteral hydrochloride, it is the relation of competition, the little molecules in inhibiting of CLB antibody of clenbuteral hydrochloride be combined with the probe CLB-BSA of chip surface, in sample, the concentration of CLB and response is varied to inverse ratio.If CLB concentration is little in sample, more antibody of clenbuteral hydrochloride is combined with chip surface probe, and SPR response is that the variation of resonance angle is larger.
Advantage of the present invention and effect are as follows:
(1) the present invention does not need sample mark, environmentally safe.
(2) the present invention only needs monospecific antibody, and sample preparation is simple and consumption is few, has simplified operation steps, contracting
Short detection time.
(3) the present invention adopts CLB-BSA as probe, and application SPR technology is carried out input, does not need expensive detecting instrument, has reduced use cost and the experiment condition of chip.
(4) the present invention can realize with portable SPR detector the field quick detection of sample, can be widely used in common lab, is 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.
The present invention can 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.Direct Detection Method detects antibody of clenbuteral hydrochloride can Effect of Anti antigen-antibody affinity and kinetic reaction process, is applicable to the screening of fundamental research and antibody etc.Competition suppresses detection method can detect the little molecule of the Clenbuterol extracting in pork liver, highly sensitive, identical with ELISA method detection limit with at present conventional HPLC, can be used for the fields such as food inspection, drug screening, medical diagnosis on disease, will produce good economic benefit and social benefit.
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.
Fig. 3 is that immunoreactive surface plasma resonance kinetic curve figure occurs standard solution (5mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L and 50mg/L) prepared by embodiment 2.
Fig. 4 is that immunoreactive surface plasma resonance kinetic curve figure occurs standard solution prepared by embodiment 3.
Fig. 5 is the recurrence canonical plotting that embodiment 3 sets up.
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.
Clenobuterol hydrochloride standard items (ethanolic solution of 100g/mL) are provided by Inst. of Environment Protection & Scientific Research Monitor, Ministry of Agric, and molecular weight is 313.7; Clenbuterol-bovine serum albumin(BSA) (CLB-BSA) coupling matter, antibody of clenbuteral are purchased from Fu Sai bio tech ltd, Guangzhou; 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 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 preparation of embodiment 1 surface plasma body resonant vibration biochip
(1) using diameter as 20mm, thickness is as the circular glass sheet of 1mm is as substrate, the golden film that deposit thickness is 50nm in substrate of glass is as the solid phase carrier of surface plasma body resonant vibration biochip;
(2) in double dish, inject 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 damping fluid and fully clean 3 times, each 3min, 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 3min adds the mixed liquor 150 μ L of N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide (EDC) after baseline stability, activates chip 15min; Then pass into PBS buffer solution for cleaning 3 times, each 3min; 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) pass into the CLB-BSA100 μ L of 83mg/L in biochip surface, fixing biological probe, the set time is 30min; 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 3 times, each 3min;
(6) add ethanolamine solutions (pH=8.5) the 150 μ L of 1mol/L, the remaining ester bond 6min of sealing deactivation; Then pass into PBS buffer solution for cleaning 3 times, each 3min.
Embodiment 2 utilizes surface plasma body resonant vibration biochip to detect antibody of clenbuteral hydrochloride by direct method
(1) Criterion curve:
Be mixed with the CLB antibody standard solution of variable concentrations with PBS damping fluid: concentration is respectively 0.5mg/L, 1mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L, 50mg/L and 100mg/L; Take PBS damping fluid as benchmark, each standard solution is got respectively 100 μ L and injects successively micro-flow cell of surface plasma resonance instrument, probe generation immune response on the surface plasma body resonant vibration biochip preparing with embodiment 1, the reaction time is set as 3min; Biochip reaction district is carried out to surface plasma body resonant vibration scanning, and sweep limit is 1～2 °; Record the variation of SPR resonance angle, obtain each standard solution immunoreactive surface plasma body resonant vibration kinetic curve (Fig. 3) 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 biochip, and the reaction time is set as 3min; Effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, and sweep limit is 1～2 °; 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 CLB antibody in unknown sample;
(3) next sample detection:
After a sample detection finishes, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, cleans 3min at every turn; Pass into again SDS-HCl solution-treated 1～3 time, Ag-Ab bond is dissociated, process 3min at every turn; 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 CLB antibody standard solution of preparation variable concentrations, sample concentration is respectively: 1mg/L, 5mg/L, 10mg/L, 30mg/L, be designated as its actual value; Utilize surface plasma body resonant vibration biochip to detect the content of CLB antibody in above-mentioned CLB antibody standard solution by direct method: the recurrence typical curve that the data integrating step (1) of measuring by SPR detector obtains, calculates the detected value of CLB antibody concentration in sample 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 surface plasma body resonant vibration biochip direct method to detect antibody of clenbuteral hydrochloride interpretation of result
Carry out along with immunoreactive, biochip surface Ag-Ab bond increases, and SPR response increases.Raise with CLB antibody concentration in sample, immune response speed speeds, and SPR resonance angle increases, and resonance curve moves to right.While detecting each sample, immune response first quick and back slow, is progressively tending towards saturated.
Embodiment 3 utilizes surface plasma body resonant vibration biochip to detect clenobuterol hydrochloride by A competitive inhibition method
(1) Criterion curve:
CLB standard solution mixes with CLB antibody-solutions, and in mixed solution, the final concentration of CLB antibody is 20mg/L; The final concentration of CLB standard items is respectively 0.5 μ g/L, 1 μ g/L, 5 μ g/L, 10 μ g/L, 15 μ g/L, 20 μ g/L; Mixed solution leaves standstill 5min, take PBS damping fluid as benchmark, the mixed solution of each standard solution and antibody-solutions is got respectively micro-flow cell of 100 μ L injection surface plasma resonance instruments, probe generation immune response on the surface plasma body resonant vibration biochip preparing with embodiment 1, the reaction time is 3min; Effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, and sweep limit is 1～2 °; Record the variation of SPR resonance angle in immunoreaction process, obtain the surface plasma resonance kinetic curve (Fig. 4) of each CLB 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 (Fig. 5);
(2) detection of Clenbuterol
Unknown sample extract and CLB antibody-solutions are mixed, and the final concentration of CLB antibody in mixed solution is 20mg/L; Mixed solution leaves standstill 5min, the mixed solution of 100 μ L is injected to micro-flow cell and carry out immune response, and the reaction time is 3min; Effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, and sweep limit is 1～2 °; Record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of testing sample, the recurrence typical curve that integrating step (1) obtains, calculates the micromolecular concentration of CLB in unknown sample;
(3) detection of next sample
After a sample detection finishes, micro-flow cell first passes into PBS buffer solution for cleaning 3 times, cleans 3min at every turn; Pass into again SDS-HCl solution-treated 1～3 time, Ag-Ab bond is dissociated, process 3min at every turn; 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 CLB standard solution of preparation variable concentrations, sample concentration is respectively: 2 μ g/L, 10 μ g/L, 20 μ g/L, 50 μ g/L, be designated as its actual value; Utilize surface plasma body resonant vibration biochip to detect the content of CLB in above-mentioned CLB standard solution by A competitive inhibition method: the recurrence typical curve that the data integrating step (1) of measuring by SPR detector obtains, calculates the detected value of the little molecular conecentration of CLB in sample 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 surface plasma body resonant vibration biochip to detect Clenbuterol interpretation of result by A competitive inhibition method
In the time that in sample, CLB concentration is large, the CLB antibody quantity that can be combined with chip surface probe is few, and immune response speed is slow, and SPR response is low, and the little molecular conecentration of CLB and SPR response are inversely proportional to.Each biochip at least can 50 groups of samples of continuous detecting after measured, exceed 50 groups of sample SPR responses and obviously decline, and illustrate that the fixing probe of biochip surface is impaired.At this moment the HCl solution that chip surface passes into 0.1mol/L can be realized chip regeneration, self assembly fixing biological probe again.
A competitive inhibition method detects approximate " S " type that is down of the micromolecular typical curve of CLB, the standard deviation of each concentration being tested repeatedly to (3 times) is less than 1%, detection limit is less than 2 μ g/L, lower than the CLB detection limit 10 μ g/L of commercialization instrument Biacore2000, identical with the CLB detection limit 2 μ g/L of HPLC and ELISA.IC50 value is 10 μ g/L.By the testing sample SPR response of unknown concentration, query criteria curve, can draw the concentration of CLB in sample, can be used for food quality monitoring and on-the-spotly detects in real time.
Embodiment 4 utilizes surface plasma body resonant vibration biochip to detect the clenobuterol hydrochloride in pork liver by A competitive inhibition method, and step is:
(1) preparation of Pig Liver extract
1. claim 2g pork liver;
2. add the HCl6mL of 0.1M, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
3. get supernatant, add the NaOH2mL of 0.1M, add 6mL ethyl acetate, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
4. getting the supernatant that 3. step obtain dries up in 50 ℃ of nitrogen;
5. add 1mL tri-distilled water to redissolve, obtain Pig Liver extract;
(2) adopt A competitive inhibition method, detect the pork liver extract that contains CLB and the pork liver extract (control group) that does not contain CLB.
Table 3 utilizes surface plasma body resonant vibration biochip to detect the clenobuterol hydrochloride interpretation of result in pork liver by A competitive inhibition method
Experimental result shows, the pork liver that contains CLB extracts sample and control group SPR response has obvious difference, prove that the method is to detecting pork, pork liver extract, there are close result and detection limit (table 3) with enzyme linked immunosorbent assay, but surface plasma body resonant vibration biochip method step is simpler, do not need two anti-and fluorescent dyes etc., SPR biochip can, for the Site Detection of Clenbuterol sample, have promotional value.
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 (10)
1. a preparation method for surface plasma body resonant vibration biochip, is characterized in that comprising following steps:
(1) the golden film that deposit thickness is 45～55nm in substrate of glass is as the solid phase carrier of surface plasma body resonant vibration biochip;
(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 pass into 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) at the fixing clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter of biochip surface; The variation of recording surface plasma resonance resonance angle, the process that monitoring bio probe is fixing, in the time that resonance angle no longer raises, probe fixation procedure finishes; Then inject PBS buffer solution for cleaning;
(6) add the remaining ester bond of ethanolamine solutions sealing deactivation; Then inject PBS buffer solution for cleaning; Obtain surface plasma body resonant vibration biochip.
2. the preparation method of surface plasma body resonant vibration biochip according to claim 1, is characterized in that:
Substrate of glass described in step (1) is that diameter is the circular glass sheet that 20mm, thickness are 1mm; The thickness of described golden film 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 concentration of the clenobuterol hydrochloride-bovine serum albumin(BSA) coupling matter described in step (5) is 83mg/L; Described regular 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 3min;
Described 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, pH value is 7.4.
3. a surface plasma body resonant vibration biochip, is characterized in that being prepared by method described in claim 1 or 2.
4. the application of surface plasma body resonant vibration biochip claimed in claim 3 in biochip field.
5. the application of surface plasma body resonant vibration biochip according to claim 4 in biochip field, is characterized in that: comprise and utilize surface plasma body resonant vibration biochip detect the application of antibody of clenbuteral hydrochloride by direct method and utilize surface plasma body resonant vibration biochip to detect the application of clenobuterol hydrochloride by A competitive inhibition method.
6. the application of surface plasma body resonant vibration biochip according to claim 5 in biochip field, is characterized in that: described utilize surface plasma body resonant vibration biochip to detect antibody of clenbuteral hydrochloride by direct method to comprise following steps:
(1) Criterion curve:
The antibody of clenbuteral hydrochloride of concentration known is mixed with the standard solution of variable concentrations with PBS damping fluid, take PBS damping fluid as benchmark, each standard solution injects respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration biochip, biochip reaction district is carried out to surface plasma body resonant vibration scanning, 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 biochip, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, 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 concentration of antibody of clenbuteral hydrochloride in unknown sample;
(3) next sample detection:
After immune response in step (two) 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, and 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 biochip according to claim 6 in biochip field, is characterized in that:
The concentration range of the antibody of clenbuteral hydrochloride standard solution described in step () is 0.5mg/L～100mg/L;
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 Na that final concentration 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 biochip according to claim 5 in biochip field, is characterized in that: described utilize surface plasma body resonant vibration biochip to detect clenobuterol hydrochloride by A competitive inhibition method to comprise following steps:
(I) Criterion curve:
Clenobuterol hydrochloride standard items are mixed with the standard solution of variable concentrations with PBS damping fluid, the clenobuterol hydrochloride standard solution of variable concentrations mixes with quantitative salt Clenbuterol antibody-solutions respectively, leave standstill, obtain the mixed solution of each standard solution and quantitative antibody of clenbuteral hydrochloride, take PBS damping fluid as benchmark, each standard solution and the quantitatively mixed solution of antibody of clenbuteral hydrochloride inject respectively micro-flow cell of surface plasma resonance instrument, with the probe generation immune response on surface plasma body resonant vibration biochip, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, 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:
The mixed solution of unknown sample extract and quantitative antibody of clenbuteral hydrochloride is injected to micro-flow cell and carry out immune response, effects on surface plasma resonance biochip reaction district carries out surface plasma body resonant vibration scanning, record the variation of SPR resonance angle, obtain the surface plasma body resonant vibration kinetic curve of testing sample, the recurrence typical curve that integrating step (I) obtains, calculates the micromolecular concentration of clenobuterol hydrochloride in unknown sample;
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 biochip according to claim 8 in biochip field, is characterized in that unknown sample extract described in step (II), comprises following concrete steps:
1. claim 2g pork or pork liver;
2. add the HCl6mL of 0.1M, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
3. get supernatant, add the NaOH2mL of 0.1M, add 6mL ethyl acetate, vibration 10min, the centrifugal 10min of 4000r/min under room temperature;
4. getting the supernatant that 3. step obtain dries up in 50 ℃ of nitrogen;
5. add 1mL tri-distilled water to redissolve, obtain unknown sample extract.
10. the application of surface plasma body resonant vibration biochip according to claim 8 in biochip field, is characterized in that:
Described in step (I), quantitatively antibody of clenbuteral hydrochloride final concentration in mixed solution is 20mg/L;
The little molecule molecular weight of clenobuterol hydrochloride described in step (I) is 313.7;
The concentration range of the standard solution described in step (I) is 0.5～100 μ g/L;
Time of repose described in step (I) is 5min;
The detection limit of the mixed solution of the clenobuterol hydrochloride standard items described in step (I) and antibody of clenbuteral hydrochloride is 100 μ L; The described immunoreactive reaction time is 3min; Described surface plasma body resonant vibration sweep limit is 1～2 °;
Described in step (II), quantitatively antibody of clenbuteral hydrochloride final concentration in mixed solution is 20mg/L; Unknown sample extract is 100 μ L with the mixed solution detection limit of quantitative antibody of clenbuteral hydrochloride; 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 (III), the massfraction of SDS is 1%, HCl is 0.01mol/L;
PBS buffer solution for cleaning number of times described in step (III) 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 Na that final concentration is 2mmol/L
2hPO
4, final concentration be 150mmol/L NaCl and water, pH value is 7.4.
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