CN105548085A - Preparation method of surface plasma resonance instrument chip based on amphiphilic heptapeptide modification - Google Patents
Preparation method of surface plasma resonance instrument chip based on amphiphilic heptapeptide modification Download PDFInfo
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- CN105548085A CN105548085A CN201510889569.2A CN201510889569A CN105548085A CN 105548085 A CN105548085 A CN 105548085A CN 201510889569 A CN201510889569 A CN 201510889569A CN 105548085 A CN105548085 A CN 105548085A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
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- G01N21/553—Attenuated total reflection and using surface plasmons
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Abstract
The invention relates to a preparation method of a surface plasma resonance instrument chip based on amphiphilic heptapeptide modification. The method includes: using disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate to prepare a 10mM phosphate buffer solution with pH of 7.4; using the phosphate buffer solution to prepare a 1-10mg/mL amphiphilic heptapeptide CYSYSYS solution; immersing a cleaned SPR (surface plasma resonance) chip into the amphiphilic heptapeptide solution, conducting soaking for 5-24h at room temperature and then taking the chip out; washing the chip with ethanol and deionized water to make the chip surface clean, and blowing the chip dry with nitrogen. The chip provided by the invention has excellent antipollution performance in single protein (bovine serum albumin, lysozyme, beta-lactoglobulin), and diluted complex system (serum, soya-bean milk and milk with 1mg/ml protein concentration). The chip provided by the invention has excellent antipollution performance, and has a simple preparation method and easily synthesizable and controllable raw materials, thus having very good feasibility and practicability.
Description
Technical field
The present invention relates to a kind of preparation method of the surface plasma resonance instrument chip based on amphipathic seven peptides modifications, belong to design and the preparation method of surface plasma resonance spectrometer antipollution chip.
Background technology
Surface plasma resonance (SurfacePlasmonResonance, be called for short SPR) instrument be the eighties in 20th century occur a kind of biosensor technology, when incident light incides low refractive index dielectric from high refractive index medium, can be totally reflected, if now at dielectric interface place plating layer of metal film (gold or silver), the evanescent wave that incident light produces can cause metal surface free electron generation collective oscillation, and then formation plasma, when evanescent wave is equal with wave number with the frequency of surface plasmon oscillations, namely resonance effect (SPR) is produced, energy is caused to transfer to surface plasma-wave from evanescent wave, the intensity of reflected light is weakened greatly, present attenuated total reflection phenomenon, incident angle when the intensity of reflected light is zero is called resonance angle.Resonance angle is relevant with metallic film interfacial medium refractive index, and refractive index changes with the molecular mass being combined in metal film surfaces, therefore the information of intermolecular interaction is obtained by analyzing resonance angle, to detect fast and without the need to features such as marks because it has, be widely used in the fields such as proteomics, medicament research and development, clinical diagnosis, food security and environmental monitoring, and demonstrate wide application prospect.
SPR chip is the assembly of surface plasma resonance instrument core the most, provides the necessary physical condition producing spr signal, mainly comprises coupled apparatus, metal film and surface matrix.But, when application surface plasma resonance instrument carries out analytical test, the surface contamination of sensing chip is a ubiquitous problem, and the non-specific adsorption from the biomolecule in sample or microorganism will reduce the accuracy quantitatively detected, and produces false positive results.Therefore, the surface of non-specific adsorption is effectively resisted in exploitation is the matter of utmost importance improving surface plasma resonance sensor sensitivity and degree of accuracy.
Summary of the invention
The object of the invention is the preparation method providing a kind of surface plasma resonance instrument chip based on amphipathic seven peptides modifications.This chip preparation method is easy and have good anti-albumen ability.
The present invention is realized by the following technical programs.A kind of preparation method of the surface plasma resonance instrument chip based on amphipathic seven PEPC YSYSYS:
Based on the surface plasma resonance instrument chip antipollution surface method for making that amphipathic seven peptides are modified, it is characterized in that: the 10mM phosphate buffer preparing pH=7.4 with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate; With the amphipathic seven PEPC YSYSYS solution that phosphate buffered saline concentration is 1-10mg/mL; The SPR chip cleaned up is immersed in above-mentioned amphipathic seven peptide solutions, takes out after soaking 5-24h under normal temperature; Make chip surface cleaning with ethanol and deionization cleaning, and dry up with nitrogen.
The naked golden chip of SPR for applying one deck layers of chrome and one deck gold film or silver/golden composite membrane in substrate.
SPR chip cleaning method is: immersed by chip in 98% concentrated sulphuric acid and 30% hydrogen peroxide mixed solution that volume ratio is 7:3, soak 2h under normal temperature; Then taking-up with washed with de-ionized water repeatedly, dry up with nitrogen.
Phosphate buffer carries out degassed process in advance.
Substrate is glass sheet, optical fiber.
Amphipathic seven peptides by end be halfcystine (C), tyrosine (Y) and serine (S) alternately form (being abbreviated as CYSYSYS).Be connected to golden film surface by the sulfydryl on halfcystine, form self assembled monolayer, thus obtain a kind of surface plasma resonance instrument chip modified based on amphipathic seven peptides.Chip of the present invention is at single albumen (bovine serum albumin, lysozyme, beta lactoglobulin), and the complex system (serum of 1mg/mL protein concentration, soya-bean milk, milk) of dilution all has excellent antifouling property.Chip of the present invention has excellent antifouling property, and preparation method is easy, and raw material is easy to synthesis and regulation and control, has good feasibility and practicality.
Beneficial effect of the present invention
1) the present invention's development has good antifouling property based on amphipathic seven PEPC YSYSYSSPR chips, and in lysozyme, bovine serum albumin, beta lactoglobulin, non-Characteristic Adsorption amount is respectively 11.78ng/cm
2, 7.75ng/cm
2, 11.61ng/cm
2, see accompanying drawing 2.In the serum of 1mg/mL protein concentration, soya-bean milk, milk dilution, non-specific adsorption amount is respectively 106.11ng/cm
2, 189.11ng/cm
2, 228.86ng/cm
2, see accompanying drawing 3, far below the non-specific adsorption amount of each albumen at the naked golden chip surface of SPR, see attached list 1.
2) the present invention's development has biological functional effect based on amphipathic seven PEPC YSYSYSSPR chips, as by immobilized bovine serum albumin antibody and then detect its antigen.
3) the present invention's development has high stability based on amphipathic seven PEPC YSYSYSSPR chips, and under drying regime, storage 3 months or pH are that after storing 7 days in 7.4 phosphate buffers, antifouling property is unchanged.
4) preparation method based on amphipathic seven PEPC YSYSYSSPR chips of the present invention's development is fast easy, solves the preparation process that multistep is in the past loaded down with trivial details, drastically increases the efficiency that chip surface is modified.
5) the present invention's development has biodegradability based on amphipathic seven PEPC YSYSYS as the antipollution material of SPR chip surface.
Accompanying drawing explanation
The SPR chip that Fig. 1 modifies based on amphipathic seven PEPC YSYSYS; Wherein, at the bottom of 1-glass chip, 2-layers of chrome, 3-golden membranous layer, the amphipathic seven PEPC YSYSYS self assembled monolayers of 4-.
The amphipathic seven PEPC YSYSYSSPR chips of Fig. 2 are to the non-specific adsorption amount of albumen in the lysozyme (Lysozyme) of 1mg/mL, bovine serum albumin (BSA), solution.
The amphipathic seven PEPC YSYSYSSPR chips of Fig. 3 are to the non-specific adsorption amount of albumen in 1mg/mL protein concentration.
Embodiment
Embodiment 1
Concrete operation method is as follows:
1) naked golden chip preparation
As shown in Figure 1, adopt electron beam evaporation deposition technology at BK7 substrate of glass (1) upper coating one deck 2-3nm thick chromium layer (2) and one deck 45-50nm thick gold membrane (3), obtain naked golden chip.
2) naked golden chip pre-service
By step 1) the naked golden chip that obtains immerses in 98% concentrated sulphuric acid and 30% hydrogen peroxide (volume ratio 7:3) mixed solution, soaks 2h under normal temperature.Then take out and use washed with de-ionized water 3 times, dry up with nitrogen for subsequent use.
3) amphipathic seven PEPC YSYSYS modify
The 10mM phosphate buffer of pH=7.4 is prepared with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate.With amphipathic seven PEPC YSYSYS (synthesis of gill biochemical corp, the Shanghai) solution that phosphate buffered saline concentration degassed is in advance 1mg/mL.By step 2) chip that obtains immerses in above-mentioned amphipathic seven peptide solutions, takes out after soaking 12h under normal temperature.Clean 3 times successively with ethanol and deionized water, and dry up with nitrogen for subsequent use, namely obtain amphipathic seven peptides; The SPR chip of the modification as shown in the amphipathic seven PEPC YSYSYS layers (4) in accompanying drawing 1.
Amphipathic seven peptide SPR chip surface protein adsorption quantity detect
The SPR chip pitch that the amphipathic seven PEPC YSYSYS obtained modify is fitted on the prism of SPR system.Take pH as the phosphate buffer of 7.4 be mobile phase, flow velocity is 10 μ L/min.After baseline is steady, the bovine serum albumin(BSA) of 100 μ L1mg/mL or serum, soya-bean milk, the milk dilution of lysozyme or beta lactoglobulin or 1mg/mL protein concentration is injected in quantitative loop, promote protein solution through mobile phase and arrive chip surface, by SPR system measurement resonance angle real-time change curve, read SPR resonance angle changing value after 20min, and calculate non-specific adsorption amount.
Embodiment 2
Concrete operation method is as follows:
1) naked golden chip preparation
As shown in Figure 1, adopt electron beam evaporation deposition technology at BK7 substrate of glass (1) upper coating one deck 2-3nm thick chromium layer (2) and one deck 45-50nm thick gold membrane (3), obtain naked golden chip.
2) naked golden chip pre-service
By step 1) the naked golden chip that obtains immerses in 98% concentrated sulphuric acid and 30% hydrogen peroxide (volume ratio 7:3) mixed solution, soaks 2h under normal temperature.Then take out and use washed with de-ionized water 3 times, dry up with nitrogen for subsequent use.
3) amphipathic seven PEPC YSYSYS modify
The 10mM phosphate buffer of pH=7.4 is prepared with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate.With the amphipathic seven PEPC YSYSYS solution that phosphate buffered saline concentration degassed is in advance 4mg/mL.By step 2) chip that obtains immerses in above-mentioned amphipathic seven peptide solutions, takes out after soaking 12h under normal temperature.Clean 3 times successively with ethanol and deionized water, and dry up with nitrogen for subsequent use, namely obtain the SPR chip that amphipathic seven peptides are modified.
4) amphipathic seven peptide SPR chip surface protein adsorption quantity detect
By step 3) obtain amphipathic seven PEPC YSYSYS modify SPR chip pitch fit on the prism of SPR system.Take pH as the phosphate buffer of 7.4 be mobile phase, flow velocity is 10 μ L/min.After baseline is steady, the bovine serum albumin(BSA) of 100 μ L1mg/mL or serum, soya-bean milk, the milk dilution of lysozyme or beta lactoglobulin or 1mg/mL protein concentration is injected in quantitative loop, promote protein solution through mobile phase and arrive chip surface, by SPR system measurement resonance angle real-time change curve, SPR resonance angle changing value is read after 20min, as shown in accompanying drawing 2,3, surperficial non-specific adsorption amount is respectively 7.75ng/cm
2, 11.78ng/cm
2, 11.61ng/cm
2, 106.11ng/cm
2, 189.11ng/cm
2, 228.86ng/cm
2, far below the non-specific adsorption amount of each albumen of naked gold surface, in table 1.
Table 1 albumen is in the non-specific adsorption amount of the naked golden chip surface of SPR
Table 1SPR naked golden chip is to the non-specific adsorption amount of albumen in the lysozyme (Lysozyme) of 1mg/mL, bovine serum albumin (BSA), beta lactoglobulin (β-lactoglobulin), serum (Bloodserum), soya-bean milk (Soybeanmilk), milk (Cowmilk) dilution.
Embodiment 3
Concrete operation method is as follows:
1) naked golden chip preparation
As shown in Figure 1, adopt electron beam evaporation deposition technology at BK7 substrate of glass (1) upper coating one deck 2-3nm thick chromium layer (2) and one deck 45-50nm thick gold membrane (3), obtain naked golden chip.
2) naked golden chip pre-service
By step 1) the naked golden chip that obtains immerses in 98% concentrated sulphuric acid and 30% hydrogen peroxide (volume ratio 7:3) mixed solution, soaks 2h under normal temperature.Then take out and use washed with de-ionized water 3 times, dry up with nitrogen for subsequent use.
3) amphipathic seven PEPC YSYSYS modify
The 10mM phosphate buffer of pH=7.4 is prepared with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate.With the amphipathic seven PEPC YSYSYS solution that phosphate buffered saline concentration degassed is in advance 10mg/mL.By step 2) chip that obtains immerses in above-mentioned amphipathic seven peptide solutions, takes out after soaking 12h under normal temperature.Clean 3 times successively with ethanol and deionized water, and dry up with nitrogen for subsequent use, namely obtain the SPR chip that amphipathic seven peptides are modified.
4) amphipathic seven peptide SPR chip surface protein adsorption quantity detect
By step 3) obtain amphipathic seven PEPC YSYSYS modify SPR chip pitch fit on the prism of SPR system.Take pH as the phosphate buffer of 7.4 be mobile phase, flow velocity is 10 μ L/min.After baseline is steady, the bovine serum albumin(BSA) of 100 μ L1mg/mL or serum, soya-bean milk, the milk dilution of lysozyme or beta lactoglobulin or 1mg/mL protein concentration is injected in quantitative loop, promote protein solution through mobile phase and arrive chip surface, by SPR system measurement resonance angle real-time change curve, read SPR resonance angle changing value after 20min, and calculate non-specific adsorption amount.
Embodiment 4
Concrete operation method is as follows:
1) naked golden light fibre preparation
Adopt electron beam evaporation deposition technology in optical fiber substrate, apply one deck 2-3nm thick chromium layer and one deck 45-50nm thick gold membrane, obtain naked golden light fine.
2) the fine pre-service of naked golden light
By step 1) the naked golden light fibre that obtains immerses in 98% concentrated sulphuric acid and 30% hydrogen peroxide (volume ratio 7:3) mixed solution, soaks 2h under normal temperature.Then take out and use washed with de-ionized water 3 times, dry up with nitrogen.
3) amphipathic seven PEPC YSYSYS modify
The 10mM phosphate buffer of pH=7.4 is prepared with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate.With the amphipathic seven PEPC YSYSYS solution that phosphate buffered saline concentration degassed is in advance 10mg/mL.By step 2) the Fibre Optical Sensor district that obtains immerses in above-mentioned amphipathic seven peptide solutions, takes out after soaking 12h under normal temperature.Clean 3 times successively with ethanol and deionized water, and dry up with nitrogen, namely obtain the optical fiber SPR sensor that amphipathic seven peptides are modified.
4) amphipathic seven peptide optical fiber surface protein adsorbances detect
By above-mentioned 3) obtained optical fiber SPR sensor by jiont treatments such as SMA905 to SPR spectrometer TT&C system.Set integral time, average time, smoothness, after signal stabilization, preserve half-light spectrum; Open light source, after signal stabilization, preserve reference spectra; Interface is adjusted to reflectivity patterns, the sensor of preparation is immersed in PBS damping fluid, after signal stabilization, superposes action spectrum, obtain the SPR of sensor in PBS damping fluid and reflect spectrogram; Then sensor is immersed in the serum of 1mg/mL bovine serum albumin(BSA) or lysozyme or beta lactoglobulin or 1mg/mL protein concentration, soya-bean milk, milk dilution, obtain the SPR of sensor in protein solution and reflect spectrogram, calculated the non-specific adsorption amount of albumen by the wavelength shift of reflection spectrogram.
Open and a kind of preparation method of the surface plasma resonance instrument chip modified based on amphipathic seven peptides that proposes of the present invention, those skilled in the art are by using for reference present disclosure, the links such as suitable change condition route realize, although method of the present invention and technology of preparing are described by preferred embodiment, person skilled obviously can change Method and Technology route as herein described or reconfigure not departing from content of the present invention, spirit and scope, realizes final technology of preparing.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are deemed to be included in spirit of the present invention, scope and content.
Claims (5)
1., based on the surface plasma resonance instrument chip antipollution surface method for making that amphipathic seven peptides are modified, it is characterized in that: the 10mM phosphate buffer preparing pH=7.4 with disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate; With the amphipathic seven PEPC YSYSYS solution that phosphate buffered saline concentration is 1-10mg/mL; The SPR chip cleaned up is immersed in above-mentioned amphipathic seven peptide solutions, takes out after soaking 5-24h under normal temperature; Make chip surface cleaning with ethanol and deionization cleaning, and dry up with nitrogen.
2. method according to claim 1, is characterized in that at the amphipathic seven PEPC YSYSYS of golden film finishing, but is equally applicable to silver/golden composite film surface.
3. method according to claim 1, is characterized in that SPR chip cleaning method is: immersed by chip in 98% concentrated sulphuric acid and 30% hydrogen peroxide mixed solution that volume ratio is 7:3, soak 2h under normal temperature; Then taking-up with washed with de-ionized water repeatedly, dry up with nitrogen.
4. method according to claim 1, is characterized in that phosphate buffer carries out degassed process in advance.
5. method according to claim 1, is characterized in that substrate is glass sheet or optical fiber.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106932366A (en) * | 2017-03-31 | 2017-07-07 | 天津大学 | The preparation method on the prism-type surface plasma resonance chip antipollution surface based on hyaluronic acid coupling lubrication fibroin modification |
| CN107091821A (en) * | 2017-03-31 | 2017-08-25 | 王利兵 | The preparation method on the surface plasma resonance instrument chip antipollution surface modified based on mucoprotein |
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| CN104155453A (en) * | 2014-07-11 | 2014-11-19 | 天津大学 | Hyaluronic acid modified surface plasma resonance spectrometer chip and preparation method thereof |
| KR20150073283A (en) * | 2013-12-20 | 2015-07-01 | 연세대학교 산학협력단 | Method for measuring biomolecule using localized surface plasmon resonance |
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| CN105021575A (en) * | 2015-07-21 | 2015-11-04 | 青岛大学 | Photoelectric sensor for detection of kinase activity on the basis of local area surface plasma resonance |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106932366A (en) * | 2017-03-31 | 2017-07-07 | 天津大学 | The preparation method on the prism-type surface plasma resonance chip antipollution surface based on hyaluronic acid coupling lubrication fibroin modification |
| CN107091821A (en) * | 2017-03-31 | 2017-08-25 | 王利兵 | The preparation method on the surface plasma resonance instrument chip antipollution surface modified based on mucoprotein |
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