CN103995104A - Protein sensing membrane, and making method and use thereof - Google Patents
Protein sensing membrane, and making method and use thereof Download PDFInfo
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- CN103995104A CN103995104A CN201410153514.0A CN201410153514A CN103995104A CN 103995104 A CN103995104 A CN 103995104A CN 201410153514 A CN201410153514 A CN 201410153514A CN 103995104 A CN103995104 A CN 103995104A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses a protein sensing membrane, and a making method and a use thereof. The protein sensing membrane comprises a three-layer structure, and the three-layer structure comprises a bottom layer which is a substrate, an interface layer which is a nano-silver particle layer and an upper layer which is a polymer film without fluorescence groups. The making method of the protein sensing membrane comprises the following steps: silylanizing the substrate by a mercapto silane coupling agent; immersing the silylanized substrate in a nano-silver sol to form a nano-silver substrate; and dissolving a polymer without fluorescence groups, and spin-coating the surface of the nano-silver particle layer with the obtained polymer solution to form the polymer layer in order to obtain the protein sensing membrane based on self-assembled nano-silver particles. The protein sensing membrane enables the detection signal of some substances of proteins having the fluorescence characteristic and the like to be enhanced times, and can be used for the rapid detection of the proteins having the fluorescence characteristic. The making method has the advantages of simplicity, easy enforcement and strong maneuverability.
Description
Technical field
The present invention relates to a kind of protein sensing film based on self-assembled nanometer silver particles and its production and use.
Background technology
Sensing technology, as one of three large pillars of modern information technologies, plays an important role at aspects such as national security, scientific experimentation, environmental monitoring and health cares.Biosensor technique is an important branch of sensing technology, and it is in the crossing domain of life science and information science together with the subjects such as bioinformatics, bionics, biocomputer.The research emphasis of biosensor technique is that bioactive materials and sensor are organically combined, and becomes the field of giving priority to of novel analytical instrument and detection technique.
Traditional biosensor technique needs mark conventionally, sensitivity is low, process is loaded down with trivial details, efficiency is low, wherein most workload is for mark, use radioactive isotope, fluorophor to do marker, safety and stability is poor, can not meet quick, sensitive, oligosaprobic demand, in the urgent need to novel high sensitivity, efficient, convenient, economic biochemical molecular detecting method and technology.Therefore, the unmarked sensing technology such as elliptically polarized light, the micro-balance of quartz crystal oscillator, surface plasma resonance technology is developed rapidly in recent years.
The metal nanoparticles such as gold, silver have highly active outer-shell electron, when light wave is radiated particle diameter much smaller than the metallic nanoparticle period of the day from 11 p.m. to 1 a.m of its excitation wavelength, the surface plasma wave producing near nanostructured, is produced resonance by confinement when incident light frequency is suitable with free electron collective concussion frequency.When metallics and fluorophor spacing are within the specific limits time, metallics can make the attenuation speed of fluorophor increase or resonance energy occurs to shift, and the fluorescence intensity of fluorophor is strengthened, and has greatly improved the sensitivity that object is detected.The multinomial feature such as convenient and swift with it, sensitivity is high, is subject to researchist's favor deeply.Current research is attached directly to metal particle layer surface by the large molecule of the biologicals such as protein mostly, exist metallics to be exposed in air and biomacromolecule and metallics between the problem such as distance is non-adjustable, cause many technical matterss also not solve, limited its application.
Summary of the invention
The object of the present invention is to provide a kind of protein sensing film and its production and use.Main technical schemes is on nano silver particles layer, to cover the separation layer of one deck without fluorophore, by the local surface plasma resonance effect of Nano Silver, utilize separation layer adsorbed proteins, by the fluorescence spectrum enhancing of the fluorescent materials such as protein, realize quick, the Sensitive Detection of these protein.
A kind of protein sensing film provided by the invention, it is comprised of three-decker: bottom is substrate, and middle layer is nano silver particles layer, and upper strata is the thin polymer film of without fluorophore, and described substrate is glass sheet or piezoid.
The present invention also provides a kind of preparation method of described protein sensing film, comprises the steps:
(1) Nano Silver presoma, reductive agent are dissolved in respectively to distilled water, obtain Nano Silver precursor solution and reductant solution; The heating of Nano Silver precursor solution is boiled a period of time, and then stir on limit, and limit is added dropwise to reductant solution wherein, continues to stop heating after heating a period of time, and ice-water bath is down to room temperature, and the centrifugal supernatant liquor of removing, obtains nano silver colloidal sol;
(2) coupling agent is dissolved in to the formation of methanol coupling agent solution, clean substrate, by Surface Treatment with Plasma 30min, is then placed in to coupling agent solution and soaks a period of time under normal temperature, make substrate surface silanization, after natural air drying, obtain silanization substrate;
(3) the silanization substrate obtaining in step (2) is immersed in to a period of time in the nano silver colloidal sol that step (1) obtains, then takes out, after vacuum drying, at silanization substrate surface, form nano silver particles layer, obtain Nano Silver substrate;
(4) polymer dissolution of without fluorophore is obtained to spin coating liquid in organic solvent, and then prepare thin polymer film on the Nano Silver substrate that adopts spin-coating method to obtain in step (3), after natural air drying, obtain protein sensing film.
Nano Silver presoma described in step (1) is a kind of in silver nitrate, silver acetate, and in precursor solution, the mass ratio of presoma and distilled water is 0.1 ~ 0.5:500; Precursor solution heating boiling temps is 120 ℃～140 ℃, and heating boiling time is 0.5 ~ 1h; Described reductive agent is sodium citrate, and in reductant solution, the mass ratio of reductive agent and distilled water is 1 ~ 10:500, after precursor solution is added dropwise to reductant solution, keeps temperature-resistant, and the continuation heat time is 5 ~ 20min; Precursor solution and reductant solution volume ratio 1:1.
Coupling agent described in step (2) is a kind of in γ-mercaptopropyl trimethoxysilane, gamma-mercaptopropyltriethoxysilane, and the mass ratio of coupling agent and methyl alcohol is 1～2:100; Substrate is glass sheet or piezoid, and substrate soak time in coupling agent solution is 0.5 ~ 2h.
Soak time described in step (3) is 0.5 ~ 1h.
In step (4) spin coating liquid, the mass ratio of without fluorophore polymkeric substance and organic solvent is 0.01 ~ 0.2:1000, described without fluorophore polymkeric substance is a kind of in polymethylmethacrylate, polyvinyl alcohol (PVA), and polymethylmethacrylate number-average molecular weight is 1.0 * 10
5~ 1.6 * 10
5, polyvinyl alcohol (PVA) average degree of polymerization is 1600 ~ 3000, alcoholysis degree>=98%, and organic solvent is a kind of in tetrahydrofuran, ethanol; The rotating speed that spin-coating method is prepared thin polymer film is 1000 ~ 3000rpm, and the time is 20 ~ 60s.
The present invention also provides a kind of purposes of protein sensing film, is applied to have the fast detecting of fluorescent characteristic protein, is specifically applied to the fast detecting of bone morphogenetic protein or bovine serum albumin.
Beneficial effect of the present invention:
The present invention utilizes sulfydryl and silver-colored interaction, adopt self-assembly method on microslide or piezoid load one deck there is the nano silver particles of certain ordered, become the substrate with local surface plasma resonance effect.Hydrophilic polymer is spin-coated on to nano silver-group basal surface film forming, with at the bottom of nano silver-group, compare, the distance between adjustable nano silver particles and fluorescent material not only, and then adjust enhancement effect of fluorescence, but also can completely cut off Nano Silver and air, hinder Nano Silver oxidized.Preparation technology of the present invention is simple, strong operability.
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Accompanying drawing explanation
Fig. 1 is the structural representation that the present invention is based on the protein sensing film of self-assembled nanometer silver (wherein: 1 is substrate, and 2 is nano silver particles layer, 3 be without fluorophore thin polymer film).
Fig. 2 is the scanning electron microscope (SEM) photograph of nano silver particles on embodiment 1 silanization substrate in the present invention.
Fig. 3 is the scanning electron microscope (SEM) photograph of nano silver particles on embodiment 2 silanization substrates in the present invention.
Fig. 4 is the scanning electron microscope (SEM) photograph of nano silver particles on embodiment 3 silanization substrates in the present invention.
Fig. 5 is the fluorescence spectrum comparison diagram (Fig. 5 a, b are respectively the fluorescent spectrum curve of bone morphogenetic protein in quartz substrate and sensing membrane) of measuring bone morphogenetic protein on the embodiment of the present invention 1 sensing membrane and quartz substrate.
Fig. 6 is the fluorescence spectrum comparison diagram (Fig. 6 c, d are respectively the fluorescent spectrum curve of bovine serum albumin in quartz substrate and sensing membrane) of measuring bovine serum albumin on the embodiment of the present invention 2 sensing membrane and quartz substrate.
Fig. 7 is the fluorescence spectrum comparison diagram (Fig. 7 e, f are respectively the fluorescent spectrum curve of bovine serum albumin in quartz substrate and sensing membrane) of measuring bovine serum albumin on the embodiment of the present invention 3 sensing membrane and quartz substrate.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to the following example, should comprise the full content in claims.
embodiment 1
(1) Nano Silver presoma silver nitrate, reductive agent sodium citrate are dissolved in respectively to distilled water, obtain Nano Silver precursor solution and reductant solution, in precursor solution, quality is than silver nitrate: distilled water is 0.5:500, and in reductant solution, quality is than sodium citrate: distilled water is 10:500; Precursor solution is heated to 130 ℃ and boils 1h, then stir on limit, and limit is added dropwise to reductant solution wherein, keeps temperature to continue heating 20min; Ice-water bath is down to room temperature, and the centrifugal supernatant liquor of removing, obtains nano silver colloidal sol.Precursor solution and reductant solution volume ratio 1:1.
(2) coupling agent γ-mercaptopropyl trimethoxysilane is dissolved in to the formation of methanol coupling agent solution, mass ratio γ-mercaptopropyl trimethoxysilane: methyl alcohol is 1:100; Clean piezoid, by Surface Treatment with Plasma 30min, is then placed in to coupling agent solution and soaks 2h under normal temperature, make piezoid surface silicon alkanisation, after natural air drying, obtain silanization substrate.
(3) in the nano silver colloidal sol silanization substrate obtaining in step (2) being obtained in step (1), soak 0.5h, then take out, after vacuum drying, at silanization substrate surface, form nano silver particles layer, obtain Nano Silver substrate.As shown in Figure 2.
(4) by polymethylmethacrylate, (number-average molecular weight is 1.0 * 10
5) be dissolved in tetrahydrofuran and obtain spin coating liquid, quality is than polymethylmethacrylate: tetrahydrofuran is 0.2:1000; And then prepare thin polymer film on the Nano Silver substrate that adopts spin-coating method to obtain in step (3), after natural air drying, obtain protein sensing film.The rotating speed that spin-coating method is prepared thin polymer film is 1000rpm, and the time is 30s.
(5) on the protein sensing film of quartz substrate and above-mentioned preparation, drip respectively the aqueous solution of bone morphogenetic protein (BMP-2), measure fluorescence emission spectrum after vacuum drying, result is as accompanying drawing 5(a), figure (b) as shown in.
embodiment 2
(1) Nano Silver presoma silver nitrate, reductive agent sodium citrate are dissolved in respectively to distilled water, obtain Nano Silver precursor solution and reductant solution, in precursor solution, quality is than silver nitrate: distilled water is 0.1:500, and in reductant solution, quality is than sodium citrate: distilled water is 1:500; Precursor solution is heated to 120 ℃ and boils 0.5h, then stir on limit, and limit is added dropwise to reductant solution wherein, keeps temperature to continue heating 5min; Ice-water bath is down to room temperature, and the centrifugal supernatant liquor of removing, obtains nano silver colloidal sol.Precursor solution and reductant solution volume ratio 1:1.
(2) gamma-mercaptopropyltriethoxysilane is dissolved in to the formation of methanol coupling agent solution, quality is than gamma-mercaptopropyltriethoxysilane: methyl alcohol is 2:100; Clean glass sheet, by Surface Treatment with Plasma 30min, is then placed in to coupling agent solution and soaks 1h under normal temperature, make glass sheet surface silanization, after natural air drying, obtain silanization substrate.
(3) in the nano silver colloidal sol silanization substrate obtaining in step (2) being obtained in step (1), soak 1h, then take out, after vacuum drying, at silanization substrate surface, form nano silver particles layer, obtain Nano Silver substrate.As shown in Figure 3.
(4) polyvinyl alcohol (PVA) (average degree of polymerization is 2400, alcoholysis degree >=98%) is dissolved in to organic solvent ethanol and obtains spin coating liquid, quality is than polyvinyl alcohol (PVA): ethanol is 0.01:1000; And then prepare thin polymer film on the Nano Silver substrate that adopts spin-coating method to obtain in step (3), after natural air drying, obtain protein sensing film.The rotating speed that spin-coating method is prepared thin polymer film is 2000rpm, and the time is 60s.
(5) on the protein sensing film of quartz substrate and above-mentioned preparation, drip respectively the aqueous solution of bovine serum albumin (BSA), measure fluorescence emission spectrum after vacuum drying, result is as accompanying drawing 6(c), as shown in Fig. 6 (d).
embodiment 3
(1) Nano Silver presoma silver acetate, reductive agent sodium citrate are dissolved in respectively to distilled water, obtain Nano Silver precursor solution and reductant solution, in precursor solution, quality is than silver acetate: distilled water is 0.3:500, and in reductant solution, quality is than sodium citrate: distilled water is 5:500; Precursor solution is heated to 120 ℃ and boils 1h, then stir on limit, and limit is added dropwise to reductant solution wherein, keeps temperature to continue heating 20min; Ice-water bath is down to room temperature, and the centrifugal supernatant liquor of removing, obtains nano silver colloidal sol.Precursor solution and reductant solution volume ratio 1:1.
(2) coupling agent γ-mercaptopropyl trimethoxysilane is dissolved in to the formation of methanol coupling agent solution, mass ratio γ-mercaptopropyl trimethoxysilane: methyl alcohol is 1.5:100; Clean piezoid, by Surface Treatment with Plasma 30min, is then placed in to coupling agent solution and soaks 1h under normal temperature, make piezoid surface silicon alkanisation, after natural air drying, obtain silanization substrate.
(3) in the nano silver colloidal sol silanization substrate obtaining in step (2) being obtained in step (1), soak 0.8h, then take out, after vacuum drying, at silanization substrate surface, form nano silver particles layer, obtain Nano Silver substrate.As shown in Figure 4.
(4) polyvinyl alcohol (PVA) (average degree of polymerization is 1600, alcoholysis degree >=98%) is dissolved in to ethanol and obtains spin coating liquid, quality is than polyvinyl alcohol (PVA): ethanol is 0.2:1000; And then prepare thin polymer film on the Nano Silver substrate that adopts spin-coating method to obtain in step (3), after natural air drying, obtain protein sensing film.The rotating speed that spin-coating method is prepared thin polymer film is 2000rpm, and the time is 40s.
(5) on the protein sensing film of quartz substrate and above-mentioned preparation, drip respectively the aqueous solution of bovine serum albumin (BSA), measure fluorescence emission spectrum after vacuum drying, result is as accompanying drawing 7(e), as shown in Fig. 7 (f).
Claims (7)
1. a protein sensing film, is characterized in that: it is comprised of three-decker: bottom is substrate, and middle layer is nano silver particles layer, and upper strata is the thin polymer film of without fluorophore, and described substrate is glass sheet or piezoid.
2. a preparation method for protein sensing film claimed in claim 1, is characterized in that comprising the steps:
(1) Nano Silver presoma, reductive agent are dissolved in respectively to distilled water, obtain Nano Silver precursor solution and reductant solution; The heating of Nano Silver precursor solution is boiled a period of time, and then stir on limit, and limit is added dropwise to reductant solution wherein, continues to stop heating after heating a period of time, and ice-water bath is down to room temperature, and the centrifugal supernatant liquor of removing, obtains nano silver colloidal sol;
(2) coupling agent is dissolved in to the formation of methanol coupling agent solution, clean substrate, by Surface Treatment with Plasma 30min, is then placed in to coupling agent solution and soaks a period of time under normal temperature, make substrate surface silanization, after natural air drying, obtain silanization substrate;
(3) the silanization substrate obtaining in step (2) is immersed in to a period of time in the nano silver colloidal sol that step (1) obtains, then takes out, after vacuum drying, at silanization substrate surface, form nano silver particles layer, obtain Nano Silver substrate;
(4) polymer dissolution of without fluorophore is obtained to spin coating liquid in organic solvent, and then prepare thin polymer film on the Nano Silver substrate that adopts spin-coating method to obtain in step (3), after natural air drying, obtain protein sensing film.
3. preparation method as claimed in claim 2, is characterized in that: Nano Silver presoma described in step (1) is a kind of in silver nitrate, silver acetate, and in precursor solution, the mass ratio of presoma and distilled water is 0.1 ~ 0.5:500; Precursor solution heating boiling temps is 120 ℃～140 ℃, and heating boiling time is 0.5 ~ 1h; Described reductive agent is sodium citrate, and in reductant solution, the mass ratio of reductive agent and distilled water is 1 ~ 10:500, after precursor solution is added dropwise to reductant solution, keeps temperature-resistant, and the continuation heat time is 5 ~ 20min; Precursor solution and reductant solution volume ratio 1:1.
4. preparation method as claimed in claim 2, is characterized in that: coupling agent described in step (2) is a kind of in γ-mercaptopropyl trimethoxysilane, gamma-mercaptopropyltriethoxysilane, and the mass ratio of coupling agent and methyl alcohol is 1～2:100; Substrate is glass sheet or piezoid, and substrate soak time in coupling agent solution is 0.5 ~ 2h.
5. preparation method claimed in claim 2, is characterized in that: the soak time described in step (3) is 0.5 ~ 1h.
6. preparation method claimed in claim 2, it is characterized in that, in step (4) spin coating liquid, the mass ratio of without fluorophore polymkeric substance and organic solvent is 0.01 ~ 0.2:1000, described without fluorophore polymkeric substance is a kind of in polymethylmethacrylate, polyvinyl alcohol (PVA), and polymethylmethacrylate number-average molecular weight is 1.0 * 10
5~ 1.6 * 10
5, polyvinyl alcohol (PVA) average degree of polymerization is 1600 ~ 3000, alcoholysis degree>=98%, and organic solvent is a kind of in tetrahydrofuran, ethanol; The rotating speed that spin-coating method is prepared thin polymer film is 1000 ~ 3000rpm, and the time is 20 ~ 60s.
7. a purposes for protein sensing film claimed in claim 1, is characterized in that: be applied to have the fast detecting of fluorescent characteristic protein, be specifically applied to the fast detecting of bone morphogenetic protein or bovine serum albumin.
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