CN100334231C - Multilayer colloid crystal based biomolecule detecting method - Google Patents
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- CN100334231C CN100334231C CNB2005100407396A CN200510040739A CN100334231C CN 100334231 C CN100334231 C CN 100334231C CN B2005100407396 A CNB2005100407396 A CN B2005100407396A CN 200510040739 A CN200510040739 A CN 200510040739A CN 100334231 C CN100334231 C CN 100334231C
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Abstract
The present invention relates to a biomacromolecule detection method based on multilayer colloidal crystals, which belongs to a simple convenience and high flux detection method used for the biomacromolecule detection in biological samples. In the biomolecule detection process, a multilayer colloidal crystal membrane is used as a flowing carrier for the biomacromolecule detection, and the biomacromolecule detection method has the steps that 1, the multilayer colloidal crystal membrane is prepared; 2, nucleic acid or protein probe molecules are fixed to the multilayer colloidal crystal membrane; 3, target molecules are hybridized; 4, the biomolecular types are recognized through determining reflectance spectrum bands of the multilayer colloidal crystal membrane by an optical fiber spectrograph, and then, the existence of the target molecules is determined according to fluorescence signals or the light absorption degree on the target molecules. The present invention has the advantages of high hybridization speed and low cost, and is a simple fast and high flux biological detection method.
Description
Technical field
The present invention is used for the method that biological sample carries out a kind of high throughput testing simply and easily of biomacromolecule detection.Can be widely used in clinical diagnosis and detection, drug screening, fields such as the detection of microbiological specimens in the environment, legal medical expert identify, customs's agricultural-food are imported and exported inspection and quarantine, gene order and functional analysis, microorganism is fractal, animals and plants breeding.
Background technology
Along with the arrival of genome epoch and genome times afterwards comprehensively, high-throughout gene test and proteinic detection technique basic means and instrument to become this life science fundamental research day by day.The parallel detection of high-throughput biomolecules can new functional gene and the protein group of high efficiency discovery, be widely used in the clone of new functional gene, in the research of expression and protein group, and in exploitation new medicine, clinical detection and the Animal or Plant Quarantine, all have wide practical use.At present in the detection of high-throughput biomacromolecule, it is main that what rely on is be the position encoded biochip of carrier dependence and be the flowing carrier liquid-phase chip technology with the fluorescent microsphere with sheet glass etc., but all there is certain defective in these two kinds of technology, though the biochip technology encoding amount is big, need expensive point sample system greatly, and hybridization speed is slow; And liquid-phase chip technology is carrier with the fluorescent microsphere, adopts dynamic hybridization technique, but because technical problem, encoding amount is little.It is problem anxious to be solved that thereby the platform of setting up the high-throughput biomacromolecule detection technology that hybridization speed is fast and encoding amount is big promotes the progress of life science.We have proposed with the colloidal crystal colloidal crystal as flowing carrier as macromolecular identify label in the biological detection, thereby are expected to make the problems referred to above to be resolved.
Colloidal crystal is a kind of of photonic crystal. thereby the periodic arrangement of spheroidal particle can form the propagation therein of optical band gap structure control light in the colloidal crystal. in colloidal crystal, having with the light of optical band gap same frequency and can't be propagated. these incident lights will be reflected by colloidal crystal. on reflection spectrum, the optical band gap of colloidal crystal shows as a very strong reflection peak. and the position of reflection peak changes with optical band gap. with respect to the colloidal crystal with ordered 3 D structure of other kind, it is easy that colloidal crystal has preparation, advantage such as film forming in the big area scope easily. we will utilize this optical characteristics of colloidal crystal to realize coding to flowing carrier. and ultimate principle is as follows: at first modify different types of probe molecule on different colloidal crystal carriers, because the position of optical band gap changes with the size of the spheroidal particle of forming colloidal crystal, therefore the kind of fixed biomolecules can be determined by measuring the optical band gap position on any colloidal crystal carrier.When detecting, the colloidal crystal carrier of probe molecule modified is mixed with the detection liquid that contains the target molecule that fluorescent mark crosses, after probe molecule and target molecule reaction, the kind of probe molecule is determined by the reflection spectrum of colloidal crystal platelet, and the existence of target molecule and otherwise definite by fluorescence spectrum.We also need existing opticinstrument is improved in the research in this stage, and it can be detected reflection spectrum and fluorescence simultaneously.
Summary of the invention
Technical problem: the present invention is a kind of method that is used for carrying out the parallel detection of biomacromolecule high-throughput.Adopt the multilayer colloid crystal film to replace fluorescent microsphere as flowing carrier and for fixing certain probe coding on it.After the hybridization, determine the kind of probe, whether according to the existence that has or not to determine target molecule of hybridization signal according to the feature reflectance spectrum of colloidal crystal film.It is fast that speed is hybridized in the present invention, with low cost.It is a kind of biological detecting method of high-throughput simply fast.
Technical scheme: the biomacromolecule detection method based on multilayer colloid crystal of the present invention is in the process of biomolecule detection, and is as follows as the biomacromolecule detection method of flowing carrier with the multilayer colloid crystal film:
The first step, the multilayer colloid crystal film and preparation: bottom polydimethylsiloxane film is after surface hydrophilic is handled, assembling bottom colloidal crystal film, cover middle layer polydimethylsiloxane film on the bottom colloidal crystal film, be assembled with top layer colloid crystal film on the polydimethylsiloxane film of middle layer again, be coated with top layer polydimethylsiloxane film on the top layer colloid crystal film; Top layer colloid crystal film is after hydrophilic treatment, assemble colloidal crystal again, and then covering polydimethylsiloxane film, then form trilaminar crystal film with photon, repeat above process successively, then can form four layers, five layers of colloidal crystal film, the particle diameter difference of forming the colloidal particle of every layer of colloidal crystal film, the feature reflectance spectrum difference of every layer of colloidal crystal film adopts the coding of the combination of different reflectance spectrums as biomacromolecule between multilayer colloid crystal film middle level and the layer;
Second step, nucleic acid or albumen probe molecule fixing on the multilayer colloid crystal film: nucleic acid or albumen probe stationary are at the lower surface of bottom polydimethylsiloxane film and the upper surface of top layer polydimethylsiloxane film, PDMS surface after UV ozone or the Cement Composite Treated by Plasma produces hydroxyl, utilize silane reagent that probe molecule is coupled at the multilayer colloid crystal film then, the fixing different probe molecule of the multilayer colloid crystal of different reflection bands of a spectrum, thus the coding of the feature reflection bands of a spectrum realization of multilayer colloid crystal film utilized to biomacromolecule;
The 3rd step, the hybridization of target molecule: target molecule is put on fluorescence or enzyme later on through pre-treatment in biological sample, then be fixed on different multilayer colloid crystals on probe molecule hybridization, nucleic acid or albumen different in the target molecule are special on the different probe molecule;
In the 4th step, the identification of target molecule: utilize fiber spectrometer to determine that the reflection bands of a spectrum of multilayer colloid crystal film discern the kind of biomolecules with this, the existence of determining target molecule according to the fluorescent signal on the target molecule or absorbance whether then.
The feature reflectance spectrum of employing multilayer colloid crystal film carries out Methods for Coding to biomacromolecule and is, every layer of colloidal crystal forming multilayer colloid crystal all has the feature reflection peak, utilize the various combination of every layer of colloidal crystal reflection peak in the multilayer colloid crystal that biomacromolecule is encoded, realize the expansion of encoding amount.
Bottom polydimethylsiloxane film, middle layer polydimethylsiloxane film, top layer polydimethylsiloxane film can be by polystyrene, or poly-polymethacrylic acid methyl ester substitutes.Bottom colloidal crystal film, the colloidal crystal in the top layer colloid crystal film is the polystyrene colloid crystal, or poly-polymethacrylic acid methyl ester colloidal crystal, or the silicon dioxide colloid crystal.The Silanization reaction that probe stationary adopts when the upper surface of the lower surface of bottom polydimethylsiloxane film and top layer polydimethylsiloxane film after being UV ozone or plasma body is as the macromolecular method of fixed biologically.
As flowing carrier, fixed biologically macromole probe is hybridized target material and macromole thereon with the colloidal crystal film in the present invention.After the wash-out, utilize the feature reflection spectrum of mirror based fiber optica spectrophotometer colloidal crystal film, as the sign of identification bioprobe molecule.Detect the signal hybridize on the bioprobe (for example fluorescent signal, chemiluminescent signal, electrochemical signals etc.) then, whether according to the existence that has or not to determine target molecule of hybridization signal.
1. the preparation of colloidal crystal film: the colloidal crystal film with feature reflectance spectrum can be divided into two kinds of opal type (being made of particle alignment) and counter opals (counter opal structure) by structure.Specify the preparation of 3 kinds of films below.
(1) preparation of single polymer layer colloidal crystal film: in 10: 1: 10 ratios the preceding aggressiveness of PDMS (polydimethylsiloxane), solidifying agent, normal hexane are mixed, be poured on substrate of glass or the silicon base hot setting.Hydrophilicity-imparting treatment is carried out on PDMS surface after the curing, and (UV ozone is handled, and utilizes the silane reagent of band unsaturated link(age) to handle then, carries out graft reaction again; Or directly initiation grafting reaction under UV-light; Oxygen plasma treatment).Utilize solvent evaporation method or vertical crystal pulling method self-assembly on the PDMS film in the polystyrene colloid crystal of different-grain diameter.Then in 5: 1: 5 ratio with PDMS before aggressiveness, solidifying agent, normal hexane mix, be poured on the colloidal crystal film, standing over night is then with its curing.
(2) preparation of multiple layer polymer colloidal crystal film (present method is an example with ELECTRODE WITH BILAYER POLYMERIC thing colloidal crystal film): in 10: 1: 10 ratios with PDMS before aggressiveness, solidifying agent, normal hexane mix, be poured on substrate of glass or the silicon base hot setting.Hydrophilicity-imparting treatment is carried out on bottom PDMS surface after the curing, and (UV ozone is handled, and utilizes the silane reagent of band unsaturated link(age) to handle then, carries out graft reaction again; Or directly initiation grafting reaction under UV-light; Oxygen plasma treatment).Utilize solvent evaporation method or vertical crystal pulling method self-assembly on the PDMS film colloidal crystal (PS colloidal crystal, PMMA colloidal crystal, silicon dioxide colloid crystal etc.) of a certain particle diameter, form bottom colloidal crystal film.Then in 5: 1: 5 ratio with PDMS before aggressiveness, solidifying agent, normal hexane mixes, be poured on the colloidal crystal film, standing over night, then with its curing, form middle layer PDMS film. middle layer PDMS film is used the same method carry out hydrophilicity-imparting treatment, use self-assembly on the PDMS film of middle layer the colloidal crystal of another kind of particle diameter or the different colloidal crystals of same particle size, form top layer colloid crystal film. in 10: 1: 10 ratios with PDMS before aggressiveness, solidifying agent, normal hexane mixes, be poured on the colloidal crystal film, standing over night, with its curing, form top layer PDMS film then.
2. probe is fixing: the fixing means of (1) PDMS individual layer and multilayer colloid crystal film is as follows: UV ozone is handled the PDMS film, APTES (aminopropyl triethoxysilane) and PDMS film reaction, by glutaraldehyde with amido modified nucleic acid probe or antigen, antibodies on the PDMS film.
3. the hybridization of target molecule: through purify, amplification back and target molecule that identifiable marker arranged be fixed on different films on the different probe molecular mixing after, the concussion acceleration is hybridized.
4 detect. and utilize the reflection spectrum of reflecting spectrograph detection colloidal crystal film, determine the kind of probe molecule.Utilize fluorescent microscope or spectrophotometric to keep the score not detect the target molecule of fluorescent mark or chemiluminescent labeling, then whether according to the existence that has or not to determine target molecule of signal.
Beneficial effect: the present invention utilizes colloidal crystal that biomolecules is encoded, thus the dynamic hybridization can realize that target molecule detects the time, and this method is simple.
1, substitutes fluorescent microsphere with colloidal crystal and encode and reduced the cost of manufacture of coding material, and simplified detecting instrument, reduced the detection cost.
2. the colloidal crystal coding is compared to the fluorescent microsphere coding, and the color that colloidal crystal presents is a schemochrome, stable in properties, and photobleaching easily takes place in fluorescent microsphere,
3. the colloidal crystal reflection spectrum is narrow, and can utilize the combination of the different reflectance spectrums of multilayer colloid crystal film to enlarge encoding amount.Up to ten thousand kinds of biomolecules of codified in theory, hundreds of biomolecules and fluorescent microsphere can only be encoded at present.
4. a kind of as flowing carrier of colloidal crystal compares with planar chip, can accelerate hybridization speed, thereby improve the efficient that detects.
Description of drawings
Fig. 1 is preparation and the probe stationary and the hybridization schema of multilayer colloid crystal film.
Fig. 2 is the structure of multilayer colloid crystal film (is example with double-deck colloidal crystal film) and the synoptic diagram after probe stationary and the hybridization.
Among the above figure bottom PDMS film 1, bottom colloidal crystal film 2, middle layer PDMS film 3, top layer colloid crystal film 4, top layer PDMS film 5, probe 6, target molecule 7 are arranged.
Embodiment
Fig. 1 is preparation and the probe stationary and the hybridization schema of multilayer colloid crystal film: the first step is the perfusion of bottom PDMS1 on sheet glass or silicon chip, after the curing, stripping down from sheet glass. second step was the preparation and the surface modification of bottom PDMS film, the 3rd step was the self-assembly of colloidal crystal 2 at bottom PDMS film, the 4th step was perfusion and the curing of top layer PDMS3, second step of repetition caused for the 4th step, form two layers of colloidal crystal film then, repeat secondary and form three layers of colloidal crystal film, and the like, the 5th step was the fixing of probe, and the 6th step was the hybridization of target molecule.
Fig. 2 is the structure of multilayer colloid crystal film (is example with double-deck colloidal crystal film) and the figure after probe stationary and the hybridization: be assembled with bottom colloidal crystal film 2 on the bottom PDMS film 1, cover middle layer PDMS film 3 on the bottom colloidal crystal film 2, be assembled with top layer colloid crystal film 4 on the PDMS film of middle layer again, be coated with top layer PDMS film 5 on the top layer colloid crystal film 4, probe 6 is fixed on the lower surface of bottom PDMS film 1 and the upper surface of top layer PDMS film 5, after the hybridization, target molecule 7 is combined on the probe molecule 6.
Fixing and the hybridization of preparation of the multilayer colloid crystal film of multiple reflectance spectrum (is example with double-deck colloidal crystal film with two kinds of reflectance spectrums) and probe:
(I) has the preparation (is example with the colloidal crystal film with two kinds of reflectance spectrums) of the multilayer colloid crystal film of multiple reflectance spectrum: the preceding aggressiveness of PDMS (polydimethylsiloxane), solidifying agent, normal hexane are mixed by 10: 1: 10 mass ratios, be poured on substrate of glass or the silicon base hot setting.Bottom PDMS1 surface after the curing carry out hydrophilicity-imparting treatment (UV ozone is handled, make the surface-CH
3Be converted into-OH; Produce hydroxyl after perhaps UV ozone is handled, utilize the silane reagent of band unsaturated link(age) to react with it then, cause the graft reaction of acrylic acid etc. again with initiator; Or directly initiation grafting reaction under UV-light; Oxygen plasma treatment produces hydroxyl, perhaps produces the hydrophilization result after the Silanization reaction again.)。Colloidal crystal (polystyrene colloid crystal, polymethyl acrylate colloidal crystal, the silicon dioxide colloid crystal etc.) self-assembly of a certain particle diameter on the PDMS film, is formed bottom colloidal crystal film 2.Then in 5: 1: 5 ratio with PDMS before aggressiveness, solidifying agent, normal hexane mix, be poured on the colloidal crystal film, standing over night then with its curing, forms middle layer PDMS film 3.Middle layer PDMS film 3 used the same method carries out hydrophilicity-imparting treatment, with the different colloidal crystals of the colloidal crystal of another kind of particle diameter or same particle size with solvent evaporation method or vertical crystal pulling method self-assembly on the PDMS film of middle layer, form top layer colloid crystal film 4.In 10: 1: 10 ratios with PDMS before aggressiveness, solidifying agent, normal hexane mix, be poured on the colloidal crystal film, standing over night then with its curing, forms top layer PDMS film 5.
(II) probe is fixing: the fixing means of double-deck colloidal crystal film is as follows: UV ozone or Cement Composite Treated by Plasma PDMS film, APTES and PDMS film reaction are combined in probe 6 (amido modified nucleic acid or antigen, antibody) on the upper surface of the little surface of bottom PDMS film 1 and top layer PDMS film 5 by glutaraldehyde.
(III) hybridization of target molecule: through purify, amplification back and target molecule 7 that identifiable marker arranged be fixed on different films on the different probe molecular mixing after, the concussion acceleration is hybridized.
(IV) detect. utilize two kinds of reflection spectrums of reflecting spectrograph detection colloidal crystal film, determine the kind of probe molecule.Utilize fluorescent microscope or spectrophotometric to keep the score not detect the target molecule of fluorescent mark or chemiluminescent labeling, then whether according to the existence that has or not to determine target molecule of signal.
Claims (5)
1, a kind of biomacromolecule detection method based on multilayer colloid crystal is characterized in that in the process of biomolecule detection, and is as follows as the biomacromolecule detection method of flowing carrier with the multilayer colloid crystal film:
The first step, the preparation of multilayer colloid crystal film: bottom polydimethylsiloxane film (1) is after surface hydrophilic is handled, assembling bottom colloidal crystal film (2), bottom colloidal crystal film (2) is gone up and is covered middle layer polydimethylsiloxane film (3), be assembled with top layer colloid crystal film (4) on the polydimethylsiloxane film of middle layer again, be coated with top layer polydimethylsiloxane film (5) on the top layer colloid crystal film (4); Top layer colloid crystal film is after hydrophilic treatment, assemble colloidal crystal again, and then covering polydimethylsiloxane film, then form trilaminar crystal film with photon, repeat above process successively, then can form four layers, five layers of colloidal crystal film, the particle diameter difference of forming the colloidal particle of every layer of colloidal crystal film, the feature reflectance spectrum difference of every layer of colloidal crystal film adopts the coding of the combination of different reflectance spectrums as biomacromolecule between multilayer colloid crystal film middle level and the layer;
Second step, nucleic acid or albumen probe molecule fixing on the multilayer colloid crystal film: nucleic acid or albumen probe (6) are fixed on the lower surface of bottom polydimethylsiloxane film (1) and the upper surface of top layer polydimethylsiloxane film (5), PDMS surface after UV ozone or the Cement Composite Treated by Plasma produces hydroxyl, utilize silane reagent that probe molecule is coupled at the multilayer colloid crystal film then, the fixing different probe molecule of the multilayer colloid crystal of different reflection bands of a spectrum, thus the coding of the feature reflection bands of a spectrum realization of multilayer colloid crystal film utilized to biomacromolecule;
The 3rd step, the hybridization of target molecule: target molecule (7) is put on fluorescence or enzyme later on through pre-treatment in biological sample, then be fixed on different multilayer colloid crystals on probe molecule (6) hybridization, nucleic acid or albumen different in the target molecule (7) are special on different probe molecule (6);
In the 4th step, the identification of target molecule: utilize fiber spectrometer to determine that the reflection bands of a spectrum of multilayer colloid crystal film discern the kind of biomolecules with this, the existence of determining target molecule according to the fluorescent signal on the target molecule (7) or absorbance whether then.
2. the biomacromolecule detection method based on multilayer colloid crystal as claimed in claim 1, it is characterized in that adopting the feature reflectance spectrum of multilayer colloid crystal film that biomacromolecule is carried out Methods for Coding being, every layer of colloidal crystal forming multilayer colloid crystal all has the feature reflection peak, utilize the various combination of every layer of colloidal crystal reflection peak in the multilayer colloid crystal that biomacromolecule is encoded, realize the expansion of encoding amount.
3. the biomacromolecule detection method based on multilayer colloid crystal as claimed in claim 1, it is characterized in that bottom polydimethylsiloxane film (1), middle layer polydimethylsiloxane film (3), top layer polydimethylsiloxane film (5) are by polystyrene, or poly-polymethacrylic acid methyl ester substitutes.
4. the biomacromolecule detection method based on multilayer colloid crystal as claimed in claim 1, it is characterized in that bottom colloidal crystal film (2), colloidal crystal in the top layer colloid crystal film (4) is the polystyrene colloid crystal, or poly-polymethacrylic acid methyl ester colloidal crystal, or the silicon dioxide colloid crystal.
5. the biomacromolecule detection method based on multilayer colloid crystal as claimed in claim 1, what it is characterized in that probe (6) adopts when being fixed on the upper surface of the lower surface of bottom polydimethylsiloxane film (1) and top layer polydimethylsiloxane film (5) is Silanization reaction behind UV ozone or the plasma body.
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| 改性聚苯乙烯微球的制备及其胶体晶体的组装 黄忠兵等，物理化学学报，第20卷第6期 2004 * |
| 改性聚苯乙烯微球的制备及其胶体晶体的组装 黄忠兵等，物理化学学报，第20卷第6期 2004;胶体晶体和基于胶体晶体的纳米结构 曹丙强等，物理，第33卷第2期 2004 * |
| 胶体晶体和基于胶体晶体的纳米结构 曹丙强等，物理，第33卷第2期 2004 * |
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