CN1811427A - Active carrier for biological chip, producing method and application thereof - Google Patents
Active carrier for biological chip, producing method and application thereof Download PDFInfo
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- CN1811427A CN1811427A CN 200610020226 CN200610020226A CN1811427A CN 1811427 A CN1811427 A CN 1811427A CN 200610020226 CN200610020226 CN 200610020226 CN 200610020226 A CN200610020226 A CN 200610020226A CN 1811427 A CN1811427 A CN 1811427A
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Abstract
The present invention relates to an active carrier for biological chip. It contains solid-phase carrier and derivative chemically bonded on said solid-phase carrier surface. The described derivative at least contains coupling group and functional group combined on the coupling group by means of covalent bond, the molecular weight of the described functional group is greater than 50 and less than 1000. Said active carrier for biological chip has high efficiency and high stability. Besides, said invention also provides the preparation method of said active carrier.
Description
Technical field
The present invention relates to be used for biochip active carrier, be used for biochip active carrier the preparation method and be used for the application (bio-chip substrate, biochip, biochip kit, and biochip test) of the active carrier of biochip.
Background technology
Among the present invention, biochip kit (" Biochip Kit ", be called for short " chip agent box " again) be qualitative and/or quantitative test in a kind of pick-up unit, it comprises microchannel chip agent box (" Micro-tunnel BiochipKit ") and micro-array chip reagent kit (" Bio-array Kit " or " Micro-array Kit ").The scope because its high flux and microminiaturized characteristics, biochip kit have a wide range of applications comprises gene expression detection, genescreen, drug screening, medical diagnosis on disease treatment, environmental monitoring and fields such as improvement, judicial expertise.Biochip kit contains biochip (" Biochip " is called for short " chip " again) and Mk system usually.In our patented claim (PCT/CN2004/000437), Mk system contains aglucon, mark substance and nanostructured (active carrier that is used for the nanostructured label).
The core of chip is the reactor on it.Most important three part is sheet base (solid phase carrier that is used for chip), is fixed on probe and structure of reactor on the sheet base in the chip reactor.Probe be in the reactor by with sample in the interaction of object and the material of fixed object.What use the earliest is to be the nucleic acid chip of probe with nucleic acid.Grew up afterwards include the polypeptide probe (for example contain polypeptide chip, polypeptide chip, polypeptide/nucleic acid chip, polypeptide/carbohydrate chip, or the like), owing in detection reaction, comprise the polypeptide interphase interaction, make its to stability, space reaction efficiency, or the like higher requirement all arranged.The sheet base mainly is the carrier that contains active deriveding group of rectangle, circle or other shape of glass, metal, plastic or other material and derivant making thereof.Structure of reactor comprises reactor isolation structure, reactor stream line structure, reactor isolating construction, reactor reaction cell structure, or the like.Probe can directly be combined on the sheet base, also can form affine nanostructured and is combined in the sheet base again (with reference to our patented claim: PCT/CN2004/000437) by combining with active nano structure (active carrier that is used for affine nanostructured).
Usually, sheet base lozenge base solid phase carrier and the derivant that is fixed on the sheet base solid phase carrier.Form derivant in the sheet base dual mode is arranged: bag quilt and finishing.Contain functional group in the derivant in order to stationary probe.Functional group is the key factor that influences reaction mass with being connected of solid phase carrier.For example, the reacting dynamics condition of effective preservation of chip biological activity, solid phase probe (for example probe significance bit dot density, spatial movement degree of freedom, or the like), or the like all connect therewith relevant.This connection is very abundant also very complicated.For example, the covalent bond connection has different solid phase probe stability with the absorption connection; Different functional groups has different solid phase probe reaction efficient; Or the like.
Sheet base solid phase carrier is carried out polymer coating to introduce the work of functional group, comprising: application number be 20020128234 United States Patent (USP), or the like.These work are based on ion pairing mechanism, the homogeneity of sheet base activity and stable aspect have serious problems.
At present, comprise by the work of sheet base solid phase carrier being carried out surface chemical modification: 1). direct stationary probe (for example, the work among the Chinese patent application CN200310107946.X) behind the introducing coupling group; 2). the introducing coupling group is introduced functional group again and is removed stationary probe earlier.In the one class work of back, the micromolecule functional group of introducing comprises amino, aldehyde radical (Chinese patent application CN01105795.5, CN03132420.7), amino hydrazine (Application No. US2004235049), epoxy radicals and other micromolecule group (U.S. Patent number 5,474,895); The big molecular function group of introducing comprise contain amino polymkeric substance (for example: US patent 6,413,722, Chinese patent application numbers 200410027316.6, or the like).Generally speaking, introduce the sheet base of micromolecule functional group preparation, the efficient that the efficient of its stationary probe and probe fixed thereon are caught object all haves much room for improvement; And introduce the sheet base of big molecular function group preparation, its stability have much room for improvement or/and its preparation method complicated (for example crosslinked) be unfavorable for suitability for industrialized production.
For the active nano structure that is used for affine nanostructured or be used for the active nano structure of nanostructured label, also have and problem like the above-mentioned base class.
Summary of the invention
The purpose of this invention is to provide a kind of solid phase carrier that is used for biochip that has high-level efficiency and high stability and be easy to prepare.Technical scheme of the present invention based on chemical modification, and makes its functionalization based on introduce medium sized functional group on the surface of solid phase carriers coupling group.
So, first aspect of the present invention, relate to a kind of active carrier that is used for biochip, it is characterized in that: it contains the derivant of chemical bonding on solid phase carrier and the surface of solid phase carriers, described derivant contains on coupling group and the coupling group functional group with covalent bonds at least, and the molecular weight of described functional group greater than 50 less than 1000.The active carrier that is used for biochip of the present invention, the advantage that has high-level efficiency and high stability and be easy to prepare.
Second aspect of the present invention relates to a kind of preparation method who is used for the active carrier of biochip of the present invention, and it comprises at least: 1). described coupling group is combined on the described surface of solid phase carriers with chemical bonded refractory; 2). with functional mass and 1) prepared product in described coupling group contact and react, described functional mass contains described functional group; 3). described functional group is fixed on the described coupling group.
The 3rd aspect of the present invention relates to the application that is used for the active carrier of biochip of the present invention, comprises bio-chip substrate, biochip, biochip kit based on active carrier of the present invention, and biochip test.A kind of multiple reactor bio-chip substrate of the present invention is characterized in that: it contains biological chip base of the present invention at least and is fixed on described structure of reactor on the base.A kind of biochip of the present invention is characterized in that: its contain biological chip base of the present invention at least and be fixed on the described base two or more probe or contain bio-chip substrate of the present invention at least and be fixed on two or more probe on the sheet base of described substrate.A kind of biochip kit of the present invention, its feature be at its lozenge base or bio-chip substrate of the present invention, and be fixed on two or more the probe on the described base.A kind of biochip test method of the present invention comprises the following steps: A at least). provide to contain the sample that detects object, described object comprises the polypeptide object; B). biochip of the present invention is provided; C). described sample is contacted go forward side by side the line correlation reaction with described probe.
Below will the present invention be described in more detail by embodiment.
Embodiment
First aspect of the present invention, relate to a kind of active carrier that is used for biochip, it is characterized in that: it contains the derivant of chemical bonding on solid phase carrier and the surface of solid phase carriers, described derivant contains on coupling group and the coupling group functional group with covalent bonds at least, and the molecular weight of described functional group greater than 50 less than 1000.
Among the present invention, term " active carrier " be meant can by the functional group on it effectively the fixation reaction thing (for example probe, mark with aglucon, or the like) solid phase carrier, biological example chip slapper base, be used for affine nanostructured the active nano structure, be used for the nanostructured label the active nano structure, or the like.Although only provided the example of polypeptide in the embodiments of the invention as reactant, other biological substance (for example nucleic acid, cell, carbohydrate, or the like), also can use active carrier of the present invention and prepare biochip constituent of the present invention (for example, biological chip base, nanostructured label, or the like).It is to be noted especially, be suitable for the fixing active carrier of polypeptide and be generally suitable for nucleic acid and fix, be suitable for the fixing active carrier of nucleic acid and then be not necessarily suited for polypeptide and fix.Among the present invention, term " functional group " is meant in the active carrier by (comprise affinity interaction, ion-exchange, oleophilic function, covalent bonding, the or the like) group in order to the fixation reaction thing that interacts; Term " coupling group " is meant in the derivant of chemical bonding on the surface of solid phase carriers group in order to connection group and solid phase carrier.
In an embodiment that is used for the active carrier of biochip of the present invention, the molecular weight of described functional group greater than 75 less than 500.
In an embodiment that is used for the active carrier of biochip of the present invention, described functional group comprises amino acid group.
In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises deprotection amino acid.
In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises that side chain contains more than two or two-NH
2Amino acid.In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises that side chain contains more than three or three-NH
2Amino acid.
In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises lysine.In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises serine.In an embodiment that is used for the active carrier of biochip of the present invention, described amino acid comprises cystine.
In an embodiment that is used for the active carrier of biochip of the present invention, described coupling group comprises organosilicon radical.For example, by adding the coupling group that following one or more coupling agents are introduced: methacryloxy silane, vinyl silanes, amino silane, epoxy radicals silicone hydride, sulfenyl silane, urea groups silane, isocynate silane, 3-aminopropyltriethoxywerene werene, or the like.
In an embodiment that is used for the active carrier of biochip of the present invention, described covalent bond comprises amido link.
In an embodiment that is used for the active carrier of biochip of the present invention, described active carrier comprises biological chip base.Among the present invention, term " sheet base " is meant a kind of parts of biochip, promptly is used for the active carrier of biochip, and it has the function of effective stationary probe.Of the present invention base, at least lozenge base solid phase carrier, be fixed on the coupling group on the sheet base solid phase carrier and be fixed on functional group on the described coupling group.Sheet base solid phase carrier comprises conventional sheet base carrier and nanostructured sheet base carrier.Described conventional sheet base carrier is not fixed with the sheet base solid phase carrier of nanostructured for the surface.Although what examination of the present invention executed that example provides is with the example of glass as sheet base solid phase carrier, other solid phase carrier (for example silicon, gold, plastic sheet or sheet metal) also can be used method of the present invention and prepare biological chip base of the present invention.Described nanostructured sheet base carrier is the sheet base solid phase carrier that is fixed with nanostructured.
In an embodiment that is used for the active carrier of biochip of the present invention, described active carrier comprises the active nano structure that is used for affine nanostructured.Affine nanostructured of the present invention, at least nanostructure-containing (for example, nano particle, nanotube, or the like), be fixed on coupling group on the nanostructured, be fixed on the functional group on the described coupling group and be fixed on probe on the described functional group.
In an embodiment that is used for the active carrier of biochip of the present invention, described active carrier comprises the active nano structure that is used for the nanostructured label.Nanostructured label of the present invention, at least contain mark substance, nanostructured (for example, nano particle, nanotube, or the like), be fixed on coupling group on the nanostructured, be fixed on the functional group on the described coupling group and be fixed on aglucon on the described functional group.
Second aspect of the present invention relates to a kind of preparation method who is used for the active carrier of biochip of the present invention, 1). described coupling group is combined on the described surface of solid phase carriers with chemical bonded refractory; 2). with functional mass and 1) prepared product in described coupling group contact and react, described functional mass contains described functional group; 3). described functional group is fixed on the described coupling group.
In an embodiment of method of the present invention: A). described coupling group contains-NH
2Or-the COOH group; B). described functional mass contains functional group, be combined in the functional group blocking group and can with described coupling group reaction-NH
2Or-the COOH group; C). described functional group is fixed on the described coupling group and is undertaken by forming amido link; D). after described functional group was fixed on the described coupling group, described method was optionally sloughed described blocking group before also being included in stationary probe.An embodiment of method of the present invention is optionally sloughed described blocking group after also being included in stationary probe.An embodiment of method of the present invention also comprises and repeats described 1), 2), 3).
The 3rd aspect of the present invention relates to the application that is used for the active carrier of biochip of the present invention.
A kind of multiple reactor bio-chip substrate of the present invention is characterized in that: it contains biological chip base of the present invention at least and is fixed on described reactor isolation structure on the base.
Term of the present invention " substrate " be meant finger in order to the preparation chip, the intermediate product of stationary probe not as yet, it has or not other structure (for example isolation structure) and is fixedly forming chip behind the aglucon based on sheet base, combination.On the substrate one or more basal cells can be arranged.Usually do not have isolation structure on the monolithic basal cell substrate, this moment, substrate was sheet base (a for example commercially available amino slide).On the multi-disc basal cell substrate isolation structure is arranged, this moment, substrate comprised sheet base and isolation structure.The sheet basal cell forms reactor behind the aglucon on fixing, multi-disc basal cell substrate forms the multiple reactor chip.Term of the present invention " structure of reactor " is meant the necessary sheet base that forms reactor, other structure beyond the probe, as isolation structure, flow passage structure etc.; Term " reactor isolation structure " is meant at least the structure of avoiding occurring between the reactor cross pollution when adding sample, comprises the structure in order to isolated part or total reactor structure (for example reaction tank, reaction chamber, stream, feed liquor structure, fluid structure, or the like).The reactor isolation structure comprises open isolation structure and closed isolation structure.Open isolation structure is meant that isolating superstructure when chip uses does not have obducent reactor isolation structure.Closed isolation structure is meant that isolating superstructure when chip uses has obducent reactor isolation structure.
In an embodiment of bio-chip substrate of the present invention, described isolation structure comprises high hydrophobic material coating.In an embodiment of bio-chip substrate of the present invention, more than big 40 degree of surface static water contact angle of the surface static water contact angle of described high hydrophobic material than described solid phase carrier.Term of the present invention " surface static water contact angle " is meant the contact angle of Static Water on the surface.For a long time, with a drop of liquid at the contact angle θ of solid matter surface as the moistening quantification test of particular fixed, this point is widely approved.If liquid disperses to form film from the teeth outwards fully, if being liquid pearl on 0 material surface, contact angle θ has certain angle, this surface is considered to nonwetting.The most frequently used solid phase carrier substrate material is a glass in the chip, and its surface static water contact angle is about 45 degree.
In an embodiment of biological chip base of the present invention, hydrophobic/oleophobic material coating that described isolation structure comprises.
A kind of biochip of the present invention, it contains biological chip base of the present invention or bio-chip substrate of the present invention at least.An embodiment of biochip of the present invention, it contains biological chip base of the present invention at least and is fixed on two or more probe on the described base.An embodiment of biochip of the present invention, it is the multiple reactor biochip, it contains bio-chip substrate of the present invention at least and is fixed on two or more probe on the described base.Among the present invention, term " probe " is meant in order to catch the material of object in detection reaction, for example: the aptamer molecule of antigen, antibody, part, part index enhanced system evolution technology screening, polypeptide, polysaccharide, common enzyme, co-factor, microbiotic, steroids, virus, cell, biotin, Avidin etc.In an embodiment of biological chip base of the present invention, described biochip comprises the chip that contains the polypeptide probe.Term of the present invention " polypeptide " is equivalent to " polypeptide " in the English, comprise natural or synthetic protein, protein fragments, synthetic peptide, or the like, in the immune detection common object and detect in general probe, for example antigen, antibody, or the like all belong to polypeptide
A kind of biochip kit of the present invention is characterized in that: it contains the active carrier that is used for biochip of the present invention at least.
A kind of biochip test method of the present invention comprises the following steps: A at least). provide to contain the sample that detects object, described object comprises the polypeptide object; B). biochip of the present invention is provided; C). described sample is contacted go forward side by side the line correlation reaction with described probe.The use of biochip of the present invention in following one or more activities: as a kind of diagnostic tool, as a kind of diagnostic tool, as a kind of molecule scanning tools, as a kind of intermolecular relationship analysis instrument, as a kind of testing tool.
Embodiment
Embodiment 1 example (1) that is used for the active carrier of biochip of the present invention
In the present embodiment, the active carrier that is used for biochip of the present invention is a biological chip base.In the present embodiment, used basic solid phase carrier is slide, comprises that conventional slide and nanostructured slide are (with reference to our another patent application PCT/CN2004/000437).
In the present embodiment, used functional mass is selected from amino acid, preferably contains-NH
2The amino acid of side group protecting group (for example Fmoc).Used amino acid is respectively: lysine, serine, cystine and their potpourri.According to method of the present invention, other amino acid also can be used to prepare biological chip base of the present invention.
In the present embodiment, used coupling agent is selected from organosilicon, preferred silane compounds (for example 3-aminopropyltriethoxywerene werene).Use silane compound to form the coupling group that contains amino or hydroxyl in surface of glass slide.
In the present embodiment, a kind of preparation method of biological chip base of the present invention, it comprises at least: 1). bonding forms described coupling group on described surface of solid phase carriers, and described coupling group contains-NH
2Or-the COOH group; 2). with functional mass and 1) surface of solid phase carriers that contains coupling group of preparation contact and reacts, wherein: A). described functional mass contains described functional group and is combined in the interior blocking group of functional group; B). described functional mass also contains except that functional group-NH
2Or-the COOH group; 3). by forming amido link described functional group is fixed on the above-mentioned solid phase carrier surface with described coupling group.
The sheet base of present embodiment preparation, it is after stationary probe (with reference to following), at room temperature place the obvious decline of not seeing the probe activity in 12 months, existing sheet base (for example by the amino slide of known method preparation, aldehyde slide with according to the polylysine slide comparison of present embodiment method preparation) then in 11 months to see the obvious decline of probe activity.Compare with existing semicarbazides slide, the sheet base of present embodiment preparation demonstrates littler difference between batch.
Embodiment 1.1 coupling group contain the preparation example (1) of amino biological chip base
In the present embodiment, a kind of preparation method of biological chip base of the present invention is:
1). clean slide and fixing silanes group
Undertaken by known method.
2). the fixed amino acid group
The slide that is fixed with the silanes group after the oven dry is immersed in the above-mentioned amino acid whose aqueous solution of preferred concentration, at room temperature reacted 1 hour, the loose material of flush away surface of glass slide is standby after the oven dry then.
Embodiment 1.2 coupling group contain the preparation example (2) of amino biological chip base
In the present embodiment, a kind of preparation method of biological chip base of the present invention is:
1). clean slide and fixing silanes group
Undertaken by known method.
2). the fixed amino acid group
The slide that is fixed with the silanes group after the oven dry is immersed in the protectant amino acid whose DFM solution of the above-mentioned Fmoc of containing of preferred concentration, at room temperature reacted 1 hour.Slough the Fmoc protective agent by known method then, the loose material of flush away surface of glass slide is standby after the oven dry again.
Embodiment 1.3 coupling group contain the preparation example (3) of amino biological chip base
In the present embodiment, a kind of preparation method of biological chip base of the present invention is:
1). prepare amino slide by known method
2). the fixed amino acid group
Amino slide after the oven dry is immersed the above-mentioned of preferred concentration contain in the protectant amino acid whose DFM solution of Fmoc, at room temperature reacted 1 hour.Slough the Fmoc protective agent by known method then, the loose material of flush away surface of glass slide is standby after the oven dry again.
The preparation example of the carboxylic biological chip base of embodiment 1.4 coupling group
In the present embodiment, a kind of preparation method of biological chip base of the present invention is:
1). clean slide and fixing silanes group
Undertaken by existing known method.
2). fixing carboxylic group
Undertaken by existing known method.
3). the fixed amino acid group
The slide that is fixed with carboxyl after the oven dry is immersed in the protectant amino acid whose DFM solution of the above-mentioned Fmoc of containing of preferred concentration, at room temperature reacted 1 hour.Slough the Fmoc protective agent by known method then, the loose material of flush away surface of glass slide is standby after the oven dry again.
Embodiment 2 examples (2) that are used for the active carrier of biochip of the present invention
In the present embodiment, the active carrier that is used for biochip of the present invention is the active nano structure that is used for the active nano structure of affine nanostructured or is used for the nanostructured label.
In the present embodiment, used solid phase carrier is nano particle (for example monox nanometer particulate STN-3, LUDOXAS-40, an and TiOx nano particulate.
In the present embodiment, used functional mass is selected from amino acid, preferably contains-NH
2The amino acid of side group protecting group (for example Fmoc).Used amino acid is respectively: lysine, serine, cystine and their potpourri.According to method of the present invention, other amino acid also can be used to prepare biological chip base of the present invention.
In the present embodiment, used coupling agent is selected from organosilicon, preferred silane compounds (for example 3-aminopropyltriethoxywerene werene).Use silane compound to form the coupling group that contains amino or hydroxyl in surface of glass slide.
In the present embodiment, a kind of preparation method of biological chip base of the present invention, it comprises at least:
1). bonding forms described coupling group on described surface of solid phase carriers, and described coupling group contains-NH
2Or-the COOH group.Bonding method is identical with the preparation method of existing amination nano silicon oxide.
2). with above-mentioned functions material and 1) surface of solid phase carriers that contains coupling group of preparation contacts and reacts.Temperature of reaction is a room temperature, 2 hours reaction time.
3). by forming amido link described functional group is fixed on the above-mentioned solid phase carrier surface with described coupling group.
The active nano structure of present embodiment preparation, it at room temperature places the obvious decline of not seeing probe on the nanostructured or label aglucon activity in 1 month after stationary probe or label aglucon.
The preparation example of embodiment 3 multiple reactor bio-chip substrates of the present invention
In the present embodiment, used biological chip base is the sheet base of the foregoing description 1 preparation.
The preparation of embodiment 3.1 high hydrophobic isolation structure substrates
In the present embodiment, used high hydrophobic material is respectively " polyacrylic acid grease coating material ", and (morning twilight chemical design institute, Chinese Chengdu provides, Static Water order of contact 85 degree), (morning twilight chemical design institute, Chinese Chengdu provides " organic silicon water-proofing coating ", Static Water order of contact 116 degree), (morning twilight chemical design institute, Chinese Chengdu provides " the hydrophobic latex paint of superelevation ", Static Water order of contact 123 degree) and " high hydrophobic monox coating " (Chinese Zhoushan nano material tomorrow company provides, and Static Water order of contact 151 is spent).Although the isolation structure of reactor of the present invention can partly be, also can all be the hydrophobic isolation structure of height of the present invention, only provided the simplest situation in the present embodiment as an example.
Preparation method in the present embodiment is: with the hydrophobic liquid applying material of above-mentioned height on position, isolation strip on the sheet base of the foregoing description 1 preparation, operation instruction by the supplier is solidified after drying at room temperature, forms the high hydrophobic convex body of height 25-115 μ m, width 2.0-2.5mm.High hydrophobic convex body can have different how much patterns, only gets band shape (convex body is a band) and line combination (for example convex body is spaced 2 lines) 2 kinds in this example.Various how much patterns can be got in the surface that high hydrophobic convex body surrounds, and only get 3mm * 3mm rectangle in this example.On this surface of substrate, laterally have 12 sheet Ji Chi, 4 sheet Ji Chi are vertically arranged, have 48 sheet Ji Chi.
The preparation of embodiment 3.2 hydrophobic/oleophobic isolation structure substrates
In the present embodiment, used hydrophobic-oleophobic material is the hydrophobic-oleophobic material that can buy on the market, be respectively the clean treasured in city (Shenzhen Anyclean Environment ﹠ Science Co., Ltd.), high hydrophobic monox coating (Chinese Zhoushan nano material tomorrow company) and two thin coating (China blue photoinitiator chemical research institute).Although the isolation structure of reactor of the present invention can partly be, also can all be the hydrophobic isolation structure of height of the present invention, only provided the simplest situation in the present embodiment as an example.
Preparation method in the present embodiment is: with above-mentioned hydrophobic-oleophobic material spreads upon on the sheet base of the foregoing description 1 preparation on the position, isolation strip, operation instruction by the supplier is solidified after drying at room temperature, forms hydrophobic-oleophobic convex body of height 25-115 μ m, width 2.0-2.5mm.Various how much patterns can be got in the surface that hydrophobic-oleophobic convex body surrounds, and only get 3mm * 3mm rectangle in this example.On this surface of substrate, laterally have 12 sheet Ji Chi, 4 sheet Ji Chi are vertically arranged, have 48 sheet Ji Chi.
The example of embodiment 4 biochips of the present invention
Biochip in the present embodiment contains the substrate of the foregoing description 3 preparations or/and the active nano structure that is used for affine nanostructured of the foregoing description 2 preparations.The substrate of embodiment 3 preparations contains the sheet base of the foregoing description 1 preparation.The used probe of present embodiment is HCV antigen (the People's Hospital, BeiJing, China hepatopathy research institute) and HIV
1+2Antigen (the People's Hospital, BeiJing, China hepatopathy research institute).
In the present embodiment, the preparation method of biochip of the present invention is: in a sheet base pond of above-mentioned substrate, in sheet base pond, 3 diameters of every kind of liquid each point are the point of 80 μ m to the liquid that will contain probe respectively according to general point sample method point sample, between point apart from be 600-700 μ m, formation 2 * 3 arrays.Aglucon density on the reaction tank sheet base is greater than 30 points/cm
2The used liquid that contains probe is respectively: HCV antigen (1.0mg/ml), HIV
1+2Antigenic solution (1.0mg/ml), HCV antigen/active nano structural composites (HCV antigen concentration 0.1mg/ml), HIV
1+2Antigen/active nano structural composites (HIV
1+2Antigen concentration).HCV antigen/active nano structural composites and HIV
1+2Antigen/active nano structural composites is affine nanostructured, and it contains the active nano structure and is fixed on the structural antigen of active nano, and its preparation method is with reference to our another patent application PCT/CN2004/000437.
The example of embodiment 5 biochip kits of the present invention
The present embodiment kit is respectively: 1). contain the kit of the biochip of embodiment 4 preparations; 2). the kit of nanostructure-containing label; 3). contain the biochip of embodiment 4 preparations and the kit of nanostructured label.In the present embodiment, the nanostructured label contains rhodamine, active nano structure and is fixed on the structural goat-anti people two of active nano and resists, and its preparation method is with reference to our another patent application PCT/CN2004/000437.
The example of embodiment 6 biological detecting methods of the present invention
In the present embodiment, No. 1 sample is a HCV antibody positive serum, and No. 2 samples are HIV
1+2The antibody positive human serum, No. 3 negative testers of sample (HCV antibody and HIV
1+2The serum tester that antibody is all negative).All samples are all through using classical ELISA method to detect in advance under 20 times of diluting reaction conditions of serum.
In the present embodiment, used biochip kit is the kit of embodiment 5 preparations.In the contrast agents box, chip for respectively on amino slide and epoxy radicals slide (Microarray Technology) routinely point sample method be fixed with the chip of identical aglucon preparation, label is rhodamine mark goat-anti people two anti-(U.S. Jackson ImmunoRresearch Laboratories company).During experiment, aforementioned 3 kinds of samples add respectively in the reaction tank of biochip.The application of sample amount is 15 μ l, reacts after 30 minutes and washs 5 times, and the each addition of cleansing solution is 25 μ l.The label addition is 15 μ l, reaction back washing 5 times, and the each addition of cleansing solution is 25 μ l, dry back is scanned at 35/50 time.Scanner is confocal laser scanner (GMS418 of an Afymetrix company chip scanner), scanning excitation wavelength 532nm, wavelength of transmitted light 570nm, the treated software of the signal that reads (JAGUARII) is handled, and the result that cloudy (-) sun (+) property obtains is judged according to the Cut-off value in the back of averaging then.The result that gained result and ELISA method detect under 20 times of diluting reaction conditions of serum in advance is consistent and the detection lower limit is lower.Biochip kit of the present invention and contrast agents box are relatively, and be stable high more than 20% in the time of 37 ℃, and detect lower limit and will hang down more than 15%.
The use of biochip kit of the present invention in following one or more activities: as a kind of diagnostic tool, as a kind of molecule scanning tools, as a kind of intermolecular relationship analysis instrument, as a kind of testing tool.
Should be understood that, for a person skilled in the art, obviously can make various changes and modification embodiment preferred of the present invention described here.Reaching without departing from the spirit and scope of the present invention under the situation that does not reduce its advantage, can carry out these modifications and change.Therefore, these modifications and change are included in the scope of appended claim.
Claims (14)
1. active carrier that is used for biochip, it is characterized in that: it contains the derivant of chemical bonding on solid phase carrier and the surface of solid phase carriers, described derivant contains on coupling group and the coupling group functional group with covalent bonds at least, and the molecular weight of described functional group greater than 50 less than 1000.
2. active carrier according to claim 1 is characterized in that: the molecular weight of described functional group greater than 75 less than 500.
3. active carrier according to claim 1 is characterized in that: described functional group comprises amino acid group.
4. active carrier according to claim 1 is characterized in that: described coupling group comprises organosilicon radical.
5. active carrier according to claim 1 is characterized in that: described covalent bond comprises amido link.
6. active carrier according to claim 1 is characterized in that: described active carrier comprises biological chip base.
7. active carrier according to claim 1 is characterized in that: described active carrier comprises the active nano structure that is used for affine nanostructured.
8. active carrier according to claim 1 is characterized in that: described active carrier comprises the active nano structure that is used for the nanostructured label.
9. the described preparation method who is used for the active carrier of biochip of one of claim 1-8 comprises the following steps: at least
1). described coupling group is combined on the described surface of solid phase carriers with chemical bonded refractory;
2). with functional mass and 1) prepared product in described coupling group contact and react, wherein said functional mass contains described functional group;
3). with described 2) described in functional group be fixed on the described coupling group.
10. method according to claim 9, wherein:
A). described coupling group contains-NH
2Or-the COOH group;
B). described functional mass contains functional group, be combined in the functional group blocking group and can with described coupling group reaction-NH
2Or-the COOH group;
C). described functional group is fixed on the described coupling group and is undertaken by forming amido link;
D). after described functional group was fixed on the described coupling group, described method was optionally sloughed described blocking group before also being included in stationary probe.
11. a multiple reactor bio-chip substrate is characterized in that: it contains right at least and requires 6 described biological chip bases and be fixed on described reactor isolation structure on the base.
12. a biochip is characterized in that: its contain at least right require 6 described biological chip bases and be fixed on the described base two or more probe or contain right at least and require 11 described bio-chip substrates and be fixed on two or more probe on the sheet base of described substrate.
13. a biochip kit is characterized in that: it contains right at least and requires one of the 1-8 described active carrier that is used for biochip.
14. a biochip test method comprises the following steps: at least
A). provide to contain the sample that detects object, described object comprises the polypeptide object;
B). provide claim 13 described biochip kit;
C). make the probe on the biochip in described sample and the described kit contact the line correlation reaction of going forward side by side.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610020226 CN1811427A (en) | 2006-01-25 | 2006-01-25 | Active carrier for biological chip, producing method and application thereof |
| CN2006800516076A CN101443661B (en) | 2006-01-25 | 2006-10-11 | Active constituent, nanostructure composition containing the active constituent and preparation method thereof |
| PCT/CN2006/002659 WO2007085156A1 (en) | 2006-01-25 | 2006-10-11 | Active component, nanostructured composition comprising active components and its preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610020226 CN1811427A (en) | 2006-01-25 | 2006-01-25 | Active carrier for biological chip, producing method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1811427A true CN1811427A (en) | 2006-08-02 |
Family
ID=36844469
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610020226 Pending CN1811427A (en) | 2006-01-25 | 2006-01-25 | Active carrier for biological chip, producing method and application thereof |
| CN2006800516076A Expired - Fee Related CN101443661B (en) | 2006-01-25 | 2006-10-11 | Active constituent, nanostructure composition containing the active constituent and preparation method thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006800516076A Expired - Fee Related CN101443661B (en) | 2006-01-25 | 2006-10-11 | Active constituent, nanostructure composition containing the active constituent and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN1811427A (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100399475B1 (en) * | 1998-02-12 | 2003-09-29 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
| US6221602B1 (en) * | 1998-11-10 | 2001-04-24 | Bio-Pixels Ltd. | Functionalized nanocrystals and their use in labeling for strand synthesis or sequence determination |
| AU2005241112B2 (en) * | 2001-07-13 | 2009-07-30 | Nanosphere, Inc. | Method for preparing substrates having immobilized molecules and substrates |
| WO2003006676A2 (en) * | 2001-07-13 | 2003-01-23 | Nanosphere, Inc. | Method for immobilizing molecules onto surfaces |
| CN1250969C (en) * | 2003-03-13 | 2006-04-12 | 成都夸常科技有限公司 | Test device and method for making qualitative and/or quantitative analysis to object |
| WO2004089818A1 (en) * | 2003-04-14 | 2004-10-21 | Centre National De La Recherche Scientifique | Functionalized carbon nanotubes, a process for preparing the same and their use in medicinal chemistry |
| EP1624306A4 (en) * | 2003-04-30 | 2008-12-24 | Chengdu Kuachang Medical Ind L | Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof |
-
2006
- 2006-01-25 CN CN 200610020226 patent/CN1811427A/en active Pending
- 2006-10-11 CN CN2006800516076A patent/CN101443661B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101443661B (en) | 2012-11-14 |
| CN101443661A (en) | 2009-05-27 |
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