CN1514246A - Plane substrate packed polymer composition and its application - Google Patents
Plane substrate packed polymer composition and its application Download PDFInfo
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- CN1514246A CN1514246A CNA031356184A CN03135618A CN1514246A CN 1514246 A CN1514246 A CN 1514246A CN A031356184 A CNA031356184 A CN A031356184A CN 03135618 A CN03135618 A CN 03135618A CN 1514246 A CN1514246 A CN 1514246A
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Abstract
The present invention relates to an encapsulation composition for encapsulating plane substrate, a polymer encapsulating chip base, a reactor containing polymer encapsulation and a device including the reactor especially chip device. The encapsulation polymer provided by the present invention consists of surface activated agent of high polymer and optimization substituted polymer which have enough high activity of combination genin and minimizing activity of non-specific combination.
Description
Technical field
The present invention relates to be used to wrap by the bag of planar substrate formed, the device of polymer coating sheet base, polymer coating reactor and specific reaction device, particularly quantitatively or/and qualitative detection device, especially biochip.
Background technology
Contain the device of specific reaction device, particularly quantitatively or/and the qualitative detection device, one of them day by day important classification is the device that contains based on the specific reaction device of planar substrate.At present, the preparation of this class reactor, main path is as follows: earlier planar substrate is carried out prepared by surface modification and have sheet base in conjunction with the aglucon activity, then aglucon is fixed on the sheet base.Difference according to used matrix and aglucon fixed form, different types of reactor and device can be arranged, for example aglucon is fixed on the enzyme mark microwell plate, the aglucon that form in the polystyrene micropore plate in the mode of stochastic distribution and is fixed on sheet base (for example activating slide) with dot matrix way and goes up the chip that forms, or the like.Be that example illustrates the problem that waits to solve in existing sheet base and the device simply below with the chip.
Chip is that micro-aglucon is fixed on the pick-up unit that forms on the sheet base in addressable mode.At present widespread use be biochip.The scope because its high flux and microminiaturized characteristics, biochip 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.The activated centre of biochip is wherein fixing aglucon.The aglucon of biochip comprises that all can be fixed on the material of the biologically active on the solid phase carrier, for example antigen, antibody, strand and multichain DNA, RNA, nucleotide, part, affinity ligand, polypeptide, cell, be organized into the biotic component that grades.The most frequently used biochip is polypeptide chip and genetic chip.Aglucon fixing on the polypeptide chip is polypeptide (comprising protein).Aglucon fixing on the genetic chip comprises nucleic acid and nucleotide.
In chip, except aglucon, the mode of aglucon-object effect and condition, the immobilization chemistry of aglucon also is an important aspect in other words.Aglucon be immobilized in physics, the chemical property that is decided by the sheet primary surface to a great extent, the latter is decided by the activation of matrix again to a great extent.At present, the matrix of chip application mainly is the matrix of glass, metal, plastic or other material.Present matrix activation has dual mode: finishing and living polymer bag quilt.Finishing promptly derives linear activated group in stromal surface.For example, commercially available amido slide, aldehyde slide and epoxy radicals slide all is by slide being carried out the sheet base of prepared by surface modification at present.Because but the number and the activity of deriveding group on the planar substrate is all limited, utilize the sheet base of surface modification method preparation at present, its kind, number and spatial activity in order to the fixing active group of aglucon is all very limited.
Matrix is carried out living polymer bag quilt, is to introduce more a, important method of the active group of more number and greater room activity more.Present polymer coating is based on ion pairing mechanism.This method requires usually to wrap and is aggregated thing and has group with the opposite polarity polarity of matrix.Although prepare the sheet base existing the reasonable time with the method, present bag is formed far from ideal.Develop new bag and be still active subject in this field by composition.New bag is by an important problem in the composition research, be to guarantee under the stable and suitable condition of aglucon of encrusting substance reason in conjunction with activity, satisfy other requirement, its nonspecific action, absorption or bonding to the material beyond the object that is contacted is minimized.In fact, present bag is aggregated thing by the bag in forming, and mainly is overactivity degree polymkeric substance, and its exemplary is a polyelectrolyte, polyaminoacid for example, and the polarity of its active lateral group (amino) is higher and replacement rate active lateral group is also very high (almost 100%).Though overactivity degree polymkeric substance has very high aglucon in conjunction with activity, this high activity often shows as high non-specific binding activity again.In addition, polyaminoacid peridium patch base also has the problem of being stabilized property of bag.Be aggregated the overactive problem of thing in order to solve bag, someone adopts process for copolymerization to introduce the dead polymer chain in overactivity degree polymkeric substance, yet as if this solution is too complicated, cost too high (is 20020128234 with reference to the disclosed application number of United States Patent Office (USPO)).
Summary of the invention
The purpose of this invention is to provide a kind of planar substrate bag and be aggregated thing composition and application thereof.Bag of the present invention is formed, and that the material that is contacted under guaranteeing encrusting substance reason stability and the suitable condition of aglucon in conjunction with activity, in also having application has is less, the minimized nonspecific action of preferred version, absorption or bonding.The invention still further relates to polymer coating sheet base, polymer coating reactor and the specific reaction device formed based on this bag, particularly quantitatively or/and qualitative detection device, especially chip.The invention still further relates to a kind of method of polymer coating sheet base and a kind of method of preparation feedback device of preparing.
Bag of the present invention is consisted of:
A kind of planar substrate bag is aggregated the thing composition and uses, it comprises at least a appropriate activated polymer, described appropriate activated polymer comprises high molecular surfactant and optimizes substituted polymer that the replacement rate of active lateral group is that 3%-50%, preferred version are 3%-23%, more preferably scheme is 3%-13% in the described optimization substituted polymer.Described appropriate activated polymer, its activity level is guaranteeing that under the stable and suitable condition of aglucon in conjunction with activity of encrusting substance, the material that also has being contacted in using has minimized nonspecific action, absorption or bonding.The acquisition of the present invention appropriateness activated polymer is selected from two technology paths: the one, introduce and the distinguishing mechanism of action of ion pairing mechanism (high molecular surfactant), and the one, the replacement rate of active lateral group is optimized (optimization substituted polymer).High molecular surfactant has hydrophobic group (normally nonpolar group) and hydrophilic group (normally polar group) (reference: high molecular surfactant, Shen Yiding writes, Chemical Industry Press, in Dec, 2002, Beijing).Present polymer coating is based on ion pairing mechanism, and high molecular surfactant bag quilt of the present invention, owing to the existence of its hydrophobic group makes bag be enlarged by mechanism.Simultaneously, the effect of high molecular surfactant and matrix and other material of being contacted is its double-basis (nonpolar group and polar group) (normally in the aqueous solution) coefficient result in reaction environment.In embodiment 4 and 5, we are surprised to find, and high molecular surfactant polyvinylpyrrolidone bag is had tangible sensitivity to improve by slide and ELISA Plate than slide that does not wrap quilt and polystyrene ELISA Plate, does not improve but observe tangible specificity.Selecting active lateral group to optimize substituted polymer, is because in the active lateral group replacement scope that it had, and can satisfy the purpose that bag of the present invention is formed.For example, in the invention process 4, DEAE replacement rate has reached this purpose at the DEAE-of 6-12% glucosan.In embodiment 4, the chip of this type of polymer coating compares with the slide that does not wrap quilt, and sensitivity is significantly improved, and improves but observe tangible specificity.
On the one hand, a kind of planar substrate bag of the present invention is aggregated the thing composition and uses, wherein appropriate activated polymer is a high molecular surfactant, and preferred version is nonionic or the weak ion high molecular surfactant that comprises polyvinylpyrrolidone and polyacrylamide.
Another aspect, a kind of planar substrate bag of the present invention are aggregated the thing composition and use, and wherein appropriate activated polymer is for optimizing substituted polymer, and the active lateral group of described optimization substituted polymer comprises chemical bonding base, cation radical, anion base and hydrophobic group.
A kind of planar substrate bag of the present invention is aggregated the thing composition and uses, and the active lateral group of wherein optimizing in the substituted polymer comprises: amino, epoxy radicals, aldehyde radical, hydrazide group (CO-NHNH
2), diethyl amino ethyl group (DEAE-), diethyl-(2-hydroxypropyl) aminoethyl (QAE-), ethyloic (CM-), sulfonic acid propyl group (SP-), amino urea groups (H2N-NH-CONH-), mercapto ethylpyridine (MEP-), siloxy group, mercapto and combination thereof.Wherein, amino, epoxy radicals, aldehyde radical, siloxy group and amino urea groups are the at present common reactive groups that finishing is introduced on stromal surface that passes through.Diethyl amino ethyl group (DEAE-), hydrazide group (CO-NHNH
2), diethyl-(2-hydroxypropyl) aminoethyl (QAE-), ethyloic (CM-), sulfonic acid propyl group (SP-), mercapto ethylpyridine (MEP-), mercapto then be that the present invention introduces bag and is aggregated thing.
Another aspect, a kind of planar substrate bag of the present invention are aggregated that thing is formed and use, and wherein optimize active lateral group in the substituted polymer and have at least one to be that general formula is-(CH2) the long-armed reactive group of nR, 2＜n＜20 wherein, R is described active lateral group.Only pipe range arm reactive group oneself to be proved to be on the affinity chromatography carrier be useful (with reference to Spacer arms in affinity chromatography:the need for a morerigorous approach.Biochem.Soc.trans.1 (1973) 289-290), but in work hereto, be not mentioned about the planar chip base.
Another aspect, a kind of planar substrate bag of the present invention is aggregated the thing composition and uses, and wherein optimizes substituted polymer and comprises that active lateral group replacement rate is that 3%-50%, preferred version are 3%-23%, more preferably scheme is poly-polysaccharide derivates, polyacrylamide derivative and the polylysine derivant of 3%-13%.The selected poly-polysaccharide derivates of the present invention, at first be because the linear long chain structure of poly-polysaccharide is easy to wrap quilt and better biocompatibility is arranged on planar substrate, easily introduce (the reference: Chemical coupling of peptides and proteins to polysaccharides bymeans of cyanogens halides.Nature 214 (1967) 1302-1304 of the various active lateral group of kind quantitatively on the poly-then polysaccharide; U.S. Pat 5372820).Select polyacrylamide derivative and polylysine derivant, also be based on similar consideration.At this, be in order to the number of the active lateral group that replaces poly-polysaccharide, polyacrylamide and polylysine rather than the intrinsic group of polymkeric substance (for example poly-polysaccharide, polyacrylamide and polylysine-OH base, amide group and amino), determine its whether optimization substituted polymer of the present invention.
A kind of planar substrate bag of the present invention is aggregated the thing composition and uses, and wherein bag is comprised glucan derivative, agarose derivative and cellulose derivative by poly-polysaccharide derivates.
Of the present invention base is:
A kind of polymer coating sheet base is coated with on all surfaces of its matrix, part surface or the some zone and is selected from above-mentioned bag and is aggregated thing by the bag in forming, and described bag is aggregated the thing functionalization by regional by described bag.Polymer coating sheet base of the present invention, its functional character depend mainly on the bulk property that comprises polymer backbone, whole side group and active lateral group replacement rate that bag is aggregated thing.Polymer coating sheet base of the present invention, wherein polymer coating is bread quilt or some bag quilt.Bread is comprised the bag quilt of matrix all surfaces and part surface.Bread is formed easily, for example matrix is soaked in polymer liquid.Point bag is carried out (for example with sample applicator the polymer liquid point sample is wrapped to the matrix and reacted) by difficulty, but product has its characteristics, and for example bag is lower by the adsorptive power in the zone outside the point, or the like.
Another aspect, polymer coating sheet base of the present invention, wherein bag is aggregated thing and has cross-linked structure.The introducing of cross-linked structure is in order to improve the stability of polymer coating.
Polymer coating sheet base of the present invention wherein wraps the cross-linked structure cross-linked structure that is aggregated thing and realizes by following one or more crosslinking chemicals: epichlorokydrin, 2,3 dibromo-propanols, methine bisacrylamide, butylene glycol bisglycidyl ether.
On the other hand, polymer coating sheet base of the present invention, its host material be selected from not chemical modification or chemical modification glass, metal oxide, metal and polymeric material, or the like.
Reactor of the present invention is:
A kind of reactor is coated with polymkeric substance and aglucon on its matrix, described polymkeric substance has at least and a kind ofly is selected from above-mentioned bag and is aggregated thing by the bag in forming, and described bag is aggregated between thing and the aglucon and combines.Described bag be aggregated combining between thing and the aglucon comprise interaction, absorption, bonding, or the like.
Reactor of the present invention, polymkeric substance and the aglucon bag quilt on the sheet base wherein, its generation type is wrapped by aglucon for bag earlier is aggregated thing again, or bag is aggregated thing and aglucon simultaneously, or bag is wrapped by other polymkeric substance and aglucon more simultaneously by partial polymer earlier.A bag example being aggregated thing and aglucon is simultaneously, and polymkeric substance and aglucon are mixed, and wraps to the matrix by the sample applicator point sample and is reacted.
Another aspect, reactor of the present invention, wherein bag is aggregated thing and has cross-linked structure.The introducing of cross-linked structure is in order to improve the stability of polymer coating.
Reactor of the present invention wherein wraps the cross-linked structure that is aggregated thing and realizes by following one or more crosslinking chemicals: epichlorokydrin, 2,3 dibromo-propanols, methine bisacrylamide, butylene glycol bisglycidyl ether.
On the other hand, reactor of the present invention, wherein aglucon is one or more materials that are selected from following group: antigen, antibody, part, aglucon, polypeptide and strand or multichain DNA, RNA, nucleotide, polynucleotide (polynucleotide), sugar, common enzyme, co-factor (cofactor), microbiotic (antibiotic), steroids (steroid).In fact, can fix so various aglucon, also be one of purpose of the present invention.For example, the professional of the industry should know, sugar can be fixed on and wrap on reactive group amino, epoxy and the AH that is aggregated on the thing, and common enzyme, co-factor (cofactor), microbiotic (antibiotic) and steroids (steroid) can be fixed on the bag that contains long-armed reactive group and be aggregated on the thing.
On the other hand, reactor of the present invention, its host material be selected from not chemical modification or chemical modification glass, metal oxide, metal and polymeric material, or the like.
Analysis and detection device of the present invention is:
Analysis and detection device of the present invention, it contains above-mentioned reactor.
Analysis and detection device of the present invention, it is that it is chip, chip agent box and the check and analysis device that contains chip.
Analysis and detection device of the present invention, it is enzyme mark hole, ELISA Plate, enzyme marking reagent box and the check and analysis device that contains enzyme mark hole.
The method for preparing the sheet base of the present invention is:
The method for preparing the sheet base of the present invention comprises the following steps:
(a) preparing to contain the bag that bag is aggregated thing is formed;
(b) matrix of preparing not chemical modification or carrying out chemical modification;
(c) described bag is contacted and forms bread quilt or some bag quilt by composition and described matrix;
(d) described bag is carried out crosslinked between the polymkeric substance with stable bag quilt; And
(e) if necessary, described bag is carried out activation.
The method for preparing the sheet base of the present invention, wherein above-mentioned bag by, crosslinked and/or activation can be carried out synchronously or with different order proceed step by step.
The method of preparation feedback device of the present invention comprises the following steps:
(a) preparing to contain the bag that bag is aggregated thing is formed;
(b) matrix of preparing not chemical modification or carrying out chemical modification;
(c) described bag is formed mixed with aglucon, described polymkeric substance must be with described aglucon combination but must be with described matrix combination;
(d) potpourri that obtains in the step (c) is contacted with described matrix and carry out immobilized reactant; And (e) if necessary, described crosslinked polymer is wrapped quilt to stablize.
" aglucon " of the present invention: be meant and be fixed on the solid phase carrier in order to discern the material of the object in the sample, as antigen, antibody, nucleic acid etc.
Of the present invention base: a kind of activation solid phase carrier, it has the feature on macroscopical plane in order to the fixing surface of aglucon, for example at the bottom of chip slapper base, the ELISA Plate micropore hole, or the like.
Matrix of the present invention: in order to the solid phase carrier of preparation activation solid phase carrier, for example in order to slide of preparation chip slapper base or the like.
Bag quilt of the present invention: be meant polymkeric substance or/and aglucon is fixed on process (when using as verb) or the formed structure (when using as noun) on matrix or the sheet base.
Bread quilt of the present invention: with polymkeric substance or/and aglucon is fixed on formed structure on all or part of surface on matrix or the sheet base.
Point bag quilt of the present invention: with polymkeric substance or/and aglucon is fixed on formed structure on the some zone on matrix or the sheet base, described zone be separate, distribution density on the sheet primary surface greater than the zone of 1 point/cm2, the aglucon bag quilt that aglucon solution point sample is formed to the sheet base by the chip sample applicator for example.
Replacement rate of the present invention: the ratio that is meant the unit sum on unit sum that the side group on the substituted polymer main chain replaced by a kind of group and the main polymer chain.
Activation of the present invention: be meant and on polymkeric substance, introduce no arm active lateral group or/and the process of long-armed active lateral group.
Of the present invention crosslinked: as to refer to form between the polymkeric substance process that chemical bond connects.
Reactor of the present invention: be the place of confession under directions aglucon and object generation specific reaction and other dependency structure that is communicated with it, the hole in the reaction tank in for example open multiple reactor biochip and relevant isolation structure and liquid in-out structure etc., the 96 hole ELISA Plate, reagent strip of quick detection kit or the like.
Device of the present invention: be meant the parts or the equipment that include reactor; Described pick-up unit is meant the relevant apparatus that includes reactor in qualitative and/or the quantitative analysis method, wherein is fixed with in the reactor in order to the aglucon with the target molecule generation idiosyncrasy in the sample.For example, biochip or biochip kit, quick detection reagent bar or quick detection kit, ELISA Plate or ELISA Plate kit, or the like.
Chip of the present invention: be equivalent to " Biochip ", " Microarray ", " Bioarray " in the English, be meant a kind of device that aglucon in the reactor can be discerned in addressable mode with the result of the target molecule generation idiosyncrasy in the sample.
Advantage of the present invention is: of the present invention be used to wrap by the bag of planar substrate comprised suitable activated polymer (high molecular surfactant and optimize substituted polymer) by composition, have the activity and the minimized non-specific bond activity of sufficiently high aglucon with the polymer coating sheet base of its bag quilt, the device, particularly chip apparatus that contains the reactor of polymer coating and contain this reactor.
Embodiment:
Embodiment 1: optimize the preparation of substituted polymer
(1) .DEAE-glucosan/DEAE-Preparation of Agarose:
Glucosan is available from Shanghai chemical reagents corporation, and agarose is available from Sigma company, and DEAE is available from Baoding adduction Fine Chemical Co., Ltd.
The preparation method is with reference to one of inventor's article (COATED SILICA SUPPORTS FORHIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY OF PROTEIN, Journal ofChromatography, 476, (1989) 195-203).
DEAE replacement rate through determination of acid-basetitration is: 7-14%.
(2). hydrazides-Preparation of Polyacrylamide:
Hydrazides-Preparation of Polyacrylamide is (reference: The derivatization of cross-linked polyacrylamidebeads, Am.J.Epidemiol.143:263-268,1969) that the method for root a tree name JK.Inman and HM Dintzis is carried out.The used non-ionic polyacrylamide of present embodiment originates from Beijing Xitao Development Tech Co., Ltd., and used hydrazine hydrate originates from Zhuzhu Chemical Group Co., Ltd., Hunan Prov..The precipitation agent of the used non-ionic polyacrylamide of present embodiment is an alcohol.
Hydrazides replacement rate is 13% through determination of acid-basetitration.
To be aggregated thing as shown in table 1 for used bag in the embodiment of the invention.
Some bag of table 1. is by superpolymer
| Bag is by superpolymer | Manufacturer | The superpolymer type |
| DEAE-glucosan (low) | Self-control | Optimize substituted polymer |
| DEAE-agarose (low) | Self-control | Optimize substituted polymer |
| Hydrazides-polyacrylamide | Self-control | Optimize substituted polymer |
| Polyvinylpyrrolidone | Tianjin, Tianjin space Fine Chemical Co., Ltd | High molecular surfactant |
Embodiment 2: the preparation of polymer coating sheet base
It is as shown in table 1 that the used bag of present embodiment is aggregated thing, used matrix is respectively that (U.S. TeleChem International is Inc) with in order to the 96 hole microwell plates (the bright magnificent Industrial Co., Ltd. of Shenzhen gold) of preparation ELISA Plate sheet base in order to the microslide of preparation chip slapper base.
1). the bag quilt of polymkeric substance on matrix
Bag in the table 1 is aggregated thing (comprising polyvinylpyrrolidone, polysaccharide derivant and polyacrylamide derivative) dissolves in the PBS damping fluid respectively, acquisition concentration is that the bag of 1% (W/V) is aggregated the thing aqueous solution.Then it is diluted 40-100 respectively doubly.The bag that microslide is placed on acquisition is aggregated in the dilution of thing, and the bag quilt that at room temperature spends the night is cleaned oven dry, and the qualified back of quality inspection is standby.The bag that adds the 100-200ul acquisition in each hole of 96 hole microwell plates is aggregated the dilution of thing, and the bag quilt that at room temperature spends the night is cleaned oven dry, and the qualified back of quality inspection is standby.
2). bag is aggregated the crosslinked of thing
If necessary, the peridium patch base of above-mentioned preparation can wrap and be aggregated the crosslinked of thing.The cross-linking method of polysaccharide derivant can be with reference to one of inventor's article (COATED SILICA SUPPORTS FORHIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY OF PROTEIN, Journal ofChromatography, 476, (1989) 195-203).
In the present embodiment, after the abundant drying of slide with above-mentioned DEAE-glucosan bag quilt, in the epichlorokydrin (Xi'an chemical reagent factory) that joins concentration and be, be warming up to 90 ℃ of reactions 30 minutes.After reaction is finished, clean the back oven dry with alcohol, sterile distilled water, the qualified back of quality inspection is standby.
3). bag is aggregated the activation of thing
If necessary, the peridium patch base of above-mentioned preparation (bag is aggregated thing not to be had crosslinked or crosslinked peridium patch base arranged) can activate with the aglucon that improves polymer coating selectively in conjunction with activity.The activation method of the polysaccharide derivant of bag quilt and polyacrylamide can be with reference to activation method (preparation of Ago-Gel and chemical modification research, " application chemical industry ", the 32nd the 1st phase of volume, 2003 days of similar chromatography glue on the matrix; Gel chromatography and application thereof, Yuan Jingming, Science Press).The activation of present embodiment is for utilizing carbodiimides (Carbodiimide) to wrapping by the activation of DEAE-glucosan, one of activation method and inventor's article (COATEDSILICA SUPPORTS FOR HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY OF PROTEIN, Journal of Chromatography, 476, (1989) 195-203) in DEAE-glucosan bag identical by the activation method of chromatography carrier.
4). some peridium patch base
In the present embodiment, putting the peridium patch base is by concentration is aggregated thing solution with putting the model machine point on matrix at the bag of four per milles to one ten thousand/(w/v), then by to present embodiment 1) in bread by the preparation of similar immobilization process, prepared sheet base is the sheet base 3 ' in the table 2.
Some sheet base of table 2
| The peridium patch base | Bag is aggregated thing | Activator | Crosslinking chemical | Matrix |
| Sheet base 1 | The DEAE-glucosan | Slide | ||
| Sheet base 2 | Hydrazides-polyacrylamide | Slide | ||
| Sheet base 3 | Polyvinylpyrrolidone | Slide | ||
| Sheet base 3 ' | Polyvinylpyrrolidone | Slide | ||
| Sheet base 4 | The DEAE-glucosan | Carbodiimides | Slide | |
| Sheet base 5 | The DEAE-glucosan | Epichlorokydrin | Slide | |
| Sheet base 6 | Polyvinylpyrrolidone | Microwell plate |
Embodiment 3: the preparation of chip
The used bag of present embodiment is aggregated thing and is selected from table 1, and used base is selected from table 2.Microslide and conduct as matrix are joined the amino slide of photo-based available from U.S. TeleChem International, Inc.The used aglucon of present embodiment comprises: HCV antigen (hepatopathy research institute of Beijing people hospital), HIV
1+2Antigen (hepatopathy research institute of Beijing people hospital) and EBV antigen.EBV antigen is synthetic peptide antigen (Detection of human antibodies using " convergent " combinatorial peptide libraries or " mixotopes " the designed from anonvariable antigen:application to the EBVviral capsid antigen p18 of method of root a tree name D.Trabchand-Bunel, D.Trabchand-Bunel et al., J.Peptide Res, 52.1998, pp495-508).
Before bag is by aglucon or aglucon/polymkeric substance, coat high hydrophobic organosilicon coating (Chengdu morning twilight chemical design institute) at select location and form high hydrophobic layer isolation structure (with reference to our another invention " a kind of biochip and preparation method of reactor isolation structure minimized height ", application number 03117397.7), thereby form 8 reaction tanks of 2 row, reaction tank is of a size of 4.5mm * 4.5mm.
1). method A: elder generation's bag is aggregated thing and wraps by aglucon
This method uses the sheet Ji Bao of preparation among the embodiment 2 by aglucon.With concentration is the antigenic solution of 1.5mg/ml, and on the activation sheet base and comparison film base of manual some model machine point sample in the table 3,2 points of each point form 2 * 2 probe arrays.After antigen coated, standby with bovine serum albumin solution sealing back.Prepared chip is the chip 1-5 (comprising chip 3 ') in the table 3.
2). method B: bag is aggregated thing and aglucon simultaneously
With concentration is that the different antigen of 1.5mg/ml mixes with the polyvinylpyrrolidone and the hydrazides-polyacrylamide solution of concentration two per milles to five/one (w/v) respectively, at room temperature react half an hour, and then with described bag be aggregated thing-aglucon solution with the form point of array on microslide.After bag is finished fully, standby with bovine serum albumin solution sealing back.Prepared chip is the chip 6 and 7 in the table 3.Table 3 chip
| Chip | Bag is aggregated thing | Matrix | Aglucon | The preparation method | Remarks |
| Chip 1 | The DEAE-glucosan | Slide | EBV | ?A | |
| Chip 2 | Hydrazides-polyacrylamide | Slide | HCV、HIV | ?A | |
| Chip 3 | Polyvinylpyrrolidone | Slide | EBV | ?A | |
| Chip 3 ' | Polyvinylpyrrolidone | Slide | EBV | ?A | Point bag quilt |
| Chip 4 | The DEAE-glucosan | Slide | HCV、HIV | ?A | Bag is activated |
| Chip 5 | The DEAE-glucosan | Slide | HCV、HIV | ?A | Bag is crosslinked |
| Chip 6 | Polyvinylpyrrolidone | Slide | HCV、HIV | ?B |
| Chip 7 | Hydrazides-polyacrylamide | Slide | HCV、HIV | ?B |
Embodiment 4: the application of chip
In the present embodiment, the contrast chip is chip A, B and C.The sheet base of its chips A is a microslide, and the sheet base of chip B and C is that amino slide and height replace DEAE-glucosan bag by slide.Microslide and amino slide are available from U.S. TeleChem International, Inc.High replacement DEAE-glucosan is the DEAE-glucosan through secondary DEAE replacement, replacement rate 23%.The preparation of these chips is the same with embodiment 3.
In the present embodiment, 6 kinds of samples are respectively HCV antibody positive serum (HCV+), HIV
1+2Antibody positive human serum, HCV+HIV positive control (HCV antibody and HIV
1+2The potpourri of antibody positive serum tester) and HCV+HIV negative control thing (HCV antibody and HIV
1+2The serum tester that antibody is all negative), EBV IgA antibody positive human serum and EBV IgA negative antibody human serum.All samples all detect with classical ELISA method in advance and determine its positive and negative.
During experiment, 4 kinds of samples add the chip (table 3) of above-mentioned preparation respectively, and every kind of sample adds 2 reaction tanks.Sample is done suitably dilution during application of sample, and the application of sample amount is 10ul, 37 ℃ of temperature of reaction, 5 minutes reaction time.The each addition of cleansing solution is 15ul, washs 5 times.Label is the goat-anti people two anti-(JacksonImmunoRresearch Laboratories company) of rhodamine mark, and addition is 10ul, 37 ℃ of temperature of reaction, 5 minutes reaction time.The reaction back is with 15ul cleansing solution washing 5 times.Dry laggard line scanning (GMS of Afymetrix company 418 chip scanners) obtains result such as table 4 after the data processing (software is).
The testing result of table 4 chip
| Chip | Bag is aggregated thing | Aglucon | HCV+ | HCV- | HIV+ | HIV- | EBV+ | EBV- |
| Chip A | Do not have | HCV、HIV | ?1163 | ?17 | ?425 | ?13 | ||
| Chip B | Do not have | HCV、HIV | ?6970 | ?119 | ?1171 | ?17 | ||
| Chip C | The high DEAE-glucosan that replaces | EBV | ?6325 | ?524 | ||||
| Chip 1 | The DEAE-glucosan | EBV | ?4526 | ?285 | ||||
| Chip 2 | Hydrazides-polyacrylamide | HCV、HIV | ?8021 | ?237 | ?23?16 | ?32 |
| Chip 3 | Polyvinylpyrrolidone | HCV、HIV | ?7761 | ?239 | ?1803 | ?35 | ||
| Chip 3 ' | Polyvinylpyrrolidone | HCV、HIV | ?7023 | ?241 | ?1703 | ?33 | ||
| Chip 4 | The DEAE-glucosan | HCV、HIV | ?8706 | ?253 | ?2431 | ?45 | ||
| Chip 5 | The DEAE-glucosan | HCV、HIV | ?7031 | ?243 | ?1675 | ?53 | ||
| Chip 6 | Polyvinylpyrrolidone | HCV、HIV | ?7542 | ?235 | ?1765 | ?34 | ||
| Chip 7 | Hydrazides-polyacrylamide | HCV、HIV | ?7980 | ?237 | ?2154 | ?37 |
Embodiment 5: the preparation of ELISA Plate and application
In this example, the sheet base of used ELISA Plate is embodiment 2 prepared (the sheet base 6 in the table 2), and used label is the goat-anti people two anti-(Beijing Tiantan Bio-pharmaceuticals goods joint stock company limited) of enzyme labeling, and used aglucon is an EBV antigen.EBV antigen is synthetic peptide antigen (Detectionof human antibodies using " convergent " combinatorial peptidelibraries or " mixotopes " the designed from a nonvariableantigen:application to the EBVviral capsid antigen p18 of method of root a tree name D.Trabchand-Bunel, D.Trabchand-Bunel et al., J.Peptide Res, 52.1998, pp495-508).
The EBV antigen of concentration 0.3ug/ml is wrapped respectively by on the sheet base 8 (the polyvinylpyrrolidone bag is by microwell plate) and comparison film base (microwell plate) of table 5, and each sheet Ji Bao is by 8 holes.Bag is standby with bovine serum albumin solution sealing back by the back.
In the present embodiment, specimen in use is EBV VCA IgA antibody positive serum and EBV VCA IgA negative antibody serum.All samples all detect with classical ELISA method in advance and determine its positive and negative.
2 kinds of samples add respectively in described control enzyme target of table 5 and the ELISA Plate during experiment, and every kind of sample adds 4 reaction tanks.Sample is done 100 times of dilutions during application of sample, and the application of sample amount is 100ul, 37 ℃ of temperature of reaction, 30 minutes reaction time.The each addition of cleansing solution is 300ul, washs 3 times.Label is the goat-anti people IgA two anti-(Beijing Tiantan Bio-pharmaceuticals goods joint stock company limited) of enzyme labeling, and addition is 100ul, 37 ℃ of temperature of reaction, 30 minutes reaction time.The reaction conditions that adds substrate is with identical with the ELISA method of classics.Utilize microplate reader (Thermo Labsystems Shanghai Lei Bo Analytical Instrument Co., Ltd) to carry out colorimetric analysis, averaging of income OD value sees Table 5.
Table 5 ELISA testing result
| ELISA Plate | The sheet base | Positive serum | Negative serum |
| ELISA Plate 0 | The comparison film base | ????0.11 | ????0.07 |
| ELISA Plate 1 | Sheet base 8 | ????0.26 | ????0.09 |
Claims (23)
1, a kind of planar substrate bag is aggregated the thing composition, it is characterized in that: it comprises at least a appropriate activated polymer, described appropriate activated polymer comprises high molecular surfactant and optimizes substituted polymer that the replacement rate of active lateral group is that 3%-50%, preferred version are 3%-23%, more preferably scheme is 3%-13% in the described optimization substituted polymer.
2, a kind of planar substrate bag according to claim 1 is aggregated the thing composition, and it is characterized in that: the preferred version of described high molecular surfactant is nonionic or the weak ion high molecular surfactant that comprises polyvinylpyrrolidone and polyacrylamide.
3, a kind of planar substrate bag according to claim 1 is aggregated the thing composition, and it is characterized in that: the active lateral group in the described optimization substituted polymer comprises chemical bonding base, cation radical, anion base and hydrophobic group.
4, be aggregated the thing composition according to claim 1 or 3 described a kind of planar substrate bags, it is characterized in that: the active lateral group in the described optimization substituted polymer comprises amino, epoxy radicals, aldehyde radical, amino urea groups (H2N-NH-CONH-), hydrazide group (CO-NHNH
2), a kind of or any two kinds and above combination in the diethyl amino ethyl group (DEAE-), diethyl-(2-hydroxypropyl) ammonia second (QAE-), ethyloic (CM-), sulfonic acid propyl group (SP-), mercapto ethylpyridine (MEP-), siloxy group, mercapto.
5, be aggregated the thing composition according to claim 1 or 3 described a kind of planar substrate bags, it is characterized in that: the active lateral group in the described optimization substituted polymer has at least one to be that general formula is-(CH2) the long-armed active gene of nR, 2＜n＜20 wherein, R is described active lateral group.
6, be aggregated the thing composition according to claim 1 or 3 described a kind of planar substrate bags, it is characterized in that: described optimization substituted polymer comprises that active lateral group replacement rate is that 3%-50%, preferred version are 3%-23%, more preferably scheme is poly-polysaccharide derivates, polyacrylamide derivative and the polylysine derivant of 3%-13%.
7, a kind of planar substrate bag according to claim 6 is aggregated the thing composition, and it is characterized in that: described poly-polysaccharide comprises glucosan, agarose and cellulose.
8, a kind of planar substrate bag is aggregated the application that thing is formed, it is characterized in that: it is to contain described planar substrate bag to be aggregated the sheet base that thing is formed, be coated with by the bag in forming on all surfaces of its matrix, part surface or the some zone and be aggregated thing, and described bag is aggregated the thing functionalization by the zone by described bag.
9, a kind of planar substrate bag according to claim 8 is aggregated the application that thing is formed, and it is characterized in that: the bag of described polymer coating sheet base is aggregated thing and has cross-linked structure.
10, according to Claim 8 or 9 described a kind of planar substrate bags be aggregated the application that thing is formed, it is characterized in that: the cross-linked structure of described polymer coating sheet base realizes by following one or both and two or more crosslinking chemicals: epichlorokydrin, 2,3 dibromo-propanols, methine bisacrylamide, butylene glycol bisglycidyl ether.
11, a kind of planar substrate bag according to claim 8 is aggregated the application that thing is formed, it is characterized in that: one of following or any two kinds and two or more combination that the matrix of described polymer coating sheet base is selected from unmodified or has modified: glass, metal oxide, metal and polymeric material, described modification comprises finishing and polymer coating.
12, a kind of planar substrate bag according to claim 8 is aggregated the application that thing is formed, and it is levied and is: the preparation method of described polymer coating sheet base may further comprise the steps:
(a) preparing to contain the bag that bag is aggregated thing is formed;
(b) matrix of preparing not chemical modification or carrying out chemical modification;
(c) described bag is contacted and forms bread quilt or some bag quilt by composition and described matrix;
(d) described bag is carried out crosslinked between the polymkeric substance with stable bag quilt; And
(e) if necessary, described bag is carried out activation.
13, a kind of planar substrate bag is aggregated the application that thing is formed, it is characterized in that: it is to contain described planar substrate bag to be aggregated a kind of reactor that thing is formed, be coated with polymkeric substance and aglucon on its matrix, described polymkeric substance has at least a kind of being selected to be used for wrapping the bag of being formed by the bag of planar substrate and to be aggregated thing, and described bag is aggregated between thing and the aglucon and combines.
14, a kind of planar substrate bag according to claim 13 is aggregated the application that thing is formed, it is characterized in that: the polymkeric substance of described reactor and the aglucon bag quilt on the sheet base, its generation type is wrapped by aglucon for elder generation's bag is aggregated thing again, or bag is aggregated thing and aglucon simultaneously, or bag is wrapped by other polymkeric substance and aglucon more simultaneously by partial polymer earlier.
15, be aggregated the application that thing is formed according to claim 13 or 14 described a kind of planar substrate bags, it is characterized in that: the polymkeric substance of described reactor has cross-linked structure.
16, a kind of planar substrate bag according to claim 15 is aggregated the application that thing is formed, it is characterized in that: the crosslinked polymer structure of described reactor realizes by following one or both and two or more crosslinking chemicals: epichlorokydrin, 2,3 dibromo-propanols, methine bisacrylamide, butylene glycol bisglycidyl ether.
17, a kind of planar substrate bag according to claim 13 is aggregated the application that thing is formed, and it is characterized in that: the aglucon in the described reactor is the material that is selected from one or both and two or more combination in any in following group: antigen, antibody, part, aglucon, polypeptide and strand or multichain DNA, RNA, nucleotide, polynucleotide (polynucleotide), sugar, common enzyme, co-factor (cofactor), microbiotic (antibiotic), steroids (steroid).
18, a kind of planar substrate bag according to claim 13 is aggregated the application that thing is formed, it is characterized in that: one of following or two or more the combination arbitrarily that the matrix of described reactor is selected from unmodified or has modified: glass, metal oxide, metal and polymeric material, described modification comprises finishing and polymer coating.
19, a kind of planar substrate bag according to claim 13 is aggregated the application that thing is formed, and it is characterized in that: the method for described reactor comprises the following steps:
(1) preparing to contain the bag that bag is aggregated thing is formed;
(2) matrix of preparing not chemical modification or carrying out chemical modification;
(3) described bag is formed mixed with aglucon, described polymkeric substance must be with described aglucon combination but must be with described matrix combination;
(4) potpourri that obtains in the step (3) is contacted with described matrix and carry out immobilized reactant; And
(5) if necessary, described crosslinked polymer is wrapped quilt to stablize.
20, a kind of planar substrate bag is aggregated the application that thing is formed, and it is characterized in that: it is to contain described planar substrate bag to be aggregated the analysis and detection device that thing is formed.
21, a kind of planar substrate bag according to claim 20 is aggregated the application that thing is formed, and it is characterized in that: described analysis and detection device is chip, chip agent box or the check and analysis device that contains chip.
22, be aggregated the application that thing is formed according to the described a kind of planar substrate bag of claim 20, it is characterized in that: described analysis and detection device is enzyme mark hole, ELISA Plate, enzyme marking reagent box or the check and analysis device that contains enzyme mark hole.
23, be aggregated the application that thing is formed according to claim 20 or 22 described a kind of planar substrate bags, it is characterized in that: in the method for described analysis and detection device, bag by, crosslinked and/or activation can be carried out synchronously or with different order proceed step by step.
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| CNA031356184A CN1514246A (en) | 2003-08-18 | 2003-08-18 | Plane substrate packed polymer composition and its application |
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| CNA031356184A CN1514246A (en) | 2003-08-18 | 2003-08-18 | Plane substrate packed polymer composition and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101643321B (en) * | 2009-09-01 | 2012-07-18 | 博奥生物有限公司 | High-polymer three-dimensional amino-group substrate as well as preparation method and application thereof |
| CN103149364A (en) * | 2011-12-06 | 2013-06-12 | 贵州金玖生物技术有限公司 | Protein chip and manufacturing method |
| CN104039438A (en) * | 2011-11-02 | 2014-09-10 | 考利达基因组股份有限公司 | Treatment for stabilizing nucleic acid arrays |
| CN107255717A (en) * | 2017-07-07 | 2017-10-17 | 北京倍肯恒业科技发展股份有限公司 | Du-6859a analyte detection ELISA kit is developed and detection method |
| CN110078163A (en) * | 2019-04-30 | 2019-08-02 | 北京科技大学 | A kind of application of polylysine derivative as demulsifier |
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- 2003-08-18 CN CNA031356184A patent/CN1514246A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643321B (en) * | 2009-09-01 | 2012-07-18 | 博奥生物有限公司 | High-polymer three-dimensional amino-group substrate as well as preparation method and application thereof |
| CN104039438A (en) * | 2011-11-02 | 2014-09-10 | 考利达基因组股份有限公司 | Treatment for stabilizing nucleic acid arrays |
| CN104039438B (en) * | 2011-11-02 | 2016-03-09 | 考利达基因组股份有限公司 | For the processing method of stabilization of nucleic acids array |
| US10837879B2 (en) | 2011-11-02 | 2020-11-17 | Complete Genomics, Inc. | Treatment for stabilizing nucleic acid arrays |
| US11835437B2 (en) | 2011-11-02 | 2023-12-05 | Complete Genomics, Inc. | Treatment for stabilizing nucleic acid arrays |
| CN103149364A (en) * | 2011-12-06 | 2013-06-12 | 贵州金玖生物技术有限公司 | Protein chip and manufacturing method |
| CN107255717A (en) * | 2017-07-07 | 2017-10-17 | 北京倍肯恒业科技发展股份有限公司 | Du-6859a analyte detection ELISA kit is developed and detection method |
| CN110078163A (en) * | 2019-04-30 | 2019-08-02 | 北京科技大学 | A kind of application of polylysine derivative as demulsifier |
| CN110078163B (en) * | 2019-04-30 | 2020-10-23 | 北京科技大学 | Application of polylysine derivative as demulsifier |
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