CN106383224A - Biological detection element and preparation method thereof - Google Patents
Biological detection element and preparation method thereof Download PDFInfo
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- CN106383224A CN106383224A CN201610789401.9A CN201610789401A CN106383224A CN 106383224 A CN106383224 A CN 106383224A CN 201610789401 A CN201610789401 A CN 201610789401A CN 106383224 A CN106383224 A CN 106383224A
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- ketone
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- oxazoline
- testing element
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
The invention provides a biological detection element, wherein a substrate is modified with an oxazolone polymer. The biological detection element provided by the invention can be loaded with various biological probes through clicking-like chemical properties of an oxazolone functional group, the reaction is simple, rapid and efficient, no activators or catalysts are needed, and moreover, nonspecific adsorption can be reduced. In addition, the surface of the substrate modified by the oxazolone polymer has good hydrophilicity and anti-biofouling performance, and thus accurate and sensitive analysis and detection of complex samples such as whole blood, plasma, serum, urine, saliva, tears and the like is achieved. Therefore, the biological detection element has broad application prospects in real-time detection equipment, microfluidic chips, gene chips and other analysis and detection advanced fields.
Description
Technical field
The present invention relates to technical field of biological, more particularly, to a kind of biological testing element and preparation method thereof.
Background technology
Biological detection, including immune detection and gene test, typically utilizes specific recognition between biomolecule to make
With, thus realize quick to object, accurately and sensitive detect, therefore in disease diagnosis and therapy, environmental monitoring and food
All there is good application prospect in inspection, also obtain the extensive attention of researcher in the last few years.However, due to actually detected sample
Product, the such as composition such as whole blood, blood plasma, serum, urine, saliva, and tear is sufficiently complex, and the clinical practice of biological detection still faces
Very big challenge, mainly include the false positive because non-specific adsorption causes and probe load capacity little cause low sensitive
Degree.
Surface texture determines many physical propertys and the final use of biomaterial with composition.Change surface nature in bag
Include biological detection, in interior biological technical field, there is special importance, wherein biocompatibility especially receives publicity.For this reason,
Researchers generally detection substrate surface modify one layer of hydrophilic polymer, thus reduce nonspecific proteins absorption and
Keep carrying probe activity.Generally, the polymer modified is obtained by two kinds of monomer copolymerizations, i.e. a kind of stable against biological contamination monomer,
A kind of monomer with probe binding site, thus realize stable against biological contamination and the probe fixing function on detection surface simultaneously.
But the content that such copolymerization inevitably results in two kinds of components reduces, relatively so that respective function is weakened.Additionally, mesh
Front two the most frequently used class probe securing units, including acrylic compounds and as methyl propenoic acid glycidyl ether class contains end-rings
The monomer of oxygen functional group.But for acrylic monomer, when with probe reaction, generally first need to carry out priming reaction, and should
Priming reaction is sensitive to PH, and therefore reaction efficiency is relatively low.And end is had to the monomer of epoxy-functional, though can directly with
Probe reaction, but this reactivity is low, reaction rate is slow, be subject in addition the required reaction temperature of biomacromolecule reaction with
The restriction of time, makes final probe fixed efficiency undesirable.Therefore, exploring one kind both can be with high-efficient carrier probe, simultaneously again can
It is particularly important with the bi-functional monomer reducing non-specific adsorption.
Chinese invention patent 101824477B provides one kind and is made up of polyamidoamine dendritic macromole modification substrate
Gene chip, wherein on dendrimer, amino, through glutaraldehyde activated, can load DNA probe.But by glutaraldehyde activated
It is easy to make glutaraldehyde two ends all react with surface amino groups during amino, thus losing and DNA probe binding ability.Chinese invention
Patent 104820093A provides a kind of method that poly-dopamine biological detection surface carries out Detection of antigen, by dopamine autohemagglutination
Be combined in substrate surface and modify a strata DOPA amine layer, followed by poly-dopamine chemical reactivity can directly and antibody response,
By antibody modification on surface.However, this poly-dopamine layer modification of surfaces therefore can also can cause non-spy with other albumino reactions
Opposite sex absorption.
Content of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of biological testing element and preparation method thereof,
Described biological testing element can have relatively low non-specific adsorption with high-efficient carrier probe simultaneously.
The invention provides a kind of biological testing element, substrate is modified with oxazole ketone polymer.
Preferably, described substrate includes orifice plate, glass, fiber, quartz, silicon, gold, one of tin indium oxide and plastics or
Several.
Preferably, described oxazole ketone polymer is obtained by oxazolone monomers polymerization, and described oxazolone monomers have shown in formula 1
Structure：
Wherein, R1And R2Independent selected from H or the alkyl with 1～4 carbon atom；R3And R4Independent selected from having 1～
The alkyl of 6 carbon atoms or the cycloalkyl with 5～6 carbon atoms.
Preferably, described oxazolone monomers are selected from 2- vinyl -4,4- dimethyl -1,3-oxazoles quinoline -5- ketone, 2- ethylene
Base -4,4- diethyl -1,3- oxazoline -5- ketone, 2- vinyl -4,4- dibutyl -1,3- oxazoline -5- ketone, 2- isopropenyl -
4,4- dimethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4- diethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -
One of 4,4- bicyclohexane base -1,3- oxazoline -5- ketone and 2- isopropenyl -4,4- dibutyl -1,3- oxazoline -5- ketone
Or it is several.
Present invention also offers a kind of preparation method of biological testing element, comprise the following steps：
A) by light trigger, the mixed solution of cross-linking agent and oxazolone monomers, it is added drop-wise to substrate surface, drying and volatilizing；
B) it is irradiated using the substrate surface that ultraviolet light obtains to above-mentioned steps a), carry out polyreaction, obtain surface
It is modified with the substrate of oxazole ketone polymer；
C) substrate that step b) obtains is reacted with bioprobe, obtain that surface modification has an oxazole ketone polymer has life
The substrate of quality testing brake；
D) close unreacted avtive spot, obtain biological testing element.
Preferably, described step d) is specially：
The substrate that step c) is obtained is reacted with hydrophilic polymer, closes unreacted avtive spot, obtains biological detection
Element.
Preferably, described hydrophilic polymer includes Amino End Group Polyethylene Glycol, holds mercapto-polyglycol, Amino End Group polyethylene
One or more of ketopyrrolidine and end sulfydryl Polyvinylpyrrolidone.
Preferably, described light trigger includes benzophenone, 1- hydroxycyclohexylphenylketone, 2- hydroxy-2-methyl -1- benzene
Base -1- acetone, 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone, 2- methyl isophthalic acid-(4- methyl mercapto phenyl) -2- morpholine
One or more of base -1- acetone and 2,2- dimethoxy-phenylf 1-Phenylethanone..
Preferably, described cross-linking agent includes polyethylene glycol dimethacrylate, N,N methylene bis acrylamide, N, N-
Vinyl bisacrylamide, allyl ether, Isosorbide-5-Nitrae-pentadiene -3- alcohol, diallyl carbonate, 1,5- hexadiene -3- alcohol, 1,3- bis-
One or more of allylurea.
Preferably, described light trigger, cross-linking agent, the mol ratio of oxazolone monomers is (0.1～5):(0.5～10):
100.
Compared with prior art, the invention provides a kind of biological testing element, substrate is modified with oxazole ketone polymer.This
The biological testing element that invention provides, by the class click chemistry characteristic of oxazole ketone, can load various bioprobes,
Reaction is simple, quickly, efficiently, need not activate or catalyst, can reduce non-specific adsorption simultaneously.And through oxazolone polymerization
The substrate surface that thing is modified, has good hydrophilic and stable against biological contamination performance, thus realizing to complex sample such as whole blood,
Blood plasma, serum, urine, saliva, and tear etc. not only accurately but also had sensitively analyzed detection.Thus in instant testing equipment, miniflow
The control analysis detection Disciplinary Frontiers such as chip and gene chip have broad application prospects.
Test result indicate that, the biological testing element probe load-reaction speed that the present invention provides is faster, in hgher efficiency, bears
Carrying capacity is bigger, and non-specific adsorption amount is little simultaneously, may finally carry out Sensitive Detection to complex sample.
Brief description
Fig. 1 is embodiment 1, in each sample under the comparative example 1 differential responses time sessile antibody fluorogram；
Fig. 2 is the sub-optimal fusion algorithm block diagram of embodiment 2, comparative example 2；
Fig. 3 is the absorbance curve figure of embodiment 3；
Fig. 4 is the fluorescence intensity curves figure of embodiment 4.
Specific embodiment
The invention provides a kind of biological testing element, substrate is modified with oxazole ketone polymer.
The present invention to described substrate and is not particularly limited, and can be applied to biological detection for well known to those skilled in the art
The substrate of element, present invention preferably comprises glass, fiber, quartz, silicon, gold, in the electrode material and plastics such as tin indium oxide
Plant or several.In some embodiments of the invention, described substrate is nitrocellulose filter, cyclic polyolefin (COP) film,
Filter paper, glass.Described substrate can be orifice plate.
Described oxazole ketone polymer is obtained by oxazolone monomers polymerization, and described oxazolone monomers have structure shown in formula 1：
Wherein, R1And R2It is preferably H or the alkyl with 1～4 carbon atom；R3And R4Preferably there is 1～6 carbon former
The alkyl of son or the cycloalkyl with 5～6 carbon atoms.
Described oxazolone monomers are more preferably 2- vinyl -4,4- dimethyl -1,3- oxazoline -5- ketone, 2- vinyl -4,
4- diethyl -1,3- oxazoline -5- ketone, 2- vinyl -4,4- dibutyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4-
Dimethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4- diethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4-
One of bicyclohexane base -1,3- oxazoline -5- ketone and 2- isopropenyl -4,4- dibutyl -1,3- oxazoline -5- ketone or several
Kind.
In some embodiments of the invention, described oxazolone monomers are 2- vinyl -4,4- dimethyl -1, and 3- dislikes
Oxazoline -5- ketone, 2- vinyl -4,4- diethyl -1,3-oxazoles quinoline -5- ketone, 2- isopropenyl -4,4- dimethyl -1,3-oxazoles
Any one or more in quinoline -5- ketone and 2- vinyl -4,4- dibutyl -1,3- oxazoline -5- ketone.
Substrate surface is modified with the biological testing element of oxazole ketone polymer, by the class click chemistry of oxazole ketone
Characteristic, can load various bioprobes, and reaction is simple, quickly, efficiently, need not activate or catalyst, can reduce non-simultaneously
Specific adsorption.And through the polymer-modified substrate surface of oxazolone, there is good hydrophilic and stable against biological contamination
Can, thus realizing complex sample such as whole blood, blood plasma, serum, urine, saliva, and tear etc. not only accurately but also were sensitively analyzed
Detection.Thus in instant testing equipment, the analysis detection Disciplinary Frontiers such as micro-fluidic chip and gene chip have wide answering
Use prospect.
Test result indicate that, the biological testing element probe load-reaction speed that the present invention provides is faster, in hgher efficiency, bears
Carrying capacity is bigger, and non-specific adsorption amount is little simultaneously, may finally carry out Sensitive Detection to complex sample.
Present invention also offers the preparation method of above-mentioned biological testing element, comprise the following steps：
A) by light trigger, the mixed solution of cross-linking agent and oxazolone monomers, it is added drop-wise to substrate surface, drying and volatilizing；
B) it is irradiated using the substrate surface that ultraviolet light obtains to above-mentioned steps a), carry out polyreaction, obtain surface
It is modified with the substrate of oxazole ketone polymer；
C) substrate that step b) obtains is reacted with bioprobe, obtain that surface modification has an oxazole ketone polymer has life
The substrate of quality testing brake；
D) close unreacted avtive spot, obtain biological testing element.
Described light trigger is preferably benzophenone, 1- hydroxycyclohexylphenylketone, 2- hydroxy-2-methyl -1- phenyl -1-
Acetone, 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone, 2- methyl isophthalic acid-(4- methyl mercapto phenyl) -2- morpholinyl -1-
One or more of acetone and 2,2- dimethoxy-phenylf 1-Phenylethanone..
The present invention produces free radical by light trigger under ultraviolet light, thus causing oxazole ketone monomer to be gathered
Close reaction.
Described cross-linking agent is preferably polyethylene glycol dimethacrylate, N,N methylene bis acrylamide, N, N- ethylene
Base bisacrylamide, allyl ether, Isosorbide-5-Nitrae-pentadiene -3- alcohol, diallyl carbonate, 1,5- hexadiene -3- alcohol, 1,3- bis- allyl
One or more of base urea.
The present invention makes described oxazolone compound coating of birdsing of the same feather flock together more stable by cross-linking agent, difficult for drop-off.
Described oxazolone monomers ibid, will not be described here.
In the present invention, described light trigger, cross-linking agent, the mol ratio of oxazolone monomers is preferably (0.1～5):(0.5～
10):100, more preferably (0.1～3):(0.5～8):100, most preferably (0.5～3):(1～8):100.
First by described light trigger, cross-linking agent, oxazolone monomers are mixed to get mixed solution to the present invention, and described mixing is molten
The solvent of liquid is preferably methanol, ethanol, acetone, oxolane, one or more of dichloromethane and chloroform；More preferably first
Alcohol, acetone, oxolane, dichloromethane or chloroform；Most preferably methanol, acetone, dichloromethane or oxolane.
Then described mixed solution is added drop-wise to substrate surface, drying and volatilizing, prepared coating.The present invention is preferred, in room temperature
Lower volatile dry.
Then using ultraviolet light, substrate surface obtained above is irradiated, carries out polyreaction.
Currently preferred, above-mentioned substrate surface is placed under uviol lamp and irradiates, carry out cross-linked polymeric.In the present invention,
The light source of described uviol lamp is preferably low pressure mercury lamp, medium pressure mercury lamp, high voltage mercury lamp, iodine-tungsten lamp and adds one of optical filter or several
Kind, the described ultraviolet light time is preferably 2～30min, more preferably 5～30min, most preferably 10～30min.The present invention
Preferably, after completing described ultraviolet light, gained sample is placed in cleaning in acetone, finally giving surface modification has oxazolone
The substrate of polymer.
After completing above-mentioned ultra-vioket radiation polyreaction, by what gained surface modification had an oxazole ketone polymer, there is biological detection
The substrate of function and bioprobe react.
Specifically, it is placed in bioprobe solution and reacted, fixing biological probe.
In the present invention, the time of described reaction be preferably 5～120min, more preferably 5～80min, most preferably 8～
60min.The temperature of described reaction is preferably 25～45 DEG C, more preferably 37 DEG C.
In the present invention, the concentration of described bioprobe solution is preferably 1pg/mL～100 μ g/mL, more preferably 1pg/
ML～80 μ g/mL, most preferably 0.1ng/mL～100 μ g/mL.Described bioprobe includes various antibody, antigen, DNA, RNA,
Polypeptide, albumen and hormone etc., specifically include Mouse IgG, the kind antibody such as Rabbit IgG, Goat IgG, and anti-first
Plant fetoprotein, carcinoembryonic antigen, prostate specific antigen, the other diseases mark antibody such as thyroxin, identification is thin
Bacterium, the DNA of cell, RNA detection sequence and polypeptide chain etc..
Currently preferred, by what the above-mentioned surface modification preparing had an oxazole ketone polymer, there is biological detecting function
Substrate surface be carried out, the present invention preferably water-bath vibration under conditions of, with the phosphate-buffered containing 0.05%Tween
Liquid is carried out, rear kept dry.
After completing the fixation of above-mentioned bioprobe, close its unreacted avtive spot.
Specifically, above-mentioned biological detection substrate is reacted with hydrophilic polymer.It is placed on containing hydrophilic polymer
Aqueous solution in reacted, close unreacted oxazole ketone.In the present invention, described hydrophilic polymer can be
The hydrophilic polymer being capable of capping avtive spot well known to those skilled in the art, the present invention is preferably the poly- second of Amino End Group
Glycol, holds mercapto-polyglycol, one or more of Amino End Group Polyvinylpyrrolidone and end sulfydryl Polyvinylpyrrolidone.
The number-average molecular weight of described hydrophilic polymer is preferably 300～30000, more preferably 1000～20000, most preferably 3000
～10000.In the present invention, the reaction density of described hydrophilic polymer preferably 0.1～100mg/mL, more preferably 1～
50mg/mL, most preferably 1～10mg/mL；The described response time is preferably 5～30min, more preferably 5～15min；Described anti-
Temperature is answered to be preferably 25～45 DEG C, most preferably 37 DEG C.
Currently preferred, after reaction terminates, above-mentioned prepared biological detection surface is carried out in phosphate buffer
Cleaning, after cleaning, kept dry is standby, finally gives biological testing element.
The above-mentioned preparation method that the present invention provides, reaction condition is gentle, and reaction rate is fast, no other side reactions, and required
Equipment is simple, and method is easy to operate.
In order to further illustrate the present invention, the biological testing element present invention being provided with reference to embodiment and its preparation
Method is described in detail.
Embodiment 1
Take 0.5mmol benzophenone, 1mmol N,N methylene bis acrylamide, 100mmol 2- vinyl -4,4- bis-
Methyl isophthalic acid, 3- oxazoline -5- ketone, add in 10mL oxolane, be sufficiently stirred for dissolving, be made into mixed solution.Take 1mL above-mentioned mixed
Close solution, be added drop-wise to 10 × 10cm2On nitrocellulose filter, room temperature volatile dry.
Above-mentioned dried nitrocellulose filter is placed in irradiation 2min under uviol lamp, is subsequently placed in acetone, shaking table is clear
After washing 30min, drying for standby.
Nitrocellulose filter after above-mentioned cleaning is placed in and resists (RBITC-Mouse containing 100 μ g/mL rhodamine labelling Mus
IgG in phosphate buffer).5min, 20min, 40min, 60min, 80min, 100min is reacted respectively at 37 DEG C,
120min.After completion of the reaction, above-mentioned sample is carried out with the phosphate buffer of 0.05%Tween, rear kept dry.
The above-mentioned sample securing fluorescent antibody is carried out fluorescence intensity scanning, research antibody fixed amount changes over time
Relation.Fluorescent intensity scan adopts laser confocal microscope (ZEISS, LSM700).Each sample under the differential responses time
The fluorescence picture of upper sessile antibody as shown in figure 1, Fig. 1 be embodiment 1, fixing in each sample under the comparative example 1 differential responses time
The fluorogram of antibody.
Test result indicate that, with the increase in response time, on sample, fluorescence intensity gradually strengthens, and shows antibody fixed amount
Gradually increase.After only reaction 5min, just there are antibodies in substrate, after reaching 40～60min, reaction tends to balance.With
Go up test result indicate that the reaction between amino has very high reactivity, reaction rate on oxazole ketone and antibody
Hurry up, fixed amount is big.
Comparative example 1
Take 0.5mmol benzophenone, 1mmol N,N methylene bis acrylamide, 100mmol Glycidyl methacrylate is sweet
Oily ether, adds in 10mL oxolane, is sufficiently stirred for dissolving, is made into mixed solution.Take the above-mentioned mixed solution of 1mL, be added drop-wise to 10
×10cm2On nitrocellulose filter, room temperature volatile dry.
Above-mentioned dried nitrocellulose filter is placed in irradiation 2min under uviol lamp, is subsequently placed in acetone, shaking table is clear
After washing 30min, drying for standby.
Nitrocellulose filter after above-mentioned cleaning is placed in the phosphate-buffered containing 100 μ g/mL RBITC-Mouse IgG
In liquid.5min, 20min, 40min, 60min, 80min, 100min, 120min is reacted successively at 37 DEG C.After completion of the reaction, will be upper
The phosphate buffer stating sample 0.05%Tween20 is carried out, rear kept dry.
The above-mentioned sample securing fluorescent antibody is carried out fluorescence intensity scanning, research antibody fixed amount changes over time
Relation.Concrete grammar is same as Example 1, will not be described here.Result such as Fig. 1, Fig. 1 are that embodiment 1, comparative example 1 are different anti-
The fluorogram of sessile antibody in lower each sample between seasonable.
It will be noted from fig. 1 that for comparative example 1, epoxy-functional is slow with antibody response, increases with the response time,
Fluorescence intensity slowly strengthens, and when reaching 120min, it is substantially weak that fluorescence intensity compares embodiment 1.
Above test result indicate that, the antibody fixed rate of embodiment 1 is considerably more rapid, and fixed amount is also bigger, oxazole ketone
In functional group and antibody, the reaction between amino has very high reactivity, and reaction rate is fast, and fixed amount is big.
Embodiment 2
Take 1.0mmol 2- hydroxy-2-methyl -1- phenyl -1- acetone, 2mmol N, N- vinyl bisacrylamide,
100mmol 2- vinyl -4,4- diethyl -1,3-oxazoles quinoline -5- ketone, add in 10mL acetone, be sufficiently stirred for dissolving, be made into
Mixed solution.Take the above-mentioned mixed solution of 1mL, be added drop-wise to 10 × 10cm2On cyclic polyolefin (COP) film, room temperature volatile dry.
Above-mentioned dried cyclic polyolefin (COP) film is placed in irradiation 10min under uviol lamp, is subsequently placed in acetone,
After shaking table cleaning 30min, drying for standby.
Cyclic polyolefin (COP) film after above-mentioned cleaning is placed in the phosphate-buffered containing 100 μ g/mL Rabbit IgG
In liquid.5min, 20min, 40min, 60min, 80min, 100min, 120min is reacted successively at 37 DEG C.After completion of the reaction, will be upper
The phosphate buffer stating sample 0.05%Tween20 is carried out, rear kept dry.
By the above-mentioned cyclic polyolefin film securing antibody, and pure polyolefin film puts into the isosulfocyanic acid fluorescence of 1mg/mL
In plain labelling bovine serum albumin (FITC-BSA) solution, adsorb 2h at 37 DEG C, cleaned with phosphate buffer three times, subsequently use
Laser confocal microscope scans, and calculates the fluorescence intensity of each sample, and every kind of sample is repeated 5 times.With non-specific on pure COP film
Protein adsorption result is 100%, obtains the sub-optimal fusion algorithm of embodiment 2, result such as Fig. 2, Fig. 2 are embodiment 2, comparative example 2
Sub-optimal fusion algorithm block diagram.
It can be observed from fig. 2 that with respect to pure COP film, embodiment 2, the i.e. polymer-modified substrate of oxazolone, showing excellent
Different anti-non-specific adsorption performance, reduces by 85%.
Comparative example 2
Take 1.0mmol 2- hydroxy-2-methyl -1- phenyl -1- acetone, 2mmol N, N- vinyl bisacrylamide,
100mmol NVP, adds in 10mL acetone, is sufficiently stirred for dissolving, is made into mixed solution.Take 1mL above-mentioned mixed
Close solution, be added drop-wise to 10 × 10cm2On cyclic polyolefin film, room temperature volatile dry.
Above-mentioned dried cyclic polyolefin film is placed in irradiation 10min under uviol lamp, is subsequently placed in acetone, shaking table is clear
After washing 30min, drying for standby.
Identical with embodiment 2 step, the above-mentioned cyclic polyolefin film having modified NVP compound is put into
In the FITC-BSA solution of 1mg/mL, at 37 DEG C, adsorb 2h, cleaned with phosphate buffer three times, subsequently shown with laser co-focusing
Micro mirror scans, and calculates the fluorescence intensity of each sample, and every kind of sample is repeated 5 times.Result such as Fig. 2, Fig. 2 are embodiment 2, comparative example 2
Sub-optimal fusion algorithm block diagram.
Figure it is seen that with respect to pure COP film, the nonspecific proteins absorption of comparative example 2 reduces, this mainly by
In NVP it is verified that, because it has excellent hydrophilic, can give modified surface good anti-non-
Specific adsorption performance.
Embodiment 2 is close with the anti-non-specific adsorption effect of comparative example 2.Therefore, result above shows, oxazolone is birdsed of the same feather flock together
Compound modify biological testing element substrate surface not only can high-efficient carrier bioprobe, can effectively suppress non-specific simultaneously
Property absorption, give detection surface good stable against biological contamination performance.
Embodiment 3
Take 2.0mmol 2,2- dimethoxy-phenylf 1-Phenylethanone., 3mmol polyethylene glycol dimethacrylate, 100mmol
2- isopropenyl -4,4- dimethyl -1,3-oxazoles quinoline -5- ketone, add in 10mL acetone, be sufficiently stirred for dissolving, be made into mixing molten
Liquid.Take the above-mentioned mixed solution of 1mL, be added drop-wise to 10 × 10cm2On Whatman4# filter paper, room temperature volatile dry.
It is placed in irradiation 20min under uviol lamp after above-mentioned filter paper is dried, be subsequently placed in acetone, after shaking table cleaning 30min,
Drying for standby.
By the phosphate buffer Deca containing the anti-alpha fetoprotein (AFP) antibody of 100 μ g/mL for the 5 μ L after polymer-modified
Wahtman 4# filter paper on.5min is reacted, after completion of the reaction, by the above-mentioned sample phosphoric acid of 0.05%Tween20 at 37 DEG C
Salt buffer is carried out, drying for standby.
Sample after above-mentioned sessile antibody is reacted with amino-polyethyleneglycols (Mn=5000), at 37 DEG C, reacts 10min, envelope
Close unreacted avtive spot.After reaction, sample is cleaned with phosphate buffer, rear drying for standby.
5 μ L are taken to contain 0ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL first kind respectively
The human plasma of fetoprotein (AFP) and phosphate buffer are added drop-wise on above-mentioned load antibodies filter paper.5min is identified at 37 DEG C,
Add the anti-alpha fetoprotein (AFP) antibody of the horseradish peroxidase-labeled of 10ng/mL afterwards, phosphate buffer cleans 3 times,
After add 3 μ L tmb substrates colour developing 10min, test the absorbance of each sample under each concentration, testing result as shown in figure 3,
Fig. 3 is the absorbance curve figure of embodiment 3.
From figure 3, it can be seen that in phosphate buffer, by elisa, the detection to AFP is limited to
1.69ng/mL；And when detecting in forming extremely complex human plasma, the detection to AFP is limited to 1.62ng/mL, this result
With the no significant difference of the test limit in phosphate buffer.Therefore, above test result indicate that the present invention provide oxazolone
The biological testing element that compound of birdsing of the same feather flock together is modified is big by institute's load antibodies amount, and activity is high, and can suppress non-specific adsorption,
It still is able to determinand is carried out sensitive and accurate detection eventually in complex component, blood plasma.
Embodiment 4
Take 3.0mmol 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone, 5mmol diallyl carbonate,
100mmol 2- vinyl -4,4- dibutyl -1,3-oxazoles quinoline -5- ketone, add in 10mL acetone, be sufficiently stirred for dissolving, be made into
Mixed solution.Take the above-mentioned mixed solution of 1mL, be added drop-wise to 10 × 10cm2On glass, room temperature volatile dry.
Above-mentioned dried glass is placed in irradiation 15min under uviol lamp, is subsequently placed in acetone, shaking table cleans 30min
Afterwards, drying for standby.
Glass after above-mentioned cleaning is placed in the-NH containing 100 μ g/mL2DNA(5’-
GGATTATTGTTAAATAATGATAAGGAT-NH2-3 ') phosphate buffer in.20min is reacted at 37 DEG C.Reaction finishes
Afterwards, above-mentioned sample is carried out with the phosphate buffer of 0.05%Tween20, rear kept dry.
The glass sample of above-mentioned fixed trapped DNA probe is reacted with mercapto-polyglycol (Mn=10000), anti-at 37 DEG C
Answer 5min, close unreacted avtive spot.After reaction, sample is cleaned with phosphate buffer, rear drying for standby.
The glass sample of above-mentioned fixed trapped DNA probe is put into DNA (the 5 '-FITC- to be measured containing variable concentrations
ATCCTTATCATTATTTAACAATAATCC-3 ') phosphate buffer in, concentration is respectively 0ng/mL, 10ng/mL,
Identify 30min at 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL, 100ng/mL, 37 DEG C, clean three with phosphate buffer
Secondary, subsequently scanned with laser confocal microscope, calculate the fluorescence intensity of each sample, every kind of sample is repeated 5 times.Result such as Fig. 4
Shown, Fig. 4 is the fluorescence intensity curves figure of embodiment 4.
From fig. 4 it can be seen that increasing to 100ng/mL with target DNA concentration from 10ng/mL, above-mentioned sample surfaces capture
Probe amount also increases therewith, and green fluorescence intensity strengthens, and shows that above-mentioned constructed DNA detection surface can effectively capture target
DNA.Therefore, the oxazolone that the present invention provides birds of the same feather flock together compound modification biological testing element detection range wide, can be to various anti-
Body, antigen includes DNA and carries out sensitive identification.
From above example and comparative example result, the biological testing element that the present invention provides passes through oxazolone sense
Roll into a ball and the amino on bioprobe, the reaction of sulfydryl and hydroxyl has class click chemistry characteristic, therefore react simple, quickly,
Efficiently, need not activate or catalyst.Simultaneously the present invention provide oxazolone birds of the same feather flock together compound modification biological testing element can assign
Give the good hydrophilic in detection surface and stable against biological contamination performance, thus realizing complex sample not only accurately but also was sensitively analyzed
Detection.The present invention provide oxazolone birds of the same feather flock together compound modification biological detection surface only need 5min can load bioprobe,
On simultaneously constructed biological detection surface, non-specific adsorption can reduce by 85%, and still can keep in undiluted human plasma
Sensitive and accurate detection to antigen.
The construction method that the present invention provides is simple and quick, mild condition, low cost, suitable large-scale production.
The explanation of above example is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that it is right
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1. a kind of biological testing element is it is characterised in that substrate is modified with oxazole ketone polymer.
2. biological testing element according to claim 1 is it is characterised in that described substrate includes orifice plate, glass, fiber,
Quartz, silicon, gold, one or more of tin indium oxide and plastics.
3. biological testing element according to claim 1 is it is characterised in that described oxazole ketone polymer is by oxazolone monomers
Polymerization obtains, and described oxazolone monomers have structure shown in formula 1：
Wherein, R1And R2Independent selected from H or the alkyl with 1～4 carbon atom；R3And R4Independent selected from having 1～6 carbon
The alkyl of atom or the cycloalkyl with 5～6 carbon atoms.
4. biological testing element according to claim 3 it is characterised in that described oxazolone monomers be selected from 2- vinyl-
4,4- dimethyl -1,3- oxazoline -5- ketone, 2- vinyl -4,4- diethyl -1,3- oxazoline -5- ketone, 2- vinyl -4,4-
Dibutyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4- dimethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4-
Diethyl -1,3- oxazoline -5- ketone, 2- isopropenyl -4,4- bicyclohexane base -1,3- oxazoline -5- ketone and 2- isopropenyl -
One or more of 4,4- dibutyl -1,3- oxazoline -5- ketone.
5. a kind of preparation method of biological testing element, comprises the following steps：
A) by light trigger, the mixed solution of cross-linking agent and oxazolone monomers, it is added drop-wise to substrate surface, drying and volatilizing；
B) it is irradiated using the substrate surface that ultraviolet light obtains to above-mentioned steps a), carry out polyreaction, obtain surface modification
There is the substrate of oxazole ketone polymer；
C) substrate that step b) obtains is reacted with bioprobe, the biology that has that obtaining surface modification has oxazole ketone polymer is examined
The substrate of brake；
D) close unreacted avtive spot, obtain biological testing element.
6. preparation method according to claim 5 is it is characterised in that described step d) is specially：
The substrate that step c) is obtained is reacted with hydrophilic polymer, closes unreacted avtive spot, obtains biological testing element.
7. preparation method according to claim 6 is it is characterised in that described hydrophilic polymer includes the poly- second of Amino End Group two
Alcohol, holds mercapto-polyglycol, one or more of Amino End Group Polyvinylpyrrolidone and end sulfydryl Polyvinylpyrrolidone.
8. preparation method according to claim 5 is it is characterised in that described light trigger includes benzophenone, 1- hydroxyl
Cyclohexyl-phenyl ketone, 2- hydroxy-2-methyl -1- phenyl -1- acetone, 2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone,
2- methyl isophthalic acid-one of (4- methyl mercapto phenyl) -2- morpholinyl -1- acetone and 2,2- dimethoxy-phenylf 1-Phenylethanone. or several
Kind.
9. preparation method according to claim 5 is it is characterised in that described cross-linking agent includes Polyethylene Glycol dimethyl allene
Acid esters, N,N methylene bis acrylamide, N, N- vinyl bisacrylamide, allyl ether, Isosorbide-5-Nitrae-pentadiene -3- alcohol, carbonic acid
Diallyl, 1,5- hexadiene -3- alcohol, one or more of 1,3- diallyl urea.
10. preparation method according to claim 5 is it is characterised in that described light trigger, cross-linking agent, oxazolone monomers
Mol ratio be (0.1～5):(0.5～10):100.
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