CN104897617A - Micro-array biochip, and preparation method and application of micro-array biochip - Google Patents
Micro-array biochip, and preparation method and application of micro-array biochip Download PDFInfo
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Abstract
The invention relates to a micro-array biochip, and a preparation method and an application of the micro-array biochip. The micro-array biochip comprises a glass chip substrate and a modification layer. The modification layer comprises a chromium layer, a silver and/or gold layer, a self-assembly monomolecular layer, and a photo-cross-linking agent and high-molecular layer, which are overlaid in sequential on the surface of the glass chip substrate. The invention also provides a protein micro-array, a method for preparing the protein micro-array, and an application of the protein micro-array to detect micromolecule medicine. According to the invention, three-dimensional high-molecular surface modification of the micro-array biochip is completed by the method, and the fixation amount of protein on the surface of the micro-array biochip can be effectively improved, so that micromolecule, hard to detect in the prior art, can be detected without marking, in accuracy, and in high flux. The micro-array biochip is of great significance to the field, such as molecular biology study and medicinal development.
Description
Technical field
The present invention relates to biochip technology field, particularly relate to a kind of micro-array biochip and its preparation method and application.
Background technology
Research small-molecule drug and protein targets target interact, to basic scientific research and drug development significant.But in actual applications, there is a kind of medicine for the interactional situation of many targets, as mixed to medicine, general property and the research of spinoff, the optimization of lead compound and old medicine are newly used.Therefore, a kind of Small molecular-target interaction detection method efficiently of exploitation will have impetus to target activity rating and drug screening etc.
In molecular biology aspect, drug development often needs to gather the interaction information (as dynamics and affinity) between lead compound/medicine and albumen target and evaluate.In recent years, surface plasma resonance biological sensor (surface plasmon resonance, SPR) in order to avoid advantages such as mark, highly sensitive and Real-Time Monitorings, the important tool becoming research Small molecular and albumen target interaction dynamics and affinity is developed rapidly.At present, SPR detection technique is mainly the method (based on BIAcore series of products) detected based on angle, and the method is by proteopexy at chip surface, and circulation small-molecule drug, carries out Real-Time Monitoring to optical signalling.But along with detecting the diversity of sample with the complicacy of its interactive network, require that detection means has high flux and high efficiency feature, the detection method of this traditional " one to one " cannot meet application demand.
The appearance of surface plasma resonance imaging technology (SPR imaging, SPRi) solves above flux problems well.SPRi technology adopts microarray format and directional light to detect, and can obtain the interaction information of chip surface in surveyed area once, has " one-to-many " the even high flux of " multi-to-multi " and detects advantage.Utilize SPRi technology, researchers have developed high flux and detect the interactional method of protein-protein, polypeptide-polypeptide and DNA-DNA.But, for the detection of Small molecular in protein arrays, still there is challenge: 1. because the sensitivity of the current SPRi technology based on intensity detection is lower than angle detection technique, in addition when Small molecular detects, because its molecular weight low generation signal response itself is very little, make the detectability of high flux SPRi instrument lower; 2. the quality of data of SPRi is relevant with collection area, and the higher meeting of flux causes sample spot area to reduce, and noise increases, and makes to detect difficulty; 3. current SPRi chip surface many employings mercaptan is modified, and proteopexy amount is lower, and make interactional small-molecule drug with it also less, signal is difficult to detect naturally.
For above problem, researchers strengthen the detection sensitivity of SPRi mainly for instrument improvement and chip chemical modification two aspects, detect micromolecular ability to obtain on arrays of immobilized protein.Such as, N.J.Tao etc. utilize SPRi and electrochemical impedance to combine, and improve the detection sensitivity of SPRi, success detects the micromolecular combination of SB203580 on P38 kinases array, and obtain dynamics and affinity constant (Anal.Chem.2014,86,9860-9865); But the method is just applicable to the instrument of this team exploitation, and SPRi main flow instrument does not have versatility.In addition, in chip chemical modification, the people such as Y.M.Wang construct three-dimensional polyacrylic acid (PAA) polymer brush at SPRi chip surface, and adopt the space length between micro-contact printing regulation and control macromolecule, obtain higher proteopexy amount (AppliedSurfaceScience266 (201) 313-318); H.W.Ma etc. utilize the method for surface initiation polymerization to construct three-dimensional macromolecule surface at SPR chip surface, and obtain the Ag-Ab detection signal (Chem.Commun., 2011,47,1190-1192) optimized.But the above method for chip chemical modification does not all successfully detect small-molecule drug in protein arrays.Therefore, the interactional method of high flux detection small molecule-protein on SPRi instrument of developing is extremely important.
Summary of the invention
The object of the present invention is to provide a kind of be applied to micro-array biochip of surface plasma resonance system and preparation method thereof, the arrays of immobilized protein utilizing it to prepare and utilize described arrays of immobilized protein to carry out detecting method and application in small-molecule drug context of detection.
For reaching above object, the present invention adopts following technology path:
First aspect, the invention provides a kind of micro-array biochip, comprise glass-chip substrate and decorative layer, described decorative layer comprise be superimposed upon described glass-chip substrate surface successively layers of chrome, silver and/or layer gold, self assembled monolayer, photocrosslinking agent and macromolecule layer.
Surface plasma resonance imaging technology utilizes the resonance that in light wave and metal (gold and/or silver), free electron ripple produces, determine metal surface bio-molecular interaction situation (combine or dissociate) by detecting resonance signal, therefore gold and/or silver are necessary conditions on surface.Between glass-chip substrate and gold and/or silver, plate one deck chromium is that chromium plays and adheres to cushion effect because the adhesiveness of gold and/or silver and glass-chip substrate is bad.
In addition, the method for modifying of chip roughly can be divided into two classes: Physical layer (glass-chromium-Jin and/or silver), and Main Function is excitating surface Plasmon Resonance; Chemosphere (self assembled monolayer-photocrosslinking agent-macromolecule), it is mainly used in the fixing of biomolecule and the protection to metallic substrates.Wherein, Physical layer is the routine techniques means of SPRi sensing chip; And chemosphere is emphasis of the present invention, through the chip that method of the present invention is modified, effectively can improve the fixed amount of biomolecule in biochip surface, the small-molecule drug being originally difficult to detect is detected.
In the present invention; the realization that small-molecule drug detects is mainly through improving protein molecular fixed amount; and protein molecular fixed amount mainly relies on the modification of three-dimensional macromolecule surface; self assembled monolayer is for the protection of substrate and provide terminal reactive group, and photocrosslinking agent plays bridging unimolecular layer and high molecular effect.
Preferably, the thickness of described layers of chrome is 0.1-9nm, such as, can be 0.1nm, 1nm, 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm or 9nm, is preferably 3nm.For the thickness of layers of chrome, those skilled in the art can carry out suitable selection according to the adhesiveness of metal and glass.If good adhesion, also layers of chrome can not be added; But in existing technology, the adhesiveness of the two does not generally also reach the degree can saving layers of chrome; Therefore, layers of chrome should not be too thin, to ensure film forming, increases adhesiveness; If layers of chrome thickness is too large, be then unfavorable for the conduction of light wave.
Preferably, the thickness of described silver and/or layer gold is 40-60nm, can be such as 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, 51nm, 52nm, 53nm, 54nm, 55nm, 56nm, 57nm, 58nm, 59nm or 60nm, be preferably 47-50nm.If thickness exceedes this scope (be greater than 60nm or be less than 40nm), sensitivity can be caused to decline, affect the sensitivity that small-molecule drug is detected.
Preferably, the reagent of described self assembled monolayer is the mercaptan of end-functionalization.
Preferably, the mercaptan of described end-functionalization is single mercaptan and/or dithiol.
Preferably, the mercaptan of described end-functionalization is alkanethiol and/or the mercaptan with PEG block.
Preferably, the group of described end-functionalization is hydroxyl, carboxyl, amino, aldehyde radical, epoxy radicals or methoxyl.
The general formula of mercaptan is: HS-R1-R2;
Wherein, R1 is alkane chain, and R2 is generally the PEG chain of end-functionalization.Such as, mercaptan mainly relies on the Van der Waals force between Au-S key and R1 in gold surface self assembly, to form fine and close monofilm; R2 is biocompatiblity molecules, is mainly used in anti-non-specific adsorption, reduces background signal.The end-functionalization group of mercaptan is mainly used in further chemical modification, and as connected photocrosslinking agent etc., different end group can have different chemical modification methods, but reaction principle is all basic chemical covalent combination.
Preferably, described photocrosslinking agent is for containing photaesthesia group, the reagent of chemical reaction can be carried out under specific wavelength (energy) illumination, be preferably the potpourri of any one or at least two kinds in azirine, diazonium, acetophenone, benzophenone or Anthraquinones, it can be such as azirine, diazonium, the potpourri of acetophenone and benzophenone, or the potpourri of azirine, acetophenone, benzophenone and Anthraquinones.Photaesthesia group, i.e. photo-crosslinking group, under light illumination can aitiogenic chemical group.The present invention mainly utilizes photochemical reaction activation light sensitive group, makes it quick, non-selective and covalently macromolecule is combined in its surface.
Preferably, described macromolecule refers to have biocompatibility and has the functional polymer of active end group, including but not limited to the synthesis macromolecule with good anti-non-specific binding performance, such as, can be polyglycol and derivant, fluoropolymer, polyacrylic acid and derivant thereof, polymethylacrylic acid and derivant, polystyrene, PLA, both sexes betaines polymkeric substance etc.; Can also be the natural polymer with good biocompatibility, as nitrocellulose, shitosan or glucosan and derivant etc. thereof.
Second aspect, the invention provides the preparation method of micro-array biochip as described in relation to the first aspect, comprises the following steps:
(1) plate layers of chrome in glass-chip substrate, then on the plated surface of layers of chrome silver and/or layer gold;
(2) completing steps (1) cleans chip afterwards, forms self assembled monolayer on surface;
(3) photocrosslinking agent is covalently bonded in the surface of self assembled monolayer;
(4) Polymer Solution is spin-coated on the chip surface of completing steps (3);
(5) after the solvent volatilization of step (4) described Polymer Solution, make described macromolecular grafted at chip surface by UV-irradiation;
Optionally, step (6) is carried out:
(6) macromolecule being grafted on chip surface is carried out functionalization.
Preferably, the method plating layers of chrome and silver and/or layer gold described in step (1) is that metal fever steams and/or ion sputtering; The coating particle adopting above method to be formed is less and thickness even, is conducive to the application of imaging sensing.
Wherein, the mode of step (2) described cleaning is the combination of any one or at least two kinds in solvent cleaning, ultrasonic cleaning and plasma cleaning method, it can be such as solvent cleaning, ultrasonic cleaning, the combination of plasma cleaning and ultrasonic cleaning, or solvent cleaning, ultrasonic cleaning and plasma cleaning combination.Described solvent cleaning solvent for use is deionized water, ethanol, acetone or N, the mixing of any one or at least two kinds in dinethylformamide, it can be such as deionized water, the potpourri of ethanol and acetone, deionized water and N, the potpourri of dinethylformamide, or the potpourri of ethanol, acetone and DMF.
Preferably, the described covalently bound mode of step (3) is amidation, esterification, open loop or nucleophilic substitution, make photocrosslinking agent be grafted to the end of self assembled monolayer, concrete grammar can be selected according to the functional end-group of biochip substrate surface functionalization group and photocrosslinking agent.Such as, the present invention's adoptable photocrosslinking agent structural formula is as follows, for (a) structure, adopts esterification to be grafted to self assembled monolayer end; For (b) structure, amidation process is adopted to be grafted to self assembled monolayer end.
Preferably, the solvent of step (4) described Polymer Solution is deionized water, ethanol, methyl alcohol, dimethyl sulfoxide (DMSO) or N, the combination of any one or at least two kinds in dinethylformamide, it can be such as deionized water, the potpourri of ethanol and methyl alcohol, the potpourri of dimethyl sulfoxide (DMSO) and deionized water, or the potpourri of DMF, dimethyl sulfoxide (DMSO), ethanol and deionized water.
Preferably, the wavelength of step (5) described ultraviolet light is 200-400nm, can be such as 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm or 400nm, be preferably 365nm.Under the ultraviolet wavelength of this scope, can ensure that the reaction efficiency of photocrosslinking agent is in higher level.
Preferably, step (6) described end-functionalization is also nonessential, can carry out functionalization after macromolecular grafted; Also can select the macromolecule with end-functionalization, directly use after being grafted to surface; Wherein, the group of step (6) described functionalization is hydroxyl, carboxyl, aldehyde radical, amino, epoxy radicals, cyano group, alkynyl, azido, azirine or biotin.The main binding biomolecules of terminal reactive group, refer in particular to conjugated protein in the present invention, different protein needs different active end groups to be fixed, as hydroxyl, carboxyl, aldehyde radical or isonitrile acidic group, or even the active biomolecule modified, as biotin or antibody label etc.End-functionalization described herein is nonessential, refers to that some macromolecule carries the end of functionalization or carried out functionalization in the liquid phase, namely can directly use after being connected to surface; And some macromolecules itself are not suitable for fixing biological molecules, need to carry out aftertreatment, molecular end is changed over reaction active groups.
In the present invention, can by regulation and control photocrosslinking agent density (molar percentage 0.01% ~ 100%), high molecular number-average molecular weight (100,000-2,000,000Da) and the parameter such as spin speed (1,000-8,000rpm), the density of accurate control micro-array biochip finishing coat and thickness, thus micro-array biochip of the present invention can be prepared simply, fast and efficiently.
The third aspect, the invention provides a kind of arrays of immobilized protein, and it comprises micro-array biochip as described in the first aspect of the invention.
Fourth aspect, the invention provides the preparation method of the arrays of immobilized protein as described in the third aspect, and described preparation method is that after protein is mixed with solution, point sample is also hatched and makes.
Preferably, described protein is mixed with solution for be dissolved in damping fluid by protein, makes it keep the biologically active of protein.
Preferably, described damping fluid is PBS damping fluid, HEPES damping fluid, Tris-HCl damping fluid or HBS-EP damping fluid.
Preferably, the pH value opsin biologically active of described damping fluid and preserving as requested, preferable ph is below described isoelectric points of proteins 0-1 unit, such as, can be 0 unit, 0.1 unit, 0.2 unit, 0.3 unit, 0.4 unit, 0.5 unit, 0.6 unit, 0.7 unit, 0.8 unit, 0.9 unit or 1 unit.
Wherein, the mode of described point sample is for utilizing point sample instrument and/or manual point sample, and the present invention does not make concrete restriction to it, and those skilled in the art can rule of thumb select voluntarily; Typical case but without limitation, can be undertaken printing by commercialization printer, carried out manual point sample by liquid-transfering gun or by means point samples such as dimethyl silicone polymer (PDMS) punching are auxiliary, form protein array.
Preferably, described in the temperature of hatching be 3-8 DEG C, can be such as 3 DEG C, 3.5 DEG C, 4 DEG C, 4.5 DEG C, 5 DEG C, 5.5 DEG C, 6 DEG C, 6.5 DEG C, 7 DEG C, 7.5 DEG C or 8 DEG C, be preferably 4 DEG C.
Preferably, described in the relative humidity of hatching be 50-80%, can be such as 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78% or 80%, be preferably 70%.
5th aspect, the invention provides a kind of detection method, and described method utilizes the arrays of immobilized protein described in fourth aspect present invention.
Preferably, said method comprising the steps of:
(1) cleaning is fixed with the chip of arrays of immobilized protein, puts into surface plasmon resonance assay imager after drying up;
(2) in surface plasmon resonance assay imager, damping fluid is added;
(3) treat baseline stability, with living again, live again in the surface of liquid by described chip;
(4) pass into detection liquid, live again;
(5) pass into correcting fluid, detect protein signal.
Preferably, step (2) described damping fluid is PBS damping fluid, HEPES damping fluid, Tris-HCl damping fluid or HBS-EP damping fluid.
Preferably, also dimethyl sulphoxide solution is added in step (2) described damping fluid.
Preferably, the volume fraction of described dimethyl sulphoxide solution is 0.5-5%, such as, can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.
Preferably, liquid of living again described in step (3) is phosphoric acid solution, glycine-HCI damping fluid, ethylene glycol solution or sodium hydrate aqueous solution; In described phosphoric acid solution, the volume ratio of phosphoric acid and water is 1:(100-400), can be such as 1:100,1:125,1:150,1:175,1:200,1:225,1:250,1:275,1:300,1:325,1:350,1:375 or 1:400; The pH of described glycine-HCI damping fluid is 1.5-2.5, such as, can be 1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4 or 2.5; The volume fraction of described ethylene glycol solution is 10-40%, such as, can be 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38% or 40%; The concentration of described sodium hydrate aqueous solution is 7-13mmol/L, such as, can be 7mmol/L, 7.5mmol/L, 8mmol/L, 8.5mmol/L, 9mmol/L, 9.5mmol/L, 10mmol/L, 10.5mmol/L, 11mmol/L, 11.5mmol/L, 12mmol/L, 12.5mmol/L or 13mmol/L.
6th aspect, the invention provides micro-array biochip as described in the first aspect of the invention or the arrays of immobilized protein described in fourth aspect is detecting the application in small-molecule drug.
The medicine of described small-molecule drug to be molecular weight be 500-1,000Da, SPRi detection signal becomes positive correlation with the quality of surface molecular.Why small-molecule drug is difficult to is detected, and is that signal response is little because the mass difference great disparity of its (mobile phase) molecular weight and albumen (Stationary liquid).And the present invention utilizes the modification of three-dimensional surface, make chip surface can the enough albumen of fixing foot, these albumen can be combined with abundant Small molecular in the detection, make SPRi obtain desirable corresponding signal.
Compared with prior art, the present invention at least has following beneficial effect:
Although SPRi has unmarked, the advantage such as high flux and kinetic measurement, traditional arrays of immobilized protein can't detect small-molecule drug on SPRi, and reason is that proteopexy amount is too low.For the deficiencies in the prior art; the present invention is by two-step approach modified biological chip; first carry out Physical layer modification (glass-chromium-Jin and/or silver); carry out chemosphere modification (self assembled monolayer-photocrosslinking agent-macromolecule) again, while excitating surface plasma primitive, reach the effect of fixing protein and protection metallic substrates.
The biochip of three-dimensional macromolecular surface modification is completed through method of the present invention, effectively can improve the fixed amount of albumen in biochip surface, enable the Small molecular that is difficult in prior art detect by unmarked, accurately and high flux detect, to the field such as molecular biology research and drug development, there is realistic meaning.
In addition, method of the present invention accurately can control the proteopexy amount of biochip surface, ensures that chip surface has the detection that small-molecule drug is carried out in enough spaces simultaneously; Preparation method is simple, quick, cost is low and easily carry out quality control, is applicable to large-scale production and popularization.
Accompanying drawing explanation
Fig. 1 is the result figure detecting biotin in embodiment 1 on the biochip containing SA, BSA and FKBP12 arrays of immobilized protein;
Fig. 2 is the result detecting tacrolimus (FK506) in embodiment 2 on FKBP12 arrays of immobilized protein, wherein, a () is arrays of immobilized protein figure, (b) is the result figure detecting tacrolimus (FK506) on the biochip containing H-IgG, BSA and FKBP12 arrays of immobilized protein;
Fig. 3 is the result detecting tacrolimus (FK506) in embodiment 3 on FKBP12 and mutant protein microarray thereof, wherein, a () is arrays of immobilized protein figure, (b) is the result figure detecting tacrolimus (FK506) on the arrays of immobilized protein biochip containing FKBP12 and mutant thereof;
Fig. 4 is the result figure detecting peptide T 7 in embodiment 4 on amyloid A β 42 and mutant microarray thereof.
Embodiment
Technical scheme of the present invention is further illustrated below by embodiment.Those skilled in the art should understand, described embodiment is only help to understand the present invention, should not be considered as concrete restriction of the present invention.
Embodiment 1 detects biotin (Biotin) on Streptavidin (SA) microarray
1, the modification of biochip substrate:
(1) the thick layers of chrome of one deck 3nm and the thick layer gold of one deck 47nm is prepared by the method for hot evaporation on the glass substrate;
(2) the ethanolic solution HS-(CH of C-terminal mercaptan is prepared
2)
11-EG
6-OH, concentration is 1mM;
(3) biochip substrate ethanol or washed with de-ionized water is clean, then biochip is put into plasma clean instrument cleaning 3 minutes;
(4) be immersed in by biochip in ready thiol solution, hatch 12 hours, after reaching the schedule time at 4 DEG C, taken out by biochip, the alternately cleaning of ethanol and deionized water, nitrogen dries up;
(5) prepare esterification and connect solution 20mL needed for photocrosslinking agent, carboxyl terminal photocrosslinking agent 10mM, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 10mM, DMAP (DMAP) 1mM, solvent is DMF (DMF);
(6) immersed by biochip in the esterification solution prepared, room temperature lucifuge reacts 4 hours, after reaching the schedule time, and cleaned with DMF, ethanol and deionized water successively by biochip, nitrogen dries up for subsequent use;
(7) be the aqueous solution that the glucosan of 500KDa is mixed with mass concentration 40% by molecular weight, stir, removing bubble is to water white uniform solution;
(8) dextran solution prepared is paved with in biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute, after spin coating, by biochip at room temperature lucifuge leave standstill within 1 hour, dry;
(9) biochip is put into ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm UV-irradiation 15 minutes (2.4J/cm
2);
(10) after reaching the schedule time, repeatedly shaken with the hot water of 50 DEG C by biochip and wash 1 hour, remove the dextran molecule that the non-covalency in surface is fixing, period changes water 3-5 time, and the rear nitrogen of biochip cleaning dries up for subsequent use;
(11) biochip is immersed succinic anhydride (10mg/mL) and DMAP (DMAP, in DMF solution 15mg/mL), room temperature (25 DEG C) reaction 16 hours, after reaching predetermined reaction time, biochip is taken out, use DMF, ethanol and washed with de-ionized water successively, nitrogen dries up.
2, the preparation of arrays of immobilized protein
(1) prepare Streptavidin (SA) sodium acetate buffer that pH=4.5 and concentration are 1mg/mL respectively, FKBP12 albumen sodium acetate buffer that bovine serum albumin(BSA) (BSA) sodium acetate buffer that pH=4.5 and concentration are 1mg/mL and pH=4.5 and concentration are 1mg/mL, for subsequent use;
(2) EDC (0.4M) and N-hydroxy-succinamide (NHS is immersed in the biochip substrate prepared, in mixed solution 0.1M), (1:1, v/v) activates 30 minutes, takes out chip, deionized water Rapid Cleaning, nitrogen dries up;
(3) three kinds of albumen buffer solution that chip is prepared with step (1) are respectively put into the relevant position of printer, adjustment parameter, print, ambient humidity is 70%, needle diameter 300 μm, duration of contact 1s, array is 6 × 6;
(4) by printed chip under 70% humidity environment, hatch 2 hours at 4 DEG C, then use PBST (0.05%Tween 20, v/v) to clean, dry up rapidly, at-20 DEG C preserve.
3, on arrays of immobilized protein, biotin is detected
(1), after being cleaned by the biochip containing SA, BSA and FKBP12 arrays of immobilized protein, SPRi system is put into respectively;
(2) System Solution is changed to PBS damping fluid (DMSO containing 1%), adjustment optical position, passes into the biotin-PBS solution (DMSO containing 1%) containing 4nM, in conjunction with the 300s that dissociates after 300s;
(3) after living again with the phosphate aqueous solution that volume fraction is 1%, then live again by the NaOH aqueous solution of 10mM.
Testing result as shown in Figure 1, due to biotin can with Streptavidin specific binding, and not with BSA and FKBP12 protein combination, if therefore biochip of the present invention can fix a large amount of SA, the signal difference being obviously different from BSA and FKBP12 albumen should be had.As can be seen from the result of Fig. 1, the chip of load SA has stronger signal intensity, peak value, in the stable fluctuation of 0.08AU, illustrates that biochip prepared by method of the present invention has very high albumen (SA) fixed amount, is enough to the Small molecular biotin be combined with SA be detected.And two negative controls, namely the signal of the chip of load BSA and the chip of load FKBP12 albumen is substantially in 0AU fluctuation, there is no obvious signal intensity.
Embodiment 2 detects tacrolimus (FK506) on FKBP12 arrays of immobilized protein
1, the modification of biochip substrate
(1) the thick layers of chrome of one deck 3nm and the thick layer gold of one deck 47nm is prepared, as the substrate of biochip by the method for hot evaporation on the glass substrate;
(2) the ethanolic solution HS-(CH of C-terminal mercaptan is prepared
2)
11-EG
6-OH and HS-(CH
2)
11-EG
6-COOH, concentration is 1mM, and OH terminal mercaptan and COOH terminal mercaptan by volume 999:1 mix;
(3) biochip substrate ethanol or washed with de-ionized water is clean, then biochip is put into plasma clean instrument cleaning 3 minutes;
(4) be immersed in by biochip in ready thiol solution, hatch 12 hours, after reaching the schedule time at 4 DEG C, taken out by biochip, the alternately cleaning of ethanol and deionized water, nitrogen dries up;
(5) prepare surface active aqueous solution 20mL, EDC (0.4M) and NHS (0.1M) is according to 1:1 volume mixture;
(6) immersed by biochip in the activated solution prepared, room temperature reaction 15 minutes, after reaching the schedule time, got express developed with deionized water successively by biochip, nitrogen dries up;
(7) chip surface is soaked into the DMF solution of 10mM amino terminal photocrosslinking agent, room temperature lucifuge reacts 4 hours, and clean with DMF, ethanol and deionized water after reaching the reaction time, nitrogen dries up for subsequent use;
(8) be the aqueous solution that the glucosan of 2000KDa is mixed with mass concentration 20% by molecular weight, stir, removing bubble is to water white uniform solution;
(9) dextran solution prepared is paved with in biochip surface, with the rotating speed of 4000rpm, spin coating 45 seconds, spin coating;
(10) biochip is put into ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm UV-irradiation 15 minutes (2.4J/cm
2);
(11) after reaching the schedule time, repeatedly shaken with the hot water of 50 DEG C by biochip and wash 1 hour, remove the dextran molecule that the non-covalency in surface is fixing, period changes water 3-5 time, and the rear nitrogen of biochip cleaning dries up for subsequent use;
(12) biochip is immersed in the DMF solution of succinic anhydride (10mg/mL) and DMAP (15mg/mL), room temperature (25 DEG C) reaction 2 hours, after reaching predetermined reaction time, biochip is taken out, use DMF, ethanol, washed with de-ionized water successively, nitrogen dries up.
2, the preparation of arrays of immobilized protein
(1) prepare phosphate buffer that pH=7.4 and concentration are the immunoglobulin G while (H-IgG) of 1mg/mL respectively, phosphate buffer that phosphate buffer that pH=7.4 and concentration are the bovine serum albumin(BSA) (BSA) of 1mg/mL and pH=7.4 and concentration are the FKBP12 albumen of 1mg/mL, for subsequent use;
(2) chip base prepared is immersed (1:1, v/v) in the mixed solution of EDC (0.4M) and NHS (0.1M) and activate 15 minutes, take out chip, deionized water Rapid Cleaning, nitrogen dries up;
(3) three kinds of albumen buffer solution that chip is prepared with step (1) are respectively put into the relevant position of printer, adjustment parameter, prints, ambient humidity is 70%, needle diameter 300 μm, duration of contact 1s, array be 6 × 6 (as Fig. 2 a);
(4) by printed chip under 70% humidity environment, hatch 2 hours at 4 DEG C, then use PBST (0.05%Tween 20, v/v) to clean, dry up rapidly, at-20 DEG C preserve.
3, on arrays of immobilized protein, detect tacrolimus (FK506)
(1), after being cleaned by the biochip containing H-IgG, BSA and FKBP12 arrays of immobilized protein, SPRi system is put into respectively;
(2) System Solution is changed to PBS damping fluid (DMSO containing 1%), adjustment optical position, passes into tacrolimus (the FK506)-PBS solution (DMSO containing 1%) containing 10nM, in conjunction with the 300s that dissociates after 300s;
(3) live again with the phosphate aqueous solution that volume fraction is 1%.
Testing result is as shown in Fig. 2 (b), because tacrolimus can be combined with FKBP12 protein-specific, and be not combined with H-IgG and BSA, if therefore biochip of the present invention can fix a large amount of FKBP12 albumen, the signal difference being obviously different from H-IgG and BSA albumen should be had.As can be seen from the result of Fig. 2 (b), the chip of load FKBP12 albumen has stronger signal intensity, peak value is in the stable fluctuation of 100RU, illustrate that biochip prepared by method of the present invention has very high albumen (FKBP12) fixed amount, be enough to detect the protein bound Small molecular FK506 with FKBP12.And two negative controls, namely the signal of the chip of load H-IgG and the chip of load BSA albumen is substantially in 0RU fluctuation, there is no obvious signal intensity.
Embodiment 3 detects FK506 on FKBP12 and mutant protein microarray thereof
1, the modification of biochip substrate
With embodiment 2.
2, the preparation of arrays of immobilized protein
(1) phosphate buffer that pH=7.4 and concentration are FKBP12 (wild type) albumen of 0.5mg/mL is prepared respectively, and the phosphate buffer of 5 Single amino acid mutations bodies (M1-M5) of identical pH and concentration, for subsequent use;
(2) chip base prepared is immersed (1:1, v/v) in the mixed solution of EDC (0.4M) and NHS (0.1M) and activate 15 minutes, take out chip, deionized water Rapid Cleaning, nitrogen dries up;
(3) six kinds of albumen buffer solution that chip is prepared with step (1) are respectively put into the relevant position of printer, adjustment parameter, prints, ambient humidity is 70%, needle diameter 300 μm, duration of contact 1s, array be 6 × 12 (as Fig. 3 a);
(4) by printed chip under 70% humidity environment, hatch 2 hours at 4 DEG C, then use PBST (0.05%Tween 20, v/v) to clean, dry up rapidly, at-20 DEG C preserve.
3, on arrays of immobilized protein, detect tacrolimus (FK506)
(1), after being cleaned by the arrays of immobilized protein biochip containing FKBP12 and mutant thereof, SPRi system is put into respectively;
(2) System Solution is changed to PBS damping fluid (DMSO containing 1%), adjustment optical position, passes into tacrolimus (the FK506)-PBS solution (DMSO containing 1%) containing 10nM, in conjunction with the 300s that dissociates after 300s;
(3) live again with the phosphate aqueous solution that volume fraction is 1%.
Testing result is as shown in Fig. 3 (b), can find out, the chip of load FKBP12 wild-type protein has stronger signal intensity, and the signal intensity of five kinds of mutant is obvious not as FKBP12 wild-type protein, illustrate that the biochip utilizing method of the present invention to prepare has very high proteopexy amount, be enough to specific binding FK506 and FKBP12 being detected.And mutant is due to the sudden change of just monamino acid, also have combination to a certain degree, but combination degree is not as wild type with FK506, signal intensity is the weak FKBP12 with wild type also.
Embodiment 4 detects peptide T 7 on amyloid A β 42 and mutant microarray thereof
1, the modification of biochip substrate
With embodiment 2.
2, the preparation of arrays of immobilized protein
(1) phosphate buffer that pH=7.4 and concentration are the amyloid A β 42 (wild type) of 0.5mg/mL is prepared respectively, and the phosphate buffer of 3 mutant of identical pH and concentration (E type, T-shaped and P type), for subsequent use;
(2) chip base prepared is immersed (1:1, v/v) in the mixed solution of EDC (0.4M) and NHS (0.1M) and activate 15 minutes, take out chip, deionized water Rapid Cleaning, nitrogen dries up;
(3) 3 kinds of albumen buffer solution that chip is prepared with step (1) are respectively put into the relevant position of printer, adjustment parameter, prints, ambient humidity is 70%, needle diameter 300 μm, duration of contact 1s, array be 6 × 12 (as Fig. 3 a);
(4) by printed chip under 70% humidity environment, hatch 2 hours at 4 DEG C, then use PBST (0.05%Tween 20, v/v) to clean, dry up rapidly, at-20 DEG C preserve.
3, on arrays of immobilized protein, peptide T 7 is detected
(1), after being cleaned by the arrays of immobilized protein biochip containing amyloid A β 42 and mutant thereof, SPRi system is put into respectively;
(2) System Solution is changed to PBS damping fluid (DMSO containing 1%), adjustment optical position, passes into the micromolecule polypeptide T7-PBS solution containing 50 μMs, in conjunction with the 300s that dissociates after 300s;
(3) live again with the phosphate aqueous solution that volume fraction is 1%.
Testing result as shown in Figure 4, can find out that the chip of load wild type starch sample albumin A β 42 has stronger signal intensity, and the signal intensity of three kinds of mutant is obvious not as wild-type protein, illustrate that the biochip utilizing method of the present invention to prepare has very high proteopexy amount, be enough to specific binding wild type starch sample albumin A β 42 and peptide T 7 being detected.And three kinds of mutant are the sudden change in some sites, therefore itself and peptide T 7 also have combination in various degree; Wherein T-shaped and mutational site that is E type may not be critical sites, therefore also has combination to a certain degree with peptide T 7; And P type is the sudden change of critical sites, therefore disease and peptide T 7 do not combine; Although the combination degree of saltant type and peptide T 7 is different, be all not so good as wild type, signal intensity is also weaker than the amyloid A β 42 of wild type.
Can draw based on the above results, the biochip of three-dimensional macromolecular surface modification is completed through method of the present invention, effectively can improve the fixed amount of albumen in biochip surface, enable the Small molecular that is difficult in prior art detect by unmarked, accurately and high flux detect, to the field such as molecular biology research and drug development, there is realistic meaning.In addition, method of the present invention accurately can control the proteopexy amount of biochip surface, ensures that chip surface has the detection that small-molecule drug is carried out in enough spaces simultaneously; Preparation method is simple, quick, cost is low and easily carry out quality control, is applicable to large-scale production and popularization.
Applicant states, the present invention illustrates process of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not namely mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (10)
1. a micro-array biochip, comprises glass-chip substrate and decorative layer, it is characterized in that, described decorative layer comprise be superimposed upon described glass-chip substrate surface successively layers of chrome, silver and/or layer gold, self assembled monolayer, photocrosslinking agent and macromolecule layer.
2. micro-array biochip according to claim 1, is characterized in that, the thickness of described layers of chrome is 0.1-9nm, is preferably 3nm;
Preferably, the thickness of described silver and/or layer gold is 40-60nm, is preferably 47-50nm;
Preferably, the reagent of described self assembled monolayer is the mercaptan of end-functionalization;
Preferably, the mercaptan of described end-functionalization is single mercaptan and/or dithiol;
Preferably, the mercaptan of described end-functionalization is alkanethiol and/or the mercaptan with PEG block;
Preferably, the group of described end-functionalization is hydroxyl, carboxyl, amino, aldehyde radical, epoxy radicals or methoxyl;
Preferably, described photocrosslinking agent is the reagent containing photaesthesia group, is preferably the potpourri of any one or at least two kinds in azirine, diazonium, acetophenone, benzophenone or Anthraquinones;
Preferably, described macromolecule is the potpourri of any one or at least two kinds in polyglycol and derivant, fluoropolymer, polyacrylic acid and derivant thereof, polymethylacrylic acid and derivant, polystyrene, PLA, both sexes betaines polymkeric substance, nitrocellulose, shitosan or glucosan and derivant thereof.
3. the preparation method of micro-array biochip according to claim 1 and 2, is characterized in that, comprises the following steps:
(1) plate layers of chrome in glass-chip substrate, then on the plated surface of layers of chrome silver and/or layer gold;
(2) completing steps (1) cleans chip afterwards, forms self assembled monolayer on surface;
(3) photocrosslinking agent is covalently bonded in the surface of self assembled monolayer;
(4) Polymer Solution is spin-coated on the chip surface of completing steps (3);
(5) after the solvent volatilization of step (4) described Polymer Solution, make described macromolecular grafted at chip surface by UV-irradiation;
Optionally, step (6) is carried out:
(6) macromolecule being grafted on chip surface is carried out functionalization.
4. preparation method according to claim 3, is characterized in that, the method plating layers of chrome and silver and/or layer gold described in step (1) is that metal fever steams and/or ion sputtering;
Preferably, the described covalently bound mode of step (3) is amidation, esterification, open loop or nucleophilic substitution;
Preferably, the solvent of step (4) described Polymer Solution is the combination of any one or at least two kinds in deionized water, ethanol, methyl alcohol, dimethyl sulfoxide (DMSO) or DMF;
Preferably, the wavelength of step (5) described ultraviolet light is 200-400nm, is preferably 365nm;
Preferably, the group of step (6) described functionalization is hydroxyl, carboxyl, aldehyde radical, amino, epoxy radicals, cyano group, alkynyl, azido, azirine or biotin.
5. an arrays of immobilized protein, is characterized in that, comprises micro-array biochip as claimed in claim 1 or 2.
6. the preparation method of arrays of immobilized protein according to claim 5, is characterized in that, described preparation method is that after protein is mixed with solution, point sample is also hatched and makes;
Preferably, described protein is mixed with solution for be dissolved in damping fluid by protein;
Preferably, described damping fluid is PBS damping fluid, HEPES damping fluid, Tris-HCl damping fluid or HBS-EP damping fluid;
Preferably, the pH value of described damping fluid is below described isoelectric points of proteins 0-1 unit;
Preferably, described in the temperature of hatching be 3-8 DEG C, be preferably 4 DEG C;
Preferably, described in the relative humidity of hatching be 50-80%, be preferably 70%.
7. a detection method, is characterized in that, described method utilizes the arrays of immobilized protein described in claim 5.
8. method according to claim 7, is characterized in that, comprises the following steps:
(1) cleaning is fixed with the chip of arrays of immobilized protein, puts into surface plasmon resonance assay imager after drying up;
(2) in surface plasmon resonance assay imager, damping fluid is added;
(3) treat baseline stability, with living again, live again in the surface of liquid by described chip;
(4) pass into detection liquid, live again;
(5) pass into correcting fluid, detect protein signal.
9. method according to claim 8, is characterized in that, step (2) described damping fluid is PBS damping fluid, HEPES damping fluid, Tris-HCl damping fluid or HBS-EP damping fluid;
Preferably, also dimethyl sulphoxide solution is added in step (2) described damping fluid;
Preferably, the volume fraction of described dimethyl sulphoxide solution is 0.5-5%;
Preferably, liquid of living again described in step (3) is phosphoric acid solution, glycine-HCI damping fluid, ethylene glycol solution or sodium hydrate aqueous solution; In described phosphoric acid solution, the volume ratio of phosphoric acid and water is 1:(100-400); The pH of described glycine-HCI damping fluid is 1.5-2.5; The volume fraction of described ethylene glycol solution is 10-40%; The concentration of described sodium hydrate aqueous solution is 7-13mmol/L.
10. micro-array biochip according to claim 1 and 2 or arrays of immobilized protein according to claim 5 are detecting the application in small-molecule drug.
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