CN103792345B - A kind of small-molecular micro-array and preparation method thereof - Google Patents
A kind of small-molecular micro-array and preparation method thereof Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/168—Specific optical properties, e.g. reflective coatings
Abstract
The invention discloses a kind of preparation method of small-molecular micro-array: the C-terminal of anti-non-specific adsorption material that chip base is modified and the carboxyl terminal of Small molecular photocrosslinking agent are carried out bonding by esterification, then makes Small molecular be fixed thereon at random and get final product by photo-crosslinking.The method adopts the mode of direct esterification, and fast and efficiently Small molecular photocrosslinking agent is coupled to the surface of anti-non-specific adsorption material, chip modification is simple and convenient, normal temperature condition can complete, both save modification cost, turn improved modification efficiency, ensure that higher detection signal; Unreacted group still keeps higher anti-non-specific adsorption ability, and background signal is reduced; Prepared small-molecular micro-array detection signal is comparatively strong, background signal is more weak, can be used for the interaction of high flux screening small-molecule drug with target.
Description
Technical field
The present invention relates to biochip technology field, particularly relate to a kind of small-molecular micro-array and preparation method thereof.
Background technology
Small-molecular micro-array is a kind of high throughput screening drug developed rapidly nearly ten years.Then small-molecular micro-array refers to fixes by point sample (or print) high-density micro-array that each organic micromolecule formed on solid state surface---be similar to pandemic DNA microarray, glass (or plastics) substrate can print the microarray of up to ten thousand different compounds, have benefited from the microminiaturization of analytical instrument, can disposable, analyze thousands of biochemical interactions single step simultaneously.1999, the reported first such as Schreiber array of small molecules, this array is by as probe, be successfully applied to the detection (Koehler of FKBP12 and aglucon thereof, A.N., A.F.Shamji, and S.L.Schreiber, Discovery of an inhibitor of atranscription factor using small molecule microarrays and diversity-orientedsynthesis.Journal of the American Chemical Society, 125 (28): pp.8420-8421.2003.).In the last few years, array of small molecules had been successfully applied at key areas such as analysis of protein and lead compound exploitations.
Because small-molecular micro-array needs the Small molecular of a large amount of, different chemical structures or biochemical structure to be fixed on carrier surface, how to fix Small molecular expeditiously, and not affecting Small molecular with albumen target in conjunction with biochemical activity, is the challenging difficult problem of small-molecular micro-array (SMM) technology most always.Emerge in large numbers a variety of convenience, the process for fixation that stable, cost is low nearly ten years, wherein, firmly fix by covalent bond the main flow that Small-molecule probe is Small molecular fixing means.At present, multiple choices coupling reaction has been used in covalently bound aspect, and meanwhile, polyfunctional group has also successfully been fixed on surface and has formed non-selective coupling.Selectivity coupling agent is of a great variety, can need design coupling agent end, in order to the fixing Small molecular with special groups according to difference.But the technology path that selectivity or directionality fix Small-molecule probe has the defect of essence: the avtive spot of Small-molecule probe is probably fixed, thus lose the biochemical activity with target effect, the reactive compound that sieve is good may be leaked.Therefore, non-selective, random mode, the pervasive fixing means in all Small-molecule probes will improve the universality of small-molecular micro-array technology in drug screening greatly, for raising drug screening and research and development ability, there is revolutionary meaning, therefore, therefore the research of association area become a study hotspot.
In numerous progress, especially with trifluoromethyl azirine (Trifluoromethylaryldiazirine, TFMAD) the photo-crosslinking surface chemistry based on functional group attracts most attention (Kanoh, N., et al., Immobilization of Natural Products on Glass Slides by Using a PhotoaffinityReaction and the Detection of Protein – Small-Molecule Interactions.AngewandteChemie, 115 (45): pp.5742-5745.2003).Compared with other azo Cabbeen (carbene) photo-crosslinking chemistry, TFMAD functional group has higher chemical stability, and the Cabbeen intermediate produced has high reaction activity, resets probability low, carbon intercalation reaction productive rate advantages of higher.What is more important, only needs relatively low-energy 365nm ultraviolet light just can excite the generation of Cabbeen, thus reduces Small-molecule probe by the probability of ultraviolet degradation.TFMAD photochemical fixation mild condition, controllability are strong, modify in appointed area by mask, can be widely used in the fixing of different kind organism molecule.In addition, under solid phase conditions, carry out photo-crosslinking can avoid molecule specific orientation and functional group may being caused to select by solvent, molecule contacts with the randomness of azo and is guaranteed.
2006, the long field of Japan Chemical research institute abundant etc. report non-selection photo-crosslinking small-molecular micro-array (Naoki Kanoh, Motoki Kyo based on TFMAD, Kazuki Inamori, Ami Ando, AyaAsami, Aiko Nakao, and Hiroyuki Osada, Analytical Chemistry, Vol.78, No.7, April1,2006).In this work, they have prepared the small-scale array having been prepared 8 kinds of female hormone and male sex hormone compound by TFMAD photo-crosslinking surface chemistry, confirm the interaction between itself and estrogen receptor a by surface plasma resonance technology, but in flux and in signal intensity, all there is very large deficiency.In recent years, there is again the small-molecular micro-array combined based on three-dimensional polyethylene glycol polymer brush surface and photocrosslinking agent, this microarray breaches two-dimensional surface in the past, utilize surface initiation polymerization technology in substrate grown macromolecule brush structure, utilize highdensity end group fixed light crosslinking chemical thus non-selective fixing small-molecule drug, utilize surface plasma resonance imaging technology to carry out high-throughout rapid screening to target.But the method for traditional connection photocrosslinking agent makes this microarray there is certain defect---the problem of non-specific adsorption.
Non-specific adsorption is a bottleneck of biochip applications always, and the high background signal caused by non-specific adsorption can produce interference to detection signal, especially for fatal especially small-molecular micro-array.Due in classic method, utilize carboxymethylated polymethyl acid polyethylene glycol surface coupling diamido end PEG, then utilize EDC and N-hydroxy-succinamide (EDC/NHS) activated terminus, final and photocrosslinking agent coupling.Such three-step reaction, ultimate yield is the product often walking reaction yield, causes ultimate yield significantly to decline, thus also diminishes to micromolecular fixed amount, and detection signal can be caused to reduce; Meanwhile, the carboxylated end having neither part nor lot in reaction can allow surface in detection solution with a certain amount of electric charge, easily causes Electrostatic Absorption, increases the source of non-specific adsorption.So the ability of the anti-non-specific adsorption of retention surface, increases the target that array of small molecules detection signal becomes researcher's pursuit simultaneously.
Summary of the invention
The object of the invention is to propose the small-molecular micro-array and preparation method thereof of a kind of high detection signal, low detection background.The preparation method of small-molecular micro-array of the present invention adopts single stage method, fast and efficiently Small molecular photocrosslinking agent is coupled to the surface of anti-non-specific adsorption material, chip modification is simple and convenient, normal temperature condition can complete, both save modification cost and turn improved modification efficiency, ensure that higher detection signal; Unreacted group still keeps the ability of the high resistance non-specific adsorption on polyglycol surface, and detection background is reduced.
For reaching this object, the present invention by the following technical solutions:
First aspect, the invention provides a kind of preparation method of small-molecular micro-array, comprising:
A preparation method for small-molecular micro-array, comprising:
(1) in chip base, modify the anti-non-specific adsorption material of hydroxyl end;
(2) the Small molecular photocrosslinking agent of band carboxyl terminal and Photocrosslinkable functional group is bonded on the anti-non-specific adsorption material of described hydroxyl end by esterification;
(3) by small molecule solution point sample to step (2) gained chip surface, after drying, carry out photo-crosslinking, obtain small-molecular micro-array chip.
As preferably, step (1) described chip base is selected from glass, silicon chip, quartz, high score subclass film, metallic film and metal-oxide film;
Preferably, described high score subclass film is any one in dimethyl silicone polymer, polystyrene, polycarbonate or polymethylmethacrylate;
Preferably, described metallic film is golden film or silverskin;
Preferably, described metal-oxide film is di-aluminium trioxide film.
As preferably, the anti-non-specific adsorption material of the described hydroxyl end of step (1) is alkyl sulfhydryl, the macromolecule surface of hydroxyl end or Supramolecular self assembly surface;
Preferably, described alkyl sulfhydryl is the alkanethiol with polyglycol segment;
Preferably, described macromolecule surface is the amphoteric ion polymer surface of polyglycol or derivatives thereof, the fluoropolymer of hydroxyl end, glucosan, cellulose or derivatives thereof or hydroxyl end;
Preferably, described Supramolecular self assembly surface is poly-rotaxane surface.
About the concrete mode of anti-non-specific adsorption material of modifying hydroxyl end step (1) Suo Shu in chip base, all modes well known by persons skilled in the art can be used.Common modification mode mainly contains following three kinds: 1, at surface initiation polymerization, namely first spread initiating agent at substrate surface, then trigger monomer is at surface aggregate, thus obtains anti-nonspecific surface; 2. surface grafting, namely first obtains the macromolecular material with anti-non-specific adsorption character, then is connected to surface by specific radical reaction; 3. carry out Supramolecular self assembly on surface, namely first modify chain host molecule at substrate surface, then guest molecule is assembled into surface, finally form Supramolecular self assembly body.
In a specific embodiment, in step (1), the anti-non-specific adsorption material of described hydroxyl end is brush PEG; The method that chip base is modified brush PEG is:
(1 ') uses the substrate of initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halogenated thiols solution; Further preferably, the concentration of described single sulfydryl halogenated thiols solution is 0.1 ~ 100mM, preferably 0.1mM ~ 10mM, more preferably 1mM; Still more preferably, described single sulfydryl halogenated thiols solution also comprises single mercapto-polyglycol;
In particular embodiments, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM, 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM.
Preferably, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively 0.1 ~ 100mM, preferably single mercapto-polyglycol of 0.1mM ~ 10mM, more preferably 1mM and the ethanolic solution of single sulfydryl halogenated thiols, with (0 ~ 999): 1, preferably (0 ~ 99): 1, the volume ratio Homogeneous phase mixing of more preferably 99:1, is taped against described substrate surface;
In particular embodiments, the ethanolic solution of described single mercapto-polyglycol is identical with the concentration of the ethanolic solution of single sulfydryl halogenated thiols and be 0.1mM, 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, and the volume ratio of the two is 0:1,1:1,9:1,49:1,99:1,199:1,299:1,399:1,499:1,599:1,699:1,799:1,899:1,999:1.
(2 ') step (1 ') gained chip is immersed in the solution containing the vinyl monomer of organic reducing agent, polymerisation catalysts and PEG, carries out monomer polymerization reactions and get final product under oxygen-free environment;
Preferably, described organic reducing agent is glucose, ascorbic acid or stannous octoate; Further preferably, the content of described organic reducing agent is 1nM ~ 1mM, is preferably 1nM ~ 0.1mM, is more preferably 0.04mM;
Preferably, described polymerisation catalysts is the potpourri of transition metal salt and sequestrant, the preferred molysite of described transition metal salt or mantoquita; Described sequestrant is preferably second bipyridine; Further preferably, the content of described transition metal salt is 1nM ~ 1mM, is preferably 1nM ~ 0.1mM, is more preferably 0.04mM; The content of described sequestrant is 1nM ~ 100mM, is preferably 1nM ~ 1mM, is more preferably 0.8mM;
Preferably, the vinyl monomer of described PEG is methacrylic acid macrogol ester; Further preferably, the content of the vinyl monomer of described PEG is 1mM ~ 1M, is preferably 1mM ~ 100mM, is more preferably 5mM;
Preferably, the time of described macromolecule growth is 1 ~ 40 hour, preferably 1 ~ 20 hour, more preferably 18 hours.
In another embodiment, in step (1), the anti-non-specific adsorption material of described hydroxyl end is hyperbranched PEG; Preferably, the method for chip base being modified hyperbranched PEG is:
(1 ' ') use the substrate of initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halogenated thiols solution; Further preferably, the concentration of described single sulfydryl halogenated thiols solution is 0.1 ~ 100mM, preferably 0.1 ~ 10mM, more preferably 1mM; Still more preferably, described single sulfydryl halogenated thiols solution also comprises single mercapto-polyglycol;
In particular embodiments, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM, 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM.
Preferably, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively 0.1 ~ 100mM, preferably single sulfydryl halogenated thiols of 0.1mM ~ 10mM, more preferably 1mM and the ethanolic solution of single mercapto-polyglycol, with (0 ~ 999): 1, preferably (0 ~ 99): 1, the volume ratio Homogeneous phase mixing of more preferably 99:1, is taped against described substrate surface;
In particular embodiments, the ethanolic solution of described single mercapto-polyglycol is identical with the concentration of the ethanolic solution of single sulfydryl halogenated thiols and be 0.1mM, 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, and the volume ratio of the two is 0:1,1:1,9:1,49:1,99:1,199:1,299:1,399:1,499:1,599:1,699:1,799:1,899:1,999:1.
(2 ' ') step (1 ' ') gained chip is immersed in the solution containing the vinyl monomer of organic reducing agent, polymerisation catalysts and PEG, carries out monomer polymerization reactions, then take out chip under oxygen-free environment;
Preferably, described organic reducing agent is glucose, ascorbic acid or stannous octoate; Further preferably, the content of described organic reducing agent is 1nM ~ 1mM, is preferably 1nM ~ 0.1mM, is more preferably 0.04mM;
Preferably, described polymerisation catalysts is the potpourri of transition metal salt and sequestrant, the preferred molysite of described transition metal salt or mantoquita; Described sequestrant is preferably second bipyridine; Further preferably, the content of described transition metal salt is 1nM ~ 1mM, is preferably 1nM ~ 0.1mM, is more preferably 0.04mM; The content of described sequestrant is 0.8mM;
Preferably, the vinyl monomer of described PEG is methacrylic acid macrogol ester; Further preferably, the content of the vinyl monomer of described PEG is 1mM ~ 1M, is preferably 1mM ~ 100mM, is more preferably 5mM;
Preferably, the time of described macromolecule growth is 1 ~ 40 hour, preferably 1 ~ 20 hour, more preferably 6 hours;
(3 ' ') carries out end-blocking with 2-bromine isobutyl acylbromide to the polymkeric substance on step (2 ' ') gained chip, forms macromole evocating agent;
(4 ' ') repeats step (2 ' ') and step (3 ' ') to gained chip, so repeats 3-4 time and get final product.
In another specific embodiments, in step (1), the anti-non-specific adsorption material of described hydroxyl end is poly-rotaxane; Preferably, the method for chip base being modified poly-rotaxane is:
Chip base is put into the ethanolic solution of the mercapto-polyglycol of two kinds of different molecular weights by (1 ' ' '), 0 ~ 30 DEG C hatch 1 ~ 12 hour after, clean, dry up;
The mercapto-polyglycol of described two kinds of different molecular weights be respectively molecular weight be 40 ~ 400 C-terminal or methoxyl terminal mercaptan and molecular weight be 1000 ~ 20000 carboxyl terminal mercaptan;
Step (1 ' ' ') gained chip immerses in the aqueous solution of the alpha-cyclodextrin of more than 10mM and hatches 1 ~ 20 hour by (2 ' ' '), then add EDC and N-hydroxy-succinamide, react 10 ~ 60 minutes;
Step (2 ' ' ') gained chip is put into the saturated alpha-cyclodextrin aqueous solution of the benzyloxycarbonyl group-TYR (Z-Tyr-OH) containing 1 ~ 10mM by (3 ' ' '), reacts 1 ~ 2 hour and get final product.
In another specific embodiments, in step (1), the anti-non-specific adsorption material of described hydroxyl end is glucosan; Preferably, the method for chip base being modified glucosan is:
Concentration is that the C-terminal alkanethiol ethanol of 1 ~ 100mM and/or aqueous solution are layered on chip surface by (1 ' ' ' '), and incubated at room was taken out after 1 ~ 20 hour;
The mixed solution that step (1 ' ' ' ') gained chip is put into NaOH containing epichlorokydrin and diethylene glycol dimethyl ether by (2 ' ' '), reacts under room temperature after 1 ~ 10 hour and takes out;
Step (2 ' ' ' ') gained chip is put into the sodium hydroxide solution of glucosan by (3 ' ' '), and room temperature reaction is after 0.1 ~ 10 hour and get final product.
In above-mentioned preparation method, as preferably, in step (2), described Photocrosslinkable functional group is selected from acetophenone, Benzophenone, quinone anthracene class, aryl azides compound and fragrant azirine, is preferably fragrant azirine;
Preferably, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of step (1) resulting tape C-terminal is modified is put into the solution containing described Small molecular photocrosslinking agent, organic alkali catalyst and dewatering agent, at 18 ~ 32 DEG C, preferably at 22 ~ 28 DEG C, more preferably reaction 1 ~ 40 hour at 25 DEG C, preferably 1 ~ 20 hour, more preferably 18 hours and obtain;
Further preferably, the solvent of described solution is organic solvent; More preferably, described organic solvent is selected from methylene chloride, tetrahydrofuran and DMF;
Further preferably, described organic alkali catalyst is selected from DMAP, DIPEA and triethylamine;
Further preferably, described dewatering agent is selected from 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and dicyclohexylcarbodiimide;
Further preferably, in described solution, the mol ratio of the terminal carboxyl group of Small molecular photocrosslinking agent and dewatering agent, organic alkali catalyst is 1:(1 ~ 3): (1 ~ 3), more preferably 1:(1 ~ 2): (1 ~ 2), is further preferably 1:1.5:1.5;
Further preferably, in described solution, the end carboxyl group content of Small molecular photocrosslinking agent is 1nM ~ 100mM, is preferably 1mM ~ 100mM, is more preferably 10mM.
Step (2) described esterification is the esterification based on solid-liquid interface, by conditions such as adjustment reaction mass proportioning, reaction time, the grafting amount of Small molecular photocrosslinking agent at chip surface can be controlled, thus ensure to obtain relatively high detection signal and lower background signal.
In a particular embodiment, step (3) described small molecule solution can be arbitrarily small molecule, such as, be small-molecule drug or the Small molecular having potential medical value;
Preferably, the condition of described photo-crosslinking is: be 0.1 ~ 100J/cm in light intensity
2, be preferably 0.1 ~ 10J/cm
2, be more preferably 1J/cm
2, wavelength is irradiate 1 ~ 30 minute, preferably 1 ~ 20 minute, more preferably 10 minutes under the ultraviolet light of 200 ~ 400nm, preferably 300 ~ 400nm, more preferably 365nm.
Second aspect, the invention provides a kind of small-molecular micro-array, is obtained by preparation method described in any one of first aspect.
Preparation method's schematic diagram of small-molecular micro-array of the present invention as shown in Figure 1, this preparation method is by the mode of direct esterification, the C-terminal of anti-non-specific adsorption material that chip base is modified and the carboxyl terminal of Small molecular photocrosslinking agent are carried out bonding, then by photo-crosslinking, Small molecular is fixed thereon at random, thus the small-molecular micro-array of the three-dimensional surface of acquisition high detection signal, low detection background, then utilize high flux detection technique to carry out small-molecule drug with the interactional screening of target.
The advantage that the three-dimensional surface small-molecular micro-array manufacture method that small-molecular micro-array preparation method of the present invention is more traditional is outstanding is, adopt single stage method, fast and efficiently Small molecular photocrosslinking agent is coupled to the surface of anti-non-specific adsorption material, chip modification is simple and convenient, normal temperature condition can complete, both save modification cost, turn improved modification efficiency, ensure that higher detection signal; Unreacted group still keeps higher anti-non-specific adsorption ability, and detection background is reduced; The surface modified through direct esterification method keeps electroneutral surface nature, and surface nature is homogeneous.
Accompanying drawing explanation
Fig. 1 is preparation method's schematic diagram of small-molecular micro-array of the present invention;
Fig. 2 be three-dimensional PEG macromolecule surface esterification coupling photocrosslinking agent forward and backward x-ray photoelectron power spectrum contrast;
Fig. 3 be three-dimensional PEG macromolecule surface esterification coupling photocrosslinking agent forward and backward FT-IR collection of illustrative plates contrast;
Fig. 4 is that the detection signal of the microarray prepared of small-molecular micro-array of the present invention and classic method when surface plasma resonance detects and background intensity contrast.
Embodiment
Technical scheme of the present invention is further illustrated below by embodiment.
the preparation of embodiment 1 brush PEG surface small-molecular micro-array
Concrete steps:
(1) layers of chrome of one deck 3.5nm thickness and the layer gold of one deck 50nm thickness is prepared, as the substrate of biochip by the method for hot evaporation on the glass substrate.
(2) prepare 400 μ L concentration be single sulfydryl halogenated thiols solution of 1mM as initiating agent, prepare single mercapto-polyglycol of 400 μ L same concentrations, by two kinds of solution in 1:99 ratio mixing, be taped against chip surface, under 4 DEG C of environment, hatch 12 hours.
(3) gold plaque ethanol and deionized water are alternately cleaned up.
(4) prepare monomer solution: monomer solution altogether 20mL, wherein ascorbic acid 0.04mM, cupric chloride 0.04mM, second bipyridine 0.8mM, methacrylic acid macrogol ester (molecular weight 360) 5mM, solvent is first alcohol and water 1:1(v/v) mixed solvent.
(5) gold plaque being immersed in the monomer solution of preparation, under being placed on oxygen-free environment, growing macromolecule, growth time 18 hours, regulating the thickness of polymeric membrane by controlling growth time.
(6), after reaching predetermined growth time, gold plaque is taken out cleaning.
(7) photocrosslinking agent solution needed for the esterification of position: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(8) immersed by chip in esterification solution, load suitable vessel, sealing, hatches 18 hours under room temperature environment.After reaching the schedule time, taken out by chip, alternately rinse well with second alcohol and water, nitrogen dries up.
(9) by being dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip prepared, after to be dried, chip is irradiated 10min under 365nm ultraviolet light, light intensity is 1J/cm
2, taken out by chip after reaching the schedule time, clean successively with dimethyl sulfoxide (DMSO), tetrahydrofuran, DMF, ethanol, deionized water equal solvent, nitrogen dries up, and obtains small-molecular micro-array of the present invention.
In above-mentioned preparation process, also have detected the forward and backward x-ray photoelectron power spectrum of chip macromolecule surface esterification coupling Small molecular photocrosslinking agent and FT-IR collection of illustrative plates respectively, respectively as shown in Figure 2 and Figure 3.
As seen from Figure 2, the forward and backward x-ray photoelectron power spectrum of esterification there occurs change, due in photocrosslinking agent molecule containing there being F and N two kinds of atoms, so there are F and N two kinds of elements that original surface does not have in elemental map after esterification, the success of explanation esterification process, and can ultimate analysis be passed through, judge the photocrosslinking agent grafting degree on surface.
As seen from Figure 3, before esterification, after (a) and esterification there is certain difference in the infared spectrum of (b).Semi-quantitatively, in the collection of illustrative plates after esterification, 1732cm
-1the stretching vibration peak grow of place C=O, 3467cm
-1the stretching vibration peak of place O-H weakens, this is because esterification makes surface dewatering condensation form ester carbonyl group, and decreases the hydroxyl of end; Meanwhile, at 1610cm
-1and 1453cm
-1having there is the characteristic peak of phenyl ring stretching vibration in left and right, illustrates and have the coupling of photocrosslinking agent molecule to get on the surface; In addition, the also difference to some extent of the fingerprint region in the forward and backward infared spectrum of esterification, this is because photocrosslinking agent contains the chemical bond of specific groups, as N=N, C-F etc.More than characterize and again illustrate that esterification coupling photocrosslinking agent successfully reacts.
This small-molecular micro-array mating surface plasma resonance imaging technology is used for detecting small-molecule drug with the interaction of target proteins, has lower background signal, and higher detection signal.
the preparation of embodiment 2 hyperbranched PEG surface small-molecular micro-array
Concrete steps are as follows:
(1) prepare substrate of glass chip, be paved with containing a certain amount of initiator solution after chip cleaning is dried up.
(2) after reaching predetermined reaction time, glass is taken out, cleaning.
(3) prepare monomer solution: monomer solution altogether 20mL, wherein ascorbic acid 0.04mM, cupric chloride 0.04mM, second bipyridine 0.8mM, methacrylic acid macrogol ester (molecular weight 360) 5mM, solvent is first alcohol and water 1:1(v/v) mixed solvent.
(4) gold plaque being immersed in the monomer solution of preparation, under being placed on oxygen-free environment, growing macromolecule, growth time 6 hours, regulating the thickness of polymeric membrane by controlling growth time.
(5), after reaching predetermined growth time, gold plaque is taken out, alternately rinses well with second alcohol and water.
(6) chip is put into the mixed solution of 1.5mL pyridine and 50mL dry ether, drip the diethyl ether solution 30mL of 2-bromine isobutyl acylbromide (BiBB) containing 10% volume ratio, stir 2 hours at 0 DEG C, more at room temperature react 10 hours, then with ethanol and ultrapure water cleaning, nitrogen dries up.
(7) repeat step 4 ~ 7, after repeating 3 ~ 4 times, obtain hyperbranched polyglycol C-terminal surface.
(8) photocrosslinking agent solution needed for the esterification of position: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(9) immersed by chip in esterification solution, load suitable vessel, sealing, hatches 18 hours under room temperature environment.After reaching the schedule time, taken out by chip, alternately rinse well with second alcohol and water, nitrogen dries up.
(10) by being dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip prepared, irradiated 10 minutes by chip after to be dried under 365nm ultraviolet light, light intensity is 1J/cm
2, taken out by chip after reaching the schedule time, clean successively with dimethyl sulfoxide (DMSO), tetrahydrofuran, DMF, ethanol, deionized water equal solvent, nitrogen dries up and obtains small-molecular micro-array of the present invention.
embodiment 3 three-dimensional poly-rotaxane surface small-molecular micro-array
(1) as the step 1 in embodiment 1, the substrate of gold plaque as biochip is prepared.
(2) prepare the ethanolic solution of mercapto-polyglycol that two kinds of concentration are 1mM, different molecular weight, one is HS-(CH
2)
11-EG6, another kind is HS-EGn-COOH(molecular weight 2000).Two kinds of solution are pressed the mixing of 10:1 volume ratio, then chip is put into mixed solution, at 4 DEG C, hatch 12 hours.After reaching the schedule time, ethanol and deionized water alternately cleaning after taking out, nitrogen dries up.
(3) chip is immersed in (20mL) in the aqueous solution of the alpha-cyclodextrin of 128mM and hatches 4 hours, then in solution, add EDC 750mg and N-hydroxy-succinamide 115mg, reaction 15min.
(4) take out chip, directly put into the saturated alpha-cyclodextrin aqueous solution of the benzyloxycarbonyl group-TYR (Z-Tyr-OH) containing 3.2mM, then react 2 hours, rear ultrapure water cleaning, nitrogen dries up.
(5) photocrosslinking agent solution needed for the esterification of position: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(6) immersed by chip in esterification solution, load suitable vessel, sealing, hatches 18 hours under room temperature environment.After reaching the schedule time, taken out by chip, alternately rinse well with second alcohol and water, nitrogen dries up.
(7) by being dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip prepared, irradiated 10 minutes by chip after to be dried under 365nm ultraviolet light, light intensity is 1J/cm
2, taken out by chip after reaching the schedule time, clean successively with dimethyl sulfoxide (DMSO), tetrahydrofuran, DMF, ethanol, deionized water equal solvent, nitrogen dries up and obtains small-molecular micro-array of the present invention.
This small-molecular micro-array is used for the interaction of on-line checkingi small-molecule drug with target proteins in conjunction with QCM (Quartz Crystal Microbalance) (QCM) technology, has low background signal, and high detection signal, and can carry out real time on-line monitoring high flux.
embodiment 4 dextran surface small-molecular micro-array
(1) as the step 1 in embodiment 1, the substrate of gold plaque as biochip is prepared.
(2) compound concentration is 1mM C-terminal alkanethiol (HS-(CH
2)
11-EG3) ethanolic solution, solution is layered on chip surface, at 4 DEG C, hatches 12 hours.Take out after reaching the time, by ethanol and ultrapure water clean surface, nitrogen dries up.
(3) solution of the epichlorokydrin of 0.6M is put into 1:1(v/v) the 0.4M sodium hydroxide solution that mixes and diethylene glycol dimethyl ether, chip is put into solution and soaks, at room temperature react 4 hours.Alternately rinse with second alcohol and water after reaction.
(4) prepare dextran solution (3g Dextran T 500 is dissolved in the sodium hydroxide solution of the 0.1M of 10ml), chip is immersed in dextran solution, room temperature reaction 20 hours.Taken out by chip after reaching predetermined reaction time, ultrapure water cleans, and nitrogen dries up.
(5) photocrosslinking agent solution needed for the esterification of position: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(6) immersed by chip in esterification solution, load suitable vessel, sealing, hatches 18 hours under room temperature environment.After reaching the schedule time, taken out by chip, alternately rinse well with second alcohol and water, nitrogen dries up.
(7) by being dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip prepared, irradiated 10 minutes by chip after to be dried under 365nm ultraviolet light, light intensity is 1J/cm
2, reach after the schedule time and taken out by chip, to hocket cleaning with dimethyl sulfoxide (DMSO), tetrahydrofuran, DMF, ethanol, deionized water equal solvent, nitrogen dries up and obtains small-molecular micro-array of the present invention.
This small-molecular micro-array mating surface plasma resonance imaging technology is used for detecting small-molecule drug with the interaction of target proteins, has low background signal, and high detection signal, and can carry out real time on-line monitoring high flux.
embodiment 5 uses the detection analysis of embodiment 1 gained small-molecular micro-array
Based on surface plasma resonance imaging technology, compare the detection signal machine background signal of small-molecular micro-array of the present invention (prepared by embodiment 1) and traditional small-molecular micro-array; Detect and use classical Interactions Mode FKBP12 and RAPA(rapamycin), this interacts as known, usually used as positive control.Detailed process is:
(1) be put on SPRi instrument by the traditional small-molecular micro-array chip with RAPA point, adjustment optical position is 12.Traditional small-molecular micro-array chip herein for contrasting is the chip that there is the anti-non-specific adsorption material of brush PEG on surface, carries out, after acidifying, polymer brushes and photocrosslinking agent being coupled together with aminoterminal PEG through succinic anhydride; Again through the array of small molecules of Small molecular point sample, drying, ultraviolet irradiation formation.
(2) point with RAPA and blank point is selected.
(3) pass into the PBST solution of the FKBP12 of 100nm/ml, record combines and dissociation curve.
(4) again small-molecular micro-array of the present invention such as said method is detected, contrast detection signal and background signal.
As shown in Figure 4, Fig. 4 result shows result: from RAPA and FKBP12 interaction situation, compared with traditional small-molecular micro-array chip, and small-molecular micro-array detection signal of the present invention is comparatively strong, and background signal is lower.
Applicant states, the present invention illustrates the present invention by above-described embodiment, but the present invention is not limited to above-mentioned, does not namely mean that the present invention must rely on above-mentioned could enforcement.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 (100)
1. a preparation method for small-molecular micro-array, is characterized in that, comprising:
(1) in chip base, modify the anti-non-specific adsorption material of hydroxyl end;
(2) the Small molecular photocrosslinking agent of band carboxyl terminal and Photocrosslinkable functional group is bonded on the anti-non-specific adsorption material of described hydroxyl end by esterification;
(3) by small molecule solution point sample to step (2) gained chip surface, after drying, carry out photo-crosslinking, obtain small-molecular micro-array chip;
Wherein, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of step (1) resulting tape C-terminal is modified is put into the solution containing described Small molecular photocrosslinking agent, organic alkali catalyst and dewatering agent, reacts 1 ~ 40 hour at 18 ~ 32 DEG C; The mol ratio of the terminal carboxyl group of Small molecular photocrosslinking agent and dewatering agent, organic alkali catalyst is 1:(1 ~ 3): (1 ~ 3).
2. preparation method according to claim 1, it is characterized in that, step (1) described chip base is selected from glass, silicon chip, quartz, high score subclass film, metallic film and metal-oxide film.
3. preparation method according to claim 2, is characterized in that, described high score subclass film is any one in dimethyl silicone polymer, polystyrene, polycarbonate or polymethylmethacrylate.
4. preparation method according to claim 2, it is characterized in that, described metallic film is golden film or silverskin.
5. preparation method according to claim 2, it is characterized in that, described metal-oxide film is di-aluminium trioxide film.
6. preparation method according to claim 1, is characterized in that, the anti-non-specific adsorption material of the described hydroxyl end of step (1) is alkyl sulfhydryl, the macromolecule surface of hydroxyl end or Supramolecular self assembly surface.
7. preparation method according to claim 6, it is characterized in that, described alkyl sulfhydryl is the alkanethiol with polyglycol segment.
8. preparation method according to claim 6, is characterized in that, described macromolecule surface is the amphoteric ion polymer surface of polyglycol or derivatives thereof, the fluoropolymer of hydroxyl end, glucosan, cellulose or derivatives thereof or hydroxyl end.
9. preparation method according to claim 6, is characterized in that, described Supramolecular self assembly surface is poly-rotaxane surface.
10. preparation method according to claim 1, it is characterized in that, the anti-non-specific adsorption material of hydroxyl end described in step (1) is brush PEG.
11. preparation methods according to claim 10, it is characterized in that, the method that chip base is modified brush PEG is:
(1 ') uses the substrate of initiator solution process chip;
(2 ') step (1 ') gained chip is immersed in the solution containing the vinyl monomer of organic reducing agent, polymerisation catalysts and PEG, carries out monomer polymerization reactions under oxygen-free environment, and growth macromolecule, to obtain final product.
12., according to preparation method described in claim 11, is characterized in that, described initiating agent is single sulfydryl halogenated thiols solution.
13., according to preparation method described in claim 12, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM ~ 100mM.
14., according to preparation method described in claim 13, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM ~ 10mM.
15., according to preparation method described in claim 14, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 1mM.
16., according to preparation method described in claim 12, is characterized in that, described single sulfydryl halogenated thiols solution also comprises single mercapto-polyglycol.
17. according to preparation method described in claim 12, it is characterized in that, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively the ethanolic solution of the mono-mercapto-polyglycol of 0.1mM ~ 100mM and single sulfydryl halogenated thiols with (0 ~ 999): the volume ratio Homogeneous phase mixing of 1, is taped against described substrate surface.
18., according to preparation method described in claim 17, is characterized in that, described single mercapto-polyglycol is identical with the concentration of single sulfydryl halogenated thiols and be respectively 0.1mM ~ 10mM.
19., according to preparation method described in claim 18, is characterized in that, described single mercapto-polyglycol is identical with the concentration of single sulfydryl halogenated thiols and be respectively 1mM.
20., according to preparation method described in claim 17, is characterized in that, the volume ratio of the ethanolic solution of described single mercapto-polyglycol and single sulfydryl halogenated thiols is (0 ~ 99): 1.
21., according to preparation method described in claim 20, is characterized in that, the volume ratio of the ethanolic solution of described single mercapto-polyglycol and single sulfydryl halogenated thiols is 99:1.
22., according to preparation method described in claim 11, is characterized in that, described organic reducing agent is glucose, ascorbic acid or stannous octoate.
23., according to preparation method described in claim 11, is characterized in that, the content of described organic reducing agent is 1nM ~ 1mM.
24., according to preparation method described in claim 23, is characterized in that, the content of described organic reducing agent is 1nM ~ 0.1mM.
25., according to preparation method described in claim 24, is characterized in that, the content of described organic reducing agent is 0.04mM.
26., according to preparation method described in claim 11, is characterized in that, described polymerisation catalysts is the potpourri of transition metal salt and sequestrant.
27., according to preparation method described in claim 26, is characterized in that, described transition metal salt is molysite or mantoquita.
28., according to preparation method described in claim 26, is characterized in that, the content of described transition metal salt is 1nM ~ 1mM.
29., according to preparation method described in claim 28, is characterized in that, the content of described transition metal salt is 1nM ~ 0.1mM.
30., according to preparation method described in claim 29, is characterized in that, the content of described transition metal salt is 0.04mM.
31., according to preparation method described in claim 26, is characterized in that, described sequestrant is second bipyridine.
32., according to preparation method described in claim 26, is characterized in that, the content of described sequestrant is 1nM ~ 100mM.
33., according to preparation method described in claim 32, is characterized in that, the content of described sequestrant is 1nM ~ 1mM.
34., according to preparation method described in claim 33, is characterized in that, the content of described sequestrant is 0.8mM.
35., according to preparation method described in claim 11, is characterized in that, the vinyl monomer of described PEG is methacrylic acid macrogol ester.
36., according to preparation method described in claim 11, is characterized in that, the content of the vinyl monomer of described PEG is 1mM ~ 1M.
37., according to preparation method described in claim 36, is characterized in that, the content of the vinyl monomer of described PEG is 1mM ~ 100mM.
38., according to preparation method described in claim 37, is characterized in that, the content of the vinyl monomer of described PEG is 5mM.
39., according to preparation method described in claim 11, is characterized in that, the time of described macromolecule growth is 1 ~ 40 hour.
40., according to preparation method described in claim 39, is characterized in that, the time of described macromolecule growth is 1 ~ 20 hour.
41., according to preparation method described in claim 40, is characterized in that, the time of described macromolecule growth is 18 hours.
42. preparation methods according to claim 1, it is characterized in that, the anti-non-specific adsorption material of hydroxyl end described in step (1) is hyperbranched PEG.
43., according to preparation method described in claim 42, is characterized in that, the method that chip base is modified hyperbranched PEG is:
(1 ") use the substrate of initiator solution process chip;
(2 ") step (1 ") gained chip is immersed in the solution containing the vinyl monomer of organic reducing agent, polymerisation catalysts and PEG, and carry out monomer polymerization reactions under oxygen-free environment, growth macromolecule, then takes out chip;
(3 ") carry out end-blocking with 2-bromine isobutyl acylbromide to the polymkeric substance on step (2 ") gained chip, form macromole evocating agent;
(4 ") repeat step (2 ") and step (3 ") to gained chip, so repeat 3-4 time and get final product.
44., according to preparation method described in claim 43, is characterized in that, described initiating agent is single sulfydryl halogenated thiols solution.
45., according to preparation method described in claim 44, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM ~ 100mM.
46., according to preparation method described in claim 45, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 0.1mM ~ 10mM.
47., according to preparation method described in claim 46, is characterized in that, the concentration of described single sulfydryl halogenated thiols solution is 1mM.
48., according to preparation method described in claim 44, is characterized in that, described single sulfydryl halogenated thiols solution also comprises single mercapto-polyglycol.
49. according to preparation method described in claim 44, it is characterized in that, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively the ethanolic solution of the mono-mercapto-polyglycol of 0.1mM ~ 100mM and single sulfydryl halogenated thiols with (0 ~ 999): the volume ratio Homogeneous phase mixing of 1, is taped against described substrate surface.
50., according to preparation method described in claim 49, is characterized in that, described single mercapto-polyglycol is identical with the concentration of single sulfydryl halogenated thiols and be respectively 0.1mM ~ 10mM.
51., according to preparation method described in claim 50, is characterized in that, described single mercapto-polyglycol is identical with the concentration of single sulfydryl halogenated thiols and be respectively 1mM.
52., according to preparation method described in claim 51, is characterized in that, the volume ratio of the ethanolic solution of described single mercapto-polyglycol and single sulfydryl halogenated thiols is (0 ~ 99): 1.
53., according to preparation method described in claim 52, is characterized in that, the volume ratio of the ethanolic solution of described single mercapto-polyglycol and single sulfydryl halogenated thiols is 99:1.
54., according to preparation method described in claim 43, is characterized in that, described organic reducing agent is glucose, ascorbic acid or stannous octoate.
55., according to preparation method described in claim 43, is characterized in that, the content of described organic reducing agent is 1nM ~ 1mM.
56., according to preparation method described in claim 55, is characterized in that, the content of described organic reducing agent is 1nM ~ 0.1mM.
57., according to preparation method described in claim 56, is characterized in that, the content of described organic reducing agent is 0.04mM.
58., according to preparation method described in claim 43, is characterized in that, described polymerisation catalysts is the potpourri of transition metal salt and sequestrant.
59., according to preparation method described in claim 58, is characterized in that, described transition metal salt is molysite or mantoquita.
60., according to preparation method described in claim 59, is characterized in that, the content of described transition metal salt is 1nM ~ 1mM.
61., according to preparation method described in claim 60, is characterized in that, the content of described transition metal salt is 1nM ~ 0.1mM.
62., according to preparation method described in claim 61, is characterized in that, the content of described transition metal salt is 0.04mM.
63., according to preparation method described in claim 58, is characterized in that, described sequestrant is second bipyridine.
64., according to preparation method described in claim 58, is characterized in that, the content of described sequestrant is 1nM ~ 100mM.
65., according to preparation method described in claim 64, is characterized in that, the content of described sequestrant is 1nM ~ 1mM.
66., according to preparation method described in claim 65, is characterized in that, the content of described sequestrant is 0.8mM.
67., according to preparation method described in claim 43, is characterized in that, the vinyl monomer of described PEG is methacrylic acid macrogol ester.
68., according to preparation method described in claim 43, is characterized in that, the content of the vinyl monomer of described PEG is 1mM ~ 1M.
69., according to preparation method described in claim 68, is characterized in that, the content of the vinyl monomer of described PEG is 1mM ~ 100mM.
70., according to preparation method described in claim 69, is characterized in that, the content of the vinyl monomer of described PEG is 5mM.
71., according to preparation method described in claim 43, is characterized in that, the time of described macromolecule growth is 1 ~ 40 hour.
72., according to preparation method described in claim 43, is characterized in that, the time of described macromolecule growth is 1 ~ 20 hour.
73., according to preparation method described in claim 43, is characterized in that, the time of described macromolecule growth is 6 hours.
74. preparation methods according to claim 1, is characterized in that, the anti-non-specific adsorption material of hydroxyl end described in step (1) is poly-rotaxane.
75., according to preparation method described in claim 74, is characterized in that, the method that chip base is modified described poly-rotaxane is:
Chip base is put into the ethanolic solution of the mercapto-polyglycol of two kinds of different molecular weights by (1 " '), 0 ~ 30 DEG C hatch 1 ~ 12 hour after, clean, dry up;
The mercapto-polyglycol of described two kinds of different molecular weights be respectively molecular weight be 40 ~ 400 C-terminal or methoxyl terminal mercaptan and molecular weight be 1000 ~ 20000 carboxyl terminal mercaptan;
Step (1 " ') gained chip immerses in the aqueous solution of the alpha-cyclodextrin of more than 10mM and hatches 1 ~ 20 hour by (2 " '), then add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and N-hydroxy-succinamide, react 10 ~ 60 minutes;
Step (2 " ') gained chip is put into the saturated alpha-cyclodextrin aqueous solution of the benzyloxycarbonyl group-TYR Z-Tyr-OH containing 1mM ~ 10mM by (3 " '), reacts 1 ~ 2 hour and get final product.
76. preparation methods according to claim 1, it is characterized in that, the anti-non-specific adsorption material of hydroxyl end described in step (1) is glucosan.
77., according to preparation method described in claim 76, is characterized in that, the method that chip base is modified glucosan is:
Concentration is that the C-terminal alkanethiol ethanol of 1mM ~ 100mM and/or aqueous solution are layered on chip surface by (1 " "), and incubated at room was taken out after 1 ~ 20 hour;
Step (1 " ") gained chip is put into the mixed solution of NaOH containing epichlorokydrin and diethylene glycol dimethyl ether by (2 " "), reacts after 1 ~ 10 hour and take out under room temperature;
Step (2 " ") gained chip is put into the sodium hydroxide solution of glucosan by (3 " "), and room temperature reaction is after 0.1 ~ 10 hour and get final product.
78. preparation methods according to claim 1, it is characterized in that, Photocrosslinkable functional group described in step (2) is selected from acetophenone, Benzophenone, quinone anthracene class, aryl azides compound and fragrant azirine.
79., according to preparation method described in claim 78, is characterized in that, described Photocrosslinkable functional group is fragrant azirine.
80. preparation methods according to claim 1, it is characterized in that, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of step (1) resulting tape C-terminal is modified is put into the solution containing described Small molecular photocrosslinking agent, organic alkali catalyst and dewatering agent, reacts 1 ~ 20 hour and obtain at 22 ~ 28 DEG C.
81. preparation methods described in 0 according to Claim 8, it is characterized in that, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of step (1) resulting tape C-terminal is modified is put into the solution containing described Small molecular photocrosslinking agent, organic alkali catalyst and dewatering agent, reacts at 25 DEG C.
82. preparation methods described in 1 according to Claim 8, it is characterized in that, step (2) specifically comprises: reacted 18 hours by the solution that the chip that the anti-non-specific adsorption material of step (1) resulting tape C-terminal is modified is put into containing described Small molecular photocrosslinking agent, organic alkali catalyst and dewatering agent.
83. preparation methods according to claim 1, it is characterized in that, the solvent of described solution is organic solvent.
84. preparation methods described in 3 according to Claim 8, it is characterized in that, described organic solvent is selected from methylene chloride, tetrahydrofuran and DMF.
85. preparation methods according to claim 1, it is characterized in that, described organic alkali catalyst is selected from DMAP, DIPEA and triethylamine.
86. preparation methods according to claim 1, it is characterized in that, described dewatering agent is selected from 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide and dicyclohexylcarbodiimide.
87. preparation methods according to claim 1, is characterized in that, the mol ratio of the terminal carboxyl group of described solution small molecular photocrosslinking agent and dewatering agent, organic alkali catalyst is 1:(1 ~ 2): (1 ~ 2).
88. preparation methods described in 7 according to Claim 8, is characterized in that, the mol ratio of the terminal carboxyl group of described solution small molecular photocrosslinking agent and dewatering agent, organic alkali catalyst is 1:1.5:1.5.
89. preparation methods according to claim 1, it is characterized in that, the end carboxyl group content of described solution small molecular photocrosslinking agent is 1nM ~ 100mM.
90. preparation methods described in 9 according to Claim 8, it is characterized in that, the end carboxyl group content of described solution small molecular photocrosslinking agent is 1mM ~ 100mM.
91., according to preparation method described in claim 90, is characterized in that, the end carboxyl group content of described solution small molecular photocrosslinking agent is 10mM.
92. preparation methods according to claim 1, it is characterized in that, step (3) described Small molecular is small-molecule drug or the Small molecular having potential medical value.
93., according to preparation method described in claim 92, is characterized in that, the condition of described photo-crosslinking is: be 0.1 ~ 100J/cm in light intensity
2, wavelength is irradiate 1 ~ 30 minute under the ultraviolet light of 200 ~ 400nm.
94., according to preparation method described in claim 93, is characterized in that, the light intensity of described photo-crosslinking is 0.1 ~ 10J/cm
2.
95., according to preparation method described in claim 94, is characterized in that, the light intensity of described photo-crosslinking is 1J/cm
2.
96., according to preparation method described in claim 93, is characterized in that, the wavelength of described photo-crosslinking is 300 ~ 400nm.
97., according to preparation method described in claim 96, is characterized in that, the wavelength of described photo-crosslinking is 365nm.
98., according to preparation method described in claim 93, is characterized in that, the irradiation time of described photo-crosslinking is 1 ~ 20 minute.
99., according to preparation method described in claim 98, is characterized in that, the irradiation time of described photo-crosslinking is 10 minutes.
100. one kinds of small-molecular micro-arrays, is characterized in that, are obtained by preparation method described in any one of claim 1-99.
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| CN104897617A (en) * | 2015-05-19 | 2015-09-09 | 国家纳米科学中心 | Micro-array biochip, and preparation method and application of micro-array biochip |
| CN105137064B (en) * | 2015-07-24 | 2016-11-30 | 中国人民大学 | A kind of method of small organic molecule modified biological sensing element |
| CN105067821B (en) * | 2015-08-10 | 2017-02-22 | 国家纳米科学中心 | Method for realizing target fishing and characterization through small molecule microarray |
| CN105548577B (en) * | 2016-02-04 | 2017-09-19 | 安徽医科大学 | A kind of Lyme disease detection protein-chip of succinyl beta cyclodextrin modification and its preparation and application |
| CN107202893B (en) * | 2016-03-16 | 2019-08-13 | 广州高通生物技术有限公司 | A kind of small molecule chip, its construction method, its application and its detection method |
| CN107202892B (en) * | 2016-03-16 | 2019-06-04 | 广州高通生物技术有限公司 | A kind of small molecule chip, preparation method and application |
| CN106039320A (en) * | 2016-05-25 | 2016-10-26 | 广州高通生物技术有限公司 | Anti-nonspecific capture magnetic bead, and preparation method and application thereof |
| CN106117453A (en) * | 2016-06-08 | 2016-11-16 | 广州高通生物技术有限公司 | A kind of macromolecule mother solution and the manufacture method of 3D biochip |
| CN106990079B (en) * | 2017-03-20 | 2019-06-25 | 中国科学院化学研究所 | A kind of surface multifunctional coating and the preparation method and application thereof |
| CN108722506B (en) * | 2018-05-29 | 2020-05-19 | 北京化工大学 | Method for controlling hydrophilization modification effect inside micro-fluidic chip |
| CN113945653B (en) * | 2021-09-27 | 2023-08-18 | 深圳职业技术学院 | Chip type living body solid phase microextraction device system and analysis method thereof |
| CN115677922B (en) * | 2022-09-28 | 2024-01-26 | 深圳市曙芯生物科技有限公司 | Dual-functional polymer and modification method |
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