CN103792345A - Small-molecule microarray and preparation method thereof - Google Patents
Small-molecule microarray and preparation method thereof Download PDFInfo
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Images
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
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Dispersion Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a preparation method for a small-molecule microarray. The preparation method comprises the steps of bonding the hydroxyl terminal of a nonspecific adsorption resistance material modified on a chip substrate with the hydroxyl terminal of a small-molecule light crosslinking agent through an esterification reaction, and randomly fixing small molecules on the hydroxyl terminals through light crosslinking. By the adoption of a direct esterification mode, the small-molecule light crosslinking agent is quickly and efficiently coupled to the surface of the nonspecific adsorption resistance material; a chip modification process is simple and convenient and can be implemented under a normal temperature condition; therefore, the modification cost is reduced, and the modification efficiency is improved; a high detection signal is guaranteed; a non-reacted group still keeps high nonspecific adsorption resistance capacity, so that a background signal is reduced; the prepared small-molecule microarray is high in detection signal and low in background signal and can be used for mutual action between high-flux screened small-molecule medicaments and targets.
Description
Technical field
The present invention relates to biochip technology field, relate in particular 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 developing rapidly nearly ten years.Then small-molecular micro-array refers on solid state surface fixes by point sample (or printing) high-density micro-array that each organic micromolecule forms---be similar to pandemic DNA microarray, on a glass (or plastics) substrate, can print the microarray of up to ten thousand different compounds, have benefited from the microminiaturization of analytical instrument, can analyze thousands of biochemical interactions disposable, single step simultaneously.1999, the reported first such as Schreiber little molecular array, this array is used 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 a transcription factor using small molecule microarrays and diversity-oriented synthesis.Journal of the American Chemical Society, 125 (28): pp.8420-8421.2003.).In the last few years, little molecular array had been successfully applied at key areas such as analysis of protein and lead compound exploitations.
Because small-molecular micro-array need to be fixed on carrier surface by the little molecule of a large amount of, different chemical structures or biochemical structure, how to fix expeditiously little molecule, and do not affect little molecule and be combined biochemical activity with albumen target, be the most challenging difficult problem of small-molecular micro-array (SMM) technology always.The process for fixation that emerge in large numbers nearly ten years a variety of convenience, stablize, cost is low, wherein, firmly fixes by covalent bond the main flow that Small-molecule probe is little molecule 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 to design coupling agent end according to difference, in order to the fixing little molecule with special groups.But the technology path of the fixing Small-molecule probe of selectivity or directionality has the defect of essence: the avtive spot of Small-molecule probe is probably fixed, thereby lose and the biochemical activity of target effect, may leak the reactive compound that sieve is good.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, there is revolutionary meaning for improving drug screening and research and development ability, therefore, therefore the research of association area become a study hotspot.
In numerous progress, especially with trifluoromethyl azirine (Trifluoromethyl aryldiazirine, TFMAD) functional group is the basic photo-crosslinking surface chemistry (Kanoh that attracts most attention, N., et al., Immobilization of Natural Products on Glass Slides by Using a Photoaffinity Reaction and the Detection of Protein – Small-Molecule Interactions.Angewandte Chemie, 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 producing has high reaction activity, resets probability low, and carbon inserts reaction yield advantages of higher.What is more important, only needs relatively low-energy 365nm ultraviolet light just can excite the generation of Cabbeen, thereby has reduced Small-molecule probe by the probability of ultraviolet degradation.TFMAD photochemical fixation mild condition, controllability are strong, can in appointed area, modify by mask, can be widely used in the fixing of different kind organism molecule.In addition, carry out photo-crosslinking and can avoid causing that molecule specific orientation and functional group select by solvent under solid phase condition, molecule contacts and is guaranteed with the randomness of azo.
2006, the abundant grade in long field of Japanese RIKEN was reported non-selection photo-crosslinking small-molecular micro-array (Naoki Kanoh, the Motoki Kyo based on TFMAD, Kazuki Inamori, Ami Ando, Aya Asami, Aiko Nakao, and Hiroyuki Osada, Analytical Chemistry, Vol.78, No.7, April1,2006).In this work, they have prepared the small-scale array of having 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 aspect flux and signal intensity aspect all exist very large deficiency.In recent years, there is again the small-molecular micro-array based on three-dimensional polyethylene glycol polymer brush shape surface and photocrosslinking agent combination, this microarray has been broken through two-dimensional surface in the past, utilize surperficial initiated polymerization technology in substrate grown polymer brush shape structure, thereby utilize the non-selective fixing small-molecule drug of highdensity end group fixed light crosslinking chemical, 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 exist certain defect---the problem of non-specific adsorption.
Non-specific adsorption is a bottleneck of biochip applications always, and the high background signal being caused by non-specific adsorption can produce and disturb detection signal, especially fatal especially for small-molecular micro-array.In classic method, utilize carboxymethylated polymethyl acid polyethylene glycol surface coupling diamido end PEG, then utilize EDC and N-hydroxy-succinamide (EDC/NHS) activation end, final and photocrosslinking agent coupling.Such three-step reaction, ultimate yield is the product of every step reaction yield, causes ultimate yield significantly to decline, thereby micromolecular fixed amount is also diminished, and can cause detection signal to reduce; Meanwhile, the carboxylated end that has neither part nor lot in reaction can allow surface detecting in solution with a certain amount of electric charge, easily causes Electrostatic Absorption, has increased the source of non-specific adsorption.So the ability of the anti-non-specific adsorption of retention surface, increases little molecular array detection signal and becomes the target that researcher pursues simultaneously.
Summary of the invention
The object of the invention is to small-molecular micro-array proposing a kind of high detection signal, low detection background and preparation method thereof.The preparation method of small-molecular micro-array of the present invention adopts single stage method, fast and efficiently little molecule photocrosslinking agent is coupled to the surface of anti-non-specific adsorption material, chip modification is simple and convenient, normal temperature condition can complete, not only save modification cost but also improved modification efficiency, guaranteed 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 little molecule photocrosslinking agent with carboxyl terminal and Photocrosslinkable functional group is bonded to by esterification on the anti-non-specific adsorption material of described hydroxyl end;
(3) little molecular solution point sample is arrived to step (2) gained chip surface, after being dried, carry out photo-crosslinking, obtain small-molecular micro-array chip.
As preferably, the described chip base of step (1) 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 macromolecule surface that the anti-non-specific adsorption material of the described hydroxyl end of step (1) is alkyl sulfhydryl, hydroxyl end or supermolecule self assembly surface;
Preferably, described alkyl sulfhydryl is the alkanethiol with polyglycol segment;
Preferably, described macromolecule surface is the amphoteric ion polymer surface of fluoropolymer, glucosan, cellulose or derivatives thereof or the hydroxyl end of polyglycol or derivatives thereof, hydroxyl end;
Preferably, described supermolecule self assembly surface is poly-rotaxane surface.
About the concrete mode of modifying the anti-non-specific adsorption material of hydroxyl end step (1) Suo Shu in chip base, can use all modes well known by persons skilled in the art.Common modification mode mainly contains following three kinds: 1, at surperficial initiated polymerization, first spread initiating agent at substrate surface, then trigger monomer is at surface aggregate, resists nonspecific surface thereby obtain; 2. surface grafting, first obtains and has the macromolecular material of anti-non-specific adsorption character, then be connected to surface by specific radical reaction; 3. carry out supermolecule self assembly on surface, first modify chain host molecule at substrate surface, then guest molecule is assembled into surface, finally form supermolecule self-assembly.
In a specific embodiments, in step (1), the anti-non-specific adsorption material of described hydroxyl end is brush shape PEG; The method of modifying brush shape PEG in chip base is:
(1 ') used the substrate of initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halo thiol solution; Further preferably, the concentration of described single sulfydryl halo thiol solution is 0.1～100mM, preferably 0.1mM～10mM, more preferably 1mM; Still more preferably, described single sulfydryl halo thiol solution also comprises single mercapto-polyglycol;
In specific embodiment, the concentration of described single sulfydryl halo thiol 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 0.1mM～10mM, more preferably single mercapto-polyglycol of 1mM and the ethanolic solution of single sulfydryl halo mercaptan, with (0～999): 1, preferably (0～99): 1, more preferably the volume ratio of 99:1 is evenly mixed, and is taped against described substrate surface;
In specific embodiment, the concentration of the ethanolic solution of described single mercapto-polyglycol and the ethanolic solution of single sulfydryl halo mercaptan is identical 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, 0.04mM more preferably;
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, 0.04mM more preferably; The content of described sequestrant is 1nM～100mM, be preferably 1nM～1mM, 0.8mM more preferably;
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, 5mM more preferably;
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; The method of preferably, modifying hyperbranched PEG in chip base is:
(1 ' ') substrate of use initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halo thiol solution; Further preferably, the concentration of described single sulfydryl halo thiol solution is 0.1～100mM, preferably 0.1～10mM, more preferably 1mM; Still more preferably, described single sulfydryl halo thiol solution also comprises single mercapto-polyglycol;
In specific embodiment, the concentration of described single sulfydryl halo thiol 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 0.1mM～10mM, more preferably single sulfydryl halo mercaptan of 1mM and the ethanolic solution of single mercapto-polyglycol, with (0～999): 1, preferably (0～99): 1, more preferably the volume ratio of 99:1 is evenly mixed, and is taped against described substrate surface;
In specific embodiment, the concentration of the ethanolic solution of described single mercapto-polyglycol and the ethanolic solution of single sulfydryl halo mercaptan is identical 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 under oxygen-free environment, then takes out chip;
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, 0.04mM more preferably;
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, 0.04mM more preferably; 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, 5mM more preferably;
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, in chip base, the method for the poly-rotaxane of modification is:
(1 ' ' ') puts into chip base the ethanolic solution of the mercapto-polyglycol of two kinds of different molecular weights, hatches after 1～12 hour for 0～30 ℃, cleans, dries up;
It is the carboxyl terminal mercaptan that 40～400 C-terminal or methoxyl end mercaptan and molecular weight are 1000～20000 that the mercapto-polyglycol of described two kinds of different molecular weights is respectively molecular weight;
(2 ' ' ') immerses step (1 ' ' ') gained chip in the aqueous solution of alpha-cyclodextrin more than 10mM and hatches 1～20 hour, 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) that contains 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; The method of preferably, modifying glucosan in chip base is:
C-terminal alkanethiol ethanol and/or aqueous solution that (1 ' ' ' ') is 1～100mM by concentration are layered on chip surface, and incubated at room was taken out after 1～20 hour;
(2 ' ' ') puts into step (1 ' ' ' ') gained chip containing the NaOH of epichlorokydrin and the mixed solution of diethylene glycol dimethyl ether, 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.
In above-mentioned preparation method, as preferably, in step (2), described Photocrosslinkable functional group is selected from acetophenone, Benzophenone, quinone anthracene class, fragrant azide and fragrant azirine, is preferably fragrant azirine;
Preferably, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of the hydroxyl end of step (1) gained is modified is put into the solution containing described little molecule photocrosslinking agent, organic alkali catalyst and dewatering agent, at 18～32 ℃, preferably at 22～28 ℃, more preferably react at 25 ℃ 1～40 hour, 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 little molecule 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 terminal carboxyl group content of little molecule photocrosslinking agent is 1nM～100mM, be preferably 1mM～100mM, 10mM more preferably.
The described esterification of step (2) is the esterification based on solid-liquid interface, by adjusting the conditions such as reaction mass proportioning, reaction time, the grafting amount of little molecule photocrosslinking agent at chip surface be can control, thereby relatively high detection signal and lower background signal guaranteed to obtain.
In specific embodiments, the described little molecular solution of step (3) can be arbitrarily small molecule, for example, be small-molecule drug or the little molecule that has 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, 1J/cm more preferably
2, wavelength be 200～400nm, preferably 300～400nm, more preferably under the ultraviolet light of 365nm, irradiate 1～30 minute, preferably 1～20 minute, more preferably 10 minutes.
Second aspect, the invention provides a kind of small-molecular micro-array, is made by preparation method described in first aspect any one.
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 carboxyl terminal of the C-terminal of the anti-non-specific adsorption material of modifying in chip base and little molecule photocrosslinking agent is carried out to bonding, then by photo-crosslinking, little molecule is fixed thereon at random, thereby the small-molecular micro-array that obtains the three-dimensional surface of high detection signal, low detection background, utilizes high flux detection technique to carry out small-molecule drug with the interactional screening of target then.
The outstanding advantage of three-dimensional surface small-molecular micro-array manufacture method that small-molecular micro-array preparation method of the present invention is more traditional is, adopt single stage method, fast and efficiently little molecule 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, improved again modification efficiency, guaranteed higher detection signal; Unreacted group still keeps higher anti-non-specific adsorption ability, and detection background is reduced; The surface of modifying through direct esterification method keeps electroneutral surface nature, and surface nature homogeneous.
Accompanying drawing explanation
Fig. 1 is preparation method's schematic diagram of small-molecular micro-array of the present invention;
Fig. 2 is the forward and backward x-ray photoelectron power spectrum contrast of three-dimensional PEG macromolecule surface esterification coupling photocrosslinking agent;
Fig. 3 is the forward and backward FT-IR collection of illustrative plates contrast of three-dimensional PEG macromolecule surface esterification coupling photocrosslinking agent;
Fig. 4 is detection signal and the background intensity contrast of the microarray prepared of small-molecular micro-array of the present invention and classic method in the time that surface plasma resonance detects.
Embodiment
Further illustrate technical scheme of the present invention below by embodiment.
embodiment 1 brushes the preparation of shape PEG surface small-molecular micro-array
Concrete steps:
(1) in substrate of glass, prepare the chromium layer of one deck 3.5nm thickness and the gold layer of one deck 50nm thickness by the method for hot evaporation, as the substrate of biochip.
(2) single sulfydryl halo thiol solution that preparation 400 μ L concentration are 1mM is as initiating agent, and single mercapto-polyglycol of preparation 400 μ L same concentrations, mixes two kinds of solution in 1:99 ratio, be taped against chip surface, under 4 ℃ of environment, hatches 12 hours.
(3) gold plaque is alternately cleaned up with ethanol and deionized water.
(4) preparation monomer solution: altogether 20mL of monomer solution, 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 is immersed in the monomer solution of preparation, is placed on the macromolecule of growing under oxygen-free environment, growth time 18 hours, regulates the thickness of polymeric membrane by controlling growth time.
(6) reach after predetermined growth time, gold plaque is taken out and cleaned.
(7) the required photocrosslinking agent solution of position esterification: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(8) chip is immersed in esterification solution, pack suitable vessel into, sealing, hatches under room temperature environment 18 hours.Reach after the schedule time, chip is taken out, alternately rinse well with second alcohol and water, nitrogen dries up.
(9) will be dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip preparing, chip is irradiated to 10min under 365nm ultraviolet light after to be dried, light intensity is 1J/cm
2, reach after the schedule time chip is taken out, 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 detect respectively forward and backward x-ray photoelectron power spectrum and the FT-IR collection of illustrative plates of the little molecule photocrosslinking agent of chip macromolecule surface esterification coupling, respectively as shown in Figure 2 and Figure 3.
As seen from Figure 2, there is variation in the forward and backward x-ray photoelectron power spectrum of esterification, there are F and two kinds of atoms of N owing to containing in photocrosslinking agent molecule, so there are F and two kinds of elements of N of not having on original surface in the elemental map after esterification, esterification process success is described, and can be by ultimate analysis, judge surperficial photocrosslinking agent grafting degree.
As seen from Figure 3, before esterification, after (a) and esterification there is certain difference in the infared spectrum of (b).Sxemiquantitative ground, in the collection of illustrative plates after esterification, 1732cm
-1the stretching vibration peak grow of the C=O of place, 3467cm
-1the stretching vibration peak of the O-H of place weakens, and this is because esterification makes surface dewatering condensation form ester carbonyl group, and has reduced 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 on surface and have the coupling of photocrosslinking agent molecule to get on; In addition, also difference to some extent of the fingerprint region in the forward and backward infared spectrum of esterification, this is the chemical bond that contains specific groups due to photocrosslinking agent, 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 the interaction of small-molecule drug with target proteins, has lower background signal, and higher detection signal.
the preparation of the hyperbranched PEG of embodiment 2 surface small-molecular micro-array
Concrete steps are as follows:
(1) prepare substrate of glass chip, after chip cleaning is dried up, be paved with and contain a certain amount of initiator solution.
(2) reach after predetermined reaction time glass is taken out, clean.
(3) preparation monomer solution: altogether 20mL of monomer solution, 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 is immersed in the monomer solution of preparation, is placed on the macromolecule of growing under oxygen-free environment, growth time 6 hours, regulates the thickness of polymeric membrane by controlling growth time.
(5) reach after predetermined growth time, gold plaque is taken out, alternately rinse well with second alcohol and water.
(6) chip is put into the mixed solution of 1.5mL pyridine and 50mL dry ether, the diethyl ether solution 30mL of the 2-bromine isobutyl acylbromide (BiBB) that dropping contains 10% volume ratio, stirs 2 hours at 0 ℃, more at room temperature reacts 10 hours, then clean with ethanol and ultrapure water, nitrogen dries up.
(7) repeating step 4～7, repeats, after 3～4 times, to obtain hyperbranched polyglycol C-terminal surface.
(8) the required photocrosslinking agent solution of position esterification: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(9) chip is immersed in esterification solution, pack suitable vessel into, sealing, hatches under room temperature environment 18 hours.Reach after the schedule time, chip is taken out, alternately rinse well with second alcohol and water, nitrogen dries up.
(10) will be dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip preparing, chip is irradiated 10 minutes under 365nm ultraviolet light after to be dried, light intensity is 1J/cm
2, reach after the schedule time chip is taken out, 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.
the three-dimensional poly-rotaxane of embodiment 3 surface small-molecular micro-array
(1), as the step 1 in embodiment 1, prepare the substrate of gold plaque as biochip.
(2) two kinds of concentration of preparation are the ethanolic solution of the mercapto-polyglycol of 1mM, different molecular weight, and one is HS-(CH
2)
11-EG6, another kind is HS-EGn-COOH(molecular weight 2000).Two kinds of solution are pressed to 10:1 volume ratio and mix, then chip is put into mixed solution, hatch 12 hours at 4 ℃.Reach after the schedule time, after taking out, ethanol and deionized water alternately clean, and nitrogen dries up.
(3) chip is immersed in to (20mL) in the aqueous solution of 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) that contains 3.2mM, then react 2 hours, rear with ultrapure water cleaning, nitrogen dries up.
(5) the required photocrosslinking agent solution of position esterification: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(6) chip is immersed in esterification solution, pack suitable vessel into, sealing, hatches under room temperature environment 18 hours.Reach after the schedule time, chip is taken out, alternately rinse well with second alcohol and water, nitrogen dries up.
(7) will be dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip preparing, chip is irradiated 10 minutes under 365nm ultraviolet light after to be dried, light intensity is 1J/cm
2, reach after the schedule time chip is taken out, 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 detecting online the interaction of 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 glucosan surface small-molecular micro-arrays
(1), as the step 1 in embodiment 1, prepare the substrate of gold plaque as biochip.
(2) compound concentration is 1mM C-terminal alkanethiol (HS-(CH
2)
11-EG3) ethanolic solution, solution is layered on to chip surface, hatch 12 hours at 4 ℃.After reaching the time, take out, 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) mix 0.4M sodium hydroxide solution and diethylene glycol dimethyl ether, chip is put into solution and soaks, at room temperature react 4 hours.After reaction, alternately rinse with second alcohol and water.
(4) preparation dextran solution (3g Dextran T 500 is dissolved in the sodium hydroxide solution of 0.1M of 10ml), is immersed in chip in dextran solution room temperature reaction 20 hours.After reaching predetermined reaction time, chip is taken out, ultrapure water cleans, and nitrogen dries up.
(5) the required photocrosslinking agent solution of position esterification: solvent is N, dinethylformamide (10mL), photocrosslinking agent TFMAD10mM, dewatering agent EDC (EDC) 15mM, catalyzer DMAP (DMAP) 15mM in reactant liquor.
(6) chip is immersed in esterification solution, pack suitable vessel into, sealing, hatches under room temperature environment 18 hours.Reach after the schedule time, chip is taken out, alternately rinse well with second alcohol and water, nitrogen dries up.
(7) will be dissolved with micromolecular dimethyl sulphoxide solution point sample on the chip preparing, chip is irradiated 10 minutes under 365nm ultraviolet light after to be dried, light intensity is 1J/cm
2, reach after the schedule time chip is taken out, with the cleaning that hockets of 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 the interaction of small-molecule drug with 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, the relatively detection signal machine background signal of small-molecular micro-array of the present invention (embodiment 1 is prepared) and traditional small-molecular micro-array; Detect and use classical Interactions Mode FKBP12 and RAPA(rapamycin), this interacts for known, usually used as positive control.Detailed process is:
(1) traditional small-molecular micro-array chip of ordering with RAPA is put on SPRi instrument, adjusting optical position is 12.Be the chip that there is brush shape PEG anti-non-specific adsorption material on surface for the traditional small-molecular micro-array chip contrasting herein, after succinic anhydride carries out acidifying, polymer brushes and photocrosslinking agent coupled together with aminoterminal PEG; Again through too small molecule point sample, the little molecular array that dry, ultraviolet irradiation forms.
(2) select point and the blank point with RAPA.
(3) pass into the PBST solution of the FKBP12 of 100nm/ml, record combination and dissociation curve.
(4) again small-molecular micro-array of the present invention is detected to contrast detection signal and background signal as said method.
As shown in Figure 4, Fig. 4 result shows result: from RAPA and FKBP12 interaction situation, compared with traditional small-molecular micro-array chip, small-molecular micro-array detection signal of the present invention is stronger, and background signal is lower.
Applicant statement, the present invention illustrates the present invention by above-described embodiment, but that the present invention is not limited to is above-mentioned, does not 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, and the selections of the equivalence replacement to the selected raw material of the present invention and the interpolation of auxiliary element, concrete mode etc., within all dropping on protection scope of the present invention and open scope.
Claims (10)
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 little molecule photocrosslinking agent with carboxyl terminal and Photocrosslinkable functional group is bonded to by esterification on the anti-non-specific adsorption material of described hydroxyl end;
(3) little molecular solution point sample is arrived to step (2) gained chip surface, after being dried, carry out photo-crosslinking, obtain small-molecular micro-array chip.
2. preparation method according to claim 1, is characterized in that, the described chip base of step (1) 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.
3. according to preparation method described in claim 1 or 2, it is characterized in that the macromolecule surface that the anti-non-specific adsorption material of the described hydroxyl end of step (1) is alkyl sulfhydryl, hydroxyl end or supermolecule self assembly surface;
Preferably, described alkyl sulfhydryl is the alkanethiol with polyglycol segment;
Preferably, described macromolecule surface is the amphoteric ion polymer surface of fluoropolymer, glucosan, cellulose or derivatives thereof or the hydroxyl end of polyglycol or derivatives thereof, hydroxyl end;
Preferably, described supermolecule self assembly surface is poly-rotaxane surface.
4. according to preparation method described in claim 1-3 any one, it is characterized in that, in step (1), the anti-non-specific adsorption material of described hydroxyl end is brush shape PEG;
Preferably, in chip base, the method for modification brush shape PEG is:
(1 ') used the substrate of initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halo thiol solution; Further preferably, the concentration of described single sulfydryl halo thiol solution is 0.1～100mM, preferably 0.1mM～10mM, more preferably 1mM; Still more preferably, described single sulfydryl halo thiol solution also comprises single mercapto-polyglycol;
Preferably, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively 0.1～100mM, preferably 0.1mM～10mM, more preferably single mercapto-polyglycol of 1mM and the ethanolic solution of single sulfydryl halo mercaptan, with (0～999): 1, preferably (0～99): 1, more preferably the volume ratio of 99:1 is evenly mixed, and is taped against described substrate surface;
(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, 0.04mM more preferably;
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, 0.04mM more preferably; The content of described sequestrant is 1nM～100mM, be preferably 1nM～1mM, 0.8mM more preferably;
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, 5mM more preferably;
Preferably, the time of described macromolecule growth is 1～40 hour, preferably 1～20 hour, more preferably 18 hours.
5. according to preparation method described in claim 1-3 any one, it is characterized in that, in step (1), the anti-non-specific adsorption material of described hydroxyl end is hyperbranched PEG;
The method of preferably, modifying hyperbranched PEG in chip base is:
(1 ' ') substrate of use initiator solution process chip;
Preferably, described initiating agent is single sulfydryl halo thiol solution; Further preferably, the concentration of described single sulfydryl halo thiol solution is 0.1～100mM, preferably 0.1～10mM, more preferably 1mM; Still more preferably, described single sulfydryl halo thiol solution also comprises single mercapto-polyglycol;
Preferably, use the method for initiator solution process chip substrate as follows: by same concentrations and be respectively 0.1～100mM, preferably 0.1mM～10mM, more preferably single mercapto-polyglycol of 1mM and the ethanolic solution of single sulfydryl halo mercaptan, with (0～999): 1, preferably (0～99): 1, more preferably the volume ratio of 99:1 is evenly mixed, and is taped against described substrate surface;
(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, then takes out chip;
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, 0.04mM more preferably;
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, 0.04mM more preferably; The content of described sequestrant is 1nM～100mM, be preferably 1nM～1mM, 0.8mM more preferably;
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, 5mM more preferably;
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.
6. according to preparation method described in claim 1-3 any one, it is characterized in that, in step (1), the anti-non-specific adsorption material of described hydroxyl end is poly-rotaxane;
Preferably, in chip base, the method for the poly-rotaxane of modification is:
(1 ' ' ') puts into chip base the ethanolic solution of the mercapto-polyglycol of two kinds of different molecular weights, hatches after 1～12 hour for 0～30 ℃, cleans, dries up;
It is the carboxyl terminal mercaptan that 40～400 C-terminal or methoxyl end mercaptan and molecular weight are 1000～20000 that the mercapto-polyglycol of described two kinds of different molecular weights is respectively molecular weight;
(2 ' ' ') immerses step (1 ' ' ') gained chip in the aqueous solution of alpha-cyclodextrin more than 10mM and hatches 1～20 hour, 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) that contains 1～10mM by (3 ' ' '), reacts 1～2 hour and get final product.
7. according to preparation method described in claim 1-3 any one, it is characterized in that, in step (1), the anti-non-specific adsorption material of described hydroxyl end is glucosan;
The method of preferably, modifying glucosan in chip base is:
C-terminal alkanethiol ethanol and/or aqueous solution that (1 ' ' ' ') is 1～100mM by concentration are layered on chip surface, and incubated at room was taken out after 1～20 hour;
(2 ' ' ') puts into step (1 ' ' ' ') gained chip containing the NaOH of epichlorokydrin and the mixed solution of diethylene glycol dimethyl ether, 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.
8. according to preparation method described in claim 1-7 any one, it is characterized in that, in step (2), described Photocrosslinkable functional group is selected from acetophenone, Benzophenone, quinone anthracene class, fragrant azide and fragrant azirine, is preferably fragrant azirine;
Preferably, step (2) specifically comprises: the chip that the anti-non-specific adsorption material of the hydroxyl end of step (1) gained is modified is put into the solution containing described little molecule photocrosslinking agent, organic alkali catalyst and dewatering agent, at 18～32 ℃, preferably at 22～28 ℃, more preferably react at 25 ℃ 1～40 hour, 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 little molecule 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 terminal carboxyl group content of little molecule photocrosslinking agent is 1nM～100mM, be preferably 1mM～100mM, 10mM more preferably.
9. according to preparation method described in claim 1-8 any one, it is characterized in that, the described little molecule of step (3) is small-molecule drug or the little molecule that has 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, 1J/cm more preferably
2, wavelength be 200～400nm, preferably 300～400nm, more preferably under the ultraviolet light of 365nm, irradiate 1～30 minute, preferably 1～20 minute, more preferably 10 minutes.
10. a small-molecular micro-array, is characterized in that, is made by preparation method described in claim 1-9 any one.
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