CN109609333B - Biological chip and preparation method thereof - Google Patents
Biological chip and preparation method thereof Download PDFInfo
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- CN109609333B CN109609333B CN201811191571.2A CN201811191571A CN109609333B CN 109609333 B CN109609333 B CN 109609333B CN 201811191571 A CN201811191571 A CN 201811191571A CN 109609333 B CN109609333 B CN 109609333B
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- 238000012986 modification Methods 0.000 claims abstract description 112
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 111
- 230000004048 modification Effects 0.000 claims abstract description 111
- 150000003384 small molecules Chemical class 0.000 claims abstract description 106
- 238000006243 chemical reaction Methods 0.000 claims abstract description 91
- 238000000018 DNA microarray Methods 0.000 claims abstract description 83
- 239000000758 substrate Substances 0.000 claims abstract description 72
- 238000001179 sorption measurement Methods 0.000 claims abstract description 65
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 42
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
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- 238000002161 passivation Methods 0.000 claims description 123
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 14
- 235000013922 glutamic acid Nutrition 0.000 claims description 14
- 239000004220 glutamic acid Substances 0.000 claims description 14
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 7
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- 238000003556 assay Methods 0.000 claims description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 2
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 claims description 2
- ZLDPNFYTUDQDMJ-UHFFFAOYSA-N n-octadecyloctadecan-1-amine;hydrobromide Chemical compound Br.CCCCCCCCCCCCCCCCCCNCCCCCCCCCCCCCCCCCC ZLDPNFYTUDQDMJ-UHFFFAOYSA-N 0.000 claims description 2
- QBVXKDJEZKEASM-UHFFFAOYSA-M tetraoctylammonium bromide Chemical compound [Br-].CCCCCCCC[N+](CCCCCCCC)(CCCCCCCC)CCCCCCCC QBVXKDJEZKEASM-UHFFFAOYSA-M 0.000 claims description 2
- MQAYPFVXSPHGJM-UHFFFAOYSA-M trimethyl(phenyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)C1=CC=CC=C1 MQAYPFVXSPHGJM-UHFFFAOYSA-M 0.000 claims description 2
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- -1 small molecule compound Chemical class 0.000 description 8
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 7
- 230000035484 reaction time Effects 0.000 description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 5
- 238000007385 chemical modification Methods 0.000 description 5
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- SNKZJIOFVMKAOJ-UHFFFAOYSA-N 3-Aminopropanesulfonate Chemical compound NCCCS(O)(=O)=O SNKZJIOFVMKAOJ-UHFFFAOYSA-N 0.000 description 4
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- 125000000542 sulfonic acid group Chemical group 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- YDYSEBSNAKCEQU-UHFFFAOYSA-N 2,3-diamino-n-phenylbenzamide Chemical compound NC1=CC=CC(C(=O)NC=2C=CC=CC=2)=C1N YDYSEBSNAKCEQU-UHFFFAOYSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- NHAWMXFBSOSFJK-BXRBKJIMSA-N OC(=O)[C@@H](N)COP(O)(O)=O.OC(=O)[C@@H](N)COP(O)(O)=O Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O.OC(=O)[C@@H](N)COP(O)(O)=O NHAWMXFBSOSFJK-BXRBKJIMSA-N 0.000 description 2
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- RSDQBPGKMDFRHH-UHFFFAOYSA-N Taurin Natural products C1CC2(C)C(=O)CCC(C)=C2C2C1C(C)C(=O)O2 RSDQBPGKMDFRHH-UHFFFAOYSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
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Abstract
The application discloses a biochip and a preparation method thereof. The biochip is a DNA chip or a protein chip, the surface of a substrate of the biochip is provided with an anti-nonspecific adsorption layer formed by combining a small molecule modification reagent on the surface of the substrate, and the small molecule modification reagent is at least one of compounds of a general formula shown in a formula I; in the formula I, R1、R2And R3Are respectively selected from hydrophilic groups or hydrogen atoms. According to the biochip, a compound of the formula is used as a small molecule modification reagent to form the nonspecific adsorption resistance layer, the adsorption layer can effectively prevent nonspecific adsorption of nucleotide used for sequencing, the characteristic detection signal ratio is improved, nonspecific signal interference is reduced, and the nonspecific adsorption resistance layer of the biochip is high in connection strength and not easy to fall off; moreover, the small molecule modification reagent has controllable reaction and good repeatability, so that the biochip has high quality, good repeatability and high availability, and is particularly suitable for single molecule detection.
Description
Technical Field
The application relates to the field of biochips, in particular to a biochip and a preparation method thereof.
Background
The single molecule detection technology can achieve the following two points: 1) recognition of the signal of a single molecule, rather than the average signal of a population of molecules, 2) tracking of the kinetic processes of a single molecule, provides details of molecular motion or reaction. Thanks to the excellent properties mentioned above, single molecule-based detection and identification techniques have received a high degree of attention from both academic and industrial sectors. Among them, the single-molecule DNA sequencing technology has been receiving much attention and research. Currently, the mainstream DNA single molecule sequencing technologies include: TIRF technology of the vast sea gene, ZMW technology of Paciffic, and Nanopore technology of oxford. The implementation of these sequencing technologies relies on highly available sequencing chips, i.e., DNA chips; thus, the performance requirements for DNA chips include at least: 1) good and reproducible single molecule modification efficiency, e.g. DNA in TIRF; 2) controllable, very low non-specific adsorption. Among them, controllable nonspecific adsorption is an important factor for experimental success or failure. In the DNA sequencing process, specific single-molecule signals and non-specific adsorption signals can be recognized and collected by an instrument, and the non-specific adsorption signals can seriously interfere with the recognition of the specific signals, so that a great obstacle is formed for the later-stage signal processing of the instrument.
Therefore, the preparation of the chip with nonspecific adsorption resistance has important value in the field of molecular detection.
Disclosure of Invention
It is an object of the present application to provide an improved biochip and a method for preparing the same.
The following technical scheme is adopted in the application:
the biochip is a DNA chip or a protein chip, the substrate surface of the biochip is provided with an anti-nonspecific adsorption layer, the anti-nonspecific adsorption layer is formed by combining a small molecule modification reagent on the substrate surface, and the small molecule modification reagent is at least one of compounds of a general formula shown in a formula I;
is like
Wherein R is1、R2And R3Are respectively selected from hydrophilic groups or hydrogen atoms.
The key point of the biochip is that the compound represented by the general formula I is used as a small molecule modification reagent to form a non-specific adsorption resistant layer of the biochip. Compared with the existing small molecule modification reagent, the small molecule modification reagent adopted by the application has the advantages of good controllability, good repeatability and the like, and can accurately control nonspecific adsorption, thereby obtaining a highly available DNA chip or protein chip for single molecule detection. The small molecule modification reagent of the present application means a small molecule compound having a relative molecular mass of 1000 or less.
Optionally, in the compound shown in the formula I, the hydrophilic group is a negative electricity group.
Optionally, the hydrophilic group contains a phosphoric acid group, a phosphate group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group, or an amide group.
Optionally, the compound shown in the formula I is at least one of taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine.
Optionally, the substrate surface of the biochip has an epoxy silane modification.
It is understood that the small molecule modifying agent may be bound to the substrate surface either by covalent or non-covalent bonds. In one implementation of the present application, the small molecule modification reagent is bound to the substrate surface via covalent bonds, thereby forming a stable and controllable anti-nonspecific adsorption layer.
The preparation method comprises the steps of adopting at least one of compounds of a general formula I as a micromolecule modification reagent, combining the micromolecule modification reagent with the surface of a substrate, and fixing the micromolecule modification reagent on the surface of the substrate to form an anti-nonspecific adsorption layer;
is like
Wherein R is1、R2And R3Are respectively selected from hydrophilic groups or hydrogen atoms.
It should be noted that, the key point of the preparation method of the present application is to use at least one of the compounds of the general formula i as the small molecule modification reagent, and as for how to bind the specific small molecule modification reagent to the substrate surface, reference may be made to the existing preparation process of the anti-nonspecific adsorption layer, which is not limited herein. However, in order to achieve a better non-specific adsorption resistant layer, in a preferred embodiment of the present application, the preparation method of the biochip is improved and optimized, and the details are shown in the following schemes.
Optionally, in the preparation method of the present application, the hydrophilic group is a negatively charged group.
Optionally, in the preparation method of the present application, the hydrophilic group contains a phosphoric acid group, a phosphoric acid ester group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group or an amide group.
Optionally, in the preparation method of the present application, the compound represented by formula one is at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid, and phosphoserine.
Optionally, in one implementation of the present application, the non-specific adsorption resistant layer is formed during the passivation process by using a passivation reaction solution containing a small molecule modification reagent.
It should be noted that the passivation reaction solution for passivation treatment may be a passivation solution conventionally used for passivation treatment, and is not specifically limited herein; the reaction conditions of the passivation treatment refer to the existing passivation treatment process.
Optionally, the concentration of the small molecule modification reagent in the passivation reaction solution is 0.01-0.2 mol/L.
The purpose of the small molecule modification reagent in the reaction solution of the passivation treatment is to bind to the surface of the substrate to form a non-specific adsorption resistant layer; it is understood that the concentration of the small molecule modification reagent in the optional passivation reaction solution of the present application is 0.01 to 0.2mol/L for the purpose of providing a better effect of the formed nonspecific adsorption layer, as long as the small molecule modification reagent of the present application is added. It is understood that outside this range, for example, too low a concentration may not form an effective nonspecific adsorption layer, too high a concentration may result in a waste of reagents, and the effect of improving the nonspecific adsorption layer is not so great.
Optionally, a reaction promoter is further added to the reaction solution of the passivation treatment to promote the binding reaction of the small molecule modification reagent and the substrate surface.
Optionally, the reaction promoter is at least one of benzyltriethylammonium chloride, phenyltrimethylammonium chloride and tetrabutylammonium chloride.
Optionally, the concentration of the reaction accelerator in the passivation reaction solution is 10-50 mmol/L.
It should be noted that the reaction promoter is used to promote the binding reaction between the small molecule modification reagent and the substrate surface, and under the action of the reaction promoter, the binding efficiency and quality of the small molecule modification reagent and the substrate surface are further improved, so that the reaction is more controllable and has better repeatability, and therefore, the reaction solution subjected to passivation treatment in the optional scheme is further added with the reaction promoter. It will be appreciated that depending on the manufacturing or product design requirements, for example where the controllability of the reaction is relatively low, the reaction promoter may not be used.
It should also be noted that benzyltriethylammonium chloride is only one reaction promoter that has proven useful in one implementation of the present application, and it is not excluded that other reaction promoters may also be used, as long as they are capable of promoting the binding reaction of the small molecule anti-non-specific adsorption reagent to the substrate surface.
Optionally, the preparation method of the application also comprises weak passivation treatment before passivation treatment; the weak passivation treatment comprises the steps of adding a small molecule modification reagent into weak passivation reaction liquid, and fixing the small molecule modification reagent on the surface of the substrate in advance by utilizing the combination reaction of the small molecule modification reagent and the surface of the substrate.
One of the functions of the weak passivation treatment is to fix a small molecule modification reagent on the surface of the substrate in advance by adding the small molecule modification reagent, and then further consolidate the small molecule modification reagent by the passivation treatment to form a stable nonspecific adsorption resistant layer.
Optionally, the concentration of the small molecule modification reagent in the weak passivation reaction solution is 15-45mmol/L, preferably 30 mmol/L.
Optionally, the reaction conditions of the weak passivation treatment are 35-40 ℃ for 2-5 h.
Optionally, the small molecule modification reagent used in the weak passivation treatment and the passivation treatment is the same.
Optionally, a surfactant is further added to the weak inactivation reaction solution for promoting the formation of covalent bonds between the DNA or protein and the substrate surface, so that the DNA or protein is sufficiently immobilized on the substrate surface.
Optionally, the surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctylammonium bromide.
Optionally, the concentration of the surfactant in the weakly passivating reaction solution is 1 to 25mmol/L, preferably 10 mmol/L.
It should be noted that, the step of weak passivation treatment is an optional modification of the present application, and in a preferred embodiment of the present application, a surfactant and a small molecule modification reagent are added to the weak passivation reaction solution of the weak passivation treatment; the weak passivation treatment has an important function besides the function of fixing the small molecule modification reagent on the surface of the substrate in advance, namely, the weak passivation treatment is used for improving the reaction efficiency of covalent bonds between DNA or protein and the surface of the substrate and enabling the DNA or protein to be more fully combined on the surface of the substrate, so that the quantity of the DNA or protein fixed on the biochip has good correlation with the concentration of the DNA or protein initially added in a fixing solution, and the quantity control and good repeatability of the DNA or protein fixed on the biochip are realized.
It should be further noted that the weakly passivating reaction solution of the weak passivation treatment provides a reaction environment for the surfactant and the small molecule modification reagent, and in an implementation manner of the application, for convenience of operation, the fixing solution is directly used as the weakly passivating reaction solution, and other reaction buffer solutions can also be used without exclusion. The fixing solution is a reaction buffer solution for fixing treatment commonly used in biochip preparation, and is not specifically limited herein.
Optionally, in one implementation of the present application, the preparation method of the present application specifically comprises the following steps,
weak passivation treatment, which comprises contacting weak passivation reaction liquid containing small molecule modification reagent or containing small molecule modification reagent and surfactant with a substrate, and performing weak passivation under constant temperature; the weak passivation treatment of the present application is intended to fix a small molecule modification reagent on the surface of a substrate in advance, and in the case of having a surfactant, it also has a function of sufficiently binding a DNA or a protein to an epoxy group; it will be appreciated that both the concentration of the small molecule modification agent and the treatment time will affect the amount of the small molecule modification agent immobilized on the substrate surface, with higher concentrations and longer treatment times corresponding to immobilization of the small molecule modification agentThe greater the amount at the substrate surface; similarly, the higher the concentration of the surfactant and the longer the treatment time, the better the effect of sufficiently binding the DNA or protein to the epoxy group is; the specific method can be determined according to production or product requirements, and is not particularly limited herein; in one implementation mode of the application, the weak passivation reaction solution for weak passivation treatment is 0.25mol/L Na containing 10nM surfactant and 30mM small molecule modification reagent2CO3/NaHCO3Introducing a reaction solution into a chip channel by using fluid equipment to perform weak passivation treatment when the pH is 9.58-10.53, wherein the volume of the flowing solution is 1mL, the fluid speed is 1mL/min, the reaction time is 3h, and the reaction temperature is 37 ℃; the above conditions are for reference and are not specifically limited herein;
passivating, including adopting passivating reaction liquid to wash the substrate which is weakly passivated, then contacting the passivating reaction liquid containing small molecule modification reagent or containing small molecule modification reagent and reaction accelerator with the washed substrate, and passivating under the condition of constant temperature; in general, the passivation reaction solution, i.e., the passivation solution, has a K content of 1mol/L2HPO4/KH2PO4pH 9.0; wherein "K" is2HPO4/KH2PO4"denotes a group consisting of2HPO4And KH2PO4The proportion of the passivation solution and the conventional DNA chip passivation solution is referred, and the passivation solution is not particularly limited; in one implementation manner of the present application, specifically, 0.25mol/L Na is adopted for the reaction solution containing the small molecule modification reagent or containing both the small molecule modification reagent and the reaction promoter2CO3/NaHCO3pH is 9.58-10.53, the concentration of the small molecule modification reagent is 0.01-0.2M, a reaction solution is introduced into a chip channel by utilizing a fluid device for reaction, the passivation treatment condition is that the number of times of flowing the passivation solution is 3-4, the volume of each flowing is 500 mu L, the speed of the fluid is 1mL/min, the interval time of each flowing is 1800s, the temperature is kept at 37 ℃ in the whole passivation process, and the conditions are not particularly limited.
The key point of the application is that a special small molecule modification reagent is adopted during passivation treatment; in further improvement, weak passivation treatment is added; as for other steps, the existing DNA chip or protein chip preparation process can be referred to.
Optionally, the preparation method further comprises a washing step, namely washing the passivated chip, wherein the washing step comprises washing the passivated substrate sequentially by adopting three washing liquids, each washing liquid is washed at least once, and the three washing liquids are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer, RI-06 is mixed solution of HEPES buffer and NaCl solution, and RI-07 is double distilled water. Washing of DNA chips after the inactivation treatment is well known in the art, and RI-05, RI-06 and RI-07 are also conventional washing solutions, and generally, each washing solution needs to be repeated 3 times. Among them, HEPES is 4-hydroxyethyl piperazine ethanesulfonic acid.
Optionally, the preparation method of the present application further comprises a fixation treatment before the weak passivation treatment, specifically comprising contacting a fixation solution containing DNA or protein with the substrate surface, and performing DNA or protein fixation under a constant temperature condition. Typically, the fixative is 0.25mol/L Na2CO3/NaHCO3pH9.78, wherein the concentration of DNA is generally 0.01-0.4nmol/L, the temperature of the fixation treatment is about 37 ℃, the treatment time is about 30min, and the above conditions are not specifically limited herein; wherein "Na" is2CO3/NaHCO3"means represented by Na2CO3And NaHCO3The ratio of the two fixing solutions is referred to the conventional DNA chip fixing solution, and is not limited herein.
The application also discloses an application of the small molecular compound in preparing a DNA chip or a protein chip, which comprises the following steps of taking the small molecular compound as a small molecular modification reagent, combining the small molecular compound on the surface of a substrate to form an anti-nonspecific adsorption layer, wherein the small molecular compound is at least one of compounds shown in a general formula I;
is like
Wherein R is1、R2And R3Respectively, a hydrophilic group or a hydrogen atom.
Optionally, in the small molecule compound, the hydrophilic group is a negatively charged group.
Optionally, in the small molecule compound, the hydrophilic group contains a phosphoric acid group, a phosphoric acid ester group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group, or an amide group.
Optionally, the small molecule compound is at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.
The key point of the application lies in finding some novel small molecule compounds which are particularly suitable for being used as small molecule modification reagents of DNA chips or protein chips, when the small molecule compounds are used as the small molecule modification reagents, the small molecule compounds not only have the advantages of the existing small molecule modification reagents, but also have controllable reaction and good repeatability, and have important value for preparing DNA chips and protein chips for highly available single molecule detection; therefore, the application provides a new application of the small molecule compounds as small molecule modification reagents for preparing DNA chips or protein chips to form an anti-nonspecific adsorption layer on the surface of a substrate.
In yet another aspect, the present application discloses the use of the biochip of the present application in nucleic acid or protein detection assays. The nucleic acid or protein detection analysis includes sequencing analysis, hybridization analysis, immunoassay, SNP detection analysis, and the like.
It should be noted that, the DNA chip and the protein chip in the present application refer to the chip with DNA or protein immobilized on the substrate surface, and in the present application, if the type of DNA and protein is not specified, the DNA refers to a substance containing DNA sequence or amino acid sequence, for example, the DNA immobilized on the DNA chip may contain nucleotide derivatives, nucleotide analogs, fluorescent labels, or both nucleotide sequence and amino acid sequence; proteins refer to a class of substances that contain amino acid sequences.
In the present application, the substrate surface of the chip has a chemical modification containing a reactive group capable of reacting with DNA or protein, and the DNA or protein is immobilized on the substrate surface by the reaction between the reactive group and the DNA or protein.
The beneficial effect of this application lies in:
according to the biochip, a compound with a structure shown in the formula I is used as a small molecule modification reagent to form an anti-nonspecific adsorption layer of the biochip, the adsorption layer can effectively prevent nonspecific adsorption of nucleotide used for sequencing, the ratio of characteristic detection signals is improved, interference of nonspecific signals is reduced, and the anti-nonspecific adsorption layer of the biochip is high in connection strength and not easy to fall off; moreover, the small molecule modification reagent has controllable reaction and good repeatability, so that the biochip has high quality, good repeatability and high availability, and is particularly suitable for single molecule detection.
Drawings
FIG. 1 is a schematic structural diagram of a packaged chip substrate according to an embodiment of the present application;
FIG. 2 is a comparison graph of the results of testing the number of nonspecific adsorption points of a DNA chip that has been weakly inactivated with a small molecule anti-nonspecific adsorption reagent having a single negatively charged group and a small molecule anti-nonspecific adsorption reagent having multiple hydrophilic groups in the examples of the present application;
FIG. 3 is a comparison graph of the results of nonspecific adsorption number tests of DNA chips that were weakly inactivated with small molecule anti-nonspecific adsorption reagents having multiple negatively charged groups and small molecule anti-nonspecific adsorption reagents having single negatively charged groups in the examples of the present application;
FIG. 4 is a comparison graph of the results of the nonspecific adsorption number test of DNA chips passivated with or without the addition of a reaction promoter in the examples of the present application;
FIG. 5 is a schematic structural view of a glass substrate in an example of the present application.
Detailed Description
The formation of the nonspecific adsorption-resistant layer by specific small molecule modification is a technology which is already in use, and the small molecule nonspecific adsorption-resistant layer has the remarkable advantages of high connection strength, difficult shedding, broad-spectrum nonspecific adsorption resistance and the like, and is widely applied. However, the existing small molecule modification generally has the problems of poor controllability, poor repeatability and the like, so that the nonspecific adsorption resistance effect of small molecule modifications of different batches or even the same batch is different, and therefore, the highly available DNA chip and protein chip for single molecule detection are difficult to prepare stably and reliably.
In the long-term mass research process of DNA chips and protein chips, the finding shows that the micromolecule modifying reagent with the general formula shown in the formula I, such as taurine, aminopropyl sulfonic acid, serine, glutamic acid, phosphoserine and the like, has high-efficiency and strong-controllability combining capacity with a substrate, and the micromolecule modifying reagent has good repeatability in combination with the substrate, can stably and reliably prepare a high-availability DNA chip or protein chip with good nonspecific adsorption resistance effect, and is particularly suitable for single-molecule detection.
In a further improvement of the present application, the present application further creatively provides that the surfactant is introduced during the preparation of the DNA chip or protein chip by utilizing the principle that the surfactant can make DNA or protein react with the chemical modification group on the surface of the substrate more fully, so that the DNA or protein can be immobilized on the substrate more effectively. Due to the use of the surfactant, the DNA or protein reacts with the chemical modification group on the surface of the substrate more fully, so that the efficiency and the quality of the DNA or protein fixed on the surface of the substrate are improved, and the quantity of the DNA or protein fixed on the biochip has good correlation with the concentration of the DNA or protein initially added in the fixing solution, thereby realizing the controllable quality and good repeatability of the DNA or protein fixed on the biochip. The method not only lays a foundation for preparing high-quality biochips, but also can meet the production requirements of customization.
In the biochip of the present application, the substrate surface has chemical modifications containing reactive groups capable of reacting with DNA or proteins. In one embodiment herein, the amino group of the DNA or protein reacts with a reactive group that is one of an epoxy group, an aldehyde group, a carboxyl group, N-hydroxysuccinimide, and diaminobenzanilide.
In one embodiment of the present application, the chemically modified structure of the substrate surface is shown in fig. 5, wherein R1 represents an alkane chain molecule terminally linked to a reactive group, wherein the reactive group is preferably at least one of an epoxy group, an aldehyde group, a carboxyl group, N-hydroxysuccinimide, and diaminobenzanilide.
In one embodiment of the present application, the chemical modification of the substrate surface can be combined with the small molecule modification reagent of the present application to immobilize the small molecule modification reagent on the substrate surface, thereby forming a non-specific adsorption resistant layer.
Some words referred to in the examples of the present application are explained as follows:
AT-01: the component is 0.25M Na2CO3/NaHCO30.6mM CTAB, pH 9.78; wherein CTAB is an abbreviation for cetyltrimethylammonium bromide;
RI-04: the component is 1M K2HPO4/KH2PO4，pH 9.0
RI-05: the component is PH7.4PBS solution;
RI-06: a mixed solution consisting of 150mM HEPES buffer solution and 150mM NaCl solution;
RI-07: the component is double distilled water;
Dot/FOV: the observation region was defined as the number of bright spots in the range of 110X 110. mu.m.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example one
In this example, DNA was immobilized on a glass substrate having epoxy silane on the surface thereof via amino groups of the DNA, thereby forming a DNA chip of this example. And in the preparation process, after the fixing treatment and before the passivation treatment, weak passivation treatment is added. This example compares the effect of using different small molecule modification reagents on a DNA chip. Specifically, this example compares the nonspecific adsorption resistance of a single negatively charged group small molecule modification reagent with that of a multiple hydrophilic group small molecule modification reagent.
In this embodiment, the DNA is fixed on the chip substrate by "in-channel" fixing, i.e., the chip substrate is packaged, and then various reagents are introduced into the packaged chip channels by using a fluid device, thereby realizing chemical reactions such as fixing treatment and passivation treatment. As shown in fig. 1, chip substrates are packaged to form independent chip channels, each chip channel can independently perform each reaction, fig. 1 shows a packaged chip substrate with 8 channels, the specification of the channels is length × width × height × 90mm × 1.8mm × 0.1mm, according to different packaging processes or fluidic devices, the chip substrates can be packaged into 16 channels, and 16 kinds of differently modified DNAs are independently manufactured on one DNA chip.
The method for preparing the DNA chip of this example is as follows:
(1) fixing treatment, namely introducing a fixing reaction solution into a channel of a chip substrate to perform fixing reaction, wherein the fixing reaction solution in the embodiment is a fixing solution AT-01 containing 0.05nM DNA and 0.6mM CTAB; wherein the 3' end of the DNA contained in the DNA simultaneously carries amino-modified NH2And Cy3 fluorophore modification; AT-01 has Na content of 0.25M2CO3/NaHCO30.6mM CTAB, pH9.78, the volume of the flow-through solution is 1mL, the flow rate is 1mL/min, the reaction time is 30min, and the reaction temperature is 37 ℃;
(2) weak passivation treatment, introducing weak passivation reaction solution containing 10mM CTAB and 30mM small molecule modification reagent 0.25M Na for performing weak passivation2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; for comparison, the small molecule modification reagent added to the weak passivation reaction solution adopted in the present embodiment is a small molecule modification reagent with a single negative charge group, specifically Taurine (Taurine), and the other part of the channels is not added with the small molecule modification reagent and only adopts the weak passivation reaction solution to perform weak passivation treatment; setting the volume of the circulating solution to be 1mL in all channels, setting the fluid speed to be 1mL/min, setting the reaction time to be 3h and setting the reaction temperature to be 37 ℃;
(3) washing to remove the weak passivation reaction solution, specifically, using a passivation solution RI-04, washing for 3 times, wherein the volume of each time is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the RI-04 component of this example is K of 1M2HPO4/KH2PO4，pH 9.0；
(4) Passivation treatment, specifically, passivation reaction liquid is introduced for passivation treatment, and the passivation reaction liquid in the embodiment is 0.25M Na containing 30mM small molecule modification reagent2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; the number of times of flowing passivation reaction liquid is 3-4, the volume of each flowing is 500 mu L, the speed of the fluid is 1mL/min, the interval time of each flowing is 1800s, and the temperature is kept at 37 ℃ in the whole passivation process; wherein, the micromolecule modification reagent in the passivation reaction liquid adopts corresponding Taurine during weak passivation treatment, that is, a channel of the Taurine is adopted during the weak passivation treatment, and the Taurine is also added into the corresponding passivation reaction liquid;
(5) washing the passivated chip by adopting three solutions, wherein each solution is washed for 3 times, the volume of each solution is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer solution with pH7.4, RI-06 is mixed solution consisting of HEPES buffer solution with 150mM and NaCl solution with 150mM, and RI-07 is double distilled water.
After washing with the three solutions, the DNA chip of this example is obtained after normal air drying or oven drying. The nonspecific adsorption of the DNA chip prepared in this example was evaluated by a monomolecular fluorescence imaging technique. The detection method of nonspecific adsorption comprises the steps of introducing Cy5 fluorescence-labeled nucleotide on the surface of the prepared DNA chip, detecting Cy5 fluorescence adsorbed on the surface of the DNA chip, and representing the number of nonspecific adsorption points of the DNA chip by the number of Cy5 fluorescence points in unit area. In this example, the number of fluorescence spots in a 110X 110 μm region was counted to characterize the number of nonspecific adsorption spots.
The results of the number of non-specific adsorption spots are shown in FIG. 2, in which the abscissa of FIG. 2 represents the number of non-specifically adsorbed fluorescence spots per unit area of 110X 110. mu.m, and the abscissa represents the number of DNA chips treated with Taurine (i.e., Taurine) and the number of DNA chips treated without small molecule modification agent (i.e., Null), respectively. The results in FIG. 2 show that after treatment with Taurine, the number of non-specific adsorption sites is about 1000Dot/FOV, which is much lower than that of DNA chips without treatment with Taurine. Therefore, the small molecule modification reagent with single negative charge group has good nonspecific adsorption resistance.
Example two
The preparation materials and procedures of the DNA chip of this example are the same as those of the first example, except that the small molecule modification reagents with multiple negative charge groups and the small molecule modification reagents with single negative charge groups are added separately, and the influence of different small molecule modification reagents on the DNA chip is compared. Specifically, the present example compares the nonspecific adsorption resistance of the small molecule modification reagent Taurine with a single negative charge group, and the small molecule modification reagents glutamic acid and phosphoserine with multiple negative charge groups.
The specific preparation method of the DNA chip of this example was as follows:
(1) fixing treatment, namely introducing a fixing reaction solution into a channel of a chip substrate to perform fixing reaction, wherein the fixing reaction solution in the embodiment is a fixing solution AT-01 containing 0.05nM DNA and 0.6mM CTAB; wherein the 3' end of the DNA contained in the DNA simultaneously carries amino-modified NH2And Cy3 fluorophore modification; AT-01 has Na content of 0.25M2CO3/NaHCO30.6mM CTAB, pH9.78, volume of flow-through solution 1mL, fluid velocity 1mL/min, reaction time 30min, reaction temperature 37 ℃;
(2) weak passivation treatment, introducing weak passivation reaction solution containing 10mM CTAB and 30mM small molecule modification reagent 0.25M Na for performing weak passivation2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; in contrast, the small molecule modification reagent added to the weak passivation reaction solution used in a part of the channels of this embodiment is a small molecule modification reagent with a single negative charge group, specifically Taurine (Taurine), and the small molecule modification reagent added to the weak passivation reaction solution used in another part of the channelsThe sub-modification reagent is a small molecule modification reagent Glutamic acid (Glutamic acid) with multiple negative charge groups, and the small molecule modification reagent added in the weak passivation reaction solution adopted by one part of channels is a small molecule modification reagent phosphoserine (Phospho-serine) with multiple negative charge groups; setting the volume of the circulating solution to be 1mL in all channels, setting the fluid speed to be 1mL/min, setting the reaction time to be 3h and setting the reaction temperature to be 37 ℃;
the point to be noted is that, in the weak passivation treatment and the subsequent passivation treatment, the added small molecule modification reagent may be the same or different; in general, the small molecule modifying agents added in both steps are identical;
(3) washing to remove the weak passivation reaction solution, specifically, using a passivation solution RI-04, washing for 3 times, wherein the volume of each time is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the RI-04 component of this example is K of 1M2HPO4/KH2PO4，pH 9.0；
(4) Passivation treatment, specifically, passivation reaction liquid is introduced for passivation treatment, and the passivation reaction liquid in the embodiment is 0.25M Na containing 30mM small molecule modification reagent2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; the number of times of flowing passivation reaction liquid is 3-4, the volume of each flowing is 500 mu L, the speed of the fluid is 1mL/min, the interval time of each flowing is 1800s, and the temperature is kept at 37 ℃ in the whole passivation process; wherein, the micromolecule modification reagent in the passivation reaction liquid adopts corresponding Taurine, glutamic acid or phosphoserine during weak passivation treatment, that is, a channel of Taurine is adopted during weak passivation treatment, Taurine is also added into the corresponding passivation reaction liquid, a channel of glutamic acid is adopted during weak passivation treatment, glutamic acid is also added into the corresponding passivation reaction liquid, a channel of phosphoserine is adopted during weak passivation treatment, and phosphoserine is also added into the corresponding passivation reaction liquid;
(5) washing the passivated chip by adopting three solutions, wherein each solution is washed for 3 times, the volume of each solution is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer solution with pH7.4, RI-06 is mixed solution consisting of HEPES buffer solution with 150mM and NaCl solution with 150mM, and RI-07 is double distilled water.
After washing with the three solutions, the DNA chip of this example is obtained after normal air drying or oven drying.
The DNA chip of this example was tested by the same test method as in example, and the results are shown in FIG. 3. In FIG. 3, the abscissa represents the number of nonspecific adsorption fluorescence spots per unit area of 110X 110. mu.m corresponding to different small molecule modification reagents, and the ordinate represents the number of nonspecific adsorption fluorescence spots per unit area corresponding to different small molecule modification reagents. The results in FIG. 3 show that the number of non-specific adsorption sites after treatment with Taurin, a small molecule modification reagent with multi-negative charge groups, is about 1000Dot/FOV, after treatment with Glutamic acid (Glutamic acid), a small molecule modification reagent with multi-negative charge groups, is about 800Dot/FOV, and after treatment with phosphoserine (Phospho-serine), a small molecule modification reagent with multi-negative charge groups, is about 650 Dot/FOV; therefore, the number of nonspecific adsorption points is respectively Taurine, Glutamic acid and Phospho-serine from large to small, which shows that the micromolecule modification reagent with multiple negative charge groups is superior to the micromolecule modification reagent with single negative charge group, and has better nonspecific adsorption resistance effect.
EXAMPLE III
The DNA chip preparation materials and procedures in this example were the same as those in the first example, except that the influence of the addition of the reaction accelerator or not, in addition to the small molecule modification reagent, on the DNA chip during the passivation treatment was compared. The reaction accelerator specifically added in this example was Benzyltriethylammonium chloride (Benzyltriethyllammonium chloride).
The specific preparation method of the DNA chip of this example was as follows:
(1) fixing treatment, namely introducing a fixing reaction solution into a channel of a chip substrate to perform fixing reaction, wherein the fixing reaction solution in the embodiment is a fixing solution AT-01 containing 0.05nM DNA and 0.6mM CTAB; wherein the 3' end of the DNA contained in the DNA simultaneously carries amino-modified NH2And Cy3 fluorophore modification; AT-01 has Na content of 0.25M2CO3/NaHCO3pH9.78, volume of the flow-through solution is 1mL, speed of the fluid is 1mL/min, reaction time is 30min, and reaction temperature is 37 ℃;
(2) weak passivation treatment, introducing weak passivation reaction solution containing 10mM CTAB and 30mM small molecule modifying reagent 0.25M Na to perform the step of' weak passivation2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; wherein, the micromolecule modifying reagent is Taurine, the volume of the flowing solution arranged in all channels is 1mL, the speed of the fluid is 1mL/min, the reaction time is 3h, and the reaction temperature is 37 ℃;
(3) washing to remove the weak passivation reaction solution, specifically, using a passivation solution RI-04, washing for 3 times, wherein the volume of each time is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the RI-04 component of this example is K of 1M2HPO4/KH2PO4，pH 9.0；
(4) Passivation treatment, specifically, passivation reaction liquid is introduced for passivation treatment, and the passivation reaction liquid in the embodiment is 0.25M Na containing 30mM micromolecule modification reagent Taurin2CO3/NaHCO3The pH is between 9.58 and 10.53, in this case specifically pH 9.78; the number of times of flowing passivation reaction liquid is 3-4, the volume of each flowing is 500 mu L, the speed of the fluid is 1mL/min, the interval time of each flowing is 1800s, and the temperature is kept at 37 ℃ in the whole passivation process; for comparison, the present example provides channels with or without the addition of the reaction accelerator Benzyltriethyllamonium chloride, respectively; the concentration of the reaction solution for inactivating the Benzyltriethyllamonium chloride was 30 mM;
(5) washing the passivated chip by adopting three solutions, wherein each solution is washed for 3 times, the volume of each solution is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer solution with pH7.4, RI-06 is mixed solution consisting of HEPES buffer solution with 150mM and NaCl solution with 150mM, and RI-07 is double distilled water.
After washing with the three solutions, the DNA chip of this example is obtained after normal air drying or oven drying.
The DNA chip of this example was tested by the same test method as in example, and the results are shown in FIG. 4. In FIG. 4, Null on the abscissa indicates a DNA chip to which no reaction promoter is added, and Add indicates a DNA chip to which a reaction promoter is added; the ordinate represents the number of non-specifically adsorbed fluorescent spots per unit area of 110X 110. mu.m. The results in FIG. 4 show that the number of non-specific adsorption fluorescence spots of the corresponding DNA chip after the reaction promoter is added is about 600Dot/FOV, while the number of non-specific adsorption fluorescence spots of the DNA chip without the reaction promoter is about 1000Dot/FOV, which indicates that the reaction promoter can further promote the small molecule modification reagent Taurine to be bonded on the substrate, thereby further reducing the non-specific adsorption of the DNA chip.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (17)
1. A biochip which is a DNA chip or a protein chip, characterized in that: the substrate surface of the biochip is provided with a non-specific adsorption layer which can prevent non-specific adsorption of nucleotides, the non-specific adsorption layer is formed by binding a small molecule modification reagent on the substrate surface, and the small molecule modification reagent is at least one of glutamic acid and phosphoserine.
2. The biochip according to claim 1, wherein: the substrate surface of the biochip has an epoxy silane modification.
3. A method of preparing the biochip according to claim 1 or 2, comprising: the method comprises the steps of enabling the small molecule modification reagent to be combined with the surface of a substrate and fixed on the surface of the substrate to form the non-specific adsorption resistant layer.
4. The method of claim 3, wherein: the method comprises the steps of passivating, wherein the passivating comprises the step of contacting a passivating reaction solution containing the small molecule modifying reagent with the surface of a substrate so as to fix the small molecule modifying reagent on the surface of the substrate;
the concentration of the micromolecule modification reagent in the passivation reaction liquid is 0.01-0.2 mol/L.
5. The method of claim 4, wherein: the passivation reaction liquid also contains a reaction promoter;
the reaction promoter is at least one of benzyltriethylammonium chloride, phenyltrimethylammonium chloride and tetrabutylammonium chloride.
6. The method of claim 5, wherein: the concentration of the reaction promoter in the passivation reaction liquid is 10-50 mmol/L.
7. The method of claim 4, wherein: the method further comprises the step of carrying out weak passivation treatment on the surface of the substrate by using weak passivation reaction liquid before passivation treatment; the weak passivation treatment comprises the step of enabling a weak passivation reaction solution to be in contact with the surface of the substrate so as to enable the small molecule modifier to be fixed on the surface of the substrate; the weak passivation reaction solution comprises the small molecule modification reagent and a buffer solution;
the concentration of the small molecule modification reagent in the weak passivation reaction liquid is 15-45 mmol/L.
8. The method of claim 7, wherein: the concentration of the small molecule modification reagent in the weak passivation reaction solution is 30 mmol/L.
9. The method of claim 7, wherein: the weak passivation treatment comprises the step of placing the surface of the substrate in the weak passivation reaction liquid at 35-40 ℃ for 2-5 h.
10. The method of claim 7, wherein: and the small molecule modification reagent in the weak passivation reaction solution and the passivation reaction solution is the same.
11. The method of claim 7, wherein: the weak passivation reaction liquid also comprises a surfactant.
12. The method of claim 11, wherein: the surfactant is at least one selected from cetyl trimethyl ammonium bromide, dioctadecyl ammonium bromide, cetyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide and tetraoctyl ammonium bromide.
13. The method of claim 11, wherein: the concentration of the surfactant is 1-25 mmol/L.
14. The method of claim 11, wherein: the concentration of the surfactant is 10 mmol/L.
15. The method according to any one of claims 7-14, wherein: the method further comprises washing the substrate surface after the weak passivation treatment, wherein the washing comprises washing the substrate surface by three washing solutions in sequence, each washing solution is washed at least once, and the three washing solutions are RI-05, RI-06 and RI-07 according to the using sequence, wherein the RI-05 is PBS buffer solution, the RI-06 is mixed solution of HEPES buffer solution and NaCl solution, and the RI-07 is double distilled water.
16. The method according to any one of claims 7-14, wherein: the method also comprises a fixation treatment before the weak passivation treatment, wherein the fixation treatment comprises contacting a fixation solution containing DNA or protein with the surface of the substrate, and performing DNA or protein fixation under constant temperature.
17. Use of a biochip according to claim 1 or 2 in nucleic acid or protein detection assays for non-diagnostic therapeutic purposes.
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| PCT/CN2019/101068 WO2020073734A1 (en) | 2018-10-12 | 2019-08-16 | Biochip and manufacturing method therefor |
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| Enhancing the Fluorescence Intensity of DNA Microarrays by Using Cationic Surfactants;Siok Lian Lai et al.;《Langmuir》;20110406;EXPERIMENTAL SECTION，CONCLUSIONS * |
| 抗蛋白质非特异性吸附材料及其在生物医学领域中的应用.抗蛋白质非特异性吸附材料及其在生物医学领域中的应用.《高分子通报》.2016, * |
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