CN109610007A - A kind of DNA chip and preparation method thereof that albumen is co-modified - Google Patents

A kind of DNA chip and preparation method thereof that albumen is co-modified Download PDF

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CN109610007A
CN109610007A CN201811191611.3A CN201811191611A CN109610007A CN 109610007 A CN109610007 A CN 109610007A CN 201811191611 A CN201811191611 A CN 201811191611A CN 109610007 A CN109610007 A CN 109610007A
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protein
modified
dna
substrate surface
sulfydryl
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高锦鸿
赵�智
赵陆洋
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SHENZHEN HANHAI GENE BIOTECHNOLOGY CO Ltd
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SHENZHEN HANHAI GENE BIOTECHNOLOGY CO Ltd
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Priority to CN201811191611.3A priority Critical patent/CN109610007A/en
Priority to CN202111182158.1A priority patent/CN113913944A/en
Publication of CN109610007A publication Critical patent/CN109610007A/en
Priority to EP19872008.8A priority patent/EP3865585A4/en
Priority to PCT/CN2019/101068 priority patent/WO2020073734A1/en
Priority to US17/227,211 priority patent/US20210301331A1/en
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
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    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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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Abstract

This application discloses a kind of DNA chips and preparation method thereof that albumen is co-modified.The co-modified DNA chip of the albumen of the application, the co-modified substrate surface for being incorporated in DNA chip by protein of albumen are formed.The co-modified DNA chip of the albumen of the application, in one implementation, proteinaceous solid is scheduled on substrate surface using covalent bond, not only have the advantages that method is easy, raw material sources are wide, work well, overall cost is low etc., and, more firm by covalent bond fixation, protein is not easy desorption, avoids non-specific adsorption resulting from.

Description

A kind of DNA chip and preparation method thereof that albumen is co-modified
Technical field
This application involves DNA chip fields, more particularly to a kind of DNA chip and preparation method thereof that albumen is co-modified.
Background technique
By DNA chain fixed in glass, gold/substrates such as silver noble metal or engineering plastics, available DNA chip is used for The recognition detection of DNA or protein biomarker object；Alternatively, further, the survey as sequence testing chip, for DNA sequence dna It is fixed.Typically, the DNA chip of High Availabitity at least needs to meet following requirement: 1) DNA modification is evenly distributed in substrate；2) Chip obtained has lower non-specific adsorption signal；3) the DNA type modified can customize, and can modify different types of DNA chain；4) preparation flow of DNA chip has good repeatability.
In DNA chip use process, the object of detection has macromolecular and small molecule, in the detection process of various molecules Will have the phenomenon that non-specific adsorption, when chip substrate surface non-specific adsorption than more serious, detection will be seriously affected As a result.In many cases, the DNA chip for preparing High Availabitity also needs to meet many more harsh requirements.Very much In usage scenario, for example, 1) the protein perhaps detection of nucleic acid or 2) complicated enzymatic system, these situations in sequencing Under, the non-specific adsorption of protein can cause to influence as follows on the performance of DNA chip: 1) absorption of slight protein can be brought non- Nonspecific signal reduces the intensity of signal, and detection is made to have poor signal-to-noise ratio；2) serious protein absorption can even seal completely The DNA chain specifically modified is closed, complete detection identification cannot.Even more serious, many adsorbed proteins bring various types Chemical group, serious interference is brought to the detection of certain chemical species, for example, protein carry polysaccharide to more The detection of sugar, the sulfydryl on protein can bring nonspecific signal in conjunction with the sulfydryl small molecule in detection architecture；Simultaneously The non-specific adsorption of nucleotide can cause the problems such as detecting the inaccuracy of signal.
Summary of the invention
The purpose of the application is to provide a kind of co-modified DNA chip of improved albumen and preparation method thereof.
The application uses following technical scheme:
The one side of the application discloses a kind of DNA chip that albumen is co-modified, and the albumen of the application is co-modified by albumen The substrate surface that matter is incorporated in DNA chip is formed.
It is appreciated that the substrate surface that protein is incorporated in DNA chip can be by Covalent bonding together, it can also be by non- Covalent bonding together, in one embodiment of the application, albumen passes through Covalent bonding together in the substrate surface of DNA chip, by egg White matter is firmly fixed to substrate surface, and for Covalent bonding together compared with physical absorption is modified, covalently bound protein is fixed more It is firm to add, and avoids desorption, and then avoids the co-modified DNA chip of albumen when in use, because non-specific caused by desorption Absorption.For example, being sequenced or detecting in identification process, it, can be to avoid because of albumen using the co-modified DNA chip of the albumen of the application Desorption caused by absorption in sequencing procedure to the absorption of enzyme and nucleotide.
Optional, in the co-modified DNA chip of the albumen of the application, the co-modified protein used of albumen is pure for ox blood Albumen.
Optional, in the co-modified DNA chip of the albumen of the application, the sulfydryl on the co-modified protein of albumen is in envelope Closed state.
It should be noted that the DNA chip that the albumen of the application is co-modified, since the sulfydryl on its protein is in closing State reduces and even avoids absorption of the DNA chip to the molecule containing sulfydryl, has the work of the anti-Molecular Adsorption containing sulfydryl With.Also, in a kind of implementation of the application, when closing to sulfydryl, used closed reagent introduces more Hydrophilic radical or negative electricity group further enhance the anti-non-specific adsorption ability of DNA chip.
Optional, in the co-modified DNA chip of the albumen of the application, it is anti-that there is substrate surface small numerator modified reagent to be formed Non-specific adsorption layer.The small numerator modified reagent of the application refers to relative molecular mass in 1000 small molecule chemical combination below Object.
It is appreciated that small numerator modified reagent, can be by Covalent bonding together in conjunction with substrate surface, it can also be by non- Covalent bonding together.In a kind of implementation of the application, small numerator modified reagent passes through Covalent bonding together in substrate surface, thus Form the anti-non-specific adsorption layer stablized, controllably.
Optional, small numerator modified reagent is in taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine At least one.
It, can be with other than protein is co-modified it should be noted that in the co-modified DNA chip of the albumen of the application Using conventional small numerator modified reagent, anti-non-specific adsorption layer is formed, the anti-non-specific adsorption of DNA chip is further increased Ability.Especially in a kind of preferred embodiment of the application, research has found that a kind of reaction controllability is good, small point favorable repeatability Son modification reagent, i.e. taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine etc., so that anti-non-specific adsorption Preparing for layer is highly controllable, effectively can react on substrate surface with protein co-modified one, it is non-to enhance resisting for DNA chip Specific adsorption ability.
The another aspect of the application discloses a kind of preparation method of DNA chip that albumen is co-modified, is included in and carries out DNA When fixing process, protein is added into fixer, makes protein that association reaction occur with substrate surface and is scheduled on proteinaceous solid Substrate surface plays the co-modified effect of albumen.
It should be noted that proteinaceous solid is scheduled on substrate surface using covalent bond in one embodiment of the application, from And it solves the problems, such as physical absorption and modifies easy desorption；It, can be with reference to existing covalent as specifically how to form covalent bond Key forming method, such as can use the amino gene of protein and the epoxy group that substrate surface is modified reacts and generates covalent bond Proteinaceous solid is scheduled on substrate surface, depending on working condition, it is not limited here.
It should also be noted that, the preparation method of in general DNA chip includes fixing process, Passivation Treatment and washing etc. Technique, the key of the application be that protein is added in the fixer of fixing process, protein and substrate surface occurs anti- It answers, proteinaceous solid is scheduled on substrate surface, as other steps, such as Passivation Treatment and subsequent washing etc. can be with reference to existing Some DNA chip preparation processes, substrate can also refer to existing conventional use of substrate, be not specifically limited herein.
Optional, the protein that the preparation method of the application uses is bovine serum albumin(BSA).
Optional, in the preparation method of the application, substrate surface is modified with epoxy silane.
It should be noted that substrate surface is modified with epoxy silane, protein is allowed to pass through its amino and epoxy Hydride modified reaction generates covalent bond and is fixed on substrate surface, so that protein be made to consolidate, effectively be fixed on substrate surface.
Optional, the preparation method of the application further includes that processing is passivated after fixing process, and in Passivation Treatment Later, sulfydryl Seal treatment is carried out to the substrate of Passivation Treatment, the sulfydryl on the fixed protein of substrate surface is closed.
It should be noted that the covalent bond ankyrin either in physical absorption protein or the embodiment of the present application All there is small molecule non-specific adsorption, the especially non-specific adsorption of the small molecule containing sulfydryl in matter；Wherein, contain The non-specific adsorption of the small molecule of sulfydryl, partly cause are introduced as caused by the sulfydryl on co-modified protein.Base In the above understanding, the proposition of the application creativeness in Passivation Treatment and then increases a sulfydryl Seal treatment, by substrate table Sulfydryl closing on the fixed protein in face；The co-modified DNA chip of albumen even is avoided to cysteine to reduce (Cys), homocysteine (Hcy) and reduced glutathione (GSH) and the nucleic acid molecule containing sulfydryl etc. are containing sulfydryl The non-specific adsorption of small-molecule substance；Also, in sulfydryl Seal treatment, introduced reagent can be co-modified in albumen DNA chip surface introduces more hydrophilic radicals or negative electricity group, further enhances the anti-non-specific adsorption ability of DNA chip.It can To understand, the key of the application is that the sulfydryl on co-modified albumen is closed in creative proposition, makes on protein Sulfydryl inactivation can refer to general protein sulfhydryl Seal treatment method as specific sulfydryl blocking test, or use Existing technology is reacted with the sulfydryl on protein makes its inactivation, is not specifically limited herein.
Those skilled in the art are understood that the nucleotide in the application refers to nucleosides, ucleotides and its class Like object, derivative, and they do not change the Watson-Crick base pairing function of nucleosides and nucleotide.
Optional, sulfydryl Seal treatment specifically includes, first using go back original reagent by the disulfide bond reduction in the protein, The sulfydryl of reproducibility is generated, then closes the sulfydryl of reproducibility generated using sulfydryl closed reagent again.
It is optional, go back original reagent be three (2- carboxyethyl) phosphines (abbreviation TCEP), three (3- hydroxypropyl) phosphines (abbreviation THPP), At least one of dithiothreitol (DTT) (abbreviation DTT) and mercaptoethanol (abbreviation ME).
Optional, sulfydryl closed reagent is at least one in iodoacetamide, iodoacetic acid, maleimide and epoxy prapanol Kind.
Optional, the preparation method of the application further includes carrying out before Passivation Treatment weak blunt after DNA fixing process Change processing；Weak Passivation Treatment, which is included in reaction solution, is added catalyst and protein, using catalyst promote DNA and protein with The combination of substrate surface makes DNA and protein be sufficiently secured within substrate surface.
It should be noted that weak Passivation Treatment step is the optional improvement project of the application, effect is to utilize Catalyst promote DNA and protein and substrate surface combination so that substrate surface combine amount of DNA or albumen quality with The DNA or protein concentration being initially added have preferably dependence and corresponding relationship, and then make DNA or egg fixed in DNA chip It is white quality controllable, improve the co-modified quality of protein and effect.If being appreciated that DNA or protein in conjunction with substrate surface It is insufficient, then it is easy to cause the amount of DNA combined in same batch or the DNA chip of different batches or albumen quality unstable, it can not Meet the production and use demand of High Availabitity DNA chip；The application, which passes through, increases weak Passivation Treatment step, and in weak Passivation Treatment It is middle to use catalyst, so that DNA and protein efficiently, is adequately combined in substrate, favorable repeatability, and DNA and egg The fixed amount of white matter is controllable.
In the application, the reaction solution of weak Passivation Treatment can using the buffer for being suitble to corresponding catalyst, if to DNA and Substrate does not have adverse effect, and can promote the combination of itself and substrate surface；The dosage of catalyst, reaction temperature, the time, PH value etc. can be adjusted according to specifically used catalyst type, be not specifically limited herein.
Optional, catalyst is surfactant.
Optional, surfactant is selected from cetyl trimethylammonium bromide, double octadecyl bromination ammoniums, cetyl three At least one of ammonio methacrylate, dodecyl trimethyl ammonium bromide and ammonium bromide and tetraoctyl ammonium bromide.
It should be noted that surfactant is cationic surface active agent in a kind of implementation of the application, tool Body is that the covalent bond of the chemical modification group of amino group and substrate surface of DNA or protein is promoted to generate, therefore, the application A kind of implementation in, specifically using surfactant cetyl trimethylammonium bromide (abbreviation CTAB), it will be understood that its It has the surfactant of similar functions, such as double octadecyl bromination ammoniums, hexadecyltrimethylammonium chloride, dodecyl Trimethylammonium bromide and ammonium bromide and tetraoctyl ammonium bromide etc. can also promote amino group and substrate surface chemical modification group to react, together Sample is suitable for the application, is not limited only to CTAB.
Optional, in the reaction solution of weak Passivation Treatment, the concentration of surfactant is 1-25mmol/L, preferably 10mmol/L。
Optional, the reaction condition of weak Passivation Treatment is 35 DEG C of -40 DEG C of reaction 2h-5h.
Optional, in the preparation method of the application, the protein added in the reaction solution of weak Passivation Treatment is also cow's serum Albumin.
Optional, in the preparation method of the application, small numerator modified reagent is also added in the reaction solution of weak Passivation Treatment, Using small numerator modified reagent and substrate surface association reaction, small numerator modified reagent is fixed on substrate surface and is formed resists non-spy Anisotropic adsorption layer.
It will be understood to those skilled in the art that the association reaction of small numerator modified reagent and substrate surface can be covalently Association reaction is also possible to Non-covalent binding reaction；A kind of implementation small molecular modification reagent of the application passes through covalent Key is incorporated in substrate surface, forms non-specific adsorption layer.
Optional, in the reaction solution of weak Passivation Treatment, the concentration of small numerator modified reagent is 15-45mmol/L, preferably 30mmol/L。
It should be noted that the application adds small numerator modified reagent in the reaction solution of weak Passivation Treatment, small molecule is repaired Decorations reagent can be incorporated in substrate surface, form anti-non-specific adsorption layer, co-modified together with albumen, play and resist non-specific suction Attached effect.Especially in the preferred embodiment of the application, using taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoric acid silk ammonia The chemical modification group of the small numerator modified reagent such as acid, these small numerator modified reagents and substrate has efficiently and controllability is strong Binding ability, also, favorable repeatability of the small numerator modified reagent in conjunction with substrate, can reliable and stable preparation resist non-spy The DNA chip of the anisotropic good High Availabitity of adsorption effect solves current small numerator modified generally existing poor controllability, can weigh The problems such as renaturation is weak.
Optional, in a kind of implementation of the application, the preparation method of the application specifically includes following steps,
Fixing process carries out under constant temperature conditions including contacting the fixer containing DNA and protein with substrate surface It is fixed；In general, fixer is the Na of 0.25mol/L2CO3/NaHCO3, pH 9.78, wherein the concentration of DNA is generally 0.01- 0.4nmol/L, the concentration of protein are generally 10-100 μm of ol/L, and the temperature of fixing process is 37 DEG C or so, and the processing time exists 30min or so, conditions above can be not specifically limited herein for reference；Wherein, " Na2CO3/NaHCO3" indicate by Na2CO3With NaHCO3The fixer of composition, the proportion of the two are not specifically limited herein with reference to conventional DNA chip fixer；
Weak Passivation Treatment including making the reaction solution containing catalyst and protein, or contains catalyst, small molecule simultaneously The reaction solution for modifying reagent and protein, contacts with the substrate after fixing process, carries out weak passivation under constant temperature conditions；The application Weak Passivation Treatment, wherein small numerator modified reagent, which is capable of fixing, forms anti-non-specific adsorption layer in substrate surface, catalysis Agent, especially surfactant can be such that DNA or protein is sufficiently combined with the modification group of substrate surface；It is appreciated that small The concentration of molecular modification reagent and processing time can all influence its amount for being fixed on substrate surface, and concentration is higher, handles the time more The amount that long corresponding small numerator modified reagent is fixed on substrate surface is bigger；Likewise, the concentration of surfactant is higher, processing Time is longer, makes DNA or protein and the well-bound effect of substrate surface chemical modification group better accordingly；Specifically may be used Be not specifically limited depending on production or product demand herein；In a kind of implementation of the application, weak Passivation Treatment Reaction solution is the 0.25mol/L of the surfactant containing 10nM, the small numerator modified reagent of 30mM and 10-100 μM of protein Na2CO3/NaHCO3, it is weak blunt that pH is passed through reaction solution progress between 9.58-10.53, using fluid device in chip channel Change processing, the volume of circulation solution are 1mL, and the speed of fluid is 1mL/min, and reaction time 3h, reaction temperature is 37 DEG C； Conditions above can be not specifically limited herein for reference；
Passivation Treatment, including being cleaned to the substrate of weak Passivation Treatment using passivating solution, then make passivating solution with it is weak blunt Change the contact of treated substrate surface, is passivated under constant temperature conditions；In general, passivating solution is the K of 1mol/L2HPO4/ KH2PO4, pH 9.0；Wherein, " K2HPO4/KH2PO4" indicate by K2HPO4And KH2PO4The passivating solution of composition, the proportion ginseng of the two Conventional DNA chip passivating solution is examined, is not specifically limited herein；In a kind of implementation of the application, existed using fluid device Be passed through in chip channel reaction solution be fixed, weak passivation, the reaction such as passivation, the condition of Passivation Treatment is, flows into passivating solution Number be 3-4 time, the volume flowed into every time is 500 μ L, and the speed of fluid is 1mL/min, and the interval time flowed into every time is 1800 seconds, in entire passivating process, temperature was kept for 37 DEG C, and conditions above can be not specifically limited herein for reference；
Sulfydryl Seal treatment, including first using go back original reagent by two in the protein adsorbed in the substrate after Passivation Treatment Sulfide linkage reduction, generates the sulfydryl of reproducibility, then again using the sulfydryl in sulfydryl closed reagent closed protein matter；At sulfydryl closing The purpose of reason is the free sulfhydryl group in closed protein matter, it will be understood that the existing closed protein matter free sulfhydryl group of capable of playing Processing scheme can be used for reference for the application；In a kind of implementation of the application, using fluid device in chip channel Reaction solution is passed through to be reacted, sulfydryl Seal treatment concrete scheme is first to be passed through in the reduction reaction liquid containing go back original reagent, Its ingredient is 150mM TrispH8.0,100mM NaCl and 30mM go back original reagent, and the volume for the solution that circulates is 1mL, fluid Speed is 1mL/min, and reaction time 10-30min, reaction temperature is 37 DEG C；Then it is passed through the envelope of the closed reagent containing sulfydryl again Reaction solution is closed, ingredient is 150mM HEPES pH 8.5,100mM NaCl and 30mM sulfydryl closed reagent, and circulate solution Volume is 1mL, and the speed of fluid is 1mL/min, and reaction time 10-30min, reaction temperature is 37 DEG C；Conditions above for With reference to being not specifically limited herein；
Washing obtains DNA chip, and including sequentially being washed to the substrate of Passivation Treatment using three kinds of cleaning solutions, every kind is washed Washing liquid at least washed once, and three kinds of cleaning solutions are sequentially RI-05, RI-06, RI-07 according to sequence is used, wherein RI-05 is phosphorus Acid buffer, RI-06 are the mixed solution of HEPES buffer solution and NaCl solution composition, and RI-07 is distilled water.After Passivation Treatment It is well known in the art for wash to DNA chip, and wherein RI-05, RI-06, RI-07 are also conventional cleaning solution, in general, Every kind of cleaning solution needs to wash repeatedly 3 times.Wherein, HEPES, that is, 4- hydroxyethyl piperazineethanesulfonic acid.
The application of the application disclosed on one side again using the DNA chip of the application in nucleic acid or Protein Detection analysis. Nucleic acid or Protein Detection analysis include sequencing analysis, hybridization analysis, immunoassay, SNP detection and analysis etc..
It should be noted that the DNA chip in the application, refers to that chip base surface secures DNA, it is possible to understand that It is that, in this application if not specializing DNA type, DNA if refers to the substance containing DNA sequence dna, as DNA can Containing nucleotide derivative, nucleotide analog, containing fluorescent marker or containing nucleotide sequence and amino acid sequence simultaneously Column.
It should be noted that chip base surface in this application has a chemical modification, which contains can be with The active group of DNA reaction, is fixed on substrate surface for DNA by reacting between active group and DNA.
The beneficial effects of the present application are as follows:
Protein is incorporated in substrate surface by the co-modified DNA chip of the albumen of the application, not only has method easy, former The advantages such as material source is wide, work well, overall cost is low, moreover, in one implementation, by covalent bond by proteinaceous solid It is fixed, it is more firm, and protein is not easy desorption, avoids non-specific adsorption resulting from.
In a kind of improvement project of the application, further by closing the sulfydryl on co-modified albumen, enhancing albumen is total Anti- non-specific adsorption function of the modifying DNA chip to the molecule containing sulfydryl；Also, it is used when carrying out sulfydryl closing Closed reagent introduces more hydrophilic radicals or negative electricity group, further enhances the anti-non-specific adsorption ability of DNA chip.
The preparation method of the co-modified DNA chip of the albumen of the application increases weak passivation step in a kind of improvement project Suddenly, using catalyst, especially surfactant, promote DNA and protein that can more fully repair with the chemistry of substrate surface Group reaction is adornd, so that DNA is effectively fixed in substrate；DNA fixed quality and efficiency are not only increased, egg is improved White co-modified effect；Moreover, making the DNA fixed amount in DNA chip highly controllable, and favorable repeatability.
In another improvement project of the application, small numerator modified reagent is added in weak passivation step, utilizes substrate pair Efficient, the controllable absorption of small numerator modified reagent is fixed, and the anti-non-specific adsorption effect of DNA chip is further enhanced.
Detailed description of the invention
Fig. 1 is the structural schematic diagram that chip base is encapsulated in the embodiment of the present application；
Fig. 2 be in the embodiment of the present application DNA chip to the absorption result comparison diagram of the protein in test system；
Fig. 3 is that DNA chip compares the absorption result of the sulfydryl small-molecule substance in test system in the embodiment of the present application Figure；
Fig. 4 is the structural schematic diagram of substrate of glass in the embodiment of the present application.
Specific embodiment
The existing co-modified DNA chip of albumen mainly utilizes physical absorption, and protein is adsorbed on substrate surface, is played Anti-protein adsorption effect；But physical absorption is not firm, is easy desorption.In this regard, the proposition of the application creativeness, passes through Proteinaceous solid is scheduled on substrate surface by covalent bond, and not only fixation is more firm in this way, but also is not easy desorption, so as to avoid because Non-specific adsorption signal caused by desorption.
In a kind of implementation of the application, DNA amino group is reacted with the active group of substrate surface.Substrate surface Chemical modification structures are as shown in figure 4, wherein R1 represents the alkane chain molecule that end is connected with active reactive group, wherein active group Group is preferably at least one of epoxy group, aldehyde radical, carboxyl, n-hydroxysuccinimide and diaminobenzene anilid.This It is covalent that the catalyst of application can promote the amino group of DNA to react generation with the active group in chemical modification shown in Fig. 4 Key, so that DNA be made to be sufficiently secured within substrate surface.
In addition, the study found that the co-modified DNA chip of existing albumen is to cysteine (Cys), homocysteine (Hcy) The relatively high adsorption of the small-molecule substances such as the nucleic acid molecule with reduced glutathione (GSH) and containing sulfydryl, mainly because To have a large amount of free sulfhydryl group therefore being capable of half Guang of nonspecific absorption on the co-modified protein as closed reagent The sulfydryls small molecule such as propylhomoserin.Based on the above understanding, the proposition of the application creativeness carries out sulfydryl envelope to the substrate after Passivation Treatment Processing is closed, the free sulfhydryl group of the protein in substrate is closed, even avoids the co-modified DNA chip of albumen to sulfydryl to reduce The absorption of small-molecule substance reduces the nonspecific signals of DNA chip.
In the further improvement project of the application, the further creative proposition of the application, using catalyst, especially Surfactant, the principle that DNA or protein can be made more fully to react with the chemical modification group of substrate surface, in DNA During prepared by chip or protein-chip, i.e., after fixing process, weak Passivation Treatment is introduced, using surfactant, DNA or protein are more effectively fixed in substrate.Due to the use of surfactant, DNA or protein and base The chemical modification group reaction of bottom surface is more abundant, not only increases DNA or proteinaceous solid is scheduled on the efficiency and matter of substrate surface Amount, moreover, making on biochip the DNA or protein compression that are initially added in the amount and fixer of fixed DNA or protein Degree has good correlation, so that it is controllable and reproducible to realize DNA or albumen quality fixed on biochip.This The biochip of only preparation high-quality is laid a good foundation, but also can satisfy the production requirement of customization.
In the further improvement scheme of the application, small numerator modified reagent also also added in weak Passivation Treatment, it will Small numerator modified reagent is fixed in substrate, the further non-specific adsorption for reducing DNA chip.Also, use taurine, Aminopropyl sulfonic acid, serine, glutamic acid and the small numerator modified reagent of phosphoserine, with the epoxy group of substrate have efficiently, And the binding ability that controllability is strong, also, favorable repeatability of the small numerator modified reagent in conjunction with substrate, solve existing small point Son modification poor controllability, the weak problem of repeatability, the height for preparing anti-non-specific adsorption and working well that can be reliable and stable Available DNA chip.
Some vocabulary involved in the embodiment of the present application carry out as described below:
AT-01: ingredient is 0.25M Na2CO3/NaHCO3, 0.6mM CTAB (i.e. cetyl trimethylammonium bromide), pH9.78；
RI-04: ingredient is 1M K2HPO4/KH2PO4, pH 9.0
RI-05: ingredient is PH7.4PBS solution；
RI-06: the mixed solution of the NaCl solution composition of HEPES buffer solution and 150mM that ingredient is 150mM；
RI-07: ingredient is distilled water；
Dot/FOV: observation area is the bright spot number of 110 × 110 μ ms.
The application is described in further detail below by specific embodiment.Following embodiment only to the application carry out into One step explanation, should not be construed as the limitation to the application.
Embodiment one
DNA is fixed on substrate table by the amino group of DNA in the substrate of glass that surface has epoxy silane by this example Face, while the co-modified DNA chip of albumen of this example is formed as co-modified albumen using bovine serum albumin(BSA).This example is right respectively It is more co-modified than addition bovine serum albumin(BSA) and do not add the co-modified influence to DNA chip of bovine serum albumin(BSA).
By the way of this example is fixed using " in channel ", DNA is fixed in chip base, i.e., first encapsulates chip base, then Various reaction reagents, washing reagent are each led into the chip channel after encapsulation using fluid device, realization fixing process, The chemical reaction of Passivation Treatment etc..As shown in Figure 1, forming mutually independent each chip channel, each core after chip base encapsulation Piece channel independent can carry out each reaction, and Fig. 1 show the encapsulation chip base in 8 channels, and the specification in channel is long × wide Chip base can also be packaged by × high 90mm × 1.8mm × 0.1mm according to different packaging technology or fluid device 16 channels can independently make the DNA of 16 kinds of different modifyings in a DNA chip.
The preparation method of the DNA chip of this example is specific as follows:
(1) fixing process is passed through fixed reaction solution in the channel of chip base, and reaction, the fixation of this example is fixed Reaction solution is the fixer AT-01 containing 0.2nM DNA and 30 μM of bovine serum albumin(BSA) (abbreviation BSA)；Wherein, contained DNA 3 ' ends simultaneous with amido modified NH2It is modified with Cy3 fluorogen；The ingredient of AT-01 is the Na of 0.25M2CO3/NaHCO3, 0.6mM CTAB, pH 9.78, the volume of the solution that circulates are 1mL, and the speed of fluid is 1mL/min, reaction time 30min, instead Answering temperature is 37 DEG C；
As control, this example is provided with the channel that BSA is not added in fixed reaction solution；
Wherein, DNA and BSA are the relationships of competitive Adsorption, and the reactivity worth on DNA and surface is much better than BSA, so even if adding The BSA for having added 15000 times of concentration, to the modification of DNA still without significant impact；In the preparation method of this example, in bearing There is addition BSA when reason and weak Passivation Treatment, the co-modified effect of such albumen is more preferable, certainly, in the case of lower requirements, Can also BSA only be added in fixing process step；
(2) weak Passivation Treatment is passed through weak passivation reaction liquid, carries out weak passivation step, and the weak passivation reaction liquid of this example is exactly The Na of 0.25M containing 10mM CTAB, 30mM taurine (Taurine) and 30 μM of BSA2CO3/NaHCO3, pH 9.58- Between 10.53, this example is specially pH 9.78；The volume that circulation solution is arranged is 1mL, and the speed of fluid is 1mL/min, reaction Time is 3h, and reaction temperature is 37 DEG C；
Wherein, it is not added with the channel of BSA when fixing process, in reaction solution, does not add BSA equally in weak Passivation Treatment, Remaining component, dosage and channel parameters setting are all identical, as control；
(3) weak passivating solution is washed away, specifically, washing times are 3 times with passivating solution RI-04, the volume being passed through every time is 1mL, the speed of fluid are 1mL/min, and temperature is kept for 37 DEG C when washing；The RI-04 ingredient of this example is the K of 1M2HPO4/KH2PO4, pH 9.0；
(4) Passivation Treatment, specifically, washing times are 3-4 times with passivating solution RI-04, the volume flowed into every time is 500 μ L, the speed of fluid are 1mL/min, and the interval time flowed into every time is 1800s, and in entire passivating process, temperature is kept for 37 DEG C； The RI-04 ingredient of this example is the K of 1M2HPO4/KH2PO4, pH 9.0；
(5) chip after Passivation Treatment is washed, including using three kinds of solution washings, every kind of solution is washed 3 times, is passed through every time Volume be 1mL, the speed of fluid is 1mL/min, and temperature is kept for 37 DEG C when washing；Three kinds of cleaning solutions according to use sequence sequentially For RI-05, RI-06, RI-07, wherein RI-05 is the phosphate buffer of pH7.4, the HEPES buffer solution that RI-06 is 150mM with The mixed solution of the NaCl solution composition of 150mM, RI-07 is distilled water.
After three kinds of solution wash, the DNA chip of this example is obtained after normally drying or dry.
Situation is adsorbed using the protein non-specific that single molecular fluorescence imaging technique evaluates the DNA chip of this example preparation, Specifically, it is passed through the Klenow Fragment of fluorescent marker on the DNA chip surface of preparation, detection DNA chip adsorption The fluorescent marker quantity of Klenow Fragment is counted with the fluorescent marker of the Klenow Fragment of unit area and is characterized The protein non-specific of DNA chip adsorbs situation.This example has specifically counted the fluorescence points in 110 × 110 μm of regions.
Test results are shown in figure 2 for this example DNA chip and its check experiment, and in Fig. 2, abscissa None is to be not added with The DNA chip of BSA, BSA indicate that addition BSA carries out the co-modified DNA chip of albumen of co-modified preparation, and ordinate indicates corresponding Fluorescence points.Fig. 2's the results show that its non-specific adsorption of the DNA chip points prepared after co-modified using BSA are about 500Dot/FOV, and it is not greater than 6000Dot/FOV using the non-specific adsorption points of the DNA chip of the co-modified preparation of BSA；It says The co-modified DNA chip that can greatly reduce of bright BSA there is good anti-protein adsorption to make the non-specific adsorption of protein With.
Embodiment two
This example DNA chip prepares material and process is the same as example 1, the difference is that increasing after Passivation Treatment Sulfydryl Seal treatment, using sulfydryl in sulfydryl closed reagent closed protein matter, playing reduces the co-modified DNA chip pair of albumen The non-specific adsorption of sulfydryl small molecule acts on.
This example DNA chip specific the preparation method is as follows:
(1) fixing process is passed through fixed reaction solution in the channel of chip base, and reaction, the fixation of this example is fixed Reaction solution is the fixer AT-01 containing 0.2nM DNA and 30 μM of bovine serum albumin(BSA) (BSA)；Wherein, the 3 ' of contained DNA End is simultaneous with amido modified NH2It is modified with Cy3 fluorogen；The ingredient of AT-01 is the Na of 0.25M2CO3/NaHCO3, 0.6mM CTAB, pH 9.78, the volume for the solution that circulates are 1mL, and the speed of fluid is 1mL/min, reaction time 30min, reaction temperature It is 37 DEG C；
(2) weak Passivation Treatment is passed through weak passivation reaction liquid, carries out weak passivation step, and the weak passivation reaction liquid of this example is exactly The Na of 0.25M containing 10mM CTAB, 30mM taurine (Taurine) and 30 μM of BSA2CO3/NaHCO3, pH 9.58- Between 10.53, this example is specially pH9.78；The volume that circulation solution is arranged is 1mL, and the speed of fluid is 1mL/min, when reaction Between be 3h, reaction temperature be 37 DEG C；
(3) weak passivating solution is washed away, specifically, washing times are 3 times with passivating solution RI-04, the volume being passed through every time is 1mL, the speed of fluid are 1mL/min, and temperature is kept for 37 DEG C when washing；The RI-04 ingredient of this example is the K of 1M2HPO4/KH2PO4, pH 9.0；
(4) Passivation Treatment, specifically, washing times are 3-4 times with passivating solution RI-04, the volume flowed into every time is 500 μ L, the speed of fluid are 1mL/min, and the interval time flowed into every time is 1800s, and in entire passivating process, temperature is kept for 37 DEG C； The RI-04 ingredient of this example is the K of 1M2HPO4/KH2PO4, pH 9.0；
(5) sulfydryl Seal treatment, a) is passed through the reaction solution containing go back original reagent TCEP, Tris that ingredient is 150mM, The TCEP of the NaCl and 30mM of pH 8.0,100mM；The purpose of this step is the disulfide bond in reduction system, generates reproducibility Sulfydryl, conducive to the closing of next step；The volume of circulation solution is 1mL, and the speed of fluid is 1mL/min, reaction time 10- 30min, reaction temperature are 37 DEG C；B) be passed through sulfydryl closed reagent reaction solution, sulfydryl closed reagent can using iodoacetamide or Person's iodoacetic acid, this example specifically use iodoacetamide, and reaction solution ingredient is HEPES, pH 8.5 of 150mM, the NaCl of 100mM and 30mM iodoacetamide；The volume of circulation solution is 1mL, and the speed of fluid is 1mL/min, reaction time 10-30min, this example 30min is specifically reacted, reaction temperature is 37 DEG C；
(6) chip after sulfydryl Seal treatment is washed, including using the washing of three kinds of solution, every kind of solution washs 3 times, every time The volume being passed through is 1mL, and the speed of fluid is 1mL/min, and temperature is kept for 37 DEG C when washing；Three kinds of cleaning solutions according to use sequence It is sequentially RI-05, RI-06, RI-07, wherein RI-05 is the phosphate buffer of pH7.4, and the HEPES that RI-06 is 150mM is buffered The mixed solution of the NaCl solution of liquid and 150mM composition, RI-07 is distilled water.
After three kinds of solution wash, the DNA chip of this example is obtained after normally drying or dry.
The DNA chip of this example preparation is evaluated to the nucleic acid molecule containing disulfide bond using single molecular fluorescence imaging technique Situation is adsorbed, specifically, being passed through the nucleic acid molecule containing disulfide bond of fluorescence Cy3 label, inspection on the DNA chip surface of preparation The Cy3 amount of fluorescence of DNA chip adsorption is surveyed, disulfide bond is contained with the Cy3 fluorescence points characterization DNA chip of unit area Nucleic acid molecule adsorb situation.This example has specifically counted the fluorescence points in 110 × 110 μm of regions.Meanwhile testing embodiment One adsorbs situation using the disulfide bond small molecule of the BSA DNA chip prepared after co-modified, as a comparison；Embodiment one uses BSA The DNA chip prepared after co-modified is not by sulfydryl Seal treatment.The nucleotide containing disulfide bond point that this example uses Son refers to EP2607369B1, and specific molecular structure refers to Fig. 2 of the patent application.
Test results are shown in figure 3, and abscissa " None " is co-modified using BSA but without carrying out sulfydryl closing in Fig. 3 The DNA chip of preparation, the i.e. co-modified DNA chip of the BSA of embodiment one are handled, " Block " is to have carry out mercapto using BSA is co-modified The DNA chip of base Seal treatment preparation, the i.e. DNA chip of this example.Fig. 3's the results show that do not carry out sulfydryl Seal treatment DNA chip, non-specific adsorption points are greater than 3000Dot/FOV, and using the non-specific of the DNA chip of sulfydryl Seal treatment Property absorption points be about 500Dot/FOV；Illustrate that sulfydryl Seal treatment can greatly reduce DNA chip to the small molecule containing sulfydryl The non-specific adsorption of substance.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off Under the premise of from the application design, a number of simple deductions or replacements can also be made.

Claims (10)

1. a kind of DNA chip that albumen is co-modified, it is characterised in that: the albumen is co-modified to be incorporated in DNA chip by protein Substrate surface is formed.
2. DNA chip according to claim 1, it is characterised in that: the protein is bovine serum albumin(BSA).
3. DNA chip according to claim 1 or 2, it is characterised in that: the sulfydryl on the protein is in closed State.
4. DNA chip according to claim 1 or 2, it is characterised in that: the substrate surface has small numerator modified reagent The anti-non-specific adsorption layer formed；
Optional, the small numerator modified reagent is in taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine At least one.
5. a kind of preparation method for the DNA chip that albumen is co-modified, it is characterised in that: including fixing process, the fixing process Including contacting the fixer containing DNA with substrate surface, DNA is fixed on substrate surface, contains albumen in the fixer It is co-modified to form albumen in conjunction with substrate surface for matter, the protein；
Optionally, the albumen is bovine serum albumin(BSA)；
Optional, the substrate surface is modified with epoxy silane, and the protein and epoxy silane reaction bonded are in substrate Surface.
6. preparation method according to claim 5, it is characterised in that: it further include sulfydryl Seal treatment, the sulfydryl closing Processing is by the sulfydryl closing on the fixed protein of substrate surface；
Optional, the sulfydryl Seal treatment specifically includes, and using go back original reagent by the disulfide bond reduction in the protein, gives birth to At the sulfydryl of reproducibility, then the sulfydryl for using sulfydryl closed reagent to close reproducibility generated；
Optionally, the go back original reagent is three (2- carboxyethyl) phosphines, three (3- hydroxypropyl) phosphine, dithiothreitol (DTT) and mercaptoethanols At least one of；
Optional, the sulfydryl closed reagent is at least one in iodoacetamide, iodoacetic acid, maleimide and epoxy prapanol Kind.
7. preparation method according to claim 5 or 6, it is characterised in that: further include weak Passivation Treatment, at the weak passivation Reason includes contacting the weak passivation reaction liquid containing catalyst and protein with the chip after fixing process, and the catalyst can Promote DNA and protein in conjunction with substrate surface, DNA and protein is made to be sufficiently secured within substrate surface；
Optionally, the catalyst is surfactant；
Optional, the surfactant is selected from cetyl trimethylammonium bromide, double octadecyl bromination ammoniums, cetyl three At least one of ammonio methacrylate, dodecyl trimethyl ammonium bromide and ammonium bromide and tetraoctyl ammonium bromide；
Optional, in the weak passivation reaction liquid, the concentration of surfactant is 1-25mmol/L；
Optional, in the weak passivation reaction liquid, the concentration of surfactant is 10mmol/L；
Optionally, the protein is bovine serum albumin(BSA).
8. preparation method according to claim 7, it is characterised in that: also repaired comprising small molecule in the weak passivation reaction liquid Reagent is adornd, the small numerator modified reagent forms anti-non-specific adsorption layer in conjunction with substrate surface；
Optional, the concentration of the small numerator modified reagent is 15-45mmol/L；
Optional, the concentration of the small numerator modified reagent is 30mmol/L；
Optional, the reaction condition of the weak Passivation Treatment is 35 DEG C of -40 DEG C of reaction 2h-5h.
9. preparation method according to claim 7, it is characterised in that: further include Passivation Treatment, the Passivation Treatment includes It is contacted using passivating solution with the substrate surface after weak Passivation Treatment, is passivated under constant temperature conditions；
Optional, it further include washing step, the washing step includes using three kinds of cleaning solutions sequentially to the substrate of Passivation Treatment It is washed, every kind of cleaning solution at least washed once, and three kinds of cleaning solutions are sequentially RI-05, RI-06, RI- according to sequence is used 07, wherein RI-05 is PBS buffer solution, and RI-06 is the mixed solution of HEPES buffer solution and NaCl solution composition, and RI-07 is double Steam water.
10. application of the DNA chip according to claim 1-4 in nucleic acid or Protein Detection analysis.
CN201811191611.3A 2018-10-12 2018-10-12 A kind of DNA chip and preparation method thereof that albumen is co-modified Pending CN109610007A (en)

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