CN109609333A - A kind of biochip and preparation method thereof - Google Patents
A kind of biochip and preparation method thereof Download PDFInfo
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- CN109609333A CN109609333A CN201811191571.2A CN201811191571A CN109609333A CN 109609333 A CN109609333 A CN 109609333A CN 201811191571 A CN201811191571 A CN 201811191571A CN 109609333 A CN109609333 A CN 109609333A
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Abstract
This application discloses a kind of biochips and preparation method thereof.The biochip of the application is DNA chip or protein-chip, and the anti-non-specific adsorption layer which there is small numerator modified reagent to be incorporated in substrate surface formation, small numerator modified reagent is at least one of the compound of general formula shown in formula one；R in formula one1、R2And R3It is respectively selected from hydrophilic radical or hydrogen atom.The biochip of the application, anti- non-specific adsorption layer is formed as small numerator modified reagent using one compound of formula, the adsorption layer can effectively prevent that the non-Characteristic Adsorption of nucleotide used is sequenced, improve Characteristics Detection signal ratio, non- special signal interference is reduced, and the anti-non-specific adsorption layer bonding strength of the biochip in the application is high, not easily to fall off；Moreover, the small numerator modified reagent reaction of the application is controllable, favorable repeatability, so that the biochip quality of the application is high, reproducible, with high availability, and especially suitable for Single Molecule Detection.
Description
Technical field
This application involves biochip fields, more particularly to a kind of biochip and preparation method thereof.
Background technique
Molecule detection can accomplish following two points: 1) identifying the signal of individual molecule, rather than molecular population is flat
Equal signal, 2) the monomolecular dynamic process of tracking, the details of molecular motion or reaction is provided.Have benefited from above-mentioned superiority
Can, the highest attention of academia and industrial circle is received based on monomolecular detection and identification technology.Wherein, single-molecule DNA
Sequencing technologies are especially by extensive concern and research.Currently, the DNA single-molecule sequencing technology of mainstream includes: big desert gene
The Nanopore technology of TIRF technology, the ZMW technology of Paciffic and Oxford company.The realization of these sequencing technologies all depends on
In the sequence testing chip of High Availabitity, i.e. DNA chip；Therefore, the performance requirement of DNA chip is included at least: 1) good and repeatable
Unimolecule modification efficiency, such as TIRF in DNA；2) controllable, extremely low non-specific adsorption.Wherein, controllable non-spy
An important factor for opposite sex absorption is experiment success or failure.During DNA sequencing, special single molecule signals and non-specific absorption are believed
It can number all be identified and collect by instrument, non-specific absorption signal can seriously interfere the identification of nonspecific signal, to the later period of instrument
Signal processing constitutes great obstacle.
Therefore, chip of the preparation with anti-non-specific adsorption has important value to Molecular Detection field.
Summary of the invention
The purpose of the application is to provide a kind of improved biochip and preparation method thereof.
The application uses following technical scheme:
The one side of the application discloses a kind of biochip, and the biochip of the application is DNA chip or protein core
The substrate surface of piece, the biochip has anti-non-specific adsorption layer, and anti-non-specific adsorption layer is by small numerator modified reagent
It is incorporated in substrate surface to be formed, which is at least one of the compound of general formula shown in formula one；
Formula one
Wherein, R1、R2And R3It is respectively selected from hydrophilic radical or hydrogen atom.
It should be noted that the biochip of the application, key is the compound conduct using general formula shown in formula one
Small numerator modified reagent forms the anti-non-specific adsorption layer of chip.Small numerator modified reagent used by the application, and it is existing
Small numerator modified reagent compare, have many advantages, such as that controllability is good, favorable repeatability, can accurately control non-specific adsorption,
To obtain the DNA chip or protein-chip of the Single Molecule Detection of High Availabitity.The small numerator modified reagent of the application, refers to phase
To molecular mass in 1000 small molecule compounds below.
Optional, in the compound of general formula shown in formula one, hydrophilic radical is negative electric group.
Optional, hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic acid group, hydroxyl or amide groups
Group.
Optional, the compound of general formula shown in formula one is taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoric acid silk
At least one of propylhomoserin.
Optional, the substrate surface of biochip is modified with epoxy silane.
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.
The another aspect of the application discloses a kind of preparation method of biochip, which is DNA chip or egg
White matter chip, the preparation method of the application include being repaired using at least one of compound of general formula shown in formula one as small molecule
Decorations reagent is fixed on substrate surface in conjunction with substrate surface, forms anti-non-specific adsorption layer；
Formula one
Wherein, R1、R2And R3It is respectively selected from hydrophilic radical or hydrogen atom.
It should be noted that the preparation method of the application, key is in the compound using general formula shown in formula one
At least one is used as small numerator modified reagent, how to be integrated to substrate surface, Ke Yican as specific small numerator modified reagent
Existing anti-non-specific adsorption layer preparation process is examined, is not specifically limited herein.But in order to reach preferably resist it is non-specific
Property adsorption layer in the preferred embodiment of the application, is improved and has been optimized to the preparation method of biochip, be detailed in subsequent side
Case.
Optional, in the preparation method of the application, hydrophilic radical is negative electric group.
Optional, in the preparation method of the application, hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic
Acid groups, hydroxyl or amide group.
Optional, in the preparation method of the application, the compound of general formula shown in formula one is taurine, aminopropyl sulfonic acid, silk
At least one of propylhomoserin, glutamic acid and phosphoserine.
Optional, in a kind of implementation of the application, anti-non-specific adsorption layer is to use to contain small numerator modified examination
The passivation reaction liquid of agent is formed during Passivation Treatment.
It should be noted that the passivation reaction liquid of Passivation Treatment can use the conventional use of passivating solution of Passivation Treatment,
This is not specifically limited；The reaction condition of Passivation Treatment refers to existing passivation process.
Optional, in passivation reaction liquid, the concentration of small numerator modified reagent is 0.01-0.2mol/L.
It should be noted that the small numerator modified reagent in the reaction solution of Passivation Treatment, its object is to be integrated to substrate
Surface forms anti-non-specific adsorption layer；It is appreciated that being capable of forming as long as the small numerator modified reagent added with the application
Anti- non-specific adsorption layer, in order to make the anti-non-specific adsorption layer to be formed that there is better effect, the optional passivation of the application
The concentration of small numerator modified reagent in reaction solution is 0.01-0.2mol/L.It is appreciated that other than the range, such as concentration
It is too low, effective anti-non-specific adsorption layer possibly can not be formed, concentration is too high to will cause reagent waste, fights non-specific inhale
Attached layer effect promoting effect is little.
Optional, reaction promoter is added in the reaction solution of Passivation Treatment, also to promote small numerator modified reagent and base
The association reaction of bottom surface.
Optional, reaction promoter is in benzyltriethylammoinium chloride, phenyl trimethicone ammonium chloride and tetrabutylammonium chloride
At least one.
Optional, concentration of the reaction promoter in passivation reaction liquid is 10-50mmol/L.
It should be noted that the effect of reaction promoter is exactly the small numerator modified reagent and substrate surface for promoting the application
Association reaction, under the action of reaction promoter, the combination for further improving small numerator modified reagent and substrate surface is imitated
Rate and quality keep reaction more controllable, repeated more preferable, therefore, also add in the reaction solution of Passivation Treatment in optional scheme
There is reaction promoter.It is appreciated that according to different production or product design demand, such as reaction controllability is required opposite
In lower situation, reaction promoter can also not used.
It should also be noted that, benzyltriethylammoinium chloride be the application a kind of implementation in confirm it is applicatory
Reaction promoter, however not excluded that can also be using other reaction promoters, as long as the anti-non-specific adsorption of small molecule can be promoted
The association reaction of reagent and substrate surface.
Optional, the preparation method of the application further includes that weak Passivation Treatment is carried out before Passivation Treatment；Weak Passivation Treatment
It is included in weak passivation reaction liquid and small numerator modified reagent is added, the combination using small numerator modified reagent and substrate surface is anti-
It answers, small numerator modified reagent is fixed on substrate surface in advance.
It should be noted that the effect of weak Passivation Treatment adds small numerator modified reagent first is that passing through, in advance by small point
Son modification reagent is fixed on substrate surface, is then further consolidated by Passivation Treatment again, forms stable anti-non-specific suction
Attached layer.
Optional, in weak passivation reaction liquid, the concentration of small numerator modified reagent is 15-45mmol/L, preferably 30mmol/
L。
Optional, the reaction condition of weak Passivation Treatment is 35 DEG C of -40 DEG C of reaction 2h-5h.
Optional, weak Passivation Treatment is identical with small numerator modified reagent used in Passivation Treatment.
Optional, surfactant is also added in weak passivation reaction liquid, for promoting DNA or protein and substrate surface
Covalent bond generate, so that DNA or protein is sufficiently secured within substrate surface.
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.
Optional, concentration of the surfactant in weak passivation reaction liquid is 1-25mmol/L, preferably 10mmol/L.
It should be noted that weak Passivation Treatment step is the optional improvement project of the application, the application's is preferred
In scheme, surfactant and small numerator modified reagent are added in the weak passivation reaction liquid of weak Passivation Treatment；At weak passivation
The effect of reason, other than small numerator modified reagent is fixed on substrate surface in advance, it to be exactly logical that there are one important roles
Weak Passivation Treatment is crossed, the covalent bond reaction efficiency of DNA or protein and substrate surface is improved, makes DNA or protein more fully
Be incorporated in substrate surface, thus make on biochip the DNA being initially added in the amount and fixer of fixed DNA or protein or
Protein concentration has good correlation, so that the amount for realizing DNA or protein fixed on biochip is controllable, and again
Renaturation is good.
It should also be noted that, it is surfactant and small numerator modified examination that the weak passivation reaction liquid of weak Passivation Treatment, which is exactly,
Agent provides a reaction environment, in a kind of implementation of the application, in order to easy to operate, directlys adopt fixer as weak blunt
Change reaction solution, however not excluded that other reaction buffers can also be used.Wherein fixer is that biochip prepares common bearing
The reaction buffer of reason, is not specifically limited herein.
Optional, in a kind of implementation of the application, the preparation method of the application specifically includes following steps,
Weak Passivation Treatment, including make to contain small numerator modified reagent and surface-active containing small numerator modified reagent or simultaneously
The weak passivation reaction liquid of agent is contacted with substrate, carries out weak passivation under constant temperature conditions；The weak Passivation Treatment of the application, purpose exist
In making small numerator modified reagent be fixed on substrate surface in advance, with surfactant, also having makes DNA or egg
White matter and the well-bound effect of epoxy group；It is appreciated that the concentration of small numerator modified reagent and processing time can all influence
Its amount for being fixed on substrate surface, concentration is higher, processing time longer corresponding small numerator modified reagent is fixed on substrate surface
Amount it is bigger；Likewise, the concentration of surfactant is higher, the processing time is longer, make DNA or protein and epoxy group accordingly
The well-bound effect of group is better；It can be specifically not specifically limited herein depending on production or product demand；The application
A kind of implementation in, the weak passivation reaction liquid of weak Passivation Treatment is the small molecule of the surfactant containing 10nM and 30mM
Modify the Na of the 0.25mol/L of reagent2CO3/NaHCO3, pH is between 9.58-10.53, using fluid device in chip channel
It is passed through reaction solution and carries out weak Passivation Treatment, the volume of circulation solution is 1mL, and the speed of fluid is 1mL/min, and the reaction time is
3h, reaction temperature are 37 DEG C；Conditions above can be not specifically limited herein for reference；
Then Passivation Treatment is made including being cleaned using passivation reaction liquid to the substrate of weak Passivation Treatment containing small point
Son modifies reagent or the passivation reaction liquid simultaneously containing small numerator modified reagent and reaction promoter is contacted with the substrate after cleaning,
It is passivated under constant temperature conditions；In general, passivation reaction liquid, that is, 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 of the two is with reference to conventional DNA chip
Passivating solution is not specifically limited herein；In a kind of implementation of the application, specifically, containing small numerator modified reagent or together
The reaction solution of the small numerator modified reagent of Shi Hanyou and reaction promoter uses the Na of 0.25mol/L2CO3/NaHCO3, pH 9.58-
Between 10.53, the concentration of small numerator modified reagent is 0.01-0.2M, and reaction solution is passed through in chip channel using fluid device
Reacted, the condition of Passivation Treatment be flow into passivating solution number be 3-4 time, the volume flowed into every time be 500 μ L, flow
The speed of body is 1mL/min, and the interval time flowed into every time is 1800s, and in entire passivating process, temperature is kept for 37 DEG C, above
Condition can be not specifically limited herein for reference.
It should be noted that using special small numerator modified reagent when the key of the application is Passivation Treatment；Into
In the improvement of one step, weak Passivation Treatment is also added；As for other steps, existing DNA chip or protein core can be referred to
Piece preparation process.
Optional, the preparation method of the application further includes washing step, i.e., washs to the chip after Passivation Treatment, should
Washing step includes sequentially being washed to the substrate of Passivation Treatment using three kinds of cleaning solutions, and every kind of cleaning solution at least washs one
Secondary, three kinds of cleaning solutions are sequentially RI-05, RI-06, RI-07 according to sequence is used, wherein RI-05 is phosphate buffer, RI-06
For the mixed solution of HEPES buffer solution and NaCl solution composition, RI-07 is distilled water.DNA chip is washed after Passivation Treatment
It is well known in the art for washing, and RI-05, RI-06, RI-07 are also conventional cleaning solution, in general, every kind of cleaning solution needs to repeat
Washing 3 times.Wherein, HEPES, that is, 4- hydroxyethyl piperazineethanesulfonic acid.
Optional, the preparation method of the application further includes that processing is fixed before weak Passivation Treatment, and specifically including makes
Fixer containing DNA or protein is contacted with substrate surface, carries out DNA under constant temperature conditions or protein 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, Gu
Surely the temperature handled is 37 DEG C or so, handles the time in 30min or so, conditions above can be not specifically limited herein for reference；
Wherein, " Na2CO3/NaHCO3" indicate by Na2CO3And NaHCO3The fixer of composition, the proportion of the two is with reference to conventional DNA core
Piece fixer, is not specifically limited herein.
The application's discloses a kind of small molecule compound answering in preparation DNA chip or protein-chip on one side again
With the application includes being incorporated in substrate surface using small molecule compound as small numerator modified reagent, forms anti-non-specific suction
Attached layer, small molecule compound are at least one of the compound of general formula shown in formula one；
Formula one
Wherein, R1、R2And R3Respectively hydrophilic radical or hydrogen atom.
Optional, in small molecule compound, hydrophilic radical is negative electric group.
Optional, in small molecule compound, hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic acid group
Group, hydroxyl or amide group.
Optional, small molecule compound is in taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine
It is at least one.
It should be noted that the key of the application is to have found that some new small molecule compounds are particularly suitable for application as
The small molecule compound of the small numerator modified reagent of DNA chip or protein-chip, the application makes as small numerator modified reagent
Used time not only has the advantages that existing small numerator modified reagent, moreover, reaction is controllable, reproducible, to the list for preparing High Availabitity
The DNA chip and protein-chip of Molecular Detection have important value；Therefore, present applicant proposes these small molecule compounds works
For preparation DNA chip or the small numerator modified reagent of protein-chip, the new of anti-non-specific adsorption layer is formed in substrate surface
Purposes.
The application of the application disclosed on one side again using the application biochip in nucleic acid or Protein Detection analysis.Its
In, nucleic acid or Protein Detection analysis include sequencing analysis, hybridization analysis, immunoassay, SNP detection and analysis etc..
It should be noted that DNA chip and protein-chip in the application, refer to that the substrate surface of chip secures
DNA or protein, in this application if not specializing the type of DNA and protein, DNA is referred to containing DNA sequence dna
Or a substance of amino acid sequence, as the fixed DNA of DNA chip can containing nucleotide derivative, nucleotide analog,
Contain nucleotide sequence and amino acid sequence containing fluorescent marker or simultaneously；Protein refers to one containing amino acid sequence
Substance.
It should be noted that chip base surface in this application has chemical modification, which contains can
With the active group of DNA or albumen qualitative response, pass through reacting DNA or proteinaceous solid between active group and DNA or protein
It is scheduled on substrate surface.
The beneficial effects of the present application are as follows:
The biochip of the application forms biology using the compound of structure shown in formula one as small numerator modified reagent
The anti-non-specific adsorption layer of chip, the adsorption layer can be effectively prevented the non-Characteristic Adsorption of sequencing nucleotide used, mention
The ratio of high Characteristics Detection signal reduces the interference of non-special signal, and the anti-non-specific adsorption of the biochip in the application
Layer bonding strength is high, not easily to fall off；Moreover, the small numerator modified reagent reaction of the application is controllable, favorable repeatability, so that this Shen
Biochip quality please is high, reproducible, has high availability, and especially suitable for Single Molecule Detection.
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 is the anti-non-specific adsorption reagent of small molecule and more hydrophilic groups of single negative electrical charge group in the embodiment of the present application
The non-specific adsorption points test result comparison of the weak Passivation Treatment DNA chip of the anti-non-specific adsorption reagent of small molecule of group
Figure；
Fig. 3 is the anti-non-specific adsorption reagent of small molecule and single negative electrical charge of more negative electrical charge groups in the embodiment of the present application
The non-specific adsorption points test result comparison of the weak Passivation Treatment DNA chip of the anti-non-specific adsorption reagent of the small molecule of group
Figure；
Fig. 4 is to add or do not add the non-specific of reaction promoter Passivation Treatment DNA chip in the embodiment of the present application to inhale
Attachment number test result comparison diagram；
Fig. 5 is the structural schematic diagram of substrate of glass in the embodiment of the present application.
Specific embodiment
It is also, small point in the technology used by the special anti-non-specific adsorption layer of small numerator modified formation
For the anti-non-specific adsorption layer of son due to bonding strength height, anti-non-specific adsorption not easily to fall off and more wide spectrum etc. is significant excellent
Gesture is widely used.But current small numerator modified generally existing poor controllability, the problems such as repeatability is weak, with
As for different batches even with a batch of small numerator modified, anti-non-specific adsorption effect is all not quite similar, accordingly, it is difficult to
The DNA chip and protein-chip of the reliable and stable Single Molecule Detection for preparing High Availabitity.
The application has found during the long-term numerous studies to DNA chip and protein-chip, general formula shown in formula one
Small numerator modified reagent, for example, taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine etc., have with substrate
Efficiently and the strong binding ability of controllability, also, favorable repeatability of the small numerator modified reagent of the application in conjunction with substrate,
The DNA chip or protein-chip for preparing the High Availabitity that anti-non-specific adsorption works well that can be reliable and stable, it is especially suitable
For Single Molecule Detection.
In the further improvement project of the application, the further creative proposition of the application utilizes surfactant energy
The principle for reacting DNA or protein more fully with the chemical modification group of substrate surface, in DNA chip or protein core
During prepared by piece, surfactant is introduced, DNA or protein are more effectively fixed in substrate.Due to table
The use of face activating agent, DNA or protein react more abundant with the chemical modification group of substrate surface, not only increase DNA or
Proteinaceous solid is scheduled on the efficiency and quality of substrate surface, moreover, making the DNA of fixation or the amount of protein on biochip and consolidating
Determining the DNA being initially added in liquid or protein concentration has good correlation, to realize DNA fixed on biochip
Or albumen quality is controllable, and reproducible.This biochip for not preparing high-quality only is laid a good foundation, but also can be expired
The production requirement customized enough.
It should be noted that there are substrate surface these chemical modifications of chemical modification to contain in the biochip of the application
It can be with the active group of DNA or albumen qualitative response.In one embodiment in this application, the amino base of DNA or protein
Group reacts with active group, and active group is that epoxy group, aldehyde radical, carboxyl, n-hydroxysuccinimide and diamino benzoyl replace
One of aniline.
In one embodiment in this application, the chemical modification structures of substrate surface are as shown in figure 5, wherein R1 represents end
End is connected with the alkane chain molecule of active reactive group, and wherein active group is preferably epoxy group, aldehyde radical, carboxyl, N- hydroxyl
At least one of succinimide and diaminobenzene anilid.
In one embodiment of the application, the chemical modification of substrate surface can be with the small numerator modified reagent of the application
In conjunction with small numerator modified reagent is fixed on substrate surface, forms anti-non-specific adsorption layer.
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, pH 9.78；Wherein, CTAB is cetyl
The abbreviation of trimethylammonium bromide；
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 forms the DNA chip of this example.And during the preparation process, after fixing process, before Passivation Treatment, increase at weak passivation
Reason.This example compared the influence to DNA chip using different small numerator modified reagents.Specifically, this example is than right single negative electricity
The anti-non-specific adsorption effect of the small numerator modified reagent of the small numerator modified reagent and more hydrophilic radicals of lotus group.
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 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 0.05nM DNA and 0.6mM CTAB；Wherein, the 3 ' ends of contained DNA 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,
PH9.78, the volume for the solution that circulates are 1mL, and the speed of fluid is 1mL/min, reaction time 30min, reaction temperature 37
℃；
(2) weak Passivation Treatment, is passed through weak passivation reaction liquid, carries out " weak passivation " step, weak passivation reaction liquid be containing
The Na of the 0.25M of the small numerator modified reagent of 10mM CTAB and 30mM2CO3/NaHCO3, pH is between 9.58-10.53, this example tool
Body is pH 9.78；As a comparison, the small numerator modified reagent added in the weak passivation reaction liquid that this example passage portion uses is list
The small numerator modified reagent of one negative electrical charge group is specifically taurine (Taurine), and small molecule is not added in another part channel
It modifies reagent and carries out weak Passivation Treatment only with weak passivation reaction liquid；The volume of all channel setting circulation solution is 1mL, fluid
Speed be 1mL/min, reaction time 3h, reaction temperature be 37 DEG C；
(3) weak passivation reaction liquid is washed away, specifically, washing times are 3 times, the volume being passed through every time with passivating solution RI-04
For 1mL, the speed of fluid is 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, being passed through passivation reaction liquid is passivated processing, the passivation reaction liquid of this example be containing
The Na of the 0.25M of the small numerator modified reagent of 30mM2CO3/NaHCO3, for pH between 9.58-10.53, this example is specially pH 9.78；
The number for flowing into passivation reaction liquid is 3-4 times, and the volume flowed into every time is 500 μ L, and the speed of fluid is 1mL/min, is flowed every time
The interval time entered is 1800s, and in entire passivating process, temperature is kept for 37 DEG C；Wherein, small numerator modified in passivation reaction liquid
Reagent, corresponding Taurine when using weak Passivation Treatment, that is to say, that in weak Passivation Treatment using the channel of Taurine,
Taurine is also added in corresponding passivation reaction liquid；
(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.Using single molecular fluorescence at
As technology evaluates the non-specific adsorption of the DNA chip of this example preparation.The detection method of non-specific adsorption is, in preparation
DNA chip surface is passed through the nucleotide of Cy5 fluorescent marker, the Cy5 fluorescence of DNA chip adsorption is detected, in unit area
Cy5 fluorescence points characterization DNA chip non-specific adsorption points.This example has specifically counted the fluorescence in 110 × 110 μm of regions
Points characterization non-specific adsorption points.
Non-specific adsorption points result is as shown in Fig. 2, abscissa is respectively the DNA chip of Taurine processing in Fig. 2
(i.e. Taurine) and the DNA chip (i.e. Null) for not adding the processing of small numerator modified reagent, ordinate be unit area 110 ×
The fluorescence of non-specific adsorption in 110 μm of regions is counted.Fig. 2's the results show that single negative electrical charge group it is small numerator modified
After reagent Taurine processing, non-specific adsorption points are about 1000Dot/FOV, and non-specific adsorption points, which are far below, not to be added
Add the DNA chip of small numerator modified reagent processing.Resist well as it can be seen that the small numerator modified reagent of single negative electrical charge group has
Non-specific adsorption effect.
Embodiment two
This example DNA chip prepares material and process is the same as example 1, the difference is that this example adds more negative electricity respectively
The small numerator modified reagent of the small numerator modified reagent of lotus group and single negative electrical charge group, more different small numerator modified reagents
Influence to DNA chip.Specifically, small numerator modified reagent Taurine of this example than right single negative electrical charge group, more negative electricity
The small numerator modified reagent glutamic acid of lotus group and the anti-non-specific adsorption effect of phosphoserine.
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 0.05nM DNA and 0.6mM CTAB；Wherein, the 3 ' ends of contained DNA simultaneous with
Amido modified NH2It is modified with Cy3 fluorogen；The ingredient of AT-01 is the Na of 0.25M2CO3/NaHCO3, the CTAB of 0.6mM, pH
9.78, the volume for the solution that circulates is 1mL, and the speed of fluid is 1mL/min, and reaction time 30min, reaction temperature is 37 DEG C；
(2) weak Passivation Treatment, is passed through weak passivation reaction liquid, carries out " weak passivation " step, weak passivation reaction liquid be containing
The Na of the 0.25M of the small numerator modified reagent of 10mM CTAB and 30mM2CO3/NaHCO3, pH is between 9.58-10.53, this example tool
Body is pH 9.78；As a comparison, the small numerator modified reagent added in the weak passivation reaction liquid that this example passage portion uses is list
The small numerator modified reagent of one negative electrical charge group is specifically taurine (Taurine), the weak passivation that another part channel uses
The small numerator modified reagent added in reaction solution is the small numerator modified reagent glutamic acid (Glutamic of more negative electrical charge groups
Acid), the small numerator modified reagent added in the weak passivation reaction liquid that another passage portion uses is the small of more negative electrical charge groups
Molecular modification phosphorylating reagent serine (Phospho-serine)；The volume of all channel setting circulation solution is 1mL, fluid
Speed is 1mL/min, and reaction time 3h, reaction temperature is 37 DEG C；
Needing to illustrate shelves is, in weak Passivation Treatment and subsequent Passivation Treatment, the small numerator modified reagent of addition be can be
The same, it can also be different；In general, the small numerator modified reagent added in two steps is consistent；
(3) weak passivation reaction liquid is washed away, specifically, washing times are 3 times, the volume being passed through every time with passivating solution RI-04
For 1mL, the speed of fluid is 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, being passed through passivation reaction liquid is passivated processing, the passivation reaction liquid of this example be containing
The Na of the 0.25M of the small numerator modified reagent of 30mM2CO3/NaHCO3, for pH between 9.58-10.53, this example is specially pH 9.78；
The number for flowing into passivation reaction liquid is 3-4 times, and the volume flowed into every time is 500 μ L, and the speed of fluid is 1mL/min, is flowed every time
The interval time entered is 1800s, and in entire passivating process, temperature is kept for 37 DEG C；Wherein, small numerator modified in passivation reaction liquid
Reagent, corresponding Taurine, glutamic acid or phosphoserine when using weak Passivation Treatment, that is to say, that in weak Passivation Treatment
Using the channel of Taurine, Taurine is also added in corresponding passivation reaction liquid, using the logical of glutamic acid when weak Passivation Treatment
Road, also adds glutamic acid in corresponding passivation reaction liquid, corresponding to be passivated using the channel of phosphoserine when weak Passivation Treatment
Also phosphoserine is added in reaction solution；
(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.
The DNA chip of this example is tested using the identical test method of embodiment one, as a result as shown in Figure 3.Fig. 3
In, abscissa is different small numerator modified reagents, and ordinate is corresponding 110 × 110 μm of units of the small numerator modified reagent of difference
The non-specific adsorption fluorescence of area is counted.Fig. 3's the results show that the small numerator modified reagent Taurine of single negative electrical charge group
After processing, non-specific adsorption points are about 1000Dot/FOV, the small numerator modified reagent glutamic acid of more negative electrical charge groups
After (Glutamic acid) processing, non-specific adsorption points are about 800Dot/FOV, and the small molecule of more negative electrical charge groups is repaired
After adoring phosphorylating reagent serine (Phospho-serine) processing, non-specific adsorption points are about 650Dot/FOV；As it can be seen that
Descending non-specific adsorption points are sequentially Taurine, Glutamic acid and Phospho-serine, are illustrated mostly negative
The small numerator modified reagent of charge groups is better than the small numerator modified reagent of single negative electrical charge group, non-specific with preferably resisting
Property adsorption effect.
Embodiment three
This example DNA chip prepares material and process is the same as example 1, the difference is that compared in Passivation Treatment
When, on the basis of adding small numerator modified reagent, add or do not add influence of the reaction promoter to DNA chip.This example tool
The reaction promoter of body addition is benzyltriethylammoinium chloride (Benzyltriethylammonium chloride).
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 0.05nM DNA and 0.6mM CTAB；Wherein, the 3 ' ends of contained DNA simultaneous with
Amido modified NH2It is modified with Cy3 fluorogen；The ingredient of AT-01 is the Na of 0.25M2CO3/NaHCO3, pH 9.78, circulate solution
Volume be 1mL, the speed of fluid is 1mL/min, and reaction time 30min, reaction temperature 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
The Na of 0.25M containing the small numerator modified reagent of 10mM CTAB and 30mM2CO3/NaHCO3, pH is between 9.58-10.53, originally
Example is specially pH 9.78；Wherein, small numerator modified reagent is Taurine, and the volume of all channel setting circulation solution is 1mL,
The speed of fluid is 1mL/min, and reaction time 3h, reaction temperature is 37 DEG C；
(3) weak passivation reaction liquid is washed away, specifically, washing times are 3 times, the volume being passed through every time with passivating solution RI-04
For 1mL, the speed of fluid is 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, being passed through passivation reaction liquid is passivated processing, the passivation reaction liquid of this example be containing
The Na of the 0.25M of the small numerator modified reagent Taurine of 30mM2CO3/NaHCO3, between 9.58-10.53, this example is specially pH
pH 9.78；The number for flowing into passivation reaction liquid is 3-4 times, and the volume flowed into every time is 500 μ L, and the speed of fluid is 1mL/
Min, the interval time flowed into every time is 1800s, and in entire passivating process, temperature is kept for 37 DEG C；As a comparison, this example is set respectively
It has set addition or has not added the channel of reaction promoter Benzyltriethylammonium chloride；
Concentration in the passivation reaction liquid of Benzyltriethylammonium chloride is 30mM；
(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.
The DNA chip of this example is tested using the identical test method of embodiment one, as a result as shown in Figure 4.Fig. 4
In, abscissa Null is the DNA chip for not adding reaction promoter, and Add is the DNA chip for adding reaction promoter；Ordinate
It counts for the non-specific adsorption fluorescence of corresponding 110 × 110 μm of unit areas.Fig. 4's the results show that addition reaction promoter
Afterwards, the non-specific adsorption fluorescence points of corresponding DNA chip are about 600Dot/FOV, the DNA without adding reaction promoter
The non-specific adsorption fluorescence points of chip are about 1000Dot/FOV, illustrate that reaction promoter can further promote small molecule
Modification reagent Taurine is integrated in substrate, to further decrease the non-specific adsorption of DNA chip.
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 biochip, the biochip is DNA chip or protein-chip, it is characterised in that: the biochip
Substrate surface there is anti-non-specific adsorption layer, the anti-non-specific adsorption layer is incorporated in substrate by small numerator modified reagent
Surface is formed, and the small numerator modified reagent is at least one of the compound of general formula shown in formula one；
Formula one
Wherein, R1、R2And R3It is respectively selected from hydrophilic radical or hydrogen atom.
2. biochip according to claim 1, it is characterised in that: the hydrophilic radical is negative electric group；
Optional, the hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic acid group, hydroxyl or amide groups
Group；
Optional, the compound of general formula shown in the formula one is taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoric acid silk
At least one of propylhomoserin；
Optional, the substrate surface of the biochip is modified with epoxy silane.
3. a kind of preparation method of biochip, the biochip is DNA chip or protein-chip, it is characterised in that: packet
It includes and small numerator modified reagent is used as using at least one of compound of general formula shown in formula one, it is fixed in conjunction with substrate surface
In substrate surface, anti-non-specific adsorption layer is formed；
Formula one
Wherein, R1、R2And R3It is respectively selected from hydrophilic radical or hydrogen atom.
4. preparation method according to claim 3, it is characterised in that: the hydrophilic radical is negative electric group；
Optional, the hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic acid group, hydroxyl or amide groups
Group；
Optional, the compound of general formula shown in the formula one is taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoric acid silk
At least one of propylhomoserin.
5. preparation method according to claim 3 or 4, it is characterised in that: the anti-non-specific adsorption layer is to use to contain
There is the passivation reaction liquid of small numerator modified reagent to be formed during Passivation Treatment；
Optional, in the passivation reaction liquid, the concentration of small numerator modified reagent is 0.01-0.2mol/L.
Optional, contain reaction promoter in the passivation reaction liquid；
Optional, the reaction promoter is in benzyltriethylammoinium chloride, phenyl trimethicone ammonium chloride and tetrabutylammonium chloride
At least one；
Optional, concentration of the reaction promoter in passivation reaction liquid is 10-50mmol/L.
6. preparation method according to claim 5, it is characterised in that: further include anti-using weak passivation before Passivation Treatment
Liquid is answered to carry out weak Passivation Treatment；
Contain the small numerator modified reagent in the weak passivation reaction liquid；
Optional, the concentration of the weak passivation reaction liquid small molecular modification reagent is 15-45mmol/L；Optional, small molecule
The concentration for modifying 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；
Optional, the weak Passivation Treatment is identical with small numerator modified reagent used in Passivation Treatment.
7. preparation method according to claim 6, it is characterised in that: also include surface-active in the weak passivation reaction liquid
Agent；
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, the concentration of the surfactant is 1-25mmol/L；
Optional, the concentration of the surfactant is 10mmol/L.
8. according to the described in any item preparation methods of claim 3-7, it is characterised in that: following steps are specifically included,
Weak Passivation Treatment, including making containing small numerator modified reagent or simultaneously containing small numerator modified reagent and surfactant
Weak passivation reaction liquid is contacted with substrate, carries out weak passivation under constant temperature conditions；
Passivation Treatment, including making containing small numerator modified reagent or blunt containing small numerator modified reagent and reaction promoter simultaneously
Change reaction solution to contact with substrate, be passivated under constant temperature conditions；
Optional, the step of preparation method also includes the chip after washing Passivation Treatment, the washing includes using three kinds
Cleaning solution sequentially washs the substrate of Passivation Treatment, and every kind of cleaning solution at least washed once, and three kinds of cleaning solutions are according to using
Sequence is sequentially RI-05, RI-06, RI-07, wherein RI-05 is PBS buffer solution, and RI-06 is that HEPES buffer solution and NaCl are molten
The mixed solution of liquid composition, RI-07 is distilled water；
Optional, the preparation method also includes that processing is fixed before weak Passivation Treatment, and the fixing process includes making
Fixer containing DNA or protein is contacted with substrate surface, carries out DNA under constant temperature conditions or protein is fixed.
9. a kind of application of small molecule compound in preparation DNA chip or protein-chip, the application includes will be described small
Molecular compound is incorporated in substrate surface as small numerator modified reagent, forms anti-non-specific adsorption layer, the small molecule
Close at least one of the compound that object is general formula shown in formula one；
Formula one
Wherein, R1、R2And R3Respectively hydrophilic radical or hydrogen atom；
Optional, the hydrophilic radical is negative electric group；
Optional, the hydrophilic radical contains phosphate group, bound phosphate groups, sulfonic acid group, carboxylic acid group, hydroxyl or amide groups
Group；
Optional, the small molecule compound is in taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine
It is at least one.
10. application of the biochip according to claim 1 or 2 in nucleic acid or Protein Detection analysis.
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| CN201811191571.2A CN109609333B (en) | 2018-10-12 | 2018-10-12 | Biological chip and preparation method thereof |
| PCT/CN2019/101068 WO2020073734A1 (en) | 2018-10-12 | 2019-08-16 | Biochip and manufacturing method therefor |
| EP19872008.8A EP3865585A4 (en) | 2018-10-12 | 2019-08-16 | Biochip and manufacturing method therefor |
| US17/227,211 US20210301331A1 (en) | 2018-10-12 | 2021-04-09 | Biochip and manufacturing method therefor |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020073734A1 (en) * | 2018-10-12 | 2020-04-16 | 深圳市真迈生物科技有限公司 | Biochip and manufacturing method therefor |
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| CN111621585B (en) * | 2020-04-16 | 2021-03-02 | 大连民族大学 | Rapid detection kit for simultaneously detecting multiple transgenic rape lines and application thereof |
| CN113138249A (en) * | 2021-04-12 | 2021-07-20 | 北京蛋白质组研究中心 | Micro-sample metabolome, proteome and phosphoproteome multi-group chemical analysis method based on micropore array chip |
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