CN113913944A

CN113913944A - Protein co-modified DNA chip and preparation method thereof

Info

Publication number: CN113913944A
Application number: CN202111182158.1A
Authority: CN
Inventors: 高锦鸿; 赵�智; 赵陆洋
Original assignee: Genemind Biosciences Co Ltd
Current assignee: Genemind Biosciences Co Ltd
Priority date: 2018-10-12
Filing date: 2018-10-12
Publication date: 2022-01-11
Also published as: CN109610007A

Abstract

The application discloses a protein co-modified DNA chip and a preparation method thereof. The protein co-modified DNA chip comprises a substrate with a surface, wherein the surface is called a modified surface, and the surface is called a surface to which protein and DNA are covalently connected. The protein co-modified DNA chip has the advantages that the protein is connected to the surface through covalent bond, the source of raw materials is wide, the comprehensive cost is low, the preparation is easy, the fixation through the covalent bond is firmer, the protein is not easy to desorb, and the non-specific adsorption caused by the protein can be avoided or reduced.

Description

Protein co-modified DNA chip and preparation method thereof

Technical Field

The application relates to the field of biomolecule detection, in particular to a protein co-modified DNA chip and a preparation method thereof.

Background

Fixing the DNA chain on the surface of glass, gold/silver noble metal or engineering plastic substrate to obtain DNA chip for identifying and detecting DNA or protein biomarker; or, further, as a sequencing chip, for the determination of DNA sequences. In general, highly available DNA chips are required to satisfy at least the following requirements: 1) the DNA modification is uniformly distributed on the substrate; 2) the prepared chip has lower non-specific adsorption signals; 3) the modified DNA species can be customized, namely different kinds of DNA chains can be modified; 4) the preparation process of the DNA chip has good repeatability.

In the use process of the DNA chip, the detected objects comprise macromolecules and micromolecules, the phenomenon of non-specific adsorption can be caused in the detection process of various molecules, and when the non-specific adsorption on the surface of the substrate of the chip is serious, the detection result can be seriously influenced. In many cases, the preparation of highly useful DNA chips is also required to meet many more stringent requirements. In many application scenarios, such as 1) detection of proteins or nucleic acids, or 2) complex enzyme-catalyzed systems in sequencing, non-specific adsorption of proteins can have the following effects on the performance of DNA chips: 1) slight protein adsorption can bring non-specific signals, reduce the intensity of the signals and enable the detection to have poor signal-to-noise ratio; 2) severe protein adsorption can even completely block the specifically modified DNA strand, rendering detection recognition incomplete. More seriously, many adsorbed proteins bring various types of chemical groups, which bring serious interference to the detection of certain compounds or targets, for example, polysaccharide carried by the proteins may interfere the detection of the polysaccharide, and thiol on the proteins may be combined with thiol small molecules in the detection system, which brings non-specific signals; in addition, non-specific adsorption of nucleotides to surfaces, etc., can cause inaccurate detection signals or low signal-to-noise ratio, making target detection difficult.

Disclosure of Invention

Embodiments of the present invention aim to solve at least one of the technical problems of the prior art to some extent or to at least provide a practical alternative. Therefore, the embodiment of the invention provides a preparation method of a biochip and the biochip.

According to an embodiment of the present invention, a protein co-modified DNA chip is provided, which comprises a substrate having a surface, wherein the surface is a modified surface, and the surface is covalently connected with the protein and the DNA.

The surface of the chip is combined with protein and DNA, and the protein and the DNA are connected to the surface through covalent bonds, so that the surface of the chip is firm and stable and is difficult to remove, and the parameters/performance of the surface of the chip are stable; furthermore, when the chip is used, nonspecific adsorption due to the unlinking/adsorption of proteins and/or DNA to the surface can be avoided. For example, the chip is applied to nucleic acid sequencing or detection and identification of nucleic acid molecules, and can avoid adsorption of enzyme and/or adsorption of nucleotide in the sequencing process caused by desorption of protein.

In some embodiments, the chip of this embodiment may also have at least one of the following additional features:

in certain examples, the protein is bovine serum albumin.

In certain examples, the surface or the protein comprises a blocked thiol group.

In certain examples, the surface further comprises a small molecule modifying agent attached thereto, the small molecule modifying agent being capable of attaching to the surface to form a non-specific adsorption resistant layer on the surface. The small molecule modifier is a compound with the molecular weight not more than 1000 daltons. Specifically, the small molecule modification reagent is selected from any one, two, three, four or all five of taurine, aminopropyl sulfonic acid, serine, glutamic acid and phosphoserine.

According to an embodiment of the present invention, there is also provided a method for preparing a protein co-modified DNA chip, which can be used for preparing the protein co-modified DNA chip of any of the above embodiments. The chip comprises a substrate having a surface and one or more channels, the surface being connected to the channels, the method comprising an immobilization process comprising passing an immobilization solution comprising DNA and protein through the channels and contacting the immobilization solution with the surface for a time period such that the DNA and the protein are covalently attached to the surface.

The method can prepare the chip with stable and controllable surface performance, the surface of which is covalently connected with DNA and protein. Moreover, the method can make the immobilized liquid flow to the channel through fluid and automatic control so as to make the DNA and protein molecules connected to the designated surface of the packaged chip, thereby being beneficial to the industrial preparation of the chip. The chip is formed by stacking an upper substrate/a lower substrate or an upper substrate/a middle substrate/a lower substrate, for example, as disclosed in CN105154323A, the lower substrate or the middle substrate may have a channel, the channel of the packaged chip has a liquid inlet and outlet and has a certain length, width and height to accommodate a certain volume of liquid, and the surface of the upper substrate near the lower substrate/the middle substrate/the channel may be connected with the DNA and protein molecules.

In some embodiments, the method of this embodiment may also have at least one of the following additional technical features:

in certain examples, the protein is bovine serum albumin.

In certain examples, the surface is a surface having an epoxy silane, and the DNA and the protein are both immobilized on the surface by covalent attachment to the epoxy silane.

In some examples, the method further comprises weak passivation treatment, wherein weak passivation solution containing surfactant is fed into the channel to replace the fixing solution, and the weak passivation solution is contacted with the surface after the fixing treatment so as to promote the connection of the DNA and/or the protein with the surface.

In certain examples, the surfactant is a cationic surfactant. Specifically, the surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and tetraoctylammonium bromide.

In certain examples, the concentration of the surfactant in the weak passivating solution is 1 to 25 mmol/L. Preferably, it is 10 mmol/L.

In certain examples, the weak passivating solution further comprises a protein that is the same protein as the protein comprised in the fixing solution. For example, bovine serum albumin. Therefore, the method is beneficial to preparing the chip with stable and controllable surface performance.

In certain examples, the weak passivating solution further comprises a small molecule modification reagent capable of linking with the surface to form a non-specific adsorption resistant layer, the small molecule modification reagent being a compound having a molecular weight of no greater than 1000 Da.

In certain examples, the small molecule modifying agent is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid, and phosphoserine.

In certain examples, the concentration of the small molecule modification reagent in the weak passivation solution is 15-45 mmol/L. Preferably, it is 30 mmol/L.

In certain examples, the weak passivation treatment includes contacting the weak passivation solution with the surface at 35 ℃ to 40 ℃ for 2h to 5 h. Therefore, the method is beneficial to preparing the chip with stable and controllable surface performance.

In some examples, the passivation treatment is further included, and the passivation treatment comprises introducing a passivation solution into the channel to replace the fixing solution or the weak passivation solution, and enabling the passivation solution to be in contact with the surface after the fixing treatment or the weak passivation treatment for a certain time.

In certain examples, a thiol blocking treatment is also included, including blocking thiol groups on the surface after the immobilization treatment or after the weak passivation treatment or after the passivation treatment. Specifically, the thiol blocking treatment comprises: and introducing a reducing reagent into the channel to replace the fixing solution or the weak passivation solution or the passivation solution so as to reduce the disulfide bond on the surface into a sulfhydryl group, and introducing a sulfhydryl blocking reagent into the channel to replace the reducing reagent so as to block the sulfhydryl group on the surface.

In certain examples, the reducing agent is selected from at least one of tris (2-carboxyethyl) phosphine, tris (3-hydroxypropyl) phosphine, dithiothreitol, and mercaptoethanol; and/or the thiol blocking reagent is selected from at least one of iodoacetamide, iodoacetic acid, maleimide, and epoxypropanol. The blocking reagent adopted in the method can introduce more hydrophilic groups or electronegative groups, so that the nonspecific adsorption resistance of the chip surface is further enhanced.

In some examples, a washing step is also included, including washing the immobilized, weakly passivated, or passivated surface with three different solutions in sequence. Thus, the chip with stable and controllable surface performance is obtained.

Embodiments of the present invention also provide a protein co-modified DNA chip prepared by the method of any of the above embodiments/examples. The technical features and advantages of the chip or the chip manufacturing method in any of the above embodiments are also applicable to the chip, and are not described herein again.

The embodiment of the invention also provides the application of the chip of any one of the above embodiments in nucleic acid and/or protein detection and analysis. Such as nucleic acid molecule detection, sequencing, and the like.

Embodiments of the present application provide a protein co-modified DNA chip formed by binding a protein to a substrate surface of a DNA chip.

It can be understood that the protein can be bound to the substrate surface of the DNA chip by covalent bonds or non-covalent bonds, in one embodiment of the present application, the protein is bound to the substrate surface of the DNA chip by covalent bonds to firmly fix the protein on the substrate surface, and the covalent bonds are more firmly fixed than physical adsorption modification, so as to avoid desorption, and further avoid non-specific adsorption caused by desorption during the use of the protein co-modified DNA chip. For example, in the sequencing or detection and identification process, the protein co-modified DNA chip can avoid the adsorption of enzyme and the adsorption of nucleotide in the sequencing process caused by the desorption of protein.

In some examples, the protein co-modification in the protein co-modified DNA chip employs bovine serum albumin as the protein.

In some examples, the thiol group on the protein co-modified protein in the protein co-modified DNA chip is in a blocked state.

The protein co-modified DNA chip reduces or even avoids the adsorption of the DNA chip to the molecules containing the sulfydryl because the sulfydryl on the protein is in a closed state, and has the function of resisting the adsorption of the molecules containing the sulfydryl. In addition, in a specific embodiment, when the sulfhydryl group is blocked, more hydrophilic groups or electronegative groups are introduced into the adopted blocking reagent, so that the nonspecific adsorption resistance of the DNA chip is further enhanced.

In some examples, the protein co-modified DNA chip has a non-specific adsorption resistant layer formed by a small molecule modification reagent on the surface of the substrate. The small molecule modification reagent of the present application means a small molecule compound having a relative molecular mass of 1000 or less.

It is understood that the small molecule modifying agent may be bound to the substrate surface either by covalent or non-covalent bonds. In one embodiment, the small molecule modification agent is covalently bound to the substrate surface to form a stable, controllable anti-non-specific adsorption layer.

In certain examples, the small molecule modifying agent is selected from any one, two or more of taurine, aminopropylsulfonic acid, serine, glutamic acid, and phosphoserine.

The protein co-modified DNA chip also adopts a small molecule modification reagent to form an anti-nonspecific adsorption layer, so that the anti-nonspecific adsorption capacity of the chip surface is further improved. In particular, in some preferred embodiments, the inventors have found that a small molecule modification reagent, such as taurine, aminopropylsulfonic acid, serine, glutamic acid, phosphoserine, etc., with good reaction controllability and repeatability, makes the preparation of the non-specific adsorption layer highly controllable, and can effectively act on the substrate surface together with protein co-modification, thereby enhancing the non-specific adsorption resistance of the DNA chip.

Another embodiment of the present application discloses a method for preparing a protein co-modified DNA chip, which comprises the steps of, during DNA immobilization, further including a protein in the immobilization solution, and allowing the protein to undergo a binding reaction with the substrate surface to immobilize the protein on the substrate surface, thereby achieving a protein co-modification effect.

The method of the embodiment utilizes covalent bonds to fix the protein and the DNA on the surface of the substrate, and the connection is stable and firm and is not easy to desorb/unligate. For example, the protein can be immobilized on the substrate surface by reacting the amino group of the protein with the epoxy group modified on the substrate surface to form a covalent bond.

Generally, the preparation method of the DNA chip comprises the processes of fixing treatment, passivation treatment, washing and the like, wherein the passivation treatment, the subsequent washing and the like can be carried out by referring to the existing preparation process of the DNA chip. The substrate may be a conventional substrate, and the present application does not specifically limit the substrate.

In some examples, the protein employed is bovine serum albumin.

In certain examples, the surface of the substrate in the method of making has an epoxy silane modification. The surface of the substrate is sometimes called as the surface and is provided with epoxy silane modification, so that the protein can be immobilized on the surface of the substrate through covalent bonds generated by the reaction between amino groups of the protein and the epoxy silane modification, and the protein can be stably and effectively immobilized on the surface of the substrate.

In some examples, the preparation method further comprises performing passivation treatment after the immobilization treatment, and performing thiol blocking treatment on the passivated substrate after the passivation treatment to block thiol groups on the immobilized protein on the surface of the substrate.

It will be appreciated that the attachment of biomolecules, such as proteins and/or nucleic acids, to surfaces, whether by physical adsorption or covalent attachment, presents the problem of non-specific adsorption of small molecules, particularly of small molecules containing sulfhydryl groups; of these, the inventors believe that non-specific adsorption of thiol-containing small molecules is due in part to the presence of thiol groups on the introduced co-modified protein. Based on the above knowledge, the method of the present embodiment performs the thiol blocking treatment on the surface after the immobilization treatment or the weak passivation treatment or the passivation treatment, so as to block the thiol on the substrate surface, for example, the thiol on the protein thereon, thereby reducing or even avoiding the non-specific adsorption of the protein co-modified DNA chip to the thiol-containing small molecule substances such as cysteine (Cys), homocysteine (Hcy), reduced Glutathione (GSH), and thiol-containing nucleotide molecules; in addition, when the sulfhydryl is subjected to blocking treatment, more hydrophilic groups or negative groups can be introduced on the surface of the protein co-modified DNA chip by the introduced reagent, so that the nonspecific adsorption resistance of the DNA chip is further enhanced. Therefore, the sulfydryl on the surface of the protein co-modified DNA chip is sealed, so that the sulfydryl on the surface is inactivated, and the surface with stable and controllable performance is favorably obtained.

The term nucleotide as used herein refers to a nucleoside, nucleotide or analog, derivative thereof, which follows the base pairing center rules.

In some examples, the thiol blocking treatment specifically includes reducing a disulfide bond in the protein with a reducing agent to generate a reducing thiol, and then blocking the generated reducing thiol with a thiol blocking agent.

In certain examples, the reducing agent is at least one of tris (2-carboxyethyl) phosphine (abbreviated TCEP), tris (3-hydroxypropyl) phosphine (abbreviated THPP), dithiothreitol (abbreviated DTT), and mercaptoethanol (abbreviated ME).

In certain examples, the thiol blocking reagent is at least one of iodoacetamide, iodoacetic acid, maleimide, and epoxypropanol.

In some examples, the preparation method of the present embodiment further includes performing a weak passivation treatment after the DNA fixing treatment and before the passivation treatment; the weak passivation treatment comprises adding a surfactant catalyst and protein into the reaction solution, and promoting the combination of DNA and protein with the substrate surface by using the surfactant catalyst, so that the DNA and protein are fully fixed on the substrate surface.

The weak passivation treatment utilizes a surfactant, preferably a cationic surfactant, to catalyze and promote the combination of DNA and protein with the surface of the substrate, so that the quantity of the DNA or the protein combined with the surface of the substrate has better dependence and correspondence with the concentration of the DNA or the protein which is initially added, the quality of the DNA or the protein fixed on the DNA chip is controllable, and the quality and the effect of protein co-modification are improved. It is understood that if the reaction for forming the DNA or protein bound to the substrate surface, such as covalent bond, is insufficient, the amount of DNA or protein bound to the same or different batches of DNA chips is liable to be unstable, and the production and use requirements of the highly available DNA chips cannot be satisfied; the embodiment enables DNA and protein to be efficiently and sufficiently combined on the surface, has good repeatability and can control the fixed amount of the DNA and the protein by adding a weak passivation treatment step and using surfactant catalysis in the weak passivation treatment.

The reaction solution for the weak passivation treatment may be a buffer solution compatible with the surfactant, and in principle, the buffer solution is compatible as long as it does not adversely affect the DNA and the surface and can promote the binding with the surface; in a specific embodiment, the fixing solution for the fixing treatment is directly used as the weak passivation solution for the weak passivation treatment. Thus, the preparation of the solution is convenient, and the industrialized preparation of the chip is convenient.

In certain examples, the surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and tetraoctylammonium bromide.

Preferably, the surfactant is a cationic surfactant, which is capable of efficiently promoting the formation of covalent bonds between amino groups of DNA and/or proteins and chemically modified groups on the substrate surface. In one example, the surfactant cetyl trimethylammonium bromide (abbreviated CTAB) is used, it being understood that other surfactants having similar functions, such as dioctadecyl ammonium bromide, cetyl trimethylammonium chloride, dodecyl trimethylammonium bromide, and tetraoctyl ammonium bromide, are also effective in promoting the reaction of the amino group and the substrate surface chemistry modifying group.

In some examples, the concentration of the surfactant in the reaction liquid for the weak passivation treatment (weak passivation liquid) is 1 to 25mmol/L, preferably 10 mmol/L.

In some examples, the conditions of the weak passivation treatment are 35 ℃ to 40 ℃ for 2h to 5 h.

In some examples, the protein added to the reaction solution of the weak passivation treatment in the preparation method is also bovine serum albumin. Thus, the preparation of the solution is convenient, and the industrial production and preparation of the chip are convenient.

In some examples, a small molecule modification reagent is further added to the reaction solution of the weak passivation treatment, and the small molecule modification reagent is immobilized on the substrate surface by a binding reaction with the substrate surface to form an anti-nonspecific adsorption layer.

It is understood that the binding reaction of the small molecule modification reagent with the substrate surface may be a covalent binding reaction or a non-covalent binding reaction; in an implementation mode of the application, the small molecule modification reagent is bonded on the surface of the substrate through a covalent bond to form a non-specific adsorption layer.

In some examples, the concentration of the small molecule modification reagent in the reaction solution of the weak passivation treatment is 15-45mmol/L, preferably 30 mmol/L.

In this embodiment, the reaction solution of the weak passivation treatment contains a small molecule modification reagent, and the small molecule modification reagent can bind to the surface of the substrate to form a non-specific adsorption resistant layer, and can enhance the non-specific adsorption resistance of the surface together with the co-modification of the protein. In certain preferred embodiments, one or more of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine is/are used as a small molecule modification reagent, the small molecule modification reagent has high-efficiency and strong-controllability binding capacity with a chemical modification group on the surface of a substrate, and the repeatability of the binding of the small molecule modification reagent with the substrate is good, so that a highly available DNA chip with good nonspecific adsorption resistance can be stably and reliably prepared, and the problems of poor controllability, weak repeatability and the like existing in the conventional small molecule modification are solved.

In some examples, the method of this embodiment further comprises the step of,

a fixing treatment comprising bringing a fixing solution containing DNA and protein into contact with the surface of the substrate and fixing the solution at a constant temperature; typically, the fixative is 0.25mol/L Na₂CO₃/NaHCO₃pH9.78, wherein the concentration of DNA is generally 0.01-0.4nmol/L, the concentration of protein is generally 10-100 μmol/L, the temperature of fixation treatment is about 37 ℃, the treatment time is about 30min, the above conditions can be referred to,it is not particularly limited herein; wherein "Na" is₂CO₃/NaHCO₃"means represented by Na₂CO₃And NaHCO₃And (3) forming a stationary liquid.

Weak passivation treatment, which comprises contacting a reaction solution containing catalyst cationic surfactant and protein, or a reaction solution containing cationic surfactant catalyst, small molecule modification reagent and protein with a substrate after fixation treatment, and performing weak passivation under constant temperature; in the weak passivation treatment in the example, the small molecule modification reagent can be fixed on the surface of the substrate to form a non-specific adsorption resistant layer, and the catalyst, especially the surfactant can enable DNA or protein to be fully combined with the modification group on the surface of the substrate; it can be understood that the concentration and the treatment time of the small molecule modification reagent both affect the amount of the small molecule modification reagent immobilized on the surface of the substrate, and the higher the concentration and the longer the treatment time are, the larger the amount of the small molecule modification reagent immobilized on the surface of the substrate is; similarly, the higher the concentration of the surfactant and the longer the treatment time, the better the effect of fully bonding the DNA or protein to the surface chemical modification group of the substrate is; the specific method can be determined according to production or product requirements, and is not particularly limited herein;

in one example, the reaction solution for the weak passivation treatment is 0.25mol/L Na containing 10nM surfactant, 30mM small molecule modifying agent and 10-100. mu.M protein₂CO₃/NaHCO₃And introducing a reaction solution into a chip channel by using a fluid device to perform weak passivation treatment under the condition that the pH is between 9.58 and 10.53, wherein the volume of the flowing solution is 1mL, the fluid speed is 1mL/min, the reaction time is 3h, and the reaction temperature is 37 ℃.

Passivating, including adopting passivating solution to wash the surface of the substrate which is weakly passivated, then contacting the passivating solution with the surface of the substrate which is weakly passivated, and passivating under the condition of constant temperature; in one example, the passivation solution is 1mol/L K₂HPO₄/KH₂PO₄pH 9.0; wherein "K" is₂HPO₄/KH₂PO₄"denotes a group consisting of₂HPO₄And KH₂PO₄A passivation solution is formed;

in one example, a reaction solution is introduced into a channel of a chip by a fluid device to perform a reaction such as immobilization, weak passivation, passivation and the like, wherein the passivation treatment is performed under the conditions that the passivation solution is introduced 3-4 times, the volume of each inflow is 500 μ L, the fluid velocity is 1mL/min, the time interval of each inflow is 1800 seconds, and the temperature is maintained at 37 ℃ during the whole passivation process.

Carrying out sulfhydryl blocking treatment, namely reducing disulfide bonds in the protein adsorbed on the substrate after passivation treatment by adopting a reducing reagent to generate reductive sulfhydryl groups, and then blocking the sulfhydryl groups on the protein by adopting a sulfhydryl blocking reagent; the purpose of the thiol blocking treatment is to block free thiols on proteins or surfaces.

In one example, a fluid device is used to introduce a reaction solution into a channel of a chip for reaction, wherein the thiol blocking treatment is performed by introducing the reaction solution into a reduction reaction solution containing a reduction reagent, the components of the reduction reaction solution are 150mM Tris pH8.0, 100mM NaCl and 30mM reduction reagent, the volume of the flow-through solution is 1mL, the fluid speed is 1mL/min, the reaction time is 10-30min, and the reaction temperature is 37 ℃; then introducing a closed reaction solution containing a sulfhydryl blocking reagent, wherein the components of the closed reaction solution are 150mM HEPES pH 8.5, 100mM NaCl and 30mM sulfhydryl blocking reagent, the volume of the flow-through solution is 1mL, the speed of the fluid is 1mL/min, the reaction time is 10-30min, and the reaction temperature is 37 ℃.

Washing to obtain a DNA chip (chip) comprises washing the passivated surface sequentially with three different solutions, each solution being washed at least once. In one example, the three solutions (washing solutions) are sequentially introduced into the channel as RI-05, RI-06 and RI-07, wherein RI-05 is phosphate buffer, RI-06 is a mixed solution of HEPES buffer and NaCl solution, and RI-07 is double distilled water. Wherein HEPES represents 4-hydroxyethylpiperazine ethanesulfonic acid.

Another embodiment of the present application provides the use of the protein co-modified DNA chip of any of the above embodiments in a nucleic acid and/or protein detection assay. Nucleic acid or protein detection assays include sequencing assays, hybridization assays, immunoassays, SNP detection assays, and the like.

The DNA chip refers to a chip substrate surface fixed with DNA, the DNA is a substance containing DNA sequence, for example, the DNA can contain nucleotide derivative, nucleotide analogue, fluorescent label, or both nucleotide sequence and amino acid sequence.

In some examples, the substrate surface of the chip is a chemically modified surface having a chemical modification containing a reactive group capable of reacting with DNA to fix the DNA on the substrate surface by a reaction between the reactive group and the DNA.

The protein co-modified DNA chip or the preparation method thereof in any of the above embodiments combines proteins on the surface of the substrate, which not only has the advantages of simple method, wide raw material source, good effect, low comprehensive cost, etc., but also fixes the proteins through covalent bonds, which is more reliable, and the proteins are not easy to desorb, thereby avoiding non-specific adsorption caused by the covalent bonds.

In some examples, the nonspecific adsorption resistance of the protein-co-modified DNA chip surface to thiol-containing molecules is further enhanced by blocking the thiol groups on the surface/protein; moreover, when thiol blocking is performed, in some preferred embodiments, more hydrophilic groups or negatively charged groups are introduced into the blocking reagent, so as to further enhance the nonspecific adsorption resistance of the chip surface.

In some examples, the preparation method of the protein co-modified DNA chip comprises a weak passivation step, wherein a surfactant, especially a cationic surfactant, is used for promoting the DNA and the protein to be capable of reacting with chemical modification groups on the surface of a substrate more fully, so that the DNA is effectively fixed on the substrate; not only improves the quality and efficiency of DNA fixation, but also improves the effect of protein co-modification; moreover, the fixed amount of DNA on the surface of the chip is highly controllable, and the preparation can be repeated.

Further, in some examples, a weak passivation solution containing a small molecule modification reagent is used in the weak passivation step, so that the small molecule modification reagent is efficiently and controllably connected to the surface to form a non-specific adsorption resistant layer, and the non-specific adsorption resistance of the chip surface is further enhanced.

Additional aspects and advantages of embodiments of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of embodiments of the invention.

Drawings

The above and/or additional aspects and advantages of embodiments of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic structural diagram of a packaged chip substrate according to an embodiment of the present application;

FIG. 2 is a graph comparing the adsorption results of the surface of the chip on the protein in the test system in the examples of the present application;

FIG. 3 is a graph comparing the adsorption results of the chip surface on the thiol small molecule substance in the test system in the examples of the present application;

FIG. 4 is a schematic structural view of the surface of a glass substrate in an example of the present application.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The present invention may repeat reference numerals and/or letters in the various examples, such repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

As used herein, the singular forms "a", "an" and the like (a/an/the) include plural referents unless the context clearly dictates otherwise; "A group" or "a plurality" means two or more.

In this document, unless stated otherwise, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicit to the number of technical features indicated; features defined as "first," "second," etc. may explicitly or implicitly include one or more of the described features.

In this context, unless otherwise indicated, "attached", "fixed", and the like are to be understood in a broad sense, e.g., attached fixedly, reversibly, directly, indirectly through an intermediate, chemically, e.g., covalently, chemically, or physically.

As used herein, the term "substrate" or "solid substrate" can be any solid support that can be used to immobilize nucleic acid sequences, such as nylon membranes, glass slides, plastics, silicon wafers, magnetic beads, and the like; sometimes also referred to as a reactor, chip or flow cell.

The existing protein co-modified DNA chip mainly utilizes physical adsorption to adsorb protein on the surface of a substrate, thereby playing a role in resisting protein adsorption; however, physical adsorption is not secure and desorption is easy.

In view of the above, embodiments of the present disclosure provide a protein co-modified DNA chip, in which both protein and DNA are linked to the surface of a substrate via covalent bonds, so that the immobilization/linkage is more secure, and desorption is less likely to occur, thereby avoiding non-specific adsorption due to desorption.

In certain embodiments, the DNA amino groups react with reactive groups on the substrate surface. The chemical modification structure of the substrate surface is shown in fig. 4, wherein R1 represents an alkane chain molecule with a terminal connected with an active reaction group, wherein the active group is preferably at least one of epoxy group, aldehyde group, carboxyl group, N-hydroxysuccinimide and diaminobenzanilide. In some embodiments, the weak passivation solution containing a surfactant can catalyze and promote the reaction of the amino group of the DNA with the active group in the chemical modification shown in FIG. 4 to form a covalent bond, so that the DNA is sufficiently immobilized on the surface of the substrate.

In addition, the inventors have found that the high adsorption of small molecule substances such as cysteine (Cys), homocysteine (Hcy), reduced Glutathione (GSH), and thiol-containing nucleotide molecules to conventional protein-co-modified DNA chips is likely due to the large number of free thiol groups on the co-modified protein as a blocking reagent, and thus, the non-specific adsorption of small thiol molecules such as cysteine. Based on the above knowledge, in some preferred embodiments, the thiol blocking treatment is performed on the substrate surface after the immobilization treatment or the weak passivation treatment or the passivation treatment, so as to block the free thiol on the substrate surface, thereby reducing or even avoiding the adsorption of the protein co-modified DNA chip on the thiol small molecule substance, and reducing the non-specific adsorption on the chip surface.

In some preferred embodiments, the weak passivation solution containing the surfactant is used to weakly passivate the surface after the immobilization treatment, so that the DNA or protein can be more sufficiently reacted with the chemical modification groups on the surface of the substrate, and the DNA or protein can be more effectively immobilized on the surface of the substrate. The weak passivation solution containing the surfactant enables the DNA or protein to react with the chemical modification group on the surface of the substrate more fully, so that the efficiency and the quality of the DNA or protein fixed on the surface of the substrate are improved, and the quantity of the DNA or protein fixed on the biochip has good correlation with the concentration of the DNA or protein initially added in the fixing solution, so that the quality of the DNA or protein fixed on the biochip is controllable, and the repeatability is good. The method not only lays a foundation for preparing high-quality biochips, but also can meet the production requirements of customization.

In some preferred embodiments, the weak passivation solution further comprises a small molecule modification reagent, such that the small molecule modification reagent is linked to the substrate surface, and can further reduce non-specific adsorption on the chip surface. And taurine, aminopropyl sulfonic acid, serine, glutamic acid and/or phosphoserine are/is used as the small molecule modification reagent, and the small molecule modification reagent has high-efficiency and strong-controllability binding capacity with an epoxy group on the surface of the substrate, so that the small molecule modification reagent has good repeatability and strong operability in binding with the surface of the substrate, and can stably and reliably prepare a high-availability DNA chip with good nonspecific adsorption resistance effect.

Some abbreviations/codes/numbers involved in the embodiments refer to the following, unless otherwise specified:

AT-01：0.25M Na₂CO₃/NaHCO₃0.6mM CTAB (i.e., cetyltrimethylammonium bromide), pH 9.78;

RI-04：1M K₂HPO₄/KH₂PO₄，pH 9.0；

RI-05：PH7.4 PBS；

RI-06: a mixed solution of 150mM HEPES and 150mM NaCl;

RI-07: double distilled water;

Dot/FOV: the observation area is the number of bright spots in the range of 110X 110 μm.

Example 10

In this example, a protein-co-modified DNA chip (which may be simply referred to as a chip or a DNA chip) of this example was formed by immobilizing DNA on a glass substrate having an epoxysilane on the surface thereof via the amino group of the DNA and using bovine serum albumin as a co-modified protein. In this example, the effect of the co-modification with and without the addition of BSA on the DNA chip was compared.

In this embodiment, the DNA is fixed on the chip substrate by "in-channel" fixing, i.e., the chip substrate is packaged, and then various reagents are introduced into the packaged chip channels by using a fluid device, thereby realizing chemical reactions such as fixing treatment and passivation treatment. As shown in fig. 1, chip substrates are packaged to form independent chip channels, each chip channel can independently perform each reaction, fig. 1 shows a packaged chip substrate with 8 channels, the specification of the channels is length × width × height × 90mm × 1.8mm × 0.1mm, the chip substrates can be packaged into 16 channels according to different packaging processes or fluidic devices, and 16 kinds of differently modified DNAs can be independently manufactured on one DNA chip.

The method for preparing the DNA chip of this example is as follows:

(1) immobilization treatment, in which an immobilization reaction solution is introduced into a channel of a chip substrate to perform an immobilization reaction, the immobilization reaction solution in this example being an immobilization solution AT-01 containing 0.2nM DNA and 30. mu.M bovine serum albumin (abbreviated as BSA); wherein the 3' end of the DNA contained in the DNA simultaneously carries amino-modified NH₂And Cy3 fluorophoreModifying; AT-01 has Na content of 0.25M₂CO₃/NaHCO₃0.6mM CTAB, pH9.78, volume of flow-through solution of 1mL, fluid velocity of 1mL/min, reaction time of 30min, reaction temperature of 37 ℃;

as a control, this example was provided with a channel to which BSA was not added in the immobilization reaction solution;

the DNA and BSA are in a competitive adsorption relationship, and the reaction performance of the DNA and the surface is far better than that of the BSA, so that even the BSA with the concentration of 15000 times is added, the DNA modification is still not obviously influenced; in the preparation method of the embodiment, BSA is added during the immobilization treatment and the weak passivation treatment, so that the protein co-modification effect is better, and the BSA can be added only in the immobilization treatment step under the condition of lower requirements;

(2) weakly passivating, introducing weakly passivating reaction solution containing 10mM CTAB, 30mM Taurine (Taurine) and 30 μ M BSA and 0.25M Na for performing the weakly passivating step₂CO₃/NaHCO₃The pH is between 9.58 and 10.53, in this case specifically pH 9.78; setting the volume of a flowing solution to be 1mL, the speed of a fluid to be 1mL/min, the reaction time to be 3h and the reaction temperature to be 37 ℃;

wherein, when in immobilization treatment, a channel without BSA is added in the reaction solution, when in weak passivation treatment, BSA is not added, and the rest components, the use amount and the channel parameter setting are the same and are used as a reference;

(3) washing to remove weak passivation solution, specifically, using passivation solution RI-04, washing for 3 times, introducing volume of 1mL each time, controlling fluid speed to be 1mL/min, and keeping temperature at 37 ℃ during washing; the RI-04 component of this example is K of 1M₂HPO₄/KH₂PO₄，pH 9.0；

(4) Passivating, specifically, passivating liquid RI-04 is used, the washing times are 3-4 times, the volume of each inflow is 500 mu L, the fluid speed is 1mL/min, the interval time of each inflow is 1800s, and the temperature is kept at 37 ℃ in the whole passivating process; the RI-04 component of this example is K of 1M₂HPO₄/KH₂PO₄，pH 9.0；

(5) Washing the passivated chip by adopting three solutions, wherein each solution is washed for 3 times, the volume of each solution is 1mL, the fluid speed is 1mL/min, and the temperature is kept at 37 ℃ during washing; the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer solution with pH7.4, RI-06 is mixed solution consisting of HEPES buffer solution with 150mM and NaCl solution with 150mM, and RI-07 is double distilled water.

After washing with the three solutions, the DNA chip of this example is obtained after normal air drying or oven drying.

The non-specific adsorption condition of the protein of the DNA chip prepared in the embodiment is evaluated by adopting a single-molecule fluorescence imaging technology, specifically, Klenow Fragment with a fluorescent label is introduced to the surface of the prepared DNA chip, the number of the fluorescent labels of the Klenow Fragment adsorbed to the surface of the DNA chip is detected, and the non-specific adsorption condition of the protein of the DNA chip is represented by the number of the fluorescent labels of the Klenow Fragment in unit area. In this example, the number of fluorescence spots in a region of 110X 110. mu.m was counted.

The test results of the DNA chip and the control test thereof in this example are shown in FIG. 2, the abscissa None represents the DNA chip without BSA added thereto, BSA represents the protein co-modified DNA chip prepared by co-modification with BSA added thereto, and the ordinate represents the number of fluorescence spots. The results in FIG. 2 show that the number of nonspecific adsorption sites of the DNA chips prepared after the co-modification with BSA was about 500Dot/FOV, while that of the DNA chips prepared without the co-modification with BSA was more than 6000 Dot/FOV; the BSA co-modification can greatly reduce the nonspecific adsorption of the DNA chip to protein, and has good protein adsorption resistance.

Example 20

The preparation materials and the flow of the DNA chip of the embodiment are the same as those of the first embodiment, except that the thiol blocking treatment is added after the passivation treatment, and the thiol blocking reagent is used for blocking the thiol on the protein, so that the nonspecific adsorption effect of the protein co-modified DNA chip on thiol small molecules is reduced.

The specific preparation method of the DNA chip of this example was as follows:

(1) fixing treatment, introducing fixing reaction into the channel of chip substrateA solution for immobilization, in this example, an immobilization solution AT-01 containing 0.2nM DNA and 30. mu.M Bovine Serum Albumin (BSA); wherein the 3' end of the DNA contained in the DNA simultaneously carries amino-modified NH₂And Cy3 fluorophore modification; AT-01 has Na content of 0.25M₂CO₃/NaHCO₃0.6mM CTAB, pH9.78, volume of flow-through solution of 1mL, fluid velocity of 1mL/min, reaction time of 30min, reaction temperature of 37 ℃;

(5) A thiol blocking treatment, a) introducing a reaction solution containing a reducing agent TCEP, the reaction solution having the composition of 150mM Tris, pH8.0, 100mM NaCl and 30mM TCEP; the purpose of this step is to reduce the disulfide bond in the system, produce the reducible sulfhydryl, benefit the next closure; the volume of the flowing solution is 1mL, the speed of the fluid is 1mL/min, the reaction time is 10-30min, and the reaction temperature is 37 ℃; b) introducing a mercapto-blocking reagent reaction solution, wherein the mercapto-blocking reagent can be iodoacetamide or iodoacetic acid, in the example, iodoacetamide is specifically adopted, and the reaction solution comprises 150mM HEPES, pH 8.5, 100mM NaCl and 30mM iodoacetamide; the volume of the flowing solution is 1mL, the speed of the fluid is 1mL/min, the reaction time is 10-30min, the reaction time is 30min in the embodiment, and the reaction temperature is 37 ℃;

(6) washing the chip subjected to sulfhydryl blocking treatment, wherein three solutions are adopted for washing, each solution is washed for 3 times, the volume of each solution is 1mL, the speed of fluid is 1mL/min, and the temperature is kept at 37 ℃ during washing; the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is phosphate buffer solution with pH7.4, RI-06 is mixed solution consisting of HEPES buffer solution with 150mM and NaCl solution with 150mM, and RI-07 is double distilled water.

The adsorption condition of the DNA chip prepared in the example to the nucleotide molecules containing disulfide bonds is evaluated by adopting a single-molecule fluorescence imaging technology, specifically, the fluorescence Cy3 labeled nucleotide molecules containing disulfide bonds are introduced to the surface of the prepared DNA chip, the fluorescence quantity of Cy3 adsorbed to the surface of the DNA chip is detected, and the adsorption condition of the nucleotide molecules containing disulfide bonds of the DNA chip is represented by the number of Cy3 fluorescence points in unit area. In this example, the number of fluorescent spots (number of bright spots) in a region of 110X 110. mu.m was counted. Meanwhile, the disulfide bond small molecule adsorption condition of the DNA chip prepared by adopting BSA co-modification in the first embodiment is tested and used as a comparison; example one DNA chip prepared after co-modification with BSA was not thiol-blocked. Reference is made, for example, to the compounds disclosed in EP2607369B1, such as the compounds shown in FIG. 2, among others, to nucleotide molecules containing disulfide bonds.

As shown in FIG. 3, the abscissa "None" in FIG. 3 indicates the DNA chip co-modified with BSA but prepared without thiol-blocking treatment, i.e., the BSA co-modified DNA chip of example one, and "Block" indicates the DNA chip co-modified with BSA and prepared with thiol-blocking treatment, i.e., the DNA chip of this example. The results in FIG. 3 show that the number of non-specific adsorption sites of the DNA chip without thiol blocking treatment was more than 3000Dot/FOV, while the number of non-specific adsorption sites of the DNA chip with thiol blocking treatment was about 500 Dot/FOV; therefore, the non-specific adsorption of the DNA chip to the small molecular substance containing the sulfydryl can be greatly reduced by the sulfydryl blocking treatment.

In the description herein, references to the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "some examples," "a specific example" or "an example" or the like mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention.

Claims

1. A protein co-modified DNA chip, comprising a substrate having a surface, wherein the surface is a modified surface, and wherein the surface is covalently attached to the protein and the DNA.

2. The chip of claim 1, wherein the protein is bovine serum albumin;

optionally, the protein comprises a blocked thiol group.

3. The chip of claim 1, wherein the surface further comprises a small molecule modification agent attached thereto, wherein the small molecule modification agent is a compound having a molecular weight of not more than 1000 daltons;

optionally, the small molecule modifier is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid, and phosphoserine.

4. A method of preparing a protein co-modified DNA chip comprising a substrate having a surface and one or more channels, said surface being associated with said channels, said method comprising an immobilization process comprising passing a fixative solution comprising DNA and protein through said channels and contacting said fixative solution with said surface for a time sufficient to covalently attach said DNA and said protein to said surface.

5. The method of claim 4, wherein the protein is bovine serum albumin;

optionally, the surface is a surface with an epoxy silane to which both the DNA and the protein are immobilized by covalent attachment to the epoxy silane.

6. The method according to claim 4 or 5, further comprising a weak passivation treatment, comprising introducing a weak passivation solution containing a surfactant into the channel to replace the fixing solution, and contacting the weak passivation solution with the surface after the fixing treatment to promote the connection of the DNA and/or the protein with the surface;

optionally, the surfactant is a cationic surfactant;

optionally, the surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and tetraoctylammonium bromide;

optionally, the concentration of the surfactant in the weak passivation solution is 1-25 mmol/L;

optionally, the concentration of the surfactant in the weak passivation solution is 10 mmol/L;

optionally, the weak passivation solution further comprises a protein, which is the same protein as the protein contained in the fixation solution; optionally, the protein is bovine serum albumin;

optionally, the weak passivation solution further comprises a small molecule modification reagent, wherein the small molecule modification reagent can be connected with the surface to form a non-specific adsorption resistant layer, and the small molecule modification reagent is a compound with the molecular weight of not more than 1000 daltons;

optionally, the small molecule modifier is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine;

optionally, the concentration of the small molecule modification reagent in the weak passivation solution is 15-45 mmol/L;

optionally, the concentration of the small molecule modification reagent in the weak passivation solution is 30 mmol/L;

optionally, the weak passivation treatment comprises contacting the weak passivation solution with the surface at 35-40 ℃ for 2-5 h.

7. The method of any one of claims 4-6, further comprising passivating, comprising introducing a passivating fluid into the channel to replace the immobilizing fluid or the weak passivating fluid and bring it into contact with the immobilized or weakly passivated surface.

8. The method according to any one of claims 4 to 7, further comprising a thiol blocking treatment comprising blocking thiol groups on the surface after the immobilization treatment or after the weak passivation treatment or after the passivation treatment;

optionally, the thiol blocking treatment comprises:

introducing a reducing agent into the channel to replace the fixing solution or the weak passivation solution or the passivation solution so as to reduce the disulfide bond on the surface to a thiol group, and

introducing a thiol blocking reagent into the channel to displace the reducing reagent to block the thiol groups on the surface;

optionally, the reducing agent is selected from at least one of tris (2-carboxyethyl) phosphine, tris (3-hydroxypropyl) phosphine, dithiothreitol, and mercaptoethanol; and/or

The sulfhydryl blocking reagent is selected from at least one of iodoacetamide, iodoacetic acid, maleimide and epoxy propanol;

optionally, a washing step is also included, which comprises washing the surface after the fixing treatment, the weak passivation treatment or the passivation treatment with three different solutions in sequence.

9. A protein co-modified DNA chip prepared by the method of any one of claims 4 to 8.

10. Use of a chip according to any one of claims 1 to 3 or a chip according to claim 9 in a nucleic acid and/or protein detection assay.