Protein co-modified DNA chip and preparation method thereof

Abstract

The present application discloses a protein co-modified DNA chip and a preparation method thereof. The protein co-modified DNA chip comprises a substrate having a surface, the surface being a modified surface, and the surface being covalently linked to a protein and a DNA. The protein co-modified DNA chip, in which the protein is covalently linked to the surface, not only has the advantages of a wide source of raw materials, low overall cost, and easy preparation, but also is more firmly fixed by covalent bonds, the protein is not easy to desorb, and the non-specific adsorption caused thereby can be avoided or reduced.

Description

Technical Field

[0001] The present application relates to the field of biomolecule detection, and in particular, to a protein co-modified DNA chip and a preparation method thereof. Background Art

[0002] By fixing the DNA chain to the surface of a substrate such as glass, gold / silver precious metals or engineering plastics, a DNA chip can be obtained for the identification and detection of DNA or protein biomarkers; or, further, as a sequencing chip for the determination of DNA sequences. Generally speaking, a highly available DNA chip must at least meet the following requirements: 1) the DNA modification is evenly distributed on the substrate; 2) the prepared chip has a low non-specific adsorption signal; 3) the modified DNA type can be customized, that is, different types of DNA chains can be modified; 4) the preparation process of the DNA chip has good repeatability.

[0003] In the process of using DNA chips, the objects to be detected include macromolecules and small molecules. There will be non-specific adsorption in the detection process of various molecules. When the non-specific adsorption on the substrate surface of the chip is serious, it will seriously affect the detection results. In many cases, the preparation of highly available DNA chips also needs to meet many more stringent requirements. In many usage scenarios, such as 1) detection of proteins or nucleic acids, or 2) complex enzyme catalysis systems in sequencing, non-specific adsorption of proteins will have the following effects on the performance of DNA chips: 1) Slight protein adsorption will bring non-specific signals, reduce the intensity of the signal, and make the detection have a poor signal-to-noise ratio; 2) Severe protein adsorption can even completely block the specifically modified DNA chain, making the detection and identification impossible. More seriously, many adsorbed proteins bring various types of chemical groups, which seriously interfere with the detection of certain compounds or targets. For example, polysaccharides carried by proteins may interfere with the detection of polysaccharides, and the thiol groups on proteins may combine with thiol small molecules in the detection system to bring non-specific signals; in addition, non-specific adsorption of nucleotides on the surface, etc., will make the detection signal inaccurate or make the signal-to-noise ratio low and difficult to achieve target detection. Summary of the invention

[0004] The embodiments of the present invention aim to solve at least one of the technical problems existing in the prior art to a certain extent or at least provide a practical alternative solution. To this end, the embodiments of the present invention provide a method for preparing a biochip and a biochip.

[0005] According to an embodiment of the present invention, a protein co-modified DNA chip is provided, wherein the chip comprises a substrate having a surface, wherein the surface is a modified surface, and the protein and the DNA are covalently linked to the surface.

[0006] The surface of the chip is bound to proteins and DNA, and the proteins and DNA are connected to the surface by covalent bonds, which are firm, stable, and difficult to remove, so that the parameters / performance of the chip surface are relatively stable; moreover, when the chip is used, non-specific adsorption caused by the debonding / adsorption of proteins and / or DNA from the surface can be avoided. For example, the chip is applied to nucleic acid sequencing or the detection and identification of nucleic acid molecules, which can avoid the adsorption of enzymes and / or nucleotides during the sequencing process caused by the desorption of proteins.

[0007] In some specific embodiments, the chip of this embodiment may also have at least one of the following additional technical features:

[0008] In certain instances, the protein is bovine serum albumin.

[0009] In some examples, the surface or the protein comprises blocked thiol groups.

[0010] In some examples, the surface is also connected to a small molecule modification agent, which can be connected to the surface to form an anti-nonspecific adsorption layer on the surface. The so-called small molecule modification agent is a compound with a molecular weight of no more than 1000 Daltons. Specifically, the small molecule modification agent is selected from any one, two, three, four or all five of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.

[0011] According to an embodiment of the present invention, a method for preparing a protein co-modified DNA chip is also provided, which can be used to prepare the protein co-modified DNA chip in any of the above embodiments. The so-called chip includes a substrate having a surface and one or more channels, the surface is connected to the channel, and the method includes a fixation treatment, including passing a fixative containing DNA and protein into the channel so that the fixative is in contact with the surface for a certain period of time, so that the DNA and the protein are covalently linked to the surface.

[0012] The method can prepare a chip with stable and controllable surface properties of DNA and protein covalently linked on the surface. Moreover, the method can make the fixing liquid flow to the channel through fluid and automatic control so that the DNA and protein molecules therein are connected to the specified surface of the packaged chip, which is conducive to the industrial preparation of the chip. The so-called chip is made of, for example, two layers of upper and lower layers or three layers of upper, middle and lower substrates / substrates stacked together, for example, see the structure disclosed in CN105154323A, the lower layer or middle layer substrate can have a channel, the channel of the packaged chip has a liquid input port and an output port and has a certain length, width and height to accommodate a certain volume of liquid, and the surface of the upper substrate close to the lower substrate / middle layer substrate / channel can be connected to the said DNA and protein molecules.

[0013] In some specific embodiments, the method of this embodiment may also have at least one of the following additional technical features:

[0014] In certain instances, the protein is bovine serum albumin.

[0015] In some examples, the surface is a surface having epoxysilane, and both the DNA and the protein are immobilized on the surface by covalently bonding with the epoxysilane.

[0016] In some examples, a weak passivation treatment is further included, including introducing a weak passivation solution containing a surfactant into the channel to replace the fixative solution, so that the solution contacts the fixed surface to promote the connection between the DNA and / or the protein and the surface.

[0017] In some examples, the surfactant is a cationic surfactant. Specifically, the surfactant is selected from at least one of hexadecyltrimethylammonium bromide, dioctadecylammonium bromide, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctylammonium bromide.

[0018] In some examples, the concentration of the surfactant in the weak passivation solution is 1-25 mmol / L, preferably 10 mmol / L.

[0019] In some examples, the weak passivation solution further comprises a protein, and the protein is the same as the protein contained in the fixing solution, for example, both are bovine serum albumin. This is conducive to preparing a chip with stable and controllable surface properties.

[0020] In some examples, the weak passivation solution further comprises a small molecule modification agent, which can be connected to the surface to form an anti-nonspecific adsorption layer, and the small molecule modification agent is a compound with a molecular weight of no more than 1000 Da.

[0021] In some examples, the small molecule modifier is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.

[0022] In some examples, the concentration of the small molecule modification reagent in the weak passivation solution is 15-45 mmol / L, preferably 30 mmol / L.

[0023] In some examples, the weak passivation treatment includes contacting the weak passivation solution with the surface at 35° C.-40° C. for 2 h-5 h. This is conducive to preparing a chip with stable and controllable surface properties.

[0024] In some examples, a passivation treatment is further included, including introducing a passivation liquid into the channel to replace the fixing liquid or the weak passivation liquid, so that the passivation liquid is in contact with the surface after the fixing treatment or the weak passivation treatment for a certain period of time.

[0025] In some examples, a thiol blocking treatment is further included, including blocking the thiol groups on the surface after the fixation treatment or the weak passivation treatment or the passivation treatment. Specifically, the thiol blocking treatment includes: introducing a reducing agent into the channel to replace the fixation solution or the weak passivation solution or the passivation solution to reduce the disulfide bonds on the surface to thiol groups, and introducing a thiol blocking reagent into the channel to replace the reducing reagent to block the thiol groups on the surface.

[0026] In some 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 agent is selected from at least one of iodoacetamide, iodoacetic acid, maleimide and glycidol. The blocking agents used here can introduce more hydrophilic groups or negatively charged groups, further enhancing the anti-nonspecific adsorption ability of the chip surface.

[0027] In some examples, a washing step is also included, including using three different solutions to wash the surface after the fixation treatment, weak passivation treatment or passivation treatment in sequence, so as to obtain a chip with stable and controllable surface properties.

[0028] The embodiment of the present invention further provides a protein co-modified DNA chip, which is prepared by the method of any of the above embodiments / examples. The technical features and advantages of the chip or the chip preparation method in any of the above embodiments are also applicable to the chip, and will not be repeated here.

[0029] The embodiments of the present invention also provide the use of the chip of any of the above embodiments in nucleic acid and / or protein detection and analysis, such as nucleic acid molecule detection, sequencing, etc.

[0030] An embodiment of the present application provides a protein co-modified DNA chip, which is formed by proteins binding to the substrate surface of the DNA chip.

[0031] It can be understood that the protein can be bound to the substrate surface of the DNA chip by covalent bonding or non-covalent bonding. In one embodiment of the present application, the protein is bound to the substrate surface of the DNA chip by covalent bonding, and the protein is firmly fixed to the substrate surface. Compared with physical adsorption modification, covalent bonding is more reliable in protein fixation, avoiding desorption, thereby avoiding non-specific adsorption caused by desorption when the protein co-modified DNA chip is used. For example, in the process of sequencing or detection and identification, the protein co-modified DNA chip of the present application is used to avoid the adsorption of enzymes and nucleotides during the sequencing process caused by the desorption of proteins.

[0032] In some examples, the protein used for protein co-modification in the protein co-modification DNA chip is bovine serum albumin.

[0033] In some examples, the thiol groups on the protein co-modified with the protein in the protein co-modified DNA chip are in a blocked state.

[0034] The protein co-modified DNA chip reduces or even avoids the adsorption of molecules containing thiol groups by the DNA chip because the thiol groups on the protein are in a closed state, and has the effect of resisting the adsorption of molecules containing thiol groups. In addition, in a specific embodiment, when the thiol groups are blocked, the blocking reagent used introduces more hydrophilic groups or negatively charged groups, further enhancing the anti-nonspecific adsorption ability of the DNA chip.

[0035] In some examples, the substrate surface of the protein co-modified DNA chip has an anti-nonspecific adsorption layer formed by a small molecule modification reagent. The small molecule modification reagent of the present application refers to a small molecule compound with a relative molecular mass of less than 1000.

[0036] It is understood that the small molecule modification agent can be bound to the substrate surface by covalent bonding or non-covalent bonding. In one embodiment, the small molecule modification agent is bound to the substrate surface by covalent bonding to form a stable and controllable anti-nonspecific adsorption layer.

[0037] In some examples, the small molecule modifying agent is selected from any one, two or more of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.

[0038] The protein co-modified DNA chip also uses a small molecule modification reagent to form an anti-nonspecific adsorption layer, further improving the anti-nonspecific adsorption ability of the chip surface. In particular, in some preferred embodiments, the inventors have discovered a class of small molecule modification reagents with good reaction controllability and repeatability, such as taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine, etc., which makes the preparation of the anti-nonspecific adsorption layer highly controllable and can effectively act on the substrate surface together with the protein co-modification to enhance the anti-nonspecific adsorption ability of the DNA chip.

[0039] Another embodiment of the present application discloses a method for preparing a protein co-modified DNA chip, including when performing DNA fixation treatment, the fixative also contains protein, so that the protein reacts with the substrate surface to fix the protein on the substrate surface, thereby achieving a protein co-modification effect.

[0040] The method of this embodiment uses covalent bonds to fix the protein and DNA on the surface of the substrate, and the connection is stable and firm, and it is not easy to desorb / disconnect. For example, the amino group of the protein can react with the epoxy group modified on the surface of the substrate to generate a covalent bond to fix the protein on the surface of the substrate.

[0041] Generally speaking, the preparation method of DNA chip includes processes such as fixation treatment, passivation treatment and washing, wherein the passivation treatment and subsequent washing can be performed with reference to the existing DNA chip preparation process. The substrate can also use a conventional substrate, and this application does not make specific limitations on this.

[0042] In some examples, the protein used is bovine serum albumin.

[0043] In some examples, the substrate surface in the preparation method has epoxy silane modification. The substrate surface is sometimes also referred to as a surface, and has epoxy silane modification, so that the protein can be fixed on the substrate surface through its amino group and the epoxy silane modification to form a covalent bond, thereby making the protein firmly and effectively fixed on the substrate surface.

[0044] In some examples, the preparation method further includes performing a passivation treatment after the fixation treatment, and after the passivation treatment, performing a thiol blocking treatment on the passivated substrate to block the thiol groups on the protein fixed on the surface of the substrate.

[0045] It is understandable that the connection of biological molecules such as proteins and / or nucleic acids to the surface, whether physical adsorption or covalent connection, has the problem of non-specific adsorption of small molecules, especially non-specific adsorption of small molecules containing thiol groups; wherein, the inventors believe that the non-specific adsorption of small molecules containing thiol groups is partly caused by the thiol groups on the introduced co-modified proteins. Based on the above understanding, the method of this embodiment, after the fixation treatment or weak passivation treatment or passivation treatment, performs a thiol blocking treatment on the surface, and blocks the thiol groups on the substrate surface, such as the thiol groups on the protein thereon, thereby reducing or even avoiding the non-specific adsorption of small molecules containing thiol groups such as cysteine ​​(Cys), homocysteine ​​(Hcy) and reduced glutathione (GSH) and nucleotide molecules containing thiol groups by the protein co-modified DNA chip; and during the thiol blocking treatment, the introduced reagent can introduce more hydrophilic groups or negatively charged groups on the surface of the protein co-modified DNA chip, further enhancing the anti-non-specific adsorption ability of the DNA chip. In this way, the thiol groups on the surface of the protein co-modified DNA chip are blocked to make the thiol groups on the surface inactive / inactive, which is conducive to obtaining a surface with stable and controllable performance.

[0046] The nucleotides referred to herein refer to nucleosides, nucleotides or their analogs and derivatives, which follow the central rule of base pairing.

[0047] In some examples, the thiol blocking treatment specifically includes first reducing the disulfide bonds in the protein using a reducing agent to generate reduced thiol groups, and then blocking the generated reduced thiol groups using a thiol blocking reagent.

[0048] In certain examples, the reducing agent is at least one of tris(2-carboxyethyl)phosphine (abbreviated as TCEP), tris(3-hydroxypropyl)phosphine (abbreviated as THPP), dithiothreitol (abbreviated as DTT), and mercaptoethanol (abbreviated as ME).

[0049] In some examples, the thiol blocking reagent is at least one of iodoacetamide, iodoacetic acid, maleimide and glycidol.

[0050] In some examples, the preparation method of this embodiment also includes performing a weak passivation treatment after the DNA fixation treatment and before the passivation treatment; the weak passivation treatment includes adding a surfactant catalyst and a protein to the reaction solution, and using the surfactant catalyst to promote the binding of DNA and protein to the substrate surface, so that the DNA and protein are fully fixed on the substrate surface.

[0051] The weak passivation treatment referred to here utilizes a surfactant, preferably a cationic surfactant, to catalyze and promote the binding of DNA and protein to the substrate surface, so that the amount of DNA or protein bound to the substrate surface has a better dependence and correspondence with the initial added DNA or protein concentration, thereby making the amount of DNA or protein fixed on the DNA chip controllable, and improving the quality and effect of protein co-modification. It can be understood that if the binding reaction of DNA or protein to the substrate surface such as the formation of covalent bonds is insufficient, it is easy to cause the amount of DNA or protein bound to the same batch or different batches of DNA chips to be unstable, and it is impossible to meet the production and use requirements of highly available DNA chips; this embodiment increases the weak passivation treatment step and uses surfactant catalysis in the weak passivation treatment, so that DNA and protein can be efficiently and fully bound to the surface, with good repeatability, and the amount of DNA and protein fixed is controllable.

[0052] The reaction solution for weak passivation treatment can use a buffer compatible with the so-called surfactant. In principle, as long as it does not cause adverse effects on DNA and the surface and can promote its binding with the surface, it is a suitable buffer; in a specific embodiment, the fixative solution of the fixation treatment is directly used as the weak passivation solution for weak passivation treatment. In this way, it is convenient to prepare the solution and to prepare the chip industrially.

[0053] In certain examples, the surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and tetraoctylammonium bromide.

[0054] Preferably, the surfactant is a cationic surfactant, which can effectively promote the formation of covalent bonds between the amino groups of DNA and / or protein and the chemical modification groups on the substrate surface. In one example, the surfactant hexadecyltrimethylammonium bromide (abbreviated as CTAB) is used. It can be understood that other surfactants with similar functions, such as dioctadecyl ammonium bromide, hexadecyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctyl ammonium bromide, can also effectively promote the reaction between the amino groups and the chemical modification groups on the substrate surface.

[0055] In some examples, the concentration of the surfactant in the reaction solution of the weak passivation treatment (weak passivation solution) is 1-25 mmol / L, preferably 10 mmol / L.

[0056] In some examples, the weak passivation treatment is carried out at 35° C.-40° C. for 2 h-5 h.

[0057] In some examples, the protein added to the reaction solution of the weak passivation treatment in the preparation method is also bovine serum albumin, so as to facilitate the preparation of the solution and the industrial production of the chip.

[0058] In some examples, a small molecule modification reagent is added to the reaction solution of the weak passivation treatment, and the small molecule modification reagent is used to bind to the substrate surface and fix the small molecule modification reagent on the substrate surface to form an anti-nonspecific adsorption layer.

[0059] It can be understood that the binding reaction between the small molecule modification reagent and the substrate surface can be a covalent binding reaction or a non-covalent binding reaction; in one implementation of the present application, the small molecule modification reagent is covalently bound to the substrate surface to form a nonspecific adsorption layer.

[0060] In some examples, the concentration of the small molecule modification reagent in the reaction solution of the weak passivation treatment is 15-45 mmol / L, preferably 30 mmol / L.

[0061] It should be noted that in this embodiment, the reaction solution of the weak passivation treatment contains a small molecule modification reagent, which can be combined with the substrate surface to form an anti-nonspecific adsorption layer, and together with the protein co-modification, enhance the surface's anti-nonspecific adsorption ability. In some preferred embodiments, one or more of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine are used as small molecule modification reagents, and the small molecule modification reagent has an efficient and controllable binding ability with the chemical modification group on the substrate surface, and the small molecule modification reagent has good repeatability in binding with the substrate, and can stably and reliably prepare a highly available DNA chip with good anti-nonspecific adsorption effect, solving the problems of poor controllability and weak repeatability commonly found in current small molecule modifications.

[0062] In some examples, the method of this embodiment further includes the following steps:

[0063] The fixation treatment includes contacting a fixative containing DNA and protein with the substrate surface and fixing the substrate under constant temperature conditions; generally, the fixative is 0.25 mol / L Na 2 CO 3 / NaHCO 3 , pH 9.78, wherein the concentration of DNA is generally 0.01-0.4nmol / L, the concentration of protein is generally 10-100μmol / L, the temperature of the fixation treatment is about 37°C, and the treatment time is about 30min. The above conditions are for reference only and are not specifically limited here; wherein, "Na 2 CO 3 / NaHCO 3 " indicates that Na 2 CO 3 and NaHCO 3 Composition of the fixative.

[0064] The weak passivation treatment includes contacting a reaction solution containing a catalyst, a cationic surfactant, a surfactant and a protein, or a reaction solution containing a cationic surfactant, a surfactant catalyst, a small molecule modification agent and a protein with the substrate after the fixation treatment, and performing weak passivation under constant temperature conditions; in the weak passivation treatment in this example, the small molecule modification agent can be fixed on the surface of the substrate to form an anti-nonspecific adsorption layer, and the catalyst, especially the surfactant, can fully combine the DNA or protein with the modification group on the surface of the substrate; it can be understood that the concentration and treatment time of the small molecule modification agent will affect the amount of the small molecule modification agent fixed on the surface of the substrate, and the higher the concentration and the longer the treatment time, the greater the amount of the small molecule modification agent fixed on the surface of the substrate; similarly, the higher the concentration of the surfactant and the longer the treatment time, the better the effect of fully combining the DNA or protein with the chemical modification group on the surface of the substrate; the specific can be determined according to production or product requirements, and is not specifically limited here;

[0065] In one example, the reaction solution for the weak passivation treatment is 0.25 mol / L Na2O3 containing 10 nM surfactant, 30 mM small molecule modification reagent and 10-100 μM protein. 2 CO 3 / NaHCO 3 , pH is between 9.58-10.53, and the reaction solution is passed into the chip channel using a fluid device for weak passivation treatment. The volume of the circulating solution is 1 mL, the fluid speed is 1 mL / min, the reaction time is 3 h, and the reaction temperature is 37°C.

[0066] The passivation treatment includes cleaning the weakly passivated substrate surface with a passivation solution, and then contacting the passivation solution with the weakly passivated substrate surface to perform passivation at a constant temperature. In one example, the passivation solution is 1 mol / L K 2 HPO 4 / KH 2 PO 4 , pH 9.0; among which, “K 2 HPO 4 / KH 2 PO 4 " indicates that K 2 HPO 4 and KH 2 PO 4 Passivation solution composed of:

[0067] In one example, a fluid device is used to pass a reaction liquid through a chip channel for fixation, weak passivation, passivation and other reactions. The passivation treatment conditions are as follows: the passivation liquid is passed 3-4 times, the volume of each flow is 500 μL, the fluid speed is 1 mL / min, the interval time between each flow is 1800 seconds, and the temperature is maintained at 37°C during the entire passivation process.

[0068] The thiol blocking treatment includes first using a reducing agent to reduce the disulfide bonds in the protein adsorbed on the passivated substrate to generate reduced thiol groups, and then using a thiol blocking reagent to block the thiol groups on the protein; the purpose of the thiol blocking treatment is to block the free thiol groups on the protein or on the surface.

[0069] In one example, a reaction solution is passed through a chip channel using a fluid device for reaction, and a specific scheme for thiol blocking treatment is as follows: first, a reducing reaction solution containing a reducing reagent is passed through, wherein the components are 150mM Tris pH8.0, 100mM NaCl and 30mM reducing reagent, the volume of the circulating solution is 1mL, the fluid speed is 1mL / min, the reaction time is 10-30min, and the reaction temperature is 37°C; then, a blocking reaction solution containing a thiol blocking reagent is passed through, wherein the components are 150mM HEPES pH 8.5, 100mM NaCl and 30mM thiol blocking reagent, the volume of the circulating solution is 1mL, the fluid speed is 1mL / min, the reaction time is 10-30min, and the reaction temperature is 37°C.

[0070] Washing to obtain a DNA chip (chip) includes washing the passivated surface with three different solutions in sequence, and washing at least once with each solution. In one example, the order in which the three solutions (washing solutions) are introduced into the channel is RI-05, RI-06, and RI-07, wherein RI-05 is a phosphate buffer, RI-06 is a mixed solution of HEPES buffer and NaCl solution, and RI-07 is double distilled water. HEPES represents 4-hydroxyethylpiperazineethanesulfonic acid.

[0071] Another embodiment of the present application provides the use of the protein co-modified DNA chip in any of the above embodiments in nucleic acid and / or protein detection analysis. Nucleic acid or protein detection analysis includes sequencing analysis, hybridization analysis, immunoassay, SNP detection analysis, etc.

[0072] The so-called DNA chip refers to a chip substrate with DNA fixed on its surface. The so-called DNA is a type of substance containing a DNA sequence. For example, DNA may contain nucleotide derivatives, nucleotide analogs, fluorescent markers, or both nucleotide sequences and amino acid sequences.

[0073] In some examples, the surface of the chip substrate is a chemically modified surface, and the chemical modification on the surface contains active groups that can react with DNA, and the DNA is fixed to the substrate surface through the reaction between the active groups and the DNA.

[0074] The protein co-modified DNA chip or its preparation method in any of the above embodiments binds the protein to the surface of the substrate, which not only has the advantages of simple method, wide source of raw materials, good effect, and low overall cost, but also fixes the protein through covalent bonds, which is more reliable, and the protein is not easy to desorb, thereby avoiding the non-specific adsorption caused by it.

[0075] In some examples, the anti-nonspecific adsorption function of the protein co-modified DNA chip surface to thiol-containing molecules is further enhanced by blocking the thiol groups on the surface / protein; and when performing thiol blocking, in some preferred embodiments, the blocking reagent used introduces more hydrophilic groups or negatively charged groups, further enhancing the anti-nonspecific adsorption ability of the chip surface.

[0076] In some examples, the preparation method of protein co-modified DNA chip includes a weak passivation step, which uses surfactants, especially cationic surfactants, to catalyze DNA and proteins to more fully react with chemical modification groups on the surface of the substrate, thereby effectively fixing the DNA on the substrate; not only the quality and efficiency of DNA fixation are improved, but also the effect of protein co-modification is improved; moreover, the amount of DNA fixed on the chip surface is highly controllable and can be prepared reproducibly.

[0077] Furthermore, 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 an anti-nonspecific adsorption layer, further enhancing the anti-nonspecific adsorption ability of the chip surface.

[0078] Additional aspects and advantages of the embodiments of the present invention will be given in part in the following description and in part will become apparent from the following description or will be learned through the practice of the embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above and / or additional aspects and advantages of the embodiments of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0080] Figure 1 It is a schematic diagram of the structure of the package chip substrate in the embodiment of the present application;

[0081] Figure 2 is a comparison chart of the adsorption results of proteins in the test system on the chip surface in the embodiment of the present application;

[0082] Figure 3 is a comparison chart of the adsorption results of the thiol small molecule substances in the test system on the chip surface in the embodiment of the present application;

[0083] Figure 4 Schematic diagram of the structure of the glass substrate surface in the embodiment of the present application. DETAILED DESCRIPTION

[0084] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The present invention may repeat reference numerals and / or reference letters in different examples, and such repetition is for the purpose of simplicity and clarity, and does not itself indicate the relationship between the various embodiments and / or settings discussed. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0085] As used herein, unless otherwise indicated, the singular forms "a", "an", etc. include plural referents (more than one); "a set" or "a plurality" refers to two or more.

[0086] In this document, unless otherwise specified, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated; features specified as "first", "second", etc. may explicitly or implicitly include one or more of the said features.

[0087] In this document, unless otherwise specified, "connection", "fixation", etc. should be understood in a broad sense. For example, it can be a fixed connection or a reversible connection, a direct connection or an indirect connection through an intermediate medium, a chemical bond such as a covalent connection, or a chemical or physical adsorption, etc.

[0088] As used herein, the "substrate" or "solid phase substrate" may be any solid support that can be used to fix nucleic acid sequences, such as nylon membranes, glass sheets, plastics, silicon wafers, magnetic beads, etc.; sometimes also referred to as reactors, chips or flow cells.

[0089] Existing protein co-modified DNA chips mainly use physical adsorption to adsorb proteins on the substrate surface, which has an anti-protein adsorption effect; however, physical adsorption is not reliable and is easily desorbed.

[0090] In this regard, an embodiment of the present application provides a protein co-modified DNA chip, in which the protein and DNA are connected to the surface of the substrate through covalent bonds. This not only makes the fixation / connection more secure, but also is not easy to desorb, thereby avoiding non-specific adsorption caused by desorption.

[0091] In certain embodiments, the DNA amino groups react with reactive groups on the substrate surface. Figure 4 As shown, R1 represents an alkane chain molecule with an active reactive group connected to the end, wherein the active group is preferably at least one of an epoxy group, an aldehyde group, a carboxyl group, N-hydroxysuccinimide and diaminobenzanilide. In some embodiments, the use of a weak passivation solution containing a surfactant can catalyze and promote the amino group of DNA to Figure 4 The active groups in the chemical modifications shown react to form covalent bonds, thereby fully immobilizing the DNA on the substrate surface.

[0092] In addition, the inventors found that the existing protein co-modified DNA chip has a high adsorption capacity for small molecules such as cysteine ​​(Cys), homocysteine ​​(Hcy), reduced glutathione (GSH) and nucleotide molecules containing thiol groups, which is probably because the co-modified protein used as a blocking agent has a large number of free thiol groups, and therefore can non-specifically adsorb cysteine ​​and other thiol small molecules. Based on the above understanding, in some preferred embodiments, the substrate surface after fixation treatment or weak passivation treatment or passivation treatment is subjected to thiol blocking treatment to block the free thiol groups on the substrate surface, thereby reducing or even avoiding the adsorption of thiol small molecules by the protein co-modified DNA chip, and reducing the non-specific adsorption on the chip surface.

[0093] In some preferred embodiments, the surface after the fixation treatment is weakly passivated using a weak passivation solution containing a surfactant, which can make DNA or protein react more fully with the chemical modification groups on the substrate surface, so that DNA or protein can be more effectively fixed on the substrate surface. The use of the weak passivation solution containing a surfactant makes DNA or protein react more fully with the chemical modification groups on the substrate surface, which not only improves the efficiency and quality of DNA or protein fixed on the substrate surface, but also makes the amount of DNA or protein fixed on the biochip have a good correlation with the DNA or protein concentration initially added in the fixative, thereby achieving a controllable amount of DNA or protein fixed on the biochip with good repeatability. This not only lays the foundation for the preparation of high-quality biochips, but also can meet customized production needs.

[0094] In some preferred embodiments, the weak passivation solution further comprises a small molecule modification agent, so that the small molecule modification agent is connected to the substrate surface, which can further reduce the non-specific adsorption on the chip surface. In addition, taurine, aminopropylsulfonic acid, serine, glutamic acid and / or phosphoserine are used as small molecule modification agents, which have a highly efficient and controllable binding ability with the epoxy groups on the substrate surface, so that the binding of the small molecule modification agent to the substrate surface has good repeatability and strong operability, and can stably and reliably prepare a highly available DNA chip with good anti-non-specific adsorption effect.

[0095] Unless otherwise specified, some abbreviations / codes / numbers involved in the embodiments are referred to as follows:

[0096] AT-01: 0.25M Na 2 CO 3 / NaHCO 3 , 0.6 mM CTAB (i.e., hexadecyltrimethylammonium bromide), pH 9.78;

[0097] RI-04: 1M K 2 HPO 4 / KH 2 PO 4 , pH 9.0;

[0098] RI-05: pH 7.4 PBS;

[0099] RI-06: a mixed solution of 150 mM HEPES and 150 mM NaCl;

[0100] RI-07: double distilled water;

[0101] Dot / FOV: The number of bright spots within the observation area of ​​110×110μm.

[0102] Example 10

[0103] In this example, DNA is fixed to the surface of a glass substrate with epoxy silane on the surface through the amino group of DNA, and bovine serum albumin is used as a co-modified protein to form a protein co-modified DNA chip (sometimes referred to as a chip or DNA chip). This example compares the effects of adding bovine serum albumin co-modification and not adding bovine serum albumin co-modification on the DNA chip.

[0104] This example uses the "in-channel" fixation method to fix DNA to the chip substrate, that is, first encapsulate the chip substrate, and then use fluid equipment to pass various reaction reagents and washing reagents into the encapsulated chip channel to achieve chemical reactions such as fixation treatment and passivation treatment. Figure 1As shown, after the chip substrate is packaged, each chip channel is formed to be independent of each other, and each chip channel can independently perform each reaction. Figure 1 The figure shows a packaged chip substrate with 8 channels, the channel specifications are length × width × height 90mm × 1.8mm × 0.1mm. According to different packaging processes or fluid equipment, the chip substrate can also be packaged into 16 channels, and 16 different modified DNAs can be independently produced on a DNA chip.

[0105] The preparation method of the DNA chip in this example is as follows:

[0106] (1) Fixation treatment: a fixation reaction solution is introduced into the channel of the chip substrate to perform a fixation reaction. The fixation reaction solution in this example is a fixation solution AT-01 containing 0.2 nM DNA and 30 μM bovine serum albumin (BSA). The 3' end of the DNA contained in the fixation solution also has an amino-modified NH 2 and Cy3 fluorophore; the composition of AT-01 is 0.25M Na 2 CO 3 / NaHCO 3 , 0.6 mM CTAB, pH 9.78, the volume of the circulating solution was 1 mL, the flow rate was 1 mL / min, the reaction time was 30 min, and the reaction temperature was 37 °C;

[0107] As a control, a channel without BSA added to the immobilization reaction solution was set up in this example;

[0108] Among them, DNA and BSA are in a competitive adsorption relationship, and the reaction performance of DNA with the surface is much better than that of BSA, so even if 15,000 times the concentration of BSA is added, there is still no significant effect on the modification of DNA; in the preparation method of this example, BSA is added during the fixation treatment and the weak passivation treatment, so that the effect of protein co-modification is better. Of course, in the case of lower requirements, BSA can also be added only in the fixation treatment step;

[0109] (2) Weak passivation treatment: weak passivation reaction solution is introduced to perform a weak passivation step. The weak passivation reaction solution in this example is 0.25 M Na2SO4 containing 10 mM CTAB, 30 mM taurine and 30 μM BSA. 2 CO 3 / NaHCO 3 , pH is between 9.58-10.53, specifically pH 9.78 in this example; set the volume of the circulating solution to 1 mL, the fluid velocity to 1 mL / min, the reaction time to 3 h, and the reaction temperature to 37°C;

[0110] Among them, the channel in which BSA was not added to the reaction solution during the fixation treatment was also not added with BSA during the weak passivation treatment, and the other components, dosages and channel parameter settings were the same, serving as a control;

[0111] (3) Wash away the weak passivation solution. Specifically, use the passivation solution RI-04 for 3 washes. The volume of each wash is 1 mL. The flow rate is 1 mL / min. The temperature is maintained at 37°C during washing. The composition of RI-04 in this example is 1 M K 2 HPO 4 / KH 2 PO 4 , pH 9.0;

[0112] (4) Passivation treatment: Specifically, the passivation solution RI-04 was used for 3-4 washes, the volume of each inflow was 500 μL, the fluid speed was 1 mL / min, the interval between each inflow was 1800 s, and the temperature was maintained at 37°C during the entire passivation process; the composition of RI-04 in this example was 1 M K 2 HPO 4 / KH 2 PO 4 , pH 9.0;

[0113] (5) Washing the chip after the passivation treatment, including washing with three solutions, washing three times with each solution, each time the volume of the solution is 1 mL, the fluid speed is 1 mL / min, and the temperature is maintained at 37°C during washing; the three washing solutions are RI-05, RI-06, and RI-07 in the order of use, wherein RI-05 is a phosphate buffer solution of pH 7.4, RI-06 is a mixed solution of 150 mM HEPES buffer and 150 mM NaCl solution, and RI-07 is double distilled water.

[0114] After washing with the three solutions and then air-drying or baking normally, the DNA chip of this example is obtained.

[0115] The single-molecule fluorescence imaging technique was used to evaluate the nonspecific adsorption of proteins on the DNA chip prepared in this example. Specifically, a fluorescently labeled Klenow Fragment was introduced into the surface of the prepared DNA chip, and the number of fluorescently labeled Klenow Fragments adsorbed on the surface of the DNA chip was detected. The number of fluorescently labeled points of Klenow Fragments per unit area was used to characterize the nonspecific adsorption of proteins on the DNA chip. In this example, the number of fluorescent points in a 110×110μm area was specifically counted.

[0116] The test results of this DNA chip and its control test are as follows Figure 2 As shown, Figure 2In the figure, the horizontal axis None represents the DNA chip without adding BSA, BSA represents the protein co-modified DNA chip prepared by adding BSA for co-modification, and the vertical axis represents the corresponding number of fluorescence points. Figure 2 The results showed that the number of nonspecific adsorption points of the DNA chip prepared with BSA co-modification was about 500 Dot / FOV, while the number of nonspecific adsorption points of the DNA chip prepared without BSA co-modification was greater than 6000 Dot / FOV, indicating that BSA co-modification can greatly reduce the nonspecific adsorption of proteins on the DNA chip and has good anti-protein adsorption effect.

[0117] Embodiment 20

[0118] The materials and process for preparing the DNA chip in this example are the same as those in Example 1, except that after the passivation treatment, a thiol blocking treatment is added, and the thiol groups on the protein are blocked with a thiol blocking reagent to reduce the nonspecific adsorption of the protein co-modified DNA chip to thiol small molecules.

[0119] The specific preparation method of the DNA chip in this example is as follows:

[0120] (1) Fixation treatment: a fixation reaction solution is introduced into the channel of the chip substrate to perform a fixation reaction. The fixation reaction solution in this example is a fixation solution AT-01 containing 0.2 nM DNA and 30 μM bovine serum albumin (BSA); wherein the 3' end of the DNA contained in the fixation solution also has an amino-modified NH 2 and Cy3 fluorophore; the composition of AT-01 is 0.25M Na 2 CO 3 / NaHCO 3 , 0.6 mM CTAB, pH 9.78, the volume of the circulating solution was 1 mL, the flow rate was 1 mL / min, the reaction time was 30 min, and the reaction temperature was 37 °C;

[0121] (2) Weak passivation treatment: weak passivation reaction solution is introduced to perform a weak passivation step. The weak passivation reaction solution in this example is 0.25 M Na2SO4 containing 10 mM CTAB, 30 mM taurine and 30 μM BSA. 2 CO 3 / NaHCO 3 , pH is between 9.58-10.53, specifically pH 9.78 in this example; set the volume of the circulating solution to 1 mL, the fluid velocity to 1 mL / min, the reaction time to 3 h, and the reaction temperature to 37 °C;

[0122] (3) Wash away the weak passivation solution. Specifically, use the passivation solution RI-04 for 3 washes. The volume of each wash is 1 mL. The flow rate is 1 mL / min. The temperature is maintained at 37°C during washing. The composition of RI-04 in this example is 1 M K 2 HPO 4 / KH 2 PO 4 , pH 9.0;

[0123] (4) Passivation treatment: Specifically, the passivation solution RI-04 was used for 3-4 washes, the volume of each inflow was 500 μL, the fluid speed was 1 mL / min, the interval between each inflow was 1800 s, and the temperature was maintained at 37°C during the entire passivation process; the composition of RI-04 in this example was 1 M K 2 HPO 4 / KH 2 PO 4 , pH 9.0;

[0124] (5) Thiol blocking treatment, a) passing a reaction solution containing a reducing agent TCEP, the composition of which is 150mM Tris, pH 8.0, 100mM NaCl and 30mM TCEP; the purpose of this step is to reduce the disulfide bonds in the system and generate reducing thiol groups, which is beneficial to the next step of blocking; the volume of the circulating solution is 1mL, the flow rate is 1mL / min, the reaction time is 10-30min, and the reaction temperature is 37°C; b) passing a thiol blocking reagent reaction solution, the thiol blocking reagent can be iodoacetamide or iodoacetic acid, and in this example, iodoacetamide is specifically used, and the reaction solution composition is 150mM HEPES, pH 8.5, 100mM NaCl and 30mM iodoacetamide; the volume of the circulating solution is 1mL, the flow rate is 1mL / min, the reaction time is 10-30min, and in this example, the reaction is specifically for 30min, and the reaction temperature is 37°C;

[0125] (6) Washing the chip after thiol blocking treatment, including washing with three solutions, washing three times with each solution, each time the volume of the solution is 1 mL, the fluid speed is 1 mL / min, and the temperature is maintained at 37°C during washing; the three washing solutions are RI-05, RI-06, and RI-07 in the order of use, wherein RI-05 is a phosphate buffer solution of pH 7.4, RI-06 is a mixed solution of 150 mM HEPES buffer and 150 mM NaCl solution, and RI-07 is double distilled water.

[0126] After washing with the three solutions and then air-drying or baking normally, the DNA chip of this example is obtained.

[0127] Single-molecule fluorescence imaging technology was used to evaluate the adsorption of disulfide bond-containing nucleotide molecules by the DNA chip prepared in this example. Specifically, fluorescent Cy3-labeled nucleotide molecules containing disulfide bonds were introduced into the surface of the prepared DNA chip, and the amount of Cy3 fluorescence adsorbed on the surface of the DNA chip was detected. The number of Cy3 fluorescence points per unit area was used to characterize the adsorption of disulfide bond-containing nucleotide molecules on the DNA chip. This example specifically counted the number of fluorescence points (number of bright spots) in an area of ​​110×110μm. At the same time, the adsorption of disulfide bond small molecules on the DNA chip prepared by co-modification with BSA in Example 1 was tested for comparison; the DNA chip prepared by co-modification with BSA in Example 1 was not treated with thiol groups. The so-called disulfide bond-containing nucleotide molecules can be, for example, referenced to the compounds disclosed in EP2607369B1, such as Figure 2 The compounds shown.

[0128] Test results such as Figure 3 As shown, Figure 3 The middle horizontal axis "None" is a DNA chip prepared by co-modification with BSA but without thiol blocking treatment, that is, the BSA co-modified DNA chip of Example 1, and "Block" is a DNA chip prepared by co-modification with BSA and thiol blocking treatment, that is, the DNA chip of this example. Figure 3 The results showed that the number of nonspecific adsorption points of the DNA chip without thiol blocking treatment was greater than 3000 Dot / FOV, while the number of nonspecific adsorption points of the DNA chip with thiol blocking treatment was about 500 Dot / FOV, indicating that the thiol blocking treatment can greatly reduce the nonspecific adsorption of DNA chips to small molecules containing thiol groups.

[0129] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "certain examples", "specific examples" or "embodiments" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0130] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A protein co-modified DNA chip, characterized in that: The chip includes a substrate having a surface, wherein the surface is a modified surface, the protein and the DNA are covalently connected to the surface, and the amino groups of the protein and the DNA form covalent bonds with the chemical modification groups on the surface, wherein the chemical modification groups are at least one of epoxy groups, aldehyde groups, carboxyl groups, N-hydroxysuccinimide and diaminobenzanilide, and the protein contains a blocked thiol group.

2. The chip according to claim 1, characterized in that: The protein is bovine serum albumin.

3. The chip according to claim 1, characterized in that: The surface is also connected to a small molecule modification agent, and the small molecule modification agent is a compound with a molecular weight not greater than 1000 Daltons.

4. The chip according to claim 3, characterized in that: The small molecule modifier is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.

5. A method for preparing a protein co-modified DNA chip, characterized in that: The chip includes a substrate having a surface and one or more channels, wherein the surface is connected to the channels. The method includes a fixation treatment, including introducing a fixative containing DNA and protein into the channel so that the fixative is in contact with the surface for a certain period of time, so that the DNA and the protein are covalently connected to the surface, and the amino groups of the protein and the DNA are covalently bonded to the chemical modification groups on the surface, wherein the chemical modification groups are at least one of epoxy groups, aldehyde groups, carboxyl groups, N-hydroxysuccinimide and diaminobenzanilide, and the protein contains a blocked thiol group.

6. The method according to claim 5, characterized in that The protein is bovine serum albumin.

7. The method according to claim 5 or 6, characterized in that: It also includes a weak passivation treatment, including introducing a weak passivation solution containing a surfactant into the channel to replace the fixing solution, so that it contacts the surface after the fixing treatment to promote the connection between the DNA and / or the protein and the surface.

8. The method according to claim 7, characterized in that The surfactant is a cationic surfactant.

9. The method according to claim 7, characterized in that: The surfactant is selected from at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctylammonium bromide.

10. The method according to claim 9, characterized in that The concentration of the surfactant in the weak passivation solution is 1-25 mmol / L.

11. The method according to claim 10, characterized in that The concentration of the surfactant in the weak passivation solution is 10 mmol / L.

12. The method according to claim 7, characterized in that The weak passivation solution further comprises protein, and the protein is the same protein as the protein contained in the fixing solution.

13. The method according to claim 12, characterized in that The protein is bovine serum albumin.

14. The method according to any one of claims 10 to 13, characterized in that: The weak passivation solution further comprises a small molecule modification agent, which can be connected to the surface to form an anti-nonspecific adsorption layer, and the small molecule modification agent is a compound with a molecular weight of no more than 1000 Daltons.

15. The method according to claim 14, characterized in that The small molecule modifier is selected from at least one of taurine, aminopropylsulfonic acid, serine, glutamic acid and phosphoserine.

16. The method according to claim 15, characterized in that The concentration of the small molecule modification reagent in the weak passivation solution is 15-45 mmol / L.

17. The method according to claim 16, characterized in that The concentration of the small molecule modification reagent in the weak passivation solution is 30 mmol / L.

18. The method according to any one of claims 8 to 13, characterized in that: The weak passivation treatment comprises contacting the weak passivation solution with the surface at 35° C.-40° C. for 2 h-5 h.

19. The method according to any one of claims 8 to 13, characterized in that: It also includes a passivation treatment, including introducing a passivation liquid into the channel to replace the fixing liquid or the weak passivation liquid, so that the passivation liquid contacts the surface after the fixing treatment or the weak passivation treatment.

20. The method according to claim 19, characterized in that It also includes a thiol blocking treatment, including blocking the thiol groups on the surface after the fixation treatment or the weak passivation treatment or the passivation treatment.

21. The method according to claim 20, characterized in that 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 to reduce the disulfide bonds on the surface to thiol groups, and A thiol blocking reagent is introduced into the channel to replace the reducing reagent, so as to block the thiol groups on the surface.

22. The method according to claim 21, characterized in that 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 glycidol.

23. The method according to any one of claims 8 to 13, characterized in that: The method also includes a washing step, which includes using three different solutions to wash the surface after the fixing treatment, the weak passivation treatment or the passivation treatment in sequence.

24. A protein co-modified DNA chip, characterized in that: The chip is prepared by the method according to any one of claims 5-23.

25. Use of the chip according to any one of claims 1 to 4 or the chip according to claim 24 in nucleic acid and / or protein detection and analysis.

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