CN113913945A

CN113913945A - Method for preparing chip, chip and method for fixing biological molecule

Info

Publication number: CN113913945A
Application number: CN202111178485.XA
Authority: CN
Inventors: 高锦鸿; 赵�智; 赵陆洋; 王�琦
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: CN109610006A

Abstract

Disclosed are a method for preparing a chip, and a method for immobilizing nucleic acid molecules and/or protein molecules. The chip comprises a substrate and a channel, the substrate is provided with a surface connected with the channel, and the method for preparing the chip comprises (a) introducing a first solution into the channel, wherein the first solution comprises biomolecules so that the biomolecules are connected to the surface, and the biomolecules are selected from nucleic acid molecules and/or protein molecules; and (b) introducing a second solution into the channel to replace the first solution in the channel after (a), wherein the second solution comprises a surfactant, so as to perform weak passivation treatment on the surface after (a), wherein the weak passivation treatment comprises contacting the surface with the second solution for a certain time to promote the connection of the biomolecule and the surface.

Description

Method for preparing chip, chip and method for fixing biological molecule

Technical Field

The present application relates to the field of biological detection, and in particular, to a method for preparing a chip, and a method for immobilizing DNA or protein.

Background

DNA is an important genetic material of many organisms including humans, and contains many important information that can provide key information for physiological or pathological tests. The method can efficiently and accurately detect the DNA, particularly can quantitatively identify the base information change, and has great scientific research significance and great clinical application value. Compared with other detection means such as electrophoresis detection, PCR detection or test paper detection, the DNA chip detection based identification detection method has the advantages of high detection flux, good accuracy, convenience in detection, no need of special testers and the like. The DNA chip is an important platform for realizing the detection of the DNA chip and is the basis for the occurrence of detection reaction. Preparing a DNA chip, wherein DNA needs to be reliably and efficiently fixed on a substrate such as a glass substrate, a silicon wafer substrate or a polymer substrate; moreover, the DNA immobilization method is required to have good repeatability, and to be capable of stably and effectively producing DNA chips with consistent quality.

The DNA chip with unreliable DNA fixation is easy to have the following defects: 1) low detection rate; 2) higher false positives or false negatives; 3) giving wrong sequence information; 4) the DNA chip has poor detection repeatability. In sum, the above defects can cause problems of high detection cost, poor detection quality and the like. Therefore, the development of an efficient and convenient DNA fixing technology and the improvement of the quality and the repeatability of the preparation method have great significance for improving the usability of the DNA chip and reducing the use cost of the DNA chip.

In the current DNA chip preparation method, common ways to fix DNA to a substrate include: 1) covalent bond linkage; 2) physical adsorption connection; 3) specific linker pairs are attached, such as biotin-streptavidin linker pairs. The covalent bond connection mode has the obvious advantages of high connection strength, difficult falling of DNA and the like, and is widely applied. However, covalent bonding, which is an active chemical reaction, has many problems in terms of reaction controllability, reaction speed, and reaction reproducibility. Therefore, the well designed chip preparation method has important value for improving the quality of the DNA chip.

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. To this end, embodiments of the present invention provide a method of preparing a biochip, a method of immobilizing DNA or protein, and a chip.

A method of preparing a chip according to an embodiment of the invention, the chip comprising a substrate and one or more channels, the substrate having a surface connected to the channels, the method comprising: (a) passing a first solution into the channel, the first solution comprising a biomolecule, the biomolecule being a nucleic acid molecule or a protein molecule, such that the biomolecule is attached to the surface; and (b) passing a second solution through the channel to replace the first solution of (a), the second solution comprising a surfactant, to weakly passivate the surface after (a), comprising contacting the surface with the second solution for a period of time to promote attachment of the biomolecule to the surface.

The chip with the biomolecules uniformly distributed on the designated surface and low non-specific adsorption of the designated surface with the biomolecules can be obtained by the method; moreover, the method is beneficial to large-scale or industrial production of the chips with stable and consistent parameters/performances, and has stronger industrial practicability.

In certain embodiments, the method further comprises any one or more of the following additional features and advantages:

in some examples, the surfactant is a cationic surfactant. Specifically, for example, one or more of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, and tetraoctylammonium bromide.

In some examples, the concentration of the surfactant in the second solution is 1 to 25 mmol/L. Preferably, the concentration of the surfactant in the second solution is 10 mmol/L. In this way, the attachment/binding of biomolecules to the surface can be efficiently facilitated.

In some examples, the certain period of time is 2h-5 h. Specifically, the weak passivation treatment is carried out for 2h-5h at 35-40 ℃. In this way, the attachment/binding of biomolecules to the surface can be efficiently facilitated.

In some examples, the biomolecule comprises a nucleic acid molecule, and the nucleic acid molecule carries an amino modification. In particular, the 3 'terminus and/or the 5' terminus of the nucleic acid molecule has the amino modification. The surface is chemically modified and has at least one active group selected from the group consisting of an epoxy group, an aldehyde group, a carboxyl group, N-hydroxysuccinimide, and diaminobenzanilide. In this manner, it is facilitated that the amino modification reacts with the reactive group to form a bond to attach/immobilize the biomolecule to the surface.

It will be appreciated that in general, biochemical reactions such as the formation of a large number of covalent bonds are often difficult to control if simultaneous and efficient formation is desired. The method of this example enables more sufficient covalent bond formation, including more consistent bond formation, and a close correlation between the amount of covalent bond formation and the amount of DNA or protein added to the surface by a weak passivation treatment and performing the weak passivation treatment using a solution containing a surfactant, thus making it feasible and highly controllable to adjust the amount of DNA or protein immobilized on the surface by adjusting the amount of biomolecules, and also has good reproducibility.

In some examples, the nucleic acid molecule is provided with a fluorophore at the 3 'end or the 5' end for localization and/or for biomolecule detection applications. For example, Cy3, Cy5, Atto647N, Atto532, and the like. Of course, it is also possible to carry targeting groups, for example groups which can bind specifically to fluorescent groups; other detectable labels/groups may also be present.

In some examples, the first solution comprises a surfactant.

In some specific examples, the surfactant included in the first solution is the same type of surfactant as the surfactant included in the second solution. Thus, the preparation of the solution is convenient, and the industrial preparation of the chip is facilitated.

In some examples, the first solution comprises 0.01 to 0.10nmol/L of the surfactant. Preferably, the first solution comprises 0.05nmol/L of the surfactant. In this way, efficient attachment/binding of the biomolecules to the surface is facilitated, as well as further treatment of the surface in combination with the second solution to promote a secure attachment/immobilization of the biomolecules to the surface.

In some examples, the method further comprises (c) introducing a third solution into the channel to replace the second solution of (b), wherein the third solution does not contain the surfactant, so as to passivate the surface after (b). Therefore, the method is beneficial to preparing the chip with stable and consistent surface performance parameters.

In some examples, the method further comprises (d) sequentially passing a fourth solution, a fifth solution and a sixth solution into the channel to wash the surface after (c), wherein the fourth solution, the fifth solution and the sixth solution are all free of surfactant. Therefore, the method is beneficial to preparing the chip with stable and consistent surface performance parameters.

According to an embodiment of the present invention, there is also provided a chip prepared according to the method of any of the above embodiments. The method of any of the above embodiments has the advantages of being also applicable to the chip prepared by the corresponding method, for example, the surface parameters/properties of the prepared chip are stable and controllable, the chip can be repeatedly prepared, and the method has strong industrial applicability.

According to an embodiment of the present invention, there is also provided a use of the chip of any one of the above embodiments for detecting nucleic acids and/or proteins. The advantages of the chip of any of the above embodiments are also applicable to this application/use and will not be described herein.

There is also provided, in accordance with an embodiment of the present invention, a method of immobilizing a biomolecule on a prescribed surface, including subjecting the surface to a weak passivation treatment after the biomolecule is immobilized on the surface, the biomolecule being a nucleic acid molecule or a protein molecule, the weak passivation treatment including contacting the surface with a second solution for a time period to promote binding of the biomolecule to the surface, the second solution including a surfactant.

In some examples, the surfactant is a cationic surfactant. For example, at least one selected from the group consisting of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctylammonium bromide.

In some examples, the concentration of the surfactant in the second solution 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 to5 h.

In some examples, the immobilization is performed using a first solution, which is a second solution comprising the biomolecule.

According to an embodiment of the present invention, there is provided a method of manufacturing a biochip including performing a weak passivation process after a fixing process; the weak passivation treatment comprises the steps of contacting a weak passivation reaction solution with the chip after the fixation treatment to promote the combination of DNA or protein and the surface of the substrate of the chip so as to fully fix the DNA or the protein on the surface of the substrate; wherein the weak inactivation reaction solution contains a surfactant which can catalyze and promote the combination of DNA or protein and the surface of the substrate, so that the DNA or the protein is fully fixed on the surface of the substrate.

The method comprises the step of subjecting the surface to which the biomolecules are attached to a weak passivation treatment, which comprises promoting/accelerating the binding of the DNA or protein to the substrate surface with a solution containing a surfactant (a second solution, sometimes referred to as a weak passivation solution or a weak passivation reaction solution) to more sufficiently immobilize the DNA or protein to the substrate surface; it is to be understood that the weakly inactivating reaction solution may be a buffer system adapted to the corresponding surfactant, and this embodiment is not particularly limited to the buffer system, but is preferably a buffer system that does not adversely affect the DNA, protein or substrate surface and does not interfere with the binding of the DNA or protein to the surface.

In certain examples, the catalyst is a surfactant.

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

In one embodiment of the present invention, the catalyst is specifically a cationic surfactant that promotes the formation of covalent bonds between amino groups of DNA or protein and chemically modified groups on the substrate surface. It is understood that other catalysts, not limited to cationic surfactants, may be used for other types of covalent bonds. In one implementation of the present application, the surfactant specifically used is cetyltrimethylammonium bromide (abbreviated CTAB), and it is understood that other surfactants having similar functions, such as dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetraoctylammonium bromide, etc., can also promote the reaction between the amino group and the substrate surface chemistry modification group, and are also applicable to the present application, and are not limited to CTAB.

In some examples, the concentration of the surfactant in the weakly passivating reaction solution is 1 to 25 mmol/L.

In some examples, the concentration of the surfactant in the weakly passivating reaction solution is 10 mmol/L.

In some examples, the reaction conditions for the weak passivation treatment are 35 ℃ to 40 ℃ for 2h to5 h.

In some examples, the DNA carries an amino modification, the substrate surface has a chemical modification, the chemically modified reactive group is at least one of epoxy, aldehyde, carboxyl, N-hydroxysuccinimide, and diaminobenzanilide, and the catalyst promotes a chemical modification reaction between the amino group of the DNA or protein and the substrate surface, such that the DNA or protein is substantially immobilized on the substrate surface.

It is understood that, in the case of proteins, the proteins may also have modified chemical groups to facilitate binding to the substrate surface, as long as the proteins are capable of reacting with reactive groups that chemically modify the substrate surface and binding the proteins to the substrate surface.

It should be noted that the purpose of amino group modification is to utilize the formation of chemical bond between amino group and chemical group on the substrate surface, based on different purposes of DNA immobilization, amino group modification can be performed on different nucleotides of DNA, which can be located at both ends of DNA, or located at other positions of DNA, for example, amino group modification can be located at 5 'end, 3' end or both ends of DNA, and the number of amino groups is not limited to one; it will be appreciated that modifications in which the modified group contains an amino group and the amino group is reactive with a reactive group on the substrate surface are also suitable.

In some examples, the 3 'end or the 5' end of the DNA is further modified with a fluorophore. In certain examples, the fluorophore modification is a Cy3 fluorophore modification. Here, the presence of a fluorescent group allows the biomolecule to be localized and/or detected, so it is understood that the kind of fluorescence which does not affect the DNA immobilization and can be used for the localization detection of DNA is applicable to the method of the present embodiment.

In certain examples, the immobilization treatment of the present application includes contacting a fixing solution containing DNA with a surface of a substrate to immobilize the DNA.

In one implementation of the subject application, the fixative solution is 0.25mol/L Na₂CO₃/NaHCO₃0.6mM CTAB, pH 9.78, wherein the concentration of DNA is generally 0.01-0.4nmol/L, the temperature of the fixation treatment is about 37 ℃, the treatment time is about 30min, and the above conditions are not specifically limited herein; wherein "Na" is₂CO₃/NaHCO₃"means represented by Na₂CO₃And NaHCO₃The composition and the ratio of the two are referred to a conventional DNA chip fixing solution, and are not particularly limited herein.

In some examples, the fixing solution contains the same type of catalyst as in the weakly passivating reaction solution.

In certain examples, the concentration of the catalyst in the fixing solution is from 0.01 to 0.10 nmol/L.

In certain examples, the concentration of the catalyst in the fixation fluid is 0.05 nmol/L.

In certain examples, the preparation method further comprises passivation treatment, wherein the passivation treatment comprises passivation by using a passivation solution to contact the surface of the substrate after the weak passivation treatment.

In one implementation of the present application, the passivation solution is 1mol/L K₂HPO₄/KH₂PO₄pH 9.0; wherein "K" is₂HPO₄/KH₂PO₄"denotes a group consisting of₂HPO₄And KH₂PO₄The proportion of the passivation solution and the conventional DNA chip passivation solution is referred, and the passivation solution is not particularly limited; in one implementation manner of the present application, a fluid device is used to introduce a reaction solution into a chip channel to perform reactions such as fixing, weak passivation, and the like, and the passivation treatment conditions include that the number of times of flowing passivation solution is 3-4, the volume of flowing in each time is 500 μ L, the speed of the fluid is 1mL/min, the interval time of flowing in each time is 1800 seconds, the temperature is maintained at 37 ℃ in the whole passivation process, and the above conditions can be referred to, and are not specifically limited herein.

In some examples, the preparation method of the present application further comprises a washing step, wherein the washing step comprises washing the passivated substrate chip with three washing solutions in sequence, each washing solution is washed at least once, and the three washing solutions are RI-05, RI-06 and RI-07 in sequence according to the using sequence, wherein RI-05 is PBS buffer solution, RI-06 is mixed solution of HEPES buffer solution and NaCl solution, and RI-07 is double distilled water. It is well known in the art to wash DNA chips after the passivation process, where RI-05, RI-06, and RI-07 are also conventional washing solutions, and typically, each washing solution needs to be repeated 3 times. Among them, HEPES is 4-hydroxyethyl piperazine ethanesulfonic acid.

The other side of the application discloses a DNA or protein chip prepared by the preparation method.

It should be noted that, on one hand, the DNA or protein chip of the present application has higher binding efficiency and quality of DNA or protein and substrate, and can effectively ensure the use performance of DNA or protein chip; on the other hand, the quality of the DNA or protein fixed on the DNA or protein chip is highly controllable and has good repeatability, and the customization of the DNA or protein chips with different test requirements can be met.

In yet another aspect, the present application discloses the use of the DNA or protein chip prepared by the preparation method of the present application in nucleic acid or protein detection assays. The nucleic acid or protein detection analysis includes sequencing analysis, hybridization analysis, immunoassay, SNP detection analysis, and the like.

In another aspect of the present application, there is disclosed a method for immobilizing DNA or protein, comprising performing a weak inactivation treatment after a DNA or protein immobilization treatment; the weak passivation treatment comprises the steps of contacting a weak passivation reaction solution with the surface of the substrate after the fixation treatment to promote the combination of DNA or protein and the surface of the substrate, so that the DNA or the protein is fully fixed on the surface of the substrate; wherein the weakly passivating reaction solution contains a catalyst which promotes the binding of DNA to or to the surface of the protein substrate, so that the DNA or the protein can be sufficiently immobilized on the surface of the substrate.

In certain examples, in the DNA or protein immobilization methods of the present application, the catalyst is a surfactant. Preferably, the surfactant is at least one of cetyltrimethylammonium bromide, dioctadecylammonium bromide, cetyltrimethylammonium chloride, dodecyltrimethylammonium bromide and tetraoctylammonium bromide.

The key point of the method for preparing a DNA or protein chip of the present invention is to add a weak passivation step to allow the DNA or protein to react with the surface of the substrate more sufficiently to form a covalent bond by using a catalyst. Therefore, the present application particularly provides a DNA or protein immobilization method, which is not only suitable for immobilizing DNA or protein on a chip substrate, but also suitable for other situations where DNA or protein is immobilized on a carrier through covalent bonds, such as immobilizing DNA or protein on a microsphere carrier, or other types of carriers, according to the use requirements or product requirements, and is not limited herein.

It should be noted that, the DNA or protein chip in the present application refers to a chip substrate surface on which DNA or protein is immobilized, and it is understood that, if the kind of DNA or protein is not specifically indicated in the present application, DNA refers to a substance containing a DNA sequence, for example, DNA may contain a nucleotide derivative, a nucleotide analog, a fluorescent label, or both a nucleotide sequence and an amino acid sequence; proteins refer to a class of substances that contain amino acid sequences. The substrate surface of the chip in the present application has a chemical modification containing a reactive group capable of reacting with DNA or protein, and the DNA or protein is immobilized on the substrate surface by the reaction between the reactive group and the DNA or protein.

The beneficial effect of this application lies in:

the DNA or protein chip preparation method creatively adds a weak passivation treatment step, and makes the DNA or protein be more fully combined with the surface of the substrate by using a catalyst in the weak passivation treatment step, so that the DNA can be fully fixed on the surface of the substrate. The method not only improves the quality and efficiency of DNA or protein fixation, but also ensures that the DNA or protein fixation amount on the DNA or protein chip is highly controllable and has good repeatability; lays a foundation for preparing high-quality and high-availability DNA or protein chips.

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 showing the results of the fixed density test on the surface of a DNA chip subjected to different weak passivation treatments in the examples of the present application;

FIG. 3 is a graph showing the results of a fixed density test on the surface of a DNA chip obtained at different initial DNA concentrations in the example 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.

In the DNA chip preparation method, a technique of immobilizing DNA on a substrate by covalent bonding has been already used. However, studies have shown that whether the reaction of the DNA with the substrate surface to form covalent bonds is sufficient or not is the key to directly affect the DNA immobilization and the production stability and reproducibility of DNA chips. Taking the example that covalent bond immobilized DNA is generated by amino group of DNA and epoxy group on the surface of a substrate, the DNA chip produced based on the principle at present has poor repeatability, and the consistency of DNA immobilization amount in different batches or even the same batch of DNA chips is poor, so that the DNA chip has poor usability; therefore, how to control the fixed amount of DNA and improve the stability and repeatability of DNA chips has been the focus of research in this field.

Based on the above recognition, the embodiments of the present application provide a method for preparing a chip, which includes adding a weak passivation treatment step after a nucleic acid molecule immobilization treatment, in which covalent bond formation of DNA to a substrate surface is more sufficiently catalyzed by a surfactant, thereby enabling more efficient immobilization of DNA on the substrate surface. The method not only improves the efficiency and quality of DNA immobilization on the substrate surface, but also ensures that the amount of DNA immobilized on the DNA chip has good correlation with the concentration of DNA added into the fixing solution, thereby realizing controllable amount and good repeatability of DNA immobilized on the DNA chip. The method not only lays a foundation for preparing high-quality DNA chips, but also can meet the production requirement of customization. The method is applicable not only to immobilization of DNA on a substrate surface but also to protein chips having similar conditions.

In a certain embodiment, amino groups of the DNA or protein react with reactive groups on the surface of the substrate. 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. The surfactant of the present application can catalyze and promote the reaction of the amino group of DNA with the active group in the chemical modification shown in fig. 4 to form a covalent bond, thereby sufficiently immobilizing the DNA on the surface of the substrate.

Some words referred to in the examples of the present application are explained as follows:

AT-01: the component is 0.25M Na₂CO₃/NaHCO₃，0.6mM CTAB，pH 9.78；

RI-04: the component is 1M K₂HPO₄/KH₂PO₄，pH 9.0；

RI-05: the component is PBS solution with pH7.4;

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

RI-07: the component is double distilled water;

Dot/FOV: the observation region was defined as the number of bright spots in the range of 110X 110. mu.m.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Example 10

In this example, DNA was immobilized on a glass substrate having epoxy silane on the surface thereof via amino groups of the DNA, thereby forming a DNA chip of this example. And in the preparation process, weak passivation treatment is added after the fixation treatment, and a surfactant CTAB is adopted as the catalyst of the embodiment to carry out the weak passivation treatment. This example compares the effect on DNA fixation of CTAB, with or without addition of CTAB, during a weak passivation treatment.

In this embodiment, the DNA is fixed on the chip substrate by "in-channel" fixing, i.e., the chip substrate is packaged (the chip has a structure similar to a sandwich), and then various reagents are introduced into the channels of the packaged chip by a fluid device, so as to achieve 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) fixing treatment, namely introducing a fixing reaction solution into a channel of a chip substrate to perform fixing reaction, wherein the fixing reaction solution in the embodiment is fixing solution AT-01 containing 0.05nM DNA and 50 thymidylate nucleotides; wherein, the 5' end of the contained DNA is simultaneously modified by amino-modified NH2 and Cy3 fluorophore;AT-01 has Na content of 0.25M₂CO₃/NaHCO₃0.6mM CTAB, pH 9.78, volume of flow-through solution of 1mL, fluid velocity of 1mL/min, reaction time of 30min, reaction temperature of 37 ℃;

(2) weak passivation treatment, introducing weak passivation reaction solution containing 0.25M Na₂CO₃/NaHCO₃10mM CTAB, pH between 9.58 and 10.53, in this case specifically pH 9.78;

for comparison, in this example, a weakly passivating reaction solution was introduced into one half of the channels, and Na having a composition of 0.25M was introduced into the other half of the channels₂CO₃/NaHCO₃pH 9.78, i.e., weak passivation reaction solution without CTAB;

setting the volume of the circulating solution to be 1mL in all channels, setting the fluid speed to be 1mL/min, setting the reaction time to be 3h and setting the reaction temperature to be 37 ℃;

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

(4) 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 to obtain a DNA chip, wherein three solutions are adopted for washing, 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.

The DNA chip of this example was obtained after washing with the three solutions, and air-drying or oven-drying normally. The DNA chip prepared in this example was evaluated for the fixed density by a single-molecule fluorescence imaging technique, and specifically, the fixed density was measured by detecting Cy3 fluorescence on the DNA strand, and the number of Cy3 fluorescence spots per unit area was used to characterize the fixed amount/density of DNA molecules on the surface of the DNA chip. In this example, the number of fluorescence spots in a region of 110X 110 μm was counted to characterize the fixed density.

As shown in FIG. 2, the results of the fixed density measurement are shown in FIG. 2, wherein the abscissa "Yes" represents the DNA chip treated with the CTAB-containing weak inactivation reaction solution, and the "No" represents the DNA chip treated with the CTAB-free weak inactivation reaction solution, and the ordinate represents the number of fluorescence spots per unit area of 110X 110. mu.m. FIG. 2 is a bar graph comparing the average value of DNA chips treated with 8 channels of the weak inactivation reaction solution containing CTAB and the average value of DNA chips treated with 8 channels of the weak inactivation reaction solution containing no CTAB in DNA chips of 16 channels. The results of FIG. 2 show that, in the weak inactivation treatment, the DNA chip treated with the weak inactivation reaction solution containing CTAB had a fixed density of about 18000Dot/FOV in a region of 110X 110. mu.m, whereas the DNA chip treated with the weak inactivation reaction solution containing no CTAB had a fixed density of only about 3000Dot/FOV in the same size region; it can be seen that the weak passivation treatment with CTAB can effectively improve the density of DNA in unit area, and the addition of CTAB can promote the amino group and the epoxy group to be fully combined.

Example 20

The DNA chip preparation materials and procedures in this example were the same as in example one, except that the DNA contents in the immobilization reaction solution were 0.01nM, 0.04nM, 0.07nM and 0.1nM, respectively, during the immobilization treatment, and the other conditions were the same as in example one, thereby verifying the effect of different concentrations of DNA on the chip.

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

(1) fixing treatment, namely introducing a fixing reaction solution into a channel of a chip substrate to carry out fixing reaction, wherein the fixing reaction solution is a fixing solution AT-01 containing DNA and 50 thymidylate nucleotides; wherein, the 5' end of the contained DNA is simultaneously modified by amino-modified NH2 and Cy3 fluorophore; AT-01 contains Na₂CO₃/NaHCO₃0.6mM CTAB, pH 9.78, flow through solutionThe volume of (A) is 1mL, the speed of the fluid is 1mL/min, the reaction time is 30min, and the reaction temperature is 37 ℃;

in this example, the immobilized reaction solutions with DNA concentrations of 0.01nM, 0.04nM, 0.07nM and 0.1nM were set, and the immobilized reaction solutions with different DNA concentrations were introduced into different channels, respectively;

(2) weak passivation treatment, introducing weak passivation reaction solution containing 0.25M Na₂CO₃/NaHCO₃10mM CTAB, pH 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 ℃;

The DNA chip of this example was obtained after washing with the three solutions, and air-drying or oven-drying normally.

The DNA chip of this example was tested by the same test method as in example, and the results are shown in FIG. 3. In FIG. 3, the abscissa represents the number of fluorescence spots per unit area of 110X 110. mu.m corresponding to different DNA concentrations, and the ordinate represents the number of fluorescence spots per unit area of 110X 110. mu.m corresponding to different DNA concentrations. The results in FIG. 3 show that the number of Cy3 fluorescence spots per unit area increases with the increase of the initial DNA concentration, i.e., the immobilization density of the DNA chip increases, which indicates that the DNA chip preparation method of this example has a good dependence on the initial DNA concentration, and that the amount of DNA immobilized on the DNA chip can be controlled by adjusting the initial DNA concentration.

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 method of making a chip comprising a substrate and one or more channels, the substrate having a surface connected to the channels, the method comprising:

(a) passing a first solution into the channel, the first solution comprising a biomolecule, the biomolecule being a nucleic acid molecule or a protein molecule, such that the biomolecule is attached to the surface; and

(b) passing a second solution to the channel to replace the first solution of (a), the second solution comprising a surfactant, to weakly passivate the surface after (a), comprising contacting the surface with the second solution for a period of time to promote attachment of the biomolecule to the surface.

2. The method of claim 1, wherein the surfactant is a cationic surfactant;

optionally, the surfactant in the second solution 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 second solution is 1-25 mmol/L;

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

optionally, the certain time period is 2h-5 h;

optionally, the weak passivation treatment is performed at 35 ℃ to 40 ℃.

3. The method of claim 1, wherein the biomolecule comprises a nucleic acid molecule and the nucleic acid molecule carries an amino modification;

optionally, the surface is a chemically modified surface having at least one active group of an epoxy group, an aldehyde group, a carboxyl group, N-hydroxysuccinimide, and diaminobenzanilide;

optionally, the 3 'terminus and/or 5' terminus of the nucleic acid molecule has the amino modification;

optionally, the 3 'end or the 5' end of the nucleic acid molecule carries a fluorophore;

optionally, the fluorophore is Cy 3.

4. The method of any one of claims 1-3, wherein the first solution comprises a surfactant;

optionally, the surfactant comprised by the first solution is the same type of surfactant as the surfactant comprised by the second solution;

optionally, the first solution comprises 0.01 to 0.10nmol/L of the surfactant;

optionally, the first solution comprises 0.05nmol/L of the surfactant.

5. The method of any one of claims 1 to 4, further comprising (c) passing a third solution to the channel to replace the second solution of (b), the third solution not including the surfactant, to passivate the surface after (b);

optionally, the method further comprises (d) sequentially introducing a fourth solution, a fifth solution and a sixth solution into the channel to wash the surface after (c), wherein the fourth solution, the fifth solution and the sixth solution do not contain a surfactant.

6. A chip prepared according to the method of any one of claims 1 to 5.

7. Use of the chip of claim 6 for detecting nucleic acids and/or proteins.

8. A method for immobilizing a biomolecule on a given surface, comprising subjecting the surface to a weak passivation treatment after the biomolecule is immobilized on the surface, the biomolecule being a nucleic acid molecule or a protein molecule,

the weak passivation treatment comprises contacting the surface with a second solution comprising a surfactant for a length of time to promote binding of the biomolecule to the surface.

9. The method of claim 8, wherein 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 surfactant in the second solution is 1-25mmol/L, optionally, 10 mmol/L;

optionally, the weak passivation treatment is carried out at 35-40 ℃ for 2-5 h.

10. The method according to claim 8 or 9, wherein the immobilization is performed using a first solution, which is a second solution comprising the biomolecule.