Patent Description:
With the gradual implementation of Human Genome Project and the rapid development of related disciplines in molecular biology, more and more genome sequences of animals, plants and microorganisms have been determined, and gene sequence data has increased rapidly at an unprecedented rate. The gene chip (also called DNA chip, biochip) technology, created with the need of such scientific development, is used for preparing and/or analyzing biomolecules which specifically refer to probe molecules (not limited to nucleic acid sequences) that are immobilized on a support and then hybridized with labeled sample molecules. The quantity and sequence information of the sample molecules are obtained by detecting the hybridization signal intensity of each probe molecule.

Chips are generally made by polymer-coated substrates. The analysis and/or preparation of molecules, such as some methods for nucleic acid sequencing, depend on the bonding of nucleic acid strands to the surface of the chip substrate, and the sequence of the bonded nucleic acid strand is then determined by a number of different methods well known in the art. Existing substrates are generally flow cells. Process for coating a flow cell may include transferring the polymerized mixture into channels on the flow cell and incubating for a fixed time. The process is simple and can produce reliable coatings that are always able to support all downstream chemical processing procedures including bridge amplification and sequencing.

<CIT> discloses a high-polymer three-dimensional amino-group substrate as well as preparation method and application thereof.

<CIT> discloses to a microarray based multiplex pathogen analysis an uses thereof.

<CIT> discloses to a surface for label independent detection and method thereof.

<NPL>, discloses surface sensitization techniques and recognition receptors immobilization on biosensors and microarrays.

<CIT> discloses a manufacturing method for a hard micro-fluid chip.

However, existing chips are in need of further improvement, since they still have many defects or limitations limiting the applications thereof.

Based on this, it is necessary to provide an amino-modified chip. The amino-modified chip can carry a probe load of a higher density, and better meet the requirements of continuously developing biomolecule preparation and/or analysis with good stability.

Examples of the present disclosure provide an amino-modified chip, containing:.

In some embodiments, in the polymer, each of the structural unit contains an average of <NUM> to <NUM> identical or different amino groups.

In some embodiments, the polymer has a degree of polymerization in a range of <NUM> to <NUM>.

In some embodiments, in the polymer the structural unit contains at least one of
<CHM>
<CHM>.

In some embodiments, the polymer is selected from at least one of polylysine, polyornithine, chitosan, a polyamidoamine dendrimer and polyethyleneimine.

, The chip further contains a probe attached to the polymer.

The probe is bonded to the polymer via a linker compound, and the linker compound has a molecular structure containing a first linker compound and a second linker compound; the first linker compound is bonded to the probe, and the second linker compound is attached to the polymer through the amino group.

The second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group.

The second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group, wherein the second linker compound is made of NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, wherein n is <NUM> to <NUM>, and group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction.

In some embodiments, the linker compound is NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, where n is <NUM> to <NUM>, group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction.

In some embodiments, the probe is modified with group -DBCO or group -N<NUM>; the bonding is a covalent bonding between group -DBCO and group -N<NUM>. A method for preparing a chip, comprising:.

wherein the second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group, the linker compound is made of NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, wherein n is <NUM> to <NUM>, and group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction.

Examples of the present disclosure further provide a method for preparing a chip, containing:.

In some embodiments, in the polymer, each of the structural unit contains an average of <NUM> to <NUM> identical or different amino groups
and.

In some embodiments, in step (<NUM>), the attaching via reaction is conducted by contacting the polymer with the substrate in an alkaline solution at pH <NUM> to <NUM>.

In some embodiments, in step (<NUM>), the grating via reaction is conducted at a reaction temperature of <NUM> to <NUM> for <NUM> to <NUM>.

In some embodiments, the method further contains step (<NUM>): attaching a probe to the polymer.

In some embodiments, the probe is attached to the polymer via a linker compound, and the linker compound has a molecular structure containing a first linker compound and a second linker compound; the first linker compound is bonded to the probe, and the second linker compound is attached to the polymer through the amino group.

In some embodiments, the second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group.

In some embodiments, the probe is modified with group -DBCO or group -N<NUM>; the bonding is a covalent bonding between group -DBCO and group -N<NUM>.

In some embodiments, the attaching via reaction is conducted by contacting the linker compound with the polymer in an alkaline solution at pH <NUM> to <NUM>.

In some embodiments, the attaching via reaction is conducted at room temperature for <NUM> to <NUM>. In some embodiments, the covalent bonding is conducted by contacting the probe with the linker compound in an alkaline solution at pH <NUM> to <NUM>.

In some embodiments, the alkaline solution for covalent bonding contains a surfactant selected from at least one of tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide.

Embodiments of the present disclosure further provides use of the chip as described above, or a chip prepared by the method as described above in preparation or analysis of a biomolecule.

The amino-modified chip disclosed in one embodiment of the present disclosure has a substrate modified with the specific active group, and the substrate is attached to a polymer containing a primary amino group and/or a secondary amino group via reaction. The polymer can form a modified surface with a higher density and a high reactivity for attaching a probe. The amino-modified chip can carry a probe load of a higher density, and better meet the requirements of continuously developing biomolecule preparation and/or analysis with good stability. The method for preparing a chip disclosed in another embodiment of the present disclosure can achieve the attaching of the probe without controlling reaction conditions strictly, making the preparing process for the chip simple, easy to control and favorable for popularization and application.

The invention is further directed to the use of the chip or a chip prepared by the method as discussed above in preparation or analysis of a biomolecule.

The amino-modified chip of the present disclosure, the method for preparing the same and the use thereof are described in further details below with reference to specific examples. The present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. In contrast, these embodiments are provided for a thorough and complete understanding of the present disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. The terms used in the specification of the present disclosure herein are for the purpose of describing specific examples only and are not intended to limit the scope of the present disclosure.

The chip described herein may be a substrate to which the polymer is attached, or a substrate where a probe is further attached to the polymer. Materials for the substrate are not specified, and the substrate is formed of at least one of glass, silicon wafer, plastic, gel and nylon film.

The term "attached to" or "modified with" as used herein may refer to a direct attach to or modification with an object, or may refer to a further attach to or modification with the object via another transition group.

The amino group as used herein refers to a structural feature having a formula -N(X)<NUM>, where each "X" is independently H, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocyclyl, and the like. It will be appreciated that in the context of the present disclosure, at least one X is H. Non-limiting types of the amino group include -NH<NUM>, -NH(alkyl), -NH(cycloalkyl), -NH(heterocyclyl) and -NH(aryl).

The term "alkyl" refers to a saturated hydrocarbon containing a primary (normal) carbon atom, a secondary carbon atom, a tertiary carbon atom, a quaternary carbon atom, or a combination thereof. Suitable examples include, but are not limited to: methyl (Me, -CH<NUM>), ethyl (Et, -CH<NUM>CH<NUM>), <NUM>-propyl (n-Pr, n-propyl, -CH<NUM>CH<NUM>CH<NUM>), <NUM>-propyl (i-Pr, i-propyl, -CH(CH<NUM>)<NUM>), <NUM>-butyl (n-Bu, n-butyl, -CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-propyl (i-Bu, i-butyl, -CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-butyl (s-Bu, s-butyl, -CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-propyl (t-Bu, t-butyl, -C(CH<NUM>)<NUM>), <NUM>-pentyl (n-pentyl, -CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-pentyl (-CH(CH<NUM>)CH<NUM>CH<NUM>CH<NUM>), <NUM>-pentyl (-CH(CH<NUM>CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl (-C(CH<NUM>)<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-butyl (-CH(CH<NUM>)CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl (-CH<NUM>CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-butyl(-CH<NUM>CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-hexyl (-CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-hexyl (-CH(CH<NUM>)CH<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-hexyl (-CH(CH<NUM>CH<NUM>)(CH<NUM>CH<NUM>CH<NUM>)), <NUM>-methyl-<NUM>-pentyl (-C(CH<NUM>)<NUM>CH<NUM>CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>)CH(CH<NUM>)CH<NUM>CH<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>)CH<NUM>CH(CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-pentyl (-C(CH<NUM>)(CH<NUM>CH<NUM>)<NUM>), <NUM>-methyl-<NUM>-pentyl (-CH(CH<NUM>CH<NUM>)CH(CH<NUM>)<NUM>), <NUM>,<NUM>-dimethyl-<NUM>-butyl (-C(CH<NUM>)<NUM>CH(CH<NUM>)<NUM>), <NUM>,<NUM>-dimethyl-<NUM>-butyl (-CH(CH<NUM>)C(CH<NUM>)<NUM> and octyl (-(CH<NUM>)<NUM>CH<NUM>).

The term "cycloalkyl" refers to a non-aromatic hydrocarbon containing ring carbon atoms and may be a monocyclic, spirocycloalkyl or bridged cycloalkyl group. Suitable examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. In addition, "cycloalkyl" may also contain one or more double bonds, and representative examples of cycloalkyl groups containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl and cyclobutadienyl.

The term "aryl" refers to an aromatic hydrocarbon group derived by removing one hydrogen atom from an aromatic cyclic compound, and may be a monocyclic aryl, fused cyclic aryl or polycyclic aryl. For polycyclic species, at least one of the rings is an aromatic ring system. Suitable examples include, but are not limited to: benzene, biphenyl, naphthalene, anthracene, phenanthrene, perylene, triphenylene, and derivatives thereof.

The term "heterocyclyl" refers to a cycloalkyl with at least one carbon atom replaced by a non-carbon atom, which may be an N atom, an O atom, an S atom, etc. The heterocyclyl may be a saturated ring or a partially unsaturated ring. Suitable examples include, but are not limited to: dihydropyridinyl, tetrahydropyridyl (piperidinyl), tetrahydrothienyl, thiooxidized tetrahydrothienyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and indolinyl.

The term "structural unit" refers to a unit structure formed by one of the monomers in the polymer. The polymer may be polymerized from one monomer or two or more different monomers, and the "structural units" may be identical or different.

The relationship between degree of polymerization and number of generation for polyamidoamine dendrimer: the degree of polymerization = <NUM> ^ (number of generation + <NUM>) - <NUM>. For example, for PamamDendrimer generation <NUM>, the degree of polymerization is <NUM>.

It will be appreciated that the chip can be used for genetic testing, or can be used for protein, polypeptide or other detections.

In one of the specific examples, in the polymer, each of the structural unit contains an average of <NUM> to <NUM> identical or different amino groups. It will be appreciated that the structural unit may or may not contain amino groups. The term "average" refers to a mean value obtained by dividing the total number of amino groups by the total number of the structural units. When the average number of amino groups in each structural unit is less than <NUM>, the polymer is a copolymer obtained by polymerizing two or more monomers, at least one of which does not contain an amino group. For example, in the polymer each of the structural unit may contain an average of <NUM> amino groups, and the polymer may be composed of <NUM> structural units free of amino group and <NUM> structural unit containing amino groups (containing <NUM> amino groups). The number of amino groups can be controlled by controlling the combination of monomers in the polymerization.

Preferably, in the polymer, each of the structural unit contains an average of <NUM> to <NUM> amino groups. It is found that the number of amino groups in the polymer is associated with the reactivity of the amino groups, which directly influences the density/number of linkages, such as probes, between the amino groups and linkers connected to the polymer. In one example, the probes are linked to the surface of the chip through the linker compounds and the amino groups of the polymer. Controlling the average number of amino groups contained in each structural unit of the polymer at <NUM> to <NUM> can well regulate the linkage density/number of the probes, thus optimizing the regulation and control of the probe density. Meanwhile, the probes can be more easily attached to the polymer through the linker compounds with a higher probe density.

In one of the specific examples, the polymer has a degree of polymerization in a range of <NUM> to <NUM>. In another one of the specific examples, the polymer has a molecular weight of <NUM> kD to <NUM> kD. A higher molecular weight may result in a higher density of nucleic acid strand that can be attached. However, at the same time, it may increase the effectiveness of immobilization of the polymer on the surface of the substrate. Also, molecules with an excessively high molecular weight have undesirable effects in terms of solubility, reaction efficiency, and the like. Therefore, in the examples of the present disclosure, the molecular weight of the polymer is preferably <NUM> kD to <NUM> kD. For the polyamidoamine dendrimer, e.g., the PamamDendrimer, the number of generations may be <NUM> to <NUM>.

Preferably, the polymer contains identical or different structural units, and may be commercially available or customized, as long as the requirements of the present application for the number of amino groups contained in the polymer and/or the degree of polymerization of the polymer are met. In one of the specific examples, in the polymer the structural unit contains at least one of
<CHM>.

Specifically, in one example, the polymer contains identical structural units. For example, the polymer is a copolymer containing identical structural units of
<CHM>
<CHM>
In another example, the polymer contains different structural units. For example, the polymer is a copolymer containing different structural units of
<CHM>
and
<CHM>
as disclosed in <NPL>, including copolymers of ornithine and lysine polymerized at different ratios, or the polymer is a copolymer containing different structural units of
<CHM>
and
<CHM>.

Preferably, the polymer is selected from at least one of polylysine, polyornithine, chitosan, a polyamidoamine dendrimer and polyethyleneimine.

In one of the specific examples, the chip further contains a probe attached onto the polymer. The probe binds to a target molecule and thus captures the target molecule. For different purposes, different probes may be selected. For example, oligonucleotide fragments, polypeptide sequences, probes containing oligonucleotide fragments, or probes containing polypeptide sequences can be selected. Meanwhile, the probes can selectively label fluorescent detection molecules, antigens, biotin, streptavidin, or other detection molecules.

In one of the specific examples, the probe may be a nucleic acid strand and/or a polypeptide.

In one of the specific examples, the probe is attached onto the polymer via a linker compound, and the linker compound has a molecular structure containing a first linker compound and a second linker compound; the first linker compound at one end is bonded to the nucleic acid strand probe, and the second linker compound at the other end is attached to the polymer through the amino group. Moreover, the second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group.

In one of the specific examples, the linker compound is made of NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, where n is <NUM> to <NUM>, group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction.

Preferably, the probe is modified with group -DBCO or group -N<NUM>; the bonding is a covalent bonding between group -DBCO and group -N<NUM>. NHS is an abbreviation for succinimidyl ester, PEG for polyethylene glycol, DBCO for diphenylcyclooctyne, N<NUM> for azide. The linkage between the nucleic acid strand and the polymer via the linker compound can, on one hand, allow NHS to be stably attached to the amino, and on the other hand, allow DBCO or N<NUM> to be subjected to a Click reaction with a nucleic acid strand modified with N<NUM> or DBCO under mild conditions of room temperature and no catalyst with high efficiency.

In the above method, further definitions of the polymer are the same as those for the chip described above and are not repeated hereinafter.

Specifically, step (<NUM>) is a substrate acquisition procedure. The acquisition can be a direct purchase, that is, acquiring a substrate modified with an epoxy group by direct purchase; or the acquisition can be self-making, where the process for self-making can be modifying a compound containing an epoxy group on the carrier by a solution reaction or plasma spray-coating.

The compound containing an epoxy group may be selected from an epoxy silane, such as <NUM>-(<NUM>,<NUM>-glycidoxy)propyltrimethoxysilane. Additionally, in one of the specific examples, the material of the carrier is at least one of glass, silicon wafer, plastic, gel and nylon film.

In one of the specific examples, the reaction conditions for modifying the compound containing an epoxy group on the carrier by a solution reaction include: a reaction temperature at room temperature, and a reaction time of <NUM> to <NUM>. Moreover, after the reaction, the mixture is dried at <NUM> to <NUM>.

It will be appreciated that the carrier may require extensive washing and activation prior to the solution reaction modification. For example, the carrier surface is alternately washed with an alcohol solvent and water under ultrasonic conditions and activated with an alkaline solution. The alkaline solution activation can be conducted by processing, e.g., in a <NUM> to <NUM> aqueous NaOH solution for <NUM> to <NUM>.

Specifically, step (<NUM>) is a polymer attaching procedure. The epoxy group on the substrate is modified, and in step (<NUM>), the amino group and the epoxy group are subjected to a attaching via reaction.

Preferably, the attaching via reaction is conducted by contacting the polymer with the substrate in an alkaline solution at pH <NUM> to <NUM>. The pH values include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Preferably, the alkaline solution at pH <NUM> to <NUM> is selected from at least one of a phosphate buffer, a carbonate buffer and a borate buffer. Furthermore, the concentration of the solute pair in the buffer at pH <NUM> to <NUM> is <NUM> to <NUM>. The concentrations of the solute pairs include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Preferably, a carbonate buffer at pH <NUM> is used as the reaction solvent, where the concentration of the solute pair is <NUM>.

Additionally, in step (<NUM>), the attaching via reaction is conducted at a reaction temperature of <NUM> to <NUM> for <NUM> to <NUM>. The combination of reaction temperature and reaction time (temperature × time) includes, but is not limited to: <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM>, <NUM> × <NUM> hand <NUM> × <NUM>.

Preferably, the reaction temperature is <NUM> and the reaction time is <NUM>.

Preferably, the method for preparing the chip described above further contains step (<NUM>): attaching a probe to the polymer. Step (<NUM>) is a probe attaching procedure.

In one of the specific examples, in step (<NUM>), the probe is attached to the polymer via a linker compound, and the linker compound has a molecular structure containing a first linker compound and a second linker compound; the first linker compound is bonded to the probe, and the second linker compound is attached to the polymer through the amino group. More specifically, the second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group.

Preferably, step (<NUM>) can be conducted in two procedures:.

In one of the specific examples, the linker compound is made of NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, where n is <NUM> to <NUM>, group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction. The linkage between the probe and the polymer via the linker compound can, on one hand, allow NHS to be stably attached to the amino, and on the other hand, allow DBCO or N<NUM> to be subjected to a click reaction with a probe modified with N<NUM> or DBCO under mild conditions of room temperature and no catalyst with high efficiency.

Preferably, the active group-modified probe is modified with group -DBCO or group -N<NUM>; the bonding is a covalent bonding between group -DBCO and group -N<NUM>. More specifically, the covalent bonding is a click reaction.

In one of the specific examples, the active group-modified probe is a nucleic acid strand modified by the active group at the <NUM>' end. It will be appreciated that if the linker compound is prepared from NHS-PEGn-DBCO, i.e., the group for bonding is -DBCO, the reactive group for <NUM>' modification is -N<NUM> functional group; if the linker compound is prepared from NHS-PEGn-N<NUM>, i.e., the group for bonding is -N<NUM>, the reactive group for <NUM>' modification is -DBCO functional group.

In one of the specific examples, in step (<NUM>-<NUM>), the concentration of the compound for providing the linker compound in the reaction system is <NUM> to <NUM>. The concentrations include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In one of the specific examples, in step (<NUM>-<NUM>), the attaching via reaction is conducted by contacting the linker compound with the polymer in an alkaline solution at pH <NUM> to <NUM>. Specifically, the pH values include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. More specifically, the alkaline solution is selected from at least one of a phosphate buffer and a carbonate buffer.

In one of the specific examples, the concentration of the solute pair in the phosphate buffer or the carbonate buffer is <NUM> to <NUM>. The concentrations of the solute pairs include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Preferably, a phosphate buffer at pH <NUM> is used as the reaction solvent for the attaching via reaction, where the concentration of the solute pair is <NUM>. In another one of the specific examples, a carbonate buffer at pH <NUM> is used as the reaction solvent for the attaching via reaction, where the concentration of the solute pair is <NUM>.

In one of the specific examples, in step (<NUM>-<NUM>), the attaching via reaction is conducted at room temperature for <NUM> to <NUM>. The reaction times include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In one of the specific examples, in step (<NUM>-<NUM>), the covalent bonding is conducted by contacting the probe with the linker compound in an alkaline solution at pH <NUM> to <NUM>. Specifically, the pH values include, but are not limited to: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. More specifically, the alkaline solution is a citrate buffer. More specifically, the covalent bonding is conducted at room temperature.

In one of the specific examples, the alkaline solution contains a surfactant selected from at least one of tetradecyltrimethylammonium bromide (TTAB), cetyltrimethylammonium bromide (CTAB) and dodecyltrimethylammonium bromide (DTAB). The surfactant is used to facilitate the sedimentation of the nucleic acid strands on the surface.

Preferably, the covalent bonding is conducted by contacting the probe with the linker compound in a citrate buffer containing <NUM> TTAB at room temperature.

Additionally, in various applications, excessive functional groups, such as -NH<NUM>, -DBCO, -N<NUM>, etc., may be optionally blocked before or after bonding to the probe.

Specifically, in one of the specific examples, the method further containing: conducting a blocking process using at least one of NHS-(PEG)n, acetic anhydride, DBCO-benzoic acid and azidobenzoic acid, where n = <NUM> to <NUM>. More specifically, the blocking of -NH<NUM> can be conducted using NHS-(PEG)n (n can be <NUM> to <NUM>), acetic anhydride, etc.; specifically, for example, -NH<NUM> may be blocked using <NUM>µL of acetic anhydride + <NUM>µL of N,N-diisopropylethylamine (DIPEA) in formamide. The blocking of -N<NUM> can be conducted using DBCO-benzoic acid. The blocking of -DBCO can be conducted using azidobenzoic acid.

Examples of the present disclosure further provides use of the chip as described above, or a chip prepared by the method as described above in preparation or analysis of a biomolecule. Specifically, the preparation or analysis of the biomolecule can be, for example, nucleic acid sequencing, library hybridization, generation of DNA clusters, and the like, and can also be, for example, protein detection, polypeptide detection, and the like.

In the following specific examples provided below, unless otherwise indicated, all experimental materials are either available from commercial suppliers (e.g., Aladdin™, Sigma™, Sangon™, etc.) or prepared in house or by a contractor in accordance with the structural formula and molecular weight information provided in the examples.

Polyamidoamine dendrimers (PamamDendrimer), or Pamam Dendrimer generation <NUM> (abbreviated as PD3. <NUM>), having a molecular weight of <NUM> and the following molecular formula:
<CHM>
NHS-PEG<NUM>-N<NUM> and NHS-PEG<NUM>-DBCO, having the following molecular formulas:
<CHM>
(NHS-PEG<NUM>-N<NUM>)
<CHM>
(NHS-PEG<NUM>-DBCO) Anhydrous dimethyl sulfoxide (DMSO), purchased from Sigma™;.

The specific procedures for coating the epoxy-modified substrate with PLO (polyornithine) and modifying the PLO-coated substrate with functional groups are as follows:.

The functional group-modified substrate can be soaked in RI02 solution for storage at <NUM> for <NUM> year.

The specific procedures for coating the epoxy-modified substrate with PLL (polylysine) and modifying the PLL-coated substrate with functional groups are as follows:.

The specific procedures for the coating epoxy-modified substrate with PamamDendrimer (polyamidoamine dendrimer) and modifying the PamamDendrimer-coated substrate with functional groups are as follows:.

The specific procedures for coating the epoxy-modified substrate with PEI (polyethyleneimine) and modifying the PEI-coated substrate with functional groups are as follows:.

The specific procedures for the coating epoxy-modified substrate with chitosan and modifying the chitosan-coated substrate with functional groups are as follows:.

In this example, the chips prepared in Examples <NUM> to <NUM> were subjected to a probe density detection. The specific detection procedures are as follows:.

Detection: Images were taken on a fluorescence detection system using a <NUM>-fold objective lens, with a wavelength of <NUM>, a laser power of <NUM> mW, an exposure time of <NUM>, and were analyzed using ImageJ.

The probe density detection results of the chips prepared in Examples <NUM> to <NUM> were shown in <FIG>, where the ordinate represents the fluorescence intensity (A. ) in unit dot/FOV, i.e., the mean fluorescence intensity of <NUM> observation region of <NUM> × <NUM>, and PLL, PLO, PD3. <NUM>, CTS and PEI on the abscissa respectively correspond to the chips modified with polymers PLL, PLO, PD3. <NUM> (polyamidoamine dendrimer), CTS (chitosan) and PEI.

Library hybridization and detection were performed using the chips prepared in Examples <NUM> to <NUM>. The specific procedures are as follows:.

DNA library: DNA library of fragments with length of <NUM> to <NUM> bp and known sequences at both ends, the molecular structure of the library is shown in <FIG>; Insertion: an inserted fragment derived from phi-X174 standard strain; T20, Pe and RD are respectively sequences set forth in SEQ ID Nos. <NUM> to <NUM>.

The DNA library was mixed with <NUM>µL of deionized water before <NUM>µL of freshly prepared <NUM> NaOH solution was added. After well mixing, the mixture was let stand for denaturation at room temperature for <NUM>, and then the reaction was stopped by adding <NUM>µL of <NUM> Tris-HCl buffer, pH <NUM> to obtain <NUM>µL of <NUM> pM denatured DNA library. Hybridization of denatured DNA library with chip probes.

The denatured DNA library was diluted to <NUM> pM using a hybridization solution containing <NUM>× SSC (<NUM>× SSC buffer diluted with RNase-free water), pH <NUM>. The diluted DNA library was introduced into lanes of the chip and the chip was incubated for hybridization at <NUM> for <NUM>. <NUM> to <NUM>µL of washing reagent (<NUM>× SSC, <NUM>% Tween <NUM>, pH <NUM>) was introduced at a rate of <NUM>µL/min. The hybridization reaction was thus completed.

The amplification was carried out using the template walking technique disclosed in the article Isothermal amplification method for next-generation sequencing (<NPL>).

The results of DNA cluster detection are shown in <FIG> and Table <NUM>, and the results of DNA cluster detection corresponding to chips prepared in Examples <NUM> to <NUM> are shown in <FIG>, respectively.

As can be seen from the detection results, the chips prepared in Examples <NUM> to <NUM> were successfully used for hybridization of the library and DNA cluster generation.

Technical features in the above examples may be combined in any combinations. In order to make the description brief, all possible combinations of various technical features in the above examples are not described; however, it should be considered as being within the scope of this specification as long as there is no contradiction in the combinations of the technical features.

Claim 1:
An amino-modified chip, comprising:
a substrate modified with an epoxy group;
a polymer attached to the substrate via the epoxy group, wherein at least one structural unit of the polymer comprises an amino group, and the amino group is a primary amino group and/or a secondary amino group; and
a probe attached to the polymer, wherein the probe is attached to the polymer via a linker compound, and the linker compound has a molecular structure comprising a first linker compound and a second linker compound; the first linker compound is bonded to the probe, and the second linker compound is attached to the polymer through the amino group,
wherein the second linker compound is selected from at least one of an -NHS group, an epoxy group and an isocyanate group, wherein the second linker compound is made of NHS-PEGn-DBCO or NHS-PEGn-N<NUM>, wherein n is <NUM> to <NUM>, and group -NHS in NHS-PEGn-DBCO or NHS-PEGn-N<NUM> is attached to the amino group via reaction.