CN1811427A - Active carrier for biological chip, producing method and application thereof - Google Patents

Active carrier for biological chip, producing method and application thereof Download PDF

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CN1811427A
CN1811427A CN 200610020226 CN200610020226A CN1811427A CN 1811427 A CN1811427 A CN 1811427A CN 200610020226 CN200610020226 CN 200610020226 CN 200610020226 A CN200610020226 A CN 200610020226A CN 1811427 A CN1811427 A CN 1811427A
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biochip
group
active
present invention
coupling
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CN 200610020226
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Chinese (zh)
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邹方霖
王建霞
陈春生
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成都夸常医学工业有限公司
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Abstract

The present invention relates to an active carrier for biological chip. It contains solid-phase carrier and derivative chemically bonded on said solid-phase carrier surface. The described derivative at least contains coupling group and functional group combined on the coupling group by means of covalent bond, the molecular weight of the described functional group is greater than 50 and less than 1000. Said active carrier for biological chip has high efficiency and high stability. Besides, said invention also provides the preparation method of said active carrier.

Description

用于生物芯片的活性载体、其制备方法及其应用 Carriers for active biochips, their preparation and their use

技术领域 FIELD

本发明涉及用于生物芯片的活性载体、用于生物芯片的活性载体的制备方法、及用于生物芯片的活性载体的应用(生物芯片基片、生物芯片、生物芯片试剂盒、及生物芯片检测)。 The present invention relates to the application of active carriers for a biochip, the method for preparing the active support for a biochip, and an active carrier is a biochip (substrate biochip, the biochip, the biochip kit and biochip ).

背景技术 Background technique

本发明中,生物芯片试剂盒(“Biochip Kit”,又简称“芯片试剂盒”)是定性和/或定量分析中的一种检测装置,其包括微通道芯片试剂盒(“Micro-tunnel BiochipKit”)和微阵列芯片试剂盒(“Bio-array Kit”或“Micro-array Kit”)。 In the present invention, a biochip kit ( "Biochip Kit", and referred to as "chip kit") is the qualitative and / or quantitative analysis of a detection device which includes a micro-channel chip kit ( "Micro-tunnel BiochipKit" ) and microarray kit ( "Bio-array kit" or "micro-array kit"). 由于其高通量和微型化特点,生物芯片试剂盒有着广泛的应用范围,包括基因表达检测、基因筛选、药物筛选、疾病诊断治疗、环境监测和治理、司法鉴定等领域。 Due to its high throughput and miniaturization characteristics, biochip kit has a wide range of applications, including gene expression field testing, genetic screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, judicial identification. 生物芯片试剂盒通常含生物芯片(“Biochip”,又简称“芯片”)和标记系统。 Biochip kits usually contain a biochip ( "Biochip", and referred to as "chip") and marking systems. 在我们的一项专利申请(PCT/CN2004/000437)中,标记系统含配基、标记物质和纳米结构(用于纳米结构标记物的活性载体)。 In our patent application (PCT / CN2004 / 000437), the marking system containing ligand, the labeling substance and nanostructures (nanostructures support an active marker).

芯片的核心是其上的反应器。 Core chip is the reactor thereon. 芯片反应器中最重要的叁部分为片基(用于芯片的固相载体)、固定在片基上的探针、和反应器结构。 The most important part of the chip reactor is a three sheet group (a solid support of the chip), fixed on the probe base plate, and the reactor configuration. 探针是反应器中通过与样品中目标物的相互作用而固定目标物的物质。 Probe is fixed in the reactor by interaction with the target substance in the sample of the target substance. 最早应用的是以核酸为探针的核酸芯片。 The first application is based on nucleic acid is a nucleic acid probe chip. 后来发展起来的包括有多肽探针的含多肽芯片(例如,多肽芯片、多肽/核酸芯片、多肽/糖芯片、等等),由于在检测反应中包括多肽间相互作用,使其对稳定性、空间反应效率、等等均有更高的要求。 Developed later include polypeptide-containing chips (e.g., chips polypeptide, polypeptide / nucleic acid chips, polypeptide / sugar chips, etc.) polypeptide probe, due to the inclusion in the interaction between the polypeptide detection reaction, so that stability, the reaction space efficiency, and the like have higher requirements. 片基主要是玻璃、金属、塑料等材料及其衍生物制作的矩形、圆形或其它形状的含有活性衍生基团的载体。 Mainly rectangular base sheet of glass, metal, plastic and other materials and derivatives produced, circular or other shaped carrier containing active derivative group. 反应器结构包括反应器隔离结构、反应器流路结构、反应器分离结构、反应器反应室结构,等等。 The reactor comprises a reactor structural isolation structure, reactor flow-path structure, reactor separation structure, the reaction chamber of the reactor structure, and the like. 探针可以直接结合在片基上,也可以通过与活性纳米结构(用于亲和纳米结构的活性载体)结合形成亲和纳米结构再结合在片基上(参考我们的一项专利申请:PCT/CN2004/000437)。 Probes may be directly bonded to the base sheet, and nanostructures affinity recombination over the substrate (see our patent application may be formed by binding the active nanostructures (active carrier for affinity nanostructures): PCT / CN2004 / 000437).

通常,片基含片基固相载体和固定在片基固相载体上的衍生物。 Typically, sheet-yl group troches solid support is fixed on the sheet substrate and a solid support derivatives. 片基中形成衍生物有两种方式:包被和表面修饰。 Sheet derivative group formed in two ways: coating and surface modification. 衍生物中含用以固定探针的功能基团。 Derivatives containing a functional group for fixing the probe. 功能基团与固相载体的连接是影响反应质量的重要因素。 Functional groups attached to the solid support is an important factor affecting the quality of the reaction. 例如,芯片生物活性的有效保存、固相探针的反应动力学条件(例如探针有效位点密度、空间运动自由度、等等)、等等均与此连接有关。 For example, effective conservation of the biological activity of the chip, a solid phase probe kinetic conditions (e.g., active site density, freedom of movement space probes, etc.), and the like are related to this connection. 这一连接非常丰富也非常复杂。 This connection is very rich very complicated. 例如,共价键连接与吸附连接可能有不同的固相探针稳定性;不同的功能基团可能有不同的固相探针反应效率;等等。 For example, covalently attached to the suction connection may have different solid-phase probe stability; different functional groups may have different solid-phase probe reaction efficiency; and the like.

对片基固相载体进行聚合物包被以引入功能基团的工作,包括:申请号为20020128234的美国专利、等等。 The base sheet of polymer coated solid support is carried out to introduce functional groups in the work, including: U.S. Patent Application No. 20020128234, and the like. 这些工作基于离子配对机理,在片基活性的均匀性与稳定性方面存在严重问题。 These work based ion pairing mechanisms, there are serious problems in terms of uniformity and stability of the active base sheet.

目前,通过对片基固相载体进行表面化学修饰的工作包括:1).引入偶合基团后直接固定探针(例如,中国专利申请号CN200310107946.X中的工作);2).先引入偶合基团再引入功能基团去固定探针。 At present, the work group to a solid support substrate surface chemical modification include: 1) the introduction of direct coupling group immobilized probes (e.g., in Chinese Patent Application No. CN200310107946.X work); 2) the introduction of the first coupling. group and then to introduce functional groups immobilized probes. 在后一类工作中,引入的小分子功能基团包括氨基、醛基(中国专利申请号CN01105795.5、CN03132420.7)、氨基肼(美国专利申请号US2004235049)、环氧基及其它小分子基团(美国专利号5,474,895);引入的大分子功能基团包括含氨基的聚合物(例如:US专利6,413,722,中国专利申请号200410027316.6,等等)。 In the latter type of work, the functional group is introduced into the small molecule include amino, aldehyde (Chinese Patent Application No. CN01105795.5, CN03132420.7), amino hydrazine (U.S. Patent Application No. US2004235049), an epoxy group, and other small molecules group (U.S. Pat. No. 5,474,895); macromolecule functional group into a polymer containing an amino group include (e.g.: US Patent No. 6,413,722, Chinese Patent application No. 200410027316.6, etc.). 一般而言,引入小分子功能基团制备的片基,其固定探针的效率及固定在其上的探针捕获目标物的效率均有待改进;而引入大分子功能基团制备的片基,其稳定性有待改进、或/和其制备方法较复杂(例如交联)不利于工业化生产。 In general, the introduction of small molecules prepared film base functional groups, the efficiency of which is fixed and immobilized capture probes in a target probe which are to be improved in efficiency; group introduced sheet prepared macromolecular functional groups, its stability to be improved, and / or methods for their preparation more complicated (e.g. crosslinking) is not suitable for industrial production.

对于用于亲和纳米结构的活性纳米结构、或用于纳米结构标记物的活性纳米结构,也有与上述片基类似的问题。 Affinity for an active nanostructures and nanostructures, nanostructured or an active marker nanostructure, but also with the above-described base sheet similar problems.

发明内容 SUMMARY

本发明的目的是提供一种具有高效率和高稳定性、而又易于制备的用于生物芯片的固相载体。 Object of the present invention is to provide a high efficiency and high stability, and easy preparation of the solid support for a biochip. 本发明的技术方案,基于化学修饰,而且基于在固相载体表面偶合基团上引入中等大小的功能基团来使其功能化。 Aspect of the present invention, chemical modification, and based functional group is introduced in a medium-sized surface coupling on a solid support functionalized groups to make.

于是,本发明的第一个方面,涉及一种用于生物芯片的活性载体,其特征在于:其含固相载体和固相载体表面上化学键合的衍生物,所述衍生物至少含偶联基团和偶联基团上以共价键结合的功能基团,且所述功能基团的分子量大于50小于1000。 Accordingly, a first aspect of the invention relates to an active support for a biochip, comprising: a derivative thereof containing the solid support and the solid support chemically bonded to the surface, said derivative containing at least conjugate functional groups on the group and coupling groups covalently bound, the functional group and a molecular weight greater than 1000 is less than 50. 本发明的用于生物芯片的活性载体,具有高效率和高稳定性、而又易于制备的优点。 The advantages of the present invention is a biochip active carrier with high efficiency and high stability, and easy preparation.

本发明的第二个方面,涉及本发明的用于生物芯片的活性载体的一种制备方法,其至少包括:1).将所述偶联基团以化学键结合在所述固相载体表面上;2).将功能物质与1)的制备物中的所述偶联基团接触并进行反应,所述功能物质含所述功能基团;3).将所述功能基团固定在所述偶联基团上。 A second aspect of the present invention, the present invention relates to a method for preparing an active carrier of a biochip, comprising at least: 1) the coupling group is chemically bonded to the surface of the solid support ; 2) preparation of the coupling group and a functional substance 1) was contacted and reacted, the functional material containing the functional group; 3) the fixing of the functional group. the coupling group.

本发明的第三个方面,涉及本发明的用于生物芯片的活性载体的应用,包括基于本发明的活性载体的生物芯片基片、生物芯片、生物芯片试剂盒,以及生物芯片检测。 A third aspect of the present invention, the present invention relates to the application of an active carrier biochips, comprising a carrier based on the activity of the present invention, the substrate is a biochip, the biochip, the biochip kit and biochip. 本发明的一种多反应器生物芯片基片,其特征在于:其至少含本发明的生物芯片片基和固定在所述片基上的反应器结构。 A multi-reactor biochip substrate of the present invention is characterized in that: it contains at least the reactor biochip structure and the fixed base plate of the present invention on the base sheet. 本发明的一种生物芯片,其特征在于:其至少含本发明的生物芯片片基和固定在所述片基上的二种或二种以上的探针、或至少含本发明的生物芯片基片和固定在所述基片的片基上的二种或二种以上的探针。 A biochip according to the present invention is characterized in that: at least a base plate and a fixed biochip of the present invention containing two or more kinds of probes on the base sheet, or a group containing at least biochip of the present invention of two or more thereof immobilized on the probe sheet and the base sheet of said substrate. 本发明的一种生物芯片试剂盒,其特征在其含片基或本发明的生物芯片基片,以及固定在所述片基上的二种或二种以上的探针。 A biochip kit of the present invention, which is characterized in that its base lozenges or biochip substrate of the present invention, and two or more than two kinds of probes fixed on the base sheet. 本发明的一种生物芯片检测方法,至少包括下列步骤:A).提供可能含检测目标物的样本,所述目标物包括多肽目标物;B).提供本发明的生物芯片;C).使所述样本与所述探针接触并进行相关反应。 A biochip detection method of the present invention, comprising at least the following steps: A) providing a sample may contain a detection target substance, the target comprises a target polypeptide; B) of the present invention to provide a biochip; C) so that... contacting the sample with the probe and related reactions.

以下将通过实施例更为详细地说明本发明。 The present invention will be described in more detail by way of examples.

具体实施方式 detailed description

本发明的第一个方面,涉及一种用于生物芯片的活性载体,其特征在于:其含固相载体和固相载体表面上化学键合的衍生物,所述衍生物至少含偶联基团和偶联基团上以共价键结合的功能基团,且所述功能基团的分子量大于50小于1000。 A first aspect of the present invention, relates to an active support for a biochip, characterized in that: it contains a solid support and the solid support chemically bonded to the surface, said derivatives containing at least a coupling group and functional groups on the coupling group covalently bonded, and the molecular weight of the functional group is less than 50 greater than 1000.

本发明中,术语“活性载体”是指可通过其上的功能基团有效地固定反应物(例如探针、标记用配基、等等)的固相载体,例如生物芯片片基、用于亲和纳米结构的活性纳米结构、用于纳米结构标记物的活性纳米结构、等等。 The present invention, the term "active support" refers to the reaction product can be effectively secured (e.g., probes, labeled with a ligand, and the like) of the solid support, e.g. biochip sheet group by functional groups thereon, for pro-active nanostructures and nanostructures, the nanostructures nanostructure an active marker, and the like. 尽管本发明的实施例中只给出了多肽作为反应物的例子,其它生物物质(例如核酸、细胞、糖类、等等),也可应用本发明的活性载体制备本发明的生物芯片组成物(例如,生物芯片片基、纳米结构标记物、等等)。 Although embodiments of the present invention, given only as a reactant polypeptide example, other biological materials (e.g. nucleic acids, cells, saccharides, etc.), may also be applied biochip active carrier composition of the present invention prepared according to the present invention (e.g., the biochip sheet group, nanostructure markers, etc.). 特别要指出的是,适于多肽固定的活性载体通常适合于核酸固定,而适于核酸固定的活性载体则不一定适合于多肽固定。 It is noted that particularly suitable immobilized polypeptide generally suitable for nucleic acid active carrier is fixed, and a carrier suitable for the nucleic acid immobilized active polypeptide is not necessarily suitable for fixing. 本发明中,术语“功能基团”是指活性载体中通过相互作用(包括亲和作用、离子交换、亲油作用、共价键合、等等)用以固定反应物的基团;术语“偶联基团”是指固相载体表面上化学键合的衍生物中用以联接功能基团和固相载体的基团。 The present invention, the term "functional group" refers to the active carrier through interaction (including affinity effect, ion exchange, oleophilic effect, covalent bonding, etc.) is fixed to a group of reactants; the term " coupling group "refers to a derivative of the solid phase support surface is chemically bonded to the functional groups and coupling to a solid support group.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述功能基团的分子量大于75小于500。 In one embodiment, the active support for a biochip according to the present invention, the molecular weight of the functional group is less than 75 greater than 500.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述功能基团包括氨基酸基团。 In one embodiment, the active support for a biochip according to the present invention, the functional group include amino group.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括脱保护氨基酸。 In one embodiment, the active support for a biochip according to the present invention, the amino acid comprising deprotection of amino acids.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括侧链含二个或二个以上-NH2的氨基酸。 In one embodiment, the active support for a biochip according to the present invention, the amino acid comprising a side chain comprising the amino acid of two or more of -NH2. 在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括侧链含三个或三个以上-NH2的氨基酸。 In one embodiment, the active support for a biochip according to the present invention, the amino acid side chains containing amino acid comprising three or more of -NH2.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括赖氨酸。 In one embodiment, the active support for a biochip according to the present invention, comprises the amino acid lysine. 在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括丝氨酸。 In one embodiment, the active support for a biochip according to the present invention, comprises the amino acid serine. 在本发明的用于生物芯片的活性载体的一项实施方案中,所述氨基酸包括胱氨酸。 In one embodiment, the active support for a biochip according to the present invention, comprises the amino acid cystine.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述偶联基团包括有机硅基团。 In one embodiment, the active support for a biochip according to the present invention, the coupling group comprises an organic silicon group. 例如,通过加入下述一种或多种偶联剂引入的偶联基团:甲基丙烯酰氧基硅烷、乙烯基硅烷、氨基硅烷、环氧基硅烷、硫基硅烷、脲基硅烷、异氰酸酯硅烷、3-氨基丙基三乙氧基硅烷、等等。 For example, by adding a coupling group consists of one or more coupling agents introduced: methacryloxy silanes, vinyl silanes, aminosilanes, epoxy silanes, sulfur silanes, ureido silanes, isocyanates silane, 3-aminopropyl triethoxysilane, and the like.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述共价键包括酰胺键。 In one embodiment, the active support for a biochip according to the present invention, the covalent linkage comprises an amide bond.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述活性载体包括生物芯片片基。 In one embodiment, the active support for a biochip according to the present invention, the biochip comprises a carrier sheet active group. 本发明中,术语“片基”是指生物芯片的一种部件,即用于生物芯片的活性载体,其具有有效固定探针的功能。 The present invention, the term "base sheet" refers to a biochip member, i.e., an active carrier biochips, which has an effective function of immobilized probes. 本发明的片基,至少含片基固相载体、固定在片基固相载体上的偶联基团、以及固定在所述偶联基团上的功能基团。 The base sheet of the present invention, at least a solid support group lozenges, immobilized on a solid support sheet substrate coupling group, and the functional group fixed to the coupling group. 片基固相载体包括常规片基载体和纳米结构片基载体。 The base sheet include a conventional solid support carrier sheet substrate sheet and a nanostructure-based carrier. 所述常规片基载体为表面未固定有纳米结构的片基固相载体。 The conventional carrier base sheet is not fixed to the surface of the base sheet of the solid phase support nanostructures. 尽管本发明的试施例给出的是以玻璃作为片基固相载体的例子,其它固相载体(例如硅、金、塑料片或金属片),也可应用本发明的方法制备本发明的生物芯片片基。 Although embodiments of the present invention, the test given example is a glass plate as a solid support substrate, other solid support (e.g., silicon, gold, a plastic sheet or a metal sheet), the present invention can also be prepared in application of the method according to the present invention biochip sheet group. 所述纳米结构片基载体为固定有纳米结构的片基固相载体。 The nanostructure film base support is fixed to the base plate of the solid phase support nanostructures.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述活性载体包括用于亲和纳米结构的活性纳米结构。 In one embodiment, the active support for a biochip according to the present invention, the reactive carrier comprises an active nanostructures and nanostructured for affinity. 本发明的亲和纳米结构,至少含纳米结构(例如,纳米粒子、纳米管、等等)、固定在纳米结构上的偶联基团、固定在所述偶联基团上的功能基团、以及固定在所述功能基团上的探针。 Affinity nanostructures of the present invention, at least containing nanostructure (e.g., nanoparticles, nanotubes, etc.), the fixed coupling groups on the nano structure, fixed function groups on the conjugate group, and a probe fixed to the functional group.

在本发明的用于生物芯片的活性载体的一项实施方案中,所述活性载体包括用于纳米结构标记物的活性纳米结构。 In one embodiment, the active support for a biochip according to the present invention, the reactive carrier comprises an active nanostructures for nanostructure markers. 本发明的纳米结构标记物,至少含标记物质、纳米结构(例如,纳米粒子、纳米管、等等)、固定在纳米结构上的偶联基团、固定在所述偶联基团上的功能基团、以及固定在所述功能基团上的配基。 Nanostructure markers of the present invention, at least a marking substance-containing nanostructure (e.g., nanoparticles, nanotubes, etc.), the fixed coupling groups on the nano structure, fixed to the functional coupling group group, and is fixed to the functional group of the ligand.

本发明的第二个方面,涉及本发明的用于生物芯片的活性载体的一种制备方法,1).将所述偶联基团以化学键结合在所述固相载体表面上;2).将功能物质与1)的制备物中的所述偶联基团接触并进行反应,所述功能物质含所述功能基团;3).将所述功能基团固定在所述偶联基团上。 A second aspect of the present invention, the present invention relates to a method for preparing a carrier for active biochip, 1) the coupling group is chemically bonded to the surface of the solid support; 2). the coupling group and a functional substance prepared in 1) was contacted and reacted, the functional material containing the functional group; 3) fixed to the functional group of the coupling group. on.

在本发明的方法的一项实施方案中:A).所述偶联基团含-NH2或-COOH基团;B).所述功能物质含功能基团、结合在功能基团内的保护基团和可同所述偶联基团反应的-NH2或-COOH基团;C).所述功能基团固定在所述偶联基团上是通过形成酰胺键进行的;D).在所述功能基团固定在所述偶联基团上后,所述方法还包括在固定探针前选择性地脱去所述保护基团。 In one embodiment of the method of the present invention: A) containing the coupling group -NH2 or -COOH group; B) of the functional substance-containing functional groups, in conjunction with protection of the functional group. or -COOH group and -NH2 group can react with the coupling group; C) the functional group fixed to the coupling group is carried out by forming an amide bond;. D) on. the functional group fixed to the rear of the upper coupling group, said method further comprising selectively removing the probe before fixing the protective group. 本发明的方法的一项实施方案,还包括在固定探针后选择性地脱去所述保护基团。 One embodiment of the method of the present invention, further comprising, after the removal of immobilized probes selectively protecting groups. 本发明的方法的一项实施方案,还包括重复所述1)、2)、3)。 One embodiment of the method of the present invention, further comprising repeating said 1), 2), 3).

本发明的第三个方面,涉及本发明的用于生物芯片的活性载体的应用。 A third aspect of the present invention, the present invention relates to the application of an active support of the biochip.

本发明的一种多反应器生物芯片基片,其特征在于:其至少含本发明的生物芯片片基和固定在所述片基上的反应器隔离结构。 A multi-reactor biochip substrate of the present invention is characterized in that: a reactor which contains at least an isolation structure according to the present invention, the base sheet and the biochip fixed on the base sheet.

本发明术语“基片”是指指用以制备芯片的、尚未固定探针的中间产品,其以片基为基础、结合有无其它结构(例如隔离结构)、且在固定配基后形成芯片。 Herein, the term "substrate" is used to prepare the chip finger, yet immobilized probes intermediate product, which is based on the base sheet, with the presence or absence of other structures (e.g., isolation structure), and the chip is formed in the fixed ligand . 基片上可以有一个或多个片基室。 There may be one or more chambers on the substrate film base. 单片基室基片上通常没有隔离结构,此时基片既是片基(例如市售的氨基玻片)。 Single substrate chamber is typically not isolated structure on a substrate, the substrate at this time both groups substrate (e.g. a commercially available amino slide). 多片基室基片上有隔离结构,此时基片包括片基和隔离结构。 Isolation structures on a multi-chip substrate chamber substrate, the substrate comprising a film base case and the isolation structure. 片基室在固定上配基后形成反应器,多片基室基片形成多反应器芯片。 Sheet base chamber formed in the fixed reactor after ligand, multi-chip substrate reactor chamber substrate is formed of a multi-chip. 本发明术语“反应器结构”是指形成反应器的必须的片基、探针以外的其它结构,如隔离结构、流路结构等;术语“反应器隔离结构”是指至少在加入样品时避免反应器之间出现交叉污染的结构,包括用以隔离部分或全部反应器结构(例如反应池、反应腔室、流路、进液结构、出液结构、等等)的结构。 Herein, the term "reactor configuration" refers to the formation of the reactor must film base, other than the probe structure, isolation structure, the flow path structure and the like; the term "isolated from the reactor structure" means at least when the sample is added to avoid structure of cross-contamination occurs between the reactors, to isolate the structure including part or all of the reactor structures (e.g. reaction vessel, the reaction chamber, the flow path structure into the liquid, the liquid structure, etc.). 反应器隔离结构包括开放式隔离结构和封闭式隔离结构。 The reactor isolation structure comprises isolation structure open and closed isolation structure. 开放式隔离结构是指芯片使用时隔离结构上方无覆盖物的反应器隔离结构。 Open isolation structure refers to the isolation structure without covering over the chip using the reactor isolation structure. 封闭式隔离结构是指芯片使用时隔离结构上方有覆盖物的反应器隔离结构。 It means a closed isolation structure is isolated from the reactor over the isolation structure of the covering structure using the chip.

在本发明的生物芯片基片的一项实施方案中,所述隔离结构包括高疏水材料涂层。 In one embodiment of the biochip substrate according to the present invention, the isolation structure comprises a coating of a highly hydrophobic material. 在本发明的生物芯片基片的一项实施方案中,所述高疏水材料的表面静态水接触角比所述固相载体的表面静态水接触角大40度以上。 In one embodiment of the biochip substrate according to the present invention, the surface of the high static water contact angle of hydrophobic material than the surface of the solid support static water contact angle above 40 degrees. 本发明术语“表面静态水接触角”是指静态水在表面的接触角。 Herein, the term "surface static water contact angle" refers to a static contact angle of water on the surface. 长时间以来,用一滴液体在固体物质表面的接触角θ作为特定固定湿润的量化测试,这一点是被广为认可的。 Since a long time, the contact angle with a drop of the liquid surface of a solid substance θ specific quantitative wet test fixture, it is widely recognized. 如果液体完全分散在表面上形成膜,接触角θ为0、如果物质表面上的液珠存在一定角度,该表面被认为是不湿润的。 If the liquid is completely dispersed to form a film, the contact angle θ is 0 on the surface, if the surface material on the bead certain angle, this surface is not considered wet. 芯片中最常用的固相载体基片材料为玻璃,其表面静态水接触角约为45度。 The most commonly used chip solid support material is a glass substrate, a surface static water contact angle of about 45 degrees.

在本发明的生物芯片片基的一项实施方案中,所述隔离结构包括疏水/疏油材料涂层。 In one embodiment of the biochip sheet substrate of the present invention, the isolation structure comprises a hydrophobic / oleophobic coating material.

本发明的一种生物芯片,其至少含本发明的生物芯片片基或本发明的生物芯片基片。 A biochip according to the present invention, at least a group containing the biochip sheet of the present invention or biochip substrate of the present invention. 本发明的生物芯片的一项实施方案,其至少含本发明的生物芯片片基和固定在所述片基上的二种或二种以上的探针。 Of two or more thereof biochip probe one embodiment of the present invention, the present invention which contains at least the base sheet and the biochip fixed on the base sheet. 本发明的生物芯片的一项实施方案,其为多反应器生物芯片,其至少含本发明的生物芯片基片和固定在所述片基上的二种或二种以上的探针。 One embodiment of the present invention, a biochip, which is a multi-reactor biochip, the biochip substrate at least containing the present invention and is fixed to the base sheet of two or more thereof probes. 本发明中,术语“探针”是指用以在检测反应中捕获目标物的物质,例如:抗原、抗体、配体、配体指数增强系统进化技术筛选的适配分子、多肽、多糖、共酶、辅因子、抗生素、类固醇、病毒、细胞、生物素、亲和素等。 The present invention, the term "probe" refers to a substance to capture the object in the detection reaction, for example: antigens, antibodies, ligands, evolution of ligands by exponential enhancement system Screening adapter molecules, polypeptides, polysaccharides, co enzymes, cofactors, antibiotics, steroid, viruses, cells, biotin, avidin and the like. 在本发明的生物芯片片基的一项实施方案中,所述生物芯片包括含多肽探针的芯片。 In one embodiment of the biochip sheet substrate in the present invention, the biochip comprises a chip containing polypeptide probes. 本发明术语“多肽”相当于英语中的“polypeptide”,包括天然或合成蛋白质、蛋白质片断、合成肽、等等,免疫检测中通常的目标物和检测中通用的探针、例如抗原、抗体、等等都属于多肽本发明的一种生物芯片试剂盒,其特征在于:其至少含本发明的用于生物芯片的活性载体。 Herein, the term "polypeptide" in the English equivalent "Polypeptide", including natural or synthetic proteins, protein fragments, synthetic peptides, etc., conventional immunoassays and the detection object common to probes, such as antigens, antibodies, etc., are considered a biochip kit polypeptide according to the present invention is characterized in that: the present invention which contains at least an active carrier for the biochip.

本发明的一种生物芯片检测方法,至少包括下列步骤:A).提供可能含检测目标物的样本,所述目标物包括多肽目标物;B).提供本发明的生物芯片;C).使所述样本与所述探针接触并进行相关反应。 A biochip detection method of the present invention, comprising at least the following steps: A) providing a sample may contain a detection target substance, the target comprises a target polypeptide; B) of the present invention to provide a biochip; C) so that... contacting the sample with the probe and related reactions. 本发明的生物芯片在下述一个或多个活动中的使用:用作一种诊断工具、用作一种诊断工具、用作一种分子扫描工具、用作一种分子间关系分析工具、用作一种测试工具。 Biochip used in the present invention, one or more of the following activities: as a diagnostic tool, as a diagnostic tool, as a molecular tool for scanning, as the relationship between a molecular analysis tool, as A test tool.

实施例实施例1本发明的用于生物芯片的活性载体的例子(1)本实施例中,本发明的用于生物芯片的活性载体为生物芯片片基。 Example embodiments of the present embodiment example (1) an active carrier of a biochip according to one embodiment of the invention, the present invention is used as active carrier biochip biochip sheet group. 本实施例中,所用片基固相载体为玻片,包括常规玻片和纳米结构玻片(参考我们的另一项专利申请PCT/CN2004/000437)。 In this embodiment, the sheet substrate used as the solid phase carrier slide, the slide including a conventional slide and nanostructures (we refer to another patent application PCT / CN2004 / 000437).

本实施例中,所用功能物质选自氨基酸、优选含有-NH2侧基保护基(例如Fmoc)的氨基酸。 In this embodiment, the selected amino acid with the functional substance, preferably contains a -NH2 side group protecting groups (e.g. Fmoc) amino acids. 所用氨基酸分别为:赖氨酸、丝氨酸、胱氨酸、和它们的混合物。 The amino acids used are: lysine, serine, cysteine, and mixtures thereof. 按照本发明的方法,其它氨基酸也可用来制备本发明的生物芯片片基。 The method according to the present invention, other amino acids may also be used to prepare a biochip sheet of this invention.

本实施例中,所用偶联剂选自有机硅、优选硅烷类化合物(例如3-氨基丙基三乙氧基硅烷)。 In this embodiment, the coupling agent is selected with a silicone, preferably a silane-based compounds (e.g. 3-aminopropyltriethoxysilane). 使用硅烷类化合物可在玻片表面形成含氨基或羟基的偶合基团。 It may be formed using a silane coupling compound containing amino or hydroxyl groups in the surface of glass.

本实施例中,本发明的生物芯片片基的一种制备方法,其至少包括:1).在所述固相载体表面上键合形成所述偶联基团,所述偶联基团含-NH2或-COOH基团;2).将功能物质与1)制备的含偶联基团的固相载体表面接触并进行反应,其中:A).所述功能物质含所述功能基团和结合在功能基团内的保护基团;B).所述功能物质除功能基团外还含-NH2或-COOH基团;3).通过与所述偶联基团形成酰胺键将所述功能基团固定在上述固相载体表面上。 In this embodiment, a method of preparing a biochip-based sheet of the present invention, comprising at least: 1) bonded to the coupling groups are formed on the surface of a solid phase support, the conjugate-containing group. -NH2 or -COOH group; 2) and the functional substance 1) preparation of a carrier containing a surface in contact with the coupling group and the solid phase reaction, wherein: a) the functional group-containing material and the functional. protecting group in conjunction with functional groups;. B) in addition to the functional material further containing functional groups -NH2 or -COOH group; 3) by formation of an amide bond with the coupling group of the functional groups immobilized on the surface of the solid phase support.

本实施例制备的片基,其在固定探针(参考以下)以后,在室温下放置12个月未见探针活性的明显下降,而现有片基(例如按已知方法制备的氨基玻片、醛基玻片和按照本实施例方法制备的聚赖氨酸玻片比较)则在11个月内以见探针活性的明显下降。 Preparation sheet substrate of the present embodiment, which after fixing the probe (refer to the following), for 12 months at room temperature no significant decline in the activity of the probe, the conventional sheet group (e.g. an amino glass prepared by known methods sheet, aldehyde slides and polylysine slides prepared according to Comparative Example of the present embodiment) in the 11 months of the probe to see decreased activity. 与现有氨基脲玻片比较,本实施例制备的片基显示出更小的批间差。 Compared with the conventional slide semicarbazide, film base prepared in Example of the present embodiment exhibits a difference between the smaller batches.

实施例1.1偶联基团含氨基的生物芯片片基的制备例子(1)本实施例中,本发明的生物芯片片基的一种制备方法为:1).清洗玻片并固定硅烷类基团按公知的方法进行。 Preparation Examples (1) Example 1.1 biochip sheet coupling group containing an amino group in the present embodiment, a method of preparing a biochip-based sheet of the present invention are: 1) washing the slides and fixed silanes groups. group carried out in a known method.

2).固定氨基酸基团将烘干后的固定有硅烷类基团的玻片浸入优选浓度的上述氨基酸的水溶液中,在室温下反应1小时,然后洗去玻片表面的未固定的物质,烘干后备用。 2) fixed to amino groups fixed to the slide after drying the silane groups was immersed in an aqueous solution of the above-described preferred amino acid concentration in the reaction at room temperature for 1 hour and then washed off non-immobilized material of the slide surface, after drying standby.

实施例1.2偶联基团含氨基的生物芯片片基的制备例子(2)本实施例中,本发明的生物芯片片基的一种制备方法为:1).清洗玻片并固定硅烷类基团按公知的方法进行。 Preparation Examples (2) Example 1.2 biochip coupling group containing an amino group sheet embodiment of the present embodiment, a method of preparing a biochip-based sheet of the present invention are: 1) washing the slides and fixed silanes groups. group carried out in a known method.

2).固定氨基酸基团将烘干后的固定有硅烷类基团的玻片浸入优选浓度的上述含Fmoc保护剂的氨基酸的DFM溶液中,在室温下反应1小时。 2) fixed to amino groups fixed to the slide after the drying groups silanes immersed DFM agent solution containing Fmoc-protected amino acid is the preferred concentration in the reaction at room temperature for 1 hour. 然后按公知的方法脱去Fmoc保护剂,再洗去玻片表面的未固定的物质,烘干后备用。 According to a known method and then removing Fmoc protecting agent, and then wash away non-immobilized slide surface material, after drying standby.

实施例1.3偶联基团含氨基的生物芯片片基的制备例子(3)本实施例中,本发明的生物芯片片基的一种制备方法为:1).按已知的方法制备氨基玻片2).固定氨基酸基团将烘干后的氨基玻片浸入优选浓度的上述含Fmoc保护剂的氨基酸的DFM溶液中,在室温下反应1小时。 Preparation Examples (3) Example 1.3 biochip coupling group containing an amino group sheet embodiment of the present embodiment, a method of preparing a biochip-based sheet of the present invention are: 1) Preparation of carbamic glass according to known methods. DFM agent solution containing Fmoc-protected amino acid of the sheet 2) fixed to an amino acid group and drying the slides dipped preferred concentration of the reaction at room temperature for 1 hour. 然后按公知的方法脱去Fmoc保护剂,再洗去玻片表面的未固定的物质,烘干后备用。 According to a known method and then removing Fmoc protecting agent, and then wash away non-immobilized slide surface material, after drying standby.

实施例1.4偶联基团含羧基的生物芯片片基的制备例子本实施例中,本发明的生物芯片片基的一种制备方法为:1).清洗玻片并固定硅烷类基团按现有的已知方法进行。 EXAMPLES Example 1.4 Preparation coupling group containing a carboxyl group biochip sheet embodiment of the present embodiment, a method of preparing a biochip-based sheet of the present invention are: 1) washing the slides and fixed by current silanes groups. some known methods.

2).固定含羧基的基团按现有的已知方法进行。 2) Fixed containing carboxyl groups carried out in a conventional known method.

3).固定氨基酸基团将烘干后的固定有羧基的玻片浸入优选浓度的上述含Fmoc保护剂的氨基酸的DFM溶液中,在室温下反应1小时。 3) fixed to a fixed group of amino acid after drying slides carboxyl group-containing amino acid is immersed in a solution DFM Fmoc protecting agent concentration is preferably in a reaction at room temperature for 1 hour. 然后按公知的方法脱去Fmoc保护剂,再洗去玻片表面的未固定的物质,烘干后备用。 According to a known method and then removing Fmoc protecting agent, and then wash away non-immobilized slide surface material, after drying standby.

实施例2本发明的用于生物芯片的活性载体的例子(2)本实施例中,本发明的用于生物芯片的活性载体为用于亲和纳米结构的活性纳米结构、或用于纳米结构标记物的活性纳米结构。 Examples of (2) an active carrier biochip Example 2 of the present embodiment of the present invention embodiment, the present invention is an active support for a biochip active nanostructures and nanostructured affinity, or for the nanostructure labeled active nanostructure.

本实施例中,所用固相载体为纳米颗粒(例如氧化硅纳米微粒STN-3、LUDOXAS-40、及氧化钛纳米微粒。 In this embodiment, nanoparticles (e.g. silica nanoparticles STN-3 used solid support, LUDOXAS-40, and titanium oxide nanoparticles.

本实施例中,所用功能物质选自氨基酸、优选含有-NH2侧基保护基(例如Fmoc)的氨基酸。 In this embodiment, the selected amino acid with the functional substance, preferably contains a -NH2 side group protecting groups (e.g. Fmoc) amino acids. 所用氨基酸分别为:赖氨酸、丝氨酸、胱氨酸、和它们的混合物。 The amino acids used are: lysine, serine, cysteine, and mixtures thereof. 按照本发明的方法,其它氨基酸也可用来制备本发明的生物芯片片基。 The method according to the present invention, other amino acids may also be used to prepare a biochip sheet of this invention.

本实施例中,所用偶联剂选自有机硅、优选硅烷类化合物(例如3-氨基丙基三乙氧基硅烷)。 In this embodiment, the coupling agent is selected with a silicone, preferably a silane-based compounds (e.g. 3-aminopropyltriethoxysilane). 使用硅烷类化合物可在玻片表面形成含氨基或羟基的偶合基团。 It may be formed using a silane coupling compound containing amino or hydroxyl groups in the surface of glass.

本实施例中,本发明的生物芯片片基的一种制备方法,其至少包括:1).在所述固相载体表面上键合形成所述偶联基团,所述偶联基团含-NH2或-COOH基团。 In this embodiment, a method of preparing a biochip-based sheet of the present invention, comprising at least: 1) bonded to the coupling groups are formed on the surface of a solid phase support, the conjugate-containing group. -NH2 or -COOH groups. 键合方法与现有的氨基化纳米氧化硅的制备方法相同。 The method of bonding the same conventional method for preparing nano amination silicon oxide.

2).将上述功能物质与1)制备的含偶联基团的固相载体表面接触并进行反应。 2) The above-described functional substance in contact with the surface of a solid support) was prepared containing coupling group and reaction. 反应温度为室温,反应时间2小时。 The reaction temperature is room temperature, the reaction time 2 hours.

3).通过与所述偶联基团形成酰胺键将所述功能基团固定在上述固相载体表面上。 3) by forming an amide bond with the coupling group to the functional group fixed to the surface of the solid support.

本实施例制备的活性纳米结构,其在固定探针或标记物配基以后,在室温下放置1个月未见纳米结构上探针或标记物配基活性的明显下降。 Active nanostructures embodiment of the present embodiment, which after fixing the probe or marker ligand, 1 month at room temperature was decreased labeled probe or ligand activity structurally no nm.

实施例3本发明的多反应器生物芯片基片的制备例子本实施例中,所用生物芯片片基为上述实施例1制备的片基。 Preparation Examples of multi-reactor biochip substrate of Example 3 of the present invention in the present embodiment, the sheet substrate is a biochip with a film base prepared in Example 1 above.

实施例3.1高疏水隔离结构基片的制备在本实施例中,所用高疏水材料分别为“聚丙烯酸脂涂料”(中国成都晨光化工设计院提供,静态水接触度85度)、“有机硅防水涂料”(中国成都晨光化工设计院提供,静态水接触度116度)、“超高疏水乳胶漆”(中国成都晨光化工设计院提供,静态水接触度123度)和“高疏水氧化硅涂料”(中国舟山明日纳米材料公司提供,静态水接触度151度)。 Preparation 3.1 a highly hydrophobic isolation structure of the substrate in the present embodiment, as highly hydrophobic materials are (China Chengdu Chenguang Chemical Institute provided, the static water contact of 85 degrees) to "polyacrylate paint", a "silicone waterproofing coatings "(China Chengdu Chenguang Chemical Institute provided static water contact level 116 degrees)," ultra-hydrophobic latex paint "(China Chengdu Chenguang Chemical Institute provided static water contact ° 123 °) and" high hydrophobic silica paint " (China Zhoushan tomorrow nano-materials companies, the static water contact of 151 degrees). 尽管本发明的反应器的隔离结构可以部分是、也可以全部是本发明的高疏水隔离结构,本实施例中只给出了最为简单的情况作为例子。 Although the reactor of the isolation structure of the present invention may be part of, or may all be highly hydrophobic isolation structure of the present invention, the present embodiment is given only by way of example the simplest case.

本实施例中的制备方法为:将上述高疏水液态材料涂抹在上述实施例1制备的片基上隔离带位置上,按供货方的使用说明在室温干燥后固化,形成高度25-115μm、宽度2.0-2.5mm的高疏水凸体。 Prepared as in the present embodiment: The above-described highly hydrophobic liquid material applied on the sheet substrate prepared in Example 1 above with the isolation position, by using the supplier's instructions and dried at room temperature after curing, form highly 25-115μm, a width of 2.0-2.5mm highly hydrophobic convex body. 高疏水凸体可以有不同几何图型,本例中仅取带状(凸体为一条带)和线组合(例如凸体为有间隔的2条线)2种。 Highly hydrophobic convex body can have a different geometrical pattern, taken in the present embodiment only the tape-shaped (convex body is a strip) and line combination (e.g. the convex body is a wire with a spacing of 2) thereof. 高疏水凸体包围的表面可以取各种几何图型,本例中仅取3mm×3mm矩形。 Highly hydrophobic surface surrounding the convex body can take a variety of geometric patterns, in the present embodiment only take 3mm × 3mm rectangle. 在基片此一表面上,横向共有12个片基池,纵向有4个片基池,共有48个片基池。 On a surface of this substrate, 12 pieces total lateral groups pools, there are four longitudinal pieces yl pool, a total of 48 pools film base.

实施例3.2疏水/疏油隔离结构基片的制备在本实施例中,所用疏水-疏油材料均为市场上可购得的疏水-疏油材料,分别为城洁宝(深圳市城洁宝环保科技有限公司),高疏水氧化硅涂料(中国舟山明日纳米材料公司)和双疏涂料(中国兰光化工研究院)。 3.2 Example hydrophobic / oleophobic prepared spacer substrate structure in the present embodiment, the hydrophobic - oleophobic materials are commercially available hydrophobic - oleophobic material, respectively Jie Town (Shenzhen City Jie environmental Protection Technology Co., Ltd.), a highly hydrophobic silica coating (China Zhoushan tomorrow nano-materials company) and double thinning paint (light blue Chinese Chemical Research Institute). 尽管本发明的反应器的隔离结构可以部分是、也可以全部是本发明的高疏水隔离结构,本实施例中只给出了最为简单的情况作为例子。 Although the reactor of the isolation structure of the present invention may be part of, or may all be highly hydrophobic isolation structure of the present invention, the present embodiment is given only by way of example the simplest case.

本实施例中的制备方法为:将上述疏水-疏油材料涂抹在上述实施例1制备的片基上隔离带位置上,按供货方的使用说明在室温干燥后固化,形成高度25-115μm、宽度2.0-2.5mm的疏水-疏油凸体。 Prepared in the present embodiment to: the hydrophobic - oleophobic material is applied on the sheet substrate prepared in Example 1 above with the isolation position described by the supplier using solidified after drying at room temperature, to form highly 25-115μm , the width of 2.0-2.5mm hydrophobic - oleophobic convex bodies. 疏水-疏油凸体包围的表面可以取各种几何图型,本例中仅取3mm×3mm矩形。 Hydrophobic - oleophobic surface surrounding the convex body may take a variety of geometric patterns, in the present embodiment only take 3mm × 3mm rectangle. 在基片此一表面上,横向共有12个片基池,纵向有4个片基池,共有48个片基池。 On a surface of this substrate, 12 pieces total lateral groups pools, there are four longitudinal pieces yl pool, a total of 48 pools film base.

实施例4本发明的生物芯片的例子本实施例中的生物芯片含上述实施例3制备的基片、或/和上述实施例2制备的用于亲和纳米结构的活性纳米结构。 Examples of embodiments Example 4 biochip embodiment of the present invention, a biochip containing the above-described embodiments the substrate 3 is prepared, affinity or activity of nanostructures and nanostructured / and prepared in Example 2 above for the present embodiment. 实施例3制备的基片含上述实施例1制备的片基。 Example 3 Preparation of the substrate containing the above-described embodiment of the sheet substrate prepared in Example 1. 本实施例所用探针为HCV抗原(中国北京人民医院肝病研究所)和HIV1+2抗原(中国北京人民医院肝病研究所)。 This embodiment of the probe used for the HCV antigen (Chinese People's Hospital, Beijing Institute of Liver Diseases) HIV1 + 2 antigen (Chinese People's Hospital of Beijing Institute of Liver Diseases) and.

本实施例中,本发明生物芯片的制备方法为:在上述基片的一个片基池中,分别将含探针的液体按照通用的点样方法点样在片基池中,每种液体各点3个直径为80μm的点,间点距为600-700μm,形成2×3阵列。 In this embodiment, the biochip production method of the present invention are: a pool in the base sheet of said substrate, respectively, the liquid containing the probe according to the general method of spotting spotted cell sheet group, each of each liquid point 3 of 80μm diameter point between 600-700 dot pitch, 2 × 3 array is formed. 反应池片基上的配基密度大于30点/cm2。 Ligand density on the sheet substrate reaction vessel of greater than 30 points / cm2. 所用含探针的液体分别为:HCV抗原(1.0mg/ml)、HIV1+2抗原溶液(1.0mg/ml)、HCV抗原/活性纳米结构复合物(HCV抗原浓度0.1mg/ml)、HIV1+2抗原/活性纳米结构复合物(HIV1+2抗原浓度)。 The liquid containing the probe were used: HCV antigen (1.0mg / ml), HIV1 + 2 antigen solution (1.0mg / ml), HCV antigen / activity nanostructure composite (HCV antigen concentration 0.1mg / ml), HIV1 + 2 antigen / activity nanostructure composite (HIV1 + 2 antigen concentration). HCV抗原/活性纳米结构复合物和HIV1+2抗原/活性纳米结构复合物为亲和纳米结构,其含活性纳米结构和固定在活性纳米结构上的抗原,其制备方法参考我们的另一项专利申请PCT/CN2004/000437。 HCV antigen / activity nanostructure composite HIV1 + 2 antigen and / nanostructure composite activity and affinity for the nanostructure, and the nanostructure which contains the active antigen immobilized on the active nanostructures, their preparation refer to our other patent application PCT / CN2004 / 000437.

实施例5本发明的生物芯片试剂盒的例子本实施例试剂盒分别为:1).含实施例4制备的生物芯片的试剂盒;2).含纳米结构标记物的试剂盒;3).含实施例4制备的生物芯片和纳米结构标记物的试剂盒。 Example Kits example embodiment of a biochip kit of Example 5 of the present invention of the present embodiment are as follows: 1) The biochip kit prepared in Example 4 containing embodiment; 2) a kit containing nanostructure marker; 3). kits and biochips containing nanostructure marker of Example 4 was prepared. 本实施例中,纳米结构标记物含罗丹明、活性纳米结构和固定在活性纳米结构上的羊抗人二抗,其制备方法参考我们的另一项专利申请PCT/CN2004/000437。 In this embodiment, the nanostructure-containing rhodamine label, active nanostructures and fixed on the active nanostructures goat anti-human secondary antibody, which is prepared with reference to another patent application in our PCT / CN2004 / 000437.

实施例6本发明的生物检测方法的例子在本实施例中,1号样为HCV抗体阳性血清,2号样为HIV1+2抗体阳性人血清,3号样为阴性对照物(HCV抗体和HIV1+2抗体都为阴性的血清对照物)。 Examples of biological detection method of Example 6 of the present invention is implemented in the present embodiment, No. 1 sample of HCV positive sera, No. 2 sample is HIV1 + 2 antibody positive human serum, No. 3 sample is a negative control (HCV antibodies and HIV1 +2 antibodies were negative sera controls). 所有的样品均经使用经典的ELISA方法在血清20倍稀释反应条件下预先检测。 All samples were detected in advance under the reaction conditions was diluted 20-fold in the serum by ELISA using the classical method.

在本实施例中,所用生物芯片试剂盒为实施例5制备的试剂盒。 In the present embodiment, the kit of the biochip kit prepared in Example 5 was used. 对照试剂盒中,芯片为分别在氨基玻片和环氧基玻片(Microarray Technology)上按常规的点样方法固定有相同配基制备的芯片,标记物为罗丹明标记羊抗人二抗(美国Jackson ImmunoRresearch Laboratories公司)。 Control Kit, the chips slide respectively on the amino group and an epoxy slides (Microarray Technology) according to the conventional method of spotting fixed base was prepared with the same chip, the marker rhodamine labeled goat anti-human secondary antibody ( United States Jackson ImmunoRresearch Laboratories company). 实验时,前述3种样品分别加入生物芯片的反应池中。 Experiment, three samples were added to the reaction vessel biochip. 加样量为15μl,反应30分钟后洗涤5次,洗涤液每次加入量为25μl。 15 l of sample volume, the reaction after 30 minutes and washed five times, each washing solution was added in an amount of 25μl. 标记物加入量为15μl,反应后洗涤5次,洗涤液每次加入量为25μl,干燥后在35/50下进行扫描。 15 l was added in an amount of marker, the reaction was washed five times, each washing solution was added in an amount of 25 l, dried scanning at 35/50. 扫描仪为共聚焦激光扫描仪(Afymetrix公司GMS418芯片扫描仪),扫描激发光波长532nm,发射光波长570nm,读取的信号经处理软件(JAGUARII)处理,然后取平均值后根据Cut-off值判定阴(-)阳(+)性得到的结果。 The scanner is a confocal laser scanner (Afymetrix company GMS418 microarray scanner), the scanning light wavelength 532nm excitation, 570nm emission wavelengths, the read signal processing software (JAGUARII) processing, and then averaged in accordance with the Cut-off value determining a female (-) anode (+) of the obtained results. 所得结果与ELISA方法在血清20倍稀释反应条件下预先检测的结果一致且检测下限更低。 The results consistent with the results obtained by ELISA and diluted under the reaction conditions previously detected in the serum and 20 times lower detection limit. 本发明的生物芯片试剂盒与对照试剂盒比较,在37℃时稳定性要高20%以上,且检测下限要低15%以上。 Biochip kits and kit of the invention is a control comparison, the stability at 37 ℃ higher than 20%, and the detection limit lower than 15%.

本发明的生物芯片试剂盒在下述一个或多个活动中的使用:用作一种诊断工具、用作一种分子扫描工具、用作一种分子间关系分析工具、用作一种测试工具。 The biochip kit of the present invention is used in one or more of the following activities: as a diagnostic tool, as a molecular tool for scanning, as the relationship between a molecular analysis tool, used as a testing tool.

应当清楚,对于本领域的技术人员来说,对这里所述的本发明的优选的实施方案显然可以作出各种变动和修改。 It should be apparent to those skilled in the art, the preferred embodiments of the invention described herein will be apparent that various changes and modifications. 在不偏离本发明的精神和范围及不减小其优点的情况下,可以进行这些修改和变动。 Without departing from the spirit and scope of the present invention and the advantages thereof are not reduced, these modifications and variations may be made. 因此,这些修改和变动包括在所附的权利要求的范围内。 Accordingly, such modifications and variations are included within the scope of the appended claims.

Claims (14)

  1. 1.一种用于生物芯片的活性载体,其特征在于:其含固相载体和固相载体表面上化学键合的衍生物,所述衍生物至少含偶联基团和偶联基团上以共价键结合的功能基团,且所述功能基团的分子量大于50小于1000。 An active support for a biochip, comprising: a derivative thereof containing the solid support and the solid support chemically bonded to the surface, said derivative containing at least coupling groups and coupling groups on to covalently bonded functional groups, and functional groups having a molecular weight greater than 1000 is less than 50.
  2. 2.根据权利要求1所述的活性载体,其特征在于:所述功能基团的分子量大于75小于500。 2. The active carrier according to claim 1, wherein: said functional group is a molecular weight greater than 75 is less than 500.
  3. 3.根据权利要求1所述的活性载体,其特征在于:所述功能基团包括氨基酸基团。 The active carrier according to claim 1, wherein: the functional groups comprise acid groups.
  4. 4.根据权利要求1所述的活性载体,其特征在于:所述偶联基团包括有机硅基团。 The active carrier according to claim 1, wherein: said coupling group comprises an organic silicon group.
  5. 5.根据权利要求1所述的活性载体,其特征在于:所述共价键包括酰胺键。 The active carrier according to claim 1, wherein: said covalent linkage comprises an amide bond.
  6. 6.根据权利要求1所述的活性载体,其特征在于:所述活性载体包括生物芯片片基。 6. The active carrier according to claim 1, characterized in that: said biochip comprises a carrier sheet active group.
  7. 7.根据权利要求1所述的活性载体,其特征在于:所述活性载体包括用于亲和纳米结构的活性纳米结构。 7. The active carrier according to claim 1, wherein: said carrier comprises an active nanostructured active for affinity nanostructures.
  8. 8.根据权利要求1所述的活性载体,其特征在于:所述活性载体包括用于纳米结构标记物的活性纳米结构。 The active carrier according to claim 1, wherein: said carrier comprises an active active nanostructures for nanostructure markers.
  9. 9.权利要求1-8之一所述用于生物芯片的活性载体的制备方法,至少包括下列步骤:1).将所述偶联基团以化学键结合在所述固相载体表面上;2).将功能物质与1)的制备物中的所述偶联基团接触并进行反应,其中所述功能物质含所述功能基团;3).将所述2)中所述功能基团固定在所述偶联基团上。 9. The one of claims 1-8 for preparing active support for a biochip, comprising at least the following steps: 1) the coupling group is chemically bonded to the surface of the solid support; 2. .) the coupling group and a functional substance preparation 1) was contacted and reacted, wherein the functional substance having the functional group;. 3) the 2) in the functional group fixed to the coupling group.
  10. 10.根据权利要求9所述的方法,其中:A).所述偶联基团含-NH2或-COOH基团;B).所述功能物质含功能基团、结合在功能基团内的保护基团和可同所述偶联基团反应的-NH2或-COOH基团;C).所述功能基团固定在所述偶联基团上是通过形成酰胺键进行的;D).在所述功能基团固定在所述偶联基团上后,所述方法还包括在固定探针前选择性地脱去所述保护基团。 10. The method of claim 9, wherein: A) said coupling group containing -NH2 or -COOH group; B) of the functional substance-containing functional group, bonded to the functional group. protecting groups, and -NH2 or -COOH groups that can react with the coupling group; C) the functional group fixed to the coupling group is carried out by forming an amide bond;. D). after the functional group fixed to the coupling groups, the method further comprising selectively removing the probe before fixing the protective group.
  11. 11.一种多反应器生物芯片基片,其特征在于:其至少含权利要求6所述生物芯片片基和固定在所述片基上的反应器隔离结构。 11. A multi-reactor biochip substrate, characterized in that: it contains at least claim 6 biochip sheet is fixed on the base sheet and the base of the reactor isolation structure.
  12. 12.一种生物芯片,其特征在于:其至少含权利要求6所述生物芯片片基和固定在所述片基上的二种或二种以上的探针、或至少含权利要求11所述生物芯片基片和固定在所述基片的片基上的二种或二种以上的探针。 12. A biochip, characterized in that: it contains at least as claimed in claim 6 said group and the fixed biochip sheet of two or more kinds of the probes on the substrate sheet, or to claim 11 containing at least or two or more two kinds of probes on a biochip substrate sheet substrate and secured to said substrate.
  13. 13.一种生物芯片试剂盒,其特征在于:其至少含权利要求1-8之一所述用于生物芯片的活性载体。 A biochip kit, characterized in that: it contains at least one of the claims 1-8 for bio-chip active carrier.
  14. 14.一种生物芯片检测方法,至少包括下列步骤:A).提供可能含检测目标物的样本,所述目标物包括多肽目标物;B).提供权利要求13所述生物芯片试剂盒;C).使所述样本与所述试剂盒中的生物芯片上的探针接触并进行相关反应。 14. A method for detecting a biochip, comprising at least the following steps: A) providing a test sample which may contain the target object, said object comprising a target polypeptide; B) 13 to provide a biochip kit as claimed in claim; C.. ). contacting the probe on the sample with the kit of the biochip and related reactions.
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