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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Publication number
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
active
functional
present invention
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CN 200610020226
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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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Priority to CN 200610020226 priority Critical patent/CN1811427A/en
Publication of CN1811427A publication Critical patent/CN1811427A/en
Priority claimed from PCT/CN2006/002659 external-priority patent/WO2007085156A1/en

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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 Active carrier for 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)。 Application The present invention relates to an active support for a biochip, preparing active carrier of bio-chip, and an active carrier biochip (biochip substrate, a 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”)。 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 the nanostructure (for active carrier nanostructure marker).

芯片的核心是其上的反应器。 The core chip is the reactor on its. 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 chip reactor three portions of a sheet group (for solid support chips), fixed on the sheet substrate probe, and 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 a reactor fixed object by interacting with the sample target material. 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 chip probe. 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 a chip (e.g., a polypeptide chip, polypeptide / nucleic acid chips, polypeptide / sugar chips, etc.) polypeptide probe, due to the inclusion among polypeptide interaction detection reaction, so that stability, space of the reaction efficiency, etc. 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。 Film base is mainly rectangular glass, metal, plastic and other materials and derivatives produced, circular or other shape vector containing reactive group derived therefrom. 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。 Reactor configuration comprising a reactor isolation structure, reactor flow-path structure, reactor separation structure, the reactor chamber 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)。 The probe may be directly bonded to the film base, affinity and nanostructures 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 group troches group to a solid support and fixed on the sheet base solid support derivatives. 片基中形成衍生物有两种方式:包被和表面修饰。 Sheet group forming derivatives 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 reaction quality. 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, the effective storage chip biological activity, kinetic conditions the solid phase probe (e.g., active site density, the spatial freedom of movement of the probe, etc.) and the like are connected thereto related. 这一连接非常丰富也非常复杂。 This connection is very rich but also 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。 On film base solid support for the polymer coating in order to introduce working functional groups, comprising: Application No. U.S. Patent 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 efforts pairing mechanism of ion based, there are serious problems in terms of uniformity and stability of sheet-yl activity.

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 sheet substrate solid support surface chemical modifications include: 1) the introduction of the coupling group is directly immobilized probes (e.g., Chinese Patent Application No. CN200310107946.X in operation); 2) first introduced into the coupling. group and then introduce functional groups to the 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 small molecule functional group is introduced 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); macromer functional groups incorporated include polymers containing an amino group (for example: 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 the film base prepared from a small molecule functional groups, the efficiency of fixed probes and immobilized capture object in the probe thereon efficiency are to be improved; introduced sheet substrate 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。 For an active nanostructures affinity and nanostructures, or an active nano nanostructure marker, but also with the sheet having 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 a solid support of the 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 on the functional group into medium sized on a solid support surface of the coupling group to make it functional.

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 a reactive carrier biochip, characterized in that: it contains a derivative on a solid support and the solid support surface chemically bonded, said derivative containing at least conjugate functional groups on the group and the coupling group covalently bonded, and the molecular weight of the functional groups is greater than 50 less than 1000. 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。 Advantages of the present invention are active carrier biochip having 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, according to the present invention is a method for preparing an active carrier biochip, comprising at least: 1) the coupling group is chemically bound to said solid support surfaces ;. 2) to the coupling group prepared functional substance 1) was contacted and reacted, the functional material containing the functional group; 3) the functional group fixed to the. 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, according to the present invention is applied for active carrier biochip, comprising on an active carrier of the present invention, a biochip substrate, a 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 a reactor configuration biochip sheet substrate and the fixing of the present invention on the film base. 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 biochip sheet substrate and the fixed containing the present invention two or more kinds of probes on said film base, or at least biochip group containing the present invention two or more kinds or two kinds of the probe sheet and fixed on the sheet substrate the substrate is. 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 it lozenges group or biochip substrate of the present invention, and two or more kinds or two kinds of probes are immobilized on said film base. 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 substance comprises a polypeptide target; B) providing a biological chip of the present invention; 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 ways

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, the reactive carrier biochip relates to a method for, characterized in that: it contains a solid support and the solid support surface chemically bonded derivative of a coupling group containing at least and functional groups on the coupling group covalently bonded, and the molecular weight of the functional groups is greater than 50 less 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 effectively immobilized reactant (e.g. a probe labeled with a ligand, and the like) of the solid support, e.g. biochip sheet group by functional groups thereon, for active nanostructures affinity and nanostructures for active nano nanostructure markers, 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 polypeptide as reactants example, other biological materials (e.g. nucleic acids, cells, saccharides, etc.), may also be applied biochip composition active carrier prepared according to the present invention of 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。 Of particular note is suitable polypeptide immobilized active carrier generally suitable for nucleic acids immobilized, and adapted to the nucleic acid immobilized active carrier is not necessarily suitable for the polypeptide is immobilized. 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.) to a group fixed reactant; the term " coupling group "refers to a derivative of the solid phase support surface is chemically bonded to the to couple functional groups and a solid phase carrier 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 groups is greater than 75 less 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 comprises amino acid side chain comprising two or more -NH2's. 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 acids include side chains containing three or more -NH2's.

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, the amino acid include 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, the amino acids include 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, the amino acids include 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 reactive carrier comprises a bio-chip film base. 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 "sheet group" refers to a member of the biochip, i.e., an active carrier biochip having effective immobilized probes function. 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 film base of the present invention, at least lozenges group a solid support, fixed on the sheet base solid support 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。 Film base solid support include conventional tablets based support and nanostructured film base support. 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 sheet-based support for the surface is not fixed to the film base solid 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 the test embodiment of the present invention given is a glass example of a solid support as a sheet substrate, other solid support (e.g., silicon, gold, a plastic sheet or a metal sheet), it can also be prepared according to the present invention, 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 film base solid 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 for affinity nanostructures. 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, containing at least the labeling substance, a nanostructure (e.g., nanoparticles, nanotubes, etc.), the fixed coupling groups on the nano structure, fixed to the coupling group function 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 invention, according to the present invention is a method for preparing an active carrier biochip, 1) the coupling group is chemically bound to said solid support surfaces; 2). the coupling group prepared functional substance 1) was contacted and reacted, the functional material containing the functional group; 3) the functional group fixed to 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) the coupling group containing -NH2 or -COOH group; B) of the functional substance-containing functional groups, bound protected within the functional group. group 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) on. the functional group fixed to the rear of the upper coupling group, said method further comprising, prior to fixing the probe selectively removing 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 comprises, after fixing the probe selectively removing the protective group. 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, according to the present invention is applied for active carrier 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: the reactor isolation structure at least containing the present invention biochip plate group and fixed to the sheet base.

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 pointed used to prepare the chip, not yet immobilized probes of intermediate products, which slice group basis, in conjunction with the presence or absence of other structures (e.g., isolation structure), and forming the chip after fixation ligand . 基片上可以有一个或多个片基室。 There may be one or more sheets base chamber on the substrate. 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, then the substrate is both a substrate group (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, then the substrate comprises a sheet substrate 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 on the fixing are formed after the reactor ligand, multi-chip substrate chamber substrate is formed of a multi-reactor 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 structures than the probe, such as isolation structure, the flow path structure and the like; the term "isolated from the reactor structure" refers to avoid at least when the sample is added structure of cross-contamination occurs between a reactor comprising structure for isolating a part or all of the reactor structures (e.g. reaction vessel, the reaction chamber, the flow passage, inlet structure, the fluid 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 an open isolation structure and hermetic 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。 Closed isolation structure refers to the reactor isolation structure covering over the isolation structure the chip used.

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 bio-chip substrate according to the present invention, the isolation structure comprises a highly hydrophobic material coating. 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 bio-chip substrate according to the present invention, the surface of the static water of the highly hydrophobic material of the contact angle is larger than the surface of the solid support by static water contact angle of 40 degrees or more. 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 the contact angle of stationary 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 long, with a drop of liquid on the contact angle θ of solid material surface as a specific wet quantization 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 bead on the material surface there is a 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。 Chip is the most commonly used solid support substrate material is glass, its 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 material coating.

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 biochip sheet group containing 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。 Two or more kinds or two kinds of probe one embodiment of a biochip according to the present invention, which contains at least the present invention biochip sheet group, and fixed on the sheet base. 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 a biochip according to the present invention, which is a multi-reactor biochip, at least a biochip substrate comprising the present invention and fixed on the sheet substrate at least two kinds or two kinds of 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, ligands by exponential enhancement system evolution techniques screened 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 of 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" corresponds to the English "Polypeptide", including natural or synthetic proteins, protein fragments, synthetic peptides, etc., immunoassays generally the object and the detection common to probes, such as antigens, antibodies, etc., are considered a biochip kit polypeptide according to the present invention is characterized in that: it contains the present invention is used in at least an active carrier 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 substance comprises a polypeptide target; B) providing a biological chip of the present invention; 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。 The biochip of the present invention is used in the following one or more activities: as a diagnostic tool, as a diagnostic tool, as a molecular scan tool, as an inter-molecular relationship 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 solid phase carrier used as slides, including a conventional slide and nanostructures slides (see our 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, selected amino acid functional material used, 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。 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 from silicone used is preferably a silane compound (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。 A silane-based compound can form a coupling group containing an amino group or a hydroxyl group on 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 film base of the present invention, comprising at least: 1) bonded to form said coupling groups on the solid support surface of the coupling group-containing. -NH2 or -COOH group; 2) the functional substance 1) preparation of the support surface contacts containing coupling group of a solid phase and reacted, wherein: a) the functional material containing the functional group and. 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 group fixed to the solid support surface.

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。 Sheet substrate prepared in the present embodiment, which after fixing the probe (refer to the following), for 12 months at room temperature no probe decreased activity, whereas 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 coupling group biochip sheet group containing amino group present embodiment, a method of preparing a biochip film base 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) The fixed amino group after drying fixed slides silanes groups immersed in aqueous solution of the amino acid is preferred concentration of the reaction at room temperature for 1 hour and then the unfixed material washed slide surface, drying after the backup.

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 coupling group biochip sheet amino-containing embodiment of the present embodiment, a method of preparing a biochip film base 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) The fixed amino group after drying fixed slides silanes groups immersed DFM solution containing Fmoc protecting agent amino 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。 Then press a known method Fmoc-deprotection agent, then wash the slide surface of the non-immobilized 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 coupling group biochip sheet amino-containing embodiment of the present embodiment, a method of preparing a biochip film base of the present invention are: 1) Preparation of carbamic glass according to known methods. DFM solution containing Fmoc protecting agent amino acid of the sheet 2) fixed amino group of the amino slides after drying is immersed in a 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。 Then press a known method Fmoc-deprotection agent, then wash the slide surface of the non-immobilized 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。 Preparation Examples Example 1.4 coupling group biochip sheet groups containing a carboxyl group embodiment according to the present embodiment, a method of preparing a biochip film base of the present invention are: 1) washing the slides and fixed silane groups by current. some known methods.

2).固定含羧基的基团按现有的已知方法进行。 2) Fixed group containing a carboxyl group is performed by conventional known methods.

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) The fixed amino group fixed after drying has a carboxyl group slides dipped DFM solution of amino-containing Fmoc protecting agent is 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。 Then press a known method Fmoc-deprotection agent, then wash the slide surface of the non-immobilized 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 invention. In this embodiment, the present invention is an active support of the biochip is an active nanostructures affinity and nanostructures, 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 solid support used, 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, selected amino acid functional material used, 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。 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 from silicone used is preferably a silane compound (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。 A silane-based compound can form a coupling group containing an amino group or a hydroxyl group on 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 film base of the present invention, comprising at least: 1) bonded to form said coupling groups on the solid support surface of the coupling group-containing. -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。 Bonding the same manner as the conventional amination nano method for producing 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 solid support surface 1) 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 solid support surface.

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 according to the present embodiment, which after fixing a probe or marker ligand, 1 month at room temperature, probe or marker decreased was 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。 Example 3 of the invention is a multi-reactor biochip substrate preparation example of this embodiment, the use of bio-chip film base as in Example 1 above prepared film base.

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, the static water contact of 116 degrees)," ultra-hydrophobic latex paint "(China Chengdu Chenguang Chemical Institute provided, the static water contact of 123 degrees) 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 isolation structure of the reactor according to the present invention may be in part, may all be highly hydrophobic isolation structure of the present invention, the present embodiment only shows the most simple case as an example.

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 in the present embodiment as follows: The above-described highly hydrophobic liquid material applied on the sheet substrate above Preparation Example 1 Isolation tape position by using the supplier's instructions solidified after drying at room temperature, to form highly 25-115μm, width of the high hydrophobic convex body 2.0-2.5mm of. 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, this embodiment takes only the tape-shaped (convex body is a strip) and line combination (e.g. the convex body is spaced two lines) 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。 Surface highly hydrophobic convex body surrounded can take a variety of geometric patterns, this embodiment takes only a 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 the substrate this surface, transversely total of 12 pieces yl pool, longitudinal with four pieces yl pool, a total of 48 pieces yl pool.

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)。 Example 3.2 Hydrophobic / prepared oleophobic isolation structure of the substrate in the present embodiment, the hydrophobic - oleophobic materials are commercially available hydrophobic - oleophobic material, respectively City Jie (Shenzhen City Jie environmental Protection Technology Co., Ltd.), a highly hydrophobic silica coating (China Zhoushan tomorrow nano-materials company) and double sparse coating (Chinese blue light 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 isolation structure of the reactor according to the present invention may be in part, may all be highly hydrophobic isolation structure of the present invention, the present embodiment only shows the most simple case as an example.

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 over the substrate above Preparation Example 1 Isolation tape position by using the supplier's instructions 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 protuberance surrounding surfaces may take a variety of geometric patterns, this embodiment takes only a 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 the substrate this surface, transversely total of 12 pieces yl pool, longitudinal with four pieces yl pool, a total of 48 pieces yl pool.

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 biochip Example 4 of the present invention, the embodiment of the biochip above embodiments containing 3-prepared substrate, or activity of nanostructured affinity nanostructures / and the above-described Example 2 prepared in the present embodiment. 实施例3制备的基片含上述实施例1制备的片基。 Substrate prepared in Example 3 containing 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 (China Beijing Institute of People's Hospital of liver disease) HIV1 + 2 antigen (China Beijing Institute of People's Hospital of liver disease) 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, preparation of a biochip of the present invention are: a film base pool in said substrate, the liquid respectively containing the probe according to the general spotting method spotted sheet yl pool, each liquid in each dot 3 having a diameter of 80μm point between the dot pitch 600-700, forming 2 × 3 array. 反应池片基上的配基密度大于30点/cm2。 Ligand density on the reaction cell sheet substrate 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 containing probe used in the liquid are: 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 and HIV1 + 2 antigen / activity nanostructure composite is affinity nanostructure containing active nanostructures and 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). containing kits biochips and nanostructured marker prepared in Example 4. 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 marker containing rhodamine, active nanostructures and fixed on the active nanostructures goat anti-human secondary antibody, which is prepared with reference to another of our patent application 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 was diluted under reaction conditions at 20 times serum by the ELISA method using the classic.

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 is prepared according to Example 5 with. 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 were at the amino glass slide and epoxy slides (Microarray Technology) according to conventional spotting methods secured prepared yl same ligand chip, labeled anti-human rhodamine labeled goat 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, the three samples were added to the reaction cell 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。 Sample volume is 15 l, washed for 30 minutes the reaction 5 times washing solution each 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。 Marker is added in an amount of 15 l, washed 5 times After the reaction, washing solution each was added in an amount of 25 l, dried, and then scanned 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, emission wavelengths 570nm, the read signal processing software (JAGUARII) treatment, and then after averaging The Cut-off value determining a female (-) positive (+) result of obtained. 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。 Consistent with the results obtained results of the ELISA method was diluted under the reaction conditions previously detected in the 20 times serum and detection limit less. 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 control kits of the present invention, comparison, at 37 ℃ stability higher than 20%, and the detection limit is 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 the following one or more activities: as a diagnostic tool, as a molecular scan tool, as an inter-molecular relationship 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 herein described 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 invention and without decreasing the advantages thereof, may be made such modifications and variations. 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 in.

Claims (14)

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 carrier biochip, characterized in that: it contains a derivative on a solid support and the solid support surface chemically bonded, said derivative containing at least coupling groups and coupling groups on to covalent functional group bond, and the molecular weight of the functional groups is greater than 50 less than 1000.
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: the molecular weight of the functional groups is greater than 75 less than 500.
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。 According to claim 1 of the active carrier, wherein: the functional groups include amino groups.
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。 4. The active carrier according to claim 1, wherein: said coupling group comprises an organic silicon group.
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.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, wherein: said active support comprising a biochip sheet group.
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 active support comprising active nanostructures for affinity nanostructures.
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。 8. The active carrier according to claim 1, wherein: said active support comprising active nanostructures for nanostructure markers.
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 process according to claim one of 1-8 preparing active carrier of the bio-chip, comprising at least the following steps: 1) the coupling group is chemically bound to said solid support surfaces; 2. .) the coupling group prepared functional substance 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.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 group, said method further comprising, prior to fixing the probe selectively removing the protective group.
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 plate group and fixed to the sheet base isolated from the reactor structure.
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 as claimed in at least claim 6 biochip sheet substrate and fixing two or more kinds or two kinds of probes on said film base, or containing at least as claimed in claim 11, wherein or two or more probes two kinds of the sheet substrate biochip substrate and secured to said substrate.
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 claims 1-8 for the active support of the biochip.
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 biochip comprising at least the following steps: A) providing may contain samples tested object, said object comprising a polypeptide target; B) 13 biochip kit as claimed in claim; C.. ). the probe in contact on the sample with the kit of the biochip and related reactions.
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