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Preparation method of microcantilever modified by antibody fragments, and microcantilever immune sensing detection system based on antibody fragment modification

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CN102951599A
CN102951599A CN 201110238911 CN201110238911A CN102951599A CN 102951599 A CN102951599 A CN 102951599A CN 201110238911 CN201110238911 CN 201110238911 CN 201110238911 A CN201110238911 A CN 201110238911A CN 102951599 A CN102951599 A CN 102951599A
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microcantilever
antibody
method
fragments
detection
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CN 201110238911
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Chinese (zh)
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CN102951599B (en )
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张青川
吴尚犬
伍小平
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中国科学技术大学
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Abstract

The invention provides a microcantilever modified by antibody fragments. Antibody Fab fragments are fixed on a gold-plated surface of the microcantilever. Through the reduction of mass and volume of parts other than antigen molecule binding sites on the antibody, effective modification density of the antigen-binding sites on the surface of the microcantilever is improved, and the efficiency of the transferring of intermolecular reaction force onto the microcantilever is improved, such that microcantilever immunoassay method sensitivity is improved. The invention also discloses a preparation method of the microcantilever, and the immune sensing detection system and a detection method based on the microcantilever modified by antibody fragments.

Description

抗体片段修饰的微梁制备方法和基于抗体片段修饰的微梁免疫传感检测系统 The method of preparing the modified micro beam antibody fragments and antibody fragments based on the modified micro beam sensing the immune system

技术领域 FIELD

[0001] 本发明涉及抗体片段,具体地是Fab片段修饰的微悬臂梁(以下也简称为微梁),及其制备方法。 [0001] The present invention relates to antibody fragments, specifically modified Fab fragment microcantilever (hereinafter, simply referred to as micro beam), and its preparation method. 本发明还公开了基于所述抗体片段修饰的微梁的免疫传感检测系统和检测方法,所述系统和方法可应用于食品安全、环境污染、生物医学、科学研究和生产制造等领域的监控和检测。 The present invention also discloses an immunoassay method for detecting and sensing systems based on the micro beam modified antibody fragments, the system and method can be used in food safety, environmental pollution, biomedical research and other fields of production monitoring and detection.

背景技术 Background technique

[0002] 免疫传感技术的原理是基于检测抗原抗体的特异性配对反应,即针对需要检测的某种靶标分子(抗原),通过生物免疫(把抗原注入小动物体内产生的)方法,产生出相应的探针分子(抗体),提取、纯化出这种探针分子,再利用抗原抗体的特异性结合去检测样品中的靶分子。 [0002] immunological principles sensing technology is based on detecting the antigen-antibody reaction specific pairing, i.e., for certain target molecules (antigens), by immune (injecting the antigen produced in vivo small animal) to be detected methods, produce corresponding probe molecule (antibody), extracted, purified such a probe molecule, then an antigen-antibody binding specifically to the target molecule in the test sample. 已有的免疫传感技术如:酶联免疫吸附试验(ELISA,原理是利用抗原与抗体的特异性反应与酶标的催化放大来进行)和免疫荧光法(IF,原理是用荧光片段标记抗原或抗体),均需要标记物来示踪抗原抗体的反应结果,就是说仅有抗体,并不能建立相应的检测方法,还需要有酶标物或荧光标记物或蛋白复合物。 Conventional immunological techniques such as sensing: enzyme-linked immunosorbent assay (ELISA, using the principle of antigen and an antibody specifically reactive with the enzyme catalyzed amplification is performed) and immunofluorescence (IF, principle is a fluorescent labeled antigen or a fragment antibody), are required to label antigen-tracer antibody reaction results, that the antibody only, and can not establish the corresponding detection method further requires fluorescent label or enzyme or protein complex. 而一些难制备的靶标分子的标记物价格昂贵,或几乎不可能制备或购买。 And the prices of some marker target molecule expensive difficult to prepare, or nearly impossible to prepare or buy. 同时酶联免疫试剂盒和蛋白芯片的检测从原理操作上都要求每一步反应完后进行清洗,标记过程繁琐耗时,是事后的检测,不能实时,原位、在线的检测。 Simultaneous detection ELISA kits and protein chip from the principle operations require cleaning after every step of the reaction, the labeling process cumbersome time-consuming, is to detect afterthought, not real-time, in-situ, on-line detection.

[0003] 基于表面应力检测的微悬臂梁免疫传感技术是近年出现的一种新的免疫生化传感方法[1],其原理是:把探针(抗原或抗体)分子用直接或间接的方式固定(修饰)到微梁一侧的镀金层上,当被检测样品液中的靶分子与微梁金表面上的探针分子发生免疫生化反应时,会使微梁表面应力改变,从而导致微梁弯曲变形,通过光学或电学方法检测这种变形的过程,可得到免疫生化反应的实时信息。 [0003] Based on microcantilever immunosensor detection of surface stress is a new method of immunological and biochemical sensing emerged in recent years [1], which is the principle: the probe (antibody or antigen) directly or indirectly with a molecule fixing means (modified) to a gold plating layer on the side of the micro beam, when the probe molecule is detected in the sample liquid and the gold surface of the micro beam target molecule immunoreactive biochemical reactions, the stress will change the micro beam surface, resulting in micro beam bending deformation which is detected by the process of optical or electrical methods, real-time information obtained immune biochemical reactions. 基于免疫特异性识别建立的微梁免疫传感技术与传统的需要标记物的免疫传感方法相比,它无需使用任何酶标、荧光物质和放射性作为反应示踪剂,消除了标记过程的影响,灵敏度高(比酶联免疫试验高数倍[2_4]),还可以通过监测微梁变形来实时、定量的监测抗原抗体的反应过程,得到更丰富的免疫生化反应的信息。 Beam microprocessor-based immunosensor immunospecifically recognize established as compared with the conventional immunological method requires sensing marker, it does not require the use of any enzyme, fluorescent substance and a radioactive tracer as a reaction, to eliminate the influence marking process , high sensitivity (several times higher than the enzyme-linked immunosorbent test [2_4]), may also be modified in real time by monitoring the micro beam, quantitative monitoring of antigen-antibody reaction to obtain more informative immune biochemical reactions. 经过这些年的发展,微梁传感被作为一种新兴技术,在生物工程和环境污染监测技术等方面与传统的方法进行对比研究,如RNA转录因子、酶、汞排放及挥发性化合物等,优于常规的酶联免疫方法。 , Micro Liang Chuangan is used as an emerging technology, after years of development and bio-engineering technology and environmental pollution monitoring comparative study of conventional methods, such as RNA transcription factors, enzymes, and mercury emissions of volatile compounds, superior to conventional enzyme immunoassay method. 但即使如此,微悬臂梁免疫传感技术的检测灵敏度还不足以在成本方面获得相对于常规酶联免疫法的巨大优势,即如果微悬臂梁免疫传感技术的灵敏度或检测极限只比酶联免疫法高数倍,而微悬臂梁免疫传感技术的设备成本与酶联免疫相对较高,使得微悬臂梁免疫传感技术难以商业化。 Even so, the detection sensitivity of microcantilever immunosensor is not enough to have a big advantage compared to conventional ELISA method in terms of cost, i.e., if the microcantilever immunosensor or sensitivity than the detection limit of enzyme-linked only immunization several times, the microcantilever immunosensor enzyme immunoassay equipment cost is relatively high, so that the microcantilever immunosensor difficult to commercialize.

[0004]目前,国内外文献报道的方法主要是利用具有双功能基团的疏基化试剂的疏基(-SH)抓住微梁表面的金表面,和另一功能团抓住抗体,实现抗体在微梁镀金表面上的固定。 [0004] Currently, the method reported in the literature is the use of mercapto (-SH) group having a hydrophobic group bifunctional reagent of the surface of the micro beam to seize the gold surface, and the other functional groups seize antibody achieve antibody is immobilized on gold-plated surface of the micro beam. 例如先将巯基化试剂11-羧酸硫醇结合到微梁的金表面,活化其上的羧基,使之与抗体上的氨基结合来固定抗体(图2)。 First, for example, mercapto carboxylic acids 11- thiol-binding agent to the gold surface of the micro beam, activated carboxyl groups thereon, so that the immobilized antibody (FIG. 2) in combination with an amino group on the antibody. 或用一种巯基化试剂盐酸硫醇亚胺,通过与抗体反应,使抗体连接一个带巯基的分子基团,再通过这个巯基将抗体联结到微梁的金表面上(中国专利CN101407548)(见图3)。 Or with one sulfhydryl reagents hydrochloride thiol imine by reaction with an antibody, the antibody linker molecule group with a mercapto group, and then through the mercapto group to join the antibody onto the gold surface of the micro beam (Chinese Patent No. CN101407548) (see image 3). 这些方法固定抗体,存在如下共同的问题,(I)Y字形抗体(见图 These methods immobilized antibody, there is a common problem as follows, (I) Y-shaped antibody (see FIG.

I)的Fe段或Fab段的顶端部,会等概率的被固定在金表面上,没有方向性[5]。 I), Fe probability Fab fragment or segment of the top portion, etc. can be immobilized on the gold surface, non-directional [5]. 而当Fab段的顶端被固定在金表面上时,结合位点被遮挡,限制了抗体的抗原结合位点与抗原的充分结合,从而降低了微梁传感灵敏度;(2)抗体与微梁之间存在不同数目C原子的单链分子,降低了抗原抗体反应产生的应力传递到梁上的效率。 When the top section is Fab immobilized on a gold surface, binding sites are blocked, limit the full antigen binding site of the antigen, thus reducing the sensitivity of the micro Liang Chuangan; (2) the antibody to the micro-beam exists between single-stranded molecules with different numbers of C atoms, reducing the efficiency of the stress generated by antigen-antibody reaction to the transmitted beam. 由此可见,中国专利CN101407548中公开的方法的灵敏度与酶联免疫法相比仅提高数倍,还无法达到商业化或用于极微量被分析物的检测。 Thus, the method disclosed in Chinese Patent No. CN101407548 ELISA sensitivity compared to only increase several times, can not reach a very small amount for the commercial or the detection of the analyte.

[0005] 因此,在本领域中存在着改进微梁表面与抗体的结合和设计从而进一步提高微梁免疫传感灵敏度和效率的需求。 [0005] Accordingly, there is a need for improved design and the binding surface of the micro beam and further enhance the immune antibodies sensing sensitivity and efficiency of micro beams in the art.

发明内容 SUMMARY

[0006] 综上所述,在已有的微梁免疫传感技术报道中,最突出的问题就是检测灵敏度。 [0006] As described above, in the conventional micro beam immunosensor reported, the most prominent problem is the detection sensitivity. 影响微梁免疫传感器灵敏度的因素主要有三个方面:①抗体的灵敏度(或抗体与抗原结合的亲和性)、②微梁上抗体的固定方法和③微梁的设计与信号读出。 Effect of micro beam sensor sensitivity factors immunized three main aspects: ① Sensitivity antibody (or antigen-binding affinity of the antibody), antibody fixing method ② and ③ micro beam micro-beam design and signal readout. 由于抗体的灵敏度和微梁的规格与信号读出方式在微梁免疫传感器系统成型之后就无法改变,唯一可变的是微梁表面抗体的固定方法。 As the sensitivity of the antibody and the specifications of the micro beam and the signal readout mode can not be changed after the immunosensor micro beam forming system, the only variable is the method of fixing the surface of the micro beam antibody. 一种合适的微梁表面抗体的修饰方法,能使固定在微梁表面的抗体与样品溶液中的抗原充分结合,并能高效地将抗原抗体结合所产生的应力变化传递到微梁表面。 A suitable method of modifying the surface of the micro beam antibody, enabling the antibody to the sample solution is fixed to the micro-beam surface antigen sufficiently binding, and can change in conjunction with stress is transferred to the surface of the micro beam efficiently antigen-antibody. 抗体在微梁表面固定的方向性、密度、活性以及抗体与微梁表面之间的联接分子的长度与刚性都有可能影响最终检测的灵敏度。 Antibody molecules of the length of the rigid coupling between the fixed directional beam micro-surface density, the active surface of the micro beam and the antibody may affect the ultimate sensitivity of detection.

[0007] 鉴于上述现有技术中的问题,本发明用于解决上述问题的技术方案是通过减少抗体上抗原分子结合位点以外其它部分的质量和体积,来提高抗原结合位点在微梁表面的有效修饰密度,以及分子间反应作用力传递到微梁上的效率,达到提高微梁免疫检测方法的灵敏度目的。 [0007] In view of the problems of the prior art, the present invention is to solve the above problems by reducing the quality aspect and the volume of the other parts of the molecule on the antigen-binding site of an antibody other than the antigen binding site to enhance the micro surface of the beam between the effective modification of the density, and the reaction force is transmitted to the efficiency molecules micro beam, the sensitivity to achieve the purpose of immunodetection methods to improve micro beam.

[0008] 抗体Ig分子的结构示意图如图1,包括两个相同的抗原结合片段(Fab)和一个可结晶片段(Fe), Fab和Fe通过中间的铰链区连接,构成Y字形结构。 [0008] The schematic structure of an antibody Ig molecule 1, comprising two identical antigen binding fragment (Fab) and a crystalline fragment (Fe), and Fe by Fab & middle hinge region, constituting the Y-shaped structure. Fab的顶端为与抗原分子的结合位点。 Fab top of the antigen binding site of the molecule. 在免疫球蛋白抗体分子的“Y”字形四肽链结构(图I)中,由两条完全相同的重链和两条完全相同的轻链以二硫键连接而成。 In the "Y" shaped immunoglobulin antibody molecules tetrapeptide chain structure (FIG. I), the two identical heavy chains and two identical light chains disulfide bonded together. 对于Fe段修饰在微梁的镀金表面上时(抗体修饰的定向性好),抗体对称(Fab段)的两个上端部抗原结合位点与抗原结合后产生的应力,通过抗体Y字型的腿部(Fe段)传递到镀金微梁表面。 Fe respect to the segment on the plated surface modified micro beam (modified antibody good orientation), bound antibody produced after symmetrically (Fab segment) of the upper portion of two antigen-binding sites of the antigen stress, through the Y-shaped antibody leg portions (Fe para) to the plated surface of the micro beam. 抗体(IgG)的分子量约150kDa,如果被检测的抗原分子分子量为数百Da,抗体抗原质量比近1000。 Molecular weight antibody (IgG) about 150kDa, if the molecular weight of the antigen molecule to be detected hundreds Da, antigen-antibody ratio of nearly 1,000 mass. 参与抗原分子结合的位点由重链和轻链的高变区(VH和VL)构成,其与抗原分子的质量比约150,而抗体分子上不参加与抗原分子结合的Fe段,其与抗原分子的质量比近400。 Involved in antigenic site molecule is composed of hypervariable regions (VH and VL) of the heavy and light chains, the quality and antigen molecule ratio of about 150, but do not participate Fe segment bound to the antigen molecules on the antibody molecule with the antigen molecular mass ratio of nearly 400. 因此我们设想,如果能够减少结合位点以外的抗体分子质量和体积,保持抗体的结合位点朝向固定表面的外法线方向,就能够提高应力的传递效率和抗体在微梁表面的有效修饰密度,从而提高微梁免疫传感的灵敏度。 Therefore, we assume that if an antibody molecule capable of reducing weight and volume than the binding site to maintain normal direction toward the binding site of an antibody of the fixing surface, it is possible to increase the effective density of the modified antibody and stress transfer efficiency of the surface of the micro beam , thereby enhancing the immune micro beam sensing sensitivity.

[0009] 为此,在第一个方面,本发明提供了一种制备微悬臂梁的方法,包括以下步骤: [0009] For this purpose, in a first aspect, the present invention provides a method of making the micro-cantilever, comprising the steps of:

[0010] I)提供抗体和带有镀金层的微悬臂梁; [0010] I) providing an antibody with the micro-cantilever and a gold plating layer;

[0011] 2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,优选木瓜蛋白酶; [0011] 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, an antibody capable of cleaved into Fab fragments of any of the enzymes protease, preferably papain;

[0012] 3)固定步骤:将包含巯基官能团和羧基或氨基官能团的巯基化试剂结合至微悬臂梁的镀金层表面上; [0012] 3) fixing steps of: containing a thiol functional group and a carboxyl group or mercapto group of amino-functional reagents bound to a gold plating layer on the surface of the microcantilever;

[0013] 4)联结步骤:将步骤2)获得的Fab片段与步骤3)获得的已经结合在微悬臂梁上的巯基化试剂联结在一起,得到镀金层表面上固定有Fab片段的微悬臂梁。 [0013] 4) coupling steps of: Step 2 and Fab fragments) obtained in 3) has been obtained on the micro-cantilever bound sulfhydryl reagents coupled together to yield Fab fragments are fixed on the surface of the gold plating layer microcantilever .

[0014] 在第二个方面,本发明还提供了一种制备微悬臂梁的方法,包括以下步骤: [0014] In a second aspect, the present invention also provides a method of making the micro-cantilever, comprising the steps of:

[0015] I)提供抗体和带有镀金层的微悬臂梁; [0015] I) providing an antibody with the micro-cantilever and a gold plating layer;

[0016] 2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,优选木瓜蛋白酶; [0016] 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, an antibody capable of cleaved into Fab fragments of any of the enzymes protease, preferably papain;

[0017] 3)联结步骤:将步骤2)获得的Fab片段与包含巯基官能团和羧基或氨基官能团的疏基化试剂联结在一起; [0017] 3) coupling steps of: Step 2 Fab fragments) obtained in the thiol-containing functional group and a carboxyl group or mercapto group of a coupling agent with amino functional groups;

[0018] 4)固定步骤:将步骤3)获得的一端与Fab片段联结的所述巯基化试剂的另一端结合至微悬臂梁的镀金层表面上,得到镀金层表面上固定有Fab片段的微悬臂梁。 [0018] 4) fixing steps of: Step 3) obtained in one end of the other end of the Fab fragment of a mercapto group of the coupling agent bonded to the surface of the gold plating layer on the micro-cantilever, Fab fragments have been fixed on the surface of the gold plating layer micro cantilever.

[0019] 在第三个方面,本发明提供了一种应力传感元件,其包括基于应力的微悬臂梁,所述微悬臂梁的镀金层上固定有抗体的Fab片段。 [0019] In a third aspect, the present invention provides a stress sensing element, which comprises a micro-cantilever-based stress, Fab fragments of antibodies are fixed on the gold plating layer of the micro-cantilever. 所述微悬臂梁优选通过本发明的方法制备ίίϋ 。 The micro-cantilever ίίϋ preferably prepared by the process of the present invention.

[0020] 在第四个方面,本发明提供了一种基于应力的微悬臂梁免疫传感检测系统,其包括本发明的应力传感元件。 [0020] In a fourth aspect, the present invention provides a stress based on microcantilever sensing the immune system, which comprises a strain sensing element of the present invention.

[0021] 在第五个方面,本发明提供了一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: [0021] In a fifth aspect, the present invention provides a method of using the immune system of the sensing microcantilever sensing test sample, comprising the steps of:

[0022] I)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; [0022] I) providing an antibody specific for the target antigen and microcantilever sensing system, the sensing microcantilever system includes a reaction tank and a stress-based micro-cantilever with a gold plating layer;

[0023] 2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,例如木瓜蛋白酶; [0023] 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, an antibody capable of cleaved into Fab fragments of any of the protease enzyme, such as papain;

[0024] 3)固定步骤:将包含巯基官能团和羧基或氨基官能团的巯基化试剂结合至微悬臂梁的镀金层表面上; [0024] 3) fixing steps of: containing a thiol functional group and a carboxyl group or mercapto group of amino-functional reagents bound to a gold plating layer on the surface of the microcantilever;

[0025] 4)联结步骤:将步骤2)获得的Fab片段与步骤3)获得的已经结合在微悬臂梁上的所述巯基化试剂联结在一起,得到镀金层表面上固定有Fab片段的微悬臂梁。 [0025] 4) coupling steps of: Step 2 and Fab fragments) obtained in 3) has been obtained in conjunction with the micro-cantilever sulfhydryl reagents coupled together to yield Fab fragments are fixed on the surface of the gold plating layer micro cantilever.

[0026] 5)将步骤4)获得的固定有Fab片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; [0026] 5) The Step 4) are fixed to Fab fragments obtained microcantilever is fixed to a well microcantilever sensing system;

[0027] 6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 [0027] 6) The sample to be tested is added to the step 5) microcantilever sensing system obtained, the test sample is detected in presence or absence of the target antigen.

[0028] 在第六个方面,本发明还提供了一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤: [0028] In a sixth aspect, the present invention also provides a method of using the immune system of the sensing microcantilever sensing test sample, comprising the steps of:

[0029] I)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁; [0029] I) providing an antibody specific for the target antigen and microcantilever sensing system, the sensing microcantilever system includes a reaction tank and a stress-based micro-cantilever with a gold plating layer;

[0030] 2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,例如木瓜蛋白酶; [0030] 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, an antibody capable of cleaved into Fab fragments of any of the protease enzyme, such as papain;

[0031] 3)联结步骤:将步骤2)获得的Fab片段与包含巯基官能团和羧基或氨基官能团的疏基化试剂联结在一起; [0031] 3) coupling steps of: Step 2 Fab fragments) obtained in the thiol-containing functional group and a carboxyl group or mercapto group of a coupling agent with amino functional groups;

[0032] 4)固定步骤:将步骤3)获得的一端与Fab片段联结的所述巯基化试剂的另一端结合至微悬臂梁的镀金层表面上,得到镀金层表面上固定有Fab片段的微悬臂梁; [0032] 4) fixing steps of: Step 3) obtained in one end of the other end of the Fab fragment of a mercapto group of the coupling agent bonded to the surface of the gold plating layer on the micro-cantilever, Fab fragments have been fixed on the surface of the gold plating layer micro cantilever;

[0033] 5)将步骤4)获得的固定有Fab片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中; [0033] 5) The Step 4) are fixed to Fab fragments obtained microcantilever is fixed to a well microcantilever sensing system;

[0034] 6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 [0034] 6) The sample to be tested is added to the step 5) microcantilever sensing system obtained, the test sample is detected in presence or absence of the target antigen. 本发明的有益效果: Advantageous effects of the invention:

[0035] 鉴于微梁免疫传感检测技术中抗体固定的重要性及巯基化试剂参与抗体固定的复杂性,本发明提出在微梁镀金表面上修饰抗体片段Fab方法来实现微梁免疫检测。 [0035] In view of the micro beam sensing technique immunoassay antibody is immobilized in importance and involvement sulfhydryl reagents immobilized antibody complex, the present invention has achieved micro beam immunoassay method Fab fragments modified antibodies on gold-plated surface of the micro beam. 结果表明,本发明的方法与现有的微梁免疫方法相比较具有以下优点: The results show that the method of the present invention and the conventional method of immunization as compared to the micro beam has the following advantages:

[0036] l)Fab片段相对于完整抗体,其体积与质量要小,因此固定到微梁表面后,具有更高的抗原结合位点的密度。 [0036] l) Fab fragments with respect to an intact antibody, to small volume and mass, and therefore fixed to the surface after micro beam, having a higher density of the antigen binding sites. 2)减少抗体结合位点以外的质量和体积,从而减少了抗体的抗原结合位点到梁表面距离,提高应力的传递效率。 2) reduce the mass and volume than the antibody binding sites, thereby reducing the transmission efficiency antigen-binding site of an antibody to the distance from the surface of the beam, to improve the stress.

附图说明 BRIEF DESCRIPTION

[0037] 图I为抗体分子结构示意图。 [0037] Figure I is a schematic view of the structure of an antibody molecule.

[0038] 图2为羧酸硫醇类化合物和双功能交联剂法固定抗体示意图。 [0038] FIG. 2 is a carboxylic acid compound and the immobilized antibody thiol schematic cross-linker method.

[0039] 图3为盐酸硫醇亚胺法固定抗体示意图。 [0039] FIG. 3 is a schematic diagram of an immobilized antibody thiol imine hydrochloride method.

[0040] 图4为木瓜蛋白酶水解抗体形成抗体片段得到Fab示意图。 [0040] FIG. 4 is a papain hydrolysis of the antibody to form an antibody Fab fragment was FIG.

[0041] 图5图示了先将Fab与盐酸硫醇亚胺反应在Fab上连接一个巯基基团,再通过这个巯基来固定Fab到金表面的示意图。 [0041] FIG 5 illustrates a schematic view of a first thiol-imine hydrochloride Fab connection with a thiol group on a Fab, and then fixed by the thiol group of the Fab to the gold surface.

[0042] 图6图示了先将硫醇类巯基化试剂连接到梁上,再通过该硫醇类巯基化试剂连接Fab的示意图。 [0042] FIG. 6 illustrates a first thiol-thiol agents connected to the beam, Fab schematic reconnection via the thiol sulfhydryl reagents.

[0043] 图7为抗人参皂苷GS-Re巯基化抗体修饰微梁,检测不同浓度抗原时微梁尖端的时间位移曲线。 [0043] FIG. 7 is an anti-ginsenoside GS-Re time displacement curve when a micro tip of the beam thiolated antibody modified micro-beams, with different concentrations of antigen.

[0044] 图8为用硫醇修饰抗人参皂苷GS-Re抗体Fab片段修饰微梁,检测不同浓度抗原时微梁尖端的时间位移曲线。 [0044] FIG. 8 is a thiol modifying anti ginsenoside GS-Re antibody Fab fragments modified micro beam, different concentrations of antigen tip micro beam when the time displacement curves.

具体实施方式 detailed description

[0045] 定义 [0045] defined

[0046] 抗体:抗体(antibody)指机体的免疫系统在抗原刺激下,由B淋巴细胞或记忆细胞增殖分化成的浆细胞所产生的、可与相应抗原发生特异性结合的免疫球蛋白。 [0046] Antibody: an antibody (Antibody) refers to the body's immune system in an antigen stimulation, the B lymphocytes or plasma cell proliferation and differentiation into memory cells produced, specifically bound immunoglobulins with the corresponding antigen. 典型的抗体分子具有4条多肽链的对称结构,包括2条较长、相对分子量较大的相同的重链(H链);2条较短、相对分子量较小的相同的轻链(L链)。 A typical antibody molecule has a symmetrical structure of four polypeptide chains, including two long, relatively larger molecular weight identical heavy chains (H chain); two shorter, relatively small molecular weight identical light chains (L chain ). 链间由二硫键和非共价键联结形成一个由4条多肽链构成的单体分子。 Inter-chain disulfide bond and non-covalent coupling forming a monomer molecules consisting of four polypeptide chains. 轻链有K和λ两种,重链有μ、δ、Y、ε和α五种。 K and λ light chains have two kinds, there is a heavy chain μ, δ, Y, ε and α are five. 整个抗体分子可分为恒定区和可变区两部分。 Whole antibody molecules can be divided into two parts, the constant and variable regions. 在给定的物种中,不同抗体分子的恒定区都具有相同的或几乎相同的氨基酸序列。 In a given species, the constant region of different antibody molecules have the same or almost the same amino acid sequence. 可变区位于"Y"的两臂末端。 The variable region is located arms the "Y" end. 在可变区内有一小部分氨基酸残基变化特别强烈,这些氨基酸的残基组成和排列顺序更易发生变异区域称高变区。 In the variable region amino acid residue changes a fraction of particularly Qiang Lie, and the residues of these amino acids in the order of variation is more likely to occur, said hypervariable regions. 高变区位于分子表面,最多由17个氨基酸残基构成,少则只有2〜3个。 Hypervariable regions of the molecule surface, composed of up to 17 amino acid residues, at least 2 to 3 months only. 高变区氨基酸序列决定了该抗体结合抗原的特异性。 The amino acid sequence of the hypervariable region determines specificity of the antibody to bind antigen. 一个抗体分子上的两个抗原结合部位是相同的,位于两臂末端称抗原结合片段(antigen-binding fragment,Fab)。 Two antigen binding sites on an antibody molecule are the same, the end of said arms positioned antigen binding fragment (antigen-binding fragment, Fab). " Y"的柄部称结晶片段(crystalline fragment, Fe),糖结合在Fe 上。 "Y" shank, said crystalline segment (crystalline fragment, Fe), sugar binding on Fe.

[0047] 从结构上来说,在本发明中,提及抗体时均指全抗体,即包括4条链和Fe段的抗体结构(见图I)。 [0047] Structurally, in the present invention, reference to an antibody refer to the whole antibody, that is comprising four chain antibody structure (see Figure I) Fe segments. 此外,从功能上来说,本发明中的“抗体”或“抗体片段”是指对靶抗原特异的抗体或抗体片段,即与抗原具有特异结合能力的抗体或抗体片段,如未特别说明,本发明中的抗体一般指单克隆抗体,抗体片段包括半抗体片段、F(ab' )2片段、Fab'片段和Fab In addition, terms of functionality, in the present invention, "antibody" or "antibody fragment" refers to an antibody or antibody fragment specific for a target antigen, i.e. the antigen-specific antibody or antibody fragment having binding ability, unless otherwise indicated, the antibodies of the invention generally refers to a monoclonal antibody, an antibody fragment comprises a half-antibody fragments, F (ab ') 2 fragments, Fab' and Fab fragments

坐寸ο Sit inch ο

[0048] 半抗体片段:通过二硫键还原剂将全抗体拆分成各自带有巯基的对称的片段,每个半抗体片段包含一条完整的轻链和一条完整重链以及Fe片段。 [0048] Semi antibody fragments: split into whole antibody via a disulfide bond reducing agent is a fragment of a respective symmetrical having a mercapto group, each half full antibody fragment comprises a light chain and a complete heavy chain fragment and Fe.

[0049] F(ab')2 :Ig (immunoglobulin,免疫球蛋白)被胃蛋白酶水解在铰链区重链间二硫键近C处切断,形成一个双价抗原结合片段简称F(ab')2片段和一些小片段pFc'。 [0049] F (ab ') 2: Ig (immunoglobulin, immunoglobulins) by pepsin hydrolysis of disulfide linkages between the cut nearly at the C heavy chain hinge region to form a bivalent antigen binding fragment referred to F (ab') 2 small fragments and fragments pFc '. 由于F(ab')2片段保留了结合相应抗原的生物学活性,又避免了Fe片段的抗原性可能引起的副作用,因而被广泛用作生物制品。 Since the F (ab ') 2 fragments retain the biological activity corresponding antigen binding, but also avoid the side effects of Fe antigenic fragment may arise, which is widely used as a biological products. 如白喉抗霉素和破伤风抗霉素,经胃蛋白酶水解后,因去掉了Fe片段的抗原性而减少了超敏反应的发生。 Such as diphtheria and tetanus antimycin antimycin, pepsin after hydrolysis, by removing the antigenic fragments Fe and reduce the occurrence of hypersensitivity reactions. pFc'片段最终被降解,无生物学活性。 pFc 'fragments eventually degraded biologically inactive.

[0050] Fab (fragment of antigen binding):木瓜蛋白酶使Ig在铰链区重链间二硫键近N端处切断,形成两个相同的单价抗原结合片段简称Fab段(如图I中所示),一个可结晶的片段简称Fe (fragment crystallizable)段。 [0050] Fab (fragment of antigen binding): papain make disulfide bonds near the N-terminus of the Ig cut between the heavy chain hinge region, form two identical monovalent antigen binding Fab fragment referred to in paragraph (as shown in FIG. I) , a crystallizable fragment referred to Fe (fragment crystallizable) segment.

[0051] Fab' :是带巯基的单价抗原结合片段。 [0051] Fab ': with a mercapto group is a monovalent antigen-binding fragment thereof.

[0052] 抗原结合位点:如图I中所示,抗体分子与抗原相结合的部位,由Ig轻、重链的CDR1、CDR2 和CDR3 组成。 [0052] The antigen binding site: As shown in FIG I, the antibodies bind to the antigen molecule parts, the Ig light, the heavy chain CDRl, CDR2 and CDR3 composition.

[0053] 抗原:是一类能诱导免疫系统发生免疫应答,并能与免疫应答的产物(抗体或效应细胞)发生特异性结合的物质。 [0053] Antigen: The product is a class of immune system can induce an immune response, the immune response and to (effector cells or antibodies) that specifically binds to the substance to occur. 抗原具有免疫原性和反应原性两种性质。 Antigens having immunogenicity and reactogenicity both properties. 根据抗原性质分为两类:完全抗原和不完全抗原。 Divided into two categories according to the nature of the antigen: complete and incomplete antigen antigen. 完全抗原(complete antigen)是一类既有免疫原性,又有免疫反应性的物质。 Complete antigen (complete antigen) is a class of both the immunogenicity, but also immunoreactive substance. 如大多数蛋白质、细菌、病毒、细菌外毒素等都是完全抗原。 As most proteins, bacteria, viruses, bacterial toxins are completely foreign antigen. 不完全抗原,即半抗原(hapten)是只具有免疫反应性,而无免疫原性的物质,故又称不完全抗原。 Incomplete antigen, i.e., (hapten) the hapten is immunoreactive only, without immunogenic substance, so called antigen incomplete.

[0054] 巯基化试剂:具有巯基的能连接抗体与金的双功能交联试剂。 [0054] sulfhydryl reagents: a bifunctional crosslinking reagent with the antibody can be connected to gold mercapto group.

[0055] 二硫键还原剂:二硫键又称SS键,是2个SH基被氧化而形成的-SS-形式的硫原子间的键。 [0055] The disulfide reducing agent: SS disulfide bond also known, is a bond between the two SH groups formed by oxidation of a sulfur atom in the form -SS-. 在巯基乙胺(2-MEA)、2-巯基乙醇、二硫苏糖醇等的硫化合物存在下能与之发生作用,还原成巯基(-SH)。 Sulfur compounds mercaptoethylamine (2-MEA), 2- mercaptoethanol, dithiothreitol or the like with the presence of energy takes place, is reduced to a mercapto group (-SH). 这些硫化物就是本发明中所说的二硫键还原剂。 These sulfides present invention means that a disulfide bond reducing agent. 在微量的二硫键还原剂存在的情况下抗体的重链间的二硫键被还原而其它的二硫键不被破坏。 Disulfide bond between the heavy chains of the antibody in the presence of trace amounts of disulfide bond reducing agent is not destroyed by reduction of disulfide bonds other.

[0056] 微悬臂梁(微梁):典型的微悬臂梁由氮化硅制成,如商品化的三角形微悬臂梁(Veeco Instruments)(尺寸:长200um,腿宽20um,厚O. 6um),单侧镀有60nm的金;抗体通常通过巯基化试剂的巯基(-SH)与金的共价结合以及巯基化试剂的另外一端(含有-COOH或-NH2等活性基团)与抗体结合来固定到微梁表面。 [0056] microcantilever (micro beam): A typical micro-cantilever made of silicon nitride, as commercialization of triangular micro-cantilever (Veeco Instruments) (size: length 200um, 20um leg width, thickness O. 6um) , one side coated with 60nm gold; antibodies generally bind gold covalently sulfhydryl reagent and the other end (-NH2 -COOH or the like containing reactive group) to the antibody bound to the thiol groups via sulfhydryl reagent (-SH) to secured to the surface of the micro beam.

[0057] 基于表面应力检测的微悬臂梁免疫传感系统:微悬臂梁免疫传感系统主要由激光器、分光镜、微悬臂梁、光电位置敏感器(PSD)、温度控制系统、蠕动泵、反应池以及数据分析处理装置组成。 [0057] Immune microcantilever based sensor system detects surface stress of: sensing microcantilever immune system consists of a laser, a beam splitter, the micro-cantilever, the position of the photoelectric sensors (the PSD), the temperature control system, a peristaltic pump, the reaction pool and data analysis processing apparatus composition. 典型的微悬臂梁免疫检测方法的步骤如下:将微悬臂梁固定到反应池中,以螺动泵控制流动缓冲液通过反应池,待反应池中气泡排净后以O. lmL/min的速度流动缓冲液通过反应池。 Typical microcantilever step immunoassay method is as follows: The micro-cantilever is fixed to a well, to actuate the pump controls the flow spiro buffer through the reaction cell, the reaction cell until bubbles drained speed O. lmL / min of running buffer through the reaction cell. 反应池的温度控制在37 ±0.01 °C,室温控制在27 ±0.01 °C。 Controlling the temperature of the reaction cell 37 ± 0.01 ° C, the temperature control 27 ± 0.01 ° C. 激光器发出一束激光经分光镜后照射在微悬臂梁的尖端,经微悬臂梁反射后再经分光镜反射后照在PSD的靶面上。 The laser emits a laser beam irradiated by the beam splitter at the tip of the micro-cantilever, micro-cantilevers after reflection by the dichroic mirror and then reflected on the target surface as the PSD. 当微悬臂梁的位移信号稳定后,加入缓冲液稀释的样品溶液,PSD实时记录微悬臂梁的尖端位移。 When the signal is stable displacement of the microcantilever, dilution buffer was added to the sample solution, PSD real time recording microcantilever tip displacement.

[0058] 本发明的优选实施方案 [0058] Preferred embodiments of the present invention

[0059] 在本发明中,抗体类型可以包括IgM、IgG、IgA、IgD、IgE。 [0059] In the present invention, the antibody types may include IgM, IgG, IgA, IgD, IgE. 此外,抗体可以通过本领域中已知的任何方法制备,包括但不限于免疫法、杂交瘤法、化学合成法、基因工程法等。 Furthermore, antibodies can be prepared by any method known in the art, including, but not limited to, immunoassay, hybridoma, chemical synthesis, genetic engineering method or the like. 所述抗体还可以是基因工程修饰的杂合抗体,诸如人源化抗体,骆驼源化抗体等针对某种哺乳动物改造的抗体,例如人-鼠杂合抗体等。 The antibody may also be modified genetically engineered hybrid antibodies, such as humanized antibodies, antibodies and other antibodies against camelized transformation of some mammals, such as human - mouse hybrid antibodies.

[0060] 本发明中提到的待测样品可以是生物样品,例如来自于哺乳动物尤其是人的样品,包括组织样品(如病理组织切片、活检、毛发、拭子等)、细胞样品(如细胞涂片、血液涂片等)、体液样品(如血液、尿液、脑脊液、唾液等)、排泄物(例如呕吐物、汗液、粪便等)。 [0060] The present invention is mentioned in the test sample may be a biological sample, such as from a mammal, especially a human sample, including tissue samples (e.g., pathological tissue sections, biopsy, hair, swab, etc.), cell sample (e.g. smear, blood smears, etc.), a body fluid sample (such as blood, urine, cerebrospinal fluid, saliva, etc.), excreta (e.g. vomit, sweat, feces, etc.). 所述待测样品还可以是环境样品,诸如土壤样品、水样、浮尘等;其他生产领域中获得的样品,诸如污水样品、食品样品等。 The test sample may also be an environmental sample, such as a soil sample, a water sample, dust and the like; sample obtained in other production areas, such as water and food samples and the like.

[0061] 本发明中所提及的抗原包括但不限于完全抗原和半抗原,其可以是在本发明中所提到的待测样品中可能存在的本领域中所知晓的需要检测的任何类型的抗原。 [0061] The present invention is mentioned antigens include but are not limited to a complete antigen and a hapten, which may be any type of sample to be tested in the present art mentioned in the present invention may exist as known to be detected antigen.

[0062] 在本发明中,在酶解步骤中使用的蛋白酶可以根据要被水解的抗体的种类或其他因素任意地选择,只要其水解产物中包括Fab片段。 [0062] In the present invention, the protease digestion step may be arbitrarily selected according to the kind or other factors of the antibody to be hydrolyzed, as long as it comprises a hydrolyzate Fab fragment. 可选的蛋白酶诸如木瓜蛋白酶,因为木瓜蛋白酶的反应条件相对温和,因此对抗体的抗原结合活性的影响较小。 Alternatively proteases such as papain, since the reaction conditions are relatively mild papain, therefore less affected antigen-binding activity.

[0063] 在本发明中,采用蛋白酶水解抗体Ig的方法和反应条件是本领域公知的,本领域普通技术人员可以根据要水解的抗体的种类来确定合适的蛋白酶和相应的水解条件。 The method and the reaction conditions [0063] In the present invention, a proteolytic Ig antibodies are well known in the art, those of ordinary skill in the art can determine the appropriate and suitable protease hydrolysis conditions depending on the type of antibody to be hydrolysed. 例如,当选择木瓜蛋白酶时,木瓜蛋白酶可以发挥其活性的环境,一般是指PH 3〜9. 5和在25〜60°C下的环境。 For example, when selecting papain, papain can exert its activity environment, generally it refers PH 3~9. 5 and the environment of at 25~60 ° C. 木瓜蛋白酶的最适pH和最适温度根据其来源而定,一般为pH 6. 0-8. O左右和37〜55°C左右(可以根据市售产品的要求来确定)。 Optimum pH and temperature of the papain according to their origin, are generally. About O and about 37~55 ° C (can be determined according to the requirements of commercial products) to pH 6. 0-8. 在本发明中可以使用的木瓜蛋白酶的来源不受限制,只要其可以将抗体分子裂解为Fab片段。 Papain sources that can be used in the present invention is not limited, as long as it can be cleaved into Fab fragment of an antibody molecule. 在本发明中,抗体或抗体片段与木瓜蛋白酶的质量比不受限制,只要足以将抗体或抗体片段充分地水解为Fab片段。 In the present invention, the quality of the antibody or antibody fragment papain ratio is not limited, as long as the antibody or antibody fragment sufficient to fully hydrolyze Fab fragment. 两者的质量比可以根据常规方法来选择,也可以同时参考所选择木瓜蛋白酶的活性、所选择的抗体类型等因素,一般可以在10 : I〜200 : I的范围内选择。 Mass ratio of them may be selected according to conventional methods, may be simultaneously active papain, antibodies selected with reference to the type of selected factors, generally in range of 10: I selection: I~200.

[0064] 在本发明的方法中,本发明中使用的巯基化试剂不受限制,只要其可以与抗体的Fab片段联结,并且可以被固定在微梁的镀金层上。 [0064] In the method of the present invention, the thiolation agent used in the present invention is not limited as long as it can be coupled to Fab fragments of antibodies, and may be fixed to the gold plating layer on the micro beam. 巯基化试剂与Fab的联结的步骤以及固定至微梁的镀金层上的步骤的次序不受限制,可以先用巯基化试剂联结抗体将抗体巯基化后与金表面固定,也可以先将巯基化试剂固定在金表面上后再与抗体联结。 Mercapto Step group of coupling agent with the Fab and the order is fixed to the step of gold plating layer micro-beam is not limited, to join the antibody to the antibody is thiolated fixed gold surface using a thiol reagent may be first thiolated then coupled with the antibody reagent is immobilized on the gold surface.

[0065] 本发明中使用的巯基化试剂包含巯基官能团和羧基或氨基官能团,其中该试剂一端的巯基用于通过自组装固定在金表面上(参见图5),另一端的羧基或氨基(也可以通过活化被暴露)用于与抗体的氨基或羧基末端发生反应(参见图6)。 [0065] The present invention thiolated reagent comprises a thiol functional group and a carboxyl group or amino functional groups, wherein the mercapto group of the reagent of one end of a self-assembled immobilized on the gold surface (see FIG. 5), the other end of the carboxyl or amino (also It may be exposed by activation) used for the reaction (see FIG. 6) with the amino or carboxyl terminus of the antibody. 本发明可用的巯基化试剂包括硫醇类化合物,例如11-羧酸硫醇,2-氨基乙硫醇(AET)和3-巯基丙酸(MPA);盐酸硫醇亚胺;磺基烃基琥珀酰亚胺基_6-(3' 2-吡啶二硫-丙酰胺)_乙酸酯(Sulfo-LC-SPDP);和3,3' -二硫双磺基琥珀酰亚胺丙酸酯(DTSSP)等。 The present invention can mercapto agents include thiols, thiol acids e.g. 11-, 2-aminoethanethiol (in AET) and 3-mercaptopropionic acid (the MPA); thiol imine hydrochloride; hydrocarbyl sulfo succinate _6- imide group (3 '2-pyridyl dithio - propionamide) _ acetate (sulfo-LC-SPDP); and 3,3' - dithiobis succinimidyl propionate sulfo ( DTSSP) and so on.

[0066] 在本发明中使用的微梁可以根据本领域中的公知方法自行制备,也可以购买市售商品,对此在本发明中并无任何限制。 [0066] The micro beam used in the present invention may themselves be prepared according to methods well known in the art, may be purchased commercially available, this is not any limitation in the present invention.

[0067] 下面结合具体实施例对本发明做进一步说明,但需要说明的是下面的实施例不限制本发明的范围。 [0067] in conjunction with the following specific examples further illustrate the invention, but it should be noted that the following embodiments do not limit the scope of the invention.

[0068] 实施例 [0068] Example

[0069] 实施例I在微梁镀金层上固定抗体片段Fab [0069] Example I Fab antibody fragments fixed on the gold plating layer micro beam

[0070] 实验I抗体片段Fab的制备 Preparation of [0070] Fab antibody fragments of Experiment I

[0071] I.准确称取人参皂苷GS-Re单克隆抗体(购自美国USBiological公司)10. Omg溶于I. OmL O. IM Tris-HCl (含2. OmM EDTA, ρΗ8· O)中得到浓度为10. Og/L的抗体溶液; [0071] I. Weigh accurately ginsenoside GS-Re monoclonal antibody (purchased from USBiological Corporation) 10. Omg dissolved I. OmL O. IM Tris-HCl (containing 2. OmM EDTA, ρΗ8 · O) obtained in at a concentration of 10. Og / L of antibody solution;

[0072] 2.称取1.5mg木瓜蛋白酶(购自美国Sigma公司)溶于I. OmL水中得到浓度为1.5g/L的木瓜蛋白酶溶液; [0072] 2. Weigh 1.5mg of papain (available from Sigma, USA) was dissolved in water to give a concentration of I. OmL 1.5g / L papain solution;

[0073] 3.将I. OmL上述步骤I的抗体溶液和I. OmL上述步骤2的木瓜蛋白酶溶液混合,37°C反应I小时。 A mixed solution of papain [0073] 3. The antibody solution I. OmL above step I and I. OmL above step 2, 37 ° C Reaction I h. 得倒抗体Fab片段。 Get back antibody Fab fragment. 用甘油等体积稀释后-20°C保存。 Save -20 ° C was diluted with an equal volume of glycerol.

[0074] 实验2微梁表面抗体Fab片段的固定 Immobilized antibody Fab fragment [0074] Experiment 2 micro beam surface

[0075] I.硫醇自组装:将洗净并用氮气吹干的镀有金层的微梁(购自美国Veeco公司)放入酶标板小孔中,加入200 μ I浓度为IOmM 11_羧酸硫醇,封口室温12小时; [0075] I. thiol self-assembled: Wash and blown dry with nitrogen plated layer of gold micro-beams (available from Veeco Instruments Inc. USA) were placed in microtiter plate wells, was added at a concentration of 200 μ I IOmM 11_ thiol carboxylic acid, sealing temperature for 12 hours;

[0076] 2.清洗:用镊子小心取出微梁,用去离子水清洗微梁三次; [0076] 2. Wash: carefully removed with forceps micro beam, washed three times with deionized water micro beam;

[0077] 3.活化:将清洗过的微梁加入新的板孔中,加入O. 2Μ的EDC(l-(3_ 二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和O. 05M的NHS (N-羟基琥珀酰亚胺)各75 μ I活化梁40分钟; [0077] 3. Activation: The cleaned micro beams add new plate wells, was added O. EDC 2Μ of (l- (3_ dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and O. 05M of NHS (N- hydroxy succinimide) activated 75 μ I of each beam 40 min;

[0078] 4.清洗:用镊子小心取出微梁,用去离子水清洗微梁三次; [0078] 4. Wash: carefully removed with forceps micro beam, washed three times with deionized water micro beam;

[0079] 5.固定抗体:将清洗过的微梁放入新的板孔之中,加入150 μ L上述实验I获得的抗人参皂苷GS-Re单克隆抗体Fab片段,37°C温育I. 5小时,得到固定有抗体片段Fab的微 [0079] The immobilized antibody: The cleaned micro beams into a new hole in the plate, anti-ginsenoside GS-Re was added 150 μ L obtained in the above experiment I monoclonal antibody Fab fragment, 37 ° C incubation I After 5 hours, to give a slightly Fab fragments of antibodies are immobilized

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[0080] 实验3直接竞争ELISA方法评价Fab抗体片段与微梁金表面的结合情况具体步骤如下: [0080] Experiment 3 Evaluation of the direct competitive ELISA binding of Fab antibody fragment microbeam gold surface the following steps:

[0081] I.清洗:用镊子小心取出上述实验2固定了抗体片段的微梁,用洗涤液(每IL洗涤液中含有8. Og NaCUO. 2g KH2PO4,2. 96g Na2HPO4 · 12H20、I. OmL Tween-20,其余为水)冲洗数次,氮气吹干; [0081] I. Wash: carefully removed with forceps 2 is fixed above the micro beam experiments of antibody fragments, with a washing solution (wash solution containing per IL 8. Og NaCUO 2g KH2PO4,2 96g Na2HPO4 · 12H20, I OmL... Tween-20, the remaining water is washed several times), dried by nitrogen;

[0082] 2.封闭:将氮气吹干后的微梁分别放入酶标板中,加入100 μ L质量百分含量为5%的BSA溶液(用PBS溶解)封闭,37°C温育30分钟; [0082] 2. Blocking: micro beam after nitrogen blow were placed in microtiter plate was added 100 μ L mass percentage of 5% BSA solution (dissolved in PBS) is closed, 37 ° C and incubated for 30 minute;

[0083] 3.清洗:用镊子小心取出微梁,用洗涤液冲洗数次,氮气吹干; [0083] 3. Wash: carefully removed with forceps micro beam, washed several times with a washing solution, dried by nitrogen;

[0084] 4.竞争:将氮气吹干后的微梁分别放入酶标板的两个小孔中(做好标记,抑制孔与非抑制孔),抑制孔加入50 μ L经样品稀释液(含有终浓度为O. Imo 1/L ρΗ7. 5的PBS、 [0084] 4. Competition: micro beam after the nitrogen blow holes microtiter plates were placed in two (marked to be suppressed and non-suppressed bore hole), suppressing well of 50 μ L sample diluent was (containing a final concentration of O. Imo PBS 1 / L ρΗ7. 5, and

O. I %体积百分含量的吐温-20和O. I %质量百分含量的明胶)稀释的浓度为100mg/L的人参皂苷GS-Re溶液(购自美国Sigma公司),非抑制孔加入50 μ L样品稀释液作为对照;然后再分别加入浓度为I. Omg/L的人参皂苷GS-Re过氧化物酶联结物(购自美国Sigma公司),37°C温育30分钟; O. I% concentration of gelatin, the volume percentage of Tween-20 O. I% and the mass percentage) is diluted 100mg / L ginsenoside GS-Re solution (purchased from Sigma), non-inhibitory hole Add 50 μ L sample diluent as control; were then added at a concentration I. Omg / L ginsenoside GS-re coupling peroxidase (purchased from Sigma, USA), 37 ° C for 30 minutes;

[0085] 5.清洗:用镊子小心取出上述抑制孔与非抑制孔中的微梁,用洗涤液冲洗数次, [0085] 5. Wash: carefully removed with forceps hole and said suppressing micro beam non-inhibited wells, washed several times with a washing solution,

氮气吹干。 Dried by nitrogen. [0086] 6.显色:将氮气吹干后的两根微梁分别放入酶标板的两个小孔中,每孔加入100 μ L显色溶液(TMB底物使用液与底物缓冲液按I : 9的体积比混匀,每IOmL上述混合液中加入4. Omg过氧化脲),室温显色; [0086] 6. color: the two micro beam after nitrogen blow holes were placed in two microtiter plates, was added 100 μ L per well developing solution (TMB Substrate working solution with substrate buffer by liquid I: 9 volume ratio of mixing, the mixed solution was added per IOmL 4. Omg carbamide peroxide) at room temperature for color development;

[0087] 7.终止:15分钟后,每孔加入50uL终止液(2. OM的硫酸溶液)终止反应,取出微梁,450nm波长处分别读取OD值。 [0087] 7. Termination: After 15 minutes, added to 50uL stop solution to each well (2. OM sulfuric acid solution) to terminate the reaction, remove the micro beam, at a wavelength of 450nm are read OD.

[0088] 实验设上次重复,结果表明,抑制孔和非抑制孔的450nm波长下的吸光度值分别为O. 113和O. 841,说明抗人参皂苷GS-Re抗体Fab片段能有效结合微梁的金表面上并具有活性。 [0088] The experimental design was repeated previous results suggest that inhibition of the absorbance value at a wavelength of 450nm and the non-suppression holes, respectively holes and O. 113 O. 841, demonstrates that anti-GS-Re ginsenoside effective binding antibody Fab fragment micro beam on the gold surface and having an activity.

[0089] 实验4微梁传感器效果监测 [0089] Experiment 4 micro beam sensors to monitor the effect of

[0090] I.清洗:无水乙醇、丙酮和PBS分别流经微梁传感系统,流速O. 5mL/min ; [0090] I. Cleaning: ethanol, acetone and PBS respectively, flows through the micro Liang Chuangan system, the flow rate of O. 5mL / min;

[0091] 2.固定微梁:将实验2中固定有抗人参皂苷GS-Re抗体Fab片段的微梁固定到微梁传感系统的反应池中,流动PBS通过反应池子。 [0091] 2. Fixed micro beam: Experiment 2 is fixed to the beam by the micro-GS-Re ginsenoside antibody Fab fragment is fixed to a well of micro-Liang Chuangan system, the flow through the reactor pool PBS. 排除反应池中的气泡后以O. lmL/min流动PBS。 After the reaction vessel to remove air bubbles O. lmL / min flow of PBS.

[0092] 3.调节光路:调节激光器与PSD(光电位置敏感器)的位置使激光器发出的激光照在微梁的尖端反射后被PSD接收。 [0092] 3. Adjust the optical path: the laser and adjusting the position of PSD (photoelectric position sensor) in the laser beam emitted by the laser according to the reception after reflection in the tip of the PSD micro beam.

[0093] 4.样品测定:当PSD接收的微梁偏转信号稳定后,加入2mL PBS稀释的人参皂苷GS-Re标准样品。 [0093] 4. Determination of sample: After a stable micro beam deflection signal received PSD, was added ginsenoside GS-Re 2mL PBS diluted standard samples. 通过PSD实时记录微梁尖端位移信息。 PSD real-time recording by the micro beam tip displacement information.

[0094] 检测了三个浓度20ng/mL、lng/mL、0. 05ng/mL ;的样品得到的位移量分别约为80、45和20nm(图8)。 [0094] The three detected concentration 20ng / mL, lng / mL, 0 05ng / mL;. The displacement amount of sample obtained is about 80,45 and 20 nm, respectively (FIG. 8). 不同浓度的人参皂苷GS-Re标准样品可以产生不同程度的微梁位移,说明抗人参皂苷GS-Re抗体片段Fab能有效结合到微梁的金表面上并能对人参皂苷GS-Re进行定量检测。 Ginsenoside GS-Re standard samples with different concentrations may be generated micro beam displacement varying degrees, indicating anti ginsenoside GS-Re antibody fragment Fab can be effectively bound to the gold surface of the micro beam on and can be quantitative detection of ginsenoside GS-Re .

[0095] 实施例2在微梁镀金层上固定抗体片段Fab [0095] Example 2 antibody immobilized on the gold plated layer micro beam Fab fragments

[0096] 实验I抗体片段Fab的制备 Preparation of [0096] Fab antibody fragments of Experiment I

[0097] I.准确称取人参皂苷GS-Re单克隆抗体(购自美国USBiological公司)10. Omg溶于I. OmL O. IM Tris-HCl (含2. OmM EDTA, ρΗ8· O)中得到浓度为10. Og/L的抗体溶液; [0097] I. Weigh accurately ginsenoside GS-Re monoclonal antibody (purchased from USBiological Corporation) 10. Omg dissolved I. OmL O. IM Tris-HCl (containing 2. OmM EDTA, ρΗ8 · O) obtained in at a concentration of 10. Og / L of antibody solution;

[0098] 2.称取1.5mg木瓜蛋白酶(购自美国Sigma公司)溶于I. OmL水中得到浓度为1.5g/L的木瓜蛋白酶溶液; [0098] 2. Weigh 1.5mg of papain (available from Sigma, USA) was dissolved in water to give a concentration of I. OmL 1.5g / L papain solution;

[0099] 3.将I. OmL上述步骤⑴的抗体溶液和I. OmL上述步骤⑵的木瓜蛋白酶溶液混合,37°C反应I小时。 [0099] 3. mixing the papain solution the above step I. OmL ⑴ antibody solution and I. OmL ⑵ the above step, 37 ° C Reaction I h. 得倒抗体片段。 Get back antibody fragment. 用甘油等体积稀释后_20°C保存。 Save _20 ° C was diluted with an equal volume of glycerol.

[0100] 实验2利用盐酸硫醇亚胺将抗体片段Fab巯基化 [0100] Experiment 2 using the mercaptan imine hydrochloride thiolated antibody Fab fragment

[0101] I.称取I. Omg盐酸硫醇亚胺(购自美国sigma公司)溶于I. OmL蒸懼水中得到浓度为I. Og/L的巯基化试剂(现配现用); [0101] I. I. Omg weighed thiol imine hydrochloride (purchased from sigma Corporation) was dissolved I. OmL distilled water to give a concentration of fear I. Og / L of sulfhydryl reagents (now with the current);

[0102] 2.将I. OmL上述实验I中的抗体片段Fab和45. 8 μ L上述实验I的巯基化试剂混合,室温条件下轻微搅拌反应Ih ; [0102] 2. The thiolated reagent mixing the above-described antibody fragment is Fab I. OmL experiment I and 45. 8 μ L of the above experiment I, the reaction was stirred at room temperature for Ih is mild;

[0103] 3.用20mM 的PBS 缓冲液(含O. 15M NaCl 和I. OmM EDTA, Ph7. 2)对上述步骤2 的反应液透析48小时,期间每3小时更换一次透析液,得到巯基化抗体片段溶液 [0103] 3. Use of 20mM PBS buffer (containing O. 15M NaCl and I. OmM EDTA, Ph7. 2) dialyzed for 48 hours the reaction solution in step 2 above, every 3 hours during the replacement of a dialysate, to give thiolated antibody fragment solution

[0104] 实验3微梁表面巯基化抗体Fab片段的固定 [0104] Experiment 3-mercapto-fixing surface of the micro beam antibody Fab fragments of

[0105] 将洗净并用氮气吹干的镀有金膜的微梁(购自美国Veeco公司)放入酶标板小孔中,加入150 μ L用PBS稀释50倍后的上述实验2制得的巯基化抗体Fab片段溶液,放在37°C水浴箱中2小时,即得到固定有抗体Fab片段的微梁。 [0105] The cleaned and blown dry with nitrogen plated with gold film microbeam (available from Veeco Instruments Inc. USA) were placed in microtiter plate wells, after the addition of 150 μ L was diluted 50-fold with PBS to obtain the above experiment 2 the thiolated antibody Fab fragment solution, placed in 37 ° C water bath for 2 hours, to obtain a micro beam fixing antibody Fab fragment.

[0106] 实验4ELISA直接竞争法效果检测 [0106] Competition experiments 4ELISA direct effect detection method

[0107] I.清洗:用镊子小心取出上述固定了抗体片段Fab的微悬臂梁,用洗涤液(每IL洗涤液中含有8. Og NaCUO. 2g KH2PO4,2. 96g Na2HPO4 · 12H20、I. OmL Tween-20,其余为水)冲洗数次,氮气吹干; [0107] I. Wash: carefully removed using tweezers the fixing Fab antibody fragments microcantilever, with a washing solution (wash solution containing per IL 8. Og NaCUO 2g KH2PO4,2 96g Na2HPO4 · 12H20, I OmL... Tween-20, the remaining water is washed several times), dried by nitrogen;

[0108] 2.封闭:将氮气吹干后的微悬臂梁分别放入酶标板中,加入100μ L质量百分含量为5%的BSA溶液(用PBS溶解)封闭,37°C温育30分钟; [0108] 2. Blocking: The microcantilever dry nitrogen were placed in the microtiter plate, add 100μ L mass percentage of blocked 5% BSA solution (dissolved in PBS), 37 ° C and incubated for 30 minute;

[0109] 3.清洗:用镊子小心取出微悬臂梁,用洗涤液冲洗数次,氮气吹干; [0109] 3. Wash: carefully removed with forceps microcantilever, washed several times with a washing solution, dried by nitrogen;

[0110] 4.竞争:将氮气吹干后的微悬臂梁分别放入酶标板的两个小孔中(做好标记,抑制孔与非抑制孔),抑制孔加入50 μ L经样品稀释液(含有终浓度为O. lmol/L pH7. 5的PBS,O. I %体积百分含量的吐温-20和O. I %质量百分含量的明胶)稀释的浓度为IOOmg/L的人参皂苷GS-Re溶液(购自美国Sigma公司),非抑制孔加入50 μ L样品稀释液作为对照;然后再分别加入浓度为I. Omg/L的人参皂苷GS-Re过氧化物酶联结物(购自美国Sigma公司),37°C温育30分钟; [0110] 4. Competition: the microcantilever after nitrogen blow were placed in microtiter plate two holes (marked to be suppressed and non-suppressed bore hole), hole inhibition by diluted sample was added 50 μ L (containing a final concentration of O. lmol / L PBS pH7. 5 a, O. I% Tween-20 volume percentage and O. I% gelatin mass percent content) was diluted concentration of IOOmg / L of GS-re ginsenoside solution (purchased from Sigma), non-inhibitory well was added 50 μ L sample diluent as control; were then added at a concentration I. Omg / L ginsenoside GS-re was coupled to peroxidase (commercially available from Sigma, USA), 37 ° C for 30 minutes;

[0111] 5.清洗:用镊子小心取出上述抑制孔与非抑制孔中的微悬臂梁,用洗涤液冲洗数次,氮气吹干; [0111] 5. Wash: carefully removed with forceps hole and the micro-cantilever said suppressing non-inhibited wells, washed several times with a washing solution, dried by nitrogen;

[0112] 6.显色:将氮气吹干后的两根微悬臂梁分别放入酶标板的两个小孔中,每孔加入100 μ L显色溶液(TMB底物使用液与底物缓冲液按I : 9的体积比混匀,每IOmL上述混合液中加入4. Omg过氧化脲),室温显色; [0112] 6. color: two microcantilever after the nitrogen blow holes were placed in two microtiter plates, was added 100 μ L per well developing solution (TMB substrate is used with a substrate solution buffer according to I: 9 volume ratio of mixing, the mixed solution was added per IOmL 4. Omg carbamide peroxide) at room temperature for color development;

[0113] 7.终止:15分钟后,每孔加入50 μ L终止液(2. OM的硫酸溶液)终止反应,取出微悬臂梁,450nm波长处分别读取OD值。 [0113] 7. Termination: After 15 minutes, each well was added 50 μ L of stop solution (2. OM sulfuric acid solution) to terminate the reaction, remove the microcantilever, at a wavelength of 450nm OD values ​​were read.

[0114] 实验设三次重复,结果表明,抑制孔和非抑制孔的450nm波长下的吸光度值分别为O. 156和O. 944,说明巯基化后的抗人参皂苷GS-Re抗体Fab片段能有效结合微悬臂梁的 [0114] Experimental set up in triplicate, the results suggest that inhibition of the absorbance value at a wavelength of 450nm and the non-suppression holes, respectively holes and O. 156 O. 944, described anti ginseng saponin after thiolated antibody Fab fragments GS-Re effective combined with the microcantilever

金表面上。 On the gold surface.

[0115] 实验5微梁传感器效果检测 [0115] Experiment 5 Effect sensor detecting micro beam

[0116] I.清洗:无水乙醇、丙酮和PBS分别流经微梁传感系统,流速O. 5mL/min ; [0116] I. Cleaning: ethanol, acetone and PBS respectively, flows through the micro Liang Chuangan system, the flow rate of O. 5mL / min;

[0117] 2.固定微梁:将实验3中修饰有抗人参皂苷GS-Re抗体Fab片段的微悬臂梁固定到微悬臂梁传感系统的反应池中,流动PBS通过反应池子。 [0117] 2. Fixed micro beam: the modified anti Experiment 3 ginsenoside GS-Re antibody Fab fragment microcantilever is fixed to a well microcantilever sensing system, the flow through the reactor pool PBS. 排除反应池中的气泡后以 After the reaction vessel to remove air bubbles

O. lmL/min 流动PBS。 O. lmL / min flow of PBS.

[0118] 3.调节光路:调节激光器与PSD(光电位置敏感器)的位置使的激光器发出的激光照在微梁的尖端并被PSD接收。 [0118] 3. Adjust the optical path: the laser and adjusting the laser position as the PSD (photoelectric position sensor) so that the emitted laser beam is received in the tip of the micro and PSD.

[0119] 4.样品测定:当PSD接收的微梁偏转信号稳定后,加入2mL PBS稀释的人参皂苷GS-Re标准样品。 [0119] 4. Determination of sample: After a stable micro beam deflection signal received PSD, was added ginsenoside GS-Re 2mL PBS diluted standard samples. 通过PSD实时记录微梁尖端位移信息。 PSD real-time recording by the micro beam tip displacement information.

[0120] 检测了三个浓度20ng/mL、l. 0ng/mL、0. 05ng/mL的样品得到的微梁尖端位移幅值分别为95、50和25nm(图8);不同浓度的人参皂苷GS-Re标准样品可以产生不同程度的微悬臂梁位移,说明抗人参皂苷GS-Re抗体fab片段能有效结合到微悬臂梁的金表面上并能对人参皂苷GS-Re进行检测。 [0120] detecting the three concentration 20ng / mL, l 0ng / mL, 0 micro beam tip displacement amplitude of the sample 05ng / mL were obtained and 95,50 of 25 nm (FIG. 8);.. Different concentrations of ginsenoside GS-Re standard samples can produce different degrees of displacement of the microcantilever described anti ginsenoside GS-Re fab antibody fragments can be effectively bound to the gold surface of the micro-cantilever and can detect ginsenoside GS-Re.

[0121]结论: [0121] Conclusion:

[0122] 以人参皂苷GS-Re抗原抗体分子为例,用本发明的抗体片段Fab修饰和完整抗体 [0122] ginsenoside GS-Re antigen-antibody molecule, for example, an antibody with a modified Fab fragment of the invention and intact antibody

1修饰的微梁进行实验测试对比,检测不同浓度的人参皂苷GS-Re抗原时,微梁尖端的时间位移响应曲线(图8和图7)显示,在微梁位移响应为20纳米左右时,抗体Fab片段修饰和完整抗体修饰的检测人参皂苷GS-Re抗原浓度分别为O. 5ng/mL和O. 05ng/mL,灵敏度提高了约10倍。 1 modified micro beams experimental tests comparing different concentrations of ginsenoside GS-Re antigen, tip timeout micro beam response curve (FIG. 8 and FIG. 7) shows that the micro beam displacement response is about 20 nanometers, antibody Fab fragment and the modified modified intact antibody for detecting antigen GS-Re ginsenoside concentrations of O. 5ng / mL and O. 05ng / mL, about 10-fold increase in sensitivity. (图7为利用盐酸硫醇亚胺做为巯基化试剂来巯基化抗体后将抗体固定到梁上(中国专利CN101407548)对人参皂苷进行检测的结果)。 (FIG. 7 for the use as a thiol imine hydrochloride thiolated reagent after thiolated antibodies fixed to the beam (Chinese Patent No. CN101407548) on the result of detecting ginsenoside).

[0123] 通过上述非限制性的实施例,可以看出,本发明的方法和通过本发明的方法制备的微悬臂梁在免疫传感检测技术中与现有技术的微梁法相比可以提高20-40倍,相当于常规酶联免疫法的200-400倍,实现了本发明的目的,即在食品安全、环境污染、生物医学、科学研究和生产制造等领域中监控和检测极微量被分析物的可能性和实用性,完全可以实现实际的商业化应用。 [0123] By the above-described non-limiting embodiments, it can be seen, the method of the present invention and prepared by the process of the micro-cantilever of the present invention in an immunoassay sensing techniques and prior art micro-beam method can be improved as compared to 20 -40-fold, 200-400-fold equivalent to a conventional ELISA method, to achieve the object of the present invention, i.e., a very small amount in the monitoring and detection of food safety, environmental pollution, biomedical research and manufacturing and other areas to be analyzed the possibility and practicality matter, can achieve real business applications.

[0124] 参考文献 [0124] Reference

[0125] [l]Koutilya R. Buchapudi, Xin Huang, Xin Yang,HaiFeng Ji and ThomasThundat, Microcantilever biosensors for chemicals and bioorganisms. Analyst,2011,136,1539-1556 [0125] [l] Koutilya R. Buchapudi, Xin Huang, Xin Yang, HaiFeng Ji and ThomasThundat, Microcantilever biosensors for chemicals and bioorganisms. Analyst, 2011,136,1539-1556

[0126] [2]Weiming Tan,Yuan Huang,Tiegui Nan,Changguo Xue,Zhaohu Li,QingchuanZhang, and BaominWang, Development of Protein A Functionalized MicrocantileverImmunosensors for the Analyses ofSmaII Molecules at part per trillion Levels.Analysis Chemistry,2010,82(2),615-620 [0126] [2] Weiming Tan, Yuan Huang, Tiegui Nan, Changguo Xue, Zhaohu Li, QingchuanZhang, and BaominWang, Development of Protein A Functionalized MicrocantileverImmunosensors for the Analyses ofSmaII Molecules at part per trillion Levels.Analysis Chemistry, 2010,82 ( 2), 615-620

[0127] [3]Hongwei Zhao ;Changguo Xue ;Tiegui Nan ;Guiyu Tan ;Zhaohu Li ;QingX. Li ;Qingchuan Zhang ;Baomin Wang,Detection of Copper Ions UsingMicrocantileverImmunosensors and Enzyme-linked Immunosorbent Assay, Analytica Chimica Acta,2010,676,81-86, [0127] [3] Hongwei Zhao; Changguo Xue; Tiegui Nan; Guiyu Tan; Zhaohu Li;. QingX Li; Qingchuan Zhang; Baomin Wang, Detection of Copper Ions UsingMicrocantileverImmunosensors and Enzyme-linked Immunosorbent Assay, Analytica Chimica Acta, 2010,676 , 81-86,

[0128] [4]Changguo Xue, Hongwei Zhao, Yanyun Chen, Baomin Wang, QingchuanZhang, Xiaoping Wu, Development of suIfhydrylated antibodyfunctionalizedmicrocantilever immunosensor for taxol, Sensors and Actuators B,2011,156,863-866 [0128] [4] Changguo Xue, Hongwei Zhao, Yanyun Chen, Baomin Wang, QingchuanZhang, Xiaoping Wu, Development of suIfhydrylated antibodyfunctionalizedmicrocantilever immunosensor for taxol, Sensors and Actuators B, 2011,156,863-866

[0129] [5] A. Kausai te-Minkst imi ene,A. Ramanav ici ene,J. Kirlyte , andA. Ramanavicius. Comparative Study of Random and Oriented Antibody ImmobilizationTechniques on the Binding Capacity of Immunosensor. Analysis Chemistry,2010,82,6401-6408。 [0129] [5] A. Kausai te-Minkst imi ene, A. Ramanav ici ene, J. Kirlyte, andA. Ramanavicius. Comparative Study of Random and Oriented Antibody ImmobilizationTechniques on the Binding Capacity of Immunosensor. Analysis Chemistry, 2010,82 , 6401-6408.

Claims (8)

1. 一种制备微悬臂梁的方法,包括以下步骤:1)提供抗体和带有镀金层的微悬臂梁;2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,优选木瓜蛋白酶;3)固定步骤:将包含巯基官能团和羧基或氨基官能团的巯基化试剂结合至微悬臂梁的镀金层表面上;4)联结步骤:将步骤2)获得的Fab片段与步骤3)获得的已经结合在微悬臂梁上的巯基化试剂联结在一起,得到镀金层表面上固定有Fab片段的微悬臂梁。 1. A process for preparing micro-cantilever, comprising the following steps: 1) providing an antibody with the micro-cantilever and the gold plating layer; 2) hydrolysis step: the antibody with a protease is cleaved into a plurality of small molecules and Fab fragments fragment, the protease is capable of an antibody is cleaved into Fab fragment of any enzyme, preferably papain; 3) fixing the steps of: a thiol-containing functional group and a carboxyl group or mercapto group reagents amino functional groups bonded to the upper surface of the gold plating layer of the microcantilever; 4) the coupling steps of: step 2 and Fab fragments) obtained in 3) has been obtained on the micro-cantilever bound sulfhydryl reagents coupled together to yield Fab fragments are fixed on the surface of the gold plating layer microcantilever.
2. 一种制备微悬臂梁的方法,包括以下步骤:1)提供抗体和带有镀金层的微悬臂梁;2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,优选木瓜蛋白酶;3)联结步骤:将步骤2)获得的Fab片段与包含巯基官能团和羧基或氨基官能团的巯基化试剂联结在一起;4)固定步骤:将步骤3)获得的一端与Fab片段联结的所述巯基化试剂的另一端结合至微悬臂梁的镀金层表面上,得到镀金层表面上固定有Fab片段的微悬臂梁。 A method of making the micro-cantilever, comprising the following steps: 1) providing an antibody with the micro-cantilever and the gold plating layer; 2) hydrolysis step: the antibody with a protease is cleaved into a plurality of small molecules and Fab fragments fragment, the protease is capable of an antibody is cleaved into Fab fragment of any enzyme, preferably papain; 3) the coupling step: the Fab fragment of step 2) is obtained with a thiol-containing functional group and a carboxyl group or mercapto group reagents amino functional groups are coupled together ; 4) fixing steps of: step 3) obtained in one end of the other end of the Fab fragment of a mercapto group bound to a coupling agent on the surface of the gold plating layer microcantilever, Fab fragments have been fixed to the gold plating layer on the surface of the microcantilever .
3. 一种应力传感元件,其包括基于应力的微悬臂梁,所述微悬臂梁的镀金层上固定有抗体的Fab片段。 A strain sensing element comprises a stress-based micro-cantilever, Fab fragments of antibodies are fixed on the gold plating layer of the micro-cantilever.
4. 一种基于应力的微悬臂梁免疫传感检测系统,其包括权利要求3的应力传感元件。 A stress based on microcantilever sensing the immune system, stress which comprises a sensor element as claimed in claim 3.
5. 一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤:1)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁;2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,例如木瓜蛋白酶;3)固定步骤:将包含巯基官能团和羧基或氨基官能团的巯基化试剂结合至微悬臂梁的镀金层表面上;4)联结步骤:将步骤2)获得的Fab片段与步骤3)获得的已经结合在微悬臂梁上的所述巯基化试剂联结在一起,得到镀金层表面上固定有Fab片段的微悬臂梁。 A system using the sensing microcantilever sensing immunoassay test sample, comprising the steps of: a) providing a target antibody specific for the antigen and microcantilever sensing system, said transmission microcantilever sensing detection system comprises a reaction tank and a gold plating layer with a stress based on microcantilever; 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, the protease is an antibody capable of cleavage into Fab fragment of any enzyme, such as papain; 3) fixing the steps of: a thiol-containing functional group and a carboxyl group or mercapto group reagents amino functional groups bound on the surface of the gold plating layer to the microcantilever; 4) linked steps: step 2) obtaining Fab fragment of step 3) has been obtained in conjunction with the micro-cantilever sulfhydryl reagents coupled together to yield Fab fragments are fixed on the surface of the gold plating layer microcantilever. 5)将步骤4)获得的固定有Fab片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中;6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测样品中是否存在所述靶抗原。 Fixed 5) in step 4) obtained in micro cantilever is fixed to the Fab fragment of a reaction cell microcantilever sensing system; 6) The sample to be tested is added to the step 5) microcantilever sensing system obtained in detecting the presence or absence of the target antigen in the test sample.
6. 一种使用微悬臂梁传感检测系统免疫传感检测待测样品的方法,包括以下步骤:1)提供对靶抗原特异的抗体和微悬臂梁传感检测系统,所述微悬臂梁传感检测系统包括反应池和带有镀金层的基于应力的微悬臂梁;2)酶解步骤:用蛋白酶将所述抗体裂解成Fab片段和多个小分子片段,所述蛋白酶为能够将抗体裂解成Fab片段的任何酶,例如木瓜蛋白酶;3)联结步骤:将步骤2)获得的Fab片段与包含巯基官能团和羧基或氨基官能团的巯基化试剂联结在一起;4)固定步骤:将步骤3)获得的一端与Fab片段联结的所述巯基化试剂的另一端结合至微悬臂梁的镀金层表面上,得到镀金层表面上固定有Fab片段的微悬臂梁;5)将步骤4)获得的固定有Fab片段的微悬臂梁固定到微悬臂梁传感检测系统的反应池中;6)将待测样品加入至步骤5)获得的微悬臂梁传感检测系统中,检测待测 A system using the sensing microcantilever sensing immunoassay test sample, comprising the steps of: a) providing a target antibody specific for the antigen and microcantilever sensing system, said transmission microcantilever sensing detection system comprises a reaction tank and a gold plating layer with a stress based on microcantilever; 2) hydrolysis step: the antibody with a protease is cleaved into Fab fragments and a plurality of small molecule fragments, the protease is an antibody capable of cleavage any enzyme as Fab fragments, such as papain; 3) the coupling step: the Fab fragment of step 2) is obtained with a thiol-containing functional group and a carboxyl group or mercapto group reagents amino functional groups are coupled together; 4) fixing steps of: step 3) the other end of the sulfhydryl reagent obtained end coupled Fab fragments bound to a gold plating layer on the surface of the micro-cantilever, micro-cantilever is fixed to obtain a Fab fragment on the surface of the gold plating layer; 5) in step 4) obtained in the fixed Fab fragments have microcantilever is fixed to a well microcantilever sensing system; 6) the sample to be tested is added to the step 5) obtained microcantilever sensing system, the detection test 品中是否存在所述靶抗原。 The presence or absence of the target antigen in the article.
7.根据权利要求1-6中任一项所述的方法、应力传感元件或免疫传感检测系统,其特征在于所述巯基化试剂选自硫醇类化合物,例如11-羧基硫醇、2-氨基乙硫醇和3-巯基丙酸;盐酸硫醇亚胺;磺基烃基琥珀酰亚胺基_6-(3' 2-吡啶二硫-丙酰胺)_乙酸酯;3,3' - 二硫双磺基琥珀酰亚胺丙酸酯中的一种或多种。 7. The method as claimed in any one of claims immune stress sensing element or sensing system, wherein said agent is selected from mercapto thiols, mercaptans e.g. 11- carboxy, 2-aminoethanethiol and 3-mercaptopropionic acid; thiol imine hydrochloride; sulfo-succinimidyl _6- hydrocarbyl (3 '2-pyridyl dithio - propionamide) _ acetate; 3,3' - one or more sulfo-dithiobis succinimidyl propionate in.
8.根据权利要求1-7中任一项所述的方法、应力传感元件或免疫传感检测系统,其特征在于所述抗体为IgM、IgG、IgA、IgD、IgE中的任一种。 8. The method according to any one of claims immune stress sensing element or sensing system, wherein said antibody is an IgM, IgG, IgA, IgD, IgE any one of.
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