电子舌生成连续的识别模式的蛋白质分析
Summary
A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.
Abstract
In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.
Introduction
精确和快速蛋白检测方法是在医学诊断学和蛋白质组学非常重要。经典的蛋白检测阵列，如生物芯片，是基于“锁和钥匙”的识别原则和需要特定的受体，如适体，抗体，或其模拟物。
近年来，差分检测灵感来自于人类的嗅觉和味觉已经成为一种替代1。这种电子鼻/舌（EN / ET）的方法是基于差分分析物结合到交叉反应的受体（CRR），它们，这并不需要是高度特异性的或选择性的靶标分子的阵列从而允许克服费力开发高选择性受体的过程。这是所有的受体结合反应，创建一个独特的模式为每个样品，就像指纹一样，允许其身份。
在ELEC发展的两个关键挑战tronic的鼻/舌为有效蛋白质检测是生产具有到中间结构上相似的分析物和对于该结合事件的适当的转导系统分辨能力感测元件。到现在为止，研究已经报道的各种方法，以阵列发展2。例如，在一项研究中的蛋白的基于阵列的识别，使用从四carboxyphenylporphyrin衍生物制备CRRs通过偶联的羧基以不同的氨基酸或二肽，以提供差动受体具有疏水核的蛋白质和不同的带电周边的亲和力开发传授鉴别约束力。使用本系统时，不同的蛋白质和蛋白质的混合物进行鉴定相互作用时测量受体的荧光猝灭与待测物3,4。在另一项研究中，29 CRRs含三肽和硼酸部分库的组合合成的方式在hexasubstituted苯支架被用于检测蛋白质与指示剂摄取比色检测5,6研制。通过这样的设计，每个受体显示出与协助从糖蛋白的差异的基础上，在肽武器的变异蛋白质，和硼酸差的结合能力。最近，一种结合有阴离子性荧光聚合物聚（对苯亚乙炔）（PPE）的不同阳离子官能化的金纳米粒子组成的阵列已经创建了用于检测和鉴定蛋白质7。和蛋白质分析物之间的竞争性结合淬火再生荧光PPE /金纳米粒子复合物，从而产生不同的识别模式的蛋白质。在这项研究中，所述官能化的纳米颗粒 – 蛋白相互作用通过改变纳米颗粒的端基的物理化学性质进行调整。此外，研究结果表明，该方法是有效的在复杂和富含蛋白质的培养基，例如蛋白质分析人血清在生理学相关浓度，从而显示出在性能分析实际样品用于诊断疾病状态8等的潜力。
虽然非常有前途的，这些系统有一些固有的局限性。它们需要设计和从5到29 CRRs用相当复杂的结构合成。此外，嗅觉系统，该系统复位下列各测量不同的是，蛋白质检测需要制备每个样品的阵列。最后，监控实时结合事件是极其困难的。
在此背景下，提出了一种组合方法通过使用少量的简单和方便的分子具有不同的物理化学性质的（亲水性，疏水性，带正电，带负电荷，中性， 等等 ）作为构建块（BBS）9。上的金表面通过混合不同黑带和控制的比例，并允许该混合物的自组装棱镜，组合表面为特色的结合蛋白，相应的属性数组创建。值得注意的是，在该系统上的自组装单层允许容易地调谐的范围内的表面性质的在高度发散的方式，可实现多种组合的交叉反应性的受体（CoCRRs）是快速和有效地生产。使用光学检测系统进行蛋白质检测，表面等离子体共振成像（SPRI）。简单地说，从LED的广泛光束单色偏振光照射棱镜表面上，整个CoCRR阵区。高分辨率的CCD摄像机可以穿过CoCRR阵列的所有点的实时差分图像。它捕获所有的局部变化在CoCRR阵列提供有约束力的事件和动力学过程10的详细信息的表面。同时，随着图像处理软件的帮助下，对应点的SPR图像自动转换成反射versu的变化的时候，产生了一系列的运动结合曲线称为传感。因此，SPRI允许无标签，同步，并行和实时观测结合的事件。此外，将获得的CoCRR阵列是可再生的和可重复使用的用于蛋白质分析。
这个协议描述了电子舌的结构通过仅使用两个小分子作为构建块和表示其为基于与SPRI获得连续的识别模式常见的蛋白质分析中的应用。
Protocol
Representative Results
Discussion
专用于结构本等的最重要的步骤，以确保良好的系统的再现性。例如，清洁的棱镜的金表面在使用前用标准程序，在BB1和BB2的11纯与混合溶液中加入甘油10％时在棱镜的金表面黑带的自组装，以消除溶剂蒸发，沉积多次重复为每个[BB1] /（[BB1] + [BB2]）的比例， 等等 。作为用于蛋白检测由SPRI，工作角度的选择是至关重要的。通常，它被固定在反射率曲线的最大斜率。此外，它是使用标准?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Garçon. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| SPRi apparatus | Horiba Scientific-GenOptics | SPRi apparatus is placed in a temperature regulated incubator at 25°C. | |
| Incubator | Memmert | ||
| Syringe pump | Cavro scientific instruments | Cavro XLP 6000 | |
| Micro Elite Degasser | Alltech | AT590507 | |
| 6-port medium pressure injection valve | Upchurch Scientific | The volume of injection loop used is 500 µl. | |
| Femto plasma cleaner (version 7) | Diener Electronic | On-line degassing system with 2 channel. | |
| Spotter | Siliflow | It is a non-contact piezoelectric spotter. | |
| SPRi-Biochip | Horiba Scientific-GenOptics | 36000067 | The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes. |
| erythrina cristagalli lectin | Sigma-Aldrich | L5390 | |
| arachis hypogaea lectin | Sigma-Aldrich | L0881 | |
| myoglobin | Sigma-Aldrich | M1882 | |
| lysozyme | Sigma-Aldrich | L6876 | |
| CXCL12α | Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9 | ||
| CXCL12γ | Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9 | ||
| lactose | Provided by Prof. David Bonnaffé, for preparation details, see supporting information in reference 9 | ||
| sulfated lactose | Provided by Prof. David Bonnaffé, for preparation details, see supporting information in reference 9 | ||
| glycerol | Sigma-Aldrich | G5150 | |
| SDS | Sigma-Aldrich | L4509 | |
| tween 20 | Sigma-Aldrich | 274348 | |
| HEPES | Sigma-Aldrich | H3375 | |
| sodium phosphate monobasic | Sigma-Aldrich | S0751 | |
| sodium chloride | Sigma-Aldrich | S3014 |
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