Schirwitz Christopher, Block Ines, König Kai, Nesterov Alexander, Fernandez Simon, Felgenhauer Thomas, Leibe Klaus, Torralba Gloria, Hausmann Michael, Lindenstruth Volker, Stadler Volker, Breitling Frank, Bischoff F Ralf
German Cancer Research Center, Heidelberg, Germany.
University of Heidelberg, Kirchhoff Institute for Physics, Heidelberg, Germany.
Curr Protoc Protein Sci. 2009 Aug;Chapter 18:18.2.1-18.2.13. doi: 10.1002/0471140864.ps1802s57.
Microchips are used in the combinatorial synthesis of peptide arrays by means of amino acid microparticle deposition. The surface of custom-built microchips can be equipped with an amino-modified poly(ethylene glycol)methacrylate (PEGMA) graft polymer coating, which permits high loading of functional groups and resists nonspecific protein adsorption. Specific microparticles that are addressed to the polymer-coated microchip surface in a well defined pattern release preactivated amino acids upon melting, and thus allow combinatorial synthesis of high-complexity peptide arrays directly on the chip surface. Currently, arrays with densities of up to 40,000 peptide spots/cm(2) can be generated in this way, with a minimum of coupling cycles required for full combinatorial synthesis. Without using any additional blocking agent, specific peptide recognition has been verified by background-free immunostaining on the chip-based array. This unit describes microchip surface modification, combinatorial peptide array synthesis on the chip, and a typical immunoassay employing the resulting high-density peptide arrays.
通过氨基酸微粒沉积，微芯片被用于肽阵列的组合合成。定制微芯片的表面可配备氨基修饰的聚（甲基丙烯酸乙二醇酯）（PEGMA）接枝聚合物涂层，该涂层允许高负载官能团并抵抗非特异性蛋白质吸附。以明确模式寻址到聚合物涂层微芯片表面的特定微粒在熔化时释放预活化的氨基酸，从而允许在芯片表面直接组合合成高复杂性肽阵列。目前，通过这种方式可以生成密度高达40,000个肽点/cm²的阵列，完全组合合成所需的偶联循环最少。在不使用任何额外封闭剂的情况下，通过基于芯片的阵列上的无背景免疫染色验证了特异性肽识别。本单元描述了微芯片表面修饰、芯片上的组合肽阵列合成以及使用所得高密度肽阵列的典型免疫测定。
