Maleimide-functionalized self-assembled monolayers for the preparation of peptide and carbohydrate biochips
Maleimide-functionalized self-assembled monolayers for the preparation of peptide and carbohydrate biochips
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DOI：
10.1021/la0262304
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发表时间：
2003-03-04
期刊：
影响因子：
3.9
通讯作者：
Mrksich, M
中科院分区：
文献类型：
--
作者：
Houseman, BT;Gawalt, ES;Mrksich, M
本文报道了一种简便的将生物活性配体固定到烷基硫醇盐/金自组装膜上的方法。该方法基于存在马来酰亚胺和五（乙二醇）基团的单层。马来酰亚胺基团有效地与硫醇封端的配体反应，而五（乙二醇）基团防止蛋白质非特异性吸附到底物上。采用循环伏安法研究了二茂铁-硫醇偶联物的固定化速率和选择性。本文介绍了使用这种方法制备的生物芯片的三个例子。在第一个实施例中，四个碳水化合物-硫醇缀合物被固定到单层，并使用荧光和表面等离子体共振光谱检查底物的凝集素结合特性。第二个生物芯片用于研究酪氨酸激酶c-src对固定化肽IYGEFKKKC的酶促磷酸化。然后使用呈递该肽的单层以阵列形式研究酶的抑制。最后一类底物，其呈现三肽Arg-Gly-Asp，用于整联蛋白介导的细胞粘附的研究。这里描述的固定化方法，它结合了自组装膜的结构顺序和惰性性质与可溶性硫醇和表面结合的马来酰亚胺基团之间的有效反应，将是有用的，用于制备基板的基础科学和生物技术中的广泛应用。
This paper reports a convenient method for immobilizing biologically active ligands to self-assembled monolayers of alkanethiolates on gold (SAMs). This methodology is based on monolayers that present maleimide and penta(ethylene glycol) groups. The maleimide groups react efficiently with thiol-terminated ligands, whereas the penta(ethylene glycol) groups prevent the nonspecific adsorption of protein to the substrate. The rate and selectivity of the immobilization of a ferrocene-thiol conjugate were characterized using cyclic voltammetry. This paper presents three examples of biochips prepared using this methodology. In the first example, four carbohydrate-thiol conjugates were immobilized to monolayers and the lectin-binding properties of the substrates were examined using fluorescence and surface plasmon resonance spectroscopy. The second biochip was used to study the enzymatic phosphorylation of the immobilized peptide IYGEFKKKC by the tyrosine kinase c-src. Monolayers presenting this peptide were then used to study the inhibition of the enzyme in an array format. The final class of substrates, which presents the tripeptide Arg-Gly-Asp, was used for studies of integrin-mediated cell adhesion. The immobilization methodology described here, which combines the structural order and inert properties of SAMs with the efficient reaction between soluble thiol and surface-bound maleimide groups, will be useful for preparing substrates for a wide range of applications in basic science and biotechnology.