Su Jing, Bringer Michelle R, Ismagilov Rustem F, Mrksich Milan
Department of Chemistry, The University of Chicago, Illinois 60637, USA.
J Am Chem Soc. 2005 May 25;127(20):7280-1. doi: 10.1021/ja051371o.
This paper reports the use of microfluidic networks (muFNs) to both prepare peptide microarrays and carry out label-free enzyme assays on self-assembled monolayers (SAMs) of alkanethiolates on gold. A poly(dimethylsiloxane) (PDMS) stamp fabricated with microchannels is used to immobilize a linear array of cysteine-terminated peptides onto SAMs presenting maleimide groups. The stamp is then reapplied to the SAM in a perpendicular direction to introduce enzyme solutions so that each solution can interact with an identical linear array of immobilized peptides. The muFNs enable multiple enzyme-substrate interactions to be simultaneously evaluated at a submicroliter scale, while the use of SAMs enables the use of MALDI mass spectrometry (MS) to analyze the enzyme activities. This paper demonstrates applications of this system for assaying multiple kinases and for profiling the activities of kinases and phosphatases in human K562 cell extracts. The combination of muFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples.
本文报道了利用微流控网络（muFNs）制备肽微阵列，并在金表面的链烷硫醇自组装单分子层（SAMs）上进行无标记酶分析。用微通道制造的聚二甲基硅氧烷（PDMS）印章，将半胱氨酸末端肽的线性阵列固定在呈现马来酰亚胺基团的SAMs上。然后将印章沿垂直方向重新应用于SAMs以引入酶溶液，使得每种溶液都能与相同的固定肽线性阵列相互作用。muFNs能够在亚微升规模上同时评估多种酶 - 底物相互作用，而SAMs的使用使得能够利用基质辅助激光解吸电离质谱（MALDI MS）分析酶活性。本文展示了该系统在测定多种激酶以及分析人K562细胞提取物中激酶和磷酸酶活性方面的应用。muFN、SAMs和MS检测的结合为生物样品中酶活性的测定提供了一个灵活的平台。
