Pan Qiong, Zhao Meiping, Liu Shaorong
Key Laboratory of Bioorganic Chemistry & Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, People's Republic of China.
Anal Chem. 2009 Jul 1;81(13):5333-41. doi: 10.1021/ac9007607.
We report a highly effective on-chip preconcentration method by combining field-amplified sample injection (FASI) and bovine serum albumin (BSA) sweeping for ultrasensitive detection of green fluorescent protein (GFP) on a simple cross-channel microchip device. With the formation of a stagnant sample/running buffer boundary by balancing the hydrodynamic flow and the electro-osmotic flow (EOF), GFP molecules can be continuously injected into the sample loading channel and stacked. We have also demonstrated that BSA is a very effective pseudo-stationary phase for sweeping concentration of proteins in comparison to the commonly used micelles. The combination of FASI and BSA sweeping yields a concentration factor of 3570 and a limit of detection of 8.4 pM for GFP. Using this method, we have separated GFP and GFP-insulin-like growth factor-I (GFP-IGF-I) fusion protein. The entire assay (GFP concentration, matrix elimination, and electrophoretic separation) can be completed within <5 min. Furthermore, we have successfully applied this method for the detection of GFP expression of E. coli cells and the GFP content in single E. coli cells.
我们报告了一种高效的芯片预浓缩方法，该方法通过结合场放大进样（FASI）和牛血清白蛋白（BSA）推扫，在一个简单的交叉通道微芯片装置上对绿色荧光蛋白（GFP）进行超灵敏检测。通过平衡流体动力流和电渗流（EOF）形成停滞的样品/运行缓冲液边界，GFP分子可以连续注入样品加载通道并堆积。我们还证明，与常用的胶束相比，BSA是一种非常有效的用于蛋白质推扫浓缩的假固定相。FASI和BSA推扫的结合产生了3570的浓缩因子和8.4 pM的GFP检测限。使用这种方法，我们分离了GFP和GFP-胰岛素样生长因子-I（GFP-IGF-I）融合蛋白。整个分析（GFP浓度、基质消除和电泳分离）可在<5分钟内完成。此外，我们已成功将该方法应用于检测大肠杆菌细胞的GFP表达和单个大肠杆菌细胞中的GFP含量。
