Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Lab Chip. 2012 Mar 7;12(5):932-8. doi: 10.1039/c2lc21111d. Epub 2012 Jan 26.
We report a novel on-line electrophoretic sample clean-up approach for highly sensitive and reproducible microchip electrophoretic (μCE) immunoassay of low-abundance proteins in human serum. The method takes advantage of the differential effect of field-amplified sample stacking on molecules with different electrophoretic mobility. Large interfering proteins are removed from the loading channel by simple voltage control, resulting in selective concentration and injection of smaller target analytes to the separation channel. As a proof of concept, an antibody-free injection mode was developed for direct μCE immunoassay of human insulin-like growth factor-I (IGF-I) in serum samples without any additional purification steps. Clear and sharp peaks were obtained for IGF-I with low background and excellent reproducibility. Besides, the assay sensitivity was further increased by addition of ethanol to the sample buffer at a concentration of 50% right before performing the μCE detection. The lower limit of detection of IGF-I achieved 0.68 ng mL(-1), with an overall signal enhancement factor of 2750. The established on-line electrophoretic sample clean-up approach may find wide applications in the development of other microchip-based high-throughput analytical platforms for clinical and biological use.
我们报告了一种新颖的在线电泳样品净化方法，用于在人血清中进行低丰度蛋白质的高灵敏度和重现性微芯片电泳（μCE）免疫分析。该方法利用场放大样品堆积对具有不同电泳迁移率的分子的不同影响。通过简单的电压控制从加载通道中去除大的干扰蛋白，从而选择性地浓缩和注入较小的靶分析物到分离通道。作为概念验证，开发了一种无抗体的注入模式，用于在无需任何额外纯化步骤的情况下直接对血清样品中的人胰岛素样生长因子-I（IGF-I）进行μCE 免疫分析。对于 IGF-I，获得了低背景和出色重现性的清晰尖锐峰。此外，通过在进行 μCE 检测之前将乙醇添加到样品缓冲液中至 50%的浓度，进一步提高了测定的灵敏度。IGF-I 的检测下限达到 0.68ng mL(-1)，整体信号增强因子为 2750。所建立的在线电泳样品净化方法可能会在开发用于临床和生物学用途的其他基于微芯片的高通量分析平台方面得到广泛应用。
