Department of Electrical Engineering and Computer Science, University of California, Berkeley 94720, USA.
Anal Chem. 2010 Apr 15;82(8):3343-51. doi: 10.1021/ac100182j.
To realize efficient homogeneous electrophoretic immunoassays, we introduce discontinuous polyacrylamide gels that enable quantitative assay completion in separation lengths as short as 350 mum in <10 s. The discontinuous cross-linked gels reduce the required electrophoretic separation lengths and thereby significantly reduce the required applied electrical potentials needed to achieve 100's V/cm electric field strengths for rapid electrophoresis. To optimize the discontinuous polyacrylamide gel assay format, we demonstrate development of a two-color homogeneous electrophoretic immunoassay for concurrent quantitation of C reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) for monitoring inflammatory response. To achieve necessary pore-size control at the gel discontinuity, an optimized mask-based fabrication protocol is introduced. The fabrication approach improves electrophoretic separations using the discontinuous separation gels by eliminating two confounding phenomena: (1) smaller than desired pores at the discontinuity which result in undesired physical exclusion of large-species and (2) an associated transition from small to large pores aft of the interface which acts to "destack" analyte bands during the separation. With the use of the optimized discontinuous separation gels, both assays were linear and quantitative over a two-log detection range, with a lower limit of detection of 11 ng/mL for CRP and 40 ng/mL for TNF-alpha. An optimal single-point detector location was identified by balancing the separation resolution and assay duration constraints. The ultrashort separation distance electrophoretic assays developed here provide flexibility in chip and instrument design by relaxing electrical potential requirements and expanding the possibilities for assay multiplexing, therefore addressing important design considerations when developing field-portable diagnostic assays for near-patient environments.
为实现高效均相电泳免疫分析，我们引入了不连续的聚丙烯酰胺凝胶，可在 350 µm 以内的分离长度和 10 秒内完成定量分析。不连续的交联凝胶减少了所需的电泳分离长度，从而显著降低了实现 100's V/cm 电场强度所需的外加电势，以实现快速电泳。为了优化不连续聚丙烯酰胺凝胶分析格式，我们展示了一种双色均相电泳免疫分析的开发，用于同时定量检测 C 反应蛋白 (CRP) 和肿瘤坏死因子-α (TNF-α)，以监测炎症反应。为了在凝胶不连续处实现必要的孔径控制，引入了优化的基于掩模的制造协议。该制造方法通过消除两种干扰现象，改善了不连续分离凝胶的电泳分离：（1）在不连续处孔径小于所需孔径，导致大物种的物理排斥；（2）在界面后从小孔径到大孔径的转变，在分离过程中“解堆叠”分析物带。使用优化的不连续分离凝胶，两种分析都呈线性，在两个对数检测范围内具有定量性，CRP 的检测下限为 11 ng/mL，TNF-α 的检测下限为 40 ng/mL。通过平衡分离分辨率和分析持续时间的限制，确定了最佳的单点检测器位置。这里开发的超短分离距离电泳分析为芯片和仪器设计提供了灵活性，降低了对电势的要求，并扩大了分析物多重检测的可能性，因此在为近患者环境开发现场便携式诊断分析时，解决了重要的设计考虑因素。
