Taylor Joseph D, Linman Matthew J, Wilkop Thomas, Cheng Quan
Department of Chemistry, University of California, Riverside, California 92521, USA.
Anal Chem. 2009 Feb 1;81(3):1146-53. doi: 10.1021/ac8023137.
We report a microfabrication approach to generate well-defined, addressable, and regenerable lipid membrane arrays in poly(dimethylsiloxane) (PDMS) microchips for label-free analysis of lipid-protein interactions with surface plasmon resonance imaging (SPRi). The multiplexed detection is demonstrated with a tethered bilayer membrane array built in parallel microchannels. These channels allow multiple measurements to be carried out simultaneously, showing low deviations for element-to-element variation in quantifiable signal. Lipid-conjugated receptors were utilized as model systems for protein binding analysis, and the feasibility of regenerating the tethering sublayer after binding was investigated. The results show that the lipid membrane can be removed effectively by nonionic surfactant Triton X-100. The small variance in SPR signal for the buildup process, i.e., <4% RSD for 3 cycles of detection, removal, and regeneration, indicates the sensing interface is highly reproducible. A calibration curve was obtained for cholera toxin using the monosialoganglioside (GM1) receptor, displaying a linear relationship in the 25 to 175 microg/mL range with a limit of detection of 260 nM. In addition, interaction of a phosphatidylinositol (PIP) with its binding protein and biotin/avidin interactions were employed for array measurements. To further enhance the SPR detection signal, a layer-by-layer amplification strategy was demonstrated that uses biotinylated antibody, NeutrAvidin and biotinylated anti-avidin, and the signal for protein binding on the membrane increased by 400%. The tethered membrane array technology, in combination with SPRi, offers an attractive platform for studies of membrane proteins, and can also find a range of applications for rapid screening of drug candidates interacting with proteins embedded in the near-native environment.
我们报道了一种微加工方法，用于在聚二甲基硅氧烷（PDMS）微芯片中生成定义明确、可寻址且可再生的脂质膜阵列，以通过表面等离子体共振成像（SPRi）对脂质 - 蛋白质相互作用进行无标记分析。通过在平行微通道中构建的拴系双层膜阵列展示了多重检测。这些通道允许同时进行多次测量，在可量化信号中元素间变化的偏差较低。脂质共轭受体被用作蛋白质结合分析的模型系统，并研究了结合后拴系子层再生的可行性。结果表明，脂质膜可以被非离子表面活性剂Triton X - 100有效去除。SPR信号在积累过程中的小方差，即检测、去除和再生3个循环的相对标准偏差（RSD）<4%，表明传感界面具有高度可重复性。使用单唾液酸神经节苷脂（GM1）受体获得了霍乱毒素的校准曲线，在25至175μg/mL范围内呈线性关系，检测限为260 nM。此外，磷脂酰肌醇（PIP）与其结合蛋白的相互作用以及生物素/抗生物素蛋白的相互作用被用于阵列测量。为了进一步增强SPR检测信号，展示了一种逐层放大策略，该策略使用生物素化抗体、中性抗生物素蛋白和生物素化抗抗生物素蛋白，膜上蛋白质结合的信号增加了400%。拴系膜阵列技术与SPRi相结合，为膜蛋白研究提供了一个有吸引力的平台，并且还可以在快速筛选与近天然环境中嵌入的蛋白质相互作用的候选药物方面找到一系列应用。
