Levich Institute and Department of Chemical Engineering, The City College of the City University of New York, New York, New York 10031, USA.
Lab Chip. 2013 Aug 7;13(15):3041-60. doi: 10.1039/c3lc50083g.
Platforms which can display cell membrane ligands and receptors as a microarray library of probes for screening against a target are essential tools in drug discovery, biomarker identification, and pathogen detection. Membrane receptors and ligands require their native bilayer environment to retain their selectivity and binding affinity, and this complicates displaying them in a microarray platform. In this study, a design is developed in which the probes are first incorporated in supported lipid bilayers formed around micron-sized particles (lipobeads), and the microbeads themselves are then arrayed on a surface by hydrodynamic capture in a microfluidic obstacle course of traps. The traps are "V" shaped open enclosures, which are arranged in a wide channel of a microfluidic device, and capture the lipobeads (slightly smaller than the channel height) as they are streamed through the course. Screening assays are undertaken directly in the device after assembly, by streaming a fluorescently labeled target through the device and detecting the bead fluorescence. Conditions are first established for which the supported bilayers on the bead surface remain intact during the capture and assay steps, using fluorescent tags in the bilayer to infer bilayer integrity. Numerical calculations of the hydrodynamic drag coefficient on the entrapped beads are presented in conjunction with the stability experiments to develop criteria for the bilayer stability as a function of the screening assay perfusion rate. Simulations of the flow streamlines are also presented to quantify the trapping efficiency of the obstacle course. Screening assays are illustrated, assaying fluorescently labeled NeutrAvidin with biotin, and labeled cholera toxin with its ganglioside binding ligand, GM1. Sequential capturing of sets of lipobeads (one at a time, and with each set bearing a different probe), followed by indexing the bead positions after each set is entrapped, allows for the construction of an indexed array of multiple probes without the need for particle encoding and is illustrated using the NeutrAvidin-biotin pair. Finally, the lipobead platform is used for quantitatively measuring the kinetic rate constants for the binding of a probe (biotin) to a target (NeutrAvidin).
能够将细胞膜配体和受体显示为探针微阵列文库的平台，是药物发现、生物标志物鉴定和病原体检测中的重要工具。细胞膜受体和配体需要其天然双层环境来保持其选择性和结合亲和力，这使得它们在微阵列平台上的显示变得复杂。在这项研究中，设计了一种方案，首先将探针掺入围绕微米级颗粒（脂质体）形成的支撑脂质双层中，然后通过在微流控障碍物通道中的流体动力捕获将微珠本身排列在表面上。这些陷阱是“V”形开口外壳，它们以宽通道的形式排列在微流控装置中，当脂质体（略小于通道高度）流过通道时捕获它们。组装后直接在设备中进行筛选分析，通过将荧光标记的靶标流过设备并检测珠的荧光来进行。首先使用双层中的荧光标记物来推断双层完整性，在捕获和分析步骤中，在珠表面的支撑双层保持完整的条件下建立条件。提出了捕获的珠上的流体动力阻力系数的数值计算，并结合稳定性实验，开发了作为筛选分析灌注率函数的双层稳定性的标准。还提出了流线的模拟，以量化障碍物通道的捕获效率。进行了筛选分析，用荧光标记的中性亲和素与生物素进行分析，并用标记的霍乱毒素与其神经节苷脂结合配体 GM1 进行分析。一组一组地捕获脂质体（一次一个，并且每组携带不同的探针），然后在捕获每组后索引珠的位置，允许在不需要粒子编码的情况下构建多个探针的索引阵列，并使用 NeutrAvidin-biotin 对进行说明。最后，使用脂质体平台定量测量探针（生物素）与靶标（中性亲和素）结合的动力学速率常数。
