Mass-transport limitations in spot-based microarrays.
Mass-transport limitations in spot-based microarrays.
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DOI：
10.1364/boe.1.000983
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发表时间：
2010-09-20
影响因子：
3.4
通讯作者：
Nolte D
中科院分区：
文献类型：
--
作者：
Zhao M;Wang X;Nolte D
在固相微阵列和生物传感器中，分析物向表面固定的亲和试剂的质量传输是灵敏检测的基本瓶颈。邻近传感器的体积中的分析物耗尽导致偏离理想缔合，显著减慢反应动力学，并导致传感器表面上的不均匀结合。在本文中，我们使用高分辨率的分子干涉成像（MI2），一个无标记的光学干涉技术，直接检测分子膜，研究跨100微米直径的蛋白质斑点的斑点内结合的不均匀分布。通过测量点内结合不均匀性，当与数值三维有限元模型结合时，可以准确地确定反应动力学。为了确保跨斑点的均匀结合，根据缔合速率ka和斑点直径来确定临界流速。跨斑点的结合不均匀性可用于区分高亲和力低浓度特异性反应与背景蛋白的低亲和力高浓度非特异性结合。
Mass transport of analyte to surface-immobilized affinity reagents is the fundamental bottleneck for sensitive detection in solid-support microarrays and biosensors. Analyte depletion in the volume adjacent to the sensor causes deviation from ideal association, significantly slows down reaction kinetics, and causes inhomogeneous binding across the sensor surface. In this paper we use high-resolution molecular interferometric imaging (MI2), a label-free optical interferometry technique for direct detection of molecular films, to study the inhomogeneous distribution of intra-spot binding across 100 micron-diameter protein spots. By measuring intra-spot binding inhomogeneity, reaction kinetics can be determined accurately when combined with a numerical three-dimensional finite element model. To ensure homogeneous binding across a spot, a critical flow rate is identified in terms of the association rate ka and the spot diameter. The binding inhomogeneity across a spot can be used to distinguish high-affinity low-concentration specific reactions from low-affinity high-concentration non-specific binding of background proteins.