Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, United States.
Anal Chem. 2012 Jul 3;84(13):5556-64. doi: 10.1021/ac300478f. Epub 2012 Jun 22.
Conventional methods to probe the binding kinetics of macromolecules at biosensor surfaces employ a stepwise titration of analyte concentrations and measure the association and dissociation to the immobilized ligand at each concentration level. It has previously been shown that kinetic rates can be measured in a single step by monitoring binding as the analyte concentration increases over time in a linear gradient. We report here the application of nonlinear analyte concentration gradients for determining kinetic rates and equilibrium binding affinities in a single experiment. A versatile nonlinear gradient maker is presented, which is easily applied to microfluidic systems. Simulations validate that accurate kinetic rates can be extracted for a wide range of association and dissociation rates, gradient slopes, and curvatures, and with models for mass transport. The nonlinear analyte gradient method is demonstrated with a silicon photonic microring resonator platform to measure prostate specific antigen-antibody binding kinetics.
传统的方法来探测生物传感器表面的大分子结合动力学采用逐步滴定的分析物浓度，并测量在每个浓度水平的固定配体的缔合和解离。 先前已经表明，可以通过监测作为分析物浓度随时间在线性梯度中增加时的结合来在单个步骤中测量动力学速率。 我们在这里报告了用于在单个实验中确定动力学速率和平衡结合亲和力的非线性分析物浓度梯度的应用。 提出了一种通用的非线性梯度发生器，它很容易应用于微流控系统。 模拟验证了对于广泛的缔合和离解速率，梯度斜率和曲率以及质量传递模型，可以提取准确的动力学速率。 非线性分析物梯度法已在硅光子微环谐振器平台上进行了验证，以测量前列腺特异性抗原-抗体结合动力学。
