Abstract
Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Assigning a defined reaction mechanism to affinity binding interactions is critical to our understanding of the associated structure-function relationship, a cornerstone of biophysical characterization. Transient kinetics are currently measured using low throughput methods such as nuclear magnetic resonance, or stop-flow spectrometry-based techniques, which are not practical in many settings. In contrast, label-free biosensors measure reaction kinetics through direct binding, and with higher throughout, impacting life sciences with thousands of publications each year. Here we have developed a methodology enabling label-free biosensors to measure transient kinetic interactions towards providing a higher throughput approach suitable for mechanistic understanding of these processes. The methodology relies on hydrodynamic dispersion modeling of a smooth analyte gradient under conditions that maintain the quasi-steady-state boundary layer assumption. A transient peptide-protein interaction of relevance to drug discovery was analyzed thermodynamically using transition state theory and numerical simulations validated the approach over a wide range of operating conditions. The data establishes the technical feasibility of this approach to transient kinetic analyses supporting further development towards higher throughput applications in life science.
Highlights
Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation
We have not added additional capillaries/microchannels and instead demonstrate that rise/fall regions associated with washout of residual sub-μL dead volumes generate sub-second dispersion gradients that are well suited to the analysis of transient kinetics
A full numerical model is sophisticated and not suitable for fitting experimental data for estimation of reaction kinetics. It does generate binding curves defined by absolute kinetic values, while retaining the complexities of experimental data, thereby providing an absolute standard for quantitative testing of simpler mechanistic models
Summary
Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Microfluidic geometries employed in current flow injection analysis (FIA)-based systems are relatively large (effective hydrodynamic diameter ≥ 50 μm) in order to maintain robust low pressure sampling and instrument developers[23] have focused on minimizing microchannel “non-swept dead volumes” in order to rapidly establish a uniform analyte concentration upon injection Despite these efforts injection rise/fall regions remain >200 ms thereby limiting the uniform concentration approximation to kinetics >500 ms. We postulated that the kinetic limit of detection could be extended by including a hydrodynamic analyte dispersion term to model changing analyte concentrations within injection rise/fall regions We evaluate this approach using test data generated by multiphysical numerical simulation and we report an experimental proof-of-principle relevant to drug discovery. We employed unmodified commercially available technology for experimental work but instrument modifications will be required for optimal implementation including, parallel sensing spots that are short in the flow direction, optimized dispersion profiles and higher time resolution
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