Laminar flow-based microfluidic systems for molecular interaction analysis-Part 2: Data extraction, processing and analysis.

The rate at which fluorescently-labeled biomolecules, that are flowing at a constant speed in a microfluidic channel, diffuse into an adjacent buffer stream can be used to calculate the diffusion coefficient of the molecule, which then gives a measure of its size. Experimentally, determining the rate of diffusion involves capturing concentration gradients in fluorescence microscopy images at different distances along the length of the microfluidic channel, where distance corresponds to residence time, based on the flow velocity. The preceding chapter in this journal covered the development of the experimental setup, including information about the microscope camera detection systems used to acquire fluorescence microscopy data. In order to calculate diffusion coefficients from fluorescence microscopy images, intensity data are extracted from the images and then appropriate methods of processing and analyzing the data, including the mathematical models used for fitting, are applied to the extracted data. This chapter begins with a brief overview of digital imaging and analysis principles, before introducing custom software for extracting the intensity data from the fluorescence microscopy images. Subsequently, methods and explanations for performing the necessary corrections and appropriate scaling of the data are provided. Finally, the mathematics of one-dimensional molecular diffusion is described, and analytical approaches to obtaining the diffusion coefficient from the fluorescence intensity profiles are discussed and compared.

Laminar flow-based microfluidic systems for molecular interaction analysis-Part 1: Chip development, system operation and measurement setup.

The recent advent of laminar flow-based microfluidic systems for molecular interaction analysis has enabled transformative new profiling of proteins in regards to their structure, disordering, complex formation and interactions in general. Based on the diffusive transport of molecules perpendicular to the direction of laminar flow in a microfluidic channel, systems of this type promise continuous-flow, high-throughput screening of complex, multi-molecule interactions, while remaining tolerant to heterogeneous mixtures. Using common microfluidic device processing, the technology provides unique opportunities, as well as device design and experimental challenges, for integrative sample handling approaches that can investigate biomolecular interaction events in complex samples with readily available laboratory equipment. In this first chapter of a two-part series, we introduce system design and experimental setup requirements for a typical laminar flow-based microfluidic system for molecular interaction analysis in the form of what we call the 'LaMInA system' (Laminar flow-based Molecular Interaction Analysis system). We provide microfluidic device development advice on choice of device material, device design, including impact of channel geometry on the signal acquisition, and on design limitations and possible post-fabrication treatments to redress these. Finally. we cover aspects of fluidic actuation, such as selecting, measuring and controlling the flow rate appropriately, and provide a guide to possible fluorescent labels for proteins, as well as options for the fluorescence detection hardware, all in the context of assisting the reader in developing their own laminar flow-based experimental setup for biomolecular interaction analysis.

On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.

Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the 'holy grail' for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.