Diffusion coefficient measurement with fluorescent detection in free-diffusion based microfluidics

Microfluidic devices have been widely used to measure the diffusion coefficients and hydrodynamic radii of various molecules, especially proteins. The existing devices that use diffusion-based gradient generation apply obstacles such as membranes or hydrogels to avoid additional fluid flow affecting the evolution of concentration distribution and precise measurement. Here, a free-diffusion based microfluidic device was developed which is capable of measuring the diffusion coefficients of various, different-sized proteins and dyes without using any obstacles by minimizing pressure differences due to its symmetrical geometry. The fluorescent detection and the ease of application of the device enable accelerated measurements and interpretation of results. Time-lapse pictures of 30 s were taken of the diffusion profiles and a custom-made self-written Python program was used to fit the profiles to the theoretical functions and calculate the diffusion coefficients. Diffusion coefficients of bovine serum albumin, immunoglobulin G and rhodamine B were determined with this method and compared to their theoretical and experimental values.