Modeling, simulation, and employing dilution-dialysis microfluidic chip (DDMC) for heightening proteins refolding efficiency.

Miniaturized systems based on the principles of microfluidics are widely used in various fields, such as biochemical and biomedical applications. Systematic design processes are demanded the proper use of these microfluidic devices based on mathematical simulations. Aggregated proteins (e.g., inclusion bodies) in solution with chaotropic agents (such as urea) at high concentration in combination with reducing agents are denatured. Refolding methods to achieve the native proteins from inclusion bodies of recombinant protein relying on denaturant dilution or dialysis approaches for suppressing protein aggregation is very important in the industrial field. In this paper, a modeling approach is introduced and employed that enables a compact and cost-effective method for on-chip refolding process. The innovative aspect of the presented refolding method is incorporation dialysis and dilution. Dilution-dialysis microfluidic chip (DDMC) increases productivity folding of proteins with the gradual reduction of the amount of urea. It has shown the potential of DDMC for performing refolding of protein trials. The principles of the microfluidic device detailed in this paper are to produce protein on the dilution with slow mixing through diffusion of a denatured protein solution and stepwise dialysis of a refolding buffer flowing together and the flow regime is creeping flow. The operation of DDMC was modeled in two dimensions. This system simulated by COMSOL Multiphysics Modeling Software. The simulation results for a microfluidic refolding chip showed that DDMC was deemed to be perfectly suitable for control decreasing urea in the fluid model. The DDMC was validated through an experimental study. According to the results, refolding efficiency of denaturant Hen egg white lysozyme (HEWL) (EC 3.2.1.17) used as a model protein was improved. Regard to the remaining activity test, it was increased from 42.6 in simple dilution to 93.7 using DDMC.