Photopolymerized diffusion-defined polyacrylamide gradient gels for on-chip protein sizing

Abstract

We report on a facile diffusion-based photopatterning technique for generating linear and non-linear decreasing pore-size gradients in cross-linked polyacrylamide gels. Diffusion of low viscosity polymer precursor solutions and a two-step photopatterning process were used to define the decreasing pore-size gradient gels in a microfluidic format, thus eliminating the need for controlled mixing and delivery of polymer precursor solutions. We present an analytical model of the non-steady state diffusion process and numerically evaluate that model for direct comparison with empirical characterizations of the gradient gels. We show that the analytical model provides an effective means to predict the steepness and linearity of a desired gradient gel prior to fabrication. To assess electrophoretic assay performance in the microfluidic gradient gels, on-chip sizing of protein samples (20-116 kDa) was investigated. Baseline resolution of six proteins was demonstrated in 4 s using 3.5% to 10% polyacrylamide gradient gels. The demonstrated ability to conduct efficient protein sizing in ultra-short separation lengths (0.3 cm) means low applied electric potentials are needed to achieve the electric field strengths required for protein separations. The low required electric potentials relax operating constraints on electrical components, as is especially important for translation of the assay into pre-clinical and clinical settings. The gradient gel fabrication method reported is amenable to adaptation to non-sizing protein assays, as well as integration with upstream sample preparation steps and subsequent orthogonal downstream assays.

Microfluidic Western blotting

“…S3), with resolution similar to conventional slab-gel Western blotting. Although only uniform pore-size gels are studied here, the PACTgel pore size distributions are tunable, thus allowing enhanced resolution over specific weight ranges of interest (22)(23)(24). Importantly, the first sizing stage is observed to complete in compact 3-mm separation distances in separation times of ∼60 s. Consequently, the duration of the sizing step in μWestern blotting compares favorably to the 40-90 min required for macroscale protein sizing.…”

Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing–thawing method to investigate stem cell differentiation behaviors

“…Recently, diverse techniques have been utilized for the systematic study of the effects of a substrate's stiffness gradient on cell behaviors. They include diffusion [21], controlled dipping into crosslinking solution [22], microfluidics/photopolymerization [23], gradient mask/photopolymerization [24], convection-alternating flow/photopolymerization [25], photopolymerization using a gradually darkening mask [26], photopolymerization using a sliding mask [16,27], temperature gradients during crosslinking [28], and controlling the height of the substrate [29]. However, the toxicity from residual monomers, precursors, photoinitiators, and crosslinkers [30e33]; different surface chemistry from different densities of crosslinkers along the gradient [22]; relatively narrow ranges of stiffness gradient [29]; and the complexity of fabrication processes are also considered as potential limitations of conventional systems.…”

Structural hydrogels

“…This wok provides inspiration for the design of unique tissue engineering scaffolds based on the gradient hydrogel network by in situ encapsulation of cells. Herr et al [69] reported on the fabrication of gradient polyacrylamide with diminishing pore sizes by using diffusion-based photopatterning method. Compared with the traditional method in the fabrication of pore-size gradients [70], this approach is much easy and displays great importance for microfluidic electrophoresis [71].…”