A high-throughput flowless microfluidic single and multi-solute concentration gradient generator: Design and...

Microfluidic concentration gradient generators (μ-CGGs) are critical in various biochemical assays, including cell migration, drug screening, and antimicrobial susceptibility testing. However, current μ-CGGs rely on integration with flow systems, limiting their scalability and widespread adoption owing to limited infrastructure and technical expertise. Hence, there is a need for flowless diffusional gradient generators capable of standalone operation, thereby improving throughput and usability. In this study, we model such a diffusional μ-CGG as an infinite source-sink system to capture two characteristic timescales: (i) gradient generation dictated by the diffusion timescale and (ii) stability determined by the rate of change in reservoir concentrations. Through finite-element simulations, we explored the influence of various geometric parameters such as the channel length, cross-sectional area, node and reservoir volumes, and the solute diffusivity on these timescales, along with experimental confirmation using fluorescent tracer diffusion. Our results show that while the gradient stability strongly depends on the reservoir volumes, diffusion length, and solute diffusion coefficient, they are independent of the node shape or the shape of the channel cross section. However, gradient profiles were found to be the strong functions of the diffusion length, solute diffusivity, and the geometric pattern of the microfluidic grid. Additionally, we showcased the versatility of the design by generating discrete gradient profiles and combinatorial gradients of two and three solutes, thus improving throughput in a wide range of on-chip biological assays. These findings underscore the potential of our microfluidic device as an easy-to-use, inexpensive, efficient, and high-throughput platform for various on-chip biological assays.