Abstract
The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.
Highlights
The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population
Using COMSOL Multiphysics, we investigated the effect of the geometry and dimensions of the valve design to the sealing pressure
We explored the effect of the actuating media by substituting air with mineral oil (Sigma Aldrich M5904) and found that the sealing pressure was further reduced to 0.04 MPa ( −x = 0.042, std. dev. = 0.003)
Summary
The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. This technique is throughput limited by the number of compartments in the array. Meier et al made use of 540 microfluidic PDMS membrane valves and Armbretch et al used 1026 circular membrane valves and corresponding microchambers in their respective research works[9,10] Both works applied about 0.20 MPa as actuation pressure to seal the microchambers and isolate the samples. Tong et al needed ≈ 0.14 MPa (20 psi) to switch the valves (4 × 4 addressable ports) to a closed s tate[12]
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