Nanoliter Cell Culture Array with Tunable Chemical Gradients

Abstract

A multitude of cell screening assays for diagnostic and research applications rely on quantitative measurements of a sample in the presence of different reagent concentrations. Standard methods rely on microtiter plates of varying well density, which provide simple and standardized sample addressability. However, testing hundreds of chemical dilutions requires complex automation, and typical well volumes of microtiter plates are incompatible with the analysis of a small number of cells. Here, we present a microfluidic device for creating a high-resolution chemical gradient spanning 200 nanoliter wells. Using air-based shearing, we show that the individual wells can be compartmentalized without altering the concentration gradient, resulting in a large set of isolated nanoliter cell culture wells. We provide an analytical and numerical model for predicting the concentration within each culture chamber and validate it against experimental results. We apply our system for the investigation of yeast cell metabolic gene regulation in the presence of different ratios of galactose/glucose concentrations and successfully resolve the nutrient threshold at which the cells activate the galactose pathway.

Toward Decentralizing Antibiotic Susceptibility Testing via Ready-to-Use Microwell Array and Resazurin-Aided Colorimetric Readout

“…Second, the assay time can be reduced by tuning the bacteria and resazurin concentrations, similar to previous works. 18,20,21 Third, macrAST currently generates antibiograms from bacteria following clinical single-colony isolation. Appending a device that can filter and concentrate bacterial cells from patient samples [17][18][19] before marcAST may obviate clinical isolation and enable direct AST from samples with monomicrobial infections, such as the plurality of samples from uncomplicated UTIs.…”

“…18,20,21 Third, macrAST currently generates antibiograms from bacteria following clinical single-colony isolation. Appending a device that can filter and concentrate bacterial cells from patient samples [17][18][19] before marcAST may obviate clinical isolation and enable direct AST from samples with monomicrobial infections, such as the plurality of samples from uncomplicated UTIs. 22 Finally, colorimetric identification of bacteria [23][24][25] may be implemented within our microwell array device and be performed in tandem with marcAST.…”

Microfluidic-based observation of local bacterial density under antimicrobial concentration gradient for rapid antibiotic susceptibility testing

“…25 Recently, a number of studies have been reported using newly developed microfluidic technologies to overcome the disadvantages of conventional AST methods. [26][27][28][29][30] Since most of the microfluidic AST platforms are microscale-systems, they not only reduce the excess cells and reagents required but also make it possible to analyze the morphology and number of single cells, which significantly improve the resolution. 23,31,32 In general, these platforms of single cell analysis test the efficacy of antibiotics based on phenotypic alteration of bacterial cells, rather than growth level.…”

Antifungal susceptibility testing ofAspergillus nigeron silicon microwells by intensity-based reflectometric interference spectroscopy

“…Furthermore, microfluidic systems for single cell susceptibility testing and diagnostic applications have been developed recently. [25][26][27] Many of these new techniques rely on costly equipment, have a limited microorganism spectrum or do not yet effectively accelerate the determination of MIC values.…”