A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

Abstract

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Microfluidics for bacterial chemotaxis

“…3.3(d) These findings show that the magnitude of the chemoeffector gradient within the test channel will be less than GE (by up to 50%) and the geometry of the system must be accounted for in determining the actual gradient magnitude. This was not recognized in previous studies that reported Qo = 1 based on wide-field fluorescence microscopy measurements [93,94]. The latter, however, is inappropriate for accurate characterization of the test-channel gradients because the out-of-plane fluorescence from the agarose layer, which is considerably thicker than the test channel, can override that of the test channel.…”

“…4.8. The CMC is a dimensionless metric of the strength of chemotaxis, measured as the degree of non-uniformity in the cell distribution along a spatial gradient, and is defined as [94]:…”

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

“…It is involved in a number of important processes including immune responses, tissue repair, and tumor cell metastasis, and understanding the molecular mechanisms governing directed cell migration is important for the development of new therapeutic strategies. Even though most, if not all, physiological and pathophysiological chemotaxis occurs within 3D environments, most in vitro chemotaxis experiments have been conducted in 2D settings (Abhyankar et al 2006;Boyden 1962;Frevert et al 2006;Irimia et al 2006;Irimia et al 2007;Jiang et al 2005;Li Jeon et al 2002;Shamloo et al 2008;Diao et al 2006;Cheng et al 2007;Sun et al 2008;Zigmond 1977).…”

Modeling of chemotactic steering of bacteria-based microrobot using a population-scale approach

“…These microfluidic channel devices have been fabricated for a linear chemical gradient using various materials, such as agarose, nitrocellulose, PEG, and other variations. 39,[42][43][44] We customized this channel for our purposes and used it to evaluate the chemotaxis of the bacteriobots. In contrast to other studies about bacteriobot motility, we separately considered the effects of environmental bacteria and attached bacteria.…”