Multiplexed microfluidic screening of bacterial chemotaxis
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Curated by eLife
eLife assessment
This manuscript presents a valuable new microfluidic tool that will allow researchers from different fields to rapidly quantify the chemotactic response of microbes to chemical gradients that have different strengths. Using planktonic bacteria, this paper convincingly shows that a multiplexed microfluidic device produces similar results to previously described microfluidic devices that generate only one gradient at a time. By performing on-chip dilutions, this device allows data for six different gradient strengths to be generated simultaneously, potentially reducing both experimental effort and biological variability.
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Abstract
Microorganism sensing of and responding to ambient chemical gradients regulates a myriad of microbial processes that are fundamental to ecosystem function and human health and disease. The development of efficient, high-throughput screening tools for microbial chemotaxis is essential to disentangling the roles of diverse chemical compounds and concentrations that control cell nutrient uptake, chemorepulsion from toxins, and microbial pathogenesis. Here, we present a novel microfluidic multiplexed chemotaxis device (MCD) which uses serial dilution to simultaneously perform six parallel bacterial chemotaxis assays that span five orders of magnitude in chemostimulant concentration on a single chip. We first validated the dilution and gradient generation performance of the MCD, and then compared the measured chemotactic response of an established bacterial chemotaxis system ( Vibrio alginolyticus ) to a standard microfluidic assay. Next, the MCD’s versatility was assessed by quantifying the chemotactic responses of different bacteria ( Psuedoalteromonas haloplanktis, Escherichia coli ) to different chemoattractants and chemorepellents. The MCD vastly accelerates the chemotactic screening process, which is critical to deciphering the complex sea of chemical stimuli underlying microbial responses.
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eLife assessment
This manuscript presents a valuable new microfluidic tool that will allow researchers from different fields to rapidly quantify the chemotactic response of microbes to chemical gradients that have different strengths. Using planktonic bacteria, this paper convincingly shows that a multiplexed microfluidic device produces similar results to previously described microfluidic devices that generate only one gradient at a time. By performing on-chip dilutions, this device allows data for six different gradient strengths to be generated simultaneously, potentially reducing both experimental effort and biological variability.
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Reviewer #1 (Public Review):
The technical approach is novel, exciting, and very carefully calibrated, and can certainly lead to many interesting downstream applications, e.g. enhanced throughput and consistency for screening purposes. Compared to traditional single-assay designs, this solution eliminates some sources of human error associated with manual dilution of reagents and reproducibility and facilitates the study of a wide spectrum of concentrations particularly at the low-concentration (below nanomolar), high-sensitivity range.
However, the study itself does not generate any fundamentally novel insights or new understanding of the biology or biophysics of the chemotactic response. It mainly reproduces previously measured trends in a more efficient and controlled manner. The novelty of the paper is purely in the technology, …
Reviewer #1 (Public Review):
The technical approach is novel, exciting, and very carefully calibrated, and can certainly lead to many interesting downstream applications, e.g. enhanced throughput and consistency for screening purposes. Compared to traditional single-assay designs, this solution eliminates some sources of human error associated with manual dilution of reagents and reproducibility and facilitates the study of a wide spectrum of concentrations particularly at the low-concentration (below nanomolar), high-sensitivity range.
However, the study itself does not generate any fundamentally novel insights or new understanding of the biology or biophysics of the chemotactic response. It mainly reproduces previously measured trends in a more efficient and controlled manner. The novelty of the paper is purely in the technology, whereas the major weakness is that this new technology was not used to demonstrate or discover some new biological phenomenon.
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Reviewer #2 (Public Review):
This manuscript develops a new microfluidic platform to study how the chemotactic response of motile cells varies in relation to its strength. Typically, chemotaxis is assayed using one microfluidic channel at a time, which limits throughput when researchers want to how to resolve how chemotaxis varies with chemoeffector concentration/gradient strength. The authors have automated this process by designing a device that can logarithmically dilute a chemoaffector with a buffer "on chip", simultaneously generating five different chemical gradients in five different channels where the maximum concentration varies by five orders of magnitude (in addition to a control lacking a gradient).
Technically, this is a major feat, requiring the design of a two-layered device, the use of herringbone mixers, and the careful …
Reviewer #2 (Public Review):
This manuscript develops a new microfluidic platform to study how the chemotactic response of motile cells varies in relation to its strength. Typically, chemotaxis is assayed using one microfluidic channel at a time, which limits throughput when researchers want to how to resolve how chemotaxis varies with chemoeffector concentration/gradient strength. The authors have automated this process by designing a device that can logarithmically dilute a chemoaffector with a buffer "on chip", simultaneously generating five different chemical gradients in five different channels where the maximum concentration varies by five orders of magnitude (in addition to a control lacking a gradient).
Technically, this is a major feat, requiring the design of a two-layered device, the use of herringbone mixers, and the careful consideration of the hydraulic resistance of each section to ensure that flow splits at junctions in a defined way to achieve the desired dilutions. It is clear the authors had to overcome many challenges before obtaining the final design. The authors have achieved their intended aims and the results from the multiplexed device are consistent with that from lower throughput devices.
Strengths:
- The multiplexed device allows researchers to greatly increase their experimental throughput when mapping out how a microbe responds to chemicals at different concentrations. While such data might be useful in its own right, such a device might make it much easier to quantify how chemotaxis varies in a multidimensional parameter space using multiple runs of this device (e.g. in analyses of fold-change detection where both the background concentration and gradient strength are varied, or in analyses that compare how the sensitivity of a microbe's chemosensory system varies in response to different chemoaffectors). Currently, it is difficult to map out how multiple parameters affect chemotaxis by running only one microfluidic experiment at a time.
- The same exact cell culture can be used in simultaneous experiments. This could potentially dramatically reduce biological variability, as cells obtained from batch cultures often differ in their metabolic state and significant variability is often observed in cultures inoculated on different days. The reduction of such variability is expected to be particularly important for strains that are very difficult/slow to grow in the laboratory or when testing cells obtained directly from environmental/clinical samples.
Weaknesses:
- Given the complexity of the device, it appears difficult to validate that the concentrations within multiplexed are the ones that are expected. It is not clear whether these devices can be used directly "off the shelf" or whether each device would need to be calibrated individually with dye beforehand. In contrast, it is relatively straightforward to serially dilute chemoaffectors manually using pipettors and obtain accurate results. It is not clear whether the on-chip dilution is a distinct advantage or whether it might add additional uncertainty/complexity.
- It is not feasible to track swimming cells in six channels simultaneously, as one cannot automatically move the microscope stage from one channel to another rapidly enough (e.g. the data collected here have 8 seconds between subsequent frames). Thus, multiplexed devices are best suited to measuring independent snapshots of the distribution of track swimming cells, rather than resolving the cellular behaviours that generate chemotaxis. However, tracking the response of slower moving, surface attached cells (e.g. eukaryotes that use ameboid movement on surfaces or bacteria that chemotax using pili) might be feasible if the gradient is maintained with constant flow. This is not explored by the authors, but if feasible it would open up a completely new avenue. Surface-attached cells move ~1000 times slower than swimming cells and experiments last for ~10-15 hours. Thus, using these multiplexed devices with surface-attached cells might facilitate much larger time savings compared to swimming cell assays, which only last for several minutes.
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