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Primary Data File Description: Measured chemical concentrations generated from the dilution microchannels for various driving pressures in each observation region. This file is also available as a multi-tab Excel file downloadable from the Supplemental Files section of this metadata page.

Observation Region

Pressure: 100 mbar - 1
Pressure 150 mbar - 1
Pressure 200 mbar - 1

Pressure: 100 mbar - 0.253104826115274
Pressure 150 mbar - 0.243842839052617
Pressure 200 mbar - 0.238542975874452

Pressure: 100 mbar - 0.0185711024758138
Pressure 150 mbar - 0.243842839052617
Pressure 200 mbar - 0.238542975874452

Pressure: 100 mbar - 0.000627274066463394
Pressure 150 mbar - 0.000595383987454315
Pressure 200 mbar - 0.000581949127052584

Pressure: 100 mbar - 0.0000886886217773395
Pressure 150 mbar - 0.000077669941377651
Pressure 200 mbar - 0.0000747648560792476

1d:T56d9,

Microfluidic Device Design

1. Hydraulic circuit framework

In analogy with electrical circuits, well-established hydraulic circuit theory [1] was used to design the complex microfluidic network of the multiplexed chemotaxis device (MCD). Briefly, for incompressible, laminar flow through a constant cross-section microchannel, the pressure drop, Δp, is linearly proportional to the volumetric flow rate, Q, and is given by Hagen-Poiseuille law [1, 2]: Δp = QR. The hydraulic resistance, R, is a function of the fluid viscosity (properties of water assumed for all fluids) and the channel geometry. Fabrication of microfluidic devices via the soft lithography method [3] used here (see below) results in rectangular cross-section microchannels (height, H; width, W; length, L).

Exact expressions for R are tabulated for rectangular and other cross-section channels and provided in various resources [4]. Combined with conservation of mass, ΣQi = 0 at the junctions (nodes) between several channels, i, the Hagen-Poiseuille law enables us to design complex microfluidic networks via the solution of a set of linear equations.

2. MCD design considerations

The primary goal of the MCD was to efficiently perform several stop-flow bacterial chemotaxis assays [5, 6] simultaneously for a range of chemostimulus concentrations. The design requirements were to: (i) dilute and distribute five logarithmically spaced concentrations of chemostimulus plus one control buffer solution to each of six chemotaxis assays. (ii) Perform those six chemotaxis assays in parallel on the same microfluidic chip. And, (iii) the microfluidic device should receive minimal fluid inputs to reduce setup time. The MCD has a two-layer architecture (dilution layer and cell injection layer) with a total of four fluid inputs and one (waste) output. Each having two inputs, the dilution layer and cell injection layer are designed to be regulated by a pressure-driven flow controller operating at a pressure, pin,1−2 ≈ 100mbar and pin,3−4 ≈ 50mbar, respectively, while the lone output is at atmospheric pressure (pout = 0). The dilution layer receives a base concentration chemostimulus solution (concentration, C0; pin,1) and a buffer solution (C = 0; pin,2), and the cell injection layer receives a bacterial suspension in buffer (pin,3) and a second buffer solution (C = 0; pin,4). The serial dilution [7] process sequentially combines the chemostimulus and buffer fluids to produce separate microchannel streams having chemostimulus concentrations of Ci = 10−iC0 (i ∈ [0, 4]) and C5 = 0 (control). The resulting six diluted chemostimulus solutions are merged in separate observation channels with a flow-stratified bacterial suspension and second buffer stream, which eventually forms the chemostimulus gradient in the chemotaxis assay. The three solutions are designed to symmetrically occupy the following fractional widths of the observation channel (total width, W): w1 = 4W/9,w2 = W/9, and w3 = 4W/9. The observation channel width (W = 1mm) and height (H = 90 μm) were chosen to set the chemostimulus gradient strength based on a physically relevant length scale [5, 8, 9] and to ensure that the upper and lower microchannel walls do not impede bacterial motility, respectively. These dimensions are in line with conventional microfluidic chemotaxis assays [5], and as a consequence of the microfabrication process, the chosen H sets the height of the dilution layer channels excluding the micromixer. The initial flow rate of the three streams in the observation region was designed to be 20, 5, 20 μl min−1 for the chemostimulus (Qout), cell (QCIL,1), and buffer solution (QCIL,2), respectively. This 4:1:4 flow rate ratio maintains stratification of cells, while ensuring that the bacterial cells are not damaged by the flow. Beyond these design requirements, the geometries - and thus hydraulic resistances - of several components are set independently, including: micromixer channels RM, bridge channels RB, and observation channels R4,4. Based on these design requirements, hydraulic circuit theory was used to determine the required resistances of each microchannel in the MCD network, and subsequently the microchannel geometries [1].

Herringbone Micromixer Design

1. Mixing performance

For the serial dilution process to perform as designed, effective mixing of the chemical solution and buffer are critical. Here, we use a well-established herringbone micromixer geometry [10], where a series of ridges on the upper wall of an otherwise rectangular microchannel drive a three-dimensional flow to enhance mixing [10, 11]. A separate microchannel - having the same cross-section geometry as the MCD design - was fabricated to independently quantify mixing performance and to select the necessary mixer length. The text micromixer channel was 41.3 mm long with 29 mixing cycles (comprised of two sets of six alternating herringbone ridges each). Two aqueous solutions of fluorescein salt (Sigma; concentrations, C50 = 50 μM and C10 = 10 μM) [12] were injected individually into a MCD, and calibration images of dye intensity were captured after each herringbone mixer cycle, corresponding to the maximum (I50) and minimum (I10) dye concentrations, respectively. The region within 20 μm of the microchannel walls was excluded from the analysis due to reflection and refraction effects [10]. Subsequently, the two solutions were flowed side-by-side with images (Ii) recorded in the same locations as above and normalized as follows: [equation added as supplemental doc].

For both flow rates, this criterion is met after 9 complete herringbone ridge cycles, and a final design with 26 herringbone cycles was chosen for the MCD. The independence of mixing efficiency on flow rate, combined with a safety factor of approximately three for the number of herringbone cycles, ensures that the serial dilution portion of the MCD will perform accurately for a wide range of chemostimulants and flow speeds.

2. Micromixer hydraulic resistance

To complete the design of the MCD, it was necessary to determine the hydraulic resistance of the herringbone micromixer Rm which was measured empirically using a parallel microfluidic device [13]. Briefly, a microfluidic device was fabricated with two parallel channels connected by shared inlets and outlets. The parallel channels had identical rectangular geometeries except one had the herringbone ridges replicating the micromixer channel section Rm. Two solutions, DI water and 1 μl ml−1tracer particle suspension (0.25 μm radius; 2% solid; carboxylated FluoroSpheres, Life Technologies), were flowed through the device using glass syringes (2.5 ml; Hamilton) mounted on two separate syringe pumps (Harvard Apparatus). The particle solution was visualized using fluorescence microscopy, and the flow rates of the two streams divided equally into the parallel channels. The micromixer hydraulic resistance was determined from the resulting flow rate ratio and the known (analytical) resistance of the non-mixer channel [1] (RM = 0.0043mPa s μm−3), and the results were corroborated by COMSOL Multiphysics simulations.

C. Microfabrication

Microfluidic channel molds were fabricated using standard single and two-layer photolithography [14] to transfer the final channel designs from a photomask (Artnet Pro, formally CAD/Art Services, Inc.) onto a silicon wafer (100mm diameter; University Wafer), which was spin-coated with photoresist (SU-8; Kayaku Advanced Materials). The single assay chemotaxis devices and MCD cell injection layer were made using SU-8 2050 and 2025, respectively, and multilayer devices (micromixer validation channels, MCD dilution layer) were made using SU8-3050 and SU8-2025 for the main rectangular channels and herringbone ridges, respectively. The ridges of the micromixers [10] were applied by halting the first-layer photolithography after the first post-exposure bake (PEB), spin-coating a second layer of SU-8 photoresist onto the wafer, then completing the remainder of the photolithography processes as usual [14]. The ridges of the herringbone micromixer [10] extend over the main channel by ≈ 10 μm on both sides to account for misalignment during the multilayer photolithography. As fabricated, the final channel heights for the MCD dilution layer were 90−94.5 μm, 37−38.5 μm for the main channel and herringbone ridges, respectively, while the cell injection layer (Fig. 1e) was 71 − 73 μm high (Bruker’s DekTak).

The MCD was fabricated using two-layer soft lithography [3] with polydimethylsiloxane (PDMS; Sylgard 184) at a 10:1 (elastomer:curing agent) ratio. All channel wells were punched using a 1.5mm diameter biopsy punch (Integra). The dilution layer mold was first silanized. through vapor deposition [15] in a vacuum desiccator with 1-2 drops of tridecafluoro-1,1,2,2-tetrahydrooctyl trichlorosilane (Gelest Inc.) to help release the cast PDMS. Post-silanization, PDMS was poured onto both the cell injection layer and dilution layer molds and degassed in a vacuum chamber (≈ 1 hour) prior to curing (65◦C for ≈ 1 hour). The resulting PDMS dilution layer channel was first plasma bonded onto a standard thickness, double wide glass slide (75mm Å~ 50mm Å~ 1 mm; Fisherbrand) using a plasma oven (Plasma Etch Inc.), and subsequently heated on a hot plate at 110◦C for one hour to promote covalent bonding [3]. Next, the cell injection layer was plasma bonded on top of the dilution layer, with care taken to ensure the alignment of the fluid wells connecting the two layers). The assembled device was baked again on a hot plate at 110◦C for one hour. The PDMS-PDMS bond was found to be sufficiently strong for the relatively low pressure applications of the MCD [16]. All other devices (e.g., single assay chemotaxis devices and micromixer validation channels) were fabricated using single-layer soft lithography, where an individual PDMS device was molded and subsequently bonded to a standard microscope slide using the procedures described above.

D. MCD dilution, flow, and gradient generation performance

The performance of the fabricated MCD was validated using epifluorescence microscopy (Nikon Ti-E) with an aqueous fluorescent dye (fluorescein sodium salt, Sigma) of various concentrations (described below), and images of the dye distribution were captured at the midplane of the channels with a sCMOS camera (Zyla 5.5; Andor Technology). Fluorescein was chosen due to its similar diffusion coefficient with the chemostimulant serine [17].

1. Serial dilution

The primary function of the MCD dilution layer is to sequentially dilute the input chemical solution (C0) with buffer to generate four logarithmically decreasing concentrations (C1-4) for each of the observation channels (plus one control, C5 = 0). The dilution performance was quantified by injecting a solution with known fluorescein concentration (C0 = 1mM). The diluted concentration field, Ci(x,y), at each of the observation channels was then determined from the local measured dye intensity, Ii(x,y), which are linearly proportional, Ci(x,y) ∝ Ii(x, y). Fluorescence images were recorded upstream of the inlet before the three fluids made contact in each observation channel using a 20°ø (0.45 NA) objective. Due to the strong ten-fold dilution, pairs of images were acquired for adjacent channels with optimized exposure times to account for the finite dynamic range of the camera. The mean normalized concentrations provided for each observation region from the serial dilution process were reconstituted from the measured image intensity as follows:

[Equation in supplemental files.]

This resulting serial dilution followed the expected logarithmic (10-fold) dilution Ci/C0 = 10−i for i ∈ [0, 4] for which the system was designed. This measurement was performed for three different sets of applied driving pressures, which yielded nearly identical results and illustrated the robustness of the serial dilution process.

2. Stratification symmetry

The symmetry of the stratified chemostimulus and buffer distributions in the observation channel is critical to prevent bias in the chemotaxis measurements. As minor errors in the manufacturing process can alter this symmetry, the widths of the chemical, cell, and buffer streams in each observation channel were measured by flowing a fluorescein solution (C0 = 100 μM) in both the chemical and buffer inlets of the dilution layer as well as the buffer inlet of the cell injection layer. By flowing DI water only through the cell suspension inlet of the injection layer, a strong intensity contrast between the central cell band and the surrounding fluids was visualized in the observation channel. Images were recorded (10°ø, 0.3 NA objective), and the widths of the three fluid streams, wi, were measured based on their spatial intensity distribution. The normalized widths of the chemostimulus and buffer streams, w′i = wi/W, across all observation channels deviated from the original design width by only 2.6% and 1.8% on average, respectively, while the mean deviation in cell suspension stream width was just 3.0%.

3. Chemostimulus gradient consistency

Beyond ensuring the symmetry of the chemostimulus and buffer stratification, the time evolution of the resultant chemostimulus gradient must be consistent across each of the observation channels to accurately compare bacterial chemotactic responses. The chemical gradient evolution was measured by first flowing a fluorescein solution (C0 = 100 μM) through both the chemical and buffer inlets of the dilution layer and DI water through both inlets of the cell injection layer. Having independently verified the performance of the serial dilution process, this approach produces identical base concentrations for all observation channels, Ci = C0, and thus, enables easy comparison of the resulting concentration gradients in each channel. Upon halting the flow, an image was recorded (10Å~, 0.3 NA objective) in each observation channel every 5 s for approximately 9 min. The time evolution of the (normalized) spatial fluorescence intensity was measured to visualize the chemical gradient. The resulting concentration profiles were found to be highly consistent across the various observation regions and for various driving pressures.

E. Cell culturing

Vibrio alginolyticus (YM4; wild-type) from −80◦C stock were grown overnight in Marine 2216 media (Difco) by incubating at 30◦C and shaking at 600 revolutions per minute (RPM). The overnight culture was diluted 100-fold into fresh pre-warmed 2216 media and grown for three hours (30◦C, shaking at 600 RPM) to O.D. ≈ 0.2. 7 ml of culture was then washed and resuspended (1,500 RCF for 5 min) in 4ml of artificial seawater (ASW). Psuedoalteromonas haloplanktis (ATCC 700530) from −80◦C stock were grown overnight in Marine 2216 (Difco) media by incubating at room temperature and shaking at 100 RPM [18]. Escherichia coli (MG1655) from −80◦C LB stock were grown overnight in Terrific Broth (TB, Sigma Aldrich) by incubating at 34◦C and shaking at 220 RPM [18]. The overnight culture was diluted 100-fold into fresh prewarmed TB media, and grown for approximately three hours (34◦C, shaking at 220 RPM) to O.D. ≈ 0.5. 8 ml of culture was then washed three times and resuspended (4,000 RCF for 5 min) in 4 ml of motility buffer (10mM potassium phosphate, 0.1mM EDTA, 10mM NaCl, pH 7, filter sterilized 0.2 μm.). 16 ml of culture was washed twice and resuspended (1,200 RCF for 5 min) in 6 ml of ASW.

F. Media and chemostimulants

Artificial seawater (ASW) was prepared following the NCMA ESAW Medium recipe, which was adapted from [19] and modified [20]. ASW was used as the bugger and the chemical solvent for chemotaxis assays for both V. alginolyticus and P. haloplanktis, while motility buffer (see above) was used for chemotaxis experiments, specifically: serine (S4500), phenol (P1037), and leucine (L7875).

G. Microfluidic chemotaxis assays

Prior to use, the MCD was pre-treated by flowing a 0.5% (w/v) bovine serum albumen solution (BSA; Sigma Aldrich) to reduce cell adhesion to the microchannel surfaces. The device was flushed for over 10 min prior to first use with the cell, chemostimulus, and buffer suspensions. For chemotaxis assays, fluid flow was driven by a single pressure controller (Elveflow OB1; 1mbar = 100Pa): pin,1−2 = 200mbar (dilution layer) and pin,3−4 = 140mbar (cell injection layer). Pressures were scaled down to 100mbar and 70mbar, respectively, for P. haloplanktis experiments. Between each chemotaxis assay, the fluid inputs were flowed for a minimum of 2 min to stratify cell, chemostimulus, and buffer streams in the observation channels. Upon halting the flow, a monotonic concentration profile was established in each observation channel due to the diffusion of the chemostimulus. The spatio-temporal evolution of the bacterial distribution was determined by imaging the cells using phasecontrast microscopy (10°ø, 0.3 NA objective; Tikon Ti-E) with a sCMOS camera (Zyla 5.5, Andor Technology) for approximately 10 min. An automated computer-controlled stage was used to cyclically move the microscope field of view to each observation channel 75 times, producing an effective imaging period of 8 s for each observation channel. Each experiment was replicated at least three times with the same culture and repeated on different days with freshly grown cells. For validation of the MCD, a conventional single assay (SA) microfluidic device was designed with a similar geometry to the individual MCD observation channels. Specifically, the SA devices had three inlets (width, 0.5mm) which merged in a single observation channel. The devices were fabricated in a single layer using soft lithography (see above), and they were pre-treated with a 0.5% (w/v) BSA solution and flushed with ASW prior to experiments. The SA chemotaxis devices were used to validate the MCD for the well-established chemotactic behavior of V. alginolyticus to the chemoattractant serine (Sigma). The three inlets of the single assay device carried the chemoattractant dissolved in ASW, V. alginolyticus suspended in ASW, and ASW, respectively. The three solutions were flow stratified for a minimum of 2 min using a syringe pump (Harvard Apparatus), whereby flow rates were controlled by syringe size to maintain a 4:1:4 ratio of the stream widths. In an identical manner to the MCD, a chemostimulus gradient develops in the channel via diffusion, and the chemotactic response of the cell population was observed over time. Imaging was performed with phase-contrast microscopy (4°ø, 0.13 NA objective; Nikon Ti-E) at 1 fps over the course of approximately 10 min using a CMOS camera (Blackfly S, Teledyne FLIR).

H. TEM imaging

Prior to use, the MCD was pre-treated by flowing a 0.5% (w/v) bovine serum albumen solution (BSA; Sigma Aldrich) to reduce cell adhesion to the microchannel surfaces. The device was flushed for over 10 min prior to first use with the cell, chemostimulus, and buffer suspensions. For chemotaxis assays, fluid flow was driven by a single pressure controller (Elveflow OB1; 1mbar = 100Pa): pin,1−2 = 200mbar (dilution layer) and pin,3−4 = 140mbar (cell injection layer). Pressures were scaled down to 100mbar and 70mbar, respectively, for P. haloplanktis experiments. Between each chemotaxis assay, the fluid inputs were flowed for a minimum of 2 min to stratify cell, chemostimulus, and buffer streams in the observation channels. Upon halting the flow, a monotonic concentration profile was established in each observation channel due to the diffusion of the chemostimulus. The spatio-temporal evolution of the bacterial distribution was determined by imaging the cells using phasecontrast microscopy (10°ø, 0.3 NA objective; Tikon Ti-E) with a sCMOS camera (Zyla 5.5, Andor Technology) for approximately 10 min. An automated computer-controlled stage was used to cyclically move the microscope field of view to each observation channel 75 times, producing an effective imaging period of 8 s for each observation channel. Each experiment was replicated at least three times with the same culture and repeated on different days with freshly grown cells. For validation of the MCD, a conventional single assay (SA) microfluidic device was designed with a similar geometry to the individual MCD observation channels. Specifically, the SA devices had three inlets (width, 0.5mm) which merged in a single observation channel. The devices were fabricated in a single layer using soft lithography (see above), and they were pre-treated with a 0.5% (w/v) BSA solution and flushed with ASW prior to experiments. The SA chemotaxis devices were used to validate the MCD for the well-established chemotactic behavior of V. alginolyticus to the chemoattractant serine (Sigma). The three inlets of the single assay device carried the chemoattractant dissolved in ASW, V. alginolyticus suspended in ASW, and ASW, respectively. The three solutions were flow stratified for a minimum of 2 min using a syringe pump (Harvard Apparatus), whereby flow rates were controlled by syringe size to maintain a 4:1:4 ratio of the stream widths. In an identical manner to the MCD, a chemostimulus gradient develops in the channel via diffusion, and the chemotactic response of the cell population was observed over time. Imaging was performed with phase-contrast microscopy (4°ø, 0.13 NA objective; Nikon Ti-E) at 1 fps over the course of approximately 10 min using a CMOS camera (Blackfly S, Teledyne FLIR).

1e:T52b,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. 1f:T96c,NSF Award Abstract: Drifting photosynthetic microbes in surface ocean waters carry out nearly half of global carbon (C) fixation, both supporting the marine food web and reducing atmospheric carbon dioxide (CO2) levels. The fate of C in ocean ecosystems is controlled by myriad individual interactions within a highly interconnected planktonic food web, the sheer complexity of which has hindered predictive understanding of global C cycling. Chemical cues govern microbial interactions, and during infection, marine viruses manipulate the metabolism of phytoplankton and bacteria, facilitating the release of dissolved organic matter from infected cells. This research aims to determine how viral metabolic reprogramming of and organic matter release from intact, infected phytoplankton influences microbial interactions and C cycling. The interdisciplinary, collaborative nature of the project will enable direct training of two postdoctoral researchers, one graduate student, and undergraduate students in viral ecology, microfluidics, and metabolomics. An educational outreach program that engages middle school students in hands-on, high speed imaging of microbes will be expanded, and the project will culminate in a three-day workshop to advance the application of microfluidic devices and mass spectrometry analyses in microbial ecology. The overarching hypothesis behind this research is that viral infection alters the chemical landscape of intact, infected picophytoplankton cells, attracting neighboring chemotactic bacteria and protistan zooplankton, and altering C flux pathways. To test this idea, a series of linked multi-scale laboratory-based experiments will be run to 1) Characterize the response of diverse model marine microbes to dissolved organic matter (DOM) released from intact, virus-infected picophytoplankton using microfluidics-based chemotaxis assays, 2) Identify key viral-derived DOM compounds eliciting chemotactic responses using stable isotope labeling, metabolomics analyses, and chemotaxis assays, and 3) Quantify micron-scale cross-trophic encounter dynamics and evaluate their impact on bulk-scale C cycling using liter-scale measurements of C dynamics linked to high spatiotemporal resolution live imaging of microbial food webs. The ultimate goal of the project is to develop a mechanistic understanding of the role of intact, virus-infected cells in oceanic C cycling.6:["$","$L1b",null,{"dataset":{"_id":"885701","description":"$1c","problem":"","acquisition":"$1d","processing":"","bcodmoProcessing":"Supplemental files were reformatted from multi-tab Excel files to bundles of zipped CSVs\r\nSupplemental file tables were reformatted so that each individual spreadsheet contains one discrete data table, each containing one observation level value per cell.\r\n\r\nIn the primary data file, the original channel position-related column names were positive or negative float values. 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