Microfluidic devices for the rapid and automated processing of sample populations

Microfluidic devices for the rapid and automated processing of sample populations are provided. Described are multiplexer microfluidic devices configured to serially deliver a plurality of distinct sample populations to a sample processing element rapidly and automatically, without cross-contaminating the distinct sample populations. Also provided are microfluidic sample processing elements that can be used to rapidly and automatically manipulate and/or interrogate members of a sample population. The microfluidic devices can be used to improve the throughput and quality of experiments involving model organisms, such as C. elegans.Board of Regents, University of Texas Syste

An Automated Microfluidic Multiplexer for Fast Delivery of C. elegans Populations from Multiwells

Automated biosorter platforms, including recently developed microfluidic devices, enable and accelerate high-throughput and/or high-resolution bioassays on small animal models. However, time-consuming delivery of different organism populations to these systems introduces a major bottleneck to executing large-scale screens. Current population delivery strategies rely on suction from conventional well plates through tubing periodically exposed to air, leading to certain disadvantages: 1) bubble introduction to the sample, interfering with analysis in the downstream system, 2) substantial time drain from added bubble-cleaning steps, and 3) the need for complex mechanical systems to manipulate well plate position. To address these concerns, we developed a multiwell-format microfluidic platform that can deliver multiple distinct animal populations from on-chip wells using multiplexed valve control. This Population Delivery Chip could operate autonomously as part of a relatively simple setup that did not require any of the major mechanical moving parts typical of plate-handling systems to address a given well. We demonstrated automatic serial delivery of 16 distinct C. elegans worm populations to a single outlet without introducing any bubbles to the samples, causing cross-contamination, or damaging the animals. The device achieved delivery of more than 90% of the population preloaded into a given well in 4.7 seconds; an order of magnitude faster than delivery modalities in current use. This platform could potentially handle other similarly sized model organisms, such as zebrafish and drosophila larvae or cellular micro-colonies. The device’s architecture and microchannel dimensions allow simple expansion for processing larger numbers of populations.The authors would like to thank the National Institutes of Health (www.nih.gov) for its generous support of this research. Specifically, the grants that made this work possible are the NIH Director's Transformative Award (NIH R01 AG041135), NIH R21 NS067340, and NIH R01 NS060129. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biomedical EngineeringElectrical and Computer EngineeringMechanical Engineerin

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Automated worm population delivery sequence.

<p>A) Schematic of the device showing areas active during the sequence example as the worms are pre-staged at the first set of control valves. An image of pre-staged <i>C</i>. <i>elegans</i> worms is below the schematic (scale bar is 1 mm). B) Illustration of all steps for one full sequence cycle. Step 1: Appropriate valves open as the gasket is pressurized to send <i>Well</i> 1’s population to the main channel, where <i>Main </i><i>Channel </i><i>Flush</i> then accelerates the worms’ transport to the main exit. Step 2: Excess worms are cleared from the main channel towards the <i>Main </i><i>Outlet</i> via flow from <i>Main </i><i>Channel </i><i>Flush</i>. Step 3: Flow from <i>Exit </i><i>Flush</i> delivers the worms from the <i>Main </i><i>Outlet</i> to an off-chip location. Step 4: “Flushback”; <i>Exit </i><i>Flush</i> flow is redirected backwards to clear any remaining worms in the well channel back to <i>Well 1</i>. This step is executed on <i>Wells 1-4</i> only after finishing Steps 1-3 on each of them. C) Timings for each step.</p

Population mixing eliminated during automated delivery at 20 psi (~138 kPa).

<p>The graphs show the fraction of animals collected after delivery from a given well that are of the same strain initially loaded into the well. The actual average number of collected worms over the average number of those initially loaded is indicated above each bar. A) Four distinct strains loaded in each <b>row</b>. B) Four distinct strains loaded in each <b>column</b>. A corresponding color-coded schematic on the right of both graphs indicates into which wells the strains were loaded at the beginning of both experiments. Each color represents a single type of strain.</p

<i>Population Delivery Chip</i> design.

<p>A) A schematic of the device indicating the flow layer (blue) and control valve layer (red). There are 16 on-chip wells arranged in a 96-well plate format for initial loading of different worm populations. Columns and wells of the array are numbered according to order of delivery. Valves <i>V1-V8</i> are multiplexer control valves and <i>V9-V12</i> control flow in the main channel. B) An image of the device with its microfluidic channels loaded with food coloring dye, showing the flow layer (green) and control valve layer (orange) (scale bar ~1mm). C) A macro-scale view of the device with the 16-well array indicated by the yellow dashed lines and a schematic of worms loaded into one of the conical wells. D) A macro-scale view of the entire chip/gasket system with pressurized input lines in the experimental setup.</p

Worm population delivery as a function of applied pressure.

<p>The fraction of worm populations loaded in 4 representative on-chip wells from 4 different columns of the <i>Population </i><i>Delivery </i><i>Chip</i> that are delivered to the outlet of the device as a function of pressure applied at the gasket and the <i>Main </i><i>Channel </i><i>Flush</i>.</p