Simple, Cost-Effective 3D Printed Microfluidic Components for Disposable, Point-of-Care Colorimetric Analysis

The fabrication of microfluidic chips can be simplified and accelerated by three-dimensional (3D) printing. However, all of the current designs of 3D printed microchips require off-chip bulky equipment to operate, which hindered their applications in the point-of-care (POC) setting. In this work, we demonstrate a new class of movable 3D printed microfluidic chip components, including torque-actuated pump and valve, rotary valve, and pushing valve, which can be operated manually without any off-chip bulky equipment such as syringe pump and gas pressure source. By integrating these components, we developed a user-friendly 3D printed chip that can perform general colorimetric assays. Protein quantification was performed on artificial urine samples as a proof-of-concept model with a smartphone used as the imaging platform. The protein was quantified linearly and was within the physiologically relevant range for humans. We believe that the demonstrated components and designs can expand the functionalities and potential applications of 3D printed microfluidic chip and thus provoke more investigation on manufacturing lab-on-a-chip devices by 3D printers

P-body movement is directional and correlates with cell size.

<p>(<b>A</b>) Reversibility analysis of the movement. A reversibility rate of PB movement was calculated for each strain (wild-type, <i>she2</i>Δ, <i>she3</i>Δ and <i>myo4</i>Δ) for 11, 11, 12 and 16 cells respectively, as explained in the main text. Non-zero values for wild-type typically occur due to a few ‘miss-clustered’ points during the transition phase. (<b>B</b>) Cluster betweenness. By relating points within a cluster to points within the other cluster we can quantitatively compare the separation of PB movement between the strains (Materials and Methods). Similar to the temporal analysis by <i>R_rev</i> we see a significant difference between the wild-type and mutant strains. (<b>C</b>) Area of she2Δ cells at the time of budding that had received a PB (blue), not received a PB but later formed one <i>de novo</i> (gray), and completely lacked a detectable PB (red). The population of cells that received a PB during cell division were 33% larger than cells that did not (p<0.03).</p

P-body localization classes in wild-type, <i>myo4</i>Δ, and <i>she2</i>Δ cells (for n = 42, 48, 44 p-bodies).

<p>P-body localization classes in wild-type, <i>myo4</i>Δ, and <i>she2</i>Δ cells (for n = 42, 48, 44 p-bodies).</p

Description of the analysis of p-body dynamics, an example from one cell.

<p>(<b>A</b>) Time lapse imaging of a p-body during cell division. A wild type strain expressing Edc3-GFP grown in 2% glucose to logarithmic phase was loaded into the microfluidic device. Minimal medium containing 0.1% glucose was flowed for 10 hours and images were acquired every 60 sec in bright field and fluorescent light. A sequence of images spanning 140 min was extracted from the entire experiment. 3 typical images from the time-lapse experiment are shown for the specified time points. In the last panel, the path of the p-body during the cell division is shown. For ease of visualization, the tracks in the microscopy image show PB locations every 5 min. as opposed to every 1 min (the rate of imaging) in Fig. 2B. Scale bar is 5 µm. (<b>B</b>) Spatial coordinates of the p-body where color code identifies the observation time. Dashed circles separate the 2 clusters, cluster 1 in black and cluster 2 in red. (<b>C</b>) Cluster analysis. Points in (B) where classified in 2 clusters and the corresponding cluster number is shown over time. This clearly demonstrates that a p-body is first localized in one cluster, in this case within the mother cell, (black cluster 1) and is then unidirectional transported to the daughter cell (red cluster 2).</p

P-body transport to the daughter cell is dependent on Myo4p, She2p and She3p.

<p>(<b>A–C</b>) Sequence of images tracking p-bodies in strains lacking Myo4p, She2p or She3p. Experiments were performed and plotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099428#pone-0099428-g002" target="_blank">Fig. 2</a>. One cell is shown here for the <i>she2</i>Δ and <i>she3</i>Δ strains and two cells are shown for the <i>myo4</i>Δ strain. 3 images from the time-lapse experiment are shown. The last panel summarizes the path of the p-body during cell division. Scale bar, 2 µm. (<b>D–F</b>) Spatial coordinates of p-bodies from cells shown in (<b>A</b>) where color corresponds to time. (<b>G–I</b>) Cluster analysis performed in each cell, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099428#pone-0099428-g002" target="_blank">Fig. 2C</a>. In the mutants p-bodies do not move to the daughter cell and p-bodies change from cluster 1 to cluster 2 multiple times.</p

Microfluidics device for studying p-body localization in yeast.

<p>(<b>A</b>) The device is a simplified version of a published design <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099428#pone.0099428-Falconnet1" target="_blank">[23]</a> that consists of 16 chambers in a 4-by-4 matrix with four media inputs accessible via rows and four cell inputs accessible via columns. The device has two layers: the control layer (red) and flow layer (blue). Cells and media are transported within the flow layer channels with the direction controlled by actuating overlaying control layer valves. Parts of the device are marked as follows: 1) cell-loading inlets, 2) chambers where cells are trapped, 3) medium inputs, 4) multiplexer to deliver medium to specific chambers, 5) waste outlet. (<b>B</b>) Close up of 4 chambers with the controlling valves. (<b>C</b>) Micrograph of a chamber with cells trapped, seen at a 4x magnification in bright field light illumination. Scale bar is 20 µm. (<b>D</b>) P-body formation in response to low glucose. Graph shows results from 2 experiment controls (black and grey) in the presence of 2% glucose medium and 2 experiments (purple and red) where p-bodies were induced in the presence of 0.1% glucose medium (4 experiments, total number of cells: 400). Images were taken every 60 seconds over 200 min, in bright field and fluorescent light. The resulting cell numbers were averaged over 20 min periods to filter noise. Custom software for automated quantification of p-bodies per cell was used (see Methods for a detailed description of the analysis).</p