# A Low-Cost Laser-Prototyped Microfluidic Device for Separating Cells and Bacteria
## 2 Materials and Methods  
### Device Fabrication and Operation  
The microfluidic device is designed to have a single inlet, a serpentine focusing section, and a spiral separation section with two outlets as shown in Figure 1. As for the serpentine section, similar designs in the literature were considered in terms of the number and dimensions of the serpentines that result in satisfactory particle focusing [33]. The spiral section was designed to have three loops with at least 2 mm distance between each loop to provide rigidity which is important during the assembly of the device. The three layers of the device were laser-cut and assembled via double-sided transparent tape (3M, Maplewood, MN, USA) on the bottom and top layers. A standard 100W CO\({}_{2}\) laser plotter (LF7010, Lazerfix, Konya, Turkiye) was used for PMMA cutting. This machine has a working area of 70 \(\times\) 100 cm and features a glass CO\({}_{2}\) tube laser. The control unit of the system is a Ruida 6442 module. Motion in the x and y axes is provided by Nema 34 stepper motors. The maximum feed rate of the laser plotter is 500 mm/s, and precision of the system is 0.01 mm/meter. The PMMA sheets in this study were cast acrylic sheets that provide higher optical clarity and better laser cutting compared to extruded acrylic sheets. Thicknesses of the bottom layer, the middle layer, and the top layer were 300 \(\upmu\)m, 100 \(\upmu\)m, and 3 mm, respectively. The top layer was chosen to be much thicker to provide at least 2 mm depth for the inlet and outlet tubes. Polyethylene tubings with a 1 mm outer diameter and 0.5 mm inner diameter were used for the inlet and outlet of the device. A five-minute epoxy glue (E340, Akfix, Istanbul, Turkey) was used to permanently fix the tubings to provide a better fluid-tight fitting that could resist high-pressure flow.  
For testing the device, 5 and 1 \(\upmu\)m polystyrene particles (Sigma Aldrich, St. Louis, MO, USA) were used with 10\({}^{7}\) and 3 \(\times\) 10\({}^{7}\) particles/mL, respectively. For simulating cell and bacteria separation, _Saccharomyces cerevisiae_ yeast cells (about 10\({}^{7}\) cells/mL) and 1 \(\upmu\)m polystyrene particles were used. Micorpatrices with similar sizes to bacteria were used mimic bacteria in the separation experiments. For the characterization of particle and cell counts, a standard hemocytometer was employed. For this, the sample containing the microparticles was diluted five times to achieve a lower concentration for easier characterization. The hemocytometer was placed under a microscope, and the grid lines within the counting chamber were focused upon. The microparticles were observed and counted using both automated and manual approaches for cross-check [48; 49]. For automated counting, images were thresholded and processed to better visualize the particles [48].  
A home-built syringe pump system was used to inject the cell/particle solution [50]. An inverted microscope (OX.2053-PLPH, Euromex, Arnhem, The Netherlands) and CMOS camera (HD, Euromex, Arnhem, The Netherlands) were used to capture images. For the yeast cells, the yeast growth medium was prepared by dissolving 10 g yeast extract, 20 g peptone, and 20 g dextrose in 1 L of sterile distilled water. The _Saccharomyces cerevisiae_ yeast culture was inoculated into 10 mL of the growth medium and incubated at 30 \({}^{\circ}\)C with mild shaking. The yeast cells were then collected by centrifugation at 4000\(\times\)\(g\) for 5 min. The supernatant was discarded, and the yeast cells were resuspended in 10 mL of sterile distilled water.