# A Low-Cost Laser-Prototyped Microfluidic Device for Separating Cells and Bacteria
## 4 Discussion  
The presented device features a single inlet for sample injection and device operation which provides simplicity in terms of required syringe pumps and reduces the footprint of the separation process. In many of the cell/bacteria separation works reported in the literature sheath flows are generally required for sample focusing and separation. In our device, the first module (serpentine) did not provide an observable particle separation, but it served the purpose of focusing the particles before they entered the spiral section of the device. In the spiral section of the device, larger particles stayed mostly close to the inner wall. This is due to the fact that Dean drag acts and pushes particles towards the inner wall and for larger particles a larger force is exerted which pushes the larger particles further than the smaller particles. Different inlet flow rates were tested to quantitatively analyze the particle separation performance of the device, with particles collected from outlets A and B being analyzed. Inlet flow rates ranging from 600 to 1000 \(\upmu\)L/min demonstrated higher separation purity and efficiency. At 1000 \(\upmu\)L/min, the majority of 1 \(\upmu\)m particles exited from outlet A, while 5 \(\upmu\)m particles exited from outlet B, in line with predictions based on inertial focusing behavior. Standard hemocytometer counting was performed to characterize the collected  
Figure 6: Separation of yeast cells and 1 \(\upmu\)m particles. (**a**) Cell and particle mixture before separation; (**b**) Sample collected from the outlet A; (**c**) sample collected from the outlet B; (**d**) purity of samples collected from the outlets A and B. Lower and higher box bounds are in the 25 th and 75 th percentiles, respectively; the line within the box is the median; and the lower and upper error lines are in the 5 th and 95 th percentiles, respectively. particles. Improved purities were observed at flow rates of 600, 800, and 1000 \(\upmu\)L/min, with the best performance at 800 \(\upmu\)L/min. Purity percentages were 93% for 1 \(\upmu\)m particles and 89% for 5 \(\upmu\)m particles from outlets A and B, respectively. Outlet B exhibited a higher level of contamination from 1 \(\upmu\)m particles in comparison to the contamination observed in outlet A caused by 5 \(\upmu\)m particles. This discrepancy can be attributed to the greater dispersion exhibited by the 1 \(\upmu\)m particles in contrast to the larger 5 \(\upmu\)m particles. It is worth noting that larger particles tend to be more effectively focused by the inertial and Dean flows. Separation efficiencies were 89% for 1 \(\upmu\)m particles and 93% for 5 \(\upmu\)m particles. The device was also tested for separating yeast cells and 1 \(\upmu\)m particles, achieving purities of 91% for yeast cells and 85% for 1 \(\upmu\)m particles, with separation efficiencies of 85% and 88%, respectively. While these are separation efficiencies comparable to the results reported in the literature, the simplicity and lower cost of our device fabrication and operation are significant factors that can greatly contribute to the ecosystem of disposable on-chip applications. The presented device only costs about 2 US dollars including the operational costs. It can also be possible to further reduce this cost by batch fabrication of larger numbers of devices. This is significantly lower than traditional microfluidics devices considering the cost of using clean-room fabrication facilities. An important limitation of the presented device is delamination at high flow rates due to increased pressure inside the channels. We observed that over about 3000 \(\upmu\)L/min delamination started, and the device failed. It is also important to note that over about 2000 \(\upmu\)L/min, slight observable leakage of the fluids occurred outside the fluidic channels. As a matter of fact, separation efficiency monotonically decreased above 1000 \(\upmu\)L/min, and this is likely due to the emergence of leaking that negatively affected inertial focusing of particles.