Abstract
Isolation and concentration of fungi in the blood improves sensitivity of the polymerase chain reaction (PCR) method to detect fungi in blood. This study demonstrates a sheathless, continuous separation and concentration method of candida cells using a viscoelastic fluid that enables rapid detection of rare candida cells by PCR analysis. To validate device performance using a viscoelastic fluid, flow characteristics of 2 μm particles were estimated at different flow rates. Additionally, a mixture of 2 μm and 13 μm particles was successfully separated based on size difference at 100 μl/min. Candida cells were successfully separated from the white blood cells (WBCs) with a separation efficiency of 99.1% and concentrated approximately 9.9-fold at the center outlet compared to the initial concentration (~2.5 × 107 cells/ml). Sequential 1st and 2nd concentration processes were used to increase the final number of candida cells to ~2.3 × 109 cells/ml, which was concentrated ~92-fold. Finally, despite the undetectable initial concentration of 101 CFU/ml, removal of WBCs and the additional buffer solution enabled the quantitative reverse transcription (RT)-PCR detection of candida cells after the 1st concentration (Ct = 31.43) and the 2nd concentration process (Ct = 29.30).
Highlights
It is expected that the detection of extremely low concentration fungi in blood could be significantly improved by removing nonspecific binding with fungal primers/probes of DNA contaminants of other nucleated blood cells in the sample[13,14,15]
Compared to previous passive methods, such as inertial microfluidics performed in Newtonian fluid, viscoelastic particle/cell manipulation can be achieved over a wide range of flow rates
An increase in the flow rate increased the Weissenberg number (Wi), i.e., elastic force increased, and suspended particles were more tightly focused at the equilibrium positions. These results indicated significant consistency with previous studies in which off-center focusing into two streams was reported in numerical studies (β ≥ 0.25) and microfluidic experiments involving the use of viscoelastic fluid (β = 0.3 with El = 0.028 and 0.11)
Summary
It is expected that the detection of extremely low concentration fungi in blood could be significantly improved by removing nonspecific binding with fungal primers/probes of DNA contaminants of other nucleated blood cells in the sample[13,14,15]. The non-uniform distribution of the first normal stress difference (N1) in a non-Newtonian fluid can laterally drive suspended particles/cells into a simple straight microchannel This was applied to three-dimensional particle focusing[38] and particle separation based on size differences[40]. In our previous work, the circular shaped channel or high aspect ratio (AR = height/width) microchannel were used to align particles/cells along the inner walls at the bifurcation channel without the aid of sheath flow before the separation process[14,49,50] Using these devices, cells of different sizes could be separated with high efficiency and high purity, the channel dimension should be carefully determined considering the lateral displacement of target cells in order to achieve high separation efficiency.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
