Viscoelastic Separation and Concentration of Fungi from Blood for Highly Sensitive Molecular Diagnostics

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 × 10 7 cells/ml). Sequential 1st and 2nd concentration processes were used to increase the final number of candida cells to ~2.3 × 10 9 cells/ml, which was concentrated ~92-fold. Finally, despite the undetectable initial concentration of 10 1 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).

Three-Dimensional Numerical Simulation of Particle Focusing and Separation in Viscoelastic Fluids

“…The focusing equilibrium position of small particles is at the center of the channel, while large particles migrate towards off-center equilibrium positions. This is consistent with the results of Liu et al [ 15 ] and Nam et al [ 16 ]. The channel with smaller aspect ratios is also studied.…”

“…In the viscoelastic fluid of straight microchannel, equilibrium position of particles may differ with various diameters, which can be utilized to particle separation [ 15 , 16 ]. To study the different size particle performance in various AR microchannels, the trajectories of particle with diameters of 6 and 12 μm are calculated and systematically analyzed.…”

“…In their research, small particles are focused at the center of the channel, while large particles are focused near the side walls of the channel. Nam et al [ 16 ] realized the separation of white blood cells and candida cells in the channel with AR = 1/2, and then used the expansion channel to further enlarge the distance between the two kinds of cells.…”

Characterization of the Dielectrophoretic Response of Different Candida Strains Using 3D Carbon Microelectrodes

“…A multi-stage protocol is preferred when attempting to isolate potential targets of interest from a blood sample in clinical diagnostics. For example, centrifugation can enable a first rapid coarse separation of serum, buffy coat and red blood cells (RBC) [48], with the Candida cells expected to be in the buffy coat [49]. Buffer exchange protocols common in clinical diagnostics can then be implemented to re-suspend the cellular content of the buffy coat in a buffer optimized for DEP and any other downstream processing.…”

Sheathless High-Throughput Circulating Tumor Cell Separation Using Viscoelastic non-Newtonian Fluid

“…In this study, we show the sheathless, high-throughput separation of CTCs using a viscoelastic fluid in a low aspect ratio (AR = height/width) microchannel. Size-dependent equilibrium positions in a straight rectangular microchannel with a low AR have previously been examined using a viscoelastic fluid [37,38,39]. However, to the best of the authors’ knowledge, previous studies have not used a low AR microfluidic device for continuous separation of CTCs from lysed blood samples.…”