(179c) Enrichment of Small Cell Populations from Large Sample Volumes Using 3D Carbon-Electrode Dielectrophoresis

Enrichment of small cell populations
from large sample volumes using 3D Carbon-electrode Dielectrophoresis

Monsur Islam and Rodrigo Martinez-Duarte

Mechanical
Engineering Department, Clemson University, Clemson, SC, USA

Here
we present the enrichment of a cell population from a large sample volume using
3D carbon-electrode dielectrophoresis. Cell enrichment and purification is
needed in a number of diagnostic applications where the particle of interest is
highly diluted in the sample volume and/or is contained in a media that hinders
its detection. There is a need for technologies capable of processing large
sample volumes in order to extract few targeted particles. An immediate
application is the isolation of pathogens in patients suffering from sepsis,
where a low pathogen load can eventually lead to death in up to 30% of the
patients. Here, we take initial steps towards isolating a diluted targeted
particle population from a relatively large sample volume. We process half a
milliliter of sample featuring an increasingly diluted (10⁴-10³)
yeast cell suspension in little more than an hour. The experimental protocol
facilitates the trapping and purification of yeast cells to obtain an enriched
cell fraction in a relatively short amount of time.

The
fabrication of the 3D carbon-electrode DEP (carbonDEP) device is shown in Fig.
1 and has been detailed by Martinez-Duarte et al. [1]. Yeast cells
were grown overnight at room temperature in 0.01 M PBS. The media used for
experimental cell suspension and buffer contained 15% sucrose, 0.3% dextrose
and 0.1% BSA in DI water; and featured a conductivity 12.62 µS/cm. The
experimental samples were obtained by pelleting the cell culture using
centrifugation at 5000 rpm for 5 min and re-suspending the cells in the experimental
media. Dilution was used to obtain targeted concentrations (10³ or
10⁴).

The
yeast suspension was then flowed through the carbonDEP device at 10 µl/min
using a syringe pump. The carbon electrodes were stimulated with a sinusoidal
signal of magnitude 20 Vpp and 100 kHz frequency to implement a positiveDEP
trapping force. After processing 500 µl sample volume, the inflow was changed
to buffer media to wash the trapped cells using 100 ul of buffer. At that
point, the electrodes were turned off to release the trapped cells and elute
them for their retrieval at the end of the device. A total of 34 fractions (20
µl each) were retrieved from the chip and were divided into 1) sample, where
yeast cells were retained from the sample solution (fractions 1-25); 2) Washes,
where trapped cells were washed with  buffer (fractions 26-30); and 3) Elutes,
where trapped cells were eluted upon turning off the electric field (fractions
31-34 ).  Results are shown in Fig. 2 for both cell concentrations used (4.525
X 10³ and 4.78 X 10⁴ cells/ml). A hemocytometer, with a
limit of detection of 10⁴ cells/ml, was used to obtain cell
concentration. In the case of 10³ initial cell concentration, the
sample is enriched up to the order of 10⁴ cells/ml. Ongoing work is first
to study how much the cell population can be enriched when the initial sample
has low cell concentrations, i.e. 10² and 10¹
cells /ml; and second to determine the maximum sample volume that could be
processed in a time that is acceptable for practical applications.

Reference:

1. Martinez-Duarte, R.;
Renaud, P.; Madou, M. J. A novel approach to dielectrophoresis using carbon
electrodes. Electrophoresis 2011, 32, 2385?92.

Fig 1. Fabrication process of
Carbon-electrode DEP device

Fig 2. Yeast cell concentration
in the different fractions obtained from the experimental protocol detailed in
the text. Note the peak on fractions 30-35 showing the elution of an enriched
sample after DEP treatment, regardless of the original cell concentration
(CONTROL fraction). Dotted line represents the experiment with 10⁴
cell suspension. Red solid line denotes the experiment using the 10³
cell suspension. Lines are the average values with n=3. Error bars are
illustrated.