(113g) Separation of Candida Cells Using 3D Carbon-Electrode Dielectrophoresis

Separation of Candida Cells using 3D Carbon-electrode
Dielectrophoresis

Jordon Gilmore, Monsur Islam and Rodrigo
Martinez-Duarte

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

Here we present preliminary results
towards separation of Candida cells using 3D carbon-electrode
dielectrophoresis (DEP). In particular, two Candida species have become
the most isolated in instances of systemic candidiasis, accounting for nearly
75% of all cases.¹ These species, Candida
albicans and Candida glabrata are usually found in the oral cavity
and intestinal tract of healthy humans. However, given certain conditions, both
of these species may become pathogenic.

The goal of this work is to employ DEP
separation techniques with carbon electrodes to isolate C. albicans and C.
glabrata from a heterogeneous solution comparable to a biological sample
from the clinical setting. The authors demonstrate successful trapping of each species
by positive dielectrophoresis (pDEP). This is a preliminary step in the goal to
separate Candida cells by species as part of a point-of-care diagnostic
tool for determining the dominant Candida species during suspected
candidiasis. This is important given the discovery that all Candida species
are not responsive to the same treatments (i.e. C. glabrata, which do
not respond to the common fluconazole treatments of C. albicans – based
candidiasis ²).

Strains of C. albicans and C.
glabrata were cultured in Yeast Malt Broth (YMB) supplemented with a 40%
glucose solution at a 1:100 glucose to YMB ratio. The YMB media was prepared
and acidified to a pH of 3.5 by the addition of Citric Acid. Cells were grown
in suspension and dynamic incubation (shaking at 215 rpm) at 28°C for two days.
Average diameters of C. albicans and C. glabrata were 4.68 ± 0.97
µm and 3.01 ± 0.49 µm, respectively.

For DEP experiments, the experimental
media used for cell suspension and buffer solution was a 15% sucrose, 0.3%
dextrose, and 0.1% bovine serum albumin (BSA) mixture in DI water. Conductivity
of the media was 12.62 µS/cm. To prepare the experimental cell suspension, 150
µl of the cell culture was suspended in 4 ml of the experimental media followed
by washing and re-suspending in experimental media. The cell concentrations of
the cell suspensions were 7.18 x 10⁶ cells/ml and 1.92 x 10⁷
cells/ml for C. albicans and C. glabrata respectively.

The fabrication of
the carbon-electrode DEP device has been detailed by our group in previous
publications³. The
cells were flowed through the DEP chip at a constant flow rate of 2.5 µl/min
using a syringe pump. The electrodes were polarized with a sinusoidal signal
with constant magnitude of 20 V_pp and varying frequencies ranging
from 10 kHz to 5 MHz. The effect of the frequencies on the pDEP has been
investigated for both species using an optical microscope. The qualitative
results based on the microscopic observation were plotted in Figure 1. For C.
albicans the cross over frequency is in between 750 kHz to 1 MHz, whereas
the cross over frequency for C. glabrata is in between 2.5 MHz and 5
MHz. Also Figure 1 shows the difference in the cell trapping over the change of
the frequencies. Figure 2 shows the DEP trapping of the cells at 100 kHz
frequency.

As the size of C.
albicans and C. glabrata are close and they belong to same species,
their DEP response is quite similar.  Therefore, the ongoing work focuses on
determining the specific DEP parameters that allow for the separation of each
species. Separation of these species will allow for identification of
the primary cell type responsible for the candidiasis case, allowing physicians
to provide more optimized treatments.

Acknowledgements

The authors acknowledge Dr. Mark
Blenner’s research group from Chemical Engineering Department for assistance
with the cell culture.

References

(1)      Chakravarthi,
S.; Haleagrahara, N. A Comprehensive Review of the Occurance and Management of
Systematic Candidiasis as an Opportunistic Infection. Microbiol. J. 2011,
1 (1), 1–7.

(2)
     Hitchcock, C. A.; Pye, G. W.; Troke, P. F.; Johnson, E. M.; Warnock, D. W.
Fluconazole Resistance in Candida Glabrata. Antimicrob. Agents Chemother.
1993, 37 (9), 1962–1965.

(3)
     Martinez-Duarte, R.; Renaud, P.; Madou, M. J. A Novel Approach to
Dielectrophoresis Using Carbon Electrodes. Electrophoresis 2011, 32
(17), 2385–2392.

Figure 1: Qualitative indication of
positive DEP (pDEP) under microscopic observation for C. albicans and C.
glabrata for different frequencies

Figure 2: Trapping of (a) C. albicans
and (b) C. glabrata cells around the carbon electrodes. The DEP
parameters were 100 kHz frequency and 20 V magnitude. The images were captured
2 minutes after the electric field was turned on.