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.