425446 Enrichment of Small Cell Populations from Large Sample Volumes Using 3D Carbon-Electrode Dielectrophoresis

Monday, November 9, 2015: 3:45 PM
Ballroom E (Salt Palace Convention Center)
Monsur Islam and Rodrigo Martinez-Duarte, Mechanical Engineering, Clemson University, Clemson, SC

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.

 


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