Microsample preparation by dielectrophoresis: isolation of malaria

Abstract

An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.

Fig. 1

(A) The interdigitated electrode array, of 500 mm² active area, was comprised of 124 parallel elements having fingers with a characteristic dimension of 100 μm. Alternate elements were connected to bus lines on opposite edges of the chamber. The volume of the interdigitated section of the array was 50 μL. (B) Chamber construction for the DEP measurements. (C) The spiral microelectrode array, of 2 mm² area, was comprised of 5 complete turns of four parallel spiral elements 20 μm in width and spacing. For clarity only one-and-a-half turns of the spiral array is shown in (B). The active volume of sample above the spiral was 0.2 μL. (D) A cross-section of part of the spiral array showing the dielectrophoretic, gravitational and hydrodynamic forces acting on cells on the spiral electrode.

Fig. 2

Views of a culture of strain T9/94 RC17 P. falciparum-infected erythrocytes containing approximately 1.1% parasitised cells suspended in 8.5% sucrose + 0.3% dextrose suspending medium on an interdigitated electrode with an applied field of 5 V_p-p at 200 kHz. Approximately 5 × 10⁶ erythrocytes were injected into the chamber. Both low-level bright field and epifluorescence illumination were provided. (A) Cells containing parasites exhibited a green fluorescence due to uptake of the potentiometric dye DiOC₆ (3) into the parasite interior from the suspending medium and show brightly in the figure. (B) A magnified view under epifluorescence illumination confirmed that 95% of parasitised cells were repelled from the high field regions and could be washed free by flowing suspending medium.

Fig. 3

Erythrocytes containing approximately 5% parasitised cells on a spiral electrode array under the same suspending medium and staining conditions used in Fig. 2. Both low-level bright field and epifluorescence illumination were provided. (A) Prior to the application of a travelling electrical field, parasitised cells (arrows) were spread throughout the sample. (B) Application of four phase signals to the spiral electrode elements (3 V_p-p, 2 MHz) caused normal erythrocytes to be trapped at the electrode edges while parasitised cells were levitated and carried towards the centre of the spiral by the travelling field.

Fig. 4

(A) The frequency response of cell levitation above the spiral electrode array was measured by microscope for (○) normal and (■) parasitised erythrocytes for stationary electrical fields (the signals applied to the four arms of the spiral electrode were phased at 0°, 180°, 0°, and 180°, respectively, so that there was no travelling wave component). (B) The frequency dependence of the cell lateral velocity induced by travelling wave DEP when four-phase excitation was established on the spiral electrode. Parasitised cells exhibited a range of frequency responses revealing a large variance in their dielectric characteristics, possibly reflecting inter-cell variations and different stages of parasite development. The solid lines show the levitation and translation behaviours predicted on the basis of the mean cellular dielectric parameters derived in Fig. 5. Spectra for T-lymphocytes (----) calculated from electro rotation data (Yang et al., 1999) are shown for comparison.

Fig. 5

Mean dielectric parameters for normal and parasitised cells derived from iterative fitting of shell models as described in the text. Parasitised cells had a very low internal conductivity compared with normal erythrocytes, an indication they had suffered almost complete loss of ions to the low-conductivity suspending medium. The much higher membrane conductivity of parasitised cells compared with normal cells reflects the lowering of membrane barrier function that accounted for this loss. Despite the ion leakage out of their host cells, the parasites retained their internal ions as reflected by their high internal conductivity.