Microfluidic approaches to malaria detection

Abstract

Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.

Fig. 1

Visual representation of some of the main methodologies used in the diagnosis of malaria. While microscopic analysis is among the most competitive methods in common use today, the diagram reveals that genetic methods offer significant advantages for the future if they can be realized in an inexpensive micro total analysis format.

Fig. 2

A. Exploded view of a flow-through PCR chip showing the configuration of flow channels in the first two temperature cycling stages. As the sample slowly flows through the channel is temperature cycled and nucleic acid amplification occurs. B. Bottom view showing the central temperature zone. C. Top view of a device to provide 20 thermal cycles. Such a device is capable of completing each temperature cycle in a matter of seconds, allowing rapid real-time PCR.

Fig. 3

The biophysical and dielectric differences between normal and malarially-parasitized cells deduced from our previous studies (summarized from Gascoyne et al., 1997).

Fig. 4

Side view of a DEP–FFF particle fractionating chamber showing the parabolic hydrodynamic flow profile that forms spontaneously when fluid flow is initiated and the forces that are experienced by particles due to sedimentation and repulsive dielectrophoretic forces from electrodes on the chamber floor. These forces balance at different levitation heights for particles having different density and dielectric properties. Because they equilibriate at different heights, different particles are carried at different speeds by the hydrodynamic flow profile and emerge from the chamber at different times.

Fig. 5

Elution profile for malarially-infected blood from a DEP–FFF chamber measured by time-resolved flow cytometry. The top panel shows all erythrocytes that emerge from the chamber. The bottom panel shows those erythrocytes that fluoresce because of staining of intracellular parasites with Sybro 14 live-cell DNA stain. Most of the parasitized cells emerge quickly from the DEP–FFF chamber, while normal cells emerge later. Leucocytes emerge after the normal erythrocytes (not shown).

Fig. 6

Design for an integrated DEP–FFF front end, cell lysis stage, and flow-through, real-time PCR system. Such a system could be inexpensively mass produced and could, in principle, be made highly portable for diagnosis of malaria and other diseases in laboratories, clinics, and in the field.