Microchip Flow Cytometry Using Electrokinetic Focusing

Abstract

Flow cytometry of fluorescently labeled and unlabeled latex particles is demonstrated on a microfabricated device. The latex particles were detected and counted using laser light scattering and fluorescence coincidence measurements. Sample confinement was accomplished using electrokinetic focusing at a cross intersection, and detection occurred 50 μm downstream from the intersection. Particles with diameters of 1 and 2 μm were analyzed and distinguished from each other based on their light scattering intensity and fluorescence. A maximum sample throughput of 34 particles/s was achieved. Sample mixtures with varying proportions of fluorescently labeled and unlabeled particles were also analyzed and found to be within experimental error of the expected ratios.

Two simple and rugged designs for creating microfluidic sheath flow

“…A more robust approach, pioneered by Ramsey's group, 8,13,14 has been to confine the sample stream on either side with a particle-free aqueous solution. This design is simple and easily fabricated, leading several researchers to imitate it.…”

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

“…Historically speaking, flow cytometry was the first technique to analyze individual subcellular particles [1], so it is not surprising that many of the first analyses of large populations of individual microparticles [2][3][4][5][6][7], cells [8,9], and subcellular particles [10] on microfluidic devices also used flow cytometry. Electrophoretic separation schemes have also been used since the first glass microfluidic devices were described by Manz et al [11] and Harrison et al [12,13] and they continue to be quite popular due to ease of application.…”

3D focusing of nanoparticles in microfluidic channels

“…One example of a particularly important application is the development of a flow cytometer on a chip [1][2][3][4][5][6][7][8][9][10]. For such systems to work efficiently, it is important to be able to confine the sample into a small, spatially well-defined volume, giving as small a probe volume as possible for the detector.…”

“…Jacobson and Ramsey [9] described a device which was microfabricated from etched glass and demonstrated two-dimensional confinement of molecules with applied voltages of 3 kV. Schrum et al [2] showed how electrokinetic focusing could be used to focus 0.97 mm and 1.94 mm diameter latex particles at velocities of 1 mm s -1 . Chou et al developed a device for detecting DNA molecules [10] and Fu et al [1,3] used a similar system to construct a microfabricated fluorescence activated cell sorter.…”

“…The basic principles of dielectrophoresis (DEP) and its application to biological materials have been established by Pohl [1] and Pethig [2]. The concept of DEP is particularly attractive as it enables non-contact manipulation of particles such as cells in solution by high frequency electric fields [3], and characterisation of their passive dielectric properties [4][5][6][7][8].…”