13 5
Figure 1: The calculated distribution of electric potential at a microfluidic cross-region during a pinched injection on a chip-based electrophoretic device. (A) Potential distribution during loading. (B) Potential distribution during dispensing and separation steps. Images reproduced from reference [9]
being dependent on the ratio of electric field strengths applied during each step. Other authors have since demonstrated similar biasing phenomena using other modes of electrokinetic injection, and although some geometries and the use of large sample reservoirs alleviate the problem they do not completely eliminate it. The only injection formats not displaying sample bias are those based on pressure differences. Consequently, there is a need for alternative injection mechanisms that are simple to implement and enable reproducible delivery of specific sample volumes without bias.
Droplet-based or segmented-flow microfluidics has been the subject of much attention over the past five years.[10,11] In such systems femtoliter – nanoliter droplets can be generated at high speed by combining two immiscible phases (typically aqueous and oil-based). The formation of droplets is a spontaneous process and is normally a result of shear force and interfacial tension at the liquid-liquid interface. Importantly, the formation of droplets within microfluidic channels can be achieved at rates of up to several kHz with exquisite control over both droplet size and composition. Based on these features such platforms are ideally suited to processing millions of individual reactions in ultra-short times and with superb reproducibility.[12] It is therefore unsurprising that a number of recent studies have assessed the utility of integrating electrophoretic and chromatographic separations with droplet microfluidics, for both sample injection and post separation fraction collection. For example, a sample can be fractionated into droplets post reaction or separation and transferred to a subsequent analytical process, or defined sample volumes can be injected into separation channels using the droplet as an injector. This approach, in theory, allows injection of the representative samples required in quantitative analysis.
Whole droplet injection also ensures that sample wastage is negligible and electrokinetic bias (encountered in continuous- flow injection) is eliminated. Recently, several novel approaches have been described employing droplet microfluidics to bridge continuous-flow devices.
One of the first attempts to controllably introduce the contents of an isolated droplet into an electrophoretic channel was described by Edgar and co-workers.[13] The reported microfluidic device consisted of a droplet generation region and a separation channel separated by an immiscible partition at the point of injection. Fusion of an incoming
aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel to allow separation of a precisely defined volume (Figure 2A-F). Although separation of fluorescently labelled amino acids contained within 10 fL droplets was successfully achieved, the authors highlighted several disadvantages of their design including the need for precise pressure control and selective channel pre-treatment (to generate hydrophobic and hydrophilic surfaces) and oil contamination that results in a reduction in electroosmotic flow. Subsequently, Roman et al. described a microfluidic device employing a K-shaped element to transfer sample from segmented
Figure 2: (A-F) Sequence of images depicting the generation, transport and injection of an aqueous droplet into an electrophoretic separation channel. Images in (A-F) reproduced from reference [13]. (G) K-shaped microfluidic interface for sample injection. The device includes a segmented flow channel, a V-shaped cross- flow channel and a separation channel. (H) Micrographs illustrating the droplet-injection approach and the coalescence of plugs using a cross flow of 100 nL/min. (I) Series of electropherograms resulting from sampling and injection of droplets (containing 1µM serine derivatised with FITC) using the discrete injector and separation on a 5 cm long electrophoresis channel at 500 V/cm. Images in (G-I) reproduced from reference [14].
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