Single Cell Microinjection Using Compliant Fluidic Channels and Electroosmotic Dosage Control

Abstract

The introduction of bio-molecules into cells and embryos is required in the fields of drug development, genetic engineering and in-vitro fertilization. It has been applied to create transgenic mammals and to improve pest and mold resistance in plants. However, the efficient transfection of materials still poses a problem, and a variety of techniques, broadly classified as biochemical and physical means, are actively being developed. One technique that is promising is capillary microinjection as it offers low cytotoxicity, targeted injections and high transfection efficiency. However, this process suffers from low throughput and variability as it is an operator mediated process. Other problems associated with capillary microinjection are limitations on the minimum needle size and variability in transfected volumes due to the use of pressure driven flow for injections. In this thesis we propose a device that employs microfluidic principles to enable cell microinjections in a 'lab on a chip' format and eliminates the problems associated with capillary microinjection. The device is fabricated using poly dimethylsiloxane (PDMS) rapid prototyping and features two separate channel structures-one to supply the targets and the other to supply the reagent. Integrated into the device are a microinjection capillary (10 μm tip diameter) and a suction capillary (0.5mm ID/1mm OD) which is used to immobilize the targets in the channel prior to injection. The actuation of the injection needle into the targets is achieved by the compliant deformation of the flexible PDMS substrate as a result of an externally applied displacement. This is made possible by the selective reinforcement of the PDMS substrate. From testing it was found that the effective needle actuation is 83.8% of the externally applied displacement. The injections occur in a planar configuration therefore providing precise control over the location of injection. Furthermore, the mechanism requires only one degree of freedom to perform injections, and therefore greatly simplifies existing injection techniques which require orientation in a three dimensional space. The limitations of the use of pressure driven flow for injections are overcome by performing reagent injection by electroosmotic flow, which is induced by applying a potential to electrodes embedded in the target and reagent supply channels. The applied potential induces electroosmotic flow through the embedded needle and into the injection target. This provides precise electrical dosage control. The flow rates were obtained by measuring the velocity of the interface between a neutral fluorescent marker and a clear pH 10 buffer solution. The obtained flow rates follow a predictable linear trend and correspond well to theory. The use of electroosmotic flow enables the use of smaller injection needles as it scales more favorably (r^-2) than pressure driven flow (r^-4) and becomes increasingly dominant in smaller dimensions. Present pressure microinjection systems are limited to injection needles with tip diameters larger than 0.2μm due to the high pressures required to dose at smaller dimensions. All components of the device are fully scalable and enable further miniaturization, multiple parallel injections and autonomous functionality. The device requires smaller volumes of samples and expensive reagents and also reduces the time required for performing injections. Overall, it device maintains the advantages of microinjection, while eliminating problems of low throughput, dosage control and restrictions on the injection needle size. The device was successfully used and characterized for the injection of single-cell Xenopus Laevis eggs and Zebrafish embryos.