Droplet-based microfluidic platform for immunoassay applications using magnetic particles
The objective of this thesis is to study on a high throughput droplet-based microfluidic platform that enables an immunoassay based on magnetic particles. This research solves technical challenges in manipulating independent droplets for magnetic particle immunoassay including a washing step to remove unbound samples. Finally, concept-of-principle applications are demonstrated by integrating the additive and subtractive function into a single microfluidic system. First of all, this thesis deals with a new fabrication process of photolithography for a better microfluidic device by removing edge bead and tiny air bubbles. It is because the edge bead and air bubbles can cause an air gap between a film mask and a photoresist surface during UV exposure. The diffraction effect of UV light by the air gap leads to inaccurate and non-uniform SU-8 patterns. A simple method for a microfluidic device fabrication is demonstrated using EBR treatment to simultaneously eliminate the edge bead at the edge of wafer and tiny air bubbles inside SU-8. The average pattern uniformity of SU-8 is improved from 50.5% to 11.3% in the case of 200μm thickness. This method is simple and inexpensive, compared to a standard EBR process, because it does not require specialized equipment and it can be applied regardless of substrate geometry (e.g. circular wafer and rectangular slide glass). Second, the first microfluidic device investigated in this thesis is a continuous-flow in-droplet magnetic particle separation in a droplet-based microfluidic device for magnetic bead-based bioassays. Two functions, electrocoalescence and magnetic particle manipulation, are performed in this device. By electrostatic force, two different solutions can be merged to be mixed at a junction of droplet generation. The manipulation of magnetic particles is achieved using an externally applied magnetic field. The magnetic particles are separated by the magnetic field to one side of the droplet and extracted by splitting the droplet into two daughter droplets: one contains the majority of the magnetic particles and the other is almost devoid of magnetic particles. The applicability of the continuous-flow in-droplet magnetic particle separation is demonstrated by performing a proof-of-concept immunoassay between streptavidin-coated magnetic beads and biotin labelled with fluorescence. Our next study in this thesis is an advanced continuous flow droplet-based microfluidic platform for magnetic particle-based assays by employing in-droplet washing. The droplet-based washing was implemented by traversing functionalized magnetic particles across a laterally merged droplet from one side (containing sample and reagent) to the other (containing buffer) by an external magnetic field. Consequently, the magnetic particles were extracted to a parallel-synchronized train of washing buffer droplets and unbound reagents were left in an original train of sample droplets. To realize the droplet-based washing function, the following four procedures were sequentially carried in a droplet-based microfluidic device: parallel synchronization of two trains of droplets by using a ladder-like channel network; lateral electrocoalescence by an electric field; magnetic particle manipulation by a magnetic field; and asymmetrical splitting of merged droplets. For the stable droplet synchronization and electrocoalescence, we optimized droplet generation conditions by varying the flow rate ratio (or droplet size). Image analysis was carried out to determine the fluorescent intensity of reagents before and after the washing step. As a result, the unbound reagents in sample droplets were significantly removed by more than a factor of 25 in the single washing step while the magnetic particles were successfully extracted into washing buffer droplets. As a proof-of-principle, we demonstrate a magnetic particle-based immunoassay with streptavidin-coated magnetic particles and fluorescently labelled biotin in the proposed continuous flow droplet-based microfluidic platform. Lastly, we develop a droplet microfluidic device for tunable concentration gradients in droplet arrays by exploiting sequential dilution between moving and stationary droplets. The key idea of our approach is to add or extract reagents from static droplets trapped in a 50-well array by merging, mixing and breaking-up with moving sample droplets. To generate various concentration gradients, several parameters (e.g., volume of moving droplets, flow rate of oil carrier fluid, and dilution time) are investigated and characterized. As an application of our approach, functionalized magnetic particles are used to perform immunoassays.