Droplet-based microfluidics for continuous chemical sensing and quantitative high-throughput separations

Hassan, Sammer-Ul (2016) Droplet-based microfluidics for continuous chemical sensing and quantitative high-throughput separations. University of Southampton, Faculty of Engineering and the Environment, Doctoral Thesis, 228pp.

Record type: Thesis (Doctoral)

Abstract

By translating bioanalytical testing from the laboratory into the clinic or at home, point-of-care (POC) diagnostics enables timely diagnosis, monitoring, and treatment of patients. Fundamental to the development of POC diagnostics has been the use of microfluidics, which offers small sample volume consumption and can combine multiple sample-processing steps into a single device. However, microfluidics has provided a limited throughput due to the multiple microvalve switching of samples, slow mixing, sample dispersion and band-broadening in microfluidic channels which result in decreasing the temporal resolution of the system. Droplet-based microfluidics has emerged as an alternative technique to manipulate and analyse sample droplets in a high-throughput format. Samples are compartmentalized in nanolitre sized droplets which eliminate dispersion or band-broadening and hence increase the temporal resolution of the system.

The development of high-throughput, quantitative and rapid microfluidic-based separations has been a long-sought goal for applications in proteomics, genomics, biomarker discovery and clinical diagnostics. Using droplet-interfaced microchip electrophoresis (MCE) techniques, a novel parallel MCE platform, based on the concept of Slipchip and a newly developed ‘Gelchip’ has been presented in this Thesis. The platform consisted of two “slipping” plastic plates, with droplet wells on one plate and separation channels with preloaded/cured gel on the other. A single relative slipping of the plates enabled generation and then injection of multiple droplets in parallel into the separation channels, allowing to analyse sample droplets in parallel and in a high-throughput format.

As proof-of-concept applications, the separation of 30 sub-nanolitre sample droplets containing fluorescent dyes was performed. Theoretical plates were calculated to be 7560/s at a distance of 3.5 cm. We further determined the effect of droplet sizes on the separation efficiency and found that theoretical plates were higher (2220/s) for smallest droplets (320 picolitres) and lower (1480/s) for largest droplets (1750 picolitres) at a distance of 8 mm. For the quantitative determination of fluorescent molecules, the change in peak areas of different concentrations of the fluorescent molecule (Fluorescein-isothiocyanate) was found to be linear with a small error of 3.6% RSD. Furthermore, separation of DNA step ladder at a distance of 1.3 cm was also achieved with theoretical plates of 79800/s which is one order of magnitude higher than our previously reported droplet-interfaced platform (Niu et al. 2013). To facilitate ‘non-microfluidic users’, new protocols were also developed to pre-cure separation gels (e.g. agarose and polyacrylamide) in open channels forming a ‘Gelchip’ that can be prepared in batch and used off-the-shelf.

Droplet-based microfluidics is ideally suited to continuous biochemical analysis, requiring small sample volumes and offering a high temporal resolution. Many biochemical assays are based on enzymatic reactions, the kinetics of which can be obtained by probing droplets at multiple points over time. Real-time and continuous chemical monitoring of traumatic brain injury (TBI) or liver transplant in intensive care unit (ICU) could be life-saving and protect from further damage to the tissue. Current systems to monitor these patients are bulky and are placed at a distance from the patient (typically 30-40 cm) which introduces sample dispersion and limits the temporal resolution of the system.

A droplet-based portable continuous chemical sensing device capable of generating droplets-on-demand and analysing glucose or lactate from near patient has been developed. The primary task of the project was to calibrate the performance of the device by generating droplets by a novel screw-driven pump, calibrating droplet sizes and detecting different droplet concentrations in an absorbance based flow cell.

To measure the reactions in droplets calorimetrically, a miniaturised 7-detector flow cell to analyse enzyme kinetics, with an example application of continuous glucose assay in droplets has been developed. Reaction rates and Michaelis-Menten kinetics were quantified for each individual droplet, and unknown glucose concentrations were accurately determined (errors < 5%) with the lowest quantified concentration of 0.5 mM. Droplets can be probed continuously giving short sample-to-result time (~30 s) measurement. In contrast to previous reports of multipoint droplet analysis (all of which used bulky microscope-based setups) the flow cell presented here has a small footprint (45 by 10 by 15 mm) and uses low-powered, low-cost components, making it ideally suited for use in field-deployable devices.

The flow cell was further upgraded via fabrication of precise micromilling of the cartridge and successfully applied for detection of glucose and lactate droplets (lowest quantified concentrations of 0.175 mM and 0.1 mM respectively) for clinical applications. Another significant development of the optical flow cell is dual light paths for accurately quantifying the size and velocity of the continuously flowing droplets, as well as quantifying the composition of the droplet. The flow cell was capable of measuring droplet lengths with errors of < 5%. With all these features, a fully functional optical flow cell is built that can be readily used in wearable/portable microfluidic devices.