Fabrication and utilisation of solid-phase microdroplet arrays for nucleic acid detection and other applications

Microdroplet arrays involves the compartmentalisation of biological or chemical reactions in hundreds of thousands of single compartments spatially distributed in 2D on a surface, for example a glass substrate. These compartments are so small that single molecules or cells can be encapsulated and interrogated. This can be used to study the heterogeneity of populations not possible by traditional methods. Reactions with single molecules in these droplets are detected, and precise quantification of the molecules can be achieved by counting the number of partitions eliciting a signal. This has, for example, enabled the sensitive detection of proteins by digital immunoassay, improving the limit of detection by three orders of magnitude compared to conventional, analogue methods.
This study concerned the development of a versatile microdroplet array platform. The thesis is comprised of three major parts.
The first part includes a numerical analysis of the hybridisation of DNA targets in low concentrations on planar biosensors, an overview of protein and DNA immobilisation on glass subtrates, the description of an optical detection set-up, as well as the design and fabrication of the liquid infrastructure surrounding the droplet arrays. The numerical analysis predicted a limit of detection of 2 aM for the detection of gene transcripts on MDAs, and showed that the process was highly diffusion limited, and that the greatest improvement to the number of molecules caught on the surface was seen by increasing the screened sample volume, something which is not possible or practical using current golden standard techniques for nucleic acid quantification.
The second part described the fabrication of a droplet array based on the deposition of the teflon-like, hydrophobic compound, FDTS. Microfluidics integrated arrays were capable of supporting 50 fL droplets surrounded by air for several hours with minimal evaporation, which greatly increase the number of possible applications of the platform. On this array, digital ELISA and cell-free protein expression was conducted. Digital ELISA reached a limit of detection around 10 fM, and was capable of splitting a patient cohort in two groups based on the concentration of Aβ1−42, a biomarker for Alzheimer's disease. In the second example, proteins were directly synthesised from a single copy template DNA and immobilised in the droplets. Subsequent detection of the synthesised proteins was carried out by antibody recognition.
The third and last part of the thesis described the characterisation of the process parameters, and the fabrication of a droplet array by the direct patterning of a UV cross-linkable fluoropolymer. Immobilisation of streptavidin selectively in the droplet spots was demonstrated by binding fluorescently labelled proteins on the array functionalised with reactive epoxy groups. Secondly, streptavidin coated microspheres (beads) were immobilised on the spots and DNA hybridisation on the beads was demonstrated.