Single step aptamer selection on a digital microfluidic chip.

Over the years, we witnessed impressive progress in the field of biosensor technology with numerous successful applications for life science research, medical diagnostics and industrial processes. A lot of research effort has been made to achieve better analytical sensitivities with single-molecule resolution as the ultimate goal. Such ultrasensitive detection platforms are extremely valuable for the early detection of low-abundance disease-relevant biomarkers as well as for screening heterogenic sample populations. In particular, bead-based microwell arrays have proven its potential for target detection with single-molecule resolution, by spatially confining individual targets in microwells.

The success of ultrasensitive bead-based microwell array technology is exemplified by the commercial applications of Illumina (i.e. BeadArray™) and Quanterix (i.e. SiMoA™) for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for the success of these applications and can be enhanced by the creation of hydrophilic-in-hydrophobic (HIH) microwell arrays. However, the fabrication of HIH microwell arrays requires complex and labour-intensive procedures and consequently calls for simpler manufacturing methods.

Additionally, current bead-based microwell array systems are restricted solely to target detection, not allowing subsequent manipulation of single beads with captured targets of interest. Although various bead-based trap-and-release systems have already been developed for manipulation of interesting targets at the single-molecule level, they either release all trapped beads at once or in a sequential manner. These platforms thus require an integrated sorting unit, which separates those beads that have captured an interesting target from the rest of the beads. Hence, there is a need for a multipurpose ultrasensitive bead-based trap-and-release system that would enable both detection and manipulation of interesting target molecules. Such platforms would be of special interest for single-cell applications, bacteria screening, drug discovery and antibody therapeutics.

Therefore, the aim of this dissertation, is to (i) establish a multipurpose platform for both detection and manipulation of single molecules by integrating an optical manipulation tool (for retrieving single magnetic beads) with the digital microfluidic-based microwell array, and (ii) evaluate an innovative microwell array fabrication method for the swift manufacturing of HIH microwell arrays.

The first part of this work focused on the integration of optical tweezers with an electrowetting-on-dielectric-based digital microfluidic microwell array system for the manipulation of single magnetic beads, seeded in a microwell array. Optical manipulation of seeded magnetic beads required their Brownian motion. To this end, the optimal experimental design approach was used for studying and identifying optimal buffer conditions, which generated high fractions of seeded beads with Brownian motion and thus enabled successful manipulation of single magnetic beads. Six different buffer parameters, including bead type, ionic buffer strength, pH, non-ionic surfactant type and concentration, were investigated in this experiment. The analysis of this optimal experimental design indicated different optimal buffer conditions for carboxylated and streptavidin-coated beads, with Tween 40 and Tween 60 as the best non-ionic surfactants. Using these optimized buffer conditions, multiple bead manipulations, including bead retrieval, trapping, transporting and repositioning in another microwell, were successfully demonstrated.

In the second part, the potential of a novel hydrophobic off-stoichiometric thiol-ene-epoxy polymer formulation was explored for imprinting HIH microwell arrays, using a stamp-moulding technique. With this method, HIH microwell arrays were created with excellent dimensions and topological features, enabling highly efficient printing and seeding of superparamagnetic beads. Using streptavidin-biotin interaction and β–galactosidase as a reporter enzyme, a digital bioassay was performed. A detection limit of 17.4 attomolar was obtained, demonstrating the potential of the imprinted microwell arrays to perform sensitive digital bioassays. Hence, it can be concluded that the novel hydrophobic Off-stoichiometric Thiol-Ene-Epoxy polymer and the integration of optical tweezers in the electrowetting-on-dielectric-based digital microfluidic microwell array system have a huge potential for the development of new commercial digital ELISA and bead-based trap-and-release system, respectively.