Peter Friis Østergaard

Fabrication of combined-scale nano- and microfluidic polymer systems using a multilevel dry etching, electroplating and molding process

Microfabricated single-cell capture and DNA stretching devices have been produced by injection molding. The fabrication scheme employed deep reactive ion etching in a silicon substrate, electroplating in nickel and molding in cyclic olefin polymer. This work proposes technical solutions to fabrication challenges associated with chip sealing and demolding of polymer high-volume replication methods. UV-assisted thermal bonding was found to ensure a strong seal of the microstructures in the molded part without altering the geometry of the channels. In the DNA stretching device, a low aspect ratio nanoslit (1/200) connecting two larger micro-channels was used to stretch a 168.5 kbp DNA molecule, while in the other device single-HeLa cells were captured against a micro-aperture connecting two larger microfluidic channels. Different dry etching processes have been investigated for the master origination of the cell-capture device. The combination of a modified Bosch process and an isotropic polysilicon etch was found to ensure the ease of demolding by resulting in slightly positively tapered sidewalls with negligible undercut at the mask interface. (Some figures may appear in colour only in the online journal)

Fabrication and modelling of injection moulded all-polymer capillary microvalves for passive microfluidic control

Rapid prototyping is desirable when developing products. One example of such a product is all-polymer, passive flow controlled lab-on-a-chip systems that are preferential when developing low-cost disposable chips for point-of-care use. In this paper we investigate the following aspects of going from rapid prototyping to pilot (mass) production. (1) Fabrication of an all-polymer microfluidic system using a rapid prototyped master insert for injection moulding and ultrasonic welding, including a systematic experimental characterisation of chip featured geometric capillary microvalve test structures. (2) Numerical modelling of the microvalve burst pressures. Numerical modelling of burst pressures is challenging due to its non-equilibrium nature. We have implemented and tested the level-set method modified with a damped driving term and show that the introduction of the damping term leads to numerically robust results with limited computational demands and a low number of iterations. Numerical and simplified analytical results are validated against the experimental results. We find that injection moulding and ultrasonic welding are effective for chip production and that the experimental burst pressures could be estimated with an average accuracy of 5% using the presented numerical model.

Optical mapping of single-molecule human DNA in disposable, mass-produced all-polymer devices

We demonstrate all-polymer injection molded devices for optical mapping of denaturation–renaturation (DR) patterns on long, single DNA-molecules from the human genome. The devices have channels with ultra-low aspect ratio, only 110 nm deep while 20 μm wide, and are superior to the silica devices used previously in the field. With these polymer devices, we demonstrate on-chip recording of DR images of DNA-molecules stretched to more than 95% of their contour length. The stretching is done by opposing flows Marie et al (2013 Proc. Natl Acad. Sci. USA 110 4893–8). The performance is validated by mapping 20 out of 24 Mbp-long DNA fragments to the human reference genome. We optimized fabrication of the devices to a yield exceeding 95%. This permits a substantial economies-of-scale driven cost-reduction, leading to device costs as low as 3 USD per device, about a factor 70 lower than the cost of silica devices. This lowers the barrier to a wide use of DR mapping of native, megabase-size DNA molecules, which has a huge potential as a complementary method to next-generation sequencing.

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.