High-throughput roll-to-roll production of polymer biochips for multiplexed DNA detection in point-of-care diagnostics

Roll-to-roll UV nanoimprint lithography has superior advantages for high-throughput manufacturing of micro- or nano-structures on flexible polymer foils with various geometries and configurations. Our pilot line provides large-scale structure imprinting for cost-effective polymer biochips (4500 biochips/hour), enabling rapid and multiplexed detections. A complete high-volume process chain of the technology for producing structures like μ-sized, triangular optical out-couplers or capillary channels (width: from 1 μm to 2 mm, height: from 200 nm up to 100 μm) to obtain biochips (width: 25 mm, length: 75 mm, height: 100 μm to 1.5 mm) was described. The imprinting process was performed with custom-developed resins on polymer foils with resin thicknesses ranging between 125–190 μm. The produced chips were tested in a commercial point-of-care diagnostic system for multiplexed DNA analysis of methicillin resistant Staphylococcus aureus (e.g., mecA, mecC gene detections). Specific target DNA capturing was based on hybridisation between surface bound DNA probes and biotinylated targets from the sample. The immobilised biotinylated targets subsequently bind streptavidin–horseradish peroxidase conjugates, which in turn generate light upon incubation with a chemiluminescent substrate. To enhance the light out-coupling thus to improve the system performance, optical structures were integrated into the design. The limits-of-detection of mecA (25 bp) for chips with and without structures were calculated as 0.06 and 0.07 μM, respectively. Further, foil-based chips with fluidic channels were DNA functionalised in our roll-to-roll micro-array spotter following the imprinting. This straightforward approach of sequential imprinting and multiplexed DNA functionalisation on a single foil was also realised for the first time. The corresponding foil-based chips were able to detect mecA gene DNA sequences down to a 0.25 μM concentration.This research was supported by R2R BIOFLUIDICS project (http://www.r2r-biofluidics.eu/) under Horizon 2020 European Union (EU) Research and Innovation Programme with grant agreement no 646260. The research was also partially supported by NextGenMicrofluidics project (https:// www. nextgenmicrofluidics.eu/) under HORIZON2020 with grant agreement no 862092. The authors cordially thank Gerburg Schider & Gerhard Mohr, Markus Postl, Paul Patter and Alexander Wheeldon (JOANNEUM RESEARCH – Materials, Weiz, Austria) for revising the manuscript, preparing all the chip and R2R pilot line illustrations, taking the photographs and providing technical support, respectively. The authors are also grateful to Christian Wolf and Johannes Götz (JOANNEUM RESEARCH – Materials, Weiz, Austria) for their supports in the fluidic design and R2R UV-NIL structuring, respectively. We further kindly thank Alba Simon Munoz and Robert Fay (SCIENION AG, Berlin, Germany) for providing the illustration of the R2R micro-spotting line. PT specially thanks Ege Ozgun (NANOTAM, Bilkent University, Ankara, Turkey) for critically reading the manuscript

Surface Enhanced Raman Spectroscopy of Unilamellar Liposomes Loaded with Silver Nanoparticles

Imaging organic molecules using surface-enhanced Raman spectroscopy (SERS) has drawn attention due to its non-invasive nature and label-free approach. The SERS approach can be used in tracking organic molecules and monitoring unique Raman spectra of the organic molecules bound to metal nanoparticles (NPs). We prepared unilamellar liposomes composed of 1,2-dipalmitoylsn- glycero-3-phosphocholine (DPPC) molecules with a diameter of around 100 nm. Electrostatic binding of silver (Ag) NPs on the surface of the unilamellar liposomes was achieved by the reduction of silver nitrate (AgNO3) which produces SERS active silver colloidal particles on the unilamellar liposome surfaces. Highly enhanced electromagnetic fields localized around neighbouring Ag NPs provide hot-spot construction around the liposomes, due to the spatial distribution of SERS enhancement in the unilamellar liposomes. It was observed that the signals fluctuate on a second time scale, presumably due to conformational change of DPPC chain and local Brownian motion of liposomal spheres