Lab-on-a-foil devices with integrated retro-reflective structures for multiplexed DNA testing.

ABSTRACT: With the Covid-19-based global pandemic that started in the beginning of 2020, the vital importance of accelerated, reliable and affordable virus testing systems has once again become clearer. Besides, we all learned very well that the disposable biochips, to be used in these in vitro diagnostic (IVD) testing systems, supposed to be produced in large amounts in a very short time to be widely available for the use of humanity to save more and more lives. That is why; roll-to-roll (R2R) polymer structuring manners offer such large quantities for the production of in vitro biochips. Our technology, based on R2R UV nanoimprint lithography (UV-NIL), has superior features. Via our pilot line, robust 7500 biochip components per 100 meter of a flexible, polymer foil coated with a UV curable photo-resin (i.e., parts with capillary fluidic channels or optical structures for IVDs) can be generated. This study shows an example of a prototype of a R2R UV-NIL generated chip: a foil, capillary flow-based IVD biochip for multiplexed DNA detection purposes (i.e., a Lab-on-a-Foil device). The biochip performance was further increased dramatically by integrating UV-NIL produced retro-reflective microstructures, which reflects the light back, to its design to enhance optical signal detection in a commercial IVD device, detecting DNA on a chemiluminescent-reaction basis.

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.

Label-Free Optical Biodetection of Pathogen Virulence Factors in Complex Media Using Microtoroids with Multifunctional Surface Functionality.

Early detection of pathogens or their virulence factors in complex media has a key role in early diagnosis and treatment of many diseases. Nanomolar and selective detection of Exotoxin A, which is a virulence factor secreted from Pseudomonas aeruginosa in the sputum of Cystic Fibrosis (CF) patients, can pave the way for early diagnosis of P. aeruginosa infections. In this study, we conducted a preliminary study to demonstrate the feasibility of optical biodetection of P. aeruginosa Exotoxin A in a diluted artificial sputum mimicking the CF respiratory environment. Our surface engineering approach provides an effective biointerface enabling highly selective detection of the Exotoxin A molecules in the complex media using monoclonal anti-Exotoxin A functionalized microtoroids. The highly resilient microtoroid surface toward other constituents of the sputum provides Exotoxin A detection ability in the complex media by reproducible measurements. In this study, the limit-of-detection of Exotoxin A in the complex media is calculated as 2.45 nM.

Oligonucleotide-based label-free detection with optical microresonators: strategies and challenges.

This review targets diversified oligonucleotide-based biodetection techniques, focusing on the use of microresonators of whispering gallery mode (WGM) type as optical biosensors mostly integrated with lab-on-a-chip systems. On-chip and microfluidics combined devices along with optical microresonators provide rapid, robust, reproducible and multiplexed biodetection abilities in considerably small volumes. We present a detailed overview of the studies conducted so far, including biodetection of various oligonucleotide biomarkers as well as deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs) and proteins. We particularly advert to chemical surface modifications for specific and selective biosensing.

Real-Time and Selective Detection of Single Nucleotide DNA Mutations Using Surface Engineered Microtoroids.

Mictoroids, as optical biosensors, can provide beneficial biosensing platforms to understand DNA alterations. These alterations could have significant clinical importance, such as the case of Pseudomonas aeruginosa, which is a commonly found pathogen in Cystic Fibrosis (CF) patients-causing poor prognosis by undergoing mutations during disease steps, gaining virulence and drug resistance. To provide a preliminary diagnosis platform for early-stage bacterial mutations, biosensing with a selective microtoroid surface was suggested. For this purpose, microtoroids with high quality factors were fabricated. The microtoroid surfaces were coated with (3-aminopropyl) triethoxysilane (APTES)/trimethylmethoxysilane (TMMS) mixed silane solution followed by EDC/NHS chemistry for covalent conjugation of DNA probes. Ethanolamine capping was applied to avoid unspecific interactions. The confocal studies confirmed homogeneous functionalization of the microtoroid surface. The DNA hybridization was demonstrated to be affected from the probe length. The optical biosensors showed a significant response (∼22 pm) to the complementary strand of the mutated type P. aeruginosa DNA, while showing substantially low and late response (∼5 pm) to the point mismatch strand. The limit of detection (LOD) for the complementary strand was calculated as 2.32 nM. No significant response was obtained for the noncomplementary strand. The results showed the microtoroids possessed selective surfaces in terms of distinguishing DNA alterations.

Label-Free Biosensing with High Selectivity in Complex Media using Microtoroidal Optical Resonators.

Although label-free biosensors comprised of optical microcavities inherently possess the capability of resolving molecular interactions at individual level, this extreme sensitivity restricts their convenience for large scale applications by inducing vulnerability towards non-specific interactions that readily occur within complex media. Therefore, the use of optical microresonators for biosensing is mostly limited within strictly defined laboratory conditions, instead of field applications as early detection of cancer markers in blood, or identification of contamination in food. Here, we propose a novel surface modification strategy suitable for but not limited to optical microresonator based biosensors, enabling highly selective biosensing with considerable sensitivity as well. Using a robust, silane-based surface coating which is simultaneously protein resistant and bioconjugable, we demonstrate that it becomes possible to perform biosensing within complex media, without compromising the sensitivity or reliability of the measurement. Functionalized microtoroids are successfully shown to resist nonspecific interactions, while simultaneously being used as sensitive biological sensors. This strategy could pave the way for important applications in terms of extending the use of state-of-the-art biosensors for solving problems similar to the aforementioned.

Phosphonate based organosilane modification of a simultaneously protein resistant and bioconjugable silica surface.

A facile method to coat silica surfaces with THPMP is introduced, forming simultaneously a protein resistant and bioconjugable surface. The coating is experimentally identified and its anti-fouling and bioconjugable characteristics are demonstrated.

Label-free nanometer-resolution imaging of biological architectures through surface enhanced Raman scattering.

Label free imaging of the chemical environment of biological specimens would readily bridge the supramolecular and the cellular scales, if a chemical fingerprint technique such as Raman scattering can be coupled with super resolution imaging. We demonstrate the possibility of label-free super-resolution Raman imaging, by applying stochastic reconstruction to temporal fluctuations of the surface enhanced Raman scattering (SERS) signal which originate from biomolecular layers on large-area plasmonic surfaces with a high and uniform hot-spot density (>10¹¹/cm², 20 to 35 nm spacing). A resolution of 20 nm is demonstrated in reconstructed images of self-assembled peptide network and fibrilated lamellipodia of cardiomyocytes. Blink rate density is observed to be proportional to the excitation intensity and at high excitation densities (>10 kW/cm²) blinking is accompanied by molecular breakdown. However, at low powers, simultaneous Raman measurements show that SERS can provide sufficient blink rates required for image reconstruction without completely damaging the chemical structure.