Already at the very beginning of the COVID-19 pandemic, an extensive PCR and antigen testing strategy was considered necessary and subsequently also proved successful in order to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on international and national levels. However, equally important will be the continuous monitoring of the seroprevalence status of populations from defined regions to detect-in a timely manner-any recurrence of infections or an eventual decline in antibody levels of vaccinated individuals, especially in the emerging post-pandemic situation. The aim of this study was to estimate the prevalence of SARS-CoV-2-specific immunoglobulin G antibodies in the federal state of Upper Austria (Austria) during the period of December 2020 until April 2021. To achieve this goal, we have analyzed anonymized data on the immune status of self-referral volunteers that have been determined at local pharmacies through a low-entry-barrier point-of-care analysis approach. The seroprevalence values for immunoglobulin type G antibodies against SARS-CoV-2 antigens obtained by rapid diagnostic testing on peripheral blood from volunteers reflect the current population-based estimates reported in the literature as well as the positivity rates detected by PCR-screening analyses. In conclusion, broad-based monitoring of IgG antibodies by means of a point-of-care testing network represents a valuable tool to assess the current immune situation within regionally defined populations.

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.

Within the last decades, a growing number of miniaturized solutions for biological and chemical analysis processes have been developed by scaling these analyses down to a lab-on-a-chip (LoC) level, thanks to sensitive and rapid testing abilities of LoCs. In parallel to that, a significant need for high-throughput and cost-efficient production of LoC components arises. Our roll-to-roll (R2R) pilot-line for imprinting micro- and nano- structures onto polymers in various designs provides high-throughput manufacturing solutions for polymer based flexible LoC components. The structure imprinting (<1 μm resolution) is performed on polymer foils with ~125-200 μm thickness using a long-lasting stamp. The system also enables accelerated 19 and localized bio-functionalization with molecular probes (e.g., DNA) using low quantities of biomaterials with a photoactive, commercial linker. Without a surface pre-functionalization, efficient generation of DNA micro-arrays are done and probe DNAs can be tethered successfully onto structured polymer components in a continuous and rapid micro-spotting process.

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.

R2R-UV-nanoimprinting sets new benchmarks for the fabrication of miniaturized, low-cost and low-weight freeform micro-optical elements applicable in lighting, display, medical diagnostics, etc. We demonstrate the related process chain, combining optical design, direct laser lithography, step&repeat imprinting and R2R reproduction.

Biosensors have successfully demonstrated the capability to detect multiple pathogens simultaneously at very low levels. Miniaturization of biosensors is essential for use in the field or at the point of care. While microfluidic systems reduce the footprint for biochemical processing devices and electronic components are continually becoming smaller, optical components suitable for integration--such as LEDs and CMOS chips--are generally still too expensive for disposable components. This paper describes the integration of polymer diodes onto a biosensor chip to create a disposable device that includes both the detector and the sensing surface coated with immobilized capture antibody. We performed a chemiluminescence immunoassay on the OPD substrate and measured the results using a hand-held reader attached to a laptop computer. The miniaturized biosensor with the disposable slide including the organic photodiode detected Staphylococcal enterotoxin B at concentrations as low as 0.5 ng/mL.

Monitoring protein function with high throughput at individual cell level is of high interest both for basic research and diagnostic applications. For this, following the changes in fluorescence resonance energy transfer (FRET) between a donor/acceptor pair, genetically encoded in the proteins of interest, is a frequently used tool. As proteins attached to or located in the plasma membrane represent a considerable fraction of total proteins, there is a need for high throughput imaging techniques suited for observation of proteins in the cell membrane only. A system is presented, which allows rapid imaging of large areas via total internal reflection fluorescence microscopy (TIRFM) conditions, using a focus-hold system, multiwavelength excitation and dual color detection. The developed imaging system enables screening of large numbers of cells under TIRFM illumination combined with FRET imaging, thereby providing the means to record, e.g., FRET-efficiency of a membrane-associated protein labeled with a donor-acceptor pair. The capability of the system to perform live-FRET scanning with TIRFM on stoichiometric FRET constructs, reaching throughput of up to 1,000 cells/s at the optical resolution limit is demonstrated. A comparison with confocal microscopy shows that TIRFM offers a 4.2-fold advantage in our conditions over confocal microscopy in detecting contributions from membrane-localized proteins.

The development of commercial portable lab-on-chip (LOC) applications based on optical detection is hindered by the lack of imaging systems that can be directly integrated into the chip itself. Currently, fluorescence/luminescence signals are read out with power-hungry, bulky and expensive off-chip imaging systems, like CCD cameras or photomultiplier tubes. Here we present an enabling technology that for the first time allows cheap and easy integration of imaging systems directly into disposable lab-on-chip systems. Our technology is based on organic semiconductor materials that can be processed in liquid form by inkjet printing, in a process much faster and cheaper than the complicated fabrication of silicon-based imaging sensors. Organic photosensors can be printed on various substrate materials like plastic foil or glass or directly onto lab-on-chip systems. The ultrathin photodiodes with an overall thickness of only 300 to 500 nm show quantum efficiencies better than 0.5 and linear light-response over 6 orders of magnitude. The pixel size can range from 50 to over 1000 mu m and inkjet fabrication allows tailoring the sensor layout to the needs of the specific application.

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit.In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function. (C) 2005 Elsevier Ltd. All rights reserved.

Recent advances in the development of new microscopical techniques with single-molecule sensitivity have given access to essentially new types of information on biological systems. In this review, basic methodological concepts of ultra-sensitive microscopy are presented and characterized, with focus on their applicability for a bioanalytical instrument. Measurements on artificial lipid bilayers were used to evaluate the feasibility of this novel technology. First examples of single molecule microscopy on cell membranes revealed new basic insights into the lateral organization of the plasma membrane.

The scavenger receptor class B type I (SR-BI) plays an important role in mediating selective uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in liver and steroidogenic tissues. The molecular mechanism by which this receptor mediates selective cholesteryl ester uptake remains still enigmatic. We applied ultrasensitive fluorescence microscopy to visualize the intracellular transport routes of HDL particles taken up via SR-BI in a Chinese hamster ovarian cell line. Although diffusion of the receptor bound particles on the cell surface is slow, internalization is accompanied by a dramatic increase in the mobility of the particles. HDL particles are endocytosed as clusters and actively transported to the perinuclear region of the cell. Costaining with organelle markers confirmed the involvement of an acidic compartment and the Golgi apparatus in the uptake process; finally, resecretion of the HDL particles was observed.

The view of the plasma membrane of biological cells was dramatically changed due to the discovery of lipid microdomains. Initially found as structurally distinct areas characterized by a specific protein content, the concept of lipid microdomains was rapidly taken over as a new scheme for explaining membrane targeted cellular processes. Despite its impact on the current image of cell function, there is still a lack of knowledge on the structural origin, size, distribution and mobility of such domains. We addressed these questions through high-resolution imaging of microdomains enriched in flourescence labeled lipids with a two-photon microscope. Our findings showed that domains are of heterogeneous size close to the optical resolution limit, and are dispersed randomly over the cell surface. For studying the functional role of microdomains, we investigated the interplay of lipids and certain membrane proteins utilizing simultaneous two color imaging. While in some cases we observed colocalization between lipids and proteins, on occasion proteins were confined to domains which were clearly separated from lipid domains. This is in perfect agreement with biochemical studies showing structurally distinct sets of domains confining different types of proteins. For maintaining functional flexibility of the cell, microdomains are expected to be linked to the cytoskeleton. We studied the mobility of individual microdomains using repeated imaging of the same area of the cell surface at a rate of one image per 10s. Interestingly, most domains were almost immobile within the plasma membrane. On occasion, however, domains were transported actively over distances up to several micrometers, which is strong evidence for interactions between domains and the cytoskeleton. These results for the first time demonstrate that ultra-sensitive optical microscopy is perfectly suited to resolve the dynamical processes of the organization of the plasma membrane, which allows for much deeper insights into the details of cell response.

Imaging diffusion of single fluorescence labeled lipids by Two Photon Excitation (TPE) has been accomplished for an artificial lipid membrane [1] and on live cells [2]. This opens the perspective to utilize the advantages of TPE, e.g. the reduced background, increased lateral and axial resolution and the possibility of synchronous multicolor experiments, to study dynamical rearrangements and interactions between biomolecules in living cells. As a first realization of this new methodology we studied the lateral confinement of fluorescence labeled lipid molecules to microdomains within the plasma membrane. For this, fluorescence labeled lipids (TMR-DPPE) were introduced into the membrane of tsA201 cells [3]. The Figure shows a typical image of the top membrane of a cell. Individual microdomains enriched in saturated phospholipids are clearly resolvable as diffraction limited spots. The images were taken with a mean power of 6mW at 830 nm and a dwell time of 5 ms/pixel. While Figure A was recorded with a step size of 0.5 μm the zoom in B was obtained with a step size of 100nm.

Using simultaneous two-photon excitation of fringelite D and a fluorescence indicator embedded in a vesicle system it was demonstrated that after excitation a proton was transferred from the pigment to the indicator similarly as recently documented for hypericin. Semiempirical AM1 calculations were used to show that the radical species formed by electron transfer from the excited pigment state constitutes an acid which is therefore well suited for intermolecular proton transfer. Accordingly, this process constitutes a suited candidate for the primary photoprocess in the signal transduction cascade of the photosensory pigments of the stentorin and blepharismin type.