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.

Impact of operational temperature changes and freeze-thaw cycles on the hydraulic conductivity of borehole heat exchangers.

A large share of the primary energy is consumed to provide space heating. Geothermal energy offers a regenerative alternative. For reasons of efficiency and environmental protection, it is important to ensure the system integrity of a borehole heat exchanger (BHE). Previous investigations have focused on the individual components of the BHE or on the grout and pipe systems' integrity. This study focused on the analysis of the hydraulic system integrity of the complete subsoil-grout-pipe system as well as possible thermally induced changes. For this purpose, a pilot-scale experiment was built to test a 1-m section of a typical BHE under in situ pressure, hydraulic and temperature conditions. During the tests the hydraulic system permeability of the soil and the BHE was measured continuously and separately from each other. In addition, the temperature monitoring array was installed in a 50-cm cross-sectional area. Significant temperature-related fluctuations in the sealing performance could be observed. Hydraulic conductivity limits required by VDI 4640-2 (Thermal use of the underground-ground source heat pump systems, 2019) were exceeded without frost action. The succeeding application of freeze-thaw cycles further enhances the system permeability. The study shows that the thermally induced effects on the system integrity of the BHE are larger and more significant than the subsequent frost-induced effects. The hydrophobic character of the high-density polyethylene (PE-HD) pipes as well as its high coefficient of thermal expansion seem to be the main points of weakness in the system. Optimization research should focus on the interface connection between grout and pipe, whereby hydrophilic pipe materials such as stainless steel or aluminum should also be considered as well as manipulation of the pipe surface properties of PE-HD.

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.

A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray.

A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.

Reef Sound as an Orientation Cue for Shoreward Migration by Pueruli of the Rock Lobster, Jasus edwardsii.

The post-larval or puerulus stage of spiny, or rock, lobsters (Palinuridae) swim many kilometres from open oceans into coastal waters where they subsequently settle. The orientation cues used by the puerulus for this migration are unclear, but are presumed to be critical to finding a place to settle. Understanding this process may help explain the biological processes of dispersal and settlement, and be useful for developing realistic dispersal models. In this study, we examined the use of reef sound as an orientation cue by the puerulus stage of the southern rock lobster, Jasus edwardsii. Experiments were conducted using in situ binary choice chambers together with replayed recording of underwater reef sound. The experiment was conducted in a sandy lagoon under varying wind conditions. A significant proportion of puerulus (69%) swam towards the reef sound in calm wind conditions. However, in windy conditions (>25 m s-1) the orientation behaviour appeared to be less consistent with the inclusion of these results, reducing the overall proportion of pueruli that swam towards the reef sound (59.3%). These results resolve previous speculation that underwater reef sound is used as an orientation cue in the shoreward migration of the puerulus of spiny lobsters, and suggest that sea surface winds may moderate the ability of migrating pueruli to use this cue to locate coastal reef habitat to settle. Underwater sound may increase the chance of successful settlement and survival of this valuable species.

A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.

Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.

A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

Inducers of settlement and moulting in post-larval spiny lobster.

The rapid and often remote location of suitable habitats used by migrating organisms is often critical to their subsequent recruitment, fitness and survival, and this includes in the marine environment. However, for the non-feeding post-larval stage of spiny lobsters, effective settlement cues for habitat selection are critical to their success but are poorly described. Therefore, the current study examined whether acoustic and substrate cues have the potential to shorten the time to moulting and affect their subsequent nutritional condition in the pueruli of the southern spiny lobster, Jasus edwardsii. Individuals moulted to first instar juveniles up to 38% faster when exposed to the underwater sound from two types of typical settlement habitat (coastal kelp- and urchin-dominated reefs) compared to those with no underwater sound. The settlement delay in the post-larvae without underwater sound also resulted in juveniles in poorer survival and nutritional condition as measured by their protein and lipid contents. In a separate experiment, post-larvae presented with seaweed and rock substrates were found to complete settlement and moult to juvenile by as much as 20% faster compared to those on the sand and control treatments. Overall, the results are the first to demonstrate that the pueruli of J. edwardsii have the ability to detect and respond to underwater sound, as well as determining that both acoustic and substrate cues play a role in modulating physiological development during settlement.

Severe Plasmodium knowlesi infection with multi-organ failure imported to Germany from Thailand/Myanmar.

During the last two decades human infections with Plasmodium knowlesi are increasingly diagnosed in South East Asia and have also been reported in travellers. A severe case of imported P. knowlesi infection in a 73-year old German is presented, who had been travelling through Myanmar and Thailand for three weeks. Microscopy showed a parasitaemia of 3% and different parasite stages including band-forms resembling Plasmodium malariae. Due to the clinical picture of severe malaria and the microscopical aspect (combination of parasites resembling P. malariae and Plasmodium falciparum), P. knowlesi was suspected. The patient was treated with intravenous quinine; he was put on mechanical ventilation and catecholamines due to cardiorespiratory failure. Parasitaemia was cleared rapidly but renal function deteriorated resulting in intermittent haemodialysis. The patient was hospitalized for six weeks but he recovered completely without any physical sequelae. Plasmodium knowlesi mono-infection was confirmed by molecular methods later on.Plasmodium knowlesi infection has to be taken into account in feverish travellers returning from Thailand/Myanmar. Moreover this species can cause life-threatening or even lethal complications. Accordingly severe P. knowlesi infection should be treated like severe P. falciparum infections.

Microarrays and single molecules: an exciting combination.

Biomolecules positioned at interfaces have spawned many applications in bioanalysis, biophysics, and cell biology. This Highlight describes recent developments in the research areas of protein and DNA arrays, and single-molecule sensing. We cover the ultrasensitive scanning of conventional microarrays as well as the generation of arrays composed of individual molecules. The combination of these tools has improved the detection limits and the dynamic range of microarray analysis, helped develop powerful single-molecule sequencing approaches, and offered biophysical examination with high throughput and molecular detail. The topic of this Highlight integrates several disciplines and is written for interested chemists, biophysicists and nanotechnologists.

Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells.

The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca(2+) imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca(2+) response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca(2+) signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.

High-throughput, multiparameter analysis of single cells.

Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.

The complex function of hsp70 in metastatic cancer.

Elevated expression of the inducible heat shock protein 70 (Hsp70) is known to correlate with poor prognosis in many cancers. Hsp70 confers survival advantage as well as resistance to chemotherapeutic agents, and promotes tumor cell invasion. At the same time, tumor-derived extracellular Hsp70 has been recognized as a "chaperokine", activating antitumor immunity. In this review we discuss localization dependent functions of Hsp70 in the context of invasive cancer. Understanding the molecular principles of metastasis formation steps, as well as interactions of the tumor cells with the microenvironment and the immune system is essential for fighting metastatic cancer. Although Hsp70 has been implicated in different steps of the metastatic process, the exact mechanisms of its action remain to be explored. Known and potential functions of Hsp70 in controlling or modulating of invasion and metastasis are discussed.

Expression analysis of multiple myeloma CD138 negative progenitor cells using single molecule microarray readout.

We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138(neg)) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138(neg) cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.

Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns.

Atomic force microscopy (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics of biomolecules by probing the interactions within individual molecules, or between a surface-bound molecule and a cantilever that carries a complementary binding partner. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 Å, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level.

Identification of immobile single molecules using polarization-modulated asynchronous time delay and integration-mode scanning.

We report the development of a data acquisition method for identifying single molecules on large surfaces with simultaneous characterization of their absorption dipole. The method is based on a previously described device for microarray readout at single molecule sensitivity (Hesse, J.; Sonnleitner, M.; Sonnleitner, A.; Freudenthaler, G.; Jacak, J.; Höglinger, O.; Schindler, H.; Schütz, G. J. Anal. Chem. 2004, 76, 5960-5964). Here, we introduced asynchronous time delay and integration- (TDI-) mode imaging to record also the time course of fluorescence signals: the images thus contain both spatial and temporal information. We demonstrate the principle by modulating the signals via rotating excitation polarization, which allows for discriminating static absorption dipoles against multiple or freely rotating single absorption dipoles. Experiments on BSA carrying different numbers of fluorophores demonstrate the feasibility of the method. Protein species with an average labeling degree of 0.55 and 2.89 fluorophores per protein can be readily distinguished.

Microarray analysis at single-molecule resolution.

Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state-of-the-art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack of appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of 2-20 microm and quantifies the abundance of target molecules by determining average pixel intensities, a novel high-resolution approach directly visualizes individual bound molecules as diffraction-limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. First, it consists of a single-molecule detection step, based on undecimated wavelet transforms, and second, a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micrometer and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15, the false negatives relative error was below 15%. Separation of foreground/background is proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements.

Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection.

We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to approximately 10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.

RNA expression profiling at the single molecule level.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.