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A comprehensive overview of PowderMEMS—a novel back-end-of-line-compatible microfabrication technology—is presented in this paper. The PowderMEMS process solidifies micron-sized particles via atomic layer deposition (ALD) to create three-dimensional microstructures on planar substrates from a wide variety of materials. The process offers numerous degrees of freedom for the design of functional MEMSs, such as a wide choice of different material properties and the precise definition of 3D volumes at the substrate level, with a defined degree of porosity. This work details the characteristics of PowderMEMS materials as well as the maturity of the fabrication technology, while highlighting prospects for future microdevices. Applications of PowderMEMS in the fields of magnetic, thermal, optical, fluidic, and electrochemical MEMSs are described, and future developments and challenges of the technology are discussed.

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB.

Most current thermal MEMS use fragile structures such as thin-film membranes or microcantilevers for thermal isolation. To increase the robustness of these devices, solid thermal insulators that are compatible with MEMS cleanroom processing are needed. This work introduces a novel approach for microscale thermal isolation using porous microstructures created with the recently developed PowderMEMS wafer-level process. MEMS devices consisting of heaters on a thin-film membrane were modified with porous microstructures made from three different materials. A thermal model for the estimation of the resulting thermal conductivity was developed, and measurements for porous structures in ambient air and under vacuum were performed. The PowderMEMS process was successfully used to create microscale thermal insulators in silicon cavities at the wafer level. Measurements indicate thermal conductivities of close to 0.1 W/mK in ambient air and close to 0.04 W/mK for porous structures under vacuum for the best-performing material. The obtained thermal conductivities are lower than those reported for both glass and porous silicon, making PowderMEMS a very interesting alternative for solid microscale thermal isolation.

The full potential of the real-time reverse transcription polymerase chain reaction (RT-PCR) as a rapid and accurate diagnostic method is limited by DNA polymerase inhibitors as well as reverse transcriptase inhibitors which are ubiquitous in clinical samples. rTth polymerase has proven to be more resistant to DNA polymerase inhibitors present in clinical samples for DNA detection and also exhibits reverse transcriptase activity in the presence of Mn2+ ions. However, the capacity of rTth polymerase, which acts as DNA polymerase and reverse transcriptase, to detect RNA in the presence of various inhibitors has not been investigated in detail. Herein, the inhibitors originating from various clinical samples such as blood, urine, feces, bodily fluids, tissues and reagents used during nucleic acid extraction were employed to evaluate the capacity of rTth polymerase to detect RNA. The results show that the inhibitors have different inhibitory effects on the real-time RT-PCR reactions by rTth polymerase, and the inhibitory effects are concentration dependent. Additionally, the capacity of rTth polymerase to detect RNA in the presence of various inhibitors is better or at least comparable with its capacity to detect DNA in the presence of various inhibitors. As a consequence, RNA may be directly detected in the presence of co-purified inhibitors or even directly from crude clinical samples by rTth polymerase.

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 μl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

Abstract Background The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. Methods We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. Results The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87–27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). Conclusions With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

The state-of-the-art monitoring of drinking water hygiene is based on the cultivation and enumeration of indicator bacteria. Despite its proven reliability, this approach has the disadvantages of being (a) relatively slow and (b) limited to a small number of indicator bacteria. Ideally, alternative methods would be less time-consuming while providing information about a larger set of hygienically relevant microorganisms including viruses. In this paper, we present insights into the design of a modular concentration and detection system for bacteria, bacteriophages and viruses. Following further validation, this or similar techniques have the potential to extend and speed up the monitoring of raw and drinking water hygiene in the future. The system consists of different modules for the concentration of microorganisms, an amplification and detection unit that includes a module for the differentiation between live and dead microorganisms, and an automated system for decision support and self-diagnosis. The ongoing testing under controlled laboratory conditions and real-life conditions in the water supply industry yields further system improvements. Moreover, the increased sensitivity and broader range of microbiological parameters emphasize the need for a reconsideration of the currently used criteria for the assessment of (drinking) water hygiene.