Due to their high abundance and pharmacological relevance there is a growing demand for the efficient production of functional membrane proteins. In this context, cell-free protein synthesis represents a valuable alternative that allows for the high-throughput synthesis of functional membrane proteins. Here, we demonstrate the potential of our cell-free protein synthesis system, based on lysates from cultured Spodoptera frugiperda 21 cells, to produce pro- and eukaryotic membrane proteins with individual topological characteristics in an automated fashion. Analytical techniques, including confocal laser scanning microscopy, fluorescence detection of eYFP fusion proteins in a microplate reader and in-gel fluorescence of statistically incorporated fluorescent amino acid derivatives were employed. The reproducibility of our automated synthesis approach is underlined by coefficients of variation below 7.2%. Moreover, the functionality of the cell-free synthesized potassium channel KcsA was analyzed electrophysiologically. Finally, we expanded our cell-free membrane protein synthesis system by an orthogonal tRNA/synthetase pair for the site-directed incorporation of p-Azido-l-phenylalanine based on stop codon suppression. Incorporation was optimized by performance of a two-dimensional screening with different Mg(2+) and lysate concentrations. Subsequently, the selective modification of membrane proteins with incorporated p-Azido-l-phenylalanine was exemplified by Staudinger ligation with a phosphine-based fluorescence dye.

With the help of HW/SW codesign, system-on-chip (SoC) can effectively reduce cost, improve reliability, and produce versatile products. The growing complexity of SoC designs makes on-chip communication subsystem design as important as computation subsystem design. While a number of codesign methodologies have been proposed for on-chip computation subsystems, many works are needed for on-chip communication subsystems. This paper proposes application-specific networkson-chip (ASNoC) and its design methodology. ASNoC is used for two high-performance SoC applications. The methodology (1) can automatically generate optimized ASNoC for different applications, (2) can generate a corresponding distributed shared memory along with an ASNoC, (3) can use both recorded and statistical communication traces for cycle-accurate performance analysis, (4) is based on standardized network component library and floorplan to estimate power and area, (5) adapts an industrial-grade network modeling and simulation environment, OPNET, which makes the methodology ready to use, and (6) can be easily integrated into current HW/SW codesign flow. Using the methodology, ASNoC is generated for a H.264 HDTV decoder SoC and Smart Camera SoC. ASNoC and 2D mesh networks-on-chip are compared in performance, power, and area in detail. The comparison results show that ASNoC provide substantial improvements in power, performance, and cost compared to 2D mesh networks-on-chip. In the H.264 HDTV decoder SoC, ASNoC uses 39% less power, 59% less silicon area, 74% less metal area, 63% less switch capacity, and 69% less interconnection capacity to achieve 2X performance compared to 2D mesh networks-on-chip.

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis.  Graphical abstract

Microarray technology provides new analytical devices that allow the parallel and simultaneous detection of several thousands of probes within one sample. Microarrays, sometimes called DNA chips, are widely used in gene-expression analysis, genotyping of individuals, analysis of point mutations and single nucleotide polymorphisms (SNP) as well as other genomic or transcriptomic variations. In this chapter we give a survey of common microarray manufacturing, the selection of support material, immobilisation and hybridisation and the detection with labelled complementary strands. However, DNA arrays may also serve as the basis for more complex analysis based on the action of enzymes on the immobilized templates. This property gives DNA microarrays the potential for being the template for whole PCR and transcription experiments with high parallelism, as will be discussed in the last section of this chapter.

In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the blaCTX-M15 gene in less than 1.6E−03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.

A purely electrical sensing scheme is presented that determines the concentration of macromolecules in solution by measuring the capacitance between planar microelectrodes. Concentrations of DNA in the ng/mL range have been used in samples of 1 L volume. The method has been applied to the characterisation of the dielectrophoretic response of DNA without the need for any chemical modifications. The influence of electrical parameters like duty cycle, voltage and frequency has been investigated. The results are in good agreement with data from dielectrophoretic studies on fluorescently labelled DNA. Extension of the method down to the single molecule level appears feasible.

Loop mediated isothermal amplification (LAMP) is one of the best known and most popular isothermal amplification methods. It's simplicity and speed make the method particularly suitable for point-of-care diagnostics. Nevertheless, false positive results remain a major drawback. Many (downstream) applications are known for the detection of LAMP amplicons like colorimetric assays, in-situ LAMP or CRISPR-Cas systems. Often, modifications of the LAMP products are necessary for different detection applications such as lateral flow assays. This is usually achieved with pre-modified primer. The aim of this study is to evaluate amplicon labelling with different modified nucleotides such as Cy5-dUTP, biotin-dUTP and aminoallyl-dUTP as an alternative to pre-labelled primers. To realise this, the effects on amplification and labelling efficiency were studied as a function of molecule size and nucleotide amount as well as target concentration. This research shows that diverse labelling of LAMP amplicons can be achieved using different, modified nucleotides during LAMP and that these samples can be analysed by a wide range of downstream applications such as fluorescence spectroscopy, gel electrophoresis, microarrays and lateral flow systems. Furthermore, microarray-based detection and the ability to identify and distinguish false positives were demonstrated as proof of concept.

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