Sensitive enzymatic determination of neurotransmitters in artificial sweat.

Dopamine (DA) and epinephrine (EN) are two phenolic molecules that are used in the human body and secreted in the brain actuating as neurotransmitters. As both molecules are highly important in the brain and the central nervous system, monitoring of their concentrations would enable better understanding of their importance and role in different physiological conditions. Copper efflux oxidase from Escherichia coli was shown in the past to have the ability of oxidizing polyphenolic compounds. As such, we have engineered this enzyme for its site-specific attachment to an electrode for the detection of DA and EN in high resolution and sensitivity. Here we present an enzymatic biosensor that enables the detection of such molecules with a 10 nM resolution and a linear range of up to 100 nM in artificial sweat samples. The presented biosensor could be used for the determination of catecholamines in different bodily fluids.

Use of Protein Engineering to Elucidate Electron Transfer Pathways between Proteins and Electrodes.

Herein, we review protein engineering tools for electron transfer enhancement and investigation in bioelectrochemical systems. We present recent studies in the field while focusing on how electron transfer investigation and measurements were performed and discuss the use of protein engineering to interpret electron transfer mechanisms.

Site-specifically wired and oriented glucose dehydrogenase fused to a minimal cytochrome with high glucose sensing sensitivity.

Direct electron transfer based enzymatic biosensors are highly efficient systems where electrons are transferred directly from the enzyme's electroactive site to the electrode. One way of achieving it is by 'wiring' the enzyme to the electrode surface. The wiring of enzymes to electrode surfaces can be reached in many different ways but controlling its orientation towards the electrode surface is still a challenge. In this study we have designed a Flavin-adenine dinucleotide dependent glucose dehydrogenase that is fused to a minimal cytochrome with a site-specifically incorporated unnatural amino acid to control its orientation towards the electrode. Several site-specifically wired mutant enzymes were compared to each other and to a non-specifically wired enzyme using atomic force microscopy and electrochemical techniques. The surface and activity analyses suggest that the site-specific wiring through different sites maintains the correct folding of the enzyme and have a positive effect on the apparent electrochemical electron transfer rate constant kETapp. Electrochemical analysis revealed an efficient electron transfer rate with more than 15 times higher imax and 10-fold higher sensitivity of the site-specifically wired enzyme variants compared to the non-specifically wired ones. This approach can be utilized to control the orientation of other redox enzymes on electrodes to allow a significant improvement of their electron transfer communication with electrodes.

DNA/RNA Electrochemical Biosensing Devices a Future Replacement of PCR Methods for a Fast Epidemic Containment.

Pandemics require a fast and immediate response to contain potential infectious carriers. In the recent 2020 Covid-19 worldwide pandemic, authorities all around the world have failed to identify potential carriers and contain it on time. Hence, a rapid and very sensitive testing method is required. Current diagnostic tools, reverse transcription PCR (RT-PCR) and real-time PCR (qPCR), have its pitfalls for quick pandemic containment such as the requirement for specialized professionals and instrumentation. Versatile electrochemical DNA/RNA sensors are a promising technological alternative for PCR based diagnosis. In an electrochemical DNA sensor, a nucleic acid hybridization event is converted into a quantifiable electrochemical signal. A critical challenge of electrochemical DNA sensors is sensitive detection of a low copy number of DNA/RNA in samples such as is the case for early onset of a disease. Signal amplification approaches are an important tool to overcome this sensitivity issue. In this review, the authors discuss the most recent signal amplification strategies employed in the electrochemical DNA/RNA diagnosis of pathogens.

Context effects of genetic code expansion by stop codon suppression.

Genetic code expansion enables the incorporation of unnatural amino acids into proteins thereby augmenting their physical and chemical properties. This is achieved by the reassignment of codons from their original sense to incorporate unnatural amino acids. The most commonly used methodology is stop codon suppression, which has resulted in numerous successful studies and applications in recent years. In these studies, many observations have been accumulated indicating that stop codon suppression efficiency depends on various cellular, operon and mRNA context effects. Predominant among these are mRNA context effects: the location of the stop codon along the mRNA governs, to a large extent, the efficiency and ability to successfully incorporate unnatural amino acids. Albeit their prevalence and importance, the mechanisms that govern context effects remain largely unknown. Herein, we will review what is known and yet to be understood with the intent to advance the propagation of genetic code expansion technology and to stimulate systematic research and debate of this open question.

Highly Efficient Flavin-Adenine Dinucleotide Glucose Dehydrogenase Fused to a Minimal Cytochrome C Domain.

Flavin-adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) is a thermostable, oxygen insensitive redox enzyme used in bioelectrochemical applications. The FAD cofactor of the enzyme is buried within the proteinaceous matrix of the enzyme, which makes it almost unreachable for a direct communication with an electrode. In this study, FAD dependent glucose dehydrogenase was fused to a natural minimal cytochrome domain in its c-terminus to achieve direct electron transfer. We introduce a fusion enzyme that can communicate with an electrode directly, without the use of a mediator molecule. The new fusion enzyme, with its direct electron transfer abilities displays superior activity to that of the native enzyme, with a kcat that is ca. 3 times higher than that of the native enzyme, a kcat/KM that is more than 3 times higher than that of GDH and 5 to 7 times higher catalytic currents with an onset potential of ca. (-) 0.15 V vs Ag/AgCl, affording higher glucose sensing selectivity. Taking these parameters into consideration, the fusion enzyme presented can serve as a good candidate for blood glucose monitoring and for other glucose based bioelectrochemical systems.