Microphysiological system with integrated sensors to study the effect of pulsed electric field.

This study focuses on the use of pulsed electric fields (PEF) in microfluidics for controlled cell studies. The commonly used material for soft lithography, polydimethylsiloxane (PDMS), does not fully ensure the necessary chemical and mechanical resistance in these systems. Integration of specific analytical measurement setups into microphysiological systems (MPS) are also challenging. We present an off-stoichiometry thiol-ene (OSTE)-based microchip, containing integrated electrodes for PEF and transepithelial electrical resistance (TEER) measurement and the equipment to monitor pH and oxygen concentration in situ. The effectiveness of the MPS was empirically demonstrated through PEF treatment of the C6 cells. The effects of PEF treatment on cell viability and permeability to the fluorescent dye DapI were tested in two modes: stop flow and continuous flow. The maximum permeability was achieved at 1.8 kV/cm with 16 pulses in stop flow mode and 64 pulses per cell in continuous flow mode, without compromising cell viability. Two integrated sensors detected changes in oxygen concentration before and after the PEF treatment, and the pH shifted towards alkalinity following PEF treatment. Therefore, our proof-of-concept technology serves as an MPS for PEF treatment of mammalian cells, enabling in situ physiological monitoring.

Biosensor Based on Graphene Directly Grown by MW-PECVD for Detection of COVID-19 Spike (S) Protein and Its Entry Receptor ACE2.

Biosensors based on graphene field-effect transistors (G-FET) for detecting COVID-19 spike S protein and its receptor ACE2 were reported. The graphene, directly synthesized on SiO2/Si substrate by microwave plasma-enhanced chemical vapor deposition (MW-PECVD), was used for FET biosensor fabrication. The commercial graphene, CVD-grown on a copper substrate and subsequently transferred onto a glass substrate, was applied for comparison purposes. The graphene structure and surface morphology were studied by Raman scattering spectroscopy and atomic force microscope. Graphene surfaces were functionalized by an aromatic molecule PBASE (1-pyrenebutanoic acid succinimidyl ester), and subsequent immobilization of the receptor angiotensin-converting enzyme 2 (ACE2) was performed. A microfluidic system was developed, and transfer curves of liquid-gated FET were measured after each graphene surface modification procedure to investigate ACE2 immobilization by varying its concentration and subsequent spike S protein detection. The directly synthesized graphene FET sensitivity to the receptor ACE2, evaluated in terms of the Dirac voltage shift, exceeded the sensitivity of the transferred commercial graphene-based FET. The concentration of the spike S protein was detected in the range of 10 ag/mL up to 10 µg/mL by using a developed microfluidic system and measuring the transfer characteristics of the liquid-gated G-FETs. It was found that the shift of the Dirac voltage depends on the spike S concentration and was 27 mV with saturation at 10 pg/mL for directly synthesized G-FET biosensor, while for transferred G-FET, the maximal shift of 70 mV was obtained at 10 µg/mL with a tendency of saturation at 10 ng/mL. The detection limit as low as 10 ag/mL was achieved for both G-FETs. The sensitivity of the biosensors at spike S concentration of 10 pg/mL measured as relative current change at a constant gate voltage corresponding to the highest transconductance of the G-FETs was found at 5.6% and 8.8% for directly synthesized and transferred graphene biosensors, respectively. Thus, MW-PECVD-synthesized graphene-based biosensor demonstrating high sensitivity and low detection limit has excellent potential for applications in COVID-19 diagnostics.

Development of a Disposable Polyacrylamide Hydrogel-Based Semipermeable Membrane for Micro Ag/AgCl Reference Electrode.

Currently, Ag/AgCl-based reference electrodes are used in most electrochemical biosensors and other bioelectrochemical devices. However, standard reference electrodes are rather large and do not always fit within electrochemical cells designed for the determination of analytes in low-volume aliquots. Therefore, various designs and improvements in reference electrodes are critical for the future development of electrochemical biosensors and other bioelectrochemical devices. In this study, we explain a procedure to apply common laboratory polyacrylamide hydrogel in a semipermeable junction membrane between the Ag/AgCl reference electrode and the electrochemical cell. During this research, we have created disposable, easily scalable, and reproducible membranes suitable for the design of reference electrodes. Thus, we came up with castable semipermeable membranes for reference electrodes. Performed experiments highlighted the most suitable gel formation conditions to achieve optimal porosity. Here, Cl- ion diffusion through the designed polymeric junctions was evaluated. The designed reference electrode was also tested in a three-electrode flow system. The results show that home-built electrodes can compete with commercial products due to low reference electrode potential deviation (~3 mV), long shelf-life (up to six months), good stability, low cost, and disposability. The results show a high response rate, which makes in-house formed polyacrylamide gel junctions good membrane alternatives in the design of reference electrodes, especially for these applications where high-intensity dyes or toxic compounds are used and therefore disposable electrodes are required.

From Microorganism-Based Amperometric Biosensors towards Microbial Fuel Cells.

This review focuses on the overview of microbial amperometric biosensors and microbial biofuel cells (MFC) and shows how very similar principles are applied for the design of both types of these bioelectronics-based devices. Most microorganism-based amperometric biosensors show poor specificity, but this drawback can be exploited in the design of microbial biofuel cells because this enables them to consume wider range of chemical fuels. The efficiency of the charge transfer is among the most challenging and critical issues during the development of any kind of biofuel cell. In most cases, particular redox mediators and nanomaterials are applied for the facilitation of charge transfer from applied biomaterials towards biofuel cell electrodes. Some improvements in charge transfer efficiency can be achieved by the application of conducting polymers (CPs), which can be used for the immobilization of enzymes and in some particular cases even for the facilitation of charge transfer. In this review, charge transfer pathways and mechanisms, which are suitable for the design of biosensors and in biofuel cells, are discussed. Modification methods of the cell-wall/membrane by conducting polymers in order to enhance charge transfer efficiency of microorganisms, which can be potentially applied in the design of microbial biofuel cells, are outlined. The biocompatibility-related aspects of conducting polymers with microorganisms are summarized.

Yeast-assisted synthesis of polypyrrole: Quantification and influence on the mechanical properties of the cell wall.

In this study, the metabolism of yeast cells (Saccharomyces cerevisiae) was utilized for the synthesis of the conducting polymer - polypyrrole (Ppy).Yeast cells were modified in situ by synthesized Ppy. The Ppy was formed in the cell wall by redox-cycling of [Fe(CN)6]3-/4-, performed by the yeast cells. Fluorescence microscopy, enzymatic digestions, atomic force microscopy and isotope ratio mass spectroscopy were applied to determine both the polymerization reaction itself and the polymer location in yeast cells. Ppy formation resulted in enhanced resistance to lytic enzymes, significant increase of elasticity and alteration of other mechanical cell wall properties evaluated by atomic force microscopy (AFM). The suggested method of polymer synthesis allows the introduction of polypyrrole structures within the cell wall, which is build up from polymers consisting of carbohydrates. This cell wall modification strategy could increase the usefulness of yeast as an alternative energy source in biofuel cells, and in cell based biosensors.

Synthesis of Polypyrrole Induced by [Fe(CN)6]3- and Redox Cycling of [Fe(CN)6]4-/[Fe(CN)6]3.

Chemical synthesis of the conducting polymer polypyrrole induced by [Fe(CN)6]3- is reported. Reaction kinetics were characterized spectrophotometrically. Reaction rate was evaluated at several different pH levels in the presence of [Fe(CN)6]3- and [Fe(CN)6]4- ions. The formation of polypyrrole at aerobic and anaerobic conditions was evaluated. We report that at anaerobic conditions [Fe(CN)6]4- cannot initiate oxidative polymerization, while its oxidized form [Fe(CN)6]3- successfully initiates and maintains the pyrrole polymerization reaction. The formation of polypyrrole was also observed in the solution containing a pyrrole monomer, [Fe(CN)6]4- and dissolved oxygen due to re-oxidation (redox cycling) of [Fe(CN)6]4- into [Fe(CN)6]3- by dissolved oxygen. Experiments to determine the polymerization reaction rate were performed and showed the highest rate in the presence of 0.5 mM of [Fe(CN)6]3- at pH 9.0, while the polymerization reaction performed at pH 7.0 was determined as the slowest. This investigation opens new horizons for the application of [Fe(CN)6]4-/[Fe(CN)6]3--based redox cycling reactions in the synthesis of the conducting polymer polypyrrole and potentially in the formation of other conducting polymers which can be formed by oxidative polymerization.

Kinetic 15N-isotope effects on algal growth.

Stable isotope labeling is a standard technique for tracing material transfer in molecular, ecological and biogeochemical studies. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism metabolism and growth, which is not consistent with current theoretical and empirical knowledge on kinetic isotope effects. Here, we demonstrate profound changes in growth dynamics of the green alga Raphidocelis subcapitata grown in 15N-enriched media. With increasing 15N concentration (0.37 to 50 at%), the lag phase increased, whereas maximal growth rate and total yield decreased; moreover, there was a negative relationship between the growth and the lag phase across the treatments. The latter suggests that a trade-off between growth rate and the ability to adapt to the high 15N environment may exist. Remarkably, the lag-phase response at 3.5 at% 15N was the shortest and deviated from the overall trend, thus providing partial support to the recently proposed Isotopic Resonance hypothesis, which predicts that certain isotopic composition is particularly favorable for living organisms. These findings confirm the occurrence of KIE in isotopically enriched algae and underline the importance of considering these effects when using stable isotope labeling in field and experimental studies.

Synthesis of polypyrrole within the cell wall of yeast by redox-cycling of [Fe(CN)6](3-)/[Fe(CN)6](4-).

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)6](3-)ions, which were generated from K4[Fe(CN)6], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)6](4-). After 24h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated.