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Pulsed electric field effects induced in a membrane, as well as intracellular structures, depend on cell type, field and media parameters. To achieve desired outcomes, membranes should be permeabilized in a controlled manner, and thus efficiency of electroporation should be investigated in advance. Here, we present a framework for using mediated amperometry as a prospective method for the investigation of electroporation and its effects on cellular machinery. Whole-cell sensors with single mediator systems comprised of hydrophilic or lipophilic mediators were successfully employed to investigate membrane permeability as well as cellular responses. Exposure of yeast cells to single electric field pulse (τ = 300 µs, E = 16 kV/cm) resulted in up to tenfold increase of current strength mediated with hydrophilic mediators. Exposure to PEF resulted in decrease of menadione mediated current strength (from 138 ± 15 to 32 ± 15 nA), which could be completely compensated by supplementing electrolyte with NADH.

The design and development of a compact square-wave pulse generator for the electroporation of biological cells is presented. This electroporator can generate square-wave pulses with durations from 3 μs up to 10 ms, voltage amplitudes up to 3500 V, and currents up to 250 A. The quantity of the accumulated energy is optimized by means of a variable capacitor bank. The pulse forming unit design uses a crowbar circuit, which gives better control of the pulse form and its duration, independent of the load impedance. In such cases, the square-wave pulse form ensures better control of electroporation efficiency by choosing parameters determined in advance. The device has an integrated graphic LCD screen and measurement modules for the visualization of the current pulse, allowing for express control of the electroporation quality and does not require an external oscilloscope for current pulse recording. This electroporator was tested on suspensions of Saccharomyces cerevisiae yeast cells, during which, it was demonstrated that the application of such square-wave pulses ensured better control of the electroporation efficiency and cell viability after treatment using the pulsed electric field (PEF)., This article belongs to the Special Issue Information Technologies and Electronics, This work was partly funded by “Geozondas” Ltd. Implementing an EU structural investment project 01.2.1-MITA-T-851-02-0004 under measure “Inocekiai.”

The modelling of protein-protein binding kinetics is important for the development of affinity-sensors and the prediction of signaling protein based drug efficiency. Therefore, in this research we have evaluated the binding kinetics of several genetically designed protein models: (i) three different ligands based on granulocyte colony-stimulating factor GCSF homo-dimeric derivatives linked by differed by linkers of different length and flexibility; (ii) an antibody-like receptor (GCSF-R) based on two GCSF-receptor sites immobilized to Fc domains, which are common parts of protein structures forming antibodies. Genetically engineered GCSF-R is similar to an antibody because it, like the antibody, has two binding sites, which both selectively bind with GCSF ligands. To design the affinity sensor model studied here, GCSF-R was immobilized on a thin gold layer via self-assembled monolayer conjugated with Protein-G. Binding kinetics between immobilized GCSF-R and all three different recombinant GCSF-based homo-dimeric derivatives were evaluated by total internal reflection ellipsometry. Association constants were determined by fitting mathematical models to the experimental data. It was clearly observed that both (i) affinity and (ii) binding kinetics depend on the length and flexibility of the linker that connects both domains of a GCSF-based ligand. The fastest association between immobilized GCSF-R and GCSF-based ligands was observed for ligands whose GCSF domains were interconnected by the longest and the most flexible linker. Here we present ellipsometry-based measurements and models of the interaction kinetics that advance the understanding of bidentate-receptor-based immunosensor action and enables us to predict the optimal linker structure for the design of GCSF-based medications.

In this research we have evaluated the binding kinetics between an immobilized receptor and several genetically engineered ligands, differing by molecular mass or by the number of binding sites available for the binding to the receptor. Genetically engineered protein (GCSF-Receptor), which contains some antibody parts (Fc domain) and at some extent is similar to antibody because also has two binding sites that selectively bind another protein – glycoprotein granulocyte colony stimulating factor (GCSF), which was immobilized on a thin gold layer in order to design an immunosensor sensitive to GCSF. Three structurally different GCSF-based proteins were genetically-engineered and evaluated as ligands, which selectively bind to immobilized GCSF-Receptor: (i) GCSF monomer (mGCSF), (ii) GCSF-homodimer consisting of two via polypeptide Lα-based linker ‘fused’ GCSF molecules ((GCSF)2Lα) and (iii) GCSF-heterodimer (SCF-Lα-GCSF), which is based on a native GCSF molecule ‘fused’ via Lα-based linker with another protein – a soluble part of stem cell factor (SCF). SCF, unlike GCSF, does not contain any site suitable for GCSF-Receptor binding. The ligands differ by: (i) molecular mass – (GCSF)2Lα and SCF-Lα-GCSF F are two times heavier than mGCS, (ii) number of binding sites – mGCSF and SCF-Lα-GCSF have one binding site, while (GCSF)2Lα has two. The binding kinetics of mGCSF, (GCSF)2Lα, and SCF-Lα-GCSF with immobilized GCSF-Receptor was investigated using total internal reflection ellipsometry. The interaction kinetics of the mGCSF and SCF-Lα-GCSF are both well described using a standard Langmuir kinetics model. However, receptor-ligand association and dissociation rates in the case of SCF-Lα-GCSF ligand are about 10 times lower than that of mGCSF. The association rate of (GCSF)2Lα is about half of that of the mGCSF, which can be explained by the smaller diffusion coefficient of the larger molecule. Moreover, unlike SCF-Lα-GCSF, the (GCSF)2Lα adsorption kinetics cannot be adequately described by the standard Langmuir kinetics model and surface regeneration (induced by ‘washing’) experiments illustrate that (GCSF)2Lα, unlike the mGCSF and SCF-Lα-GCSF, is irreversibly bound to the surface modified by immobilized GCSF-Receptors. Therefore, to describe binding kinetics in the case of (GCSF)2Lα we have applied advanced kinetic model based on three protein association stages (three-stage kinetics model) in which (GCSF)2Lα forms several different intermediate complexes with GCSF-Receptor. This model precisely describes the time-varying surface concentration of (GCSF)2Lα bound to surface modified by immobilized GCSF-Receptors. In addition to the bioanalytical-aspects possible improvement of GCSF-based drugs is discussed.

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.

Prolonged storage of frozen dough worsens the structure of thawed dough. The main reason is the inhibition of yeast activity. In this study we investigated applicability of pulsed electric field treatment for introduction of cryoprotectant into yeast cells. We showed that pre-treatment of cells suspended in a trehalose solution improves freezing tolerance and results in higher viability after thawing. Viability increased with rise in electric field strength (from 3 to 4.5 kV/cm) and incubation time (from 0 to 60 min) after exposure. Pretreatment resulted in lower decrease in the viability of thawed cells, viability of untreated cells dropped to 10%, while pre-treatment with PEF and trehalose tripled the viability.

The present paper shows a green method for the formation of polypyrrole by two different MIUG 12p and MIUG 4.88 strains of Streptomyces spp. bacteria. According to our knowledge, such method has never been proposed before for pyrrole oxidation. For this experiment pyrrole monomer was added into bacterial medium after 6 days of incubation and after 4 days polypyrrole (PPy) based 10–25 μm microspheres has been observed in the medium around living cells, The microsphere diameter dependence on the bacterial strains was observed. The morphological characterization of the PPy microspheres was subsequently performed by optical microscopy (OM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy. The characterization of formed hollow microspheres confirmed that they consist of polypyrrole.

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.

Magnetic particles are promising carriers for targeted drug delivery. Granulocyte colony-stimulating factor (G-CSF) is a cytokine which stimulates neutrophil proliferation process. In this study, G-CSF molecules were successfully covalently immobilized on magnetic silica gel beads (MagBs) surface. Comparison of free and on MagBs immobilised G-CSF (MagBs-G) on proliferation efficiency and viability of mouse myeloid cell line NFS-60 was performed using XTT assay. Cytotoxic or stimulation effects of used MagBs on NFS-60 cell proliferation were not observed. However, the positive effect on proliferation of cells was noticed increasing the concentration of MagB-G in the test wells from 0.5 μg/ml to 50 μg/ml. Proliferation of cells in the presence of 50 μg/ml MagBs-G (2.7 μg/ml G-CSF) resulted in maximal proliferation response, which was reached using free G-CSF. Comparing the concentration of G-CSF that gives half-maximal response of free (3.6 pM) and on MagBs immobilized (11.4 nM) G-CSF can be concluded that immobilized G-CSF possessed the same efficiency as free G-CSF molecules, however the higher amount of immobilized G-CSF is required.