Arunas Stirke

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.

Electric field-induced effects on yeast cell wall permeabilization.

The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.

Permeabilization of yeast Saccharomyces cerevisiae cell walls using nanosecond high power electrical pulses

The electrical field-induced changes of the yeast Saccharomyces cerevisiae cells permeabilization to tetraphenylphosphonium (TPP+) ions were studied using square-shaped, nanosecond duration high power electrical pulses. It was obtained that pulses having durations ranging from 10 ns to 60 ns, and generating electric field strengths up to 190 kV/cm significantly (up to 65 times) increase the absorption rate of TPP+ ions without any detectible influence on the yeast cell viability. The modelling of the TPP+ absorption process using a second order rate equation demonstrates that depending on the duration of the pulses, yeast cell clusters of different sizes are homogeniously permeabilized. It was concluded, that nanosecond pulse-induced permeabilization can be applied to increase the operational speed of whole cell biosensors.

System for the Nanoporation of Biological Cells Based on an Optically-Triggered High-Voltage Spark-Gap Switch

This nanosecond electric pulse generator is designed for the electroporation of biological cells suspended in a liquid media. It is based on a spark-gap switch, which is optically triggered by a 0.45-ns duration and 1-mJ energy laser pulse (wavelength 1062 nm). This system can also be triggered manually by changing the distance between the spark-gap electrodes. It is able to generate in a 75- Ω impedance transmission line near-perfect square-shaped electric pulses (rise and fall times ) with durations of 10, 40, 60, or 92 ns. The maximal amplitude of such pulses is 12.5 kV. The main advantage of this system is its ability to generate single pulses, the amplitude and duration of which can be precisely set in advance. To treat the cells, a coaxial cuvette with a 0.03-mL active volume and a 1-mm distance between the 28.3- mm2 circular-shaped electrodes was used. The system was tested on human erythrocytes. It was demonstrated that for the 92- and 40-ns duration pulse, the amplitude required to electroporate 50% of the cells was 20 and 65 kV/cm, respectively.

Compact high-sensitivity potentiometer for detection of low ion concentrations in liquids

The compact potentiometer, based on an electronic circuit protected from electrostatic and electromagnetic interference, was developed for the measurement of low ion concentrations in liquids. The electronic circuit of the potentiometer, consisting of analogous and digital parts, enables the measurement of fA currents. This makes it possible to perform reliable measurements of ion concentrations in liquids that are as small as 10-8-10-7M. The instrument was tested using electrodes that were selective for tetraphenylphosphonium (TPP+) ions. It was demonstrated that the characteristic response time of the potentiometer electronic circuit to changes in the concentration of these ions in a liquid was in the order of 10 s. An investigation of TPP+ absorption by baker yeast has shown that this device can be successfully used for long term (several hours) measurements with zero signal drift, which was about 1 μV/s. Finally, due to the small dimensions of the electronic circuit (7.5 × 2 × 1.5 cm), this potentiometer can be easily installed at a large apparatus in the laboratory condition (≈25 °C), such as high pulsed electrical generators of magnetic fields that are used in electroporation studies of biological cells.

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.