One of the plausible reactions occurring during high-voltage pulses, which are used to electroporate the cells, is the oxidation of the metal ions of the anode resulting in the dissolution of the anode. In the case of the anode made from stainless steel, which is one of the most popular electrode materials, iron ions ( Fe²⁺ and Fe³⁺) are released from the anode. Here, this process and its consequences have been studied. A single square-wave electric pulse with the duration of 2 ms and the amplitude of 1.2 kV/cm increased the concentration of iron ions in solution by over 0.5 mM. Iron ions released from the anode behave as a Lewis acid and hydrolyze the water molecules in the solution, reducing the pH of a solution and might play a role in the changes of the medium conductivity. In addition, the roughness of the stainless steel anode increased progressively, in proportion with the total amount of the electric charge that had passed through the unit area of the electrode. The reduction of the viability of cells by iron ions has been demonstrated. The iron ions quench the fluorescence of anticancer drugs, which are used when photodynamic tumor therapy is combined with electroporation, such as porphyrin sulfonate and Adriamycin.

Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new μ-Tip-Enhanced Raman Spectroscopy (μ-TERS). The enhancing mechanism for μ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel μ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.

Due to the harmful effects of chemical fertilizers and pesticides, the need for an eco-friendly solution to improve soil fertility has become a necessity, thus microbial biofertilizer research is on the rise. Plant endophytic bacteria inhabiting internal tissues represent a novel niche for research into new biofertilizer strains. However, the number of species and strains that need to be differentiated and identified to facilitate faster screening in future plant-bacteria interaction studies, is enormous. Surface enhanced Raman spectroscopy (SERS) may provide a platform for bacterial discrimination and identification, which, compared with the traditional methods, is relatively rapid, uncomplicated and ensures high specificity. In this study, we attempted to differentiate 18 bacterial isolates from two oaks via morphological, physiological, biochemical tests and SERS spectra analysis. Previous 16S rRNA gene fragment sequencing showed that three isolates belong to Paenibacillus, 3—to Pantoea and 12—to Pseudomonas genera. Additional tests were not able to further sort these bacteria into strain-specific groups. However, the obtained label-free SERS bacterial spectra along with the high-accuracy principal component (PCA) and discriminant function analyses (DFA) demonstrated the possibility to differentiate these bacteria into variant strains. Furthermore, we collected information about the biochemical characteristics of selected isolates. The results of this study suggest a promising application of SERS in combination with PCA/DFA as a rapid, non-expensive and sensitive method for the detection and identification of plant-associated bacteria.