Exploring the Living Cell: Applications and Advances of Scanning Electrochemical Microscopy.

A living cell is a complex network of molecular, biochemical and physiological processes. Cellular activities, such as ion transport, metabolic processes, and cell-cell interactions can be determined electrochemically by detecting the electrons or ions exchanged in these processes. Electrochemical methods often are noninvasive, and they can enable the real-time monitoring of cellular processes. Scanning electrochemical microscopy (SECM) is an advanced scanning probe electroanalysis technique that can map the surface topography and local reactivity of a substrate with high precision at the micro- or nanoscale. By measuring electrochemical signals, such as redox reactions, ion fluxes, and pH changes, SECM can provide valuable insights into cellular activity. As a result of its compatibility with liquid medium measurements and its nondestructive nature, SECM has gained popularity in living cell research. This review aims to furnish an overview of SECM, elucidating its principles, applications, and its potential to contribute significantly to advancements in cell biology, electroporation, and biosensors. As a multidisciplinary tool, SECM is distinguished by its ability to unravel the intricacies of living cells and offers promising avenues for breakthroughs in our understanding of cellular complexity.

Application of Prussian Blue in Electrochemical and Optical Sensing of Free Chlorine.

In this paper, an electrochemical free chlorine (FCL) sensor was formed by modifying a fluorine-doped tin oxide-coated glass slide (glass|FTO) with a layer of Prussian blue (glass|FTO|PB). The glass|FTO|PB sensor exhibited a wide linear detection range from 1.7 to 99.2 µmol L-1 of FCL with a sensitivity of ~0.8 µA cm-2 µmol-1 L and showed high selectivity for FCL. However, ClO3-, ClO4- and NO3- ions have induced only a negligible amperometric response that is highly beneficial for a real-life sample analysis as these ions are commonly found in chlorine-treated water. Moreover, in this work, optical absorption measurement-based investigations of partially reduced PB were carried out as a means to characterize PB catalytic activity towards FCL and to investigate the possibility of applying PB for the optical detection of FCL.

Scanning electrochemical microscopy based irreversible destruction of living cells.

In this research, scanning electrochemical microscopy combined with electrochemical impedance spectroscopy has been applied to irreversible electroporation of active yeast cells by causing cell death. This finding is important for the development of irreversible electroporation technique, which could be suitable for the curing of cancerous tissues, because during this research cell death has been achieved using relatively low ultramicro-electrode (UME) voltage, precisely of 2.0 V vs Ag/AgCl,Cl-sat. It was determined that the irreversibly electroporated area of immobilized yeast cells was located directly below the UME and was of approximately 20 times larger width than the diameter of the UME, leaving undamaged cells out of this area. The ability of SECM to move the UME with high accuracy in x, y, and z directions and the ability to use electrodes of various diameters as well as the fact that the diameter of the electroporated area depends on the diameter of the UME and on the distance between the UME and the surface, what offers the possibility to establish targeted electroporation systems for selective treatment of tissues.

Biosensors Based on Bio-Functionalized Semiconducting Metal Oxides.

Immobilization of biomaterials is a very important task in the development of biofuel cells and biosensors. Some semiconducting metal-oxide-based supporting materials can be used in these bioelectronics-based devices. In this article, we are reviewing some functionalization methods that are applied for the immobilization of biomaterials. The most significant attention is paid to the immobilization of biomolecules on the surface of semiconducting metal oxides. The improvement of biomaterials immobilization on metal oxides and analytical performance of biosensors by coatings based on conducting polymers, self-assembled monolayers and lipid membranes is discussed.

Scanning electrochemical microscope as a tool for the electroporation of living yeast cells.

In this study, a scanning electrochemical microscope (SECM) was for the first time adapted to perform the electroporation process of living yeast cells. We have demonstrated that relatively low voltage pulses of 1-2 V vs. Ag/AglCl,Cl-sat applied to gold-based ultramicroelectrode (Au-UME) are performing reversible electroporation of yeast cells immobilized on fluorine-doped tin oxide (FTO)/glass surface. SECM and electrochemical impedance spectroscopy (EIS) were used for the determination of quantitative electrochemical characteristics before and after the electroporation. The electrochemical impedance spectroscopy (EIS) illustrated significant electrochemical changes of electroporated yeast cells, while SECM feedback mode surface vertical scan current-distance curves showed that the diameter of the area affected by the electrical pulse is about 25 times larger than the diameter of the Au-UME used for the electroporation process. The results presented in this research open up a possibility to develop a targeted electroporation system which will affect only the selected area of tissue or some other cell-covered surface. Such model is promising for the selective treatment of selected cells in tissues and/or other sensitive biological systems while selecting the location and size of electroporated areas.

Hybrid bilayer membranes on metallurgical polished aluminum.

In this work we describe the functionalization of metallurgically polished aluminum surfaces yielding biomimetic electrodes suitable for probing protein/phospholipid interactions. The functionalization involves two simple steps: silanization of the aluminum and subsequent fusion of multilamellar vesicles which leads to the formation of a hybrid bilayer lipid membrane (hBLM). The vesicle fusion was followed in real-time by fast Fourier transform electrochemical impedance spectroscopy (FFT EIS). The impedance-derived complex capacitance of the hBLMs was approximately 0.61 µF cm-2, a value typical for intact phospholipid bilayers. We found that the hBLMs can be readily disrupted if exposed to > 400 nM solutions of the pore-forming peptide melittin. However, the presence of cholesterol at 40% (mol) in hBLMs exhibited an inhibitory effect on the membrane-damaging capacity of the peptide. The melittin-membrane interaction was concentration dependent decreasing with concentration. The hBLMs on Al surface can be regenerated multiple times, retaining their dielectric and functional properties essentially intact.

Electroporation of a hybrid bilayer membrane by scanning electrochemical microscope.

A novel method, suitable for targeted electroporation of hybrid bilayer membranes (hBLMs) by scanning electrochemical microscope (SECM) is introduced by this work. A redox-probe-free system was applied for (i) SECM-based electroporation of a hBLM and for (ii) SECM-based visualization of pores formed by SECM-based electroporation in the hBLM. The hBLM was formed on a glass substrate modified by fluorine-doped tin oxide, and the structure (glass/FTO/hBLM) was used for further investigations. A specific 'constant-current region' at 1-30 µm distances between the UME and the hBLM surface was observed in the approach curves, which were registered while a Pt-based ultramicroelectrode (UME) was approaching the glass/FTO/hBLM surface. This 'constant-current region' was used as the characteristic feature for characterisation of the hBLM, and by assessment of the approach curves it was possible to distinguish whether an area of the hBLM was electroporated. SECM-based electroporation of the hBLM was performed by using increased potential difference between the reference electrode and the UME. Depending on the duration of the applied potential-pulse and on the distance between the UME and the hBLM surface, irreversible or reversible electroporation of the hBLM was achieved. The data shows that SECM can be successfully applied for both electroporation and characterisation of the hBLM.

Mixed hybrid bilayer lipid membranes on mechanically polished titanium surface.

Mixed self-assembled monolayers of octadecyltrichlorosilane (OTS) and methyltrichlorosilane (MTS) were deposited via simple silanization procedure on a mechanically polished titanium surface. The monolayers act as molecular anchors for mixed hybrid bilayer lipid membranes (mhBLM) which were accomplished via vesicle fusion. A variation of the MTS concentration in silanization solutions significantly affects properties of mhBLMs composed of a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol). The bilayers become less insulating following an increase of the MTS content. On the other hand, an increase of the MTS concentration provides flexibility of the mhBLM membranes necessary for the functional reconstitution of membrane proteins. The optimal molar ratio of MTS in silanization solution is 40% providing anchors for intact mhBLMs as confirmed by their specific capacitance of 0.86 µF cm-2. We found that the bilayers containing 40% (mol) of cholesterol bind cholesterol dependent pneumolysin (PLY). However, we did not observe functional reconstitution of PLY. While α-hemolysin almost fully disrupts mhBLMs assembled from 100% diphytanoyl. An important advantage of the titanium/OTS/MTS molecular anchor systems is their ability of repetitive regeneration of phospholipid bilayers without losing functional properties as demonstrated in the current study. This creates a possibility for the multiple-use phospholipid membrane biosensors which have a potential of decreasing the cost of such electrochemical/electroanalytical devices.

Silane-based self-assembled monolayer deposited on fluorine doped tin oxide as model system for pharmaceutical and biomedical analysis.

Recently, self-assembled monolayers (SAMs) are gaining a lot of interest due to their simplicity of preparation and wide applicability in the development of model systems used in pharmaceutical and biomedical analysis. The most efficient methods used for the investigation of SAM-based structures usually include cyclic voltammetry and electrochemical impedance spectroscopy. Scanning electrochemical microscopy (SECM) could be also used as an alternative method for SAM investigations, because this method enables to map modified surface. In this work, the surface of fluorine doped tin oxide (FTO) was modified with octadeciltrichlorosilane (OTS) based SAM and investigated using SECM. Measurements, which were carried out by SECM, lead to conclusion that highly heterogeneous and distributed monolayer has been formed on FTO surface.

Electrochemical Deposition and Investigation of Poly-9,10-Phenanthrenequinone Layer.

In this research, a 9,10-phenanthrenequinone (PQ) was electrochemically polymerized on a graphite rod electrode using potential cycling. The electrode modified by poly-9,10-phenanthrenequinone (poly-PQ) was studied by means of cyclic voltammetry, electrochemical impedance spectroscopy, atomic force microscopy and scanning electron microscopy. The poly-PQ shows variations in growth pattern depending on the number of potential cycles for the initiation of polymerization. Formed poly-PQ layer demonstrates good electric conductivity, great degree of electrochemical capacitance and unique oxidation/reduction properties, which are suitable for broad technological applications, including applicability in biosensors, supercapacitors and in some other electrochemical systems.

Redox-probe-free scanning electrochemical microscopy combined with fast Fourier transform electrochemical impedance spectroscopy.

Scanning electrochemical microscopy (SECM) hybridized with fast Fourier transform-based electrochemical impedance spectroscopy (FFT-EIS) seems to be a powerful variation of scanning electrochemical impedance microscopy (SEIM), wherein both state-of-the-art techniques are combined (FFT-SEIM) and can be used for the investigation and treatment of tissues at single cell level. However, in most EIS-based experiments, harmful redox mediators are applied, which affect the functioning of living cells and tissues. Therefore, the development of a redox-probe-free FFT-SEIM is still a very important challenge in electrochemistry. For this reason, in this research, we have demonstrated a redox-probe-free evaluation of conducting and non-conducting surfaces by combining scanning electrochemical microscopy with FFT-EIS. It was demonstrated that using the fast Fourier transform-based FFT-EIS technique, EIS spectra could be registered much faster compared to experiments performed using the conventional EIS equipment. An ultramicroelectrode (UME) was used as a scanning electrode to ensure high spatial resolution. We have performed FFT-SEIM measurements in a redox-probe-free mode (without any additional redox probes) and have investigated several surfaces with different conductivities. The FFT-EIS equipment and the built-in software help to avoid the influence of possible formation of hydrogen bubbles on the UME. This research opens up a new avenue for the application of FFT-SEIM in the investigation of samples that are unstable and very sensitive towards redox mediators (e.g., tissues and/or living cells).