Development of an Activity-Based Ratiometric Electrochemical Switch for Direct, Real-Time Sensing of Pantetheinase in Live Cells, Blood, and Urine Samples.

Pantetheinase is a key biomarker for the diagnosis of acute kidney injury and the monitoring of malaria progression. Currently, existing methods for sensing pantetheinase, also known as Vanin-1, show considerable potential but come with certain limitations, including their inability to directly sense analytes in turbid biofluid samples without tedious sample pretreatment. Here, we describe the first activity-based electrochemical probe, termed VaninLP, for convenient and specific direct targeting of pantetheinase activity in turbid liquid biopsy samples. The probe was designed such that cleavage of the pantetheinase amide linkage, triggered by a self-immolative reaction, simultaneously ejects an amino ferrocene reporter. Among the distinctive properties of the VaninLP probe for sensing pantetheinase are its high selectivity, sensitivity, and enzyme affinity, a wide linear concentration range (8-300 ng/mL), and low limit of detection (2.47 ng/mL). The designed probe precisely targeted pantetheinase and was free of interference by other electroactive biological species. We further successfully applied the VaninLP probe to monitor and quantify the activity of pantetheinase on the surfaces of HepG2 tumor cells, blood, and urine samples. Collectively, our findings indicate that VaninLP holds significant promise as a point-of-care tool for diagnosing early-stage kidney injury, as well as monitoring the progression of malaria.

Interplay between Electric Field Strength and Number of Short-Duration Pulses for Efficient Gene Electrotransfer.

Electroporation is a method that shows great promise as a non-viral approach for delivering genes by using high-voltage electric pulses to introduce DNA into cells to induce transient gene expression. This research aimed to evaluate the interplay between electric pulse intensity and 100 µs-duration pulse numbers as an outcome of gene electrotransfer efficacy and cell viability. Our results indicated a close relationship between pulse number and electric field strength regarding gene electrotransfer efficacy; higher electric pulse intensity resulted in fewer pulses needed to achieve the same gene electrotransfer efficacy. Subsequently, an increase in pulse number had a more negative impact on overall gene electrotransfer by significantly reducing cell viability. Based on our data, the best pulse parameters to transfect CHO cells with the pMax-GFP plasmid were using 5 HV square wave pulses of 1000 V/cm and 2 HV of 1600 V/cm, correspondingly resulting in 55 and 71% of transfected cells and maintaining 79 and 54% proliferating cells. This shows ESOPE-like 100 µs-duration pulse protocols can be used simultaneously to deliver cytotoxic drugs as well as immune response regulating genetically encoded cytokines.

Localised Electrochemical Impedance Spectroscopy of Gold Nanoparticles Labelled Antibodies Probed by Platinum Microstructured Ultramicroelectrode.

This research is focused on enhancing the capabilities of scanning electrochemical impedance microscopy (SEIM) for detecting gold nanoparticle-labelled antibodies using electrochemically modified platinum ultramicroelectrode. The primary objective was to address the high resistance issue encountered in previous measurements with SEIM via the utilization of SEIM probes based on micro-electrodes modified by platinum microstructures, which improved the sensitivity and precision of the detection of targeted biomolecules. The modified probe resulted in a lowered charge transfer resistance by over ten times and a decrease in detection to around 100 fg/mL. We suggest potential applications in various biotechnological and biomedical fields, with future research expected to further refine this technique.

Fast detection of micro-objects using scanning electrochemical microscopy based on visual recognition and machine learning.

Scanning electrochemical microscopy (SECM) is a scanning probe microscope with an ultramicroelectrode (UME) as a probe. The technique is advantageous in the characterization of the electrochemical properties of surfaces. However, the limitations, such as slow imaging and many functions depending on the user, only allow us to use some of the possibilities. Therefore, we applied visual recognition and machine learning to detect micro-objects from the image and determine their electrochemical activity. The reconstruction of the image from several approach curves allows it to scan faster and detect active areas of the sample. Therefore, the scanning time and presence of the user is diminished. An automated scanning electrochemical microscope with visual recognition has been developed using commercially available modules, relatively low-cost components, design, software solutions proven in other fields, and an original control and data fusion algorithm.

Characteristics and Functionality of Cantilevers and Scanners in Atomic Force Microscopy.

In this paper, we provide a systematic review of atomic force microscopy (AFM), a fast-developing technique that embraces scanners, controllers, and cantilevers. The main objectives of this review are to analyze the available technical solutions of AFM, including the limitations and problems. The main questions the review addresses are the problems of working in contact, noncontact, and tapping AFM modes. We do not include applications of AFM but rather the design of different parts and operation modes. Since the main part of AFM is the cantilever, we focused on its operation and design. Information from scientific articles published over the last 5 years is provided. Many articles in this period disclose minor amendments in the mechanical system but suggest innovative AFM control and imaging algorithms. Some of them are based on artificial intelligence. During operation, control of cantilever dynamic characteristics can be achieved by magnetic field, electrostatic, or aerodynamic forces.

Microbial Biofuel Cells: Fundamental Principles, Development and Recent Obstacles.

This review focuses on the development of microbial biofuel cells to demonstrate how similar principles apply to the development of bioelectronic devices. The low specificity of microorganism-based amperometric biosensors can be exploited in designing microbial biofuel cells, enabling them to consume a broader range of chemical fuels. Charge transfer efficiency is among the most challenging and critical issues while developing biofuel cells. Nanomaterials and particular redox mediators are exploited to facilitate charge transfer between biomaterials and biofuel cell electrodes. The application of conductive polymers (CPs) can improve the efficiency of biofuel cells while CPs are well-suitable for the immobilization of enzymes, and in some specific circumstances, CPs can facilitate charge transfer. Moreover, biocompatibility is an important issue during the development of implantable biofuel cells. Therefore, biocompatibility-related aspects of conducting polymers with microorganisms are discussed in this review. Ways to modify cell-wall/membrane and to improve charge transfer efficiency and suitability for biofuel cell design are outlined.

Development of Electrostatic Microactuators: 5-Year Progress in Modeling, Design, and Applications.

The implementation of electrostatic microactuators is one of the most popular technical solutions in the field of micropositioning due to their versatility and variety of possible operation modes and methods. Nevertheless, such uncertainty in existing possibilities creates the problem of choosing suitable methods. This paper provides an effort to classify electrostatic actuators and create a system in the variety of existing devices. Here is overviewed and classified a wide spectrum of electrostatic actuators developed in the last 5 years, including modeling of different designs, and their application in various devices. The paper provides examples of possible implementations, conclusions, and an extensive list of references.

Deep Q-Learning in Robotics: Improvement of Accuracy and Repeatability.

Recent industrial robotics covers a broad part of the manufacturing spectrum and other human everyday life applications; the performance of these devices has become increasingly important. Positioning accuracy and repeatability, as well as operating speed, are essential in any industrial robotics application. Robot positioning errors are complex due to the extensive combination of their sources and cannot be compensated for using conventional methods. Some robot positioning errors can be compensated for only using machine learning (ML) procedures. Reinforced machine learning increases the robot's positioning accuracy and expands its implementation capabilities. The provided methodology presents an easy and focused approach for industrial in situ robot position adjustment in real-time during production setup or readjustment cases. The scientific value of this approach is a methodology using an ML procedure without huge external datasets for the procedure and extensive computing facilities. This paper presents a deep q-learning algorithm applied to improve the positioning accuracy of an articulated KUKA youBot robot during operation. A significant improvement of the positioning accuracy was achieved approximately after 260 iterations in the online mode and initial simulation of the ML procedure.

Electrochemically Deposited Molecularly Imprinted Polymer-Based Sensors.

This review is dedicated to the development of molecularly imprinted polymers (MIPs) and the application of MIPs in sensor design. MIP-based biological recognition parts can replace receptors or antibodies, which are rather expensive. Conducting polymers show unique properties that are applicable in sensor design. Therefore, MIP-based conducting polymers, including polypyrrole, polythiophene, poly(3,4-ethylenedioxythiophene), polyaniline and ortho-phenylenediamine are frequently applied in sensor design. Some other materials that can be molecularly imprinted are also overviewed in this review. Among many imprintable materials conducting polymer, polypyrrole is one of the most suitable for molecular imprinting of various targets ranging from small organics up to rather large proteins. Some attention in this review is dedicated to overview methods applied to design MIP-based sensing structures. Some attention is dedicated to the physicochemical methods applied for the transduction of analytical signals. Expected new trends and horizons in the application of MIP-based structures are also discussed.

Evaluation of a Yeast-Polypyrrole Biocomposite Used in Microbial Fuel Cells.

Electrically conductive polymers are promising materials for charge transfer from living cells to the anodes of electrochemical biosensors and biofuel cells. The modification of living cells by polypyrrole (PPy) causes shortened cell lifespan, burdens the replication process, and diminishes renewability in the long term. In this paper, the viability and morphology non-modified, inactivated, and PPy-modified yeasts were evaluated. The results displayed a reduction in cell size, an incremental increase in roughness parameters, and the formation of small structural clusters of polymers on the yeast cells with the increase in the pyrrole concentration used for modification. Yeast modified with the lowest pyrrole concentration showed minimal change; thus, a microbial fuel cell (MFC) was designed using yeast modified by a solution containing 0.05 M pyrrole and compared with the characteristics of an MFC based on non-modified yeast. The maximal generated power of the modified system was 47.12 mW/m2, which is 8.32 mW/m2 higher than that of the system based on non-modified yeast. The open-circuit potentials of the non-modified and PPy-modified yeast-based cells were 335 mV and 390 mV, respectively. Even though applying a PPy layer to yeast increases the charge-transfer efficiency towards the electrode, the damage done to the cells due to modification with a higher concentration of PPy diminishes the amount of charge transferred, as the current density drops by 846 µA/cm2. This decrease suggests that modification by PPy may have a cytotoxic effect that greatly hinders the metabolic activity of yeast.

Scanning Electrochemical Impedance Microscopy in Redox-Competition Mode for the Investigation of Antibodies Labelled with Horseradish Peroxidase.

Scanning electrochemical microscopy enhanced by electrochemical impedance spectroscopy (SEIM) was applied to detect immobilized antibodies labelled with horseradish peroxidase (Ab-HRP). The localized HRP activity was investigated by the SEIM redox competition (RC-SEIM) mode using hydrogen peroxide as a substrate and hexacyanoferrate as a redox mediator. Electrochemical impedance shows to be related to the consumption of hydrogen peroxide at the ultramicroelectrode. For the evaluation of impedimetric results, an equivalent electric circuit was applied with solution resistance, double-layer capacitance, and charge-transfer resistance. These equivalent circuit characteristics depend on the distance between the sample and ultramicroelectrode, and the concentration of substrate. From the gathered data, the charge-transfer resistance appeared to be the parameter describing the behavior of HRP catalyzed reaction as it showed a linear dependence on H2O2 concentration. The RC-SEIM mode suitability for the studying of HRP catalyzed reactions and for the evaluation of Ab-HRP bound to the surface was demonstrated. Additionally, the applicability of RC-SEIM mode for the determination of Ab-HRP affinity bound to the target analyte was discussed.

Wearable Feet Pressure Sensor for Human Gait and Falling Diagnosis.

Human falls pose a serious threat to the person's health, especially for the elderly and disease-impacted people. Early detection of involuntary human gait change can indicate a forthcoming fall. Therefore, human body fall warning can help avoid falls and their caused injuries for the skeleton and joints. A simple and easy-to-use fall detection system based on gait analysis can be very helpful, especially if sensors of this system are implemented inside the shoes without causing a sensible discomfort for the user. We created a methodology for the fall prediction using three specially designed Velostat®-based wearable feet sensors installed in the shoe lining. Measured pressure distribution of the feet allows the analysis of the gait by evaluating the main parameters: stepping rhythm, size of the step, weight distribution between heel and foot, and timing of the gait phases. The proposed method was evaluated by recording normal gait and simulated abnormal gait of subjects. The obtained results show the efficiency of the proposed method: the accuracy of abnormal gait detection reached up to 94%. In this way, it becomes possible to predict the fall in the early stage or avoid gait discoordination and warn the subject or helping companion person.

Baker's Yeast-Based Microbial Fuel Cell Mediated by 2-Methyl-1,4-Naphthoquinone.

Microbial fuel cell (MFC) efficiency depends on charge transfer capability from microbe to anode, and the application of suitable redox mediators is important in this area. In this study, yeast viability experiments were performed to determine the 2-methyl-1,4-naphthoquinone (menadione (MD)) influence on different yeast cell species (baker's yeast and Saccharomyces cerevisiae yeast cells). In addition, electrochemical measurements to investigate MFC performance and efficiency were carried out. This research revealed that baker's yeast cells were more resistant to dissolved MD, but the current density decreased when yeast solution concentration was incrementally increased in the same cell. The maximal calculated power of a designed baker's yeast-based MFC cell anode was 0.408 mW/m2 and this power output was registered at 24 mV. Simultaneously, the cell generated a 62-mV open circuit potential in the presence of 23 mM potassium ferricyanide and the absence of glucose and immobilized MD. The results only confirm that MD has strong potential to be applied to microbial fuel cells and that a two-redox-mediator-based system is suitable for application in microbial fuel cells.

Polyethylene-Carbon Composite (Velostat®) Based Tactile Sensor.

The progress observed in 'soft robotics' brought some promising research in flexible tactile, pressure and force sensors, which can be based on polymeric composite materials. Therefore, in this paper, we intend to evaluate the characteristics of a force-sensitive material-polyethylene-carbon composite (Velostat®) by implementing this material into the design of the flexible tactile sensor. We have explored several possibilities to measure the electrical signal and assessed the mechanical and time-dependent properties of this tactile sensor. The response of the sensor was evaluated by performing tests in static, long-term load and cyclic modes. Experimental results of loading cycle measurements revealed the hysteresis and nonlinear properties of the sensor. The transverse resolution of the sensor was defined by measuring the response of the sensor at different distances from the loaded point. Obtained dependencies of the sensor's sensitivity, hysteresis, response time, transversal resolution and deformation on applied compressive force promise a practical possibility to use the polyethylene-carbon composite as a sensitive material for sensors with a single electrode pair or its matrix. The results received from experimental research have defined the area of the possible implementation of the sensor based on a composite material-Velostat®.

Towards Microorganism-Based Biofuel Cells: The Viability of Saccharomyces cerevisiae Modified by Multiwalled Carbon Nanotubes.

This research aimed to evaluate the toxic effect of multi-walled carbon nanotubes (MW-CNTs) on yeast cells in order to apply MW-CNTs for possible improvement of the efficiency of microbial biofuel cells. The SEM and XRD analysis suggested that here used MW-CNTs are in the range of 10-25 nm in diameter and their structure was confirmed by Raman spectroscopy. In this study, we evaluated the viability of the yeast Saccharomyces cerevisiae cells, affected by MW-CNTs, by cell count, culture optical density and atomic force microscopy. The yeast cells were exposed towards MW-CNTs (of 2, 50, 100 µg/mL concentrations in water-based solution) for 24 h. A mathematical model was applied for the evaluation of relative growth and relative death rates of yeast cells. We calculated that both of the rates are two times higher in the case if yeasts were treated by 50, 100 µg/mL of MW-CNTs containing solution, comparing to that treated by 0 and 2 µg/mL c of MW-CNTs containing solution. It was determined that the MW-CNTs have some observable effect upon the incubation of the yeast cells. The viability of yeast has decreased together with MW-CNTs concentration only after 5 h of the treatment. Therefore, we predict that the MW-CNTs can be applied for the modification of yeast cells in order to improve electrical charge transfer through the yeast cell membrane and/or the cell wall.

Scanning electrochemical microscopy in the development of enzymatic sensors and immunosensors.

Scanning electrochemical microscopy (SECM) is very useful, non-invasive tool for the analysis of surfaces pre-modified with biomolecules or by whole cells. This review focuses on the application of SECM technique for the analysis of surfaces pre-modified with enzymes (horseradish peroxidase, alkaline phosphatase and glucose oxidase) or labelled with antibody-enzyme conjugates. The working principles and operating modes of SECM are outlined. The applicability of feedback, generation-collection and redox competition modes of SECM on surfaces modified by enzymes or labelled with antibody-enzyme conjugates is discussed. SECM is important in the development of miniaturized bioanalytical systems with enzymes, since it can provide information about the local enzyme activity. Technical challenges and advantages of SECM, experimental parameters, used enzymes and redox mediators, immunoassay formats and analytical parameters of enzymatic SECM sensors and immunosensors are reviewed.

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).

Single-walled carbon nanotube based coating modified with reduced graphene oxide for the design of amperometric biosensors.

In this study a polycarbonate filter membrane (PcFM) with 400 nm diameter holes was covered/protruded by single walled carbon nanotubes (SWCNT) and then formed PcFM/SWCNT structure was covered by thin layer of graphene oxide (GO) or reduced graphene oxide (rGO) in order to get the multilayered PcFM/SWCNT/GO and PcFM/SWCNT/rGO coatings, respectively. It was determined that the SWCNTs filaments were able to form a layer on the polycarbonate membrane having a number of carbon nanotube arranged in different orientations. A fraction of SWCNT filaments protruded through the holes of polycarbonate membrane and in such way significantly enhanced the adhesion of SWCNT-based layer and provided electrical conductivity across the PcFM. Atomic force microscopy (AFM), scanning electron microscopy (SEM) images and Raman spectroscopy-based evaluation revealed the characteristic morphology features: wide distribution of height profile, separate GO/rGO flakes on the top of PcFM/SWCNT/GO structure and close attachment of rGO flakes on the top of multilayered PcFM/SWCNT/rGO coating. Performed contact angle measurement (CAM) enabled to determine the surface energy components and wettability data of prepared coatings. Both PcFM/SWCNT/GO and PcFM/SWCNT/rGO coatings were modified with glucose oxidase (GOx). Amperometric measurements revealed that multilayered PcFM/SWCNT/rGO/GOx coating is the most suitable structure for glucose biosensor design.

Modification of the AFM Sensor by a Precisely Regulated Air Stream to Increase Imaging Speed and Accuracy in the Contact Mode.

Increasing the imaging rate of atomic force microscopy (AFM) without impairing of the imaging quality is a challenging task, since the increase in the scanning speed leads to a number of artifacts related to the limited mechanical bandwidth of the AFM components. One of these artifacts is the loss of contact between the probe tip and the sample. We propose to apply an additional nonlinear force on the upper surface of a cantilever, which will help to keep the tip and surface in contact. In practice, this force can be produced by the precisely regulated airflow. Such an improvement affects the AFM system dynamics, which were evaluated using a mathematical model that is presented in this paper. The model defines the relationships between the additional nonlinear force, the pressure of the applied air stream, and the initial air gap between the upper surface of the cantilever and the end of the air duct. It was found that the nonlinear force created by the stream of compressed air (aerodynamic force) prevents the contact loss caused by the high scanning speed or the higher surface roughness, thus maintaining stable contact between the probe and the surface. This improvement allows us to effectively increase the scanning speed by at least 10 times using a soft (spring constant of 0.2 N/m) cantilever by applying the air pressure of 40 Pa. If a stiff cantilever (spring constant of 40 N/m) is used, the potential of vertical deviation improvement is twice is large. This method is suitable for use with different types of AFM sensors and it can be implemented practically without essential changes in AFM sensor design.

Atomic force microscopy as a tool for the investigation of living cells.

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.