Development of a Disposable Polyacrylamide Hydrogel-Based Semipermeable Membrane for Micro Ag/AgCl Reference Electrode.

Currently, Ag/AgCl-based reference electrodes are used in most electrochemical biosensors and other bioelectrochemical devices. However, standard reference electrodes are rather large and do not always fit within electrochemical cells designed for the determination of analytes in low-volume aliquots. Therefore, various designs and improvements in reference electrodes are critical for the future development of electrochemical biosensors and other bioelectrochemical devices. In this study, we explain a procedure to apply common laboratory polyacrylamide hydrogel in a semipermeable junction membrane between the Ag/AgCl reference electrode and the electrochemical cell. During this research, we have created disposable, easily scalable, and reproducible membranes suitable for the design of reference electrodes. Thus, we came up with castable semipermeable membranes for reference electrodes. Performed experiments highlighted the most suitable gel formation conditions to achieve optimal porosity. Here, Cl- ion diffusion through the designed polymeric junctions was evaluated. The designed reference electrode was also tested in a three-electrode flow system. The results show that home-built electrodes can compete with commercial products due to low reference electrode potential deviation (~3 mV), long shelf-life (up to six months), good stability, low cost, and disposability. The results show a high response rate, which makes in-house formed polyacrylamide gel junctions good membrane alternatives in the design of reference electrodes, especially for these applications where high-intensity dyes or toxic compounds are used and therefore disposable electrodes are required.

Molecularly Imprinted Polymers for the Determination of Cancer Biomarkers.

Biomarkers can provide critical information about cancer and many other diseases; therefore, developing analytical systems for recognising biomarkers is an essential direction in bioanalytical chemistry. Recently molecularly imprinted polymers (MIPs) have been applied in analytical systems to determine biomarkers. This article aims to an overview of MIPs used for the detection of cancer biomarkers, namely: prostate cancer (PSA), breast cancer (CA15-3, HER-2), epithelial ovarian cancer (CA-125), hepatocellular carcinoma (AFP), and small molecule cancer biomarkers (5-HIAA and neopterin). These cancer biomarkers may be found in tumours, blood, urine, faeces, or other body fluids or tissues. The determination of low concentrations of biomarkers in these complex matrices is technically challenging. The overviewed studies used MIP-based biosensors to assess natural or artificial samples such as blood, serum, plasma, or urine. Molecular imprinting technology and MIP-based sensor creation principles are outlined. Analytical signal determination methods and the nature and chemical structure of the imprinted polymers are discussed. Based on the reviewed biosensors, the results are compared, and the most suitable materials for each biomarker are discussed.

Revealing the SARS-CoV-2 Spike Protein and Specific Antibody Immune Complex Formation Mechanism for Precise Evaluation of Antibody Affinity.

The profound understanding and detailed evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (SCoV2-S) protein and specific antibody interaction mechanism is of high importance in the development of immunosensors for COVID-19. In the present work, we studied a model system of immobilized SCoV2-S protein and specific monoclonal antibodies by molecular dynamics of immune complex formation in real time. We simultaneously applied spectroscopic ellipsometry and quartz crystal microbalance with dissipation to reveal the features and steps of the immune complex formation. We showed direct experimental evidence based on acoustic and optical measurements that the immune complex between covalently immobilized SCoV2-S and specific monoclonal antibodies is formed in two stages. Based on these findings it was demonstrated that applying a two-step binding mathematical model for kinetics analysis leads to a more precise determination of interaction rate constants than that determined by the 1:1 Langmuir binding model. Our investigation showed that the equilibrium dissociation constants (KD) determined by a two-step binding model and the 1:1 Langmuir model could differ significantly. The reported findings can facilitate a deeper understanding of antigen-antibody immune complex formation steps and can open a new way for the evaluation of antibody affinity towards corresponding antigens.

Electrochemical Immunosensor for the Determination of Antibodies against Prostate-Specific Antigen Based on ZnO Nanostructures.

In this study, ZnO nanostructures with different types of morphologies and particle sizes were evaluated and applied for the development of an immunosensor. The first material was composed of spherical, polydisperse nanostructures with a particle size in the range of 10-160 nm. The second was made up of more compact rod-like spherical nanostructures with the diameter of these rods in the range of 50-400 nm, and approximately 98% of the particles were in the range of 20-70 nm. The last sample of ZnO was made up of rod-shaped particles with a diameter of 10-80 nm. These ZnO nanostructures were mixed with Nafion solution and drop-casted onto screen-printed carbon electrodes (SPCE), followed by a further immobilization of the prostate-specific antigen (PSA). The affinity interaction of PSA with monoclonal antibodies against PSA (anti-PSA) was evaluated using the differential pulse voltammetry technique. The limit of detection and limit of quantification of anti-PSA were determined as 1.35 nM and 4.08 nM for compact rod-shaped spherical ZnO nanostructures, and 2.36 nM and 7.15 nM for rod-shaped ZnO nanostructures, respectively.

Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review).

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.

Molecularly imprinted polypyrrole based sensor for the detection of SARS-CoV-2 spike glycoprotein.

This study describes the application of a polypyrrole-based sensor for the determination of SARS-CoV-2-S spike glycoprotein. The SARS-CoV-2-S spike glycoprotein is a spike protein of the coronavirus SARS-CoV-2 that recently caused the worldwide spread of COVID-19 disease. This study is dedicated to the development of an electrochemical determination method based on the application of molecularly imprinted polymer technology. The electrochemical sensor was designed by molecular imprinting of polypyrrole (Ppy) with SARS-CoV-2-S spike glycoprotein (MIP-Ppy). The electrochemical sensors with MIP-Ppy and with polypyrrole without imprints (NIP-Ppy) layers were electrochemically deposited on a platinum electrode surface by a sequence of potential pulses. The performance of polymer layers was evaluated by pulsed amperometric detection. According to the obtained results, a sensor based on MIP-Ppy is more sensitive to the SARS-CoV-2-S spike glycoprotein than a sensor based on NIP-Ppy. Also, the results demonstrate that the MIP-Ppy layer is more selectively interacting with SARS-CoV-2-S glycoprotein than with bovine serum albumin. This proves that molecularly imprinted MIP-Ppy-based sensors can be applied for the detection of SARS-CoV-2 virus proteins.

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.

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.

Biosensors for the Determination of SARS-CoV-2 Virus and Diagnosis of COVID-19 Infection.

Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Electrochemical Biosensor for the Determination of Specific Antibodies against SARS-CoV-2 Spike Protein.

In this article, we report the development of an electrochemical biosensor for the determination of the SARS-CoV-2 spike protein (rS). A gold disc electrode was electrochemically modified to form the nanocrystalline gold structure on the surface. Then, it was further altered by a self-assembling monolayer based on a mixture of two alkane thiols: 11-mercaptoundecanoic acid (11-MUA) and 6-mercapto-1-hexanol (6-MCOH) (SAMmix). After activating carboxyl groups using a N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixture, the rS protein was covalently immobilized on the top of the SAMmix. This electrode was used to design an electrochemical sensor suitable for determining antibodies against the SARS-CoV-2 rS protein (anti-rS). We assessed the association between the immobilized rS protein and the anti-rS antibody present in the blood serum of a SARS-CoV-2 infected person using three electrochemical methods: cyclic voltammetry, differential pulse voltammetry, and potential pulsed amperometry. The results demonstrated that differential pulse voltammetry and potential pulsed amperometry measurements displayed similar sensitivity. In contrast, the measurements performed by cyclic voltammetry suggest that this method is the most sensitive out of the three methods applied in this research.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Advances and insights in the diagnosis of viral infections.

Viral infections are the most common among diseases that globally require around 60 percent of medical care. However, in the heat of the pandemic, there was a lack of medical equipment and inpatient facilities to provide all patients with viral infections. The detection of viral infections is possible in three general ways such as (i) direct virus detection, which is performed immediately 1-3 days after the infection, (ii) determination of antibodies against some virus proteins mainly observed during/after virus incubation period, (iii) detection of virus-induced disease when specific tissue changes in the organism. This review surveys some global pandemics from 1889 to 2020, virus types, which induced these pandemics, and symptoms of some viral diseases. Non-analytical methods such as radiology and microscopy also are overviewed. This review overlooks molecular analysis methods such as nucleic acid amplification, antibody-antigen complex determination, CRISPR-Cas system-based viral genome determination methods. Methods widely used in the certificated diagnostic laboratory for SARS-CoV-2, Influenza A, B, C, HIV, and other viruses during a viral pandemic are outlined. A comprehensive overview of molecular analytical methods has shown that the assay's sensitivity, accuracy, and suitability for virus detection depends on the choice of the number of regions in the viral open reading frame (ORF) genome sequence and the validity of the selected analytical method.

From Microorganism-Based Amperometric Biosensors towards Microbial Fuel Cells.

This review focuses on the overview of microbial amperometric biosensors and microbial biofuel cells (MFC) and shows how very similar principles are applied for the design of both types of these bioelectronics-based devices. Most microorganism-based amperometric biosensors show poor specificity, but this drawback can be exploited in the design of microbial biofuel cells because this enables them to consume wider range of chemical fuels. The efficiency of the charge transfer is among the most challenging and critical issues during the development of any kind of biofuel cell. In most cases, particular redox mediators and nanomaterials are applied for the facilitation of charge transfer from applied biomaterials towards biofuel cell electrodes. Some improvements in charge transfer efficiency can be achieved by the application of conducting polymers (CPs), which can be used for the immobilization of enzymes and in some particular cases even for the facilitation of charge transfer. In this review, charge transfer pathways and mechanisms, which are suitable for the design of biosensors and in biofuel cells, are discussed. Modification methods of the cell-wall/membrane by conducting polymers in order to enhance charge transfer efficiency of microorganisms, which can be potentially applied in the design of microbial biofuel cells, are outlined. The biocompatibility-related aspects of conducting polymers with microorganisms are summarized.

Evaluation of Electrochromic Properties of Polypyrrole/Poly(Methylene Blue) Layer Doped by Polysaccharides.

Polypyrrole (Ppy) and poly(methylene blue) (PMB) heterostructure (Ppy-PMB) was electrochemically formed on the indium tin oxide (ITO) coated glass slides, which served as working electrodes. For electropolymerization, a solution containing pyrrole, methylene blue, and a saccharide (lactose, sucrose, or heparin) that served as dopant was used. The aim of this study was to compare the effect of the saccharides (lactose, sucrose, and heparin) on the electrochromic properties of the Ppy-PMB layer. AFM and SEM have been used for the analysis of the surface dominant features of the Ppy-PMB layers. From these images, it was concluded that the saccharides used in this study have a moderate effect on the surface morphology. Electrochromic properties were analyzed with respect to the changes of absorbance of the layer at two wavelengths (668 nm and 750 nm) by changing the pH of the surrounding solution and the potential between +0.8 V and -0.8 V. It was demonstrated that the highest absorbance changes are characteristic for all layers in the acidic media. Meanwhile, the absorbance changes of the layers were decreased in the more alkaline media. It was determined that the Ppy-PMB layers with heparin as a dopant were more mechanically stable in comparison to the layers doped with lactose and sucrose. Therefore, the Ppy-PMB layer doped with heparin was selected for the further experiment and it was applied in the design of electrochromic sensors for the determination of three xanthine derivatives: caffeine, theobromine, and theophylline. A linear relationship of ΔA (∆A = A+0.8V - A-0.8V) vs. concentration was determined for all three xanthine derivatives studied. The largest change in optical absorption was observed in the case of theophylline determination.

Molecular Imprinting Technology for Determination of Uric Acid.

The review focuses on the overview of electrochemical sensors based on molecularly imprinted polymers (MIPs) for the determination of uric acid. The importance of robust and precise determination of uric acid is highlighted, a short description of the principles of molecular imprinting technology is presented, and advantages over the others affinity-based analytical methods are discussed. The review is mainly concerned with the electro-analytical methods like cyclic voltammetry, electrochemical impedance spectroscopy, amperometry, etc. Moreover, there are some scattered notes to the other electrochemistry-related analytical methods, which are capable of providing additional information and to solve some challenges that are not achievable using standard electrochemical methods. The significance of these overviewed methods is highlighted. The overview of the research that is employing MIPs imprinted with uric acid is mainly targeted to address these topics: (i) type of polymers, which are used to design uric acid imprint structures; (ii) types of working electrodes and/or other parts of signal transducing systems applied for the registration of analytical signal; (iii) the description of the uric acid extraction procedures applied for the design of final MIP-structure; (iv) advantages and disadvantages of electrochemical methods and other signal transducing methods used for the registration of the analytical signal; (vi) overview of types of interfering molecules, which were analyzed to evaluate the selectivity; (vi) comparison of analytical characteristics such as linear range, limits of detection and quantification, reusability, reproducibility, repeatability, and stability. Some insights in future development of uric acid sensors are discussed in this review.

Insights in the Application of Stoichiometric and Non-Stoichiometric Titanium Oxides for the Design of Sensors for the Determination of Gases and VOCs (TiO2-x and TinO2n-1 vs. TiO2).

In this review article, attention is paid towards the formation of various nanostructured stoichiometric titanium dioxide (TiO2), non-stoichiometric titanium oxide (TiO2-x) and Magnéli phase (TinO2n-1)-based layers, which are suitable for the application in gas and volatile organic compound (VOC) sensors. Some aspects related to variation of sensitivity and selectivity of titanium oxide-based sensors are critically overviewed and discussed. The most promising titanium oxide-based hetero- and nano-structures are outlined. Recent research and many recently available reviews on TiO2-based sensors and some TiO2 synthesis methods are discussed. Some promising directions for the development of TiO2-based sensors, especially those that are capable to operate at relatively low temperatures, are outlined. The applicability of non-stoichiometric titanium oxides in the development of gas and VOC sensors is foreseen and transitions between various titanium oxide states are discussed. The presence of non-stoichiometric titanium oxide and Magnéli phase (TinO2n-1)-based layers in 'self-heating' sensors is predicted, and the advantages and limitations of 'self-heating' gas and VOC sensors, based on TiO2 and TiO2-x/TiO2 heterostructures, are discussed.

Progress and Insights in the Application of MXenes as New 2D Nano-Materials Suitable for Biosensors and Biofuel Cell Design.

Recent progress in the application of new 2D-materials-MXenes-in the design of biosensors, biofuel cells and bioelectronics is overviewed and some advances in this area are foreseen. Recent developments in the formation of a relatively new class of 2D metallically conducting MXenes opens a new avenue for the design of conducting composites with metallic conductivity and advanced sensing properties. Advantageous properties of MXenes suitable for biosensing applications are discussed. Frontiers and new insights in the area of application of MXenes in sensorics, biosensorics and in the design of some wearable electronic devices are outlined. Some disadvantages and challenges in the application of MXene based structures are critically discussed.

Towards direct enzyme wiring: a theoretical investigation of charge carrier transfer mechanisms between glucose oxidase and organic semiconductors.

In this work, a general theoretical and numerical approach based on semiconductor theory, which could be applied to a study of direct enzyme wiring, has been discussed. Marcus-Hush theory was applied to evaluate the potential transfer of charge carriers (holes and electrons) between glucose oxidase (GOx) and organic semiconductors. Two mechanisms of multistep hopping of charge to/from the oxidised/reduced flavin-based moiety through residues of aromatic amino acids located in GOx and long range charge direct tunnelling from the cofactor to the organic semiconductor surface have been proposed and evaluated. It was determined that the hole-hopping mechanism is possible and proceeds at a low ionization potential of the organic semiconductor. The calculations reveal that hopping of electrons is blocked, but direct electron tunnelling between the cofactor and the organic semiconductor is still probable. The most optimal conditions and tunable characteristics of GOx-based biosensors such as the ionization potential, electron affinity of organic semiconductors and distance between the enzyme and surface were estimated for the first time.

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).

TiO2-x/TiO2-Structure Based 'Self-Heated' Sensor for the Determination of Some Reducing Gases.

In this research we report the gas-sensing properties of TiO2-x/TiO2-based hetero-structure, which was 'self-heated' by current that at constant potential passed through the structure. Amperometric measurements were applied for the evaluation of sensor response towards ethanol, methanol, n-propanol and acetone gases/vapours. The sensitivity towards these gases was based on electrical resistance changes, which were determined by amperometric measurements of current at fixed voltage applied between Pt-based contacts/electrodes deposited on the TiO2-x/TiO2-based layer. X-ray diffraction (XRD) analysis revealed the formation of TiO2-x/TiO2-based hetero-structure, which is mainly based on Ti3O5/TiO2 formed during the hydro-thermal oxidation-based sensing-layer preparation process. Additionally, photoluminescence and time-resolved photoluminescence decay kinetics-based signals of this sensing structure revealed the presence of TiO2 mainly in the anatase phase in the TiO2-x/TiO2-based hetero-structure, which was formed at 400 °C annealing temperature. The evaluation of TiO2-x/TiO2-based gas-sensing layer was performed at several different temperatures (25 °C, 72 °C, 150 °C, 180 °C) and at these temperatures different sensitivity to the aforementioned gaseous materials was determined.