Investigation of Peptides for Molecular Recognition of C-Reactive Protein-Theoretical and Experimental Studies.

We investigate the interactions between C-reactive protein (CRP) and new CRP-binding peptide materials using experimental (biological and physicochemical) methods with the support of theoretical simulations (computational modeling analysis). Three specific CRP-binding peptides (P2, P3, and P9) derived from an M13 bacteriophage have been identified using phage-display technology. The binding efficiency of the peptides exposed on phages toward the CRP protein was demonstrated via biological methods. Fibers of the selected phages/peptides interact differently due to different compositions of amino acid sequences on the exposed peptides, which was confirmed by transmission electron microscopy. Numerical and experimental studies consistently showed that the P3 peptide is the best CRP binder. A combination of theoretical and experimental methods demonstrates that identifying the best binder can be performed simply, cheaply, and fast. Such an approach has not been reported previously for peptide screening and demonstrates a new trend in science where calculations can replace or support laborious experimental techniques. Finally, the best CRP binder─the P3 peptide─was used for CRP recognition on silicate-modified indium tin oxide-coated glass electrodes. The obtained electrodes exhibit a wide range of operation (1.0-100 µg mL-1) with a detection limit (LOD = 3σ/S) of 0.34 µg mL-1. Moreover, the dissociation constant Kd of 4.2 ± 0.144 µg mL-1 (35 ± 1.2 nM) was evaluated from the change in the current. The selectivity of the obtained electrode was demonstrated in the presence of three interfering proteins. These results prove that the presented P3 peptide is a potential candidate as a receptor for CRP, which can replace specific antibodies.

Ion Transfer Voltammetry with an Electrochemical Pen.

We present a new electrochemical system that combines paper-based sensing and ion-transfer voltammetry, bringing the latter a step closer toward point-of-care applications. Studies at the interface between two immiscible electrolyte solutions (ITIES) are often performed to detect redox-inactive species; unfortunately, due to the inherent instability of the interface, it is rather poorly explored outside specialized laboratories. Here, we address this limitation by combining a pen-like device containing the gelled organic phase with a paper-supported aqueous phase. This combination makes the system more user-friendly, potentially low-cost, and easy to assemble. We show the applicability of the new cell to analyze both simple and ionophore-facilitated transfer of ions and proteins, preconcentration of species, and analysis of mixtures through combination with paper chromatography. The native ion content of the paper also enabled measurements without added electrolytes. Those studies could broaden the scope for the application of the label-free electrochemical detection of nonredox-active species at points-of-need.

Photochemical Printing of Plasmonically Active Silver Nanostructures.

In this paper, we demonstrate plasmonic substrates prepared on demand, using a straightforward technique, based on laser-induced photochemical reduction of silver compounds on a glass substrate. Importantly, the presented technique does not impose any restrictions regarding the shape and length of the metallic pattern. Plasmonic interactions have been probed using both Stokes and anti-Stokes types of emitters that served as photoluminescence probes. For both cases, we observed a pronounced increase of the photoluminescence intensity for emitters deposited on silver patterns. By studying the absorption and emission dynamics, we identified the mechanisms responsible for emission enhancement and the position of the plasmonic resonance.

Silver Island Film for Enhancing Light Harvesting in Natural Photosynthetic Proteins.

The effects of combining naturally evolved photosynthetic pigment-protein complexes with inorganic functional materials, especially plasmonically active metallic nanostructures, have been a widely studied topic in the last few decades. Besides other applications, it seems to be reasonable using such hybrid systems for designing future biomimetic solar cells. In this paper, we describe selected results that point out to various aspects of the interactions between photosynthetic complexes and plasmonic excitations in Silver Island Films (SIFs). In addition to simple light-harvesting complexes, like peridinin-chlorophyll-protein (PCP) or the Fenna-Matthews-Olson (FMO) complex, we also discuss the properties of large, photosynthetic reaction centers (RCs) and Photosystem I (PSI)-both prokaryotic PSI core complexes and eukaryotic PSI supercomplexes with attached antenna clusters (PSI-LHCI)-deposited on SIF substrates.

Electrochemical Detection of Dopamine and Serotonin in the Presence of Interferences in a Rotating Droplet System.

In this Article, a rotating droplet system is used for simultaneous detection of dopamine and serotonin. Carbon nanoparticles functionalized with sulfonic groups on the electrode surface enables potential discrimination between the neurotransmitters and the most common interferences, whereas the efficient and low-volume hydrodynamic system helps to lower the detection limit toward physiologically relevant concentrations. Here, we present results with a 10 nM limit of detection for serotonin and a 100 nM to 2 µM linear response range from the system in a sample containing an equimolar concentrations of dopamine and serotonin and 0.5 mM concentration of both uric and ascorbic acids. Demonstrating the practical applicability of this method, we measure the concentration of serotonin in 70 µL of mice blood serum samples without additional pretreatment.

Paper-Based System for Ion Transfer Across the Liquid-Liquid Interface.

While ion transfer studies were shown to be a promising method for fundamental electrochemistry, pharmacokinetic studies, and sensing, they suffer from inherent instability of the interface formed between the organic and aqueous phases. This limits to some extent the range of solvents which can be used and confines these studies to the laboratory. We propose here the use of paper, which has revolutionized the way we think of miniaturized analytical devices during the past decade, as a perfect substrate for ion transfer studies across the liquid-liquid interface. We describe the design of a simple three-phase electrode paper-based setup for redox-driven transfer of anions from an aqueous to an organic phase. Electrochemical measurements of seven different anions and concentration dependence studies are in good agreement with the results obtained with traditional setups. Additionally, we show the applicability and limitations of such setups to the analysis of anion mixtures.

Pencil Lead as a Material for Microfluidic 3D-Electrode Assemblies.

We present an electrochemical, microfluidic system with a working electrode based on an ordered 3D array of pencil leads. The electrode array was integrated into a plexiglass/PDMS channel. We tested the setup using a simple redox probe and compared the results with computer simulations. As a proof of concept application of the device we showed that the setup can be used for determination of dopamine concentration in physiological pH and ultrasensitive, although only qualitative, detection of p-nitrophenol with a limit of detection below 1 nmol L-1. The observed limit of detection for p-nitrophenol is not only much lower than achieved with similar methods but also sufficient for evaluation of exposure to pesticides such as methyl parathion through urinalysis. This low cost setup can be fabricated without the need for clean room facilities and in the future, due to the ordered structure of the electrode could be used to better understand the process of electroanalysis and electrode functionalization. To the best of our knowledge it is the first application of pencil leads as 3D electrochemical sensor in a microfluidic channel.

Generation-Collection Electrochemistry Inside a Rotating Droplet.

In this work, we explore generation-collection electrochemistry in a rotating droplet hydrodynamic system, where a 70 µL droplet containing a redox active species (ferrocyanide) is sandwiched between an upper rotating rod and bottom nonmoving generator and collector planar electrodes. In such a system, we studied the effect of the counter electrode reaction on the recorded generator current, and the effect of the generator-collector distance (ranging from 3 to ca. 500 µm) on the collection efficiencies obtained at rotation rates ranging from 50 to 1100 rpm. We found that the counter electrode reaction competes with the collector reaction for the regeneration of the electroactive species; thus, collection efficiencies of 100% are probably impossible to obtain with this system geometry. We found that the collection efficiency increases with the droplet rotation rate and decreases with the generator-collector distance. The highest collection efficiency we obtained is 62% for the generator-collector distance of 3 µm, which is more than two times higher than that for typical bulk experiments with a commercial rotating ring disk electrode. We show that the increased collection efficiency can be successfully used in epinephrine detection for filtering out signals from ascorbic acid and uric acid interferents.

Highly active 3-dimensional cobalt oxide nanostructures on the flexible carbon substrates for enzymeless glucose sensing.

The demand for electrochemical sensors with high sensitivity and reliability, fast response, and excellent selectivity has stimulated intensive research on developing highly active nanomaterials. In this work, freestanding 3D/Co3O4 thorn-like and wire-like (nanowires) nanostructures are directly grown on a flexible carbon fiber paper (CFP) substrate by a single-step hydrothermal process without using surfactants or templates. The 3D/Co3O4 thorn-like nanostructures show higher electrochemical activity than wire-like because of their high conductivity, large specific surface areas, and mesopores on their surface. The characterization of 3D/Co3O4 nanostructures is performed by using high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction analysis (XRD), and electrochemical methods. The 3D/Co3O4 thorn-like nanostructures displayed non-enzymatic higher catalytic activity towards the electrochemical detection of glucose, compared to the 3D/Co3O4 wire-like morphology. The 3D/Co3O4 thorn-like nanostructures show a wide linear range response of glucose concentration ranging from 1 to 1000 µM with a detection limit of 0.046 µM (S/N = 3). The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode selectively detects glucose in the presence of 100-fold excess of interfering compounds. The 3D/Co3O4 thorn-like nanostructure-modified CFP electrode is tested with human blood serum samples and validated with commercial glucose sensors. The newly developed sensor material shows potential for glucose monitoring in clinical and food samples.

Electrochemical Glucose Sensing: Is There Still Room for Improvement?

As diabetes is considered one of the biggest health care challenges of the coming decades substantial effort is being made to develop novel glucose monitoring systems, this includes thousands of articles which are being published every year. To the question in the title, we answer an unequivocal "yes" but maybe not necessarily in the areas where most of the published research is focused.

Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro.

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which were not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions.

Tungsten carbide nanotubes supported platinum nanoparticles as a potential sensing platform for oxalic acid.

Supported tungsten carbide is an efficient and vital nanomaterial for the development of high-performance, sensitive, and selective electrochemical sensors. In this work, tungsten carbide with tube-like nanostructures (WC NTs) supported platinum nanoparticles (PtNPs) are synthesized and explored as an efficient catalyst toward electrochemical oxidation of oxalic acid for the first the time. The WC NTs supported PtNPs modified glassy carbon (GC) electrode is highly sensitive toward the electrochemical oxidation of oxalic acid. A large decrease in the oxidation overpotential (220 mV) and significant enhancement in the peak current compared to unmodified and Pt/C modified GC electrodes have been observed without using any redox mediator. Moreover, WC NTs supported PtNPs modified electrode possessed wide linear concentration ranges from 0 to 125 nM and a higher sensitivity toward the oxidation of oxalic acid (80 nA/nM) achieved by the amperometry method. The present modified electrode showed an experimentally determined lowest detection limit (LOD) of 12 nM (S/N = 3). Further, WC NTs supported PtNPs electrode can be demonstrated to have an excellent selectivity toward the detection of oxalic acid in the presence of a 200-fold excess of major important interferents. The practical application of WC NTs supported PtNPs has also been demonstrated in the detection of oxalic acid in tomato fruit sample, by differential pulse voltammetry under optimized conditions.

Antibody modified gold nanoparticles for fast and selective, colorimetric T7 bacteriophage detection.

Herein, we report a colorimetric immunosensor for T7 bacteriophage based on gold nanoparticles modified with covalently bonded anti-T7 antibodies. The new immunosensor allows for a fast, simple, and selective detection of T7 virus. T7 virions form immunological complexes with the antibody modified gold nanoparticles which causes them to aggregate. The aggregation can be observed with the naked eye as a color change from red to purple, as well as with a UV-vis spectrophotometer. The aggregate formation was confirmed with SEM imaging. Sensor selectivity against the M13 bacteriophage was demonstrated. The limit of detection (LOD) is 1.08 × 10(10) PFU/mL (18 pM) T7. The new method was compared with a traditional plaque test. In contrast to biological tests the colorimetric method allows for detection of all T7 phages, not only those biologically active. This includes phage ghosts and fragments of virions. T7 virus has been chosen as a model organism for adenoviruses. The described method has several advantages over the traditional ones. It is much faster than a standard plaque test. It is more robust since no bacteria-virus interactions are utilized in the detection process. Since antibodies are available for a large variety of pathogenic viruses, the described concept is very flexible and can be adapted to detect many different viruses, not only bacteriophages. Contrary to the classical immunoassays, it is a one-step detection method, and no additional amplification, e.g., enzymatic, is needed to read the result.

Selective electrochemical detection of dopamine in a microfluidic channel on carbon nanoparticulate electrodes.

There is a continuous need for the construction of detection systems in microfluidic devices. In particular, electrochemical detection allows the separation of signals from the analyte and interfering substances in the potential domain. Here, a simple microfluidic device for the sensitive and selective determination of dopamine in the presence of interfering substances was constructed and tested. It employs a carbon nanoparticulate electrode allowing the separation of voltammetric signals of dopamine and common interfering substances (ascorbic acid and acetaminophen) both in quiescent conditions and in flow due to the electrocatalytic effect. These voltammograms were also successfully simulated. The limit of detection of dopamine detected by square wave voltammetry in 1 mM solutions of interfering substances in phosphate buffered saline is about 100 nM. In human serum a clear voltammetric signal could be seen for a 200 nM solution, sufficient to detect dopamine in the cerebral fluid. Flow injection analysis allows a decrease in the limit of detection down to 3.5 nM.

NFC Smartphone-Based Electrochemical Microfluidic Device Integrated with Nanobody Recognition for C-Reactive Protein.

Point-of-care testing (POCT) devices play a crucial role as tools for disease diagnostics, and the integration of biorecognition elements with electronic components into these devices widens their functionalities and facilitates the development of complex quantitative assays. Unfortunately, biosensors that exploit large conventional IgG antibodies to capture relevant biomarkers are often limited in terms of sensitivity, selectivity, and storage stability, considerably restricting the use of POCT in real-world applications. Therefore, we used nanobodies as they are more suitable for fabricating electrochemical biosensors with near-field communication (NFC) technology. Moreover, a flow-through microfluidic device was implemented in this system for the detection of C-reactive protein (CRP), an inflammation biomarker, and a model analyte. The resulting sensors not only have high sensitivity and portability but also retain automated sequential flow properties through capillary transport without the need for an external pump. We also compared the accuracy of CRP quantitative analyses between commercial PalmSens4 and NFC-based potentiostats. Furthermore, the sensor reliability was evaluated using three biological samples (artificial serum, plasma, and whole blood without any pretreatment). This platform will streamline the development of POCT devices by combining operational simplicity, low cost, fast analysis, and portability.

Spectroelectrochemical studies of TDMQ20: A potential drug against Alzheimer's disease.

Alzheimer's Disease (AD), reported for the first time in 1906, is a common disease that remains incurable to this day. In the past, a family of treatments using Cu(II) chelators failed during clinical trials, evidencing the importance of pre-clinical studies. In this work, we performed electrochemical characterisation of TDMQ20, a new potential drug against AD, using electrochemistry and spectroelectrochemistry. On the basis of voltammetry, we determined that TDMQ20 undergoes a two-step irreversible oxidation process and a one-step irreversible reduction process. Both oxidation and reduction reactions are pH-sensitive. Bidimensional UV-Vis spectroelectrochemistry (UV-Vis-SEC) allowed us to confirm that oxidation of TDMQ20 can occur both on the aliphatic chain and on the aromatic ring. The results expand the knowledge of the TDMQ20 redox activity in the human body which is important from the point of view of the toxicity of the proposed therapy.

Anomalous effect of flow rate on the electrochemical behavior at a liquid|liquid interface under microfluidic conditions.

We have investigated the oxidation of ferrocene at a flowing organic solvent|aqueous electrolyte|solid electrode junction in a microfluidic setup using cyclic voltammetry and fluorescent laser scanning confocal microscopy. At low flow rates the oxidation current decreases with increasing flow, contrary to the Levich equation, but at higher flow rates the current increases linearly with the cube root of the flow rate. This behavior is explained using a simple model postulating a smallest effective width of the three-phase junction, which after fitting to the data comes to be ca. 20 µm. The fluorescence microscopy reveals mixing of the two phases close to the PDMS cover, but the liquid|liquid junction is stable close to the glass support. This study shows the importance of the solid|liquid|liquid junctions for the behavior of multiphase systems under microfluidic conditions.

Determination of the Dissociation Constant for Polyvalent Receptors Using ELISA: A Case of M13 Phages Displaying Troponin T-Specific Peptides.

Phage-derived affinity peptides have become widespread thanks to their easy selection via phage display. Interactions between a target protein and its specific peptide are similar to those between antibodies and antigens. The strength of these non-covalent complexes may be described by the dissociation constant (Kd). In this paper, protein-specific peptides are exposed on the pIII protein present in the M13 bacteriophage virion with up to five copies. Therefore, one phage particle can bind from one to five ligands. Here, we discuss the dependences between phage-displayed peptides and their ligands in solution using a model system based on troponin T (TnT) binding phages. Moreover, a method of calculating Kd values from ELISA experiments was developed and is presented. The determined Kd values are in the picomolar range.

Formation of MnO2-coated ITO electrodes with high catalytic activity for enzymatic glucose detection.

We present the formation of a cheap and environmentally friendly working electrode material for glucose biosensors with good catalytic properties. The classic electrode in such devices consists of a conductive material modified with the enzyme glucose oxidase. The working principle is the electrochemical detection of hydrogen peroxide as a product of the enzymatic transformation of glucose. As a base material, we offer manganese dioxide; it is a natural highly selective catalyst for the decomposition of H2O2 and is electrochemically deposited onto the surface of ITO. We approached the formation of MnO2 films systematically. By changing parameters such as the deposition method, pH of the electrolyte, and the drying temperature of the precipitate, a series of electrodes were formed. These electrodes were characterized by SEM, electrochemical impedance spectroscopy, and XPS and their electrocatalytic activity was studied. Significant differences in the sensitivity of the electrodes were detected. The manganese dioxide film with the best catalytic characteristics is formed in the electrolyte with pH 1 by cyclic voltammetry and then drying at 60 °C. The surface of the electrode was then modified with a solution of GOx enzyme with a concentration of 2 mg ml-1 (100-250 units per mg solid). The sensitivity of such an electrode is 117.8 µA mmol-1 cm-2. The range of determined concentrations of glucose is from 0.1 mM to 3 mM. The sensitivity is comparable to that of electrodes based on expensive materials such as graphene and noble metals.

Fabrication of ITO microelectrodes and electrode arrays using a low-cost CO2 laser plotter.

Good electronic (Rs = ∼5 Ω sq-1) and optical properties (transmittance: >83%) make indium tin oxide (ITO) an attractive electrode substrate. Despite the commercial availability of high-quality ITO and some low-cost methods for direct deposition being in use by now, the definition of patterns is still a concern. Putting their popularity and extensive use aside, the manufacturing of ITO electrodes so far lacks a rapid, highly reproducible, flexible, cost-effective, easy patterning process that could surpass difficult, time-consuming techniques such as lithography. Herein, we present a low-cost method based on CO2 laser irradiation for preparing ITO microelectrodes and electrode arrays. Electrodes of different sizes and shapes were examined to identify the performance of the proposed methods. Direct ablation of the ITO layer was optimized for rectangular electrodes of 25, 50, and 100 µm in width, while laser cutting of scotch tape stencils and subsequent wet etching were used to create circular electrodes with a diameter of 1.75 mm. A multielectrode array system consisting 8 of these circular electrodes was fabricated on a (25 × 25) mm2 plate, characterized electrochemically through cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM), and as an example application used for monitoring the anchoring behavior of HeLa and HepG2 cell cultures through cell-based electrochemical impedance spectroscopy. Together, the direct ablation method and preparation of laser cut stencils form a complete toolbox, which allows for low-cost and fast fabrication of ITO electrodes for a wide variety of applications. To demonstrate the general availability of the method, we have also prepared a batch of electrodes using a laser plotter in a local printing shop, achieving high intra-workshop reproducibility.