Hybrid Tamm-surface plasmon polariton mode for highly sensitive detection of protein interactions.

The total internal reflection ellipsometry (TIRE) method was used for the excitation and study of the sensitivity properties of the hybrid Tamm plasmon polariton - surface plasmon polariton (TPP-SPP) and single surface plasmon resonance (SPR) modes of the GCSF receptor immobilization. Additionally, the optimized sensitivity of the hybrid TPP-SPP mode was investigated and compared with the single SPR mode when the BSA proteins formed a layer on the gold surface. The dispersion relations for the hybrid TPP-SPP and single SPR modes were used to explain the enhanced sensitivity of the ellipsometric parameters for the hybrid TPP-SPP mode over the conventional SPR. The SPP component (δΔh-SPP/δλ=53.9°/nm) of the hybrid TPP-SPP mode was about 6.4 times more sensitive than single SPR (δΔSPR/δλ=8.4°/nm) for the BSA protein layer on the gold film. It was found that the sensitivity of the hybrid plasmonic mode can be made controllable by using the strong coupling effect between the TPP and SPP components. The strong coupling regime reduces absorption and scattering losses of the metal for the SPP component in the hybrid TPP-SPP mode and, as a result, narrows the plasmonic resonance.

Compact high-sensitivity potentiometer for detection of low ion concentrations in liquids.

The compact potentiometer, based on an electronic circuit protected from electrostatic and electromagnetic interference, was developed for the measurement of low ion concentrations in liquids. The electronic circuit of the potentiometer, consisting of analogous and digital parts, enables the measurement of fA currents. This makes it possible to perform reliable measurements of ion concentrations in liquids that are as small as 10-8-10-7M. The instrument was tested using electrodes that were selective for tetraphenylphosphonium (TPP+) ions. It was demonstrated that the characteristic response time of the potentiometer electronic circuit to changes in the concentration of these ions in a liquid was in the order of 10 s. An investigation of TPP+ absorption by baker yeast has shown that this device can be successfully used for long term (several hours) measurements with zero signal drift, which was about 1 µV/s. Finally, due to the small dimensions of the electronic circuit (7.5 × 2 × 1.5 cm), this potentiometer can be easily installed at a large apparatus in the laboratory condition (≈25 °C), such as high pulsed electrical generators of magnetic fields that are used in electroporation studies of biological cells.

Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals.

Scanning electrochemical impedance microscopy for investigation of glucose oxidase catalyzed reaction.

In this research biointerface based on immobilized glucose oxidase (GOx) was evaluated by scanning electrochemical impedance microscopy (SEIM), which consisted of merged scanning electrochemical microscopy (SECM) and electrochemical impedance spectroscopy (EIS). The gluconolactone, which is quickly hydrolyzed to gluconic acid, is produced during the enzyme-catalyzed glucose oxidation reaction. Gluconic acid formed above an enzyme-modified not-conducting plastic surface, was evaluated by EIS technique. A two electrode cell consisting of a scanning probe, which was based on 10 µm diameter ultramicroelectrode and stationary platinum counter/reference electrode was applied for the measurement. Locally measured solution impedance depends on the gluconic acid concentration close to the ultramicroelectrode surface and on the ion diffusion, which is hindered when the electrode is approaching close to the GOx-modified surface. EIS results were evaluated by applying an equivalent circuit consisting of elements representing solution resistance, double-layer capacitance, charge-transfer resistance and Warburg impedance. Solution resistance was calculated and showed to be dependent on the position of ultramicroelectrode. Also it was observed that the thickness of the conducting layer and gluconic acid concentration both are changing in time. The results indicate that here proposed SEIM technique could become a valuable tool for the investigation and characterization of enzyme-modified surfaces of biosensors and biofuel cells.

The isotope method for the determination of stoichiometry between compounds forming the polypyrrole and glucose oxidase composite.

Stable isotope ratio mass spectrometry is a conventional method used in archaeology, and medical, environmental and paleoenvironmental reconstruction studies. However new insights and applicability of the equipment often open new research areas and improve our understanding of the ongoing processes. Therefore the stable isotope ratio mass spectrometry method was applied for the stoichiometry determination of the complex polypyrrole and glucose oxidase composite (PPy-GOx composite). The enzyme glucose oxidase and conducting polymer polypyrrole were reported to form a composite, which was evaluated in time using the dynamic light scattering method. The consistent enlargement of the PPy-GOx composite and the relative decrease of the spare enzyme molecules were observed in the polymerization solution. UV-VIS spectrometry was employed to follow the polymerization process. The isotope mixing model was applied for the evaluation of the constitution of the PPy-GOx composite. According to the obtained results the determination of the PPy-GOx composite stoichiometry could be more reliably determined using the nitrogen isotope ratio approach in comparison to the carbon approach. We expect that this novel work will widen the applications of stable isotope ratio mass spectrometry in research.

Immunosensor based on fluorescence quenching matrix of the conducting polymer polypyrrole.

In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity.

Comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor.

A comparative study of four different antibody immobilization techniques that are suitable for modification of surface plasmon resonance (SPR) chip (SPR-chip) is reported. Antibodies against human growth hormone (anti-HGH) were used as the model system. The evaluated SPR-chip modification techniques were (i) random immobilization of intact anti-HGH (intact-anti-HGH) via self-assembled monolayer (SAM) based on 11-mercaptoundecanoic acid (MUA); (ii) random immobilization of intact-anti-HGH within carboxymethyl dextran (CMD) hydrogel by direct covalent amine coupling technique; (iii) oriented coupling of intact-anti-HGH via Fc-fragment to protein-G layer assembled on SAM consisting of MUA (MUA/pG); (iv) oriented immobilization of fragmented anti-HGH antibodies (frag-anti-HGH) via their native thiol-groups directly coupled to the gold. To liberate these thiol groups, the intact-anti-HGH was chemically "divided" into two frag-anti-HGH fragments by chemical reduction with 2-mercaptoethylamine (2-MEA). Optimal concentration of 2-MEA for preparation of anti-HGH was 15 mM. The surface concentration of immobilized antibodies and the antigen binding capacity for all four differently modified SPR-chips was evaluated and compared. The maximum surface concentration of immobilized intact-anti-HGH was obtained by immobilizing the antibody within CMD-hydrogel. The maximal antigen binding capacity was obtained by SPR-chip based on intact-anti-HGH immobilized via MUA/pG. The immobilization based on application of frag-anti-HGH was found to be the most suitable for design of SPR-immunosensor for HGH detection, due to its sufficient antigen binding capacity, simplicity, and low cost in respect to the currently evaluated techniques.

Electrochemical impedance spectroscopy of polypyrrole based electrochemical immunosensor.

Polypyrrole (Ppy) has been shown as a matrix for label-free electrochemical immunosensor based on electrochemical impedance spectroscopy (EIS) measurements. The immunosensing system model presented here was based on bovine leukemia virus (BLV) protein (gp51) entrapped within electrochemically-synthesized polypyrrole (Ppy/gp51). This Ppy/gp51 layer interacted with antibodies against gp51 (anti-gp51-Ab) that are present in significant concentration in the blood serum of BLV infected cattle. After this interaction protein complex (Ppy/gp51/anti-gp51-Ab) was formed. The horseradish peroxidase (HRP) labeled secondary antibodies (Ab) against anti-gp51-Ab were applied as agents interacting with Ppy/gp51/anti-gp51-Ab and forming the large protein complex (Ppy/gp51/anti-gp51-Ab/Ab). The EIS study was performed for electrodes modified with different Ppy layers described here and an optimal equivalent circuit was adopted for evaluation of EIS spectra, it was a major outcome of this study.

Stabilization of (CdSe)ZnS quantum dots with polypyrrole formed by UV/VIS irradiation initiated polymerization.

Polypyrrole formation initiated by UV/VIS irradiation and stabilization of (CdSe)ZnS quantum dots is reported. Presented results demonstrate that UV/VIS irradiation is slowly destructing Q-dots that decreases quantum yield and shifts peak of photoluminescence (PL) spectra to short wavelength range. The same Q-dot solution under UV/VIS light irradiation in the presence of pyrrole is more stable, there is no PL band shifts only PL intensity of Q-dot decrease. UV/VIS induced Ppy formation process in the presence/absence of Q-dots dissolved in toluene and phosphate buffer saline (PBS) not containing Q-dots was investigated. The increase in the optical absorbance in the range of 400 nm-600 nm was exploited for the monitoring of polypyrrole formation process. Results obtained proved that in the presence of UV/VIS formed polypyrrole destruction of Q-dots is much slower if compared with bare Q-dots destruction under similar conditions. However, formed polypyrrole showed no positive effect on quantum yield of Q-dot, just the opposite-polypyrrole quenched Q-dot photoluminescence. Results presented and observation by other authors on pyrrole polymerization allow to predict that by UV/VIS induced polymerization formed polypyrrole is assembling polymeric layer on heterogeneous phase between Q-dot and toluene or water (in PBS solution). Established pi-pi conjugated chain of formed polypyrrole quenches photoluminescence of Q-dot because this polymer was formed in close proximity to Q-dot surface.

Conducting polymer based fluorescence quenching as a new approach to increase the selectivity of immunosensors.

Polypyrrole (Ppy) has been shown to be a superior matrix for fluorescence detection based immunosensors: (i) the fluorescence of polypyrrole and polypyrrole modified by entrapped proteins was almost not detectable when this polymer was excited by near UV 325 nm light; (ii) polypyrrole quenched the fluorescence of such fluorescence agents as fluoresceine 5(6)-isothiocyanate, rhodamine B and enzyme-horseradish peroxidase (HRP) by almost 100% if they were deposited in the solution as a drop at the Ppy surface followed by evaporation of the solvent. According to our knowledge, this work is first application of Ppy in the design of a fluorescence-based immunosensor, where low Ppy fluorescence background and Ppy induced fluorescence quenching were exploited. These sensors were devoted to the detection of antibodies against bovine leukemia virus (BLV) protein gp51 (anti-gp51-Ab). A biological recognition system of this fluorescence immunosensor model was based on polypyrrole with entrapped BLV proteins gp51 (gp51/Ppy). This gp51/Ppy layer was applied for the detection of anti-gp51-Ab. Secondary antibodies against anti-gp51-Ab labeled with HRP (Ab*) were applied as fluorescence-detectable labels that are able to recognize specifically and interact with the complex of gp51 proteins and anti-gp51-Ab antibodies (gp51/anti-gp51-Ab). It was demonstrated that fluorescence of non-specifically adsorbed Ab* was almost completely quenched by the Ppy substrate. In addition, enzymatic activity of HRP was exploited as a traditional reference method for verification of the formation of the immune complex gp51/anti-gp51-Ab/Ab*.

Amperometric biosensor for the determination of creatine.

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1-1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an [Fe(CN)(6)](3-) /[ Fe(CN)(6)](4-) redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris-HCl buffer (pH 8.0) at 20 degrees C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine.

Conducting polymer-based nanostructurized materials: electrochemical aspects.

New modern technologies require new materials. During the past decade, the movement towards nanodimensions in many areas of technology aroused a huge interest in nanostructurized materials. The present article reviews recent works dealing with electrochemistry-related aspects of nanostructurized conducting polymers. Electrochemical synthesis and some properties of nanostructurized conducting polymers, and nanocomposites derived from conducting polymers and metals, carbon, and inorganic and organic materials are considered. Some potential areas for electrochemistry-related applications of nanocomposites are highlighted, including batteries, supercapacitors, energy conversion systems, corrosion protection, and sensors.

Circulating immune complexes as indicators of environmental contamination.

The aim of this study was to examine the effect of environmental contamination on the concentration of circulating immune complexes (CIC) in bovine blood. We determined that in a conventionally ecologically clear district, the CIC concentration in healthy bovine blood serum was within the limits of 0.18-0.52 mg/mL, (mean = 0.31 mg/mL, V = 30.14%). In a conventionally ecologically contaminated district, the CIC concentration in healthy bovine blood serum was detected within the limits of 0.48-0.99 mg/mL (mean = 0.65 mg/mL, V = 17.97%; P < 0.05). The CIC concentration in serum of bovine leukemia virus (BLV)-infected bovines from different districts was within the limits of 0.29-1.76 mg/mL (mean = 0.81 mg/mL, V = 36.67%; P < 0.05). According to these preliminary results of the investigation, it may be supposed that the increased concentration of CIC is closely related to the environmental contamination. Therefore, studies of CIC concentration in healthy mammals are of great importance for the monitoring of anthropogenic factors and can be exploited as an indicator of environmental contamination.

Polypyrrole-entrapped quinohemoprotein alcohol dehydrogenase. Evidence for direct electron transfer via conducting-polymer chains.

It is reported for the first time that direct electron-transfer processes between a polypyrrole (PPY) entrapped quinohemoprotein alcohol dehydrogenase from Gluconobacter sp. 33 (QH-ADH) and a platinum electrode take place via the conducting-polymer network. The cooperative action of the enzyme-integrated prosthetic groups--pyrroloquinoline-quinone and hemes--is assumed to allow this electron-transfer pathway from the enzyme's active site to the conducting-polymer backbone. A hypothetical model of the electron transfer is proposed which is supported by the influence of various parameters, such as, e.g., ionic strength and nature of the buffer salts. This unusual electron-transfer pathway leads to an accentuated increase of the K M app value (102 mM) and hence to a significantly increased linear detection range of an ethanol sensor based on this enzyme.