Almira Ramanaviciene

Biocompatibility of polypyrrole particles: an in-vivo study in mice

The objectives of this study were the chemical synthesis of polypyrrole particles, the investigation and estimation of the impact of polypyrrole particle concentration, and the evaluation of the effect of duration of treatment on immune‐related haematological parameters and peritoneum cells in mice. The results showed that chemically prepared polypyrrole particles did not have any detectable cytotoxic effect on mouse peritoneum cells. Polypyrrole particles did not induce any allergic response, nor did they affect spleen, kidney or liver indexes. Moreover, no effect of polypyrrole particles on immune‐related haematological parameters was observed. No inflammation was detected in the peritoneum of mice after a 6‐week period of treatment with polypyrrole particles. In conclusion, chemically synthesized polypyrrole particles showed good biocompatibility in mice and are attractive candidates for biomedical applications in‐vivo.

Site-directed antibody immobilization techniques for immunosensors

Immunosensor sensitivity, regenerability, and stability directly depend on the type of antibodies used for the immunosensor design, quantity of immobilized molecules, remaining activity upon immobilization, and proper orientation on the sensing interface. Although sensor surfaces prepared with antibodies immobilized in a random manner yield satisfactory results, site-directed immobilization of the sensing molecules significantly improves the immunosensor sensitivity, especially when planar supports are employed. This review focuses on the three most conventional site-directed antibody immobilization techniques used in immunosensor design. One strategy of immobilizing antibodies on the sensor surface is via affinity interactions with a pre-formed layer of the Fc binding proteins, e.g., protein A, protein G, Fc region specific antibodies or various recombinant proteins. Another immobilization strategy is based on the use of chemically or genetically engineered antibody fragments that can be attached to the sensor surface covered in gold or self-assembled monolayer via the sulfhydryl groups present in the hinge region. The third most common strategy is antibody immobilization via an oxidized oligosaccharide moiety present in the Fc region of the antibody. The principles, advantages, applications, and arising problems of these most often applied immobilization techniques are reviewed.

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.

Magnetic gold nanoparticles in SERS-based sandwich immunoassay for antigen detection by well oriented antibodies.

The aim of the study was to develop an indirect, robust and simple in application method for the detection of bovine leukemia virus antigen gp51. Surface-enhanced Raman scattering (SERS) was applied as detection method. Magnetic gold nanoparticles (MNP-Au) modified by antibodies in oriented or random manner were used for the binding of gp51. The high performance liquid chromatography analysis indicated that the best antibody immobilization and antigen capturing efficiency was achieved using fragmented antibodies obtained after reduction of intact antibodies with dithiothreitol. In order to increase efficiency and sensitivity of immunoassay Raman labels consisting of gold nanorods coated by 5-thio-nitrobenzoic acid layer with covalently bounded antibodies have been constructed. The LOD and LOQ of the proposed immunoassay for antigen gp51 detection were found to be 0.95μgmL(-1) and 3.14μgmL(-1), respectively. This immunoassay was successfully applied for the detection of gp51 in milk samples in a rapid, reliable and selective manner. We believe that the proposed SERS-based immunoassay format can be applied for the detection of other proteins.

Enzymatic biofuel cell based on anode and cathode powered by ethanol.

Enzymatic biofuel cell based on enzyme modified anode and cathode electrodes are both powered by ethanol and operate at ambient temperature is described. The anode of the presented biofuel cell was based on immobilized quino-hemoprotein-alcohol dehydrogenase (QH-ADH), while the cathode on co-immobilized alcohol oxidase (AOx) and microperoxidase (MP-8). Two enzymes AOx and MP-8 acted in the consecutive mode and were applied in the design of the biofuel cell cathode. The ability of QH-ADH to transfer electrons directly towards the carbon-based electrode and the ability of MP-8 to accept electrons directly from the same type of electrodes was exploited in this biofuel cell design. Direct electron transfer (DET) to/from enzymes was the basis for generating an electric potential between the anode and cathode. Application of immobilized enzymes and the harvesting of the same type of fuel at both electrodes (cathode and anode) avoided the compartmentization of enzymatic biofuel cell. The maximal open circuit potential of the biofuel cell was 240mV.

Application of polypyrrole for the creation of immunosensors

This review focuses on the use of conducting polymer (CP) films in electrochemical affinity sensors and emphasizes innovative designs and unique applications of immunosensors. The review covers some aspects in the application of polypyrrole (Ppy) for the creation of immunosensors. Polypyrrole film fabrication methods like solvent casting, adsorption, and electropolymerization are presented. The focus, is on electrochemically synthesized Ppy as very promising material for the formation of miniaturized electrochemical immunosensors. Polypyrrole films implemented in various capacities in amperometric, conductometric, and potentiometric immunosensor design are reviewed. The acceptance of immobilization and detection approaches used recently in affinity sensors with critical analysis applied in certain techniques is discussed. The biologically active components (BAC) used for the creation of polypyrrole-based immunosensors are described briefly. Some future trends in the development of polypyrrole-based immunosensors are predicted, as well as possible directions discussed.

Enzymatically synthesized polyaniline layer for extension of linear detection region of amperometric glucose biosensor.

In this article a new method for fabrication of enzymatic electrodes suitable for design of amperometric glucose biosensor and/or anode of biofuel cell powered by glucose is presented. Glucose oxidase (GOx) E.C. 1.1.3.4. from Penicillium vitale was immobilized on the carbon rod electrode by cross-linking it with glutaraldehyde (GOx-electrode). Catalytic activity of immobilized GOx was exploited for polymerisation of aniline by taking a high concentration of hydrogen peroxide produced during the catalytic action of immobilized GOx and locally lowered pH due to the formation of gluconic acid; it created optimal conditions for the polymerisation of aniline. The GOx layer was self-encapsulated within formed polyaniline (PANI) matrix (GOx/PANI-electrode). Properties of the GOx/PANI-electrode have been studied and results were compared with GOx-electrode. The results show that the upper detection limit of glucose using GOx-electrode was dramatically changed by the formation of PANI layer. An increase in the upper detection limit, optimal pH region for operation and stability of GOx based electrode modified by PANI was detected when comparing that of an unmodified GOx-electrode.

Polymerization Model for Hydrogen Peroxide Initiated Synthesis of Polypyrrole Nanoparticles

A very simple, environmentally friendly, one-step oxidative polymerization route to fabricate polypyrrole (Ppy) nanoparticles of fixed size and morphology was developed and investigated. The herein proposed method is based on the application of sodium dodecyl sulfate and hydrogen peroxide, both easily degradable and cheap materials. The polymerization reaction is performed on 24 h time scale under standard conditions. We monitored a polaronic peak at 465 nm and estimated nanoparticle concentration during various stages of the reaction. Using this data we proposed a mechanism for Ppy nanoparticle formation in accordance with earlier emulsion polymerization mechanisms. Rates of various steps in the polymerization mechanism were accounted for and the resulting particles identified using atomic force microscopy. Application of Ppy nanoparticles prepared by the route presented here seems very promising for biomedical applications where biocompatibility is paramount. In addition, this kind of synthesis could be suitable for the development of solar cells, where very pure and low-cost conducting polymers are required.

Evaluation of cytotoxicity of polypyrrole nanoparticles synthesized by oxidative polymerization.

Polypyrrole (Ppy) is known as biocompatible material, which is used in some diverse biomedical applications and seeming to be a very promising for advanced biotechnological applications. In order to increase our understanding about biocompatibility of Ppy, in this study pure Ppy nanoparticles (Ppy-NPs) of fixed size and morphology were prepared by one-step oxidative polymerization and their cyto-compatibility was evaluated. The impact of different concentration of Ppy nanoparticles on primary mouse embryonic fibroblasts (MEF), mouse hepatoma cell line (MH-22A), and human T lymphocyte Jurkat cell line was investigated. Cell morphology, viability/proliferation after the treatment by Ppy nanoparticles was evaluated. Obtained results showed that Ppy nanoparticles at low concentrations are biocompatible, while at high concentrations they became cytotoxic for Jurkat, MEF and MH-22A cells, and it was found that cytotoxic effect is dose-dependent.

Surface Plasmon Resonance Label-free Monitoring of Antibody Antigen Interactions in Real Time*

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label‐free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label‐free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibody rabbit anti‐cow albumin IgG1 (anti‐BSA). The formation of a self‐assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non‐covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.