Hanna Radecka

Electrochemical biosensor for pesticides based on acetylcholinesterase immobilized on polyaniline deposited on vertically assembled carbon nanotubes wrapped with ssDNA.

An electrochemical biosensor for the determination of pesticides: methyl parathion and chlorpyrifos, two of the most commonly used organophosphorous insecticides in vegetable crops, is described. The self-assembled monolayers (SAMs) of single walled carbon nanotubes (SWCNT) wrapped by thiol terminated single strand oligonucleotide (ssDNA) on gold was utilized to prepare nano size polyaniline matrix for acetylcholinesterase (AChE) enzyme immobilization. The key step of this biosensor was AChE-acetylcholine enzymatic reaction which causes the small changes of local pH in the vicinity of an electrode surface. The pesticides were determined through inhibition of enzyme reaction. The dynamic range for the determination of methyl parathion and chlorpyrifos was found to be in between 1.0x10(-11) and 1.0x10(-6) M (0.6<SD<3.5) with good reproducibility and stability. The detection limit of the biosensor for both pesticides was found to be 1x10(-12) M. The biosensor has been applied for the determination of methyl parathion and chlorpyrifos in spiked river water samples.

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10 pg/ml and 1 ng/ml with limit of detection 5 pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.

A highly sensitive electrochemical genosensor based on Co-porphyrin-labelled DNA

We report the use of Co-porphyrins as electrochemical tags for a highly sensitive and selective genosensor. An avian influenza virus-based DNA sequence characteristic of H5N1 was detected at femtomolar levels from competing non-complementary sequences through hybridisation with the labeled DNA.

Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum

This paper describes the development of an immunosensor for detection of anti-hemagglutinin antibodies. Its preparation consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with EDC/NHS coupling reaction, (iii) covering with anti-His IgG monoclonal antibody, (iv) immobilization of the recombinant His-tagged hemagglutinin (His6-H5 HA), (v) filling free space with BSA. The interactions between two variants of recombinant HA (short and long) from highly pathogenic avian influenza virus H5N1 and the anti-H5 HA monoclonal antibody (Mab 6-9-1) have been explored with electrochemical impedance spectroscopy (EIS). The impedimetric immunosensor displayed a very good detection limit (LOD) of 2.1 pg/mL, the quantification limit (LOQ) of 6.3 pg/mL and a dynamic range from 4 pg/mL to 20 pg/mL. In addition, this analytical device was applied for detection of antibodies against His6-H5 HA in serum of vaccinated hen using serial 10-fold dilutions of serum. The immunosensor proposed was able to detect antibody in hen serum diluted up to 7 × 10(7)-fold. The sensitivity of immunosensor was about four orders of magnitude much better than ELISA.

Fullerene modified supported lipid membrane as sensitive element of sensor for odorants.

A supported lipid bilayer membrane (s-BLMs) formed on a freshly cleaved metallic surface by the Tien method was applied for the design of an electrochemical sensor for detection of neutral odorant molecules. The lipid bilayer was modified by saturation with fullerene C(60), which possesses electron mediator properties and facilitates a redox reaction occurring at the border of the lipid membrane and metal surface. I(2)/I(-) and ferrocenyl trimethyl bromide were used as electroactive marker ions. The smell compounds adsorb on the surface of the lipid layer and change its structure. As a consequence the ratio of marker ion penetration to the lipid membrane is altered. The magnitude of these changes depends on the amount and chemical structure of adsorbed molecules. The research presented was carried out by cyclic voltammetry. The magnitude of the electrochemical signal generated by smell compounds was correlated with other parameters describing their molecular properties such as: octanol/water partition coefficients and dipole moments.

Interaction of organic derivatives of tin (IV) and lead (IV) with model lipid membranes

Abstract Here we present the results of a comparative study on the interactions between the trialkyl-, triphenyl derivatives of tin (IV) and lead (IV) and dialkyl-, diphenyltin (IV) derivatives on the planar bilayer lipid membranes (BLMs), widely applied as a model of cell membrane. The relative degree of depolarization of the lipid membrane potential caused by the influence of the above compounds was applied as a parameter to estimate their toxicity. The research has shown, that trialkyl derivatives of tin (IV) are considered as the most active ones in relation to BLM from all alkyl tin and lead derivatives that have been studied. Dialkyltin (IV) and trialkyllead (IV) derivatives were found to be the least active. In phenyl derivatives group, the activity sequence is as follow: triphenyllead>triphenyltin>diphenyltin. Depolarization activity of compounds investigated strongly depended on the lipophilicity of their reaction with water products.

A Voltammetric Biosensor Based on Glassy Carbon Electrodes Modified with Single-Walled Carbon Nanotubes/Hemoglobin for Detection of Acrylamide in Water Extracts from Potato Crisps

The presence of toxic acrylamide in a wide range of food products such as potato crisps, French fries or bread has been confirmed by Swedish scientists from Stockholm University. The neurotoxicity, possible carcinogenicity of this compound and its metabolites compels us to control them by quantitative and qualitative assays. Acrylamide forms adduct with hemoglobin (Hb) as a result of the reaction the -NH2 group of the N-terminal valine with acrylamide. In this work we present the use of glassy carbon electrodes coated with single-walled carbon nanotubes (SWCNTs) and Hb for voltammetric detection of acrylamide in water solutions. The electrodes presented a very low detection limit (1.0×10-9 M). The validation made in the matrix obtained by water extraction of potato crisps showed that the electrodes presented are suitable for the direct determination of acrylamide in food samples.

Electroactive dipyrromethene-Cu(II) self-assembled monolayers: complexation reaction on the surface of gold electrodes.

In the work presented, thiol- and COOH-terminated dipyrromethene derivatives have been applied for gold electrode modification. Dipyrromethene deposited onto a solid support, after binding Cu2+, can act as a redox active monolayer. The complexation of Cu(II) ions has been performed on the surface of gold electrodes modified with dipyrromethene. The characterization of dipyrromethene-Cu(II) self-assembled monolayers (SAMs) has been done by cyclic voltammetry (CV), wettability contact angle measurements, and atomic force microscopy (AFM). The new electroactive monolayer could be applied for the immobilization of proteins and ssDNA or for electrochemical anion sensing without redox markers in the solution.

Association Constants of Pyridine and Piperidine Alkaloids to Amyloid ß Peptide Determined by Electrochemical Impedance Spectroscopy

Amyloid beta(1-40) peptide was immobilized on an Au-colloid modified gold electrode and an electrochemical impedance spectroscopy (EIS) system was elaborated for determining the association constants, K(a), between small molecular ligands and the peptide. The changes in the resistance of the modified electrode layer with deposited Abeta(1-40) peptide were measured with EIS in relation to a series of concentrations of the ligands studied. The association constants were calculated from Langmuir isotherms. The method is sensitive, reproducible and consumes only very little amounts of interacting species. The method was applied to determine the affinity of a series of pyridine and piperidine derivatives, mainly alkaloids of a known ability, to cross the blood-brain barrier. Along with nicotine and its main metabolite cotinine, the following agents were taken for the study: anabasine, arecoline, coniine, lobeline, pseudopelletierine, trigonelline, as well as pyridine and piperidine themselves. For the sake of comparison, two vitamins were also subjected to the study: ascorbic acid and pyridoxine. There was no association of these vitamins, which were tested as a negative control. For the compounds studied, a strong association with Abeta(1-40) was determined with K(a) ranging from 1.7 x 10(7) M(-1) for (+/-)-anabasine to 2.3 x 10(8) M(-1) for arecoline hydrobromide. As a positive control, a well known amyloid specific binder, Congo Red, was tested, displaying K(a) equal to 3.7 x 10(8) M(-1).

Surface Plasmon Resonance Based Biosensors for Exploring the Influence of Alkaloids on Aggregation of Amyloid-β Peptide

The main objective of the presented study was the development of a simple analytical tool for exploring the influence of naturally occurring compounds on the aggregation of amyloid-β peptide (Aβ40) in order to find potential anti-neurodegenerative drugs. The gold discs used for surface plasmon resonance (SPR) measurements were modified with thioaliphatic acid. The surface functionalized with carboxylic groups was used for covalent attaching of Aβ40 probe by creation of amide bonds in the presence of EDC/NHS. The modified SPR gold discs were used for exploring the Aβ40 aggregation process in the presence of selected alkaloids: arecoline hydrobromide, pseudopelletierine hydrochloride, trigonelline hydrochloride and α-lobeline hydrochloride. The obtained results were discussed with other parameters which govern the phenomenon studied such as lipophilicity/hydrophilicy and Aβ40-alkaloid association constants.