Tova Neufeld

A micro flow injection electrochemical biosensor for organophosphorus pesticides.

We describe a disposable, amperometric micro flow injection electrochemical biosensor that can be applied to the identification and quantification of highly toxic organophosphorus (OP) compounds in the environment, on the spot and in a short time. The system traces very small quantities of OP by monitoring the enzymatic reaction of acetylcholine esterase (AChE) and its inhibition. The sensor is sensitive, rapid, small, inexpensive, disposable and can be operated by non-professional technicians. The electrochemical cell consists of screen-printed electrodes covered with an enzymatic membrane and placed in a home-made flow cell. The electrodes are connected to a computer-controlled potentiostat. We quantitatively detected the OP compound, dimethyl 2,2-dichlorovinyl phosphate (DDVP), by monitoring the OP induced decrease in enzymatic degradation of the substrate, acetylthiocholine chloride (ATCh), to thiocholine and acetic acid. Thiocholine reacts with hexacyanoferrate ion in the working solution and the reduction of [Fe(CN)6](-3) to [Fe(CN)6](-4) and its subsequent reoxidization by the electrode generates very sharp, rapid and reproducible electric signals. The ability to detect low quantities is extremely important when dealing with hazardous environmental pollutants.

Recombinant single chain antibodies in bioelectrochemical sensors

Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In particular, single chain antibodies (scFvs) can be isolated and expressed in bacterial systems that also allow their in vitro manipulation at the gene level. In this work, we present for the first time results of single-chain phage displayed antibodies combined with amperometric detection and its application as an immunosensor. The scFv is immobilized on a carbon electrode and used to capture and quantify its specific target antigen. We describe the detection of the sugar milk lactose, the bacteria Listeria monocytogenes, and the enzyme MtKatG, which is expressed by Mycobacteriumtuberculosis.

Electrochemical detection of protein–protein interactions using a yeast two hybrid: 17-β-Estradiol as a model

In this work we present a modified yeast two-hybrid bioassay for the highly sensitive detection of protein-protein interactions, based on the electrochemical monitoring of beta-D-galactosidase reporter gene activity, using p-aminophenyl-beta-D-galactopyranoside (PAPG) as a synthetic substrate. In a model system, the sensitive detection of 17-beta-estradiol was achieved at concentrations as low as 10(-11)M (approx 2 pg/ml) by monitoring 17-beta-estradiol receptor dimerization after exposure to 17-beta-estradiol. The sensitivity of this system was higher than that of standard optical methods by three orders of magnitude.

A citrate-binding site in calmodulin.

Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low‐affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP‐NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C‐terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X‐ray structures of several citrate‐binding proteins by computerized molecular graphics enabled us to identify a putative citrate‐binding site (CBS) on the CaM molecule around residues Arg106‐His107. Owing to the tight proximity of this site to the third Ca2+‐binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation‐sensitive protein. Copyright

Computational fluid dynamic model of diffusion and convection processes in electrochemical sensor

Electrochemical amperometric transducers monitor the electric current through the electrochemical cell and measure, with high sensitivity, the concentration of biological or chemical species. The present study examined the physical phenomena of mass transfer in the vicinity of an electrochemical sensor operating in flow conditions using a theoretical model. A three-dimensional (3-D) geometry of the electrochemical cell with computational fluid dynamic simulations of time-dependent convection and diffusion are used. The study focused mainly on the contribution of the convection, while migration due to ion movement by the electric field and kinetics limitations due to enzyme activity have been neglected. The concentration of the electroactive species from a thin layer close to the electrode is calculated by numerical simulations. This calculated concentration has been compared with the concentration obtained from the electric current measured by an amperometric experimental system. Comparison between the simulations and experimental patterns shows good agreement. Only a short delay at the onset of the measured experimental concentration was observed, compared to the simulation data. The source for the small disagreements could be connected to the manual procedures of the incomplete bubble removal in the experimental setup and to the migration effect that was neglected in the model assumptions.

Amperometric Biosensors Based on Microflow Injection System

Novel electrochemical cells based on a microflow system combined with amperometric enzyme electrodes were developed and served for quantitative determination of various compounds, such as organophosphates and lactose. The resulting biosensors are selective and efficient owing to immobilization of the sensing elements on the electrodes. The sensors are easy to operate, and the procedures are rapid, accurate, reproducible, and inexpensive, requiring neither special skills and training nor complicated instrumentation. The use of a microflow cell ensures the continuous flux of a new substrate, thus preventing the accumulation or adsorption of products to the electrode. Miniaturization of the sensor has two main advantages: (1) it is easy to carry and therefore can be used outdoors as well, and (2) it allows working with low volumes of compounds and reagents, which is highly important when dealing with hazardous compounds.

Numerical and experimental analysis of enzymatic reaction in electrochemical sensors: electrochemical enzymatic analysis

The research examines, numerically and experimentally, the identification of substrate concentration in amperometric electrochemical flow cells. Three-dimensional numerical simulations have been preformed for predicting the mass transfer processes in the vicinity of the electrochemical cell using a realistic geometrical model. The experimental procedure included the fixed enzyme /spl beta/-galactosidase and the injected substrate, para-aminophenyl /spl beta/-D-galactopyranoside. For the optimization of the inlet flow rate, simulations and experiments have been preformed using flow rates between 0.05-250 /spl mu/l/min with an identical substrate concentration. The numerical simulation results were used to evaluate the species concentration distribution in the vicinity of the electrochemical cell for predicting the electric current through the electrode. Different substrate concentrations applied and ranged between 0.05-1.125 mg/ml at a chosen flow rate of 50 /spl mu/l/min. A good agreement was found between the numerical and the experimental electric current evolution values, especially for the higher substrate concentrations. The correlation coefficient was 0.98 in the higher substrate concentrations. A linear relationship was obtained between the inlet substrate concentration and the steady-state electric current for both the numerical and the experimental results. Once this linear relationship is established, the inlet substrate concentration based on the electric current through the electrode can be established.

Development and Application of Bioelectrochemical Sensors for On-Site Monitoring

This work describes the development and application of rapid, disposable and sensitive bioelectrochemical sensors. The sensors are based on enzyme amplification amperometric detection, with screen-printed electrodes, a microflow injection system and an enzymatic membrane placed in a microflow electrochemical cell. The basic configuration of the sensor can be adapted to and applied in various analytical determinations. Examples of sensors to detect organophosphorous pesticides, formaldehyde and lactose in raw milk are discussed.