Judith Rishpon

An electrochemical biosensor for formaldehyde

Abstract This paper reports the development of a novel detection method, based on the coupling of a biosensor measuring device and a flow-injection system, using the enzyme formaldehyde dehydrogenase and a Os(bpy) 2 -poly(vinylpyridine) (POs-EA) chemically modified screen-printed electrode. The sensor can detect 30 ng ml −1 of formaldehyde in aqueous solution (corresponding to sub-ppb atmospheric concentrations of formaldehyde). The sensor is selective, inexpensive, stable over several days, and disposable, as well as simple to manufacture and operate. The system described here can easily be adapted to other substrates using their corresponding dehydrogenases.

An amperometric enzyme-channeling immunosensor

This paper presents a new disposable amperometric, enzyme-channeling immunosensor for a quantitative, rapid, separation-free enzyme immunoassay (EIA) that can be used in clinical diagnostics, as well as in biomedical, biochemical, and environmental research. The sensor consists of a disposable, polymer-modified, carbon electrode on which enzyme 1 is coimmobilized with a specific antibody that binds the corresponding antigen in a test solution. The solution also contains a conjugate of enzyme 2. An immunological reaction brings the two enzymes into close proximity at the electrode surface, and the signal is amplified through enzyme channeling. The localization of both enzymes on the electrode surface limits the enzymatic reactions to the polymer/membrane/electrode interface. The sensor overcomes the problem of discriminating between the signal that is produced by the immuno-bound enzyme label on the electrode surface and the background level of signal that emerges from the bulk solution. Combining enzyme-channeling reactions, optimizing hydrodynamic conditions, and electrochemically regenerating mediators within the membrane layer of the antibody electrode significantly increased the signal-to-noise ratio of the sensor. The amperometric enzyme-channeling immunosensor enabled the performance of separation-free EIAs without washing steps, resulting in a relatively short assay time of 5-30 min for the complete immunoassay, compared with at least 1-3 h for ELISA methods. Model systems using peroxidase-antibody, biotin-avidin, viral antigens (CD4-gp120), and bacteria (Staphylococcus aureus) were investigated. S. aureus cells were detected in pure culture at concentrations as low as 1000 cells/ml.

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.

Characterization of peptide-nanostructure-modified electrodes and their application for ultrasensitive environmental monitoring.

Dense arrays of self-assembled nanostructures are highly important for the fabrication of high-performance sensors of large surface area. The organized incorporation of novel biocompatible organic nanostructures into extremely sensitive amperometric biosensors is demonstrated. Peptide nanoforest biosensors for phenol detection were 17-fold more sensitive than uncoated electrode and more sensitive than those modified with carbon nanotubes or combined coating. The high sensitivity reported, together with the biocompatibility and the ability to chemically and biologically modify these elements, may provide a novel platform for biosensors design and fabrication for environmental monitoring, homeland security, and other applications.

On-line monitoring of gene expression.

Gene expression in cultures of Escherichia coli has been determined in situ and on-line by the use of an electrochemical sensor. Intact bacteria were used to monitor the induction of the lacZ gene; the onset of stationary phase was also monitored, using a reporter gene fused to the RpoS-dependent promoter of the osmY gene. The technique described can in principle be used to determine the activity of any promoter, with a variety of reporter genes. This technology is non-intrusive, allows real-time monitoring of gene expression, and will be useful in the study of growth regulation and development.

Alkaline phosphatase as a label for a heterogeneous immunoelectrochemical sensor: An electrochemical study

Abstract This work describes a novel system of immunoelectrochemical assays. The system is based on the covalent binding of antibodies to glassy carbon electrodes and on the use of alkaline phosphatase as a label in an enzyme immunoassay. The electrochemical behaviour of the substrates phenyl phosphate, p-nitrophenyl phosphate, p-aminophenyl phosphate and their products was characterized. Immuno-electrochemical assays using alkaline phosphatase and the above substrates were developed and tested in the following systems: dog IgG, mouse IgG, and β-HCG.

An amperometric biosensor for real-time analysis of molecular recognition

Abstract An amperometric, enzyme-channeling immunosensor was used to study the qualitative and quantitative aspects of molecular biorecognition in real time. The immunosensor consisted of a polyethylenimine-modified carbon electrode on which glucose oxidase was coimmobilized with a specific antibody or with calmodulin. The immunological reactions were monitored electrochemically in situ, and the binding curves were directly visualized on a computer screen. This approach was applied for estimating the kinetic constants of the reaction between IgG and its specific anti-IgG antibodies, as well as for mapping of sites and the interaction of calmodulin and its target peptides and proteins via its monoclonal antibodies. The respective association and dissociation rate constants for IgG/antiIgG were calculated as 1.3×106 M−1 s−1 and 0.2×10−3 s−1. The site-mapping of phosphodiesterase, melittin, calcineurin, or mastoparan on the calmodulin molecule were estimated using two conformation-dependent mAbs, CAM1 and CAM4, whose epitopes were previously located. The data presented confirm that the amperometric, enzyme-channeling immunosensor may be used as a tool for valuable characterizing of biospecific interactions, such as determining the concentration of molecules, selecting different antibodies, and characterizing the binding sites of biomolecules.

Carbon nanotubes based electrochemical biosensor for detection of formaldehyde released from a cancer cell line treated with formaldehyde-releasing anticancer prodrugs

This paper reports the development of an electrochemical biosensor for the detection of formaldehyde in aqueous solution, based on the coupling of the enzyme formaldehyde dehydrogenase and a carbon nanotubes (CNT)-modified screen-printed electrode (SPE). We monitored the amperometric response to formaldehyde released from U251 human glioblastoma cells situated in the biosensor chamber in response to treatment with various anticancer prodrugs of formaldehyde and butyric acid. The current response was higher for prodrugs that release two molecules of formaldehyde (AN-193) than for prodrugs that release only one molecule of formaldehyde (AN-1, AN-7). Homologous prodrugs that release one (AN-88) or two (AN-191) molecules of acetaldehyde, showed no signal. The sensor is rapid, sensitive, selective, inexpensive and disposable, as well as simple to manufacture and operate.

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.

A potentiometric biosensor for pesticides based on the thiocholine hexacyanoferrate (III) reaction

Abstract A solid-state acetylcholinesterase (AChE) sensor was developed on the basis of AChE acetylthiocholine-hexacyanoferrate (III) reaction. Various properties of the sensor were investigated and possibilities for its practical use in the detection and assay of pesticides are suggested. The response of the sensor was induced by two phenomena: the Donnan effect and the redox potential. Unlike pH-sensitive sensors, this type of sensor is operated at high ionic strength and a high buffer capacity. The ability of the AChE sensor to assay pesticides was demonstrated by the characterization and quantitative determination of dichlorvos based on its inhibition of AChE. The kinetics of inhibition by dichlorvos of the reaction with AChE were studied by potentiometric and photometric methods. The bimolecular reaction constant was 7·07 10 5 M −1 min −1 and the affinity constant was 4·76 10 −6 M. The detection limits for dichlorvos were 1 μM for a flow-injection mode and 0·2 μM for stationary measurement. The AChE acetylthiocholine-hexacyanoferrate(III) system opens up possibilities for the detection and assay of toxic chemicals by the use not only potentiometric but also of other types of sensors.