Detection of pathogenic Bacteria by Electrochemical Impedance Spectroscopy: Influence of the immobilization strategies on the sensor performance

Electrochemical impedance spectroscopy (EIS) is applied to detect pathogenic E. coli O157:H7 bacteria via a label free immunoassay-based detection method. Polyclonal anti-E.coli antibodies (PAb) are immobilized onto gold electrodes following two different strategies, via chemical bond formation between antibody amino groups and a carboxylic acid containing self-assembled molecular monolayer (SAM) and alternatively by linking a biotinylated anti-E. coli to Neutravidin on a mixed-SAM. Impedance spectra for sensors of both designs for increasing concentrations of E. coli are recorded in phosphate buffered saline (PBS). The Nyquist plots can be modeled with a Randle equivalent circuit, identifying the charge transfer resistance RCT as the relevant concentration dependent parameter. Sensors fabricated from both designs are able to detect very low concentration of E. coli with limits of detection as low as 10-100 cfu/ml. The influence of the different immobilization protocols on the sensor performance is evaluated in terms of sensitivity, dynamic range and resistance against nonspecific absorption

Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 2. Immunoassay evaluation

8 pages, 5 figures, 5 tables.-- PMID: 12822926 [PubMed].-- Printed version published Jul 2, 2003.Immunoassays for 2,4,6-trichloroanisol (TCA) have been evaluated. The assays were developed after raising antibodies against three different immunizing haptens (1). Lack of reproducibility has been one of the main problems of these assays. Precision was worse on these assays, reaching lower limits of detection. The high lipophilicity of TCA and its, consequently, low water solubility have been found to be the major cause of this problem. A reliable microplate-based enzyme-linked immunosorbent assay (ELISA) has been set after consideration of the TCA physicochemical features and evaluation of important parameters affecting immunoassay performance. The immunoassay uses As78 (developed against hapten B-KLH) and C9-OVA as the coating antigen. The selectivity is high although the brominated analogue 2,4,6-TBA is also recognized. In buffered media containing 7% ethanol, the resulting assay shows a good accuracy with an IC50 value of 0.53 μg L-1 and a limit of detection of 0.044 μg L-1. Red and white wine samples caused important interferences in the immunoassay demonstrating the necessity of a cleanup procedure prior to the ELISA.This work has been supported by INIA (VIN00-053-C3-2), the EC Quality of Life Program (Contract QLRT-2000-01670), and CICYT (AGL2001-5005-E and AGL2002-04635-C04-03).Peer reviewe

Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6

11 pages, 8 figures.-- PMID: 15210718 [PubMed].-- Printed version published Sep 17, 2004.A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.This work was supported by the European Union, Fighting Blindness Ireland, and the Higher Education Authority of Ireland.Peer reviewe

Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 1. Molecular modeling studies for antibody production

8 pages, 4 figures, 2 tables.-- PMID: 12822925 [PubMed].-- Printed version published Jul 2, 2003.-- Supporting information available at: http://pubs.acs.org/doi/suppl/10.1021/jf034003hNine antisera have been raised against 2,4,6-trichloroanisole (2,4,6-TCA) by immunizing them with three different haptens. With the spacer arm at the meta position, hapten A (3-(2,4,6-trichloro-3-methoxyphenyl)propanoic acid) preserved all of the functional groups of the target analyte. In hapten B (5-(2,4,6-trichlorophenoxy)pentanoic acid), the spacer was placed in the molecule substituting the methoxy group. Finally, hapten C (3-(3,5-dichloro-4-methoxyphenyl)propanoic acid) held the spacer arm at the para position instead of the chlorine atom of the target analyte. Using theoretical models, we have studied how the molecular geometry and the electronic distribution are affected by the introduction of the linker. The evaluation of the avidity of the resulting antibodies demonstrates that the orientation produced by the spacer arm must also be considered an essential aspect. The screening for competitive assays performed after synthesizing a battery of heterologous competitors has provided with these antibodies eight indirect enzyme-linked immunosorbent assays with acceptable properties. From the number of assays obtained, their maximal absorbance, their signal-to-noise ratio, the slope, and the IC50 values obtained, it can be concluded that hapten C provided the best antibodies.This work has been supported by INIA (VIN00-053-C3-2), the EC Quality of Life Program (Contract QLRT-2000-01670), and CICYT (AGL2001-5005-E and AGL2002-04635-C04-03).Peer reviewe

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines

9 pages, 4 figures, 6 tables.-- PMID: 12502401 [PubMed].-- Available online Nov 22, 2002.The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm-1) and basic pH values (7.5−9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.This work has been supported by the EC Program (Contract QLRT-2000-01670) and by CICYT (BIO2000-0351-P4-05 and AGL2001-5005-E).Peer reviewe

The radical scavenger CR-6 protects SH-SY5Y neuroblastoma cells from oxidative stress-induced apoptosis: effect on survival pathways

13 pages, 4 figures.-- PMID: 16787420 [PubMed].Reactive oxygen species (ROS) and oxidative stress have long been linked to cell death of neurons in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are at present unclear. In the current work we have used the human neuroblastoma SH-SY5Y cell line as a model for studying the molecular events occurring after inducing apoptosis with H2O2. We show that treatment of SH-SY5Y cells with H2O2 up-regulates survival pathways during early stages of apoptosis. Subsequently, the decline of anti-apoptotic protein levels leads to the activation of the calcium-dependent proteases calpains and the cysteine proteases caspases. Additionally, we demonstrate that CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acts as a scavenger of ROS and prevents apoptosis by enhancing and prolonging up-regulation of survival pathways. Furthermore, we show that pre-treatment of SH-SY5Y cells with a cocktail containing CR-6, the pan-caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methylketone) and the calpain inhibitor SJA6017 confers almost total protection against apoptosis. In summary, the present work characterizes the molecular mechanisms involved in oxidative stress-induced apoptosis in SH-SY5Y cells. Our findings highlight the relevance of CR-6, alone or in combination with other drugs, as potential therapeutic strategy for the treatment of neurodegenerative diseases.This work was supported by the Health Research Board of Ireland.Peer reviewe