PubMed Health⌕ Search

PubMed · 16893640

Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection.

Abstract

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

S Piermarini, L Micheli, N H S Ammida, G Palleschi, D Moscone. 2006-08-08. Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection.. https://doi.org/10.1016/j.bios.2006.06.029

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis.

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.

Aflatoxin B1↗

Filtration-based tyramide amplification technique--a new simple approach for rapid detection of aflatoxin B1.

The catalyzed reporter deposition (CARD) method of signal amplification, also called "tyramide signal amplification", has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B(1) (AFB(1)) in a variety of foodstuffs with a detection limit of 12.5 microg kg(-1). The assay procedure involves sequential addition of standards or sample, AFB(1)-horseradish peroxidase (HRP) conjugate, B-T, avidin-HRP, and substrate solution over anti-AFB(1) antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB(1) at concentrations from 25 to 100 mg kg(-1) were between 95 and 105%. AFB(1) results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.

Aflatoxin B1↗

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1.

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.

Aflatoxin B1↗