Integrated optical sensors have become important in recent years since they are
the only technology which allows the direct detection of biomolecular
interactions. Moreover, silicon microelectronics technology allows mass
production as well as the fabrication of nano-/macrosystems on the same
platform by hybrid integration of sources, sensors, photodetectors and
complementary metal-oxide semiconductor electronics.For the fabrication of an
optical sensor nanodevice with an integrated Mach–Zehnder interferometric
(MZI) configuration, the optical waveguides must have two main features:
monomode behaviour and a high surface sensitivity. In this paper we
present the development of a MZI sensor based on total internal reflection
waveguides with nanometre dimensions. The aim is to use these sensors in
environmental control to detect water pollutants by immunoassay techniques.
To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently characterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterologous assay haptens. Depending on the immunoreagent combination and assay format, competitive assays with I(50) values in the 1.2-10.2 nM (0.27-2.27 ng/mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFNH, coupled either to horseradish peroxidase or to ovalbumin, was used to develop a direct and an indirect enzyme-linked immunosorbent assay, respectively. Optimized immunoassays displayed very similar analytical characteristics, with an I(50) value around 0.7 ng/mL and a limit of detection around 0.08 ng/mL. Both immunoassays were able to tolerate the presence of methanol up to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pesticides were not recognized by the MAb. These immunoassays should reasonably allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practical importance.
The effect of several physicochemical factors on the analytical characteristics of an immunoassay to carbaryl has been studied and this information used to optimize the assay. The immunoassay is based on the LIB-CNH36 monoclonal antibody (MAb) and employs a heterologous coating conjugate. The optimized enzyme-linked immunosorbent assay (ELISA), performed in a high salt concentration buffer with bovine serum albumin instead of Tween 20, has a carbaryl I 50 of 0.058 ppb and a detection limit of 0.010 ppb, which means a 4-fold improvement in the assay sensitivity with respect to the nonoptimized conditions. The assay proved to be very specific for carbaryl since not even 1-naphthol, the main carbaryl metabolite, was recognized. Water samples with very different conductivities were spiked with carbaryl at 0.05, 0.1, and 0.5 ppb and directly analyzed by the developed ELISA. The mean recovery was 112%, and the mean coefficient of variation was 10.7%, which proves the suitability of the method to determine carbaryl in waters at such low levels.
With the aim of developing a highly sensitive immunoassay for the
insecticide carbaryl, seven
compounds were synthesized, conjugated to carrier proteins, and used as
immunizing haptens in
mice and rabbits. Polyclonal and monoclonal antibodies were
subsequently obtained and characterized for their affinity to carbaryl. Three monoclonal antibodies
(MAbs) that showed the highest
affinity (I
50 < 3.0 nM) were chosen for
immunoassay development and further tested in the coating
conjugate and coating antibody enzyme-linked immunosorbert assay
(ELISA) formats, against both
homologous and heterologous conjugates. Among several immunoassays
developed, the combination
of LIB-CNH36 MAb with the heterologous conjugate
ovalbumin−N-(1-naphthoyl)-6-aminohexanoic
acid in the coating conjugate ELISA format afforded the highest
sensitivity for carbaryl determination
(I
50 = 1.1 nM). This work also provided
some insights into the influence of the immunizing hapten
structure on the affinity of the antibodies obtained, as well as into
the effect of the ELISA format
and hapten heterology on the assay sensitivity.
Keywords: ELISA; immunoassay; carbaryl; monoclonal antibody;
N-methylcarbamate; pesticide;
hapten design; hapten heterology; ELISA format
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