Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish

Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BC) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BC, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BC which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable and efficient monoclonal cell line obtained is an unlimited source of sensitive and specific antibody to MG and other triphenylmethanes. (C) 2011 Elsevier B.V. All rights reserved.

Adsorption and photocatalysed destruction of cationic and anionic dyes on mesoporous titania films: Reactions at the air-solid interface

Cationic dyes, such as methylene blue (MB), Thionine (TH) and Basic Fuschin (BF), but not anionic dyes, such as Acid Orange 7 (AO7), Acid Blue 9 (AB9) and Acid Fuschin (AF), are readily adsorbed onto mesoporous titania films at high pH (pH 11), i.e. well above the pzc of titania (pH 6.5), due to electrostatic forces of attraction and repulsion, respectively. The same anionic dyes, but not the cationic dyes, are readily adsorbed on the same titania films at low pH (pH 3), i.e. well below titania's pzc. MB appears to adsorb on mesoporous titania films at pH 11 as the trimer (lambda(max) = 570 nm) but, upon drying, although the trimer still dominates, there is an absorption peak at 665 nm, especially notable at low [MB], which may be due to the monomer, but more likely MB J-aggregates. In contrast, the absorption spectrum of AO7 adsorbed onto the mesoporous titania film at low pH is very similar to the dye monomer. For both MB and AO7 the kinetics of adsorption are first order and yield high rate constants (3.71 and 1.481 g(-1) min(-1)), indicative of a strong adsorption process. Indeed, both MB and AO7 stained films retained much of their colour when left overnight in dye-free pH 11 and 3 solutions, respectively, indicating the strong nature of the adsorption. The kinetics of the photocatalytic bleaching of the MB-titania films at high pH are complex and not well-described by the Julson-Ollis kinetic model [A.J. Julson, D.F. Ollis, Appl. Catal. B. 65 (2006) 315]. Instead, there appears to be an initial fast but not simple demethylation step, followed by a zero-order bleaching and further demethylation steps. In contrast, the kinetics of photocatalytic bleaching of the AO7-titania film give a good fit to the Julson-Ollis kinetic model, yielding values for the various fitting parameters not too dissimilar to those reported for AO7 adsorbed on P25 titania powder. (C) 2008 Elsevier B.V. All rights reserved.

Identification of dyes on ancient Chinese paper samples using the subtracted shifted Raman spectroscopy method

The Stein Collection in the British Library contains the Diamond Sutra, the world's oldest, dated, printed document. The paper of the Diamond Sutra and other documents from the Stein collection is believed to be dyed yellow by a natural extract, called huangbo, from the bark of Phellodendron amurense, which contains three major yellow chromophores: berberine, palmatine, and jatrorrhizine, Conservation of these documents requires definite information on the chemical composition of the dyes but no suitable, completely noninvasive analytical method is known. Here we report resonance Raman studies of a series of prate dyes, of plant materials and extracts, and of dyed ancient and modern paper samples. Resonance Raman spectroscopy is used to enhance the spectra of the dyes over the signals from the paper matrixes in which they are held. The samples an give resonance Raman spectra which are dominated by intense fluorescence, but by using SSRS (subtracted shifted Raman spectroscopy) we have obtained reliable spectra of the pure dyes, native bark from the Phellodendron amurense, modern paper dyed with huangbo extracted from this bark, and ancient paper samples. For both ancient paper samples whose pigment bands were detected, the relative intensities of the bands due to berberine and palmatine suggest that the ancient paper is richer in berberine than its modern counterpart, This is the first nondestructive in situ method for detection of these pigments in manuscripts, and as such has considerable potential benefit for the treatment of irreplaceable documents that are believed to be dyed with huangbo but documents on which conservation work cannot proceed without definite identification of the chemical compounds that they contain.

Development and validation of rapid disequilibrium enzyme-linked immunosorbent assays for the detection of Methyl Yellow and Rhodamine B dyes in foods

Sensitive and specific enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of two illegal synthetic dyes: Methyl Yellow (MY) and Rhodamine B (RB) in food. Polyclonal antibodies were raised against synthesised immunogens and employed in unique direct disequilibrium ELISAs. The time of the assays was only twenty minutes (five minutes for each incubation step with sample and enzyme conjugate and ten minutes with enzyme substrate). The IC50 for MY was in the range 1.4-4.2 ng mL(-1) and for RB 0.1-0.5 ng mL(-1). A simple sample preparation method was developed for the analysis of a range of sauces. In the case of spices a dispersive solid phase extraction was applied to purify the extracts. The testing of twenty samples took approximately one and a half hours (including sample preparation and analysis). Both assays were validated according to the Commission Decision 2002/657/EC criteria for use in sauces and spices. The detection capability for MY in sauces and spices was determined to be less than 15 ng g(-1) and 50 ng g(-1), respectively and for RB, 10 ng g(-1) for both types of food samples. The precision of the developed assays was determined in a repeatability study. The intra-and inter-assay coefficients of variation were less than 25% for both tests and matrix types. The simplicity and performance of both assays indicate that they will be very reliable screening methods for the detection of the illegal dyes MY and RB in a range of food products.

Effect of alkali on methylene blue (CI Basic Blue 9) and other thiazine dyes

A detailed study of the action of alkali on methylene blue (Cl. Basic Blue 9) and other thiazine dyes was carried out through a combination of UV/visible spectroscopy, thin layer chromatography, mass and NMR spectrometry and computational methods. In 0.1 M aq alkali solution, methylene blue forms a highly coloured, lipophilic species that is mainly Bernthsen's methylene violet i.e. a hydrolysis decomposition product, this being contrary to the report of a red N-hydroxy methylene blue adduct. The nature of the heterocyclic nitrogen atom in C.I. Basic Blue 9 is discussed and it is concluded there is no basis for the proposal of nucleophile addition at this site of the dye. In contrast, other thiazine dyes are deprotonated by alkali to form their neutral, highly coloured, lipophilic conjugate base forms. (C) 2010 Elsevier Ltd. All rights reserved.

Luminescence of leuco-thiazine dyes

Details of the novel luminescence of the leuco forms of the thiazine dyes, methylene blue and thionine, are reported, including their emission maxima, quantum yields and lifetimes of the luminescence. Other work shows that this luminescence is independent of reducing agent type and solution pH and is a common feature of most thiazine dyes.