Immunoassays for aflatoxins

Immunoassays for aflatoxin analysis have been regarded as valuable supplements to existing and rapidly developing chromatographic techniques. We describe six types of aflatoxin immunogens and their characteristics, reported antibodies against aflatoxins, traditional and novel labeled materials for assay signaling, three immunoassay formats, assay devices (e.g., microtiter plate and reader, lateral flow strip, electronic and optical immunosensors, and a rapid tester dedicated to aflatoxins) and applications of immunoassay in agricultural products. We show trends towards sensitivity, simplification, intelligence and portability. After setting out five challenges in developing immunoassays, we predict that techniques involving novel nanoparticle labels and non-competitive assay may become the main trends in research and that immunoassay devices will be used in many fields.     

Stability of aflatoxins in solution

The stability of aflatoxins B1, B2, G1, and G2 was studied in solutions containing different concentrations of water, acetonitrile, and/or methanol, and in autosampler vials treated with nitric acid or silanized. When stored at room temperature (20 degrees C) for 24 h, aflatoxins G1 and G2 were stable only in solutions containing 100% organic solvent, whereas aflatoxins B1 and B2 were stable in solutions of methanol-water and acetonitrile-water at greater than 60 and 40% organic content, respectively. At 5 degrees C, aflatoxins G1 and G2 showed a significant decrease in concentration only when kept in less than 20% aqueous organic solvent. Significant loss of aflatoxins was realized in standard, commercially available amber type I borosilicate autosampler vials, but chemical etching of the vials with nitric acid or with silanization prevented aflatoxin degradation. These results indicate that aflatoxins are unstable in aqueous solutions and that this instability can be counteracted by the presence of at least 20% organic solvent and keeping the solutions at 5 degrees C or by the use of treated vials.     

红霉素的生物合成与组合生物合成

组合生物合成在筛选和发展新型药物方面日益被生物、化学和医药界所关注.红霉素作为组合生物合成发展的模式化合物一直是人们研究的热点.概述了红霉素的生物合成机制及近年来在此基础上采用组合生物合成获得红霉素衍生物的研究进展,并对此方面存在的问题、应用前景作了展望.     

Fluorimetric determination of aflatoxins by flow-injection analysis

Summary A fast and inexpensive fluorimetric method for the determination of total aflatoxins (B₁, B₂, G₁, and G₂) in food of use in screening numerous samples suspectedly containing these substances is proposed. The sensitivity of the method (determination range between 0.5 and 200.0 ng ml^–1) allows these analytes to be detected at concentrations well below legal limits; hence, separation-detection techniques such as HPLC need only be used with samples in which these compounds are found to occur. The method has been applied to maize, peanut and tapioca samples, obtaining average recoveries of 100.9 with deviations of ±5% with respect to 100% recovery.

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Fluorometrische Bestimmung von Aflatoxinen durch Fließinjektionsanalyse

     

Nanoparticle-based immunosensors and immunoassays for aflatoxins

Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety.     

High-performance liquid chromatography of aflatoxins in human urine

A method was developed for the extraction and determination of unconjugated aflatoxins in human urine by high-performance liquid chromatography. The analysis is based on the elimination of lipid-soluble constituents other than unconjugated aflatoxins in urine by light petroleum extraction. The unconjugated aflatoxins were subsequently extracted from the aqueous phase with chloroform-acetone. Chromatography was performed isocratically with a silica column at 40 degrees C. The resolved aflatoxins were detected and identified by ultraviolet and fluorometric detectors. The recoveries of aflatoxins B1 and G1 added prior to the extraction were 72% and 83%, respectively. This procedure is simple, sensitive and practically useful for epidemiological survey of unconjugated aflatoxins in human urine from areas with a high risk of aflatoxin consumption.     

Determination of aflatoxins in food by overpressured-layer chromatography

Legislative measures for monitoring and control of aflatoxin levels in foods and foodstuffs have been introduced in many countries. The aim of the present work was to make developments in the field of aflatoxin analysis, focusing upon the use of overpressured-layer chromatography (OPLC) for quantitative determination. OPLC methods have been developed for the determination of aflatoxins B1, B2, G1, G2 in different foods. These methods are suitable for sample clean-up and separation as well. Using OPLC we could analyze 10 samples simultaneously. The methods were investigated with fish, corn and wheat samples spiked with 2-10 ng/g aflatoxins. Quantitative evaluation of aflatoxins was accomplished by densitometry. Average recoveries from each food were greater than 73%. The OPLC technique seems to be a rapid, reproducible and cost-effective analysis for quantitative determination of aflatoxins in foods.     

Rapid determination of aflatoxins in corn and peanuts

A rapid and simple method using ultra-high-pressure liquid chromatography with UV detection for the determination of aflatoxins B1, B2, G1 and G2 in corn and peanuts has been developed. In this method, aflatoxins were extracted with a mixture of acetonitrile and water (86:14) and then purified by solid-phase clean-up with a MycoSep#226 AflaZon(+) column. The toxins were determined by UPLC-UV without derivatizing aflatoxins in real samples, which has not been used in other studies. The mean recoveries of aflatoxins from non-infected peanut and corn samples spiked with aflatoxins B1, B2, G1 and G2 at concentrations from 0.22 to 5 microg/kg were between 83.4% and 94.7%. The detection limits (S/N=3) for B1, B2, G1 and G2 were 0.32, 0.19, 0.32 and 0.19 microg/kg, and the corresponding quantification limits (S/N=10) were 1.07, 0.63, 1.07 and 0.63 microg/kg, respectively. We also applied this method on real samples. Among 16 peanut samples, 2 (12.5% incidence) were contaminated with aflatoxin; among 18 corn samples, 4 (22% incidence) were contaminated. The proposed method is rapid, simple and accurate for monitoring aflatoxins in corn and peanuts.     

Biosynthesis of thalicarpine

The incorporation of (±)-, norlaudanosoline, ?nor-reticuline, ?N-methylcoclaurine and ?norlaudanidine into thalicarpine in Cocculus laurifolius DC has been studied and specific utilization of (±)- reticuline is demonstrated. The evidence supports that both the “halves” of thalicapine are derived from reticuline. Parallel feedings of (S)-, and (R)-, reticulines showed that the stereospecifity is maintained in the biosynthesis of thalicarpine from the 1-benzyl-tetrahydroisoquinoline precursor.A double-labelling experiment with (±)-[1-³H, 4′-O¹⁴CH₃] nor-reticuline has shown that the 4′OMe group of a nor-reticuline unit is lost in the biotransformation into thalicarpine. Feeding experiments also revealed that the plants can convert (S)-bolding and (S)-isoboldine into thalicarpine.     

Biosynthesis of isotetrandrine

The incorporation of (±)-coclaurine, (±)-N-methylcoclaurine, didehydro-N-methylcoclaurinium iodide, (+)-(S)-N-methylcoclaurine and (?)-(R)-N-methylcoclaurine into isotetrandrine in Cocculus laurifolius DC has been studied and specific utilization of (±)-, (+)-(S)- and (?)-R-N-methylcoclaurines and didehydro-N-methylcoclaurinium iodide demonstrated. The evidence supports intermolecular oxidative coupline of (+)-(S)- and (?)-(R)-N-methylcoclaurines to form isotetrandrine. Double labelling experiment with (±)-N- [¹⁴C] methyl [1 - ³H] coclaurine demonstrated that the hydrogen atom at the asymmetric centre in N-methylcoclaurine is retained in the bioconversion into isotetrandrine.     

Biosynthesis of Mikrolin

Mikrolin (8) and dechloromikrolin (9) have been shown to exist as tautomeric mixtures in solution. The structures of mono-O-trifluoroacetyl Mikrolin (10) and di-O-acetyl Mikrolin (11) have been elucidated. The products 15 to 23 from reduction of the metabolites 8 9 with Pd/C and Zn in aqueous acetic acid have been identified. The ¹³C-NMR. spectra of Mikrolin (8) and dechloromikrolin (9) and their derivatives have been completely assigned Based on the results of incorporation experiments with sodium [1-¹³C]-, [2-¹³C]- and [1, 2-¹³C]-acetate, a biosynthetic pathway is proposed for Mikrolin (8) .