RESUMEN
Gold nanoparticles (GNPs) are widely used in a variety of biomedical diagnostic assays and for imaging. Their popularity stems from key properties such as their low toxicity and high extinction coefficients, as well as straightforward synthesis methods that allow GNPs to be produced quickly and inexpensively. Here we describe the use of GNPs for visual detection in a lateral flow assay using benzodiazepine affinity assay to illustrate the methods.
Asunto(s)
Anticuerpos/química , Cromatografía de Afinidad/métodos , Oro/química , Nanopartículas del Metal/química , Anticuerpos/inmunología , Oxazepam/inmunología , Sistemas de Atención de PuntoRESUMEN
We report on the development of solid phase microextraction probes for drug analysis, prepared with antibodies specific for benzodiazepines covalently immobilized to the surface. In the technique, immobilized antibody probes are exposed to a sample containing the drug for 30 min. Extracted drugs are subsequently desorbed from the probes in 500 microL of methanolic desorption solution, which is dried, reconstituted in a small volume of injection solution and analysed by LC-MS/MS. The antibodies were characterized both before and after immobilization, to facilitate the rational selection of antibodies for such analyses. Polyclonal and monoclonal antibodies were compared as was the impact of affinity purification of the polyclonal antibody to isolate the drug-specific fraction. The probes were evaluated for utility in analyzing 7-aminoflunitrazepam at sub ng/mL concentrations in urine, which is expected to be found several days after a single oral dose of 2 mg of flunitrazepam. Such analyses are required in monitoring for abuse of this drug, both in terms of 'club drug' use and in cases of drug-facilitated sexual assault. In these cases drug concentrations in blood and urine are much lower than in chronic abuse cases and are difficult to analyse by conventional methods. The method developed has a limit of detection of 0.02 ng/mL, with accuracy ranging from 1% to 27% and precision (% R.S.D.) ranging from 2% to 10% between the lower and upper limits of quantitation for the analysis of 7-aminoflunitrazepam in urine. The dynamic range of the method is from 0.02 ng/mL, which is limited by the instrument sensitivity, to 0.5 ng/mL, which is approaching the capacity of the probes. This would allow for quantitative analysis of samples at concentrations below that measurable by many other methods for general benzodiazepines analysis from urine, and a highly selective screen for samples at higher concentrations. The method has similar limits of detection to the most sensitive literature methods specifically designed for such analysis but with the advantage of significantly simplified sample preparation. This simplification makes the technique more amenable for use by both professionals and non-professionals.
Asunto(s)
Flunitrazepam/análogos & derivados , Hipnóticos y Sedantes/orina , Algoritmos , Anticuerpos/química , Anticuerpos/aislamiento & purificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Tampones (Química) , Calibración , Cromatografía de Afinidad , Cromatografía Liquida , Flunitrazepam/inmunología , Flunitrazepam/orina , Humanos , Hipnóticos y Sedantes/inmunología , Inmunoquímica , Inmunoglobulina G/química , Indicadores y Reactivos , Oxazepam/inmunología , Oxazepam/orina , Reproducibilidad de los ResultadosRESUMEN
A simple and specific radioimmunoassay (RIA) was developed for the determination of oxazepam and other 1,4-benzodiazepines in human blood serum and urine (e.g., diazepam, desmethyldiazepam, chlorazepate). For serum a 1:10 dilution, for urine a 1:100 dilution is recommended. Blood and hemolyzed samples need prior extraction by Amberlite XAD-2. The antisera were raised by immunizing "White New Zealand"-rabbits with an oxazepam-3-hemisuccinate bovine serum albumin conjugate. Using 0.1 ml serum dilution the sensitivity is 0.01 mg/l per tube. Especially higher concentrations show a tendency toward underestimation. Being not limited to a single 1,4-benzodiazepine derivative, the specificity of the antisera is also suitable for a screening analysis. Compared to thin-layer chromatographic analysis of urine this assay shows improved sensitivity (0.05--0.1 mg/l in 0.1 ml of a 1:100 dilution = 1 microliter of urine). For forensic investigations, an analysis in the sequence of urine-RIA, blood/serum-RIA, blood/serum-"electron-capture"-gas-liquid chromatography (ECD-GLC) seems to be a helpful approach. Blood levels of diazepam and desmethyldiazepam determined by RIA and GLC after extraction are in satisfactory agreement.