The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is found worldwide in the aqueous environment. Therefore, it has raised increased public concern on potential long-term impact on human health and wildlife. The importance of DCF has been emphasized by the European Union recently by including this pharmaceutical in the first watch list of priority hazardous substances in order to gather Union-wide monitoring data. Rapid and cheap methods of analysis are therefore required for fresh and wastewater monitoring with high sample load. Here, for the first time, well-characterized monoclonal antibodies (mAbs) against DCF were generated and a highly sensitive ELISA developed. The best antibody (mAb 12G5) is highly affine (KD = 1.5 × 10(-10) M), stable to potential matrix interferences such as pH value (pH range 5.2-9.2), calcium ion concentration (up to 75 mg/L), and humic acid content (up to 20 mg/L). The limit of detection (LOD, S/N = 3) and IC50 of the ELISA calibration curve were 7.8 and 44 ng/L, respectively. The working range was defined between 11 and 180 ng/L. On average, about 10 % cross-reactivity (CR) was found for DCF metabolites 5-OH-DCF, 4'-OH-DCF, and DCF-acyl glucuronide, but other structurally related NSAIDs showed binding <1 % compared to the parent compound. While DCF concentrations at the low ppt range were measured in river and lake water, higher values of 2.9 and 2.1 μg/L were found in wastewater influents and effluents, respectively. These results could be confirmed by solid phase extraction combined with LC-MS.
We investigated the assembly of Gold nanoparticles (AuNPs) on Gold and Silicon sensors with two final objectives: (i) understanding the factors governing the interaction and (ii) building up a nanostructured piezoelectric immunosensor for diclofenac, a small-sized pharmaceutical pollutant. Different surface chemistries were devised to achieve AuNPs assembly on planar substrates. These surface chemistries included amines to immobilize AuNPs via electrostatic interaction, or a mixture of amines and thiols to covalently attach the AuNPs. We also generated PEG-amine terminated surfaces to benefit from the well-known non-biofouling properties of PEG-coated surfaces. The functional substrates and the resulting gold nanoparticle layers were characterized in detail by Surface IR, contact angle measurements and Scanning Electron Microscopy (SEM). The mechanism of adsorption is discussed herein considering the nature of the terminal groups and their charge at the pH of AuNPs adsorption. The coverage and the dispersion of AuNPs were strongly dependent on the anchoring points on the surfaces; the optimal were reached when the attachment layer offered multiple interaction points, in particular, for NH 2 /SH and PEG/NH 2 terminated surfaces, where the percentage of isolated particles was up to 78 %. In addition, PEG-coated surfaces led to a stable AuNPs layer resistant to ultrasounds and to further functionalization of the immobilized nanoparticles. These surfaces were used to engineer quartz crystal microbalance (QCM) biosensors for diclofenac detection. The AuNPs nanostructured substrates significantly enhanced the biosensor sensitivity as compared to planar substrates (up to 6 times higher). This enhancement presages a higher sensitivity in the competitive detection of diclofenac on these systems. More importantly, despite the biorecognition and the drastic regeneration conditions, SEM images show that gold nanoparticles layers are stable and reliable, which paves the way for their use as nanostructured platforms for multiple applications.
The plant toxin ricin is a lectin that binds to D-galactose or lactose moieties by multivalent interactions. In the present work, this avidity was used to develop a novel sandwich glyco-immunoassay using a carbohydrate microarray. For realization, 6-azidohexyl-lactose was immobilized on an alkyne silane surface by Cu(I) catalyzed click chemistry. This procedure is fast, and prevents any nonspecific binding on the microarray surface. Ricin binds via its B-chain to the lactose moieties, and is detected by the biotinylated anti-ricin A-chain. By adding a horseradish peroxidase-labeled streptavidin, a chemiluminescence signal can be generated. This method is described as a sandwich-type glyco-immunoassay. The signal on the glyco-chip can be regenerated for at least 10 measurements. The limit of detection was estimated to be 80 ng mL -1 . The assay was carried out on the automated microarray readout platform MCR 3. In this way, it took 20 min for one measurement, including regeneration of the chip surface.
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