Colorimetric determination of p-nitrophenol by using ELISA microwells modified with an adhesive polydopamine nanofilm containing catalytically active gold nanoparticles

A microplate method is described for the quantification of p-nitrophenol (p-NPh) in urine samples where it can be found after exposure to certain insecticides such as methyl parathion or paraoxon. The assay is based on the use of a polydopamine (PDA) film doped with gold nanoparticles (AuNPs). The latter exerts a catalytic effect on the reduction of nitrophenols by NaBH4. PDA has adhesive properties and can be used to fix the AuNPs on several solid substrates, here ELISA polystyrene microwells. The optical and catalytic properties of different populations of AuNPs spontaneously grown on PDA films were investigated, mainly in terms of the relationship between AuNPs@PDA nanocomposite preparation and its catalytic activity and stability. The reduction of o-, m-, and p-nitrophenols by NaBH4 in aqueous solution was exploited as model study. The approach demonstrates that useful kinetic information on the catalytic effect can be obtained on 96-wells simultaneously by a conventional ELISA reader at a fixed wavelength of 415 nm. The method was successfully applied to the quantification of p-NPh in (spiked) urine samples and gave high reproducibility (RSD = 3.5%) and a 6.30 μM (836 μg/L) detection limit. [Figure not available: see fulltext.]. © 2019, Springer-Verlag GmbH Austria, part of Springer Nature

Colorimetric determination of total protein content in serum based on the polydopamine/protein adsorption competition on microplates

We report a facile, low-cost and safe analytical method for estimation of total protein content, even in complex matrix like human serum, by exploiting the competition between proteins and polydopamine (PDA) for surface binding. The surface coating has been examined by using a microplate reader taking the advantage of the PDA absorbance in the visible region, obtaining new insights into the modelling of polydopamine deposition and polymer/protein adsorption competition. This is helpful for rational development of imprinted biosensors, and potentially offering a with broad applications ranging from diagnostic tools in medicine to food analysis. The isothermal adsorption of polydopamine on polystyrene surface of multi-well plate displays a Langmuir-shaped curve. It allows the determination of the parameters of polymer film formation useful for any analytical assay depending on the surface coating. Among these, the molecular imprinting and the optical and acoustic evanescent sensing

“In vitro” studies on galectin-3 in human natural killer cells

Galectin-3 (Gal-3) is a β-galactoside binding protein able to modulate both innate and adaptive immune responses. First identified in macrophages, Gal-3 has been studied widely in many mammalian immune cells, but scarcely in natural killer (NK) cells. The aim of this study was to analyze Gal-3 in human NK cells, isolated from peripheral blood mononuclear cells. Both PCR and RT-PCR analysis showed that resting human NK cells express Gal-3 mRNA, which can be modulated upon cytokine stimulation (100 U/ml IL–2 + 20 ng/ml IL-15) for different period of time (1–24 h). Western blot, cytofluorimetry, and confocal microscopy analysis clearly demonstrated that the Gal-3 gene can translate into the corresponding protein. From our results, resting NK cells, isolated from different healthy donors, can express high or low basal levels of Gal-3. In NK cells, Gal-3 was always intracellularly detected at both cytoplasm and nucleus levels, while never at the membrane surface, and its localization resulted independent from the cellular activation status. In addition, the intracellular Gal-3 can co-localize with perforin in exocytic vesicles. Cell treatment with a thiodigalactoside-based Gal-3 inhibitor (1–30 μM) slightly increased the number of degranulating NK cells, while it significantly increased the percentage of cells releasing high amounts of cytotoxic granules (+ 36 ± 3% vs. inhibitor-untreated cells at 30 μM Gal-3). In conclusion, our results demonstrate that human resting NK cells express Gal-3 at both gene and protein levels and that the Gal-3 expression can be modulated upon cytokine stimulation. In the same cells, Gal-3 always localizes intracellularly and functionally correlates with the degree of NK cell degranulation

Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA)

In this study we report the characterization of two new aptamers selected against troponin T [a biomarker for the diagnosis of acute myocardial infarction (AMI)] by surface plasmon resonance (SPR), a reference optical platform for label-free detection of molecular binding interactions, and the possible set up of an ELONA assay for TnT detection based on a single detecting aptamer adsorbed on microwell (direct ELONA), or on the couple of aptamers in a sandwich format (sandwich ELONA). The subsequent colorimetric detection is achieved in both cases by using streptavidin-horseradish peroxidase (S-HRP) as signal reporter

Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells

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