Simultaneous Detection of Thyroid-Stimulating Hormone and Free Thyroxin in Dried Spots of Human Blood by Using Phosphorescent Nanoparticles

A Phosphorescence Analysis (PHOSPHAN)-based test system was developed for simultaneous detection of thyroid-stimulating hormone (TSH) and free thyroxin (fT4), the markers of thyroid function, in dried human blood samples. The immunoassay was performed at the bottom of polystyrene 96-well microplate wells covered with analyte-specific microarrays (0.35 mm in diameter). The detection system consisted of polystyrene nanoparticles (46 ± 14 nm in diameter) with incorporated long luminescent Eu(III) chelate complexes (1960 molecules/particle). Fluorescence intensity was measured in time-resolved mode (TRF). Analytical parameter values and possible limitations of this experimental system were investigated. The system allowed the simultaneous detection of high-molecular-weight and low-molecular-weight compounds in clinically relevant concentration ranges, from 2.0 to 50 pmol/L for free T4, and 0.8 to 50 µIU for TSH. The sensitivity of the analysis was 1.0 ± 0.2 pmol/L (fT4) and 0.3 ± 0.1 µIU/mL (TSH), which corresponded to the detection thresholds of 3 × 10–18 mol T4 and 0.9 × 10–9 IU TSH. The test determined correctly the TSH and T4 concentrations in standard samples used in quality assessment programs, as well as the thyroid status of the patients included in the investigation (n = 48). Results of hormone detection in dried blood spots with our experimental test system correlated well with the results of hormone detection in serum samples using commercial kits (r = 0.9730 for TSH and r = 0.8585 for fT4). The main drawback of the system was associated with high variability of measurement results (up to 20%), which limits the possibility of its application for the quantification of the hormones level in patient blood. We believe that this system can be useful for detecting thyroid disorders both in the existing programs of neonatal screening for congenital hypothyroidism and in mass screening surveys in risk groups of the adult population, for example, in ecologically unfavorable regions.