Abstract — A Phosphorescence Analysis (PHOSPHAN)-based test system was developed for simultaneous detection of thyroid-stimulating hormone (TSH) and free thyroxin (fT 4 ), 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 T 4 , and 0.8 to 50 µIU for TSH. The sensitivity of the analysis was 1.0 ± 0.2 pmol/L (fT 4 ) and 0.3 ± 0.1 µIU/mL (TSH), which corresponded to the detection thresholds of 3 × 10 –18 mol T 4 and 0.9 × 10 –9 IU TSH. The test determined correctly the TSH and T 4 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 fT 4 ). 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.

中文翻译：

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磷光纳米颗粒同时检测人血干斑中促甲状腺激素和游离甲状腺素

摘要 — 开发了一种基于磷光分析 (PHOSPHAN) 的测试系统，用于同时检测干燥人体血液样本中的促甲状腺激素 (TSH) 和游离甲状腺素 (fT 4 )，这是甲状腺功能的标志物。在覆盖有分析物特异性微阵列（直径 0.35 毫米）的聚苯乙烯 96 孔微孔板孔底部进行免疫测定。检测系统由聚苯乙烯纳米粒子（直径为 46 ± 14 nm）与掺入长发光 Eu(III) 螯合物（1960 个分子/粒子）组成。以时间分辨模式 (TRF) 测量荧光强度。研究了该实验系统的分析参数值和可能的局限性。该系统允许同时检测临床相关浓度范围内的高分子量和低分子量化合物，游离 T 4 的浓度范围为 2.0 至 50 pmol/L，TSH 的浓度范围为 0.8 至 50 µIU。分析的灵敏度为 1.0 ± 0.2 pmol/L (fT 4 ) 和 0.3 ± 0.1 µIU/mL (TSH)，对应于 3 × 10 –18 mol T 4 和 0.9 × 10 –9 IU TSH 的检测阈值. 该测试正确确定了质量评估程序中使用的标准样品中的 TSH 和 T 4 浓度，以及调查中包括的患者的甲状腺状态 (n = 48)。使用我们的实验测试系统在干血斑中检测激素的结果与使用商业试剂盒检测血清样品中的激素结果具有很好的相关性（对于 TSH r = 0.9730，对于 fT 4 r = 0.8585）。该系统的主要缺点是测量结果的高度可变性（高达 20%），这限制了其应用于患者血液中激素水平定量的可能性。我们相信该系统可用于检测现有的先天性甲状腺功能减退症新生儿筛查计划和成人人群风险群体的大规模筛查调查中的甲状腺疾病，例如，在生态不利地区。