Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current “gold standard” enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.

中文翻译：

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利用光学检测探索异质夹心免疫测定中简单的基于颗粒的信号放大策略

异质夹心免疫分析广泛用于生物分析和医学诊断中的生物标志物检测。当前“金标准”酶联免疫吸附测定 (ELISA) 的高分析物灵敏度源自信号生成酶放大步骤，产生大量光学可检测报告分子。对于未来的即时检测 (POCT) 和即时需求应用，人们对更简单的检测策略越来越感兴趣，这些策略可以避免耗时且依赖于温度的酶反应。旨在达到与酶扩增反应相当的检测限的一个常见概念是使用用大量发色团染色的聚合物纳米颗粒 (NP)。我们探索了不同的简单的基于 NP 的信号放大策略，用于异质夹心免疫测定，该策略依赖于不同类型的光学可检测报告基因的提取触发释放步骤。因此，链霉亲和素功能化聚苯乙烯颗粒（PSP）被用作（i）荧光染料香豆素153（C153）和（ii）氯化血红素（hem）分子的载体，催化鲁米诺反应，实现化学发光（CL）检测。此外，(iii) 评估了基于血红素的微过氧化物酶 MP11 的 NP 标记。对于每种扩增方法，PSP 首先在尺寸、上样浓度和表面化学方面进行系统优化。然后，对于炎症标志物 C 反应蛋白 (CRP) 的免疫测定，将优化 PSP 系统可实现的分析物灵敏度与已建立的用于光度和 CL 检测的 ELISA 概念进行比较。经过仔细优化，MP11 标记的 PSP 和 CL 检测的检测限 (LOD) 为 0.1 ng/mL，其性能与光度 ELISA (0.13 ng/mL) 类似，这证明了我们的新颖检测概念的巨大潜力。