AbstractUpconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF4:Yb3+,Er3+ type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540 nm upon 980 nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1 ng·L−1 to 0.48 ng·L−1 in case of cTnI and from 0.070 mIU·L−1 to 0.020 mIU·L−1 in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings. Graphical abstractThe presence of free poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific protein binding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctly more sensitive assays to be performed.

中文翻译：

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通过添加游离聚（丙烯酸）减少聚（丙烯酸）涂层上转换纳米粒子的非特异性结合，提高免疫测定的灵敏度

摘要上转换纳米粒子 (UCNPs) 因其出色的可检测性而成为免疫分析中有吸引力的报告基因。然而，抗体-UCNP 偶联物在蛋白质包被的固体支持物上的非特异性结合会导致背景，这限制了免疫测定的灵敏度。因此，无法充分发挥 UCNP 作为记者的全部潜力。作者在此报告了一种通过减少抗体-UNCP 偶联物在蛋白质包被的固体支持物上的非特异性结合来提高基于 UCNP 的免疫测定灵敏度的方法。在这里研究的测定中，聚（丙烯酸）（PAA）包被的 NaYF4:Yb3+、Er3+ 型 UCNP 与两种不同的抗心肌肌钙蛋白 I (cTnI) 和促甲状腺激素 (TSH) 的抗体结合。两步异质夹心免疫测定在微量滴定孔中进行，在 980 nm 激发下，在 540 nm 处测量抗体-UCNP 偶联物的绿色发光。在将 UCNP 添加到孔中之前，通过将游离 PAA 与 PAA 包被的 UCNP 混合，可以减少抗体-UCNP 偶联物的非特异性结合。缓冲液中的游离 PAA 降低了 cTnI 和 TSH 免疫测定的背景（与不含游离 PAA 的对照测定相比）。如果将 PAA 添加到缓冲液中，检测限在 cTnI 的情况下从 2.1 ng·L-1 降低到 0.48 ng·L-1，在 TSH 的情况下从 0.070 mIU·L-1 降低到 0.020 mIU·L-1。据推测，游离 PAA 的作用是由于封闭了 PAA 包被的 UCNP 非特异性结合蛋白质的表面区域。这里介绍的方法很可能适用于其他种类的 PAA 涂层纳米粒子，可以想象类似的方法也适用于其他纳米粒子涂层。图形摘要缓冲溶液中游离聚丙烯酸 (PAA) 的存在可防止 PAA 包被的上转换纳米粒子 (UCNP) 在异质夹心免疫测定中发生聚集和非特异性蛋白质结合。非特异性结合的减少使得能够进行明显更灵敏的测定。