Abstract：
Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).
摘要：
将等温核酸扩增策略整合到免疫测定中可以显着降低分析检测限（LOD）。另一方面,扩增步骤增加了时间，并发症,试剂、和分析格式的成本。为了评估异构多步免疫测定的利弊，我们定量了有无滚环扩增(RCA)的前列腺特异性抗原(PSA).此外,我们比较了时间门控（TG）与连续波（CW）光致发光（PL）检测，使用tr配合物和荧光素染料，分别。对于直接（非扩增）和扩增测定，TGPL检测提供了大约四到八倍的低LOD，说明了自发荧光背景抑制的重要性，即使对于多次洗涤测定格式。扩增的测定需要大约2.4小时的测定时间，但导致LOD降低近100倍，降至1.3pg/mL的PSA。在RCA免疫测定中实施TG-FRET（使用Tb-Cy5.5供体-受体对）导致LOD略高（3.0pg/mL），但是比率检测格式提供了重要的好处，如更高的再现性，较低的标准偏差，和复用能力。总的来说,我们的直接比较证明了即使在非均相测定中生物背景抑制的重要性，以及使用等温RCA强烈降低分析LOD的潜力。使此类测定成为常规酶联免疫吸附测定(ELISA)的可行替代方案。
