Institut d'Electronique Fondamentale, Université Paris-Sud, 91405 Orsay Cedex, France.
ACS Nano. 2013 Aug 27;7(8):7411-9. doi: 10.1021/nn403253y. Epub 2013 Aug 7.
A myriad of quantum dot (QD) biosensor examples have emerged from the literature over the past decade, but despite their photophysical advantages, QDs have yet to find acceptance as standard fluorescent reagents in clinical diagnostics. Lack of reproducible, stable, and robust immunoassays using easily prepared QD-antibody conjugates has historically plagued this field, preventing researchers from advancing the deeper issues concerning assay sensitivity and clinically relevant detection limits on low-volume serum samples. Here we demonstrate a ratiometric multiplexable FRET immunoassay using Tb donors and QD acceptors, which overcomes all the aforementioned limitations toward application in clinical diagnostics. We demonstrate the determination of prostate specific antigen (PSA) in 50 μL serum samples with subnanomolar (1.6 ng/mL) detection limits using time-gated detection and two different QD colors. This concentration is well below the clinical cutoff value of PSA, which demonstrates the possibility of direct integration into real-life in vitro diagnostics. The application of IgG, F(ab')2, and F(ab) antibodies makes our homogeneous immunoassay highly flexible and ready-to-use for the sensitive and specific homogeneous detection of many different biomarkers.
在过去的十年中，文献中涌现出了大量的量子点 (QD) 生物传感器示例，但尽管具有光物理优势，但 QD 尚未被接受为临床诊断中的标准荧光试剂。缺乏使用易于制备的 QD-抗体缀合物进行可重复、稳定和稳健的免疫分析一直困扰着这个领域，这阻止了研究人员深入研究关于低体积血清样本的检测灵敏度和临床相关检测限的问题。在这里，我们展示了一种使用 Tb 供体和 QD 受体的比率型可复用 FRET 免疫分析，该分析克服了应用于临床诊断的所有上述限制。我们使用时间门控检测和两种不同的 QD 颜色，在 50 μL 血清样本中检测到亚纳摩尔 (1.6 ng/mL) 的前列腺特异性抗原 (PSA) 检测限。该浓度远低于 PSA 的临床截止值，这表明直接集成到实际体外诊断中的可能性。IgG、F(ab')2 和 F(ab) 抗体的应用使我们的均相免疫分析具有高度的灵活性，可用于灵敏、特异性地均相检测许多不同的生物标志物。
