Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710062, P.R. China.
Guizhou Academy of Forestry, Guiyang, 550000, P.R. China.
Analyst. 2022 Jul 22;147(15):3444-3450. doi: 10.1039/d2an00742h.
Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, = 980 nm; = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL, low detection limit of 0.06 ng mL, small sample volume (120 μL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.
热休克蛋白 70（Hsp70）属于热休克蛋白（HSP）家族，据报道是一种有潜力的诊断生物标志物。在这项工作中，通过荧光和上转换纳米粒子探针的结合，制备了侧向流动免疫层析条，用于灵敏快速地测定 Hsp70。上转换纳米粒子（UCNPs，尺寸∼39nm，λex=980nm；λem=540nm）由 NaYF4：Yb/Er 核和聚丙烯酸修饰壳组成，与 Hsp70 抗体共价偶联形成信号探针，通过动态光散射和 zeta 电位分析进行了表征。基于夹心型免疫测定法，构建了侧向流动分析（LFA），使用 PVP 背衬上的样品垫、测试垫和吸附垫。Hsp70 抗体、IgG 抗体和信号探针分别滴加在测试区、测试垫的对照区和样品垫上。在夹心 LFA 中，由于两种抗体结合到 Hsp70 抗原表位，特异性结合，提供了优越的特异性和高灵敏度，使其成为血清 Hsp70 等复杂样品的理想传感平台。优化了制备侧向流动免疫层析条的重要参数。在优化条件下，可通过上转换纳米粒子的荧光强度增加来检测 Hsp70，线性范围从 0.11 到 12ng/mL，检测限低至 0.06ng/mL，样品体积小（120μL），测定时间短（15min），重现性好。荧光法成功应用于血清样品中 Hsp70 的测定，回收率良好。通过结合侧向流动免疫层析条和上转换纳米粒子的可及性，荧光法可以作为一种具有高灵敏度和快速检测的蛋白质分析的即时检测方法。
