Ying Ling-Ling, Ma Yuan-Chun, Xu Bei, Wang Xian-Hua, Dong Lin-Yi, Wang Dong-Mei, Liu Kun, Xu Liang
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, 300070, PR China.
School of Public Health, Tianjin Medical University, Tianjin, 300070, PR China.
J Chromatogr A. 2017 Aug 4;1509:1-8. doi: 10.1016/j.chroma.2017.06.023. Epub 2017 Jun 12.
A combination between modification with nanoparticles (NP) and oriented antibody immobilization (OAI) on the inner face of capillary was for the first time developed for immunoaffinity in-tube solid-phase microextraction (SPME) to promise high antigen extraction capacity. β-microglobin (βMG) and cystatin C (Cys-C) were selected as model antigens. Poly(glycidyl methacrylate) (PGMA) NPs were chemically immobilized onto the capillary by a ring-opening reaction. Antibodies for βMG and Cys-C were immobilized on the NPs through OAI. Scanning electron micrograph of the OAI capillary clearly showed that the PGMA NPs were coated onto the inner surface of capillary in a dense monolayer. In addition, random antibody immobilized (RAI) capillaries and OAI capillaries without NP were also prepared as controls. The extraction capacities of OAI capillaries were 2.02 and 2.18mgm for βMG and Cys-C, and were about 5 and 6 times as many as RAI capillaries and OAI capillaries without NP, respectively. The resultant capillaries were used as in-tube SPME materials to enrich βMG and Cys-C for particle-enhanced turbidimetric immunoassay. When using 1.0mgL standard solutions, the recoveries of OAI capillaries, RAI capillaries and OAI capillaries without NP were 103.6% and 96.8%, 48.5% and 31.5%, and 24.2% and 25.7% for βMG and Cys-C, respectively. Furthermore, the method quantitation limit by OAI capillaries was 5 and 10 times lower than that by RAI capillaries and OAI capillaries without NP, respectively. This result indicated that the NP-coated capillaries with OAI are more suitable for using as immunoaffinity in-tube SPME materials than that with RAI.
首次开发了一种将纳米颗粒（NP）修饰与抗体在毛细管内表面的定向固定（OAI）相结合的方法，用于免疫亲和管内固相微萃取（SPME），以实现高抗原提取能力。选择β-微球蛋白（βMG）和胱抑素C（Cys-C）作为模型抗原。通过开环反应将聚甲基丙烯酸缩水甘油酯（PGMA）纳米颗粒化学固定在毛细管上。βMG和Cys-C的抗体通过OAI固定在纳米颗粒上。OAI毛细管的扫描电子显微镜图像清楚地表明，PGMA纳米颗粒以致密的单层形式涂覆在毛细管内表面。此外，还制备了随机抗体固定（RAI）毛细管和不含NP的OAI毛细管作为对照。OAI毛细管对βMG和Cys-C的提取能力分别为2.02和2.18mg/m，分别约为RAI毛细管和不含NP的OAI毛细管的5倍和6倍。所得毛细管用作管内SPME材料，用于富集βMG和Cys-C以进行颗粒增强比浊免疫分析。当使用1.0mg/L标准溶液时，OAI毛细管、RAI毛细管和不含NP的OAI毛细管对βMG和Cys-C的回收率分别为103.6%和96.8%、48.5%和31.5%、24.2%和25.7%。此外，OAI毛细管的方法定量限分别比RAI毛细管和不含NP的OAI毛细管低5倍和10倍。该结果表明，与RAI相比，具有OAI的NP涂层毛细管更适合用作免疫亲和管内SPME材料。
