【作者】 李诚予； 曹迪； 漆楚波； 庞代文； 唐宏武；

【Author】 Cheng-Yu Li;Di Cao;Chu-Bo Qi;Dai-Wen Pang;Hong-Wu Tang;College of Chemistry and Molecular Sciences, Wuhan University;

【机构】 武汉大学化学与分子科学学院；

【摘要】 本研究将单光束光镊技术和双光子荧光检测技术相结合,构建检测装置,并实现了全血清中癌胚抗原(CEA)的免分离"一步法"定量检测。对于检测装置的构建,我们以价格较为低廉的被动式调Q亚纳秒激光器(波长1064 nm)同时作为光镊光源和双光子荧光激发光源。该激光光束依次经过能量调节、扩束、二向色镜(DM)反射后充满显微镜物镜后瞳,最后由100?物镜(NA=1.30)聚焦至自制样品池内形成单光束光阱。为了提高检测灵敏度,我们以EMCCD为荧光信号检测器。以3μm羧基聚苯乙烯微球作为固相载体,采用传统的双位点免疫结合策略,实现对CEA的富集。首先通过EDC/NHS活化反应,在微球表面共价偶联CEA捕获抗体,制备得到免疫微球。然后将氨基修饰发射波长为605nm的量子点作为荧光标记材料,通过SMCC活化反应,将另一种CEA单克隆抗体的Fab片段共价结合至量子点表面,制备得到量子点标记的检测抗体。最后采用双位点一步法捕获待测CEA得到免疫复合微球。由于只有在强烈会聚的激光焦点处才能实现双光子荧光信号的激发,溶液中游离的量子点不会影响荧光信号的检测,因此无需分离多余的量子点。通过移动样品池,逐一捕获单个免疫复合微球并同时检测其表面的双光子荧光信号。通过平行测定不同浓度梯度组中50颗微球的荧光强度,将其平均值用于CEA的定量检测,在缓冲溶液中的检测限可以达到7.8 pg/m L。由于以近红外激光为光源,复杂样品体系本身不会产生背景荧光,因此本方法有很高的信背比。将本方法直接在纯血清环境中进行CEA的检测,结果和缓冲体系十分接近,检测限可达8.6 pg/m L。最后,我们分析了12例癌症病人的血清来考察该方法的可靠性和实用性,其检测结果与化学发光法的检测结果吻合。更多还原

【Abstract】 We constructed a detection setup by combing single-beam optical tweezers with two-photon fluorescence for one-step separation-free quantitative detection of carcinoembryonic antigen(CEA) in human whole serum. A low-cost 1064 nm passively Q-switched sub-nanosecond pulse laser was used as both the trapping beam and excitation source for two-photon excitation. A traditional sandwich immunization strategy was adopted to one-step capture CEA by using polystyrene microspheres as the solid carrier and 605 nm quantum dots(QDs) as the fluorescent tag. Because of two-photon fluorescent signals only originate from the focal point, the free QDs outside the focal point do not contribute to the final signals, thus it is unnecessary to separate the excessive QDs, and the detection limit reaches as low as 7.8 pg/m L in buffer solution. Simultaneously, owing to the advantage of near-infrared excitation, the background signals are negligible in human whole serum, leading to a very close detection limit(8.6 pg/m L) compared with that in buffer system. Finally, 12 cases of real samples were measured in our platform with reliable results, demonstrating its potential in clinical diagnosis.更多还原