【作者】 唐宏武； 庞代文； 李诚予； 曹迪；

【Author】 Hong-Wu Tang;Dai-Wen Pang;Cheng-Yu Li;Di Cao;Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE) & College of Chemistry and Molecular Sciences, Wuhan University;

【机构】 武汉大学化学与分子科学学院生物医学分析化学教育部重点实验室；

【摘要】 将单光束激光光镊捕获和操纵微球的技术和荧光检测相结合,构建一种悬浮式生物芯片。分别以波长473 nm的小功率连续固体激光器和波长1064 nm的被动式调Q亚纳秒激光器同时作为光镊光源和荧光激发光源,分别构建了单光子和双光子荧光检测系统。采用聚苯乙烯微球特异性定量富集待测分子,并采用荧光量子点探针特异性标记微球表面的靶分子,通过检测光阱处微球的单光子或双光子荧光信号,实现对靶分子的定量检测。分别采用单光子和双光子体系检测了禽流感病毒H7N9和H5N1的HA和NA的基因序列,结果表明单光子体系能获得更高的灵敏度,但双光子体系避免了背景荧光的干扰,具有更高的信背比,因此特别适用于复杂体系的分析^[1]。采用双光子荧光检测体系,对全血清中癌胚抗原（CEA）进行了免分离"一步法"检测,检测限达8.6 pg/mL。对十几例癌症患者血清CEA含量进行了检测,结果均与常规的化学发光法检测结果吻合。基于近红外光镊的双光子荧光检测的悬浮式生物芯片的主要优势:（1）样品用量少;（2）数据稳定可靠（量子点标记结合近红外激发,抗光漂白能力强）;（3）信背比高;（4）可直接用于含复杂生物基质的液体试样（如血清等）的分析（免分离）。更多还原

【Abstract】 We constructed suspended biochip by combing single-beam optical tweezers(OT) with fluorescence detection for biomolecules. A 473 nm low-power continuous-wave solid laser and a 1064 nm passively Q-switched nanosecond pulse laser were simultaneously used as the OT laser and excitation source for single and two-photon fluorescence, respectively. Polystyrene microspheres were applied as the solid carrier for the specific enrichment of target molecules through bioaffinity and quantum dots(QDs) were used as the fluorescence tags, and fluorescence signal from the trapped microsphere is monitored for the quantitative detection of the targets. Both HA and NA gene sequences of avian influenza virus H7N9 and H5N1 were detected by single-photon and two-photon fluorescence strategy, respectively. The results show that although higher sensitivity is obtained by single-photon fluorescence, two-photon system provides higher signal-to-background ratio owing to no autofluorescence being excited. Therefore, the two-photon system was applied for one-step separation-free quantitative detection of carcinoembryonic antigen(CEA) in human whole serum with detection limit 8.6 pg/mL. Finally, real serum samples were measured using our platform with results consistent with chemiluminescence analysis. The results demonstrate that the two-photon platform has some advantages:(1) Tiny sample volume;(2) High reliability;(3) High signal-to-background ratio;(4) Separation-free for direct analysis of complex biological fluid samples.更多还原