国家重大科学研究计划项目“生物医学纳米材料对血细胞作用的研究”工作进展与讨论

生物纳米探针对血液恶性肿瘤细胞的体外作用与检测研究

南京大学 朱俊杰 教授


1. 微流控芯片光电双通道法白血病细胞表面糖基多组分同时检测

细胞表面糖基在细胞分化,增殖,免疫应答,信号转导以及肿瘤增长和转移等过程当中发挥着重要作用。细胞表面糖基表达水平的变化与许多疾病如乳腺癌,前列腺癌等相关。所以,细胞表面糖基表达可以作为一种有效的标志物用于识别在细胞在不同生物过程中表面糖基表达的差异。发展识别细胞表面糖基表达的有效手段对于研究它们在疾病发展过程中的作用非常重要。当前用于糖基分析的分析方法主要包括质谱,核磁共振和色谱。然而这些方法常常需要相对昂贵的仪器和专业化的人员,且样品消耗量大,需要人员多。此外,由于这些方法对细胞样品的破坏性所以不能用于活细胞分析。

我们发展了一种集成型微流控芯片装置,该装置兼具电化学阻抗技术和光学显微方法的优点,利用ITO电极阵列作为电化学传感界面和光学观察窗口,用于评价肿瘤细胞K562表面糖基表达程度。通过电化学阻抗测定和光学观察可知细胞在电极界面上表现出结合上的差异从而表明细胞表面糖基不同的表达丰度。将金纳米颗粒修饰在电极表面来提高电化学细胞传感器的灵敏度。所提出的方法可以进行无标记,非破坏性检测并且四组不同的测试可以在一块5×5cm2 的芯片上一次完成。同时,电化学信号和光学信号可以同时收集,这种双检测模式可以提高糖基多组分检测的灵敏度和准确性。该平台被进一步用于评估在药物诱导下活细胞胞膜糖基多组分表达上的变化。在每一个微腔当中所需的细胞数量仅为5000个,这对于研究少量细胞样品非常重要。我们相信该光电微流控芯片平台将变成高通量细胞表面糖基多组分分析的有力工具,在癌症的诊断和治疗方面有潜在应用前景。


Fig.1 (A) Side view of one channel in the two microfluidic configurations. In configuration 1, the four circular openings of 3 mm diameter were punched on the four cell capture chamber regions of the PDMS slab for the SPCEs integrating. In configuration 2, the four circular openings of 3 mm diameter were punched in the down stream of the four cell capture chamber regions for the SPCEs integrating. (B) The construction process of the device for optical−electrochemical monitoring according to configuration 2. Inset shows photograghs of the devices. (C) Schematic representation of lectin-based array for cell surface glycan evaluation. The recognization layer consisted of specific lectins which recognize the corresponding glycan on the cell surface. The EIS assay and optical microscopic observation were performed for the glycan evaluation.


Fig. 2 (A) Nyquist diagram of electrochemical impedance spectra recorded from 0.01 to 105 Hz for [Fe(CN)6]3-/[Fe(CN)6]4-(10 mM, 1:1) in 10 mM PBS (pH 7.4, containing 0.10 M KCl) at (a) K562/BSA/AuNPs/PDDA/ITO, (b) K562/BSA/PNA/AuNPs/PDDA/ITO, (c) K562/BSA/Con A/AuNPs/PDDA/ITO, and (d) K562/BSA/WGA/AuNPs/PDDA/ITO. Cell density is 1.0×106 cells mL-1. (B) Ret values of lectin-covered electrode arrays after blocking with BSA (blue) and ΔRet values for the increase of Ret upon cell binding (red). The error bars come from three independent chips.


Fig. 3 (A) Binding patterns of K562 cells to the lectin array. (B) Binding densities represented in area coverage of K562 cells on lectin array.


Fig. 4 Effects of AZT on glycan expression of K562 cells determined using electrochemical and optical method. The percent variation (Δ%) obtained was calculated as follows: Δ% = [T/C − 1] ×100%, where T is the ΔRet value or area coverage % value obtained from lectin array incubation with AZT-treated cells and C is that from untreated cells. The error bars come from three independent chips.

2. 一步法制备适配体功能化的银纳米及其在细胞特异性成像中的应用

作为荧光金属纳米簇的一员,以寡聚核苷酸为模板制备的银纳米引起了广泛的关注且在生物相关领域展现出巨大的潜力。然而,相对较弱的荧光发射强度和细胞膜穿透性限制了银纳米簇在细胞内的进一步应用。AS1411是富含鸟嘌呤的抗增殖磷酸寡聚核苷酸链,目前作为一种抗癌药物已进入二期临床试验。我们通过一步反应制备了AS1411功能化的银纳米簇。共聚焦扫描电镜和Z轴扫描证实AS1411功能化的银纳米能够内化到MCF-7乳腺癌细胞内部并且特异性地将细胞核染成红色。特别地,MTT实验表明该银纳米细胞毒性小并且对MCF-7细胞的生长抑制效果比单纯的AS1411更好。同时,我们提出了合成适配体功能化银纳米簇的普遍方法,比如Sgc8c和MUC1适配体。由于制备过程的简单以及对目标物的特异性识别能力,该荧光平台能够扩大银纳米簇在生物传感和生物成像中的应用潜能。


Fig.1 Scheme of cell-type specific imaging by aptamer functionalized Ag NCs.


Fig. 2 The confocal laser scanning microscopy of MCF-7 human breast cancer cells (A),NIH-3T3 mouse fibriblast cells (B) and MCF-10A human normal mammary epithelial cells (C) incubated with NC-AS1411-T5 stabilized Ag NCs at 4°C for 30min. (1) bright field images; (2) Ag NCs fluorescence images (red); (A3) overlap of corresponding fluorescence image and bright field image; (B3, C3) fluorescence images with DAPI nuclear staining (blue). The Ag NCs were excited with 543 nm and DAPI with UV. Scale bar, 20 μm. Under the same procedure, no detectable emissions from the control cells.


Fig.3 Intracellular distribution of internalized NC-AS1411-T5 stabilized Ag NCs. MCF-7 cells were incubated with Ag NCs at 4 °C for 30min. After fixed with paraformaldehyde for 20min, the cells were nucleus staining with DAPI for 10min. The fluorescence imaging was then recorded by confocal laser microscopy. (A) bright field images; (B) fluorescence images of Ag NCs (red); (C) fluorescence images wih DAPI nuclear staining (blue); (D) overlap of corresponding fluorescence image and bright field image. Scale bar, 25 μm.

参考文献

  • Jun-Tao Cao, Xiao-Yao Hao, Ying-Di Zhu, Ken Sun, and Jun-Jie Zhu*, Anal. Chem. 2012, 84, 6775-6782.
  • Jingjing Li, Xiaoqin Zhong, Fangfang Cheng, Jian-Rong Zhang*, and Jun-Jie Zhu*, Anal. Chem. 2012, 84, 4140-4146.