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. 基于PDMS-金纳米粒子复合膜的微芯片构建同时检测两种心脏标志物的电化学免疫传感器

日益发展的微流控芯片免疫传感器对临床诊断的发展提供了可能。我们基于微流控芯片构建了同时检测两种心脏标志物的电化学免疫传感器,提出了在微管道中,通过流动进样模式,同时检测两种心脏标志物cTnI和CRP的电化学免疫方法。该方法的定量是基于PDMS-金纳米粒子复合膜微反应器中的免疫反应。CdTe量子点和ZnSe量子点分别与二抗结合用于免疫反应。CdTe量子点和ZnSe量子点用酸溶解后,通过用阳极溶出伏安法测溶液中的Cd2+和Zn2+的量进而能得到标志物的量。该方法也可以同时检测临床血清样品中的cTnI和CRP。cTnI和CRP的线性范围分别为0.01~50 ng/mL,0.5~200 ng/mL,检测限分别约为5 amol和307 amol(30 μL溶液)。我们用该方法检测了20个临床血清样品,该方法显示了一定的精确性,结果与传统方法相比没有明显差别。因此该方法可以成功用于临床检测两种心脏标志物。该方法精密度高、灵敏高、稳定性好,并且有amol的检测限。该方法将微流控芯片与电化学方法成功结合,是临床实验室中蛋白检测方法的有益补充。


Figure 1 Schematic diagram of in-situ synthesis of PDMS-GNPs composite microreactors and immunoassay analytical procedure based on QDs label.


Figure 2 The schematic view of the electrochemical analysis via the flow injection mode on the microchip.

2. 功能化碳纳米管/量子点纳米探针用于Caspase 3激活和抑制的检测

将碳管/量子点纳米探针的信号放大作用与半胱氨酸蛋白酶3(Caspase 3)对DEVD肽段的特异性识别与酶解相结合,研制出一种检测Caspase 3活性与抑制作用的电化学传感器。首先利用LBL静电组装的方法合成了碳纳米管/PDDA/CdTe QDs纳米复合物,随后通过碳二酰亚胺偶合反应,共价偶联链亲和素得到功能化的纳米探针。然后设计了一种Biotin标记的含有DEVD肽段的多肽,这种多肽可以特异性的识别激活的Caspase 3。组装到电极上的多肽被凋亡细胞裂解液中的Caspase 3切割后,多肽末端的biotin分子从电极表面脱落下来。随后利用量子点纳米探针,我们可以检测电极上剩余Biotin分子的比例。由于biotin分子的剩余量与Caspase 3的活性成反比,因此通过电化学检测QDs的信号,可以间接反映细胞裂解液中Caspase 3的活性。此外,这种电化学传感器为检测Caspase 3抑制剂及评估抗癌药物的药效提供了一个有力的平台。


Figure 3. (A) Preparation process of CNTs-QDs-SA bioconjugates via LbL assembly. (B) Schematic illustration of the electrochemical strategy for sensing caspase 3 activity.


Figure 4. Time course of caspase 3 activation by apoptosis inducer and concentration-dependent caspase 3 inhibition by Ac-DEVD-CHO. (A) Anodic stripping voltammetry measurements for caspase 3 activity after the HL-60 cells were incubated with apoptosis inducer for (a) 0, (b) 2h, (c) 4h, (d) 6h, (e) 12h, and (f) 24h. (B) Plot for the dependence of peak currents on the incubation time of apoptosis inducer. (C) Anodic stripping voltammetry measurements for caspase 3 inhibition. HL-60 cells were pre-incubated with (a) 0, (b) 5 μM, (c) 17 μM and (d) 21 μM Ac-DEVD-CHO for 30 min, before addition of the apoptosis inducer for 12h; (e) HL-60 cells without treatment were as control. (D) Relationship between the remaining percentage of peak currents (A) and the concentration of caspase 3 inhibitor Ac-DEVD-CHO. Error bars represent one standard deviation for three independent measurements.


Figure 5. (A) Time course of caspase 3 activation by apoptosis inducer measured using a caspase-3 cellular activity assay kit plus. Caspase-3 activity was measured according to experimental procedures. Error bars represent one standard deviation for three independent measurements. (B) Colorimetric detection of caspase 3 inhibition by Ac-DEVD-CHO. HL-60 cells were pre-incubated with 5 μM Ac-DEVD-CHO for 30 min, before addition of the apoptosis inducer for 12 h or 24 h. Error bars represent one standard deviation for three independent measurements.

3. 四氧化三铁纳米结构的控制合成与类酶活性研究

高指数晶面对于催化活性的影响已经得到广泛关注,我们结合近期四氧化三铁类酶活性的发现,利用溶剂热方法,通过选择合适的反应条件,合成了不同结构、暴露不同晶面的四氧化三铁纳米材料,以3,3’,5,5’-N四甲联苯胺(TMB)和H2O2为底物对其类酶活性进行了比较研究。结果表明类酶催化主要受晶粒尺寸以及晶面活性的影响,该发现对于生物纳米酶催化的研究具有重要意义。


Figure 6. Typical SEM (A, D, G), TEM (B, E, H) and high-resolution TEM (C, F, I) images of the obtained Fe3O4 nanocrystals with different structures. Cluster spheres (A-C); octahedra (E-F); triangular plates (G-I).


Figure 7. Typical photographs and relative activity of TMB-derived oxidation product, in the presence of H2O2, catalysed by the Fe3O4 nanocrystals with different structures or without Fe3O4: Cluster spheres (A); triangular plates (B); octahedra (C); without Fe3O4 (D).

发表的文章

  1. Zhou, F.; Lu, M.; Wang, W.; Bian, Z. P.; Zhang, J. R.*; Zhu, J. J.*, Clin.Chem.2010,56(11),1701-1707.
  2. Jing-Jing Zhang, Ting-Ting Zheng, Fang-Fang Cheng and Jun-Jie Zhu*,Chem. Commun., 2011, 47, 1178-1180.
  3. Shanhu Liu, Feng. Lu, Ruimin Xing, Jun-Jie Zhu*. Chem. Eur. J.2011,17 (2):620-625.