聚丁二炔生物传感器变色免疫法检测乳腺癌MCF-7细胞

Detection of MCF-7 Breast Cancer Cell Line Based on Polydiacetylene Chromatic Immunoassay

关键词:  聚丁二炔生物  变色免疫分析  合成  表征  乳腺癌MCF-7细胞

Key words:  polydiacetylene/phospholipids  chromatic immunoassay  synthesize  characterize  breast tumor MCF-7 cell line

摘要:

目的 合成能特异性识别肿瘤细胞的聚丁二炔生物传感器。方法 超声乳化-共价修饰法制备聚丁二炔/磷脂(polydiacetylene/Phospholipid，PDA/PC)纳米囊泡，通过共价修饰法将鼠抗人细胞角蛋白抗体CK19共价修饰固定在PDA/PC纳米囊泡表面，制备可变色的PDA/PC生物传感器，用于检测肿瘤细胞。透射电镜负染技术，激光散射粒径测定仪、紫外-可见分光光度计扫描，计算比色响应(colorimetric response，CR)等对合成的PC/PDA生物传感器进行表征，以乳腺癌细胞株MCF-7为模型，模拟考察PDA/PC生物传感器识别鉴定外周血循环肿瘤细胞(circulating tumorcells，CTCs)可行性。结果 TEM检查结果显示制备的PDA/PC生物传感器呈球形或类球形、粒径均匀，平均粒径约为(223.4±23.6)nm；激光散射粒径分析仪显示生物传感器的强均粒径和多分散系数为298.4 nm和0.184；特异性抗原加入后，纳米粒发生团聚，颜色出现由蓝至红的变化，且CR值随着抗原浓度增加而增加，与高表达的乳腺癌细胞MCF-7发生特异性结合，充分混匀后，溶液颜色产生由蓝至红的变化。结论 成功合成由单克隆抗体抗CK19修饰的PDA/PC生物传感器，通过免疫化学显色技术，可快速、有效、灵敏的检测溶液中微量乳腺癌MCF-7细胞，为PDA/PC生物传感器技术在CTC识别上的应用奠定基础。

Abstract:

OBJECTIVE To synthesize PDA/PC biosensor capable of specific recognition of cancer cells by immunoassay. METHODS polydiacetylene/phospholipids (PDA/PC) nanoparticle vesicles were prepared by sonication and subsequent EDC/NHS coupling. Anti-Cytokeratin19 monoclonal antibody was covalent linked to PDA/PC nanoparticle vesicle to fabricate a polydiacetylene (PDA)-based colorimetric biosensor for detecting tumor cells. Physicochemical property were characterized by transmission electron microscope negatively staining technique, laser scattering particle size analyzer and UV-Vis spectrophotometer. Colorimetric response CR changing with the amount of CK19 antigen were determined to assess their color change sensitivity. Breast tumour cell line MCF-7 was chosen as a model to evaluate the biosensor to determine the tumor cell with CK19 expressed. RESULTS The PDA/PC biosensors were round or oval with uniform partilcle size of (223.4±23.6) nm measured by TEM. Size by intensity of 298.4 nm and polydispersity index(PDI) of 0.184 were also determined by Malvern Zetasizer 3000HS. The PC/PDA biosensor was agglomerated with the addition of the human CK19 and CR increased with the rising of the concentration of monoclonal antibody solution. Also, the color of the solution was turned from blue to red. CONCLUSION The PDA/PC biosensor has been successfully fabricated for detection of cancer cell based on the chromatic immunoassay with the naked eye-discernible color change. This method is simple, rapid and sensitive and can be a potential technique for CTC recognition by PDA/PC biosensor.