基于核酸适体的比色技术检测肿瘤标志物血管内皮生长因子  

A colorimetric method for vascular endothelial growth factor detection based on aptamer and magnetic beads

作　　者：刘景荣[1] 潘云苓[1] 赵燕苹 刘美岑[2] 陈敬华[2] 李春艳[2] 

机构地区：[1]福州市第一医院中西医结合肿瘤科,福建福州350009 [2]福建医科大学药学院,福建福州350122

出　　处：《南方医科大学学报》2017年第2期210-215,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(21375017);福州市科技计划项目(2013-S-122-4);福建省科技厅重点项目(2013Y0045);福建省自然科学基金(2013J06003);福建省高等学校新世纪优秀人才支持计划(JA13130)~~

摘　　要：目的基于核酸适体和磁珠,提出一种检测肿瘤标志物血管内皮生长因子(VEGF)的比色方法。方法核酸适体与标记脲酶的单链结合,通过生物素-亲和素的特殊识别作用固定在链霉亲和素修饰的磁珠上;待检测的肿瘤VEGF标志物与适体结合形成茎环结构,使标记脲酶的单链脱落下来;经磁分离,转移上清液,加入到含一定量尿素和酚红试剂的溶液中。利用上清液中的脲酶水解尿素产生氨,使得溶液的p H值升高,导致溶液的颜色变化,通过肉眼或紫外分光光度计分析显色结果。结果该检测体系在最佳条件下,检测VEGF的线性范围为0.1~10 pmol/L,检测限达0.06 pmol/L。并利用此体系,检测人肺癌患者血清中的VEGF浓度,其结果与ELISA方法检测出的结果基本一致。将本方法与ELISA方法同步盲法测定10例血清样本,结果显示本方法的准确率为90%,表明其具有较高的有效性和敏感性。结论该检测方法操作简单方便,同时具有较高的灵敏度和选择性,在临床VEGF检测中具有潜在的应用前景。Objective To develop a novel colorimetric method for detecting the tumor biomarker vascular endothelial growth factor （VEGF） based on aptamer and magnetic beads. Methods The capture aptamer was hybridized to urease functionalized single-stranded DNA （ssDNA） and immobilize on the surface of magnetic beads by specific biotin-avidin binding. In the presence of VEGF, aptamers bound to VEGF to form a specific stem-loop structure to release the urease functionalized ssDNA. After separation, the supernatant was transferred to a tube and urea and phenol red were added. Urease hydrolyzed urea to produce ammonia to cause an increase of the pH value and a color change of phenol red. The results were inspected with either the naked eyes or by a UV spectrophotometer. Results Under optimized conditions, the detection system showed a good linear relationship for VEGF detection in the range of 0.1 to 10 pmol/L with a detection limit as low as 0.06 pmol/L. The results of VEGF detection in the serum of patients with lung cancer were consistent with those using an ELISA Kit. The results of examination of 10 serum samples with this aptamer-based method and ELISA kit showed that the accuracy of this method was 90%. Conclusion This aptamer-based system provides an simple and convenient method for VEGF detection with a high sensitivity and selectivity.

关 键 词：核酸适体 磁珠 脲酶 比色 血管内皮生长因子 

分 类 号：R730.43[医药卫生—肿瘤]