Terminal deoxynucleotidyl transferase based signal amplification for enzyme-linked aptamer-sorbent assay of colorectal cancer exosomes

被引：18

作者：

Huang, Zhipeng ^{[1
]}

Lin, Qiuyuan ^{[1
]}

Ye, Xin ^{[1
]}

Yang, Bin ^{[1
]}

Zhang, Ren ^{[1
]}

Chen, Hui ^{[1
]}

Weng, Wenhao ^{[2
]}

Kong, Jilie ^{[1
]}

机构：

[1] Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China

[2] Tongji Univ, Yangpu Hosp, Dept Clin Lab, Sch Med, Shanghai 200090, Peoples R China

来源：

TALANTA | 2020年 / 218卷

基金：

中国国家自然科学基金;

关键词：

CRC exosomes; TdT; Aptamer; ELASA; SENSITIVE DETECTION; LABEL-FREE; DNA; BIOMARKERS; DIAGNOSIS; HYBRIDIZATION; IMMUNOASSAY; SYSTEM;

D O I：

10.1016/j.talanta.2020.121089

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

Exosomes have received increasingly significant attention and have shown great clinical value as biomarkers for a number of diseases. However, there is still a lack of a highly sensitive and visualized method for the detection of exosomes in numerous samples simultaneously. Here, we developed a high-throughput, colorimetric and simple method to detect colorectal cancer (CRC) exosomes based on terminal deoxynucleotidyl transferase (TdT)-aided ultraviolet signal amplification. Anti-A33, a CRC exosomal protein marker, was selected as a capture probe, and a facility-prepared EpCAM (CRC exosomal protein) aptamer-Au-primer complex was used as a signal probe. After the CRC exosomes were captured onto the surface of 96-well plates, the primer was extended to the poly(biotin-adenine) chains with the help of TdT, resulting in an increase in the binding amount of avidinmodified horseradish peroxidase (Av-HRP) for H2O2-mediated oxidation of 3,3 ',5,5 '-tetramethyl benzidine (TMB) in enzyme-linked aptamer-sorbent assay (ELASA). The results showed that the incorporation of ploy (biotin-A) enabled approximately 10.4-fold signal amplification. This approach achieved a linear range of 9.75 x 10(3)-1.95 x 10(6) particles/mu L for CRC cell-derived exosomes. The feasibility of the developed assay was evaluated using clinical serum samples. CRC patients (n = 16) could be clearly and successfully distinguished from healthy individuals (n = 9). Furthermore, this proposed platform holds considerable potential for the detection of different targets, simply by changing the aptamer and antibody

引用

页数：8