Chen Chaohui, Luo Ming, Ye Tai, Li Ningxing, Ji Xinghu, He Zhike
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China.
Analyst. 2015 Jul 7;140(13):4515-20. doi: 10.1039/c5an00485c.
An ultrasensitive method for the detection of protein is critically important in fundamental research and practical applications due to the low abundance of disease markers in body fluids or tissues. To detect the trace levels of disease markers with high sensitivity and specificity, a sensitive colorimetric biosensor for protein assay was developed using gold nanoparticles (AuNPs) and rolling circle amplification (RCA). After binding the biotinylated primer/circular template to the streptavidin-conjugated sandwich ELISA immunocomplex, the biotinylated primer was isothermally extended to generate single-stranded DNA (ssDNA). Sequentially, the padlock DNA was added and hybridized with the RCA products. The aggregation of the additional AuNPs in the supernatant containing the surplus padlock DNA and a certain concentration of salt could then be observed. The established sensor allowed for the specific detection of α-fetoprotein (AFP) with a detection limit of 33.45 pg mL(-1). It was also demonstrated that this method could distinguish 500 pg mL(-1) AFP with the naked eye. In addition, this biosensor could be applied to complex sample analysis and could be further used as a universal method for any protein or virus determination by changing the corresponding antibodies.
由于体液或组织中疾病标志物的丰度较低，一种超灵敏的蛋白质检测方法在基础研究和实际应用中至关重要。为了高灵敏度和特异性地检测痕量水平的疾病标志物，利用金纳米颗粒（AuNPs）和滚环扩增（RCA）开发了一种用于蛋白质测定的灵敏比色生物传感器。将生物素化引物/环状模板与链霉亲和素偶联的夹心ELISA免疫复合物结合后，生物素化引物等温延伸以生成单链DNA（ssDNA）。随后，加入锁式DNA并与RCA产物杂交。然后可以观察到含有多余锁式DNA和一定浓度盐的上清液中额外AuNPs的聚集。所建立的传感器能够特异性检测甲胎蛋白（AFP），检测限为33.45 pg mL(-1)。还证明该方法可以用肉眼区分500 pg mL(-1)的AFP。此外，这种生物传感器可应用于复杂样品分析，通过更换相应抗体，还可进一步用作检测任何蛋白质或病毒的通用方法。
