Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Anal Chem. 2012 Feb 7;84(3):1623-9. doi: 10.1021/ac2029002. Epub 2012 Jan 24.
Highly sensitive detection of proteins is essential to biomedical research as well as clinical diagnosis. However, so far most detection methods rely on antibody-based assays and are usually laborious and time-consuming with poor sensitivity. Here, we develop a simple and sensitive method for the detection of a biomarker protein, platelet-derived growth factor BB (PDGF-BB), based on aptamer-based target-triggering two-stage amplification. With the involvement of an aptamer-based probe and an exponential amplification reaction (EXPAR) template, our method combines strand displacement amplification (SDA) and EXPAR, transforming the probe conformational change induced by target binding into two-stage amplification and distinct fluorescence signal. This detection method exhibits excellent specificity and high sensitivity with a detection limit of 9.04 × 10(-13) M and a detection range of more than 5 orders of magnitude, which is comparable with or even superior to most currently used approaches for PDGF-BB detection. Moreover, this detection method has significant advantages of isothermal conditions required, simple and rapid without multiple separation and washing steps, low-cost without the need of any labeled DNA probes. Furthermore, this method might be extended to sensitive detection of a variety of biomolecules whose aptamers undergo similar conformational changes.
高灵敏度的蛋白质检测对于生物医学研究和临床诊断至关重要。然而，迄今为止，大多数检测方法依赖于抗体检测，通常繁琐且耗时，灵敏度较差。在这里，我们开发了一种基于适体的靶触发两步放大的简单而灵敏的生物标志物蛋白血小板衍生生长因子 BB（PDGF-BB）检测方法。通过涉及基于适体的探针和指数扩增反应（EXPAR）模板，我们的方法结合了链位移扩增（SDA）和 EXPAR，将靶结合诱导的探针构象变化转化为两步放大和明显的荧光信号。该检测方法具有优异的特异性和高灵敏度，检测限为 9.04×10^(-13)M，检测范围超过 5 个数量级，与目前用于 PDGF-BB 检测的大多数方法相当，甚至更优。此外，这种检测方法具有不需要恒温条件、简单快速、无需多次分离和洗涤步骤、成本低且不需要任何标记 DNA 探针等显著优势。此外，该方法可能扩展到对各种生物分子的敏感检测，这些生物分子的适体经历类似的构象变化。
