State Key Laboratory of Chemo/Biosensing and Chemometrics, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, PR China.
Anal Chem. 2011 Apr 15;83(8):3050-7. doi: 10.1021/ac103274j. Epub 2011 Mar 29.
The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.
滚环扩增（RCA）和圆形链置换聚合。本质上，这两种技术都是基于循环核酸链置换聚合（CNDP），这限制了等温聚合在医学诊断和生物分析中的应用。在本研究中，开发了圆形通用靶分子（非核酸链）置换聚合（CCDP）来放大生物分子分析的荧光信号，将等温聚合扩展到无任何介质的适体系统中。通过将适体与常见的发夹 DNA 探针结合，我们设计了一种自阻断的荧光双功能寡核苷酸探针（信号探针），用于两种疾病标志物 PDGF-BB 和 p53 基因的均相平行检测。基于 CNDP 和 CCDP 信号放大，实现了高灵敏度（例如，检测 PDGF 的浓度低至 1.8×10^-10 M）和选择性检测（即使存在明显更高浓度（7-200 倍）的非靶蛋白也没有干扰），并大大拓宽了线性响应范围。此外，双功能信号探针具有令人印象深刻的简单性、便利性和短的检测时间。本文描述了信号探针的设计，详细研究了影响荧光信号的因素，对分析性能进行了详细表征，并验证了该测定法在复杂介质中的应用。该生物传感方案作为概念验证有望促进寡核苷酸探针在基础研究和医学诊断中的应用。
