Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University , Jinan 250100, China.
School of Pharmaceutical Sciences, Shandong University , Jinan 250012, China.
ACS Appl Mater Interfaces. 2016 Feb 3;8(4):2573-81. doi: 10.1021/acsami.5b10285. Epub 2016 Jan 25.
An ultrasensitive and highly selective electrochemical assay was first attempted by combining the rolling circle amplification (RCA) reaction with poly(thymine)-templated copper nanoparticles (CuNPs) for cascade signal amplification. As proof of concept, prostate specific antigen (PSA) was selected as a model target. Using a gold nanoparticle (AuNP) as a carrier, we synthesized the primer-AuNP-aptamer bioconjugate for signal amplification by increasing the primer/aptamer ratio. The specific construction of primer-AuNP-aptamer/PSA/anti-PSA sandwich structure triggered the effective RCA reaction, in which thousands of tandem poly(thymine) repeats were generated and directly served as the specific templates for the subsequent CuNP formation. The signal readout was easily achieved by dissolving the RCA product-templated CuNPs and detecting the released copper ions with differential pulse stripping voltammetry. Because of the designed cascade signal amplification strategy, the newly developed method achieved a linear range of 0.05-500 fg/mL, with a remarkable detection limit of 0.020 ± 0.001 fg/mL PSA. Finally, the feasibility of the developed method for practical application was investigated by analyzing PSA in the real clinical human serum samples. The ultrasensitivity, specificity, convenience, and capability for analyzing the clinical samples demonstrate that this method has great potential for practical disease diagnosis applications.
我们首次尝试将滚环扩增（RCA）反应与聚（胸腺嘧啶）模板铜纳米粒子（CuNPs）相结合，构建超灵敏、高选择性的电化学分析方法，实现级联信号放大。为了验证这一概念，我们选择前列腺特异性抗原（PSA）作为模型靶标。通过增加引物/适配体的比例，我们利用金纳米粒子（AuNP）作为载体，合成了引物-AuNP-适配体生物共轭物，用于信号放大。特异性构建的引物-AuNP-适配体/PSA/抗 PSA 三明治结构触发了有效的 RCA 反应，其中产生了数千个串联的聚（胸腺嘧啶）重复序列，并直接作为随后 CuNP 形成的特异性模板。通过溶解 RCA 产物模板化的 CuNPs 并通过差分脉冲溶出伏安法检测释放的铜离子，很容易实现信号读出。由于设计的级联信号放大策略，新开发的方法实现了 0.05-500 fg/mL 的线性范围，具有 0.020 ± 0.001 fg/mL PSA 的显著检测限。最后，通过分析实际临床人血清样本中的 PSA，研究了所开发方法在实际应用中的可行性。该方法具有超灵敏性、特异性、便利性和分析临床样本的能力，表明其在实际疾病诊断应用中具有很大的潜力。
