Division of Physical Biology and Bioimaging Center, Shanghai Institute of Applied Physics, Chinese Academy of Sciences , Shanghai 201800, China.
Chemistry Department, King Saud University , Riyadh 11451, Saudi Arabia.
ACS Appl Mater Interfaces. 2015 Nov 25;7(46):25618-23. doi: 10.1021/acsami.5b08817. Epub 2015 Nov 11.
Electrochemical DNA (E-DNA) sensors have been greatly developed and play an important role in early diagnosis of different diseases. To determine the extremely low abundance of DNA biomarkers in clinical samples, scientists are making unremitting efforts toward achieving highly sensitive and selective E-DNA sensors. Here, a novel E-DNA sensor was developed taking advantage of the signal amplification efficiency of nanoprobe-initiated enzymatic polymerization (NIEP). In the NIEP based E-DNA sensor, the capture probe DNA was thiolated at its 3'-terminal to be immobilized onto gold electrode, and the nanoprobe was fabricated by 5'-thiol-terminated signal probe DNA conjugated gold nanoparticles (AuNPs). Both of the probes could simultaneously hybridize with the target DNA to form a "sandwich" structure followed by the terminal deoxynucleotidyl transferase (TdT)-catalyzed elongation of the free 3'-terminal of DNA on the nanoprobe. During the DNA elongation, biotin labels were incorporated into the NIEP-generated long single-stranded DNA (ssDNA) tentacles, leading to specific binding of avidin modified horseradish peroxidase (Av-HRP). Since there are hundreds of DNA probes on the nanoprobe, one hybridization event would generate hundreds of long ssDNA tentacles, resulting in tens of thousands of HRP catalyzed reduction of hydrogen peroxide and sharply increasing electrochemical signals. By employing nanoprobe and TdT, it is demonstrated that the NIEP amplified E-DNA sensor has a detection limit of 10 fM and excellent differentiation ability for even single-base mismatch.
电化学 DNA（E-DNA）传感器得到了极大的发展，在不同疾病的早期诊断中发挥着重要作用。为了在临床样本中测定 DNA 生物标志物的极低丰度，科学家们正在不懈努力，以实现高灵敏度和选择性的 E-DNA 传感器。在这里，利用纳米探针引发的酶聚合（NIEP）的信号放大效率，开发了一种新型的 E-DNA 传感器。在基于 NIEP 的 E-DNA 传感器中，捕获探针 DNA 在其 3'-末端巯基化以固定在金电极上，并且纳米探针通过 5'-末端硫代信号探针 DNA 修饰的金纳米粒子（AuNPs）制成。两种探针都可以同时与靶 DNA 杂交形成“三明治”结构，然后由末端脱氧核苷酸转移酶（TdT）催化纳米探针上游离 3'-末端的 DNA 延伸。在 DNA 延伸过程中，生物素标记物被掺入到 NIEP 产生的长单链 DNA（ssDNA）触须中，导致亲和素修饰的辣根过氧化物酶（Av-HRP）的特异性结合。由于纳米探针上有数百个 DNA 探针，一次杂交事件会产生数百个长 ssDNA 触须，从而导致数万次 HRP 催化过氧化氢的还原和电化学信号的急剧增加。通过使用纳米探针和 TdT，证明了 NIEP 放大的 E-DNA 传感器具有 10 fM 的检测限和出色的单碱基错配区分能力。
