The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu Shandong 273165, PR China; State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Biosens Bioelectron. 2014 Apr 15;54:442-7. doi: 10.1016/j.bios.2013.11.041. Epub 2013 Nov 20.
In this work, by combining the enzymatic recycling reaction with the DNA functionalized gold nanoparticles (AuNPs)-based signal amplification, we have developed an electrochemical biosensor for label-free detection of DNA with high sensitivity and selectivity. In the new designed biosensor, a hairpin-structured probe HP was designed to hybridize with target DNA first, and an exonuclease ExoIII was chosen for the homogeneous enzymatic cleaving amplification. The hybridization of target DNA with the probe HP induced the partial cleavage of the probe HP by ExoIII to release the enzymatic products. The enzymatic products could then hybridize with the hairpin-structured capture probe CP modified on the electrode surface. Finally, DNA functionalized AuNPs was further employed to amplify the detection signal. Due to the capture of abundant methylene blue (MB) molecules by both the multiple DNAs modified on AuNPs surface and the hybridization product of capture DNA and enzymatic products, the designed biosensor achieved a high sensitivity for target DNA, and a detection limit of 0.6 pM was obtained. Due to the employment of two hairpin-structured probes, HP and CP, the proposed biosensor also exhibited high selectivity to target DNA. Moreover, since ExoIII does not require specific recognition sequences, the proposed biosensor might provide a universal design strategy to construct DNA biosensor which can be applied in various biological and medical samples.
在这项工作中，我们通过将酶促循环反应与基于 DNA 功能化金纳米粒子（AuNPs）的信号放大相结合，开发了一种用于高灵敏度和选择性无标记检测 DNA 的电化学生物传感器。在新设计的生物传感器中，设计了发夹结构的探针 HP 首先与靶 DNA 杂交，选择外切酶 ExoIII 进行均相酶切扩增。靶 DNA 与探针 HP 的杂交诱导 ExoIII 部分切割探针 HP 以释放酶产物。然后，酶产物可以与修饰在电极表面的发夹结构捕获探针 CP 杂交。最后，进一步使用 DNA 功能化的 AuNPs 来放大检测信号。由于 AuNPs 表面修饰的多个 DNA 以及捕获 DNA 和酶产物的杂交产物捕获了大量亚甲基蓝（MB）分子，因此设计的生物传感器对靶 DNA 具有高灵敏度，检测限为 0.6 pM。由于采用了两个发夹结构的探针 HP 和 CP，该生物传感器还表现出对靶 DNA 的高选择性。此外，由于 ExoIII 不需要特定的识别序列，因此该生物传感器可能为构建适用于各种生物和医学样本的 DNA 生物传感器提供通用设计策略。
