Yao Wu, Wang Lun, Wang Haiyan, Zhang Xiaolei, Li Ling
Anhui Key Laboratory of Chemo-Biosensing, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, PR China.
Biosens Bioelectron. 2009 Jul 15;24(11):3269-74. doi: 10.1016/j.bios.2009.04.016. Epub 2009 Apr 17.
An aptamer-based electrochemiluminescent (ECL-AB) biosensor for ATP detection with high sensitivity and specificity was developed. The biosensor was assembled based on several steps. First, a complementary DNA (cDNA) of the ATP-binding aptamer, which has six complementary bases at both its ends, was hybridized with the aptamer molecule to form a rigid, linear double-stranded DNA (ds-DNA). The ds-DNA was then labeled with a ruthenium complex at the 3' terminus of cDNA, followed by the immobilization of this ds-DNA onto Au electrode surface through the 5'-HS on the cDNA. In the presence of ATP, due to the ATP binding to the aptamer, the aptamer molecules dissociated from the ds-DNA complex, which resulted in the formation of stem-loop structure of the single-stranded cDNA and led to the increase of the ECL signal. The increased ECL intensity was found linearly to the logarithm of the concentration of ATP ranging from 0.05 nM to 10 nM with a detection limit of 0.02 nM. Different from other ECL-AB biosensors with aptamers as the probes, this sensing system proposed here is based on the utilization of the cDNA of aptamers as the probes for ECL sensing. Therefore, such sensing system could provide a promising label-free and more readily regenerated model for aptamer-based small-molecules detection.
开发了一种用于ATP检测的基于适配体的电化学发光（ECL-AB）生物传感器，具有高灵敏度和特异性。该生物传感器通过几个步骤组装而成。首先，将ATP结合适配体的互补DNA（cDNA）（其两端有六个互补碱基）与适配体分子杂交，形成刚性的线性双链DNA（ds-DNA）。然后在cDNA的3'末端用钌配合物标记ds-DNA，接着通过cDNA上的5'-HS将该ds-DNA固定在金电极表面。在ATP存在的情况下，由于ATP与适配体结合，适配体分子从ds-DNA复合物中解离，导致单链cDNA形成茎环结构，从而使ECL信号增强。发现ECL强度的增加与ATP浓度的对数在0.05 nM至10 nM范围内呈线性关系，检测限为0.02 nM。与其他以适配体为探针的ECL-AB生物传感器不同，这里提出的传感系统基于利用适配体的cDNA作为ECL传感的探针。因此，这种传感系统可以为基于适配体的小分子检测提供一个有前景的无标记且更易于再生的模型。
