Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, China.
Biosens Bioelectron. 2011 Oct 15;28(1):277-83. doi: 10.1016/j.bios.2011.07.032. Epub 2011 Jul 23.
In this study, an enzyme-amplified electrochemical biosensor was developed for detection of the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia (APL). This new sensor employs a hairpin locked nucleic acids (LNAs) probe dually labeled with biotin and carboxyfluorescein molecule (FAM). The probe is immobilized at a streptavidin-modified electrode surface via the biotin-streptavidin bridge, and FAM serves as an affinity tag for the peroxidase conjugate binding. Initially, the immobilized hairpin probe was in the "closed" state in the absence of the target, which shielded FAM from being approached by the bulky anti-FAM-HRP conjugate due to the steric effect. Target binding opens the hairpin structure of the probe, the probe undergoes a significant conformational change, forcing FAM away from the electrode. As a result, the FAM label becomes accessible by the anti-FAM-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. This new biosensor demonstrates its excellent specificity for single-base mismatch and able to detect as little as 83 fM target DNA even in the presence of human serum. We also employed this sensor to directly detect PCR real sample with satisfactory results.
在这项研究中，我们开发了一种酶放大电化学生物传感器，用于检测急性早幼粒细胞白血病（APL）中的早幼粒细胞白血病/维甲酸受体α（PML/RARα）融合基因。这种新的传感器采用双标记有生物素和羧基荧光素分子（FAM）的发夹锁核酸（LNA）探针。探针通过生物素-链霉亲和素桥固定在链霉亲和素修饰的电极表面上，FAM 作为过氧化物酶缀合物结合的亲和标签。最初，在不存在靶标的情况下，固定的发夹探针处于“闭合”状态，由于空间位阻效应，FAM 被阻止接近庞大的抗-FAM-HRP 缀合物。靶标结合打开探针的发夹结构，探针发生显著的构象变化，迫使 FAM 远离电极。结果，FAM 标记物可被抗-FAM-HRP 接近，并且可以通过酶放大的电化学电流信号灵敏地转导靶标杂交事件。这种新的生物传感器表现出对单碱基错配的优异特异性，甚至在存在人血清的情况下，也能够检测到低至 83 fM 的靶 DNA。我们还使用该传感器直接检测 PCR 实际样本，结果令人满意。
