检测急性早幼粒细胞白血病PML/RARα融合基因的电化学传感器研制

Development of Electrochemical Biosensor for Detection of PML/RARα Fusion Gene in Acute Promyelocytic Leukemia

针对急性早幼粒细胞白血病(APL)中PML/RARα融合基因的碱基序列,设计了锁核酸(LNA)修饰的发夹结构捕获探针,结合信号探针构建新型的"三明治"电化学传感模式。信号探针末端修饰的生物素可与酶上的亲和素结合,通过检测酶催化H2O2氧化底物3,3',5,5'-四甲基联苯胺(TMB)产生的电化学信号,实现对靶序列的检测。该传感器可识别和定量检测PBS缓冲液中人工合成的PML/RARα融合基因序列。结果表明,该传感器能很好地区分互补序列、单碱基及多碱基错配序列,杂交电流值与目标链浓度在1.0×10-11～1.6×10-10 mol/L范围内呈较好的线性关系,检出限为1.0×10-13 mol/L。同时,该新型传感器成功地用于无稀释人血清中PML/RARα融合基因的检测,具有特异性强、灵敏度高和重复性好的优点,有望用于临床实际样品的检测,进而实现临床上急性早幼粒细胞白血病的早期诊断及预后判断。

A novel DNA electrochemical probe（locked nucleic acid,LNA） was designed and involved in constructing an electrochemical DNA biosensor for the detection of PML/RARα fusion gene in acute promyelocytic leukemia（APL）.This biosensor was based on a ＂sandwich＂ detection strategy,which involved a pair of LNA probes,e.g.hairpin capture probe and reporter probe.Streptavidin-HRP was bound to biotin labeled at the end of reporter probe via streptavidin-biotin affinity binding.In the presence of hydrogen peroxide（H2O2）,HRP catalyzed the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidene（TMB） to offer an enzymatically amplified electrochemical current signal for the detection of target DNA.This sensor was applied in the direct quantitative detection of synthetic PML/RARα fusion gene in PBS buffer.The results indicated that the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences.A linear relationship between the amperometric signal and the target concentration was obtained in the range of 1.0×10-11-1.6×10-10 mol/L with a detection limit of 1.0×10-13 mol/L.In addition,the biosensor was used for the determination of PML/RARα fusion gene in human serum samples without dilution with high sensitivity,selectivity and good repeatability.This method would be expected to use in real sample for further solving the actural problems of early diagnosis and prognosis monitoring of APL.