| Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia. Over 95% of cases are characterized by chromosome reciprocal translocation, t(15; 17) (q22; q12), resulting in the generation of fusion gene between promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARα), which forms PML/RARαfusion gene. This single gene rearrangement plays an important role in leukemogenesis through antagonizing retinoic acid signaling and the regulatory pathways mediated by APL.Electrochemical biosensors based on the specific recognition of biomaterials present some outstanding advantages including high sensitivity, nice selectivity, low cost, and the possibility of continuous monitoring of biological and synthetic processes. Typical applications include environmental monitoring and control, clinical diagnosis, chemical measurements in the agriculture, food and drug industries.The paper was made up of three parts. Firstly, based on the combination of locked nucleic acid (LNA) probe and electrochemical enzyme immunoassay, the sandwich-type electrochemical enzymes-based LNA-modified DNA biosensor was developed for detection of PML/RARαfusion gene in PBS buffer and human serum. The results indicate that, the biosensor showed an excellent specificity both in PBS buffer and serum to distinguish the complementary sequence and different mismatch sequences. A linear relationship between the amperometric signal and the target concentration in the range of 1.0×10-12-2.0×10-11 mol/L with a detection limit of 6.9×10-14 mol/L was obtained in PBS buffer. In addition, the amperometric signal was found to be a non-linear logarithmic function related to the target concentration in the range of 1.0×10-11-1.0×10-8 mol/L in serum, and the detection limit was experimentally found to be less than 1 pmol/L. These results demonstrated this biosensor could effectively repel nonspecific adsorption of proteins in real samples.In addition, the applications of LNA sensor for the detections of dsDNA and long DNA targets were studied. The results indicate that, the rapid reannealing of denatured dsDNA during the course of hybridization was the key factor influencing the detection of dsDNA by sensor. This problem was solved in this study by adding short PML and RARαpartial oligonucleotides into the hybridization solutions to release the complementary ssDNA stand, then realizing the direct detection of dsDNA without the use of alkaline, helicase enzymes or denaturants.More importantly, we observed that the electrochemical signals of long synthetic DNA targets were different, when target sequences that were complementary to capture/reporter probes were located at different regions of long targets (i.e., 3'-end, int-, and 5'-end). The noncomplementary sequence at the 3'end of long targets could significantly affect their hybridization efficiency or even detection sensitivity. The results of this part provided useful and necessary theoretical basis for sample preparation.Finally, in the initial study of APL practical samples, combining with PCR technique, PCR amplicons of the PML/RARαfusion gene were obtained, and detected by our electrochemical method. By gel electrophoresis and sequencing, it was obtained that the PCR product was 191bp in length and had exact sequence complementary to probes. In the 100μL hybridization system, an electrochemical sequence-specific detection of PCR products was achieved, which provided a good basis of further detection research in practical samples. |
PDF Full Text Request