Experimental Studies Of Enzyme-amplified Amperometric Biosensor For The Detection Of PML/RARa Fusion Gene In Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is M3 subtype of FAB in acute myeloid leukemia (AML). 95% of APL patients are of distinctive chromosome translocation of t(15;17) which results in the formation of PML/RARαfusion gene finally. Therefore, the detection of PML/RARαfusion gene has a great significance. The traditional morphology can't quantitate. So the study of new detection technology is the current hot topic in the domanial of PML diagnosis. DNA electrochemical biosensor is a kind of totally new biosensor which is developed rapidly in recent years and it is also an important mean for analysing and testing nucleic acid structure.This paper aimed to establish highly sensitive enzyme biosensor and optimized a variety of experimental conditions then finally created a new type of enzyme-linked amplified amperometric gene biosensor. Non-specific absorption of enzyme and nucleotide is a main source leading to the background staining in enzyme-amplified amperometric detection of DNA. To eliminate the background staining in this system, the blocking procedure by mercapto-hexanol (MCH) and bovine serum albumin (BSA) was studied systematically. The results show that the anti-fouling effect of BSA is better than that of MCH. A simple and efficient blocking strategy employing BSA as the sole blocking reagent was established and applied in the detection of PML-RARαfusion gene in acute promyelocytic leukemia. It can effectively eliminate the background staining, shorten the pre-processing time, and achieve strong signal amplification. The experiment showed this method is possible to quantitatively detect PML/RARαfusion gene in low concentration. In the range of 1×10-9~ 5×10-8 M and 1×10-12~ 1×10-11 M there was a simple equation between the complementary DNA and the respond signal, with detection limit of 1.22×10-13M. It could effectively distinguish complementary sequence from non-complementary sequence of PML/RARα.With the advantages of good stability,weak background signal and low toxicity. Resveratrol (RST) was used as the substrate of horseradish peroxidase (HRP). After catalyzing reaction in pH 6.0 Britton-Robinson buffer solution (B-R) in 37℃for 5min, the oxidation current signal was collected by using DPV technology. This method could sensitively distinguish complementary sequence from non-complementary sequence of PML/RARα. In the range of 3×10-11~2×10 -10 M there was a linear relationship between the complementary DNA and the detection signal, with detection limit of 9.12×10-12M. It is possible to qualitatively and quantitatively detect PML/RARαfusion gene in APL.