Experimental Studies Of DNA Electrochemical Biosensor For Detection Of PML/RARα Fusion Gene In Acute Promyelocytic Leukemia

DNA electrochemical biosensor is a novel biosensor which developed rapidly in recent years. It is one of the important methods for analysis and detection of nucleic acid structure. As it has advantages of fast detection, simplity, sensitivity, low price, DNA electrochemical biosensor has attracted more and more attention.A novel DNA electrochemical biosensor for detection of PML/RARαfusion gene in acute promyelocytic leukemia (APL) using 2-nitroacridone (2-NAD) as the hybridization indicator was developed in this article. The experiment showed that 2-NAD was an indicator of high specificity, because it could effectively distinguish complementary sequence from noncomplementary sequence of PML/RARα. After hybridizing with 1.31×10-7M complementary sequence in pH 4.0 phosphate buffer solution (PBS) base solution containing 10μM 2-NAD in 45℃for 30min, PML/RARαprobe electrode got a good detection signal. In the range of 9.08×10-8~5.45×10-7 M, there was a simple equation between the complementary DNA and the respond signal, with detection limit of 2.1×10-8 M. It is possible to qualitatively and quantitatively detect PML/RARαfusion gene in APL.Besides, this article has developed a new method for supervising DNA hybridization directly based on electrochemical alternating current (AC) impedance technology, and many experimental conditions have been optimized. At first, 5'mercapto modified 22-bases oligonucleotide probe and mercaptoacetic acid were self-assembled on the surface of gold electrod(AuE) to form a molecular recognition layer. Then the hybridization signal was the changed value of electronic transfer resistance after hybridization by AC impedance technology. The fixed time of recognition layer, the proportion of oligonucleotide probe and mercaptoacetic acid, hybridization temperature and time, ionic strength of detection solution were optimized. Under optimal conditions, the probe DNA hybridized with 1.0×10-8M target DNA in 45℃for 30min. The obvious difference of Ret after hybridization demonstrated that AC impedance technology could detect PML/RARαfusion gene qualitatively.In the initial study of APL practical samples, the PCR analysis of cDNA of samples was conducted using upstream primer and downstream primer. After electrophoresis, the fragments of gelation were recovered and sequenced. It demonstrated that the specific probe sequence was complementary to target DNA which provided a good basis of futher detection research in practical samples.