Experimental Studies Of DNA Electrochemical Biosensor Used For Detecting Multi-drug Resistant Gene

Electrochemical DNA biosensor is a novel gene detection technology. Due to its outstanding advantages, such as high sensitivity, good selectivity, rapid response, easy operation and low cost, electrochemical DNA biosensor is widely used in life science research and clinical medicine. Because of superior conductivity, outstanding catalytic properties, good biocompatibility and some other unique physical and chemical properties, nanomaterials have got extensive concern from the biological sensing researchers. Taking advantages of these characteristics of nanomaterials, novel electrochemical DNA biosensors builded will greatly improve the selectivity, sensitivity and detection range of DNA biosensor. Multi-drug resistance (MDR) is the main cause of tumor chemotherapy failure, and the abnormity of MDR1expression is the primary reason that leads to multi-drug resistance. Detection of MDR1gene, through which the patients response to chemotherapy and tolerance degree can be predicted and reasonable individualized tumor chemotherapy regimens can be developed, has an important significance in the related fields, such as cancer clinical, therapy prognosis monitoring, new drug screening and so on. Therefore, taking advantages of the excellent characteristics of nanomaterials and electrochemical biosensing technology, this paper mainly focused on exploring and studying the preparation of functional nanocomposites, design of novel signal enhancement strategy, and construction of sensitive biosensor interface. Novel electrochemical nano-DNA biosensors were fabricated for sensitive, rapid, simple and economical detection of MDR1gene. The details are given as follows:Part One: Experimental study of a novel lable-free electrochemical DNA biosensor based on AuNPs/TB-GO nanocomposites film for detection of MDR1geneA new protocol of label-free electrochemical DNA biosensor based on Au nanoparticles/toluidine blue–grapheme oxide (Au NPs/TB–GO) modified electrode for effectively determination of MDR1gene was presented. AuNPs/TB-GO nanocomposites were composited and coated onto the glass carbon electrode (GCE), and then probe DNA sequence was immobilized on the electrode via Au-S bond to construct the DNA biosensor which detected of MDR1gene by using TB in the nanocomposites film directly indicated the electrochemical signal. Under optimal conditions, the decreased currents were proportional to the logarithm of the concentration of the target DNA in the range of1.0×1011–1.0×109M with a detection limit of2.9×1012M (at an S/N of3). In addition, the biosensor exhibited good selectivity, acceptable stability and reproducibility. The proposed method was simple, fast and inexpensive for the determination of MDR1gene at low levels.Part Two: Experimental studies of electrochemical DNA biosensor based on dual signal amplification technology for highly sensitive detection of MDR1geneDNA-AuNPs compounds, which used as signal probe, were prepared by immobilized complementary DNA fragments and non-complementary DNA fragments on AuNPs via Au-S bond. Then capture probe was fixed to the electrode surface by covalent coupling activator technology, and a sandwich-type DNA hybridization mode was structured. The DNA hybridization signals were dual amplified by AuNPs of signal DNA and silver catalytic technology, thus a highly sensitive DNA biosensor was constructed for detection of MDR1gene. Under the optimal conditions, the current signal responded to the logarithm of target DNA concentration in a wide range from1.0×10-13M~1.0×10-9M with a low limit of detection of3.3×10-14M (S/N=3). Moreover, the prepared DNA biosensor exhibited superior selectivity, high sensitivity and good reproducibility.Part Three: Experimental study of a novel electrochemical DNA biosensor based on PDA-RGO nanocomposites for highly sensitive detection of MDR1genePDA-RGO nanocomposite prepared by in-situ polymerization reduction method was coated onto GCE, and single-stranded probe DNA modified with analkylamino modifier at the5’-end (NH2-ssDNA) was grafted on PDA-RGO surface via Michael addition reaction. Then, the sandwich-type electrochemical enzymes-based DNA biosensor was fabricated for detection of MDR1gene. The results demonstated PDA-RGO nanocomposite exhibited excellent stability and superior reproducibility. Compared with the bare GCE and GO-based GCE, PDA-RGO modified GCE could accelerate the electron transfer and improve the catalytic performance. Under the given conditions, the current signal responded to the logarithm of target DNA concentration in a wide range from1.0×10-13M~1.0×10-9M with a low limit of detection of5.7×10-14M (S/N=3). The biosensor showed superior selectivity and obtained satifactory experimental results.