Experimental Studies Of Aligned Carbon Nanotubes Arrays Modified Electrochemical DNA Biosensor For Detection Of Multi-drug Resistant Gene

Malignant tumor is one of the leading causes of death and become serious threat to human health in recent years. The generation of multi-drug resistance(MDR) is the main cause of treatment failure of chemotherapy which is an important means of comprehensive treatment of cancer. The overexpression of MDR1 gene is believed to be the main reason that leads to MDR. The establisment of a rapid and accurate method for MDR1 gene detection has an important significance in cancer clinical therapy and prognosis monitoring.Electrochemical DNA biosensors are widely employed in basic medical research and clinical diagnosis due to their outstanding advantages, such as high sensitivity, good selectivity and rapid response. Nanomaterials have the characters of extraordinary electrochemical properties and excellent bio-compatibility, therefore have been widely used in the biological sensing researches. Aligned Carbon Nanotubes arrays modified electrochemical DNA biosensors were built by taking full advantage of electrochemical DNA biosensor technology and nano self-assembly technique, which were used for the rapid and sensitive detection of MDR1 gene. The main contents are as follows:Part I: Experimental studies of Aligned MWCNTs arrays modified electrochemical biosensor for detection of MDR1 geneIn this part, an Aligned Carbon Nanotubes arrays modified electrochemical DNA biosensor with needlelike protrusion structure was built to detect MDR1 gene. The sensing interface was constructed by self-assembled process. We modified the amino group on the gold electrode surface by reacting with cysteamine via Au-S bond, and then connected the carboxyl multi-walled carbon nanotubes on the gold electrode surface by amide linkage to form an aligned MWCNTs array modified electrode. Under the action of the coupling activator, 5’ end-labeled capture probe DNA with aminogroup was immobilized on the pipe end of MWCNTs by amide bond formation to construct the DNA biosensor. Chronocoulometry was employed to the detection of MDR1 gene by using ruthenium hexaammine(Ru Hex) as signal indicator. Experimental results show that the increased electric quantity are proportional to the logarithm of the concentration of the target DNA in the range of 1.0×10-12 mol/L~1.0×10-8 mol/L with a detection limit of 6.4×10-13 mol/L(S/N=3) under optimal conditions. In addition, the biosensor exhibits good selectivity, acceptable stability and reproducibility, which can be used in the qualitative and quantitative determination of MDR1 gene.Part II: Experimental studies of Aligned MWCNTs arrays-PDDA-Au NPs multilayer film modified electrochemical biosensor for detection of MDR1 geneIn this part, a uniform and stable electrochemical DNA biosensor with Aligned MWCNTs arrays-PDDA-Au NPs multilayer film was constructed on the gold electrode surface, which was based on the aligned MWCNTs arrays modified electrode we built in the first part of this article. The construction of sensing interface was based on layer-by-layer electrostatic self-assembly technique. Negatively charged carboxyl MWCNTs and gold nanoparticles(Au NPs) were bridged by positively charged cationic polymer poly diallyl dimethyl ammonium chloride(PDDA) via electrostatic force. 5’end-labeled capture probe DNA with sulfhydryl group was immobilized on the Au NPs by Au-S bond formation to construct the DNA biosensor for the detection of MDR1 gene. Experimental results show that this modified biosensor has good stability and reproducibility. Furthermore, compared to the biosensor we built in the first part, the DNA hybridization electrochemical signals of this modified biosensor can be further amplified by Au NPs. Under the optimization of experimental conditions, the increased electric quantity are proportional to the logarithm of the concentration of the target DNA in the range of 5.0×10-13 mol/L~1.0×10-8 mol/L with a detection limit of 9.4×10-14mol/L(S/N=3). It also shows excellent noise immunity in simulated human serum samples which is possible to apply in practical samples.Part III: Experimental studies of Aligned MWCNTs arrays-r GO-Au NPs modified electrochemical biosensor for detection of MDR1 geneIn this section, an electrochemical DNA biosensor with Aligned MWCNTsarrays-r GO-Au NPs composite structure was constructed on the gold electrode surface.Reduced graphene oxide-gold nanoparticles(r GO-Au NPs) nanocomposites were prepared by one-step reduction method, and were adsorbed on the surface of Aligned MWCNTs arrays electrode we built in the first part to form nano-composite with three-dimensions structures. 5’ end-labeled capture probe DNA with sulfhydryl group was immobilized on the Au NPs by Au-S bond formation to construct the DNA biosensor, which was prepare for the detection of MDR1 gene. Experimental results indicate that the synthetic nanocomposites show good stability and reproducibility.Aligned MWCNTs arrays-r GO-Au NPs modified electrode has a larger active area and a smaller charge transfer resistance which leads to its high sensitivity. Under the optimization of experimental conditions, the linear range of the concentration of the target DNA was measured as 1.0×10-12 mol/L~1.0×10-8 mol/L with a detection limit of3.5×10-13 mol/L(S/N=3).