Experimental Studies On The DNA Electrochemical Biosensor For Detection Of Marker Genes In Breast Cancer Metastasis

Objective:1. To provide the scientific basis for detection of breast cancer marker genes, a electrochemical sensor was prepared and investigated, and the performance of the sensor also was studied..2. To establish a DNA electrochemical biosensor for detection of mammaglobin(MG) in breast cancer metastasis.3. To establish a DNA electrochemical biosensor for detection of cytokeratin 19 (CK19) in breast cancer metastasis, and to achieve early diagnosis of breast cancer metastasis.Methods:1.Polybromothymol blue (BTB)-modified electrode was prepared using cyclic voltammetry in pH 5.0 PBS containing 0.2 mM BTB in the potential window of -0.2 to 1.6V at a scan rate of 100 mVs-1 for 15 cycles. The electrochemically synthesized film was investigated using electrochemical impedance spectroscopy. Using ascorbic acid, dopamine and uric acid as the model of electrical activity, the performance of the sensor was studied by simultaneous determination of these three compounds in phosphate buffer solution (pH 5.0).2. Using glassy carbon electrode as the substrate electrode, we designed a length of 25 bp single-stranded DNA fragment of mammaglobin as probe, and then we immobilized the probe on the electrode surface by covalent Bonding. After this, we detected the nature of electrode by electrochemical impedance technique before and after DNA hybridization, and optimized the experimental conditions, constructed DNA electrochemical biosensor based on the new method of detecting breast gene metastasis.we used the sener to detect the actual PCR amplification products in clinical breast cancer patient, and we also compared the fact with conventional gel electrophoresis.3. Using gold electrode as the substrate electrode, we designed a length of 25 bp single-stranded DNA fragment of cytokeratin 19 as probe,and then we immobilized the probe on the electrode surface by self-assembly method. For exploring the immobilization condition of the probe, the electrochemical impedance spectroscopy and electrochemical active indicator methelyne blue (MB) are applied.In this study, a novel hybridization indicator, Hoechst 33258 used to construct electrochemical DNA biosensor. We also investigated and optimized the hybridization conditions between the probe and complementary DNA, and studied the related linear detection.Results:.1. The poly-BTB-modified GCE shows excellent electrocatalytic activity toward the oxidation of AA, DA, and UA in phosphate buffer solution (pH5.0). The electrochemical behavior of the polymer-modified electrode depends on film thickness, i.e., the electropolymyerization time. and it also resolves the overlapped oxidation peaks of AA, DA and UA into three well-defined peaks at potentials of 94 mV, 296 mV, and 438 mV in the DPV, method respectively. In DPV determination, the detection limit of AA, DA, and UA was estimated to be on the order of 1.0×10^-6 M, 1.0×10^-8 M and 1.0×10^-6 M , respectively. In addition, this modified electrode can be successfully applied to determine AA and DA in injection and UA in urine samples without interference.2. Sodium dodecyl sulfate (SDS) was used as a non-specific adsorption removal agent, which can effectively remove the single or double-stranded DNA on the electrode surface. The best time of SDS elution was 3 min, the best time for immobilizing a probe was 30 min, The optimal hybridization temperature and time were 37?and 20 min. With this optimal experimental conditions, we used electrochemical impedance to detect the breast cancer marker gene mammaglobin. In the 1.0×10-9?2.0×10^-8 mol·L^-1 concentration range, the detection response signal was a good linear relationship (r = 0.9992), the detection limit was 5.0×10^-10 mol·L^-1 (S/N=3). We used gel electrophoresis and above electrochemical method respectively to detect the PCR amplification products of actual clinical breast cancer samples, then we found that we failed to detect significant fluorescence signal in 32-fold dilution to gel electrophoresis, while clear signals can also be observed in the electrochemical method when diluted to 512 times.3. For exploring the immobilized condition of the probe, the electrochemical impedance spectroscopy and electrochemical active indicator MB are applied. The experimental results showed that the largest amount of the gene fragment immobilized electrode is 3.0?mol·L^-1 and the best immobilized temperature and time are 4?,20 h. PL spectra show that Hoechst 33258 is a strong specific indicator that can distinguish the complementary and non-complementary sequence of gene CK19. It proved a good detection that CK19 probe ssDNA modified electrodes hybrid for 30 min at 55?in phosphate buffer solution (pH 7.0). On the range of 5.0×10^-7 5.0×10^-6 M, there was good linear relationship between the respond signal and the concentration of complementary DNA, the lowest limit is 1.8×10^-8 M.Conclusion:1. In this study, polybromothymol blue-modified electrode was used to detect electrical activity of three electro-catalytic model molecules AA, DA and UA. It is conclusioned that poly-BTB-modified electrode has good sensitivity and selectivity, it can be used to detect AA, DA, UA and their actual samples. Thus poly-BTB-modified electrode can play a greater role in biochemical analysis, as an electrochemical sensor, it can provide the scientific basis of experiments for detection of breast cancer metastasis marker genes.2. DNA electrochemical biosensor for detection of mammaglobin in breast cancer metastasis which was builded in my experiment, it can well distinguish between single and double-stranded series of artificial samples of mammaglobin in breast cancer metastasis.The detection was rapid, accurate, sensitive, simple.When it was applied to detect PCR amplification of Actual clinical samples, we found that the new method was faster, more sensitive compared to the traditional methods of gel electrophoresis. In the end, it can provide scientific experimental basis in clinical application for early diagnosis of breast cancer.3. With Hoechst 33258 as the hybridization indicator, DNA electrochemical biosensor for detection of CK19 in breast cancer metastasis which was builded in my experiment, it can well distinguish between fully complementary sequence and single base mismatch sequence, The advantages of this detection method were high sensitivity, high specificity and good reproducibility. Combined with above the DNA electrochemical biosensor for detection of mammaglobin, we can develop a multi-channel biosensing chip used to rapid diagnosis of breast cancer metastasis, And eventually used in clinical application, therefore our experimental study has great potential value and far-reaching significance.