Experimental Studies on the DNA Electrochemical Biosensor for Detection of Marker Genes in Breast Cancer Metastasis
|Keywords||Bromine thymol blue polymer film modified electrode Electrochemical analysis Ascorbic acid (AA) Dopamine (DA) Uric acid (UA) DNA electrochemical biosensors Breast cancer metastasis Mammary gland protein A (MG) Cytokeratin -19 (CK19) Hoechst 33258 Methylene blue (MB) Sentinel lymph node (SLN) AC impedance technology ( EIS )|
Objective 1. Preparation and characterization of electrochemical sensors to study the performance of electrochemical sensors for electrochemical sensors used in breast cancer metastasis detection of marker genes to provide scientific experimental basis; 2. Establish DNA electrochemical biosensor for breast cancer metastasis flag new gene MG detection methods; 3. DNA electrochemical sensor for the detection of breast cancer metastasis marker genes CK19, early diagnosis of breast cancer metastasis. Method 1. Within the potential range of -0.2 V sup> 1.6 V, scan rate of 100 mVs-1 polymerization by cyclic voltammetry (CV) 15 laps to prepare bromine thymol blue polymer film modified electrode AC impedance spectroscopy (EIS) characterization of the prepared electrode; make use of ascorbic acid (AA), dopamine (DA) and uric acid (UA) three substances as electro-active model molecules in pH 5.0 PBS solution, prepared BTB modified electrode measured three electrical activity model molecules to study the performance of electrochemical sensors prepared. 2 glassy carbon electrode as the base electrode, the design 25 bases in length metastasis in breast cancer marker gene Mg single-stranded DNA fragment as a probe, based on the technology of the coupling activator covalent bonding method, the MG single-stranded DNA sequence Probe The needle is fixed to the electrode surface, using electrochemical impedance detection technique for the detection of the electrical signal before and after DNA hybridization, and detection of the experimental conditions were optimized, the new method of detecting breast cancer metastasis marker gene construct DNA electrochemical biosensor; application establish new methods of clinical breast cancer samples PCR amplification products were detected with traditional gel electrophoresis methods controlled trial. 3 to the gold electrode as the base electrode, the design 25 bases in length breast cancer metastasis marker genes CK19 gene DNA fragment as a probe, by the self-assembly method the probe fixed on the electrode surface, using the AC impedance method and combined electrochemical activity directions MB agents to inspect the probe fixed conditions and probe stability, Hoechst 33258 for hybridization indicator the CK19 gene electrochemical biosensor constructed on the basis of this study. Investigated and optimization of the probe between the paired DNA hybridization conditions, and the linear detection. Results ascorbic acid (AA), dopamine (DA) and uric acid (UA) three electrical activity model molecular Polybrominated thymol blue modified glassy carbon electrode surface electrochemically active has been greatly improved. Polymer film thickness and electrolyte buffer the acidity of the polymer film modified glassy carbon electrode. The lowest detection limit and can solve the simultaneous determination of AA, DA and UA in the peak overlap problem, AA, DA and UA in 94 mV, 296 mV and 438 mV three separate DPV peak, AA, DA and UA were 1.0 × 10? 6 M, 1.0 × 10? 8 M, and 1.0 × 10? 6 M. Bromine thymol blue polymer film modified electrodes were also successfully measured vitamin C injection, dopamine injection, the concentration of AA, DA and UA in urine samples. 2 experiments using sodium dodecyl sulfate (SDS) as a removing agent, it is possible to effectively remove the non-specific adsorption of the mono-or double-stranded DNA on the electrode surface. After inspection, SDS elution time of 3 min, and the the probe fixed time of 30 min, the the hybrid optimal temperature and time were 37 ℃ and 20 min, under the conditions of this experiment, using electrochemical impedance detection technology measured breast cancer metastasis the marker gene MG 1.0 × 10-9 sup> 2.0 × 10 -8 sup> mol · L -1 sup> concentration range electrochemical impedance detection signal was a linear relationship (r = 0.9992), the detection limit of 5 × 10 -1 sup> 0 mol · L -1 sup> (S / N = 3); clinical breast actual samples PCR amplified product was detected by gel electrophoresis and the newly established electrochemical method, respectively, found that, when the dilution factor to 32 times when gel electrophoresis failed to detect the fluorescence signal significantly, while create dilution factor to 512 times with a new electrochemical method can also be observed that the apparent electrical signal. By AC impedance method combined the electrochemical activity indicator MB groping probe fixed conditions found in the temperature of 4 ° C, the probe in the case of a fixed time of 20 h on a gold electrode surface can form a stable self-assembled film probe immobilized concentration 3μmol · L -1 sup> sup>; fluorescence analysis results show that, Hoechst 33258 is a specific indicator may be specific with CK19 gene fragment binding, and can effectively distinguish between ssDNA and dsDNA; hybridization indicator Hoechst 33258, electrochemical detection the CK19 gene-specific sequence of experiments, 30 min, 55 ° C hybridization detection signal. Complementary DNA in 5.0 × 10 -7 / sup> . Sup> 5.0 × 10 -6 sup> M concentration range, and the detection signal into a linear relationship, linear equations Ip (μA) = 2.8734 0.25562C (μM), r = 0.9971, a detection limit of 1.8 × 10 -8 sup> M. Conclusion 1. Prepared in this experiment the electrochemical sensor modified by poly BTB electrocatalytic properties of the electrode of the three electrical activity of AA, DA and UA model molecular studies showed that poly the BTB modified electrode has good sensitivity and selectivity, can be used for both the determination of the the three electrical activity model molecules actual samples. Bromine thymol blue (BTB) polymer film modified electrode in the biochemical analysis could play a greater role in electrochemical sensors for the detection of breast cancer metastasis marker genes to provide a scientific experimental basis. DNA electrochemical biosensor 2. Experimental build for the detection of breast cancer metastasis marker genes MG, well distinguish breast cancer metastasis marker genes MG single, double-stranded series of artificial samples with fast, accurate, sensitive, operation the advantages of simple; through the determination of the the clinical actual sample PCR amplification product, compared with the traditional method of gel electrophoresis detection faster, higher sensitivity, scientific experiments eventually applied to the early diagnosis of clinical breast cancer metastasis basis. 3 The experimental build CK19 DNA hybridization indicator Hoechst 33258 electrochemical biosensor, the sensor identification hybridization solution completely complementary sequences with single base mutation sequences with high sensitivity, specificity, good reproducibility, with the second part of the experiment to build DNA electrochemical biosensor for the detection of breast cancer metastasis marker genes MG combined led to the development of multi-channel electrochemical biosensor chip, can be achieved on breast cancer metastasis rapid diagnosis, this experimental study has enormous potential clinical value and far-reaching significance.