Electrochemical Impedance Sensor For Determination Of Human Telomere DNA And Breast Cancer Gene BRCA1

Cancer is one of the most serious diseases that human beings can not compete today, which constitutes a great threat to human health and life. Each year about 7 million people worldwide suffer from malignant tumor diseases, about thousands people died of cancer. In recent years, the incidence of malignant tumors in our country is on the rise. With the progress and development of technology of medical science, the human is more familiar with various diseases, and the effective control of human beings. Since the implementation of human genome project, people gradually learn and understand disease from gene level, making unscramble gene on human physical had the new key. With the diagnostic method such as malignant tumor growing and improving, about 80%-90% of the cancer patients can make diagnosed. The world health organization made the latest authoritative conclusion: cancer patients if can early detection, diagnosis, cure rate is 80%. Early cancer detection and diagnosis, therefore, has become one of the worldwide hot topic research and breakthrough of researchers for a long time. Double chain structure and long chain polymers-DNA is also called deoxyribonucleic acid, which is the carrier of genetic information, and is the key to the prevention and cure open human life mystery. Electrochemical impedance sensor is more simple, rapid and sensitive technolgy to detect immune and nucleic acid.Nanomaterials(such as gold nanoparticles, graphene, carbon nanotubes and other materials) appear to have opened a new era for the development and application of analytical chemistry. Nanomaterials with their large specific surface area, high surface reactivity, high catalytic efficiency, and high adsorption ability, provide a new research technolgy such as biomedical research and analytical chemistry. The nucleic acid hybridization technology combines with electrochemical analysis technology for biological detection laid a solid foundation. Nanomaterials technology not only improves the electrochemical detection sensitivity, but also expands the application range of analytical chemistry.The research purpose of this paper is mainly based on nanomaterials construct nano-materials modified substrate electrode, application of new type of DNA electrochemical biosensors detect telomere DNA and breast cancer DNA for quantitative analysis, sample test, and DNA sequences in the chain fully complementary base, three base mismatch, single-base mismatch for accurate recognition. Through electrochemical deposition method and physical adsorption method such as gold nanoparticles, graphene, and carbon nanotubes modified onto the bare electrode surface as electrode modified materials and DNA carrier, which not only magnifies the genetic testing signals and improves the detection sensitivity. Using electrochemical impedance technology, the fabricated label free DNA electrochemical sensor is quantitativly detected.The following is the main research content of this article:1. The electrochemical impedance sensor based on the gold electrode modified with gold nanoparticles(GNPs)-Nafion films was developed for the detection of human telomere DNA. A new nanocomposite films(GNPs-Nafion) was obtained by mixing gold nanoparticle and nafion after ultrasonic dispersion. The GNPs-Nafion nanocomposites were further cast onto a gold electrode to fabricate a novel modified electrode(GNPs-Nafion/Au), which was a platform to immobilize the capture probe of DNA, obtained the electrochemical impedance sensor. The electrode modification processes were characterized by scanning electron microscopy(SEM). Cyclic voltammetry and electrochemical impedance technology were used to monitor the fabrication process of the electrochemical sensor and quantitative determination of the target DNA hybridization. Under the optimization of experimental conditions, the response signal of the electrochemical impedance sensor(△Rt) was linear with the logarithm of complementary DNA concentrations from 0.001 to 1.0 nmol/L with a detection limit of 3.0 pmol/L(S/N = 3). The relative standard deviation obtained was 3.5% with 7 times in parallel determination of 0.5 nmol/L target DNA. The sensor showed a wide linear range, high sensitivity, no labeling, can be used for the detection of other sequence DNA analysis.2. A novel electrochemical impedance sensor for the determination of human telomere DNA was developed on the basis of multi-walled carbon nanotubes-nafion/gold nanoparticles modified electrode. The bare Au electrode was first modified with multi-walled carbon nanotubes-nafion films by dropping, and then gold nanoparticles(GNPs) were electrodeposited onto the surface of multi-walled carbon nanotubes-nafion modified Au electrode(MWNTs-Nafion/GNPs/Au). The electrode modification processes were characterized by scanning electron microscopy(SEM) and electrochemical technology. Then a single-stranded DNA(ss DNA) probe for human telomere DNA was immobilized on the modified electrode for a specific time. The experimental conditions, such as electrochemical deposition time of GNPs, probe immobilization time and target DNA(complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical impedance methods. In the measurement of human telomere DNA, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)6]3-/4- as the probe was monitored. Under the optimized experimental conditions, the sensor response signal and the concentration of logarithm of complementary human telomere target DNA concentration was in the range of 1.0×10-13 to 5.0×10-11 mol/L had good linear relationship with a detection limit of 2.5×10-14 mol/L(S/N = 3). And the 5.0?10-12 mol/L human telomeres DNA was paralleled determination for 11 times and its RSD was 4.6%. The biosensor showed excellent selectivity for the detection of the complementary sequences from no complementary sequences, so it can be used for detection of human telomere DNA and other DNA.3. A novel DNA sensor was designed for the determination of breast cancer gene BRCA1 based on three different electrochemical probes, the negative electrochemical probe [Fe(CN)6]3-/4-, methylene blue and the positive electrochemical probe [Ru(NH3)6]3+. First a breast cancer gene BRCA1 ss DNA was immobilized onto the surface of gold nanaoparticles-MWCNT-Nafion modified gold electrode through the adsorption, which fabricated breast cancer gene BRCA1 DNA sensor. The DNA electrochemical sensor was characterized by cyclic voltammetry and differential pulse voltammetry and electrochemical impedance method. The electron transfer resistance of the DNA sensor was changed after the hybridization of complementary sequence DNA, breast cancer gene BRCA1 DNA was quantitatively detected by the change in electron transfer resistance((35)Ret). Under the optimized conditions, In 5 mmol/L K3[Fe(CN)6]- 5 mmol/L K4 [Fe(CN)6 ](p H =7.4) solution, the electrochemical current response was linear with the logarithm of the breast cancer gene BRCA1 DNA concentration from 0.1 nmol/L to 500.0 nmol/L with a detection limit of 0.03 nmol/L(S/N =3).The linear equation is(35)(40)(?(32))=16.34 logc(mol/L) +174.12.When methylene blue as electrochemical probe, the electrochemical current response was linear with the concentration of breast cancer gene BRCA1 DNA from 1.0 nmol/L to 500.0 nmol/L. The correlation coefficient of 0.9980 and the detection limit was 0.3 nmol/L. The linear equation is(35)(40)(?(32))=17.80logc(mol/L) +172.95. Based on the difference binding ability of [Ru(NH3)6]3+to ss-DNA and ds-DNA, the current of [Ru(NH3)6]3+ was linear with the concentration of breast cancer gene BRCA1 DNA from 1.0 nmol/L to 500.0 nmol/L. The detection limit was 0.3 nmol/L. The linear equation is(35)I(?(32))=19.57 logc(mol/L)+196.48.Three kinds of electrochemical probe, [Fe(CN)6]3-/4- probes had the lowest detection limit and the most wide linear range for detection of the breast cancer gene BRCA1 DNA. The sensitivity of breast cancer gene BRCA1 DNA was best by using the electrochemical probe [Ru(NH3)6]3+.4. A novel electrochemical impedance sensor for detecting the breast cancer gene BRCA1 was developed based on a multi-walled carbon nanotubes-nafion/gold nanoparticle modified Au electrode(GNPs/MWNTs-Nafion/Au). The electrode modification processes were characterized by scanning electron microscopy(SEM), displays that both MWNT and GNPs distributed homogeneously on the surface of GCE. Probe oligonucleotides(ss DNA) were immobilized on the gold nanoparticles via the strong affinity between the thiol groups and gold nanoparticles. The electrode modification processes were characterized by electrochemical technology. The experimental conditions, such as probe immobilization time and target DNA(complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical impedance methods. The electroactive complex, [Ru(NH3)6]3+, which can bind to anionic phosphates of aptamer and DNA strands completely through electrostatic interactions. [Ru(NH3)6]3+ is adsorbed on a half-duplex aptamers backbone, giving a clear detection signal in admittance. In the measurement of BRCA1, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)6]3-/4- as the probe was monitored. Under the optimized experimental conditions, the sensor response signal and the concentration of logarithm of complementary BRCA1 target DNA concentration was in the range of 0.001 to 5.0 n mol/L had good linear relationship with a detection limit of 0.3 pmol/L(S/N = 3). And the 0.1 nmol/L BRCA1 was paralleled determination for 11 times and its RSD was 3.2%. The biosensor showed excellent selectivity for the detection of the complementary sequences from no complementary sequences. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.