| Pancreatic cancer is a alimentary canal cancer which has high malignant degree and is difficult to diagnose and treat. About 90% of PC originate in pancreatic ductal adenocarcinoma of gland tubule epithelium. The morbidity and mortality of PC has increased significantly in recent years. Futhermore, the 5-year survival rate of PC <1%, so it is one of the worst prognosis of malignant tumors. When most of patients with pancreatic cancer seek medical advice, the pancreatic cancer is already terminal cancer which is unable to cure. The researches show that micro RNA-21(mi R-21) of patients with pancreatic cancer has excessively high expression and also can be used as index of prognosis of PC. And the CEA level of peripheral blood conspicuously increase in patients with pancreatic cancer. Thus, mi R-21 and CEA level in peripheral blood can act as the basis of making a definite diagnosis of early pancreatic carcinoma.In recent years, biosensor technology is rapidly developing. Fluorescent biosensor is widely used in various fields of biological research and diagnosis. It realizes the analysis of the target through translating the signal related to the analyte concentration into fluorescent signal and has the advantages of fast analysis speed,signal stability, high selectivity and high sensitivity. And electrochemical biosensor plays an increasingly important role in genetic diagnosis, antibody detection, cell detection and anti-cancer drug screening. It can detect the target quickly with high sensitivity and high accurateness through combining the biomolecule with highly sensitive electrochemical method and has the advantages of speediness, sensitivity,low cost and simple operation.Combining the biological features of mi R-21 and CEA, we designed the fluorescence sensor of signal amplification for the detection of mi R-21 and ratiometric electrochemical immunosensor for the detection of CEA in this paper. The main contents are as follows:Chapter one constructing a fluorescent sensor based on signal amplification strategy triggered by polymerase for the detection of micro RNA-21.In this section, constructing a fluorescent sensor based on signal amplification strategy triggered by double hairpin probe as capture probe and KF- polymerase as inducer for the detection of mi R-21. At first, the double hairpin probe exists in big hairpin probe structure. At the moment, the labeled fluorescein and quenching agent on opposite sides of the probe leading to the fluorescence quenching. When mi R-21 occurs, some big hairpin probe will open. Then, fluorescein and quenching agent separate, so fluorescence intensity can increase. At this point, double hairpin probe exists in the structure of the small hairpin probe. With the structural characteristics of small hairpin probe, under the action of KF- polymerase, mi R-21 that combined with the small hairpin probe will release into the reaction liquid due to the crowding out effect of the polymer chain, so as to get back in the reaction liquid and unreacted large hairpin probe reaction, so that the released mi R-21 can hybridize with big hairpin probe again making target circulation and signal amplification. There is a positive correlation between the increase of fluorescence intensity and the concentration of mi R-21, which could help realize the detection of mi R-21. Electrophoresis and imaging techniques were applied to the characterization of the experimental mechanism. Under the optimal conditions, the linearity range and LOD were given as0.01-10 n M, 6.81 p M respectively. In experiment of simulated serum sample, though the detection signal decreased, there was a linear relation between ΔF and the concentration of mi R-21 at a range of 0.01-0.5 n M. The detection results of the concentration of mi R-21 in human pancreatic cancer, liver cancer and colon cancer cells were compared with the results of RT-PCR and the trends of two detection results were consistent. The proposed method is expected to be developed for the detection of mi R-21 in cells.Chapter two constructing a fluorescent sensor based on signal amplification strategy triggered by Exo III for the detection of micro RNA-196 a.In this section, designing fluorescence labeled capture probe whose bsae numbers less than target chain according to the shear characteristic of Exo III andconstructing a signal-amplification fluorescent sensor based on GO as fluorescence quenching platform and Exo III-assisted target recycling. The fluorescence intensity of the proposed method was compared with the traditional one-to-one hybridization reaction to verify the feasibility and sensitivity of this method. Under the optimal conditions, the linearity range and LOD were given as 0.01-20 n M, 5.11 p M respectively. The proposed fluorescence detection method based on Exo III and GO is simple, rapid and high sensitive and is expected to be developed for the detection of mi R-196 a in practical samples.Chapter three constructing a ratiometric electrochemical immunosensor based on PTh- Au NPs as internal reference value for the detection of CEA.In this section, constructing a ratiometric electrochemical immunosensor based on PTh-Au NPs as internal reference signal and K3[Fe(CN)6] as indicator signal,detected the CEA by differential pulse voltammetry in the K3[Fe(CN)6] with p H 5.0.In order to verify the feasibility of the method, detecting the response DPV changes in PTh-Au composites before and after modifying with CEA in PBS solution with different p Hs. Futhermore, using TEM characterized the PTh-Au NPs composite,employing EIS characterized the preparation process. Under the optional experimental condition, the linearity range of measuring CEA was from 0.005 to 40 ng/m L, and the LOD was 2.2 pg/m L for CEA. When we detected the CEA in serum of patients with the proposed method, the detection results were close to the results of chemiluminescence. The absolute value of relative error was less than 7.2%. |
PDF Full Text Request