| Analysis of biomarkers with sensitivity, speeding, specificity, accuracy, and automatics plays a vital role in clinical diagnostics and molecular biology research. In recent years, isothermal amplification have been widely used in the preparation and application of biosensors due to its high sensitivity, simple operation, and easily application. Under this research background, in the present dissertation, we dedicate to the design and development of efficient biosensors, and its application in clinical research. By employing nanostructures such as, molecular beacon (MB), gold nanoparticle (GNP), and graphene oxide (GO), isothermal amplification is improved greatly. The main content of the dissertation are as follows:1. We designed a difunctional GNP probe, developed a bidirectional method with four detection-color states for detection of urine telomerase activity, and further applied it in diagnosis of bladder cancer. The difUnctinal GNP probes can perform four color-detection states corresponding to different concentrations and activity of telomerase. Moreover, this strategy was bidirectional according to the GNP detection states and values of A650/A520 of detection samples, like number axis. Origin was the red GNP probes with minimum A650/A520-The forward direction of number axis represented precipitated GNP probes. The negative direction of number axis represented purple and blue GNP probes. More importantly,18 urine specimens from bladder cancer patients, inflammation patients, and normal individuals were investigated to demonstrate the applicability for clinical diagnosis.2. Here we firstly demonstrated a real-time colorimetric method using gold nanoparticles to monitor the process of DNA strand displacement cascade in complex reaction environment. As the reaction proceeded, the color of solution would gradually turn red, which can be monitored through naked eyes. Interestingly,14 Single-base mismatched strands can be distinguished by naked eyes. It should be noted that the system functions well in complex mixture, fetal bovine serum. The ability of detecting DNA strand or single-base mismatch kept the same in complex mixture. The proposed method greatly widen the application of DNA strand displacement cascade.3. In order to meet the requirement of clinical diagnosis, high accuracy and low sample consumption, we configured a binary assay with sensitivity, selectivity, and simplicity. The proposed strategy can realize simultaneously detection of miRNA and telomerase. The oligonucleotide and protein detections were united via the sequence designing and the introduction of a nicking enzyme, Nt.CviPII. In addition, the GO was used to decrease background, and Nt.CviPII was also employed to trigger signal enhancement. More importantly, nearly 300 urine samples from bladder cancers and non-bladder cancers were used to test the single biomarker assay and dual biomarkers assay. The results demonstrated that, in comparison with single biomarker assay, the dual biomarkers assay had higher specificity. The proposed method gave a new thought for early diagnosis of cancer.4. In order to realize miRNA detection with high speed and sensitivity, we developed a one-pot analytic method based on the dual-cycle isothermal amplification. By designing a functional molecular probe, and employing the nicking enzyme, exonuclease, and polymerase, a quadratic amplification for miRNA detection was achieved. The proposed method exhibited ultra-high sensitivity with a detection limit of 10 fM at 37 ? and 1 aM at 4 ? that corresponds to 9 strands of miRNA in a 15 ?L sample. The most important was that we had demonstrated its potential application in clinical research with great sensitivity and selectivity to crude extractions from MCF-7, PC3 cell lines, and even patient tissues. |
PDF Full Text Request