A New Biosensor Based On Nanoparticles And Application In Assay Of P53Protein

In recent years, the mortality rate of cancer, one of the most threatening diseases of the human being, is becoming increasingly high. The accurate and sensitive detection of trace tumor marker is capable of diagnosing cancer, prevention, treatment and clinical observation. Thereinto, p53, a tumor suppressor protein and a transcription factor, plays an important role in cell growth control, DNA repair, and programmed cell death. In more than50%of cancer cases, the p53gene has been found to be mutated, therefore, the detection of p53protein is the essential need.This thesis reported new methods based on nanotechnology, bio bar code and RCA, which were used to detect wide-type p53and taotal p53protein by QCM and SERS. This thesis mainly consists of the following three parts:1We designed a novel method to detect wide-type p53protein with a quartz crystal microbalance (QCM) sensor based on the amplification of gold nanoparticles (Au NPs). To achieve the QCM assay, gold crystal chip (Au chip) was immobilized with primer antibody (Ab1) of p53proteins through the linking of cysteamine. Then the wide-type p53protein and consensus double-stranded (ds-) DNA modified with Au NPs were injected sequentially. According to the high affinity of the consensus ds-DNA to the wild-type p53protein and the antibody to p53protein, a stable framework was fabricated. The detection limit of this assay for wide-type p53protein determined is0.1pg.mL-1. The method holds great promise in the biochemical assay for the early diagnosis of cancers, which has high sensitivity and excellent selectivity.2Based on the amplification of quartz crystal microbalance (QCM) signals by gold nanoparticles, a novel and sensitive assay for the total p53protein detection has been developed by using nanoparticles, immunogold and sandwich-type techniques. To achieve the QCM assay, gold crystal chip (Au chip) was immobilized with primer antibody (Ab1) of p53proteins through the linking of cysteamine. Then the total p53 protein and Ab2modified with Au NPs were injected sequentially. According to the antibody to p53, a stable "sandwich" framework was fabricated. The detection limit of this assay for total p53protein determined is0.1pg.mL-1. Combined with the first part, the difference in the QCM signals between the total p53and wide-type p53protein reveals the extent of p53mutation, which is indicative of cancer development. Thus, the QCM sensor could potentially serve as an attractive technique for rapid, sensitive, reliable, and label-free cancer diagnoses.3The detection of wide-type p53protein is achieved by surface enhanced Raman spectroscopy (SERS), based on rolling circle amplification (RCA) and bio bar code signal amplification technology. To achieve the SERS assay, the primer DNA modified on JS, was developped into a long-chain DNA at the presence of rolling circle amplification. The long-chain DNA can hybridize with bio bar code to amplify the SERS signals. According to the high affinity of the consensus ds-DNA to the wild-type p53protein and the antibody to p53, all these parts were immobilized on the Gold-CS. At last, we can detect wild-type p53by SERS.