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The Detection of p53 Based on Gold Nanoparticle Probes

Author: HuangYunYan
Tutor: ZhaoLinChuan；JiaChunPing
School: Suzhou University
Course: Biochemistry and Molecular Biology
Keywords: Gold nanoparticle gold nanoparticle probes micromagnetic particle probes protein chip
CLC: R730.4
Type: Master's thesis
Year: 2010
Downloads: 134
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Abstract


Protein biomarker detection has become increasingly important for cancer diagnosis and monitoring as tremendous advances. The p53 protein is the important tumor marker. So far, p53 antibody in serum is usually detected occupying the immunohistochemical method (IHC) and the enzyme linked immunosorbent assay (ELISA). As the concentration of p53 is very trace, above two types can not be used for the detection of p53. In our study, capture antibody functionalized magnetic microparticle (MMP) were used as supporting substrates for determination of p53 protein. Then, p53 protein in serum was detected by gold nanoparticle (Au NP) probe coated with p53 detecting antibody and horseradish peroxidase (HRP). Moreover, the combination of Au NP probe and protein chip can be developed into a set of detection analysis system about sample preparation, detection of biochemical reactions, in which can simultaneously detect many proteins of one sample in a chip surface.1 The characterization of MMP fabricated by p53 capture antibodyThe fluorescent scan revealed that the antibody is successfully marked to the MMP and the antibody is active. Furthermore, the transmission electron microscopy (TEM) scan showed that MMP probes combined p53 capture antibody were of generally spherical appearance, and most had well distributions and magnetic response.2 The preparation of Au NPs probesAu NPs were prepared through sodium citrate reduction of chlorouric acid. From the TEM, the Au NPs had uniform, good morphology and dispersed evenly, the average diameter was 10 nm. After coating with p53 capture antibody and HRP, a shadow around the Au NPs probe was clearly observed compared to bare Au NPs. Furthermore, the peak of Au NPs absorption shifted from 520 to 524 nm (IgG-Au-HRP) and 526 nm (IgG-Au-DNA-HRP), respectively. This red shift was due to the coating with antibody and HRP.3 Optimization for preparation conditions of Au NPs probesFrom the optimum pH of solution and protein stability experiment, the optimum pH was 9.0. Besides, the optimal p53/Au NPs ratio is 1/400 (v/v). Experimental parameters involved in preparation of IgG-Au-HRP and IgG-Au-DNA-HRP probes were optimized. The ideal ratio of HRP/detecting antibody p53 was 10/1(IgG-Au-HRP), and the ratio of DNA/detecting antibody p53 was 2.5/1(IgG-Au-DNA-HRP). Based on quantification of HRP immobilized on Au NP probe, HRP/IgG-Au ratio was 11 while HRP/IgG-Au-DNA was 20.4 The p53 protein detection using Au NP probes and MMP probesThe capture antibody is immobilized onto the MMP. The detector antibody is immobilized on the Au NP. The detected antigen is sandwiched by capture antibody and detector antibody mobilized on MMP and Au NP. The assay can be finished within 2 h. The detection sensitivity of this method based IgG-Au-DNA-HRP and IgG-Au-HRP probes was 0.03 ng/mL and 0.055 ng/mL, respectively, which were more sensitive than conventional enzyme linked immuncsorbent assay.5 The p53, CEA and NSE detection using Au NP probes and protein chipCapture antibodies of p53、CEA and NSE were immobilized onto the surface of the slide, detector antibodies were immobilized on the Au NP , the presence of target proteins (p53、CEA and NSE ) caused the formation of the sandwich structures (protein chip–target proteins–AuNP probes) through the interaction between antigen and its antibody. Then, the singal of Au NP was subsequently amplified by silver staining enhancement solution. The result showed that it could be used in combined detection of tumor proteins.Our results show that the detection of p53 protein based on gold nanoparticle probes and magnetic microparticle probes has a good reproducibility and high detection sensitivity. This method can be used to detect protein markers of tumors, nervous system or other diseases for early diagnostics. Moreover, multiplexed protein detection method based on combination of gold nanoparticle probe and protein chip affords a new detect method about tumor protein combined detection.

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CLC: > Medicine, health > Oncology > General issues > Tumor diagnostics
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