Preliminary Exploration Of The Application Of Nanomaterials In Breast Cancer Molecular Classification

Breast cancer is one of the most common cancers among women in China, as well as in the majority of developed countries. Clinically, breast cancer is a remarkably heterogeneous disease with the presence of multiple molecular subtypes. Breast cancer molecular classification is based on molecular biomarkers, which paly an important role in general prognosis assessment and the prediction of therapy response of different subtypes. So measuring and looking into the properties of breast cancer biomarkers is crucial to future exploration of the cancer classification. Nanomaterials, which are measured between 1-1000 nm, are being actively developed for breast cancer imaging, biomolecular profiling of breast cancer biomarkers, and targeted drug delivery. They can be coated with antibodies, antigens, aptamers, DNA, or receptors overexpressed on the surface of cancer cells or specific to cancer biomarkers, and then used to detect or analyze the properties of breast cancer biomarkers due to their excellent properties (eg:catalytic property, small size effect, optical property and so on). In this thesis, we prepared several kinds of nanomaterials coated with different biomolecules, and explored their application in breast cancer molecular classification based on biomarkers. The main results are summarized as follow:1. Aptamer-Ag NCs and fluorophore-tagged aptamers used in the classification of breast cancer cellsFristly, MUC1-Ag NCs was prepared by one-pot process, using MUC1 aptamer as the template. The MUC1-Ag NCs can recognize the human breast cancer cell line MCF-7 which expresses the MUC1 protein at high level specifically. While considering the inherent fluorescence properties of the aptamer stabilized Ag NCs, the apamer-Ag NCs were hard to be used in distinguishing several different breast cancer lines simultaneously. Hence, we designed the (Biotin-aptamer)-(Avidin-FITC) system to investigate the binding properties between aptamers (S6, MUC1 and SYL3C) and breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231). Finally, two aptamers (SYL3C and S6) were choosed and labled with fluorophers (Cy3 and Cy5), respectively. They were incubated with three kinds of breast cancer cells simultaneously. The confocal images exhibited a significant differences of the merge colors for the different cancer cell lines. Therefore wecan recognize the breast cancer cell lines visually from the merge color for each cell line.2. Research on the anti-PR labeled fluorescent nanoparticle targetting for MCF-7 cell nucleiFluorescet carbon dots (CDs) with plentiful surface-covered amino groups were prepared by the solvothermal method.Meanwhile, SiO2@FITC-COOH nanoparticles were synthesized by the micro-emulsion method. The two fluorescent nanoparticles thus can be readily conjugated with anti-PR via traditional EDC/NHS cross-linking reaction, repectively. Then the resultant biofunctional and fluorescent CDs-(anti-PR) or SiO2@FITC-(anti-PR) were incubated with MCF-7 cells in different conditions. The following conclusions can be reached:(1) when the MCF-7 cells were fixed by 4% paraformaldehyde and blocked for 40 min in PBS containing 4% BSA and 0.1% Triton X-100, CDs-(anti-PR) can label the nuclei specifically, while SiO2@FITC-(anti-PR) were distributed in the cytoplasm; (2) Neither CDs-(anti-PR) nor SiO2@FITC-(anti-PR) can label the nuclei when they were incubated with MCF-7 cells that remain alive.3. Electrochemical detection of breast cancer SNP by using Pd/GO nanocomposite labelingPalladium/graphene oxide (Pd/GO) nanocomposites were prepared on the substrate graphene oxide. With the multiple functional groups, the clean and well-dispersed Pd/GO nanocomposites were used to label DNA, which further acted as catalysts to reduce copper ions to metallic copper to enhance the signal (denoted as "Pd/GO label copper stain"). Based on this strategy, the electrochemical detection of a single-base mutation associated with the breast cancer SNP rs 14200542 is specially studied by employing differential pulse voltammetry (DPV). The analytical performance of this system shows that after 15 min of copper staining, there is a linear relationship between the peak current resulting from the oxidative dissolution of the copper deposit and the logarithm of the target DNA concentration in the range of 10?M to 1 pM. The limit of detection can reach 1 pM, which benefits from the high catalytic activity of the Pd/GO nanocomposites along with a low background level of "Pd/GO label copper stain". Therefore, this process can be expected to be a good alternative to silver staining used as a nanomaterial-based signal amplification strategy in the future.