| One of the most important aspects to cancer treatment is the early and accurate diagnosis of the disease. Early diagnosis enables current treatments to much more effective and leads to greatly improved survival rates. In an effort to realize this, I developed two diagnostic assays based on aptamer conjugated nanoparticles. Aptamers are single stranded oligonucleotide chains that forms a three dimensional structure that can bind with high affinity and specificity to a targeted molecule. We developed a novel cell-based aptamer selection strategy called cell-SELEX to produce a group of aptamers for the specific recognition of individual cells without prior knowledge of the biomarkers on the cells. The cell-SELEX process uses whole cells as targets to select aptamers that can distinguish target from control cells. Once selected, the aptamer can be chemically synthesized and easily functionalized for bioconjugation to different nanomaterials, fluorophores, or therapeutic agents.;The first assay is based on two types of silica nanoparticles, one where a fluorescent dye has been doped inside the particle while the other has a magnetic nanoparticle doped inside the silica. The aptamers allow the nanoparticles to bind to the cell surface. After the application of a magnetic field, the magnetic nanoparticles and anything bound to them are immobilized and the 14 unbound materials can be washed away. This allows for the selective enrichment and detection of the target cells.;The second assay uses gold nanoparticles instead of silica-based nanoparticles. The gold nanoparticles are in close proximity and their surface plasmons can interact. The interaction results in a red shift of the absorption of the particles and an increase in the extinction coefficient of the particles. Using these properties of the gold nanoparticles with the selectivity and affinity of the aptamers results in colorimetric assay where a solution containing the target cells changes color.;However, detection is only one important criterion for cancer treatment. A better and more complete understanding of the disease at a biomolecular level is critical to developing more effective treatments. By microinjecting multiple molecular beacons with different fluorophores inside of single breast carcinoma cells and monitoring with advanced fluorescent microscopy, the expression of multiple genes can be simultaneously monitored inside of single living cells. The mRNA for B-actin, Manganese Superoxide Dismutase, and a control sequence were detected simultaneously using this method. Using ratiometric analysis as a basis for the measurements allows the different gene expression levels to be compared from cell to cell. Not only does this allow differentiation of individual mRNA expression levels between multiple single cells, but it also allows for mRNA expression trend analysis at the single cell level. This can be further coupled with in vivo ion monitoring experiments to allow a more complete understanding of cellular processes. |
