Determining nanoparticle size distribution and concentration for bioanalytical applications

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This proposal requests funds for a Nanoparticle Tracking Analysis (NTA) instrument that enables one to measure the size, the number distribution, and number concentration of colloidal nanoparticles (NPs). While there are many applications of this type of instrument, we are particularly interested in obtaining this information about NPs designed for high throughput multiparameter analysis of biomarkers on individual cells. Biomarkers are proteins, polysaccharides, nucleic acids and other biomacromolecules that identify the type of cell and indicate its physiological state. The analysis of biomarker expression on a cell-by-cell basis is of fundamental interest in cell biology and serves as an important diagnostic for cell based diseases such as cancer. Both the Winnik group and the Walker group are developing NP reagents that allow one to overcome the limitations of flow cytometry, a technique widely used in both the research laboratory and in the clinic. Overcoming these limitations could have a transformative impact on both single-cell biology and medicine. The Walker group uses gold NPs to carry Raman active dyes detected by their Raman signal. The Winnik group uses lanthanide NPs in conjunction with the new technique of mass cytometry commercialized by Fluidigm Canada in Markham ON. For both groups, the challenge is learning how to coat the NPs with lipids in a manner that enables us to target specific biomarkers, and more important, to suppress non-specific interaction with cells. To accomplish this goal, we have to be able to characterize the NPs in solution before and after lipid coating with state-of-the-art resolution and precision. We have to overcome the limitations of dynamic light scattering in detecting and quantifying broad particle size distributions where they occur, so they can be avoided, and we have to be able to detect and quantify small extents of particle aggregation when it happens. These measurements have to be carried out on a daily basis by students in both labs. The NTA instrument not only provides these capabilities, it also allows one to track simultaneously fluorescence from particles in solution. To attach antibodies to our lipid-coated NPs, we generate PEGylated-lipid micelles to which antibodies are attached at the distal end of the PEG chains. The lipid-PEG-antibody conjugates are transferred from the micelles to the lipid coated NPs by lipid insertion. If we use antibodies labeled with fluorescent dyes, the fluorescence capabilities of the NTA will allow us to monitor and optimize this key step. The development of nanoparticle based labeling protocols is a competitive field. Without proper characterization of our particles, it is not possible to compare their performance with particles made elsewhere, which are well characterized. This affects whether our technology will be taken up, in other labs or by industry, where quantitative comparisons are essential.