Determining nanoparticle size distribution and concentration for bioanalytical applications

该提案要求为纳米颗粒跟踪分析（NTA）仪器提供资金，该仪器能够测量胶体纳米颗粒（NP）的大小，数量分布和数量浓度。虽然这种类型的仪器有许多应用，但我们特别感兴趣的是获得有关NP的信息，这些NP设计用于对单个细胞上的生物标志物进行高通量多参数分析。生物标志物是蛋白质、多糖、核酸和其他生物大分子，它们可以识别细胞类型并指示其生理状态。在逐个细胞的基础上分析生物标志物表达是细胞生物学中的基本兴趣，并且用作基于细胞的疾病如癌症的重要诊断。Winnik小组和步行者小组都在开发NP试剂，使人们能够克服流式细胞术的局限性，流式细胞术是一种广泛用于研究实验室和临床的技术。克服这些限制可能对单细胞生物学和医学产生变革性影响。 步行者小组使用金纳米颗粒来携带通过其拉曼信号检测的拉曼活性染料。Winnik小组使用镧系元素NPs与Fluidigm Canada在Markham ON商业化的质量细胞计数新技术相结合。对于这两个小组来说，挑战是学习如何以使我们能够靶向特定生物标志物的方式用脂质包覆NPs，更重要的是，抑制与细胞的非特异性相互作用。为了实现这一目标，我们必须能够以最先进的分辨率和精度表征脂质涂层前后溶液中的NP。我们必须克服动态光散射在检测和量化宽粒度分布方面的局限性，这样才能避免它们，并且我们必须能够在发生时检测和量化小范围的颗粒聚集。这些测量必须由两个实验室的学生每天进行。 NTA仪器不仅提供这些功能，还允许同时跟踪溶液中颗粒的荧光。为了将抗体附着到我们的脂质包被的NP上，我们产生了PEG化的脂质胶束，抗体附着在PEG链的远端。脂质-PEG-抗体缀合物通过脂质插入从胶束转移到脂质包被的NP。如果我们使用荧光染料标记的抗体，NTA的荧光能力将使我们能够监测和优化这一关键步骤。 基于纳米颗粒的标记方案的开发是一个竞争激烈的领域。如果没有对我们的颗粒进行适当的表征，就不可能将它们的性能与其他地方制造的颗粒进行比较，这些颗粒具有良好的特性。这会影响我们的技术是否会被其他实验室或行业采用，因为定量比较是必不可少的。

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.