Fluorescent nanofoldamers for multiplex flow cytometry

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PROJECT SUMMARY / ABSTRACT Polychromatic flow cytometry (FC) is a vital analytical technique used in immunology, basic research, and clinical medicine. FC is an indispensable tool for understanding disease development at the cellular and subcellular levels and for monitoring the effectiveness of new immunotherapies by identifying key cellular subpopulations using multiple biomarkers within a panel. It also has a critical role in growing clinical applications including development of vaccines for infectious diseases, characterization of immunological cells and responses related to transplantation and treatment of graft-versus-host disease, and development of vaccines and immune therapies to prevent and treat HIV infections. Increasing the number of spectrally distinct fluorophores will give researchers greater flexibility and give clinicians a higher level of accuracy in the diagnosis and management of conditions where the size of the sample used for diagnosis is limited or cell number is low, including those derived from fine needle aspiration, laparoscopy, core biopsy, and cerebrospinal fluid. Improvements in optics and lasers have driven the recent introduction of new fluorophores for FC. However, most of these possess broad and often overlapping emission profiles, much like traditional fluorophores. Overlapping spectra can be resolved by the combined use of bandpass filters and mathematical compensation (overlap subtraction); however, such compensation increases experiment error, reduces sensitivity, and limits multiplexing. Thus, even with a prototype 64-color instrument, leading investigators have only been able to implement a 40-parameter panel – advances in reader technology have outstripped available capabilities in fluorophores. NIRvana Sciences is developing two families of tetrapyrrole fluorophores for use in FC. These fluorophores are excellent candidates for use in polychromatic FC due to their ultra-violet/violet excitation, long Stoke’s shifts into the red and near-infrared spectrums, and very narrow emissions. In the current project, NIRvana seeks to complete development of a new technology, referred to as FluoroPods, that allows us to fully utilize our unique portfolio of dyes. This polymer-based system enables the use of dyes that possess excellent spectral properties but lack sufficient water solubility and/or are heavily quenched in biological systems. Upon completion of the project, NIRvana Sciences will have created a palette of exceptionally bright FluoroPods that will form the basis of a platform of 22+ new colors using only the 355 and 405 nm lasers.