Development of a Nearly Universal Assay Kit for Cell Separations

This Phase I proposal will result in an assay kit called DensiSep for multiplexed separations of cells or proteins For decades scientists have used density to isolate purify and extract target cells Nature however presents a puzzle biological systems always conform to a bell curve The DensiSep kit introduces an entirely new form of biological separations that has the potential to eliminate the bell curve of biology Our kit is based on the concept of magnetic beads but expands the utility of such assays through novel multiplexing capabilities which reduces assay time and cost by up to and respectively The separation of individual cells from a complex mixture is a ubiquitous challenge in biology In many cases researchers are tasked with isolating targets in concentrations as low as one cell per mL of solution whereas there are approximately x red blood cells per mL of blood Often it is advantageous to isolate multiple targets simultaneously so called multiplexing The relatively recent rise of magnetic bead based assays reflects their value as a powerful tool to rapidly and easily isolate cells from complex mixtures such as blood Multiplexing these assays however requires either iterative separations or secondary labeling steps followed by flow cytometry Both methods are expensive and time consuming DensiSep combines magnetic polymer beads functionalized with linker groups to attach any antibody DensiSep beads and aqueous multiphase systems AMPS biocompatible mixtures of buffered polymers and salts in water that spontaneously separate into phases having sharp steps in density Three sets of DensiSep beads will be selected to have a specific density such that upon addition to the DensiSep AMPS and minutes of centrifugation each set of beads will collect at a unique interface between the phases of the AMPS irrespective of the density of the cells being separated DensiSep will enable a researcher to run multiple separations in parallel using a single centrifugation followed by an easy bead removal method via a pipette or with the aid of a magnet a process that can be easily automated with robotic pipettes or by building extraction ports into the tubes The reduction in cell manipulation and the ease of visualization provided by the DensiSep will enable higher purity separations with better yields compared to current technology Our Phase I proposal seeks to develop a rapid multiplexed biological assay kit to separate and purify nearly any cell from a complex mixture We believe that our kit will enable researchers to spend less time on preparations and more time generating meaningful data The ability to inexpensively multiplex using our kit will potentially accelerate the innovation of new medications and make existing medications more affordable to patients who require them