In this proposal we aim to develop new ?particle-drop? technology to analyze single-molecules and single-cells without complex microfluidic devices and benchtop pressure-control systems, democratizing the latest tools to explore biology at its ultimate limits. There has been increasing excitement in life science research and diagnostic development in regards to ?digital assays? that assay biology at the limit of a single molecule or cell. Such assays, including digital polymerase chain reaction (PCR), digital immunoassays, and more recently barcoded single- cell RNA-seq approaches, rely on fractionating a large sample volume into small enough volumes that cannot chemically communicate, such that a limit is reached where each volume contains either 0 or 1 molecule (or cell) of interest, following Poisson statistics. These approaches often make use of microfluidics to create an emulsion of monodisperse droplets or an array of wells or chambers that break up a volume. However, the use of microfluidics has resulted in costly benchtop instruments and chips that have limited the ability of the average lab to perform digital assays or single-cell RNA-seq. We propose an entirely new approach to break up an aqueous volume into monodisperse drops that can be performed at the benchtop using standard laboratory equipment. We employ a new fabrication approach, Optical Transient Liquid Molding, to create 3D-shaped microparticles with Janus properties that stabilize an aqueous drop in their hydrophilic patterned interiors, while also comprising a hydrophobic exterior which allows suspension in a hydrophobic continuous phase. These ?drop-carrier particles? when mixed with an aqueous solution with analyte and then oil form a monodisperse emulsion of ?particle-drops?. These particle-drops stabilize the emulsion while allowing reactions to occur within and at the particle surface, enabling for some digital assays which require separate beads to be introduced. We propose to develop the manufacturing and emulsification methods for particle-drops along with exploring low- cost optical readout approaches. We will also identify conditions to perform a digital nucleic acid amplification reaction within particle-drops and characterize the performance in comparison to standard microfluidic digital nucleic acid amplification approaches. These demonstrations would provide a clear impetus to apply particle- drops to other digital assays, such as single-cell RNA-seq and digital-ELISA, leading to cost-effective solutions to expand and democratize these assays, and ultimately accelerate biological discovery.
This proposal aims to develop new ?particle-drop? technology to analyze single molecules and single cells using standard research equipment, expanding the number of researchers able to perform detailed biological analyses and enabling simplified diagnostic tests that reach the ultimate limits of biology.
