Compositions and methods for cell-like calibration particles

Inventors

Kim, Jeffrey • Nguyen, Anh Tuan • Miller, Brandon

Assignees

Slingshot Biosciences Inc

Abstract

A method includes calibrating a cytometric device for analysis of a target cell, by inserting, into the cytometric device, a hydrogel particle. The hydrogel particle has at least one of a background fluorescent property or a spectral property that is substantially similar to the at least one of a background fluorescent property or a spectral property of the target cell. The method also includes measuring at least one property of the hydrogel particle using the cytometric device.

Core Innovation

The disclosed invention relates to calibrating a cytometric device for analysis of a target cell using cell-like hydrogel calibration particles. The calibration particle has a background fluorescent property that is substantially similar to the corresponding background fluorescent property of the target cell, and the fluorescent property of the hydrogel particle is measured using the cytometric device to calibrate the device. The hydrogel particle is configured to bind to an antibody that is bound to a conjugated fluorophore, thereby enabling background fluorescent property matching rather than using substantially different background fluorescent properties.

The calibration particle is engineered so that its background fluorescent property and related spectral profile are matched for cytometric performance. The background fluorescent property includes components such as inter-laser delay, fluorescence response, and sort timing, and the invention includes calibration and compensation/spectral unmixing workflows based on the measured hydrogel particle fluorescence. The hydrogel approach is contrasted with polystyrene beads as calibration materials with different behavior.

The hydrogel particles are described as synthetic hydrogels with tunable optical properties. Tunable optical behavior is achieved through engineered composition and crosslinking density, including independent modulation of autofluorescence versus FSC/SSC, and optional inclusion of nanoparticle additives and chemical functionalization. The document further describes hydrogel particles as optionally monodisperse particles produced by droplet polymerization and microfluidics, and describes binding surrogates that include an intercalating nucleic acid labeling reagent or an amine-reactive labeling reagent for the calibration particle.

Claims Coverage

The document includes two independent claims covering methods of calibrating a cytometric device for analysis of a target cell using hydrogel particles with substantially similar background fluorescent properties and binding capabilities to labeling reagents or fluorophore-conjugated antibodies. Across the dependent claims, the hydrogel particle is refined with quantitative physical/optical parameters and specified compositions and background fluorescent property components (e.g., inter-laser delay, fluorescence response, sort timing).

Both independent claims center on calibrating a cytometric device by measuring a hydrogel particle whose background fluorescent property is substantially similar to the target cell’s corresponding background fluorescent property. The independent claims further require hydrogel binding capability, either to an antibody bound to a conjugated fluorophore or to at least one of an intercalating nucleic acid labeling reagent and an amine-reactive labeling reagent, while dependent claims refine the matched background fluorescent property components and particle properties.

Stated Advantages

Documented Applications