Compositions and methods for cell-like calibration particles
Inventors
Kim, Jeffrey • Nguyen, Anh Tuan • Miller, Brandon
Assignees
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Publication Number
US-11726023-B2
Publication Date
2023-08-15
Expiration Date
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 invention describes synthetic hydrogel calibration particles engineered to have background fluorescent properties and spectral properties substantially similar to those of a target cell. The particles are configured to mimic optical scatter, bind labeling reagents such as intercalating nucleic acid or amine-reactive labeling reagents, or carry attached fluorophores. Particle composition and physical properties are independently tunable.
The hydrogel particles address the mismatch observed with polystyrene beads, which exhibit scatter and background fluorescence that do not match target cells. By providing cell-like fluorescence and autofluorescence across detector channels, these hydrogel particles facilitate cytometric calibration, fluorescence compensation, and spectral unmixing measurements.
Reported comparisons demonstrate that hydrogel particles show more cell-like fluorescence across detector channels than polystyrene beads. The disclosure describes a peak-to-peak spectral match between Alexa 700-stained hydrogel particles and lymphocytes, and contrasts internal scatter in translucent hydrogel particles with scatter in opaque polystyrene beads.
Claims Coverage
The claims define ten inventive features across ten independent claims (claims 1 and 20 through 28), directed to hydrogel particles having background fluorescent and spectral properties substantially similar to those of a target cell.
Hydrogel particle with a selected monomer
Claim 1 covers a composition comprising a hydrogel particle having a background fluorescent property and a spectral property substantially similar to those of a target cell, with the particle including a monomer selected from the stated group.
Modified hydrogel particle that binds labeling reagents
Claim 20 covers a composition comprising a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell, modified to bind at least one of an intercalating nucleic acid labeling reagent or an amine-reactive labeling reagent.
Hydrogel particle with refractive index greater than about 1.15
Claim 21 covers a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell and a refractive index greater than about 1.15.
Hydrogel particle with refractive index greater than about 1.3
Claim 22 covers a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell and a refractive index greater than about 1.3.
Hydrogel particle with refractive index greater than about 1.7
Claim 23 covers a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell and a refractive index greater than about 1.7.
Hydrogel particle including a polymer nanoparticle additive
Claim 24 covers a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell, including a polymer nanoparticle additive.
Hydrogel particle comprising a free amine group
Claim 25 covers a hydrogel particle comprising a free amine group and having background fluorescent and spectral properties substantially similar to those of a target cell.
Hydrogel particle comprising allylamine
Claim 26 covers a hydrogel particle comprising allylamine and having background fluorescent and spectral properties substantially similar to those of a target cell.
Hydrogel particle modified with a fluorophore or fluorochrome
Claim 27 covers a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell, modified by conjugating or attaching a fluorophore or a fluorochrome.
Hydrogel particle modified to match a target-cell fluorescence profile
Claim 28 covers a composition comprising a hydrogel particle with background fluorescent and spectral properties substantially similar to those of a target cell, modified to match the target cell's fluorescence profile.
The independent claims center on cell-like hydrogel-particle compositions and separately specify selected monomer compositions, binding to labeling reagents, refractive-index thresholds, a polymer nanoparticle additive, amine functional groups, fluorophore conjugation, or matching a target-cell fluorescence profile.
Stated Advantages
The hydrogel particles provide more cell-like fluorescence across detector channels than polystyrene beads.
The described spectral comparison reports a peak-to-peak match between Alexa 700-stained hydrogel particles and lymphocytes.
The particles address the mismatch between polystyrene-bead scatter and background fluorescence and those of target cells.
Documented Applications
Calibration of cytometers and cytometric devices using particles with target-cell-like fluorescent or spectral properties.
Fluorescence compensation measurements, including the use of modified particles configured to bind antibody–fluorophore conjugates or DNA-binding dyes.
Spectral unmixing measurements.
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