Compositions and methods for disease diagnosis using single cell analysis
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Publication Number
US-11959910-B2
Publication Date
2024-04-16
Expiration Date
Abstract
Certain embodiments of the invention are directed to evaluating and identifying cells by recording and interpreting a time-dependent signal produced by unique cell respiration and permeability attributes of isolated viable cells.
Core Innovation
The invention provides a method for identifying a cell by using at least two different reporters having identical emission spectra. A sample containing cells is contacted with the reporters to form a sample mixture, and the mixture is partitioned into a plurality of volume partitions containing one cell or one cellular aggregate.
Each volume partition is incubated for a period of time at a specified temperature or series of temperatures while optical characteristics of the volume partitions are monitored during the incubation time. Based on the optical characteristics over time, waveforms are constructed for the volume partitions.
The method identifies the cell in a volume partition based on the waveform constructed for the volume partition. In refinements, the waveforms can be compared to a library of stored waveforms, and the partitioning can be implemented as droplets dispersed in an immiscible fluid with partitioning using Laplace pressure gradients and/or shear stress. The monitoring can be performed using a camera, and the volume partitions can be arranged as a two-dimensional static array.
Claims Coverage
The independent claim is for identifying a cell using time-based optical waveforms constructed from optical characteristics of individually partitioned cells, where the partitioned volume partitions are produced after contacting a sample with at least two different reporters having identical emission spectra. Dependent claims narrow how reporters are selected, how partitioning is achieved and formatted, and how optical monitoring and waveform-based identification are performed; the inventive features across the claim set are primarily waveform-based identification and controlled incubation with monitored optical characteristics over time.
Reporter mixture with identical emission spectra
Contacting a sample containing cells with at least two different reporters having identical emission spectra, thereby forming a sample mixture.
Partitioning into volume partitions with one cell or one cellular aggregate
Partitioning the sample mixture into a plurality of volume partitions containing one cell or one cellular aggregate.
Incubation at specified temperature or series of temperatures
Incubating the volume partitions for a period of time at a specified temperature or series of temperatures.
Time-dependent monitoring of optical characteristics
Monitoring optical characteristics of the volume partitions during the incubation time.
Waveform construction from optical characteristics over time
Constructing waveforms for the volume partitions based on the optical characteristics over time.
Waveform-based identification of the cell
Identifying the cell in the volume partition based on the waveform constructed for the volume partition.
Waveform comparison to stored waveform library
Identifying the cell by comparing the waveform to a library of stored waveforms.
Selection of reporters from an explicit group
The at least two different reporters are selected from a specified group of fluorescent, photooxidizable, redox-responsive, membrane-localizing, energy-transfer, pH-indicating, and dye-based reporter options and derivatives.
Droplet partitioning in an immiscible fluid
The volume partitions are droplets dispersed in an immiscible fluid.
Partitioning using Laplace pressure gradients
The sample is further partitioned using Laplace pressure gradients.
Camera-based monitoring of optical characteristics
Monitoring the optical characteristics of the volume partitions is performed using a camera.
Overall claim coverage centers on constructing and using time-dependent waveforms derived from optical characteristics monitored during incubation of individual cells or cellular aggregates in volume partitions, where reporter mixing uses at least two different reporters having identical emission spectra. Dependent claims further specify reporter classes, droplet/immiscible partitioning and partitioning mechanisms, camera-based monitoring, and optionally comparison to a stored waveform library.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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