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Publication Number
US-11131674-B2
Publication Date
2021-09-28
Expiration Date
Abstract
Microarray compositions suitable for analysis by one or several spectrographic methods are disclosed. In an embodiment, a microarray composition includes a three-dimensional solid support and a plurality of reactive microbeads positioned on the solid support in spatially distinct and addressable locations.
Core Innovation
A composite microarray composition includes a three-dimensional microwell solid support with microwells and spatially addressable reactive microbeads positioned in the microwells. Reactive microbeads include reactive sites that release analytes into a microwell, where the released analytes localize into compact spots within the microwell. The localized analyte spots are configured for spectrographic analysis, particularly mass spectrometry.
The invention defines constraints for bead and microwell design, including bead and microwell size ratios, bead accessibility, bead-to-analytical-site spacing, and microwell spacing matched to laser probing beam focusing and instrument spatial resolution. A three-dimensional microwell array plate format, gasketed microwell plates, and related microarray materials and configurations promote localization of cells and analytes within defined microwells, and compatible surface layer coatings include conductive or charge-dissipative conductive layers such as ITO.
Microarray fabrication and decoding approaches are disclosed, including positional encoding and encoder-less or random bead arrays with optical or mass tag decoding. Downstream analysis includes repeated and kinetic measurements, mass spectrometry imaging, optical and MS imaging, and automated high-throughput whole-cell mass spectrometry for very large cell populations. The disclosed workflows extend to nucleic-acid sequencing concepts and magnetic bead workflows, and further include live-cell and composite microbead-microwell cell microarrays for whole-cell MALDI MS, including single-cell and rare-cell detection, and difference mass spectrometry for detecting changes between cell populations.
Claims Coverage
The independent-claim families cover optical spot localization of analyte release within microwells and targeted mass spectrometry over the localized analyte spot, with spatial selectivity that suppresses bead-origin signals. Additional claim-supported features include automated high-throughput whole-cell mass spectrometry, microwell spacing matched to probing beam focusing and instrument spatial resolution, and difference mass spectrometry for comparing Sample and Reference spectra.
Optical spot localization of analyte release within microwells
Using an optical imaging instrument to acquire an optical image of a microarray containing a spot produced by eluting an analyte from a bead positioned inside a microwell into the microwell, and using a computer to analyze the optical image to identify an area of the microarray that contains the analyte spot or to determine localization of the analyte spot.
Targeted mass spectrometry over localized analyte spot
Positioning an ionization beam of a mass spectrometer directly over the analyte spot and acquiring mass spectrometric data from the microarray such that a mass spectrometry signal of the analyte is obtained from the analyte spot and not from the bead, or acquiring mass spectrometric data such that a mass spectrometry signal of the analyte is detected at the location of the analyte spot and not detected at a location of the bead.
Microwell spacing matched to probing beam focusing and instrument spatial resolution
Microwell array plate formats and microwell spacing are matched to laser probing beam focusing and instrument spatial resolution so that single-cell interrogation and imaging are performed without cross-talk.
Difference mass spectrometry comparison of Sample and Reference spectra
Comparing Sample and Reference spectra and interpreting difference spectral features to detect apoptosis- and treatment-induced spectral changes.
The claim set is directed to spatially selective mass spectrometry of microarray spots by first optically determining analyte-spot localization from analyte released inside microwells, and then acquiring mass spectrometric data at the localized analyte-spot location so that the analyte signal is obtained from the spot rather than from the bead.
Stated Advantages
Obtaining a mass spectrometry signal of the analyte from the analyte spot and not from the bead.
Detecting a mass spectrometry signal of the analyte at the location of the analyte spot and not at a location of the bead.
Enables automated high-throughput whole-cell mass spectrometry (WCMS) for rapid analysis of very large cell populations.
Supports single-cell interrogation and mass spectrometric imaging with reduced cross-talk through microwell spacing matched to probing beam focusing and instrument spatial resolution.
Enables reproducible quantitative mass spectrometry by addressing peak position and intensity variation.
Detects apoptosis- and treatment-induced spectral changes using difference mass spectrometry.
Enables multiplexed fluorescence and mass spectrometry readouts.
Documented Applications
Repeated and kinetic mass spectrometry measurements on microarrays.
2D and 3D optical and mass spectrometry imaging.
Nucleic-acid sequencing concepts on bead arrays.
Magnetic bead workflows using the disclosed microarray concepts.
Live-cell composite microbead-microwell cell microarrays for whole-cell MALDI mass spectrometry, including single-cell and rare-cell detection.
Difference mass spectrometry to detect changes between cell populations.
MALDI imaging of localized bead/analyte features and associated spatial interrogation using mass spectrometric imaging in a microwell array format.
Apoptosis detection and analysis of treatment-induced spectral changes using difference mass spectrometry.
Multiplexed fluorescence and mass spectrometry readouts, including fluorescence readouts combined with mass readout measurements.
Release of active agents from microbead-microwell composite arrays in the context of downstream whole-cell mass spectrometry analysis.
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