High resolution systems, kits, apparatus, and methods for high throughput microbiology applications
Inventors
Blainey, Paul C. • Seely, Michael W. • Stocker, Roman • Zengler, Karsten • Conradson, Scott • Christey, Peter • Hallock, Alexander
Assignees
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Publication Number
US-11136621-B2
Publication Date
2021-10-05
Expiration Date
Abstract
A kit includes a microfabricated device having a top surface defining an array of microwells for receiving a sample comprising at least one cell, and a membrane for applying on the top surface of the microfabricated device to retain the at least one cell in at least one microwell of the array of microwells after the sample is loaded on the microfabricated device.
Core Innovation
The invention relates to microfabricated microwell chip systems and kits for receiving a sample comprising at least one cell and for cultivating, screening, isolating, and analyzing microbial/biological entities while maintaining well-specific locational integrity. The microfabricated device includes a top surface defining an array of microwells having a surface density of at least 500 microwells per cm^2, enabling high-density, high-throughput partitioning at the level of individual microwells.
To retain the at least one cell in the microwells after the sample is loaded, the device further includes a membrane applied to the top surface. The membrane is characterized by retained-cell functionality and can include gas-permeable and impermeable-for-liquid behaviors, or other semi-permeable or filter-like characteristics, thereby supporting controlled sampling and downstream workflows while keeping cells associated with specific microwells.
A further core aspect is the use of unique nucleic-acid location tags that associate nucleic-acid molecules with specific microwell positions. The tags include both a target-specific nucleotide sequence and a location-specific nucleotide sequence, and the workflow supports indexing and mapping between microwell location and nucleic-acid content for digital counting of species abundance.
Claims Coverage
The independent claim requires a kit with two principal elements: a microfabricated device with a high-density array of microwells on a top surface, and a membrane applied on the top surface to retain loaded cells in the microwells. The inventive features further refine the microwell array density and size constraints, specify membrane permeability behavior, and add nucleic-acid unique tags that encode both target recognition and well location for well-specific identification and indexing.
High-density microwell array microfabricated device
A microfabricated device for receiving a sample comprising at least one cell, the microfabricated device having a top surface defining an array of microwells having a surface density of at least 500 microwells per cm^2.
Membrane for retaining loaded cells in microwells
A membrane for applying on the top surface of the microfabricated device to retain the at least one cell in at least one microwell of the array of microwells after the sample is loaded.
Higher microwell surface density on the top surface
An array of microwells whose top-surface area has a surface density of at least 750 microwells per cm^2.
Microwell diameter range
Microwells having diameters in the range of about 25 μm to about 500 μm.
Gas-permeable and otherwise impermeable membrane
A membrane that is permeable only to gas and impermeable otherwise.
Unique nucleic-acid tags with target-specific and location-specific sequences
At least some microwells having unique nucleic-acid tags containing both a target-specific nucleotide sequence for annealing to a target nucleic acid fragment and a location-specific nucleotide sequence for identifying the microwell’s location on the device.
Forward and reverse PCR primer-containing unique tags
A first unique tag with a target-specific nucleotide sequence containing a forward polymerase chain reaction (PCR) primer and a second unique tag with a target-specific nucleotide sequence containing a reverse PCR primer.
Overall, the claims cover a kit architecture combining a microfabricated high-density microwell array with a cell-retaining membrane, with refinements that specify array and microwell dimensions, membrane permeability behavior, and well-specific nucleic-acid unique tags that include both target-specific and location-specific sequences, including forward- and reverse-PCR primer-containing tag constructs.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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