High resolution systems, kits, apparatus, and methods for screening microorganisms and other high throughput microbiology applications
Inventors
Hallock, Alexander • WEMMER, Kimberly
Assignees
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Publication Number
US-10865434-B2
Publication Date
2020-12-15
Expiration Date
Abstract
A microfabricated device defining a high density array of microwells is described for cultivating cells from a sample. The device may be incubated to grow a plurality of cells, which may be split into an analysis portion and a reserve portion. Methods are provided for screening a biological entity of interest using the microfabricated device, for example, for screening phosphate solubilizing bacteria or other bacteria producing acids.
Core Innovation
The invention provides microfabricated high-density microwell chip systems for screening and cultivation of microorganisms and other cells. A microfabricated device has a top surface defining an array of microwells, and the chip is incubated to grow cells from loaded cells within individual microwells while maintaining well identity.
At least one microwell is loaded with at least one cell from a sample, an indicator substance, and an amount of a nutrient. A membrane is applied to the microfabricated device to retain the at least one cell in the microwell while allowing incubation, so that the plurality of cells grows and the screen evaluates acid-influenced optical behavior at the microwell level.
Presence or absence of at least one biological entity of interest is determined based on optical properties of the microwell after incubation, where the optical property is influenced by acid produced during incubation and associated changes in the indicator substance. The document also describes time-course optical measurements, multilayer wells-within-wells, membranes for cell retention and nutrient diffusion, recovery or picking of wells using membranes, and well indexing using location-specific nucleic-acid tagging or barcoding with PCR/NGS-based identification including digital absolute abundance estimation.
Claims Coverage
The independent claims are clm-00001, clm-00017, and clm-00018, covering three inventive features that include microwell-array microfabricated devices, loading cells with indicator and nutrient, membrane retention, incubation to grow cells, and optical readouts tied to acid production to determine presence or absence.
Microwell-array microfabricated screening with acid-influenced optical evaluation
Using a microfabricated device with a top surface defining an array of microwells, loading at least one microwell with at least one cell from the sample, an indicator substance, and an amount of a nutrient, applying a membrane to retain the at least one cell, incubating to grow a plurality of cells, evaluating an optical property of the microwell after incubation, and determining a presence or absence of at least one biological entity of interest that has produced an acid influencing the optical property in the microwell during incubation based on the optical property.
Indicator-substance change after incubation to determine acid-producing biological entity presence
Loading at least one microwell of the array with at least one cell from the sample, an indicator substance, and an amount of a nutrient, applying a membrane to retain the at least one cell, incubating to grow a plurality of cells, and determining a presence or absence of at least one biological entity of interest produced an acid in the microwell during incubation based on a change of the indicator substance after incubation.
Depositing indicator per microwell and measuring acid-influenced optical property
Depositing an indicator substance into each of a plurality of microwells, loading the sample onto the microfabricated device so that at least one microwell receives at least one cell and an amount of a nutrient, applying a membrane to retain the received cell and nutrient in each of the plurality of microwells, incubating, and determining presence or absence of at least one biological entity of interest that has produced an acid in the microwell during incubation based on a measurement of an optical property of the microwell after incubation, wherein the optical property is influenced by the acid.
Across the independent claims, the core coverage centers on microfabricated microwell-array screening that retains cells with a membrane, incubates to grow plurality of cells, and determines presence or absence of an acid-producing biological entity of interest using indicator-related optical readouts.
Stated Advantages
Enables screening for presence or absence of at least one biological entity of interest based on acid-influenced optical readouts at the microwell level.
Supports cultivation and screening in high-density microwell chip systems using membrane-retained microwells.
Documented Applications
Screening phosphate-solubilizing bacteria.
Screening for acid/low-pH activity using indicator substances with optical readouts.
Media adaptation.
Mutant selection.
Bioremediation.
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