HIGH RESOLUTION SYSTEMS, KITS, APPARATUS, AND METHODS FOR SCREENING MICROORGANISMS AND OTHER HIGH THROUGHPUT MICROBIOLOGY APPLICATIONS

• US 20180051310A1
• Filed: 10/19/2017
• Published: 02/22/2018
• Est. Priority Date: 04/21/2015
• Status: Active Grant

• Current Assignee: Isolation Bio Incorporated
• Original Assignee: General Automation Lab Technologies, Inc.
• Inventors: HALLOCK, Alexander, WEMMER, Kimberly
• Granted Patent: US 10,865,434 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2200/141 Preventing contamination, tampering
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 23/24 Gas permeable parts
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 : Massive parallel sequencing
• CPC Class Codes: C12Q 2547/101 : by confinement to a single tube/container
• CPC Class Codes: C12Q 2563/159 : Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 : the label being a nucleic acid
• CPC Class Codes: G01N 2333/195 : from bacteria

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.

Claims

1. A method of screening for at least one biological entity of interest in a sample using a microfabricated device having a top surface defining an array of microwells, the method comprising:

loading into at least one microwell of the array of microwells;

(a) at least one cell from the sample;

(b) an indicator substance; and

(c) an amount of a nutrient;

applying a membrane to the microfabricated device to retain the at least one cell in the at least one microwell;

incubating the microfabricated device at predetermined conditions for a duration of time to grow a plurality of cells from the at least one cell in the at least one microwell;

evaluating an optical property of the at least one microwell; and

determining a presence or absence of at least one biological entity of interest in the at least one microwell based on the optical property.

2. The method of claim 1, wherein the optical property of the at least one microwell correlates to the amount or form of the indicator substance.

3. The method of claim 1, wherein the indicator substance is an inorganic phosphate salt that is insoluble in water.

4. The method of claim 3, wherein at least one biological entity of interest comprises a microorganism that solubilizes the inorganic phosphate salt.

5. The method of claim 1, wherein the indicator comprises a pH sensitive dye.

6. The method of claim 5, wherein the pH sensitive dye can change color in a visible light range within a selected range of pH.

7. The method of claim 5, wherein the pH sensitive dye is fluorescent in a first range of pH and not fluorescent in a second, different range of pH.

8. The method of claim 5, wherein the pH sensitive dye is a fluorescent dye whose fluorescence changes at different pH.

9. The method of claim 1, wherein the at least one biological entity of interest comprises a microorganism that produces an acid.

10. The method of claim 1, wherein the at least one biological entity of interest comprises a eukaryotic cell.

11. The method of claim 1, wherein the at least one biological entity of interest comprises bacteria.

12. The method of claim 1, wherein measuring the optical property comprises taking a plurality of measurements of the optical property at different times during the course of the incubation.

13. The method of claim 1, further comprising:

if a biological entity of interest is determined to be present in the at least one microwell, transferring at least some of the plurality of cells after incubation to a target location.

14. The method of claim 1, wherein the at least one microwell includes a plurality of microwells, and wherein loading the at least one cell comprises loading into each of the plurality of microwells, on average, one cell.

15. The method of claim 1, wherein each microwell of the array of microwells has a diameter of about 25 μ

m to about 500 μ

m.

16. The method of claim 1, wherein the surface density of the array of microwells is at least 750 microwells per cm².

17. The method of claim 1, wherein evaluating the optical property comprises:

(a) measuring the optical property of the at least one microwell after the at least one cell, the indicator substance, and the nutrient have been loaded and before incubation;

(b) measuring the optical property of the at least one microwell after incubation; and

(c) comparing the measured optical property before incubation and after incubation.

18. A method of screening for at least one biological entity of interest in a sample using a microfabricated device having a top surface defining an array of microwells, the method comprising:

loading into at least one microwell of the array of microwells;

(a) at least one cell from the sample;

(b) an indicator substance; and

(c) an amount of a nutrient;

applying a membrane to the microfabricated device to retain the at least one cell in the at least one microwell;

incubating the microfabricated device at predetermined conditions for a duration of time to grow a plurality of cells from the at least one cell in the at least one microwell; and

determining a presence or absence of at least one biological entity of interest in the at least one microwell based on a change of the indicator substance after incubation.

19. A method of screening for at least one biological entity of interest in a sample using a microfabricated device having a top surface defining an array of microwells, wherein each of the microwells has a bottom, the method comprising:

depositing an indicator substance into each of a plurality of microwells of the array of microwells;

loading the sample onto the microfabricated device such that at least one microwell of the plurality of microwells receives at least one cell from the sample and an amount of a nutrient;

applying a membrane to the microfabricated device to retain the received at least one cell and the nutrient in each of the plurality of microwells;

incubating the microfabricated device at predetermined conditions for a duration of time; and

determining a presence or absence of at least one biological entity of interest in the at least one microwell of the plurality of microwells based on a measurement of an optical property of the at least one microwell.