# US 10,865,434 B2 · High resolution systems, kits, apparatus, and methods for screening microorganisms and other high throughput microbiology applications

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- Number: US 10,865,434 B2
- Document ID: US10865434B2
- Status: Granted
- Kind: B2
- Application number: 15/787,708
- Filed: 2017-10-19
- Published: 2018-02-22
- Granted: 2020-12-15
- Earliest claimed: 2015-04-21
- Assignees: General Automation Lab Technologies Inc.
- Applicants: General Automation Lab Technologies, Inc.
- Inventors: Alexander Hallock; Kimberly Wemmer
- Web page: https://patentgrep.com/patent/US10865434B2

## CPC

- 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)}
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 23/24: {Gas permeable parts}
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6853: using modified primers or templates
- C12Q 1/689: for bacteria
- B01L 3/0244: {using pins}
- B01L 2200/025: Align devices or objects to ensure defined positions relative to each other
- B01L 2200/0689: Sealing
- B01L 2200/12: Specific details about manufacturing devices
- B01L 2200/141: Preventing contamination, tampering
- B01L 2300/021: Identification, e.g. bar codes
- B01L 2300/0636: Integrated biosensor, microarrays
- B01L 2300/0681: Filter
- B01L 2300/0829: Multi-well plates; Microtitration plates
- B01L 2300/0893: having a very large number of wells, microfabricated wells
- B01L 2300/165: Specific details about hydrophobic, oleophobic surfaces
- G01N 2333/195: from bacteria
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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 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 at least one microwell during incubation based on the optical property.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 5)

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.

### Claim 8 (depends on 5)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

The method of claim 1, wherein each microwell of the array of microwells has a diameter of about 25 μm to about 500 μm.

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 1)

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.

### Claim 17

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 that has produced an acid in the at least one microwell during incubation based on a change of the indicator substance after incubation.

### Claim 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, 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 that has produced an acid in the at least one microwell of the plurality of microwells during incubation based on a measurement of an optical property of the at least one microwell after incubation, wherein the optical property is influenced by the acid.