# US 2022/0033889 A1 · Detection and analysis of cells

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- Number: US 2022/0033889 A1
- Document ID: US20220033889A1
- Status: Published application
- Kind: A1
- Application number: 17/282,630
- Filed: 2019-10-04
- Published: 2022-02-03
- Earliest claimed: 2018-10-04
- Assignees: First Light Diagnostics, Inc.
- Applicants: First Light Diagnostics, Inc.
- Inventors: Don Straus
- Web page: https://patentgrep.com/patent/US20220033889A1

## CPC

- G01N 33/54333: {Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction}
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- C12Q 1/18: Testing for antimicrobial activity of a material
- C12Q 1/6841: In situ hybridisation
- C12Q 1/689: for bacteria
- G01N 35/0098: {involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation (magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C)}
- G01N 35/0099: {comprising robots or similar manipulators (robots per se B25J)}
- B01L 3/502738: {characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746)}
- B01L 9/00: Supporting devices; Holding devices
- B01L 2200/04: Exchange or ejection of cartridges, containers or reservoirs
- B01L 2200/0605: Metering of fluids
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2300/042: Caps; Plugs
- B01L 2300/043: Hinged closures
- B01L 2300/0636: Integrated biosensor, microarrays
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/087: Multiple sequential chambers
- B01L 2400/043: magnetic forces
- B01L 2400/065: sliding valves
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 35/025: {having a carousel or turntable for reaction cells or cuvettes}
- G01N 2035/00346: {Heating or cooling arrangements}
- G01N 2035/00356: {Holding samples at elevated temperature (incubation)}
- G01N 2035/0436: {with pre-packaged reagents, i.e. test-packs}
- G01N 2035/0446: {Combinations of the above}
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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

## Abstract

The invention provides a version of fluorescent in situ hybridization (FISH) in which all the steps are performed at physiological temperatures, i.e., body temperature, to detect and identify pathogenic bacteria in clinical samples. Methods of the invention use species-specific fluorescent probes to label clinically important infectious bacteria. A sample such as a urine sample is loaded into a cartridge, fluorescently labeled, and imaged with a microscope. Labelled bacteria are pulled down onto an imaging surface and a dye cushion is used to keep unbound probes off of the imaging surface. A microscopic image of the surface shows whether and in what quantities the infectious bacteria are present in the clinical sample.

## Claims

### Claim 1

A method for detecting a microorganism, the method comprising:
incubating, without exceeding 45 degrees C., a sample with probes specific for target nucleic acid of a species of microorganism;
separating intact cells in the sample from unbound probes; and
detecting probes bound within the intact cells to show the presence of the species in the sample.

### Claim 2 (depends on 1)

The method of claim 1, further comprising performing antimicrobial susceptibility testing directly from the sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein performing antimicrobial susceptibility testing comprises differential growth, permeabilization, hybridization, and magnetic tagging.

### Claim 4 (depends on 3)

The method of claim 3, wherein permeabilization, hybridization, and magnetic tagging are concerted.

### Claim 5 (depends on 2)

The method of claim 2, wherein the antimicrobial susceptibility is performed in a single cartridge.

### Claim 6 (depends on 1)

The method of claim 1, wherein the method further comprises using FISH for detecting, quantifying, and identifying target microbes.

### Claim 7 (depends on 6)

The method of claim 6, further comprising performing phenotypic antimicrobial susceptibility testing.

### Claim 8 (depends on 6)

The method of claim 6, further comprising detecting and quantifying target microbes in less than about 90 minutes.

### Claim 9 (depends on 8)

The method of claim 8, further comprising detecting and quantifying target microbes in less than about 60 minutes.

### Claim 10 (depends on 9)

The method of claim 9, further comprising detecting and quantifying target microbes in less than about 30 minutes.

### Claim 11 (depends on 1)

The method of claim 1, wherein the sample is maintained at or beneath about 40 degrees C. throughout all steps.

### Claim 12 (depends on 1)

The method of claim 1, wherein:
the probes comprise fluorescently-labeled oligonucleotides complementary to ribosomal RNA of the species;
the incubating step further includes exposing the cells to magnetic particles that bind to surfaces of bacterial cells;
the separating step includes using a magnetic field to pull magnetic particle-bound cells through a dye cushion and onto an imaging surface while the dye cushion holds unbound labels away from the imaging surface;
the detecting step comprising non-magnified imaging of the imaging surface to capture single cell images; and
the steps are all performed at temperatures between about 36 and 39 degrees C.

### Claim 13 (depends on 12)

The method of claim 12, wherein the incubating step includes exposing the cells to reagents that permeabilize the cells, thus allowing the probes to enter the cells and bind to targets therein.

### Claim 14 (depends on 13)

The method of claim 13, wherein the probes comprise oligonucleotides that are complementary to microbial RNAs.

### Claim 15 (depends on 13)

The method of claim 13, wherein: the reagents include one or more detergents; the probes comprise oligonucleotides; and the target nucleic acids are microbial ribosomal RNAs.

### Claim 16 (depends on 15)

The method of claim 15, wherein: the detergents include one or more of CHAPSO and SB3-12; and the probes include fluorescently-labeled oligonucleotides complementary to portions of the microbial ribosomal RNAs.

### Claim 17 (depends on 1)

The method of claim 1, wherein the probes include: a fluorescently-labeled oligonucleotide that is complementary to a segment of ribosomal RNA specific to the species.

### Claim 18 (depends on 17)

The method of claim 17, wherein the probes further include and one or more helper probes that are oligonucleotides that bind to the ribosomal RNA at a location within 1 to 30 bases from the segment where the fluorescently-labeled oligonucleotide binds.

### Claim 19 (depends on 17)

The method of claim 17, wherein the fluorescently-labeled oligonucleotide is between 10 and 18 bases in length and includes at least one conformationally-restricted nucleic acid.

### Claim 20 (depends on 1)

The method of claim 1, wherein:
the incubating step includes exposing the cells to magnetic particles that bind to surfaces of bacterial cells; and
the separating step includes using a magnetic field to pull bound cells away from the unbound probes.

### Claim at position 21

21-26. (canceled)