# US 2021/0172002 A1 · Detecting cell vitality

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- Number: US 2021/0172002 A1
- Document ID: US20210172002A1
- Status: Published application
- Kind: A1
- Application number: 17/048,389
- Filed: 2019-04-18
- Published: 2021-06-10
- Earliest claimed: 2018-04-19
- Applicants: The University of Warwick
- Inventors: James Peter Stratford; Munehiro Asari
- Web page: https://patentgrep.com/patent/US20210172002A1

## CPC

- C12Q 1/02: involving viable microorganisms
- C12Q 1/18: Testing for antimicrobial activity of a material
- C12Q 1/10: Enterobacteria
- G01N 21/64: Fluorescence; Phosphorescence
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 33/582: {with fluorescent label}
- C12N 1/20: Bacteria; Culture media therefor
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method and device for detecting cell vitality, i.e. determining whether cells are alive (vital) or dead (non-vital). The method includes detecting vitality of cells in a sample), including: providing a sample containing cells and a fluorescent dye; disposing a portion of the sample in a test volume between a pair of electrodes; applying a voltage across the pair of electrodes to generate an electric field across the portion of the sample; illuminating the test volume; measuring a fluorescence response from the test volume over a period of time after applying the voltage across the pair of electrodes; and detecting cells in the sample to be vital dependent on a change in their fluorescence response over the period of time.

## Claims

### Claim 1

A method comprising:
detecting vitality of cells in a sample, comprising:
providing a sample comprising cells and a fluorescent dye;
disposing a portion of the sample in a test volume between a pair of electrodes;
applying a voltage across the pair of electrodes to generate an electric field across the portion of the sample;
illuminating the test volume;
measuring a fluorescence response from the test volume over a period of time after applying the voltage across the pair of electrodes; and
detecting cells in the sample to be vital dependent on a change in their fluorescence response over the period of time.

### Claim 2 (depends on 1)

The method of claim 1 wherein the change in fluorescence is an increase in fluorescence response over the period of time.

### Claim 3 (depends on 1)

The method of claim 1 wherein the electric field is less than 1 kV/cm.

### Claim 4 (depends on 3)

The method of claim 3 wherein the electric field is greater than 100V/cm.

### Claim 5 (depends on 1)

The method of claim 1 wherein the electric field is alternating.

### Claim 6 (depends on 1)

The method of claim 1 wherein the voltage is applied for less than 10 s.

### Claim 7 (depends on 5)

The method of claim 5 wherein the voltage is applied for greater than 0.1 s.

### Claim 8 (depends on 6)

The method of claim 6 wherein the voltage is applied for less than 5 s.

### Claim 9 (depends on 1)

The method of claim 1 wherein the detecting comprises detecting the cells in the sample to be non-vital cells if their fluorescence response decreases over the period of time.

### Claim 10 (depends on 1)

The method of claim 1 wherein the period of time is less than 60 s.

### Claim 11 (depends on 10)

The method of claim 10 wherein the period of time is less than 10 s.

### Claim 12 (depends on 10)

The method of claim 10 wherein the period of time is greater than 0.1 s.

### Claim 13 (depends on 1)

The method of claim 1 wherein the fluorescent dye is a Nernstian dye.

### Claim 14 (depends on 1)

The method of claim 1 wherein the sample flows through the test volume from a first location to a second location of the test volume over the period of time, and wherein the illuminating the test volume with light and measuring a fluorescent response are repeated at the first and second locations.

### Claim 15 (depends on 1)

The method of claim 1 wherein the detecting comprises detecting vital cells in the sample if their fluorescence response changes by more than a predetermined amount over the period of time.

### Claim 16 (depends on 15)

The method of claim 15 wherein the predetermined amount is greater than 50%.

### Claim 17 (depends on 1)

The method according to claim 1, further comprising detecting a strain of bacteria in a sample, comprising:
treating the sample with an antibiotic; and
performing the detecting vitality of cells in the sample to detect bacteria resistant to the antibiotic.

### Claim 18 (depends on 17)

The method of claim 17 wherein the bacteria is Escherichia coli.

### Claim 19 (depends on 18)

The method of claim 18 wherein the antibiotic is vancomycin.

### Claim 20

An apparatus for detecting vitality of cells in a sample, the apparatus comprising:
a sample holder for containing a sample of cells, the sample holder comprising a pair of electrodes on opposing sides of a test volume;
a voltage generator connected to the pair of electrodes;
a light source arranged to illuminate the test volume; and
a light detector arranged to detect a fluorescent response from the test volume upon illumination by the light source.

### Claim 21 (depends on 21)

The apparatus of claim 21 wherein the sample holder comprises a transparent substrate having a surface on which the pair electrodes are provided.

### Claim 22 (depends on 20)

The apparatus of claim 20 wherein the light source is arranged to illuminate the sample from an opposing side of the test volume to the light detector.

### Claim 23 (depends on 20)

The apparatus of claim 20 comprising an optical fibre plate between the sample holder and the light detector, wherein the light detector is an image sensor.

### Claim 24 (depends on 20)

The apparatus of claim 20 wherein the light source is a first light source arranged to illuminate a first portion of the test volume and the light detector is a first light detector arranged to detect fluorescence light emitted from the first portion of the test volume, the apparatus comprising a second light source arranged to illuminate a second portion of the test volume and a second light detector arranged to detect fluorescence emitted from the second portion of the test volume, the sample holder configured to allow a sample to flow through the test volume from the first portion to the second portion.

### Claim 25

A method of detecting vitality of cells in a sample, the method comprising:
applying a voltage across a pair of electrodes in a test volume containing the sample and a fluorescent dye to generate an electric field across the portion of the sample;
illuminating the test volume;
measuring a fluorescence response from the test volume over a period of time after applying the voltage; and
detecting cells in the sample to be vital dependent on a change in their fluorescence response over the period of time.

### Claim 26

A non-transitory computer-readable medium comprising a computer program stored thereon, comprising instructions to cause a computer of an apparatus to perform a method of detecting vitality of cells in a sample when the computer executes the instructions, wherein the instructions configure the computer to:
apply a voltage across a pair of electrodes in a test volume containing the sample and a fluorescent dye to generate an electric field across the portion of the sample;
illuminate the test volume;
measure a fluorescence response from the test volume over a period of time after applying the voltage; and
detect cells in the sample to be vital dependent on a change in their fluorescence response over the period of time.