# US 2013/0280751 A1 · Method and probe for monitoring oxygen status in live mammalian cells

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- Number: US 2013/0280751 A1
- Document ID: US20130280751A1
- Status: Published application
- Kind: A1
- Application number: 13/879,933
- Filed: 2010-10-22
- Published: 2013-10-24
- Earliest claimed: 2010-10-22
- Assignees: University College Cork, National University of Ir Cork
- Inventors: Dmitri Papkovsky; Andreas Fercher; Ingo Klimant; Sergey Borisov; Alexander Zhdanov
- Web page: https://patentgrep.com/patent/US20130280751A1

## CPC

- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/582: {with fluorescent label}
- G01N 33/587: {Nanoparticles}
- G01N 33/84: involving inorganic compounds or pH
- 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)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

The invention is based on the use of photoluminescent probes for intracellular sensing of oxygen, especially assaying intracellular oxygen concentration. The photoluminescent probe comprises a suspension of polymeric particles having an average diameter in the 20 nm to 100 nm range, formed from an amphiphilic cationic co-polymer which is oriented in the formed particle to provide a hydrophobic core and a hydrophilic shell. The probe includes a hydrophobic oxygen-sensitive photoluminescent dye such as Pt-tetrakis(pentafluorophenyl)porphine, PtPFPP embedded in the hydrophobic core of the particle, and the co-polymer includes quaternary ammonium groups which provide hydrophilic and cationic character to the particle shell. The photoluminescent probe, which in use is provided in the form of an aqueous suspension of probe, is incubated with live mammalian cells in a suitable growth medium for a period of time to allow the probe particles passively load into the cells. Oxygen can then be sensed by detecting a photoluminescent signal of the photoluminescent probe, which can be correlated with oxygen status, for example oxygen concentration or changes in oxygen concentration/levels, using existing techniques.

## Claims

### Claim 1

A method of determining oxygen concentration in a live mammalian cell sample by photoluminescence quenching technique, which method employs a photoluminsecent probe, the method comprising the steps of:
incubating the live mammalian cell sample in a suitable growth medium with the photoluminescent probe to allow passive loading of probe;
detecting a photoluminescent signal of the probe from the probe-loaded cell sample; and
correlating the detected photoluminescent signal to local oxygen concentration within the cell sample,
wherein the photoluminescent probe is an aqueous suspension of polymeric particles having an average size of 20 to 100 nm and a probe concentration of 0.1 to 100 μg/ml, the particles comprising an amphiphilic cationic polymer matrix having a hydrophobic core and a hydrophilic positively charged surface provided by quaternary amino groups, and a hydrophobic oxygen-sensitive photoluminescent dye embedded in the hydrophobic core, and wherein the nanoparticulate probe is capable of penetrating the cell and providing a high loading efficiency, a stable photoluminescence intensity signal, and reliable lifetime-based sensing of intracellular oxygen.

### Claim 2 (depends on 1)

A method as claimed in claim 1 in which the photoluminescent probe is used in the form of an aqueous suspension at a working concentration of 1 to 10 μg/ml.

### Claim 3 (depends on 1)

A method as claimed in claim 1 in which the amphiplilic cationic polymer matrix comprises a co-polymer of poly(ethylacrylate, methyl-metacrylate and chloro trimethyl-ammoniethyl methacrylate) containing quaternary ammonium groups.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

A method as claimed in claim 1 in which the photoluminescent probe has low intrinsic toxicity on the cells.

### Claim 6 (depends on 1)

A method as claimed in claim 1 in which the hydrophobic oxygen sensitive photoluminescent dye is selected from Pt-porphyrin, Pd-porphyrin, Pt-porphyrin-ketone or Pd-porphyrin-ketone, Pt-benzoporphyrin or Pd-benzoporphyrin, cyclometallated complex of Ir3+, Os2+ or Ru2+, or close analogs or derivatives of these dyes.

### Claim 7 (depends on 1)

A method as claimed in claim 1 in which the hydrophobic oxygen sensitive photoluminescent dye is PtPFPP dye.

### Claim 8 (depends on 1)

A method as claimed in claim 1, in which the photoluminescent probe is capable of 1.5 to 15 fold quenching at ambient oxygen concentration of 21 kPa or 250 μM O2.

### Claim 9 (depends on 1)

A method as claimed in claim 1 in which from 0.1% to 3.0% (w/w) of the photoluminescent probe is oxygen sensitive photoluminescent dye.

### Claim 10 (depends on 1)

A method as claimed in claim 1, in which the cell sample is washed prior to measurement of the photoluminscent signal to remove extracellular probe.

### Claim 11 (depends on 1)

A method according to claim 1 which is used to monitor oxygen in cell populations.

### Claim 12 (depends on 1)

A method according to claim 1 which is used to monitor oxygen in individual cells.

### Claim 13 (depends on 1)

A method according to claim 1 in which the photoluminescent signal is analysed by time-resolved fluorometry in the microsecond domain.

### Claim 14 (depends on 1)

A method according to claim 1 in which the photoluminescent signal is analysed by fluorescence microscopy imaging technique.

### Claim 15 (depends on 1)

A method according to claim 1 in which the photoluminescence lifetime of the photoluminescent signal, or parameter related to it, is measured.

### Claim 16 (depends on 1)

A method according to claim 1 which employs ratiometric intensity based oxygen sensing or imaging, and in which the photoluminescent probe contains an additional oxygen-insensitive dye which is used as a reference or as part of a FRET pair.

### Claim 17 (depends on 1)

A method of determining the metabolic activity of a cell or a cell population, which method comprises a step of monitoring oxygen concentration of the cell or cell population according to a method of claim 1, and correlating the measured oxygen concentration, or changes in the oxygen concentration, of the cell or cell population to metabolic activity of the cell or cell population.

### Claim 18 (depends on 16)

A method of determining the effects of a test substance on the metabolic activity of a cell or cell population, the method comprising a step of stimulating a cell or cell population with the test substance, determining the metabolic activity of the cell or cell population according to a method of claim 16.

### Claim 19

(canceled)

### Claim 20

(canceled)

### Claim 21

(canceled)