# US 7,611,862 B2 · Method and apparatus for detecting and quantifying bacterial spores on a surface

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- Number: US 7,611,862 B2
- Document ID: US7611862B2
- Status: Granted
- Kind: B2
- Application number: 11/332,788
- Filed: 2006-01-12
- Published: 2006-12-28
- Granted: 2009-11-03
- Earliest claimed: 2004-11-12
- Assignees: California Institute of Technology
- Inventors: Adrian Ponce
- Web page: https://patentgrep.com/patent/US7611862B2

## CPC

- C12Q 1/06: Quantitative determination
- C12Q 1/22: Testing for sterility conditions
- G01N 1/2813: {Producing thin layers of samples on a substrate, e.g. smearing, spinning-on (G01N1/30 takes precedence)}
- 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 2001/2826: {Collecting by adsorption or absorption}
- G01N 2001/2833: {Collecting samples on a sticky, tacky, adhesive surface}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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)

## Abstract

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

## Claims

### Claim 1

A method for detecting and quantifying individual bacterial spores comprising:
capturing the bacterial spores;
transferring the bacterial spores to a test surface;
providing one or more lanthanide ions on the test surface;
releasing aromatic molecules from the bacterial spores on the test surface;
forming a complex of the one or more lanthanide ions and the aromatic molecules on the test surface;
exciting the complex to generate a characteristic luminescence of the complex on the test surface; and
detecting and quantifying the individual bacterial spores by imaging the bacterial spores via luminescence spots surrounding the bacterial spores, said luminescent spots generated on the test surface through lifetime-gated imaging, with a micrometric spatial resolution.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the capturing of the bacterial spores comprises transferring bacterial spores from a second surface by way of an adhesive polymer.

### Claim 3 (depends on 2)

The method of claim 2, wherein the adhesive polymer is selected from the group consisting of PDMS, agar and agarose.

### Claim 4 (depends on 1)

The method of claim 1, wherein the capturing of the bacterial spores comprises transferring bacterial spores from a second surface by way of a swab.

### Claim 5 (depends on 4)

The method of claim 4, further comprising transferring the bacterial spores from the swab into water.

### Claim 6 (depends on 5)

The method of claim 5 wherein the water is passed through a water filter.

### Claim 7 (depends on 1)

The method of claim 1 wherein the capturing of the bacterial spores is from a place of origin selected from the group consisting of water and air.

### Claim 8 (depends on 7)

The method of claim 7 wherein the transferring of the bacterial spores is from a place of origin to a filter selected from the group consisting of an air filter and a water filter.

### Claim 9 (depends on 1)

The method of claim 1, wherein the test surface comprises at least one adhesive polymer selected from the group consisting of PDMS, agar, agarose, PDMS together with agar, and PDMS together with agarose.

### Claim 10 (depends on 1)

The method of claim 1, wherein the test surface comprises at least one partially transparent adhesive polymer.

### Claim 11 (depends on 1)

The method of claim 1, wherein the one or more lanthanide ions are provided to the test surface prior to the bacterial spores, after the bacterial spores, or as a mixture with the bacterial spores.

### Claim 12 (depends on 1)

The method of claim 1, wherein the one or more lanthanide ions are terbium or europium or a mixture thereof.

### Claim 13 (depends on 1)

The method of claim 1, wherein the aromatic molecules are selected from the group consisting of dipicolinic acid, dipicolinate, and dipicolinic acid together with dipicolinate.

### Claim 14 (depends on 1)

The method of claim 1, wherein the aromatic molecules interact with the test surface resulting in a derivative of the aromatic molecules.

### Claim 15 (depends on 1)

The method of claim 1, wherein the aromatic molecules are released from the bacterial spores by germination of the bacterial spores.

### Claim 16 (depends on 15)

The method of claim 15, wherein the germination of the bacterial spores is induced by way of a germinating agent selected from the group consisting of L-alanine, L-asparagine and D-glucose.

### Claim 17 (depends on 1)

The method of claim 1, wherein the aromatic molecules are released from the bacterial spores by lysis of the bacterial spores.

### Claim 18 (depends on 17)

The method of claim 17, wherein the lysis of the bacterial spores is induced by way of a method of lysis selected from the group consisting of microwaving, autoclaving, sonication, plasma cleaning dry heating and hydrogen chloride gasing.

### Claim 19 (depends on 9)

The method of claim 9 wherein the test surface further comprises at least one lanthanide ion and a germinating agent.

### Claim 20 (depends on 9)

The method of claim 9 wherein the test surface further comprises at least one lanthanide ion and is subject to a method of lysis.

### Claim 21 (depends on 1)

The method according to claim 1, wherein the complex is excited by UV light.

### Claim 22 (depends on 2)

The method of claim 2 wherein the adhesive polymer is the test surface.

### Claim 23 (depends on 3)

The method of claim 3 wherein the adhesive polymer is the test surface.

### Claim 24 (depends on 8)

The method of claim 8, wherein the filter is the test surface.

### Claim 25 (depends on 1)

The method of claim 1, wherein the releasing of aromatic molecules from the bacterial spores on the test surface is by germination of the bacterial spores on the test surface;
wherein the complex is a first complex;
wherein the method further comprises:
releasing aromatic molecules from nongerminated spores on the test surface by lysis;
forming a second complex of the one or more lanthanide ions and lysis-released aromatic molecules on the test surface;
exciting the second complex to generate a characteristic luminescence of the second complex on the test surface; and
detecting and quantifying the bacterial spores by imaging the bacterial spores, via the luminescence of the second complex on the test surface by lifetime-gated imaging.

### Claim 26 (depends on 25)

The method of claim 25 wherein the test surface comprises terbium or europium ions;
wherein the aromatic molecules are DPA and/or DP molecules;
wherein the exciting of the first and second complex is with UV radiation;
the method further comprising the step of dividing the quantified luminescence of the first complex by the sum of the luminescence of the first and second complexes.

### Claim 27 (depends on 26)

A method of claim 26 wherein the quantifying of the bacterial spores represented by the generated luminescence comprises counting the number of luminescent spots and the number of spores per luminescent spot can be estimated by the spot intensity.

### Claim 28 (depends on 1)

An apparatus for detecting and quantifying individual bacterial spores according to the method of claim 1, comprising:
a test surface, wherein the test surface comprises one or more adhesive polymers, one or more lanthanide ions, bacterial spores and aromatic molecule released from said bacterial spores
an ultraviolet light radiation device adjacent to the test surface to excite a complex of lanthanide ions and aromatic molecules and generate a characteristic luminescence of the complex;
an life-time gated imaging device for imaging the luminescence.

### Claim 29 (depends on 28)

An apparatus of claim 28 wherein a microscope is connected to the life-time gated imaging device.

### Claim 30

A method for detecting and quantifying bacterial spores comprising:
providing the bacterial spores on a test surface, each bacterial spore having a spore body;
providing one or more lanthanide ions on the test surface;
releasing aromatic molecules from the spore body of a bacterial spore onto the test surface, the released aromatic molecules defining on the test surface a surrounding area around the spore body of the bacterial spore;
forming a complex of the one or more lanthanide ions and the aromatic molecules on the surrounding area;
exciting the complex to generate a characteristic luminescence of the complex on the surrounding area; and
detecting and quantifying the bacterial spores by imaging surrounding areas through lifetime-gated imaging with a micrometric spatial resolution.

### Claim 31 (depends on 30)

The method of claim 30, wherein imaging surrounding areas is performed by imaging individual surrounding areas.

### Claim 32 (depends on 30)

The method of claim 30, wherein imaging surrounding areas is performed by imaging surrounding areas of individual spores clustered together on the test surface.

### Claim 33 (depends on 30)

The method of claim 30, wherein the bacterial spores are embedded in the test surface.

### Claim 34 (depends on 30)

The method of claim 30, wherein the test surface is an adhesive polymer.

### Claim 35 (depends on 34)

The method of claim 34 wherein the adhesive polymer is optically transparent.

### Claim 36 (depends on 30)

The method of claim 30, wherein the releasing of aromatic molecules from the bacterial spores on the test surface is by germination of the bacterial spores on the test surface; and wherein the complex is a first complex;
wherein the method further comprises:
releasing aromatic molecules from nongerminated spores on the test surface by lysis the released aromatic molecules defining on the test surface a surrounding area around the spore body;
forming a complex of the one or more lanthanide ions and the aromatic molecules on the surrounding area;
exciting the complex to generate a characteristic luminescence of the complex on the surrounding area; and
detecting and quantifying the bacterial spores by imaging surrounding areas through lifetime-gated imaging.

### Claim 37

A method for detecting and quantifying bacterial spores, the method comprising
providing the bacterial spores, each bacterial spore having a spore body;
providing one or more lanthanide ions;
releasing aromatic molecules from the spore body
forming a complex of the one or more lanthanide ions and the aromatic molecules;
exciting the complex to generate a characteristic luminescence of the complex; and
detecting and quantifying bacterial spores by imaging the bacterial spores through lifetime-gated imaging,
wherein
releasing aromatic molecules from the spore body is performed on a test surface to generate on the test surface an aromatic molecules concentration region around the spore body
the complex of the one or more lanthanide ions and the aromatic molecules is formed on said region, and
detecting and quantifying bacterial spores is performed by imaging the aromatic molecules concentration regions on the test surface with a micrometric spatial resolution.

### Claim 38 (depends on 37)

The method of claim 37, wherein the imaged aromatic molecules concentration regions are individual aromatic molecules concentration regions.

### Claim 39 (depends on 37)

The method of claim 37, wherein the imaged aromatic molecules concentration regions are aromatic molecules concentration regions of individual bacterial spores clustered together on the test surface.

### Claim 40 (depends on 37)

The method of claim 37, wherein the bacterial spores are embedded in the test surface.

### Claim 41 (depends on 37)

The method of claim 37, wherein the test surface is an adhesive polymer.

### Claim 42 (depends on 41)

The method of claim 41, wherein the adhesive polymer is optically transparent.

### Claim 43 (depends on 37)

The method of claim 37, wherein the releasing of aromatic molecules from the bacterial spores on the test surface is by germination of the bacterial spores on the test surface; and wherein the complex is a first complex;
wherein the method further comprises:
releasing aromatic molecules from nongerminated spores on the test surface by lysis to generate on the test surface aromatic molecules concentration regions around each spore body;
forming a second complex of the one or more lanthanide ions and lysis-released aromatic molecules on said concentration regions;
exciting the second complex to generate a characteristic luminescence of the second complex on the concentration regions; and
detecting and quantifying the bacterial spores by imaging the aromatic molecules concentration regions on the test surface.