Method for classifying the presence or absence of a microorganism in a biological medium

Patent metadata

Field	Value
Publication number	US-9556469-B2
Application number	US-201214127939-A
Country	US
Kind code	B2
Filing date	Jun 27, 2012
Priority date	Jun 27, 2011
Publication date	Jan 31, 2017
Grant date	Jan 31, 2017

Abstract

Porous sol-gel material essentially consisting of units of one or more first polyalkoxysilanes chosen from the following compounds: (chloromethyl)triethoxysilane; 1,3-dimethyltetramethoxydisiloxane; ethyltrimethoxysilane; triethoxy(ethyl)silane; triethoxymethylsilane; triethoxy(vinyl)silane; trimethoxymethylsilane; trimethoxy(vinyl)silane; tetraethoxysilane or tetramethoxysilane (TMOS) and of units of one or more second polyalkoxysilanes chosen from the following compounds: (N-(3-(trimethoxysilyl)propyl)ethylenediamine; 3-aminopropyltriethoxysilane (APTES) and 3-aminopropyltrimethoxysilane, in a first polyalkoxysilane/second polyalkoxysilane molar ratio of 1/0.01 to 1/1, optionally comprising a probe molecule, method of preparation and applications in the trapping of monocyclic aromatic hydrocarbons and other pollutants or in their detection.

First claim

The invention claimed is:

1. A method for determining the presence or absence of at least one microorganism in a biological medium, said method comprising at least:
providing an enclosure containing:
a liquid or semi-solid phase formed in whole or in part of said biological medium capable of containing at least one living form of said microorganism, nutritional elements necessary for proliferation of said microorganism, and an enzymatic substrate that is specific to said microorganism and that can be metabolized into at least one Volatile Organic Compound (VOC) volatile metabolite; and
a gas phase adjacent to said liquid or semi-solid phase,

exposing at least said liquid or semi-solid phase to conditions that are favorable for said microorganism to metabolize said enzymatic substrate into at least one molecule of said VOC volatile metabolite; and
determining, by optical transduction, the presence or absence of said VOC metabolite, an indicator of the presence of said microorganism;

wherein:
said VOC metabolite, if formed, interacts with a nanoporous matrix which has an affinity for said VOC volatile metabolite, said matrix being in a form that is separate from said enzymatic substrate and arranged in the gas phase;
the detection by optical transduction of a change in the optical properties of said matrix indicates that said matrix interacts with said metabolite; and
VOC is a metabolite specific to the metabolization of the enzymatic substrate and representative of an enzymatic route specific to the microorganism subject of detection.

2. The method of claim 1 , wherein said VOC metabolite has intrinsic optical properties.

3. The method of claim 1 , wherein said VOC metabolite is distinct from natural metabolites generated during microbial growth.

4. The method of claim 1 , wherein said enzymatic substrate is a phenol or naphthol substrate.

5. The method of claim 1 , wherein said VOC metabolite is a phenol derivative.

6. The method of claim 5 , wherein said VOC metabolite is nitrophenol, cyanophenol, cyanonitrophenol, acetylphenol, a propionylphenol derivative, a thiophenol derivative, a naphthol derivative, an aniline derivative, or a naphthylamine derivative.

7. The method of claim 1 , wherein said VOC metabolite is p-cyanophenol, 4-nitrophenol (p-nitrophenol), m-cyano-p-nitrophenol (3-cyano-4-nitrophenol), 2, 6-dichloro-4-nitrophenol, 2,6-dichloro-4-acetylphenol, thiophenol, 2, 6-dichloro-4-propionylphenol, 2,6-difluoro-4-acetylphenol, 2,6-dibromo-4-acetylphenol, 1-naphthol, 2-naphthol, α-naphthylamine, β-naphthylamine, 4-nitroaniline (p-nitroaniline), umbelliferone, naphthazarin, 4-trifluoromethylumbelliferone, 4-methylumbelliferone, o-cyanophenol, m-cyanophenol, 6-cyano-2-naphthol, 6-hydroxyquinoline-N-oxide, 2-methyl-6-hydroxyquinoline-N-oxide, 6-hydroxyquinoline, 7-hydroxyquinoline, or 8-hydroxyquinoline.

8. The method of claim 1 , wherein the VOC metabolite is a photoacid or a photobase.

9. The method of claim 1 , wherein said VOC metabolite can be detected by absorbance and/or fluorescence.

10. The method of claim 1 , wherein said VOC metabolite can be detected by absorbance, and in that the associated nanoporous matrix is transparent or absorbs poorly in the detection zone.

11. The method of claim 1 , wherein said VOC metabolite can be detected by fluorescence, and in that the associated nanoporous matrix is void of intrinsic fluorescence or has low intrinsic fluorescence.

12. The method of claim 1 , wherein said matrix has a pore size distribution adjusted to the size of said VOC metabolite.

13. The method of claim 12 , wherein said matrix has a pore size distribution below 100 nm.

14. The method of claim 1 , wherein said matrix has a specific surface area varying from 300 to 1000 m 2 ·g −1 .

15. The method of claim 1 , wherein said matrix is constituted of an organic, inorganic or organic-inorganic hybrid substance.

16. The method of claim 1 , wherein said matrix derives from the polycondensation of alcoholates having formula
M(X) m (OR a ) n (R b ) p ,
wherein:
M corresponds to silicon, aluminum, tungsten, titanium, zirconium, niobium, vanadium, tantalum, yttrium and cerium;
R a corresponds to a C 1 to C 6 alkyl radical or to a C 5 aryl;
R b corresponds to a C 1 to C 6 alkyl, to a C 5 to C 10 aryl or to a C 3 to C 6 aminoalkyl;
n, m and p are integers, such that their sum is equal to the valence of M and n is greater than or equal to 2, where m and p may be equal to 0; and
X is a halogen.

17. The method of claim 1 , wherein said matrix comprises at least one probe molecule that can amplify the optical transduction signal via its interaction with said VOC metabolite trapped in said matrix.

18. The method of claim 17 , wherein said probe is 4-benzoylamino-2,5-diethoxybenzenediazonium chloride (Fast Blue BB), dimethylaminocinnamaldehyde (DMACA), or 5,5′-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent).

19. The method of claim 1 , wherein said matrix is arranged in the gas phase and circulation of said VOC volatile metabolite towards the gas phase is optimized.

20. The method of claim 19 , wherein circulation of said VOC volatile metabolite towards the gas phase is optimized by mechanical stirring or surface nebulization of the liquid or semi-solid phase.

21. The method of claim 20 , wherein circulation of said VOC volatile metabolite towards the gas phase is optimized by the liquid phase flowing on a divided inert solid phase to increase the surface area of the liquid-gas interface.

22. The method of claim 1 , further defined as comprised in a method of conducting an antibiogram test (AST).

Assignees

• Marcoux Pierre
• Dupoy Mathieu
• Guillemot Laure-Hélène
• Tran-Thi Thu-Hoa
• Commissariat Energie Atomique
• Centre Nat Rech Scient

Inventors

• Marcoux Pierre
• Dupoy Mathieu
• Guillemot Laure-Hélène
• Tran-Thi Thu-Hoa

Classifications

• C12Q2334/10

  p-Nitrophenol derivatives · CPC title

• C12Q1/04 Primary

  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)} · CPC title

• C12Q2334/30

  Naphthol derivatives, e.g. alpha-naphthyl-esters, i.e. alpha-NE, beta-naphthyl-esters, i.e. beta-NE · CPC title

• C12Q1/18 Primary

  Testing for antimicrobial activity of a material · CPC title