Method and device for analysing the intracellular chemical state of living cells by nuclear magnetic resonance
AssigneeBruker BioSpin GmbH
InventorsBenjamin Gonzalez, Martial Piotto, Gaspard Huber
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Abstract
The invention concerns a method for analysing the intracellular chemical state of living cells by nuclear magnetic resonance (NMR). Said method comprises a step which consists in preparing the cell sample and a step which consists in an NMR analysis of the sample, both steps being carried out at very low temperature so as to freeze the biochemical state of the living cells to obtain specific and accurate reproducible measurements of the cellular functioning in given conditions. The invention also concerns the device coupled to the NMR spectrometer for implementing the method of analysis.
Claims22
A method for analyzing the chemical state of living cells using nuclear magnetic resonance (NMR) by comparing at least one NMR spectrum and/or at least one NMR measurement value obtained on a sample of said living cells with at least one reference NMR spectrum and/or at least one reference NMR measurement value obtained on at least one reference sample of living cells, so as to identify at least one peak of said NMR spectrum and/or at least one NMR measurement value constituting a marker specific for the metabolic state of said living cells, characterized in that it comprises a step of preparing said sample under conditions which make it possible to set the chemical state of said living cells, said sample being subjected to a very low temperature for a sufficient amount of time to stop the intracellular reactions between the moment at which said sample is taken and the moment at which the chemical state of the cells is measured. and also throughout the analysis, said preparation of said sample comprising the following steps:
- a) directly and immediately immersing said sample in liquid nitrogen;
- b) lyophilizing said sample;
- c) optionally storing said sample at very low temperature;
- d) at the desired time for the analysis, mixing and redissolving said lyophilized sample, at very low temperature, with a solvent having a very low melting point.
The method as claimed in any one of the preceding claims, characterized in that the temperature throughout the NMR analysis is less than 0° C.
The method as claimed in any one of the preceding claims, characterized in that the analysis of the chemical state of said cell is carried out by HRMAS NMR, preferably by two-dimensional HRMAS NMR.
The method as claimed in any one of the preceding claims, characterized in that said sample is taken from a cell or tissue culture medium which contains molecules enriched in stable isotopes, in which said living cells are cultured or incubated.
The method as claimed in any one of the preceding claims, characterized in that said living cells are prokaryotic or eukaryotic, animal or plant, cells.
The use of a method as claimed in any one of the preceding claims, for identifying living cells the physiological state of which deviates from the norm.
A specific marker which can be obtained using the method as claimed in any one of the preceding claims.
Dependencies are inferred from the claim text.