EP2265933A1 - Method and device for high speed quantitative measurement of biomolecular targets on or in biological analysis medium - Google Patents

Abstract

The present invention relates to a device and method for the high speed quantitative measurement of biomolecular targets on the surface or in the body of a planar medium for biological analysis. The method, according to the invention, includes the following steps: a) at least two laser beams (F") are focused and overlaid on each measuring point of said medium by the simultaneous intersection of these beams to extract a contained hot plasma (P), including a measured chemical element present in the targets and another chemical element exogenous to the targets and present in a known quantity on this medium; b) luminous emission rays for each plasma, corresponding to the quantified element and exogenous element, are detected and analyzed for each measuring point while measuring the brightness of these rays; then c) the concentration in each measuring point of the quantified element is determined through prior calibration of the rays of the quantified element to determine a correlation between the brightness of the rays, specific to said element, and the concentrations of the latter in mixtures of the quantified element and the exogenous element in known proportions.

Description

 METHOD AND DEVICE FOR QUANTITATIVE HIGH-CADENCE MEASUREMENT OF BIOMOLECULAR TARGETS PRESENTED ON OR IN A BIOLOGICAL ANALYSIS MEDIUM

The present invention relates to a method of high-speed quantitative measurement of biomolecular targets on the surface or in the thickness of a biological analysis plane support, and a device for implementing this method. The invention is particularly applicable to the quantitative measurement of nucleic acids or unlabeled proteins segregated on the surface or in the thickness of a preferably planar biological analysis support, such as a biochip, for example with a matrix probes, a transfer membrane, an electrophoresis or chromatography gel or a support of glass or polyimide (eg Kapton®), without limitation.

Laser-induced breakdown spectroscopy ("Laser-induced breakdown spectroscopy" or "LIBS") is a very powerful method for determining the elemental composition of the surface of a material. This composition is obtained by measuring the emission lines originating from the atoms or ions constituting a transient plasma induced on the surface of the material by a laser beam, and the elemental analysis of this surface can be obtained by sweeping it by a laser source performing successive shots in a regular grid, so as to map the surface of this material. With each laser firing, the emission spectrum of the desired chemical elements is recorded, as well as the focus coordinates of the beam on the surface of the material. It is then possible to reconstruct a point-to-point image of the surface of the material at a selected wavelength corresponding to a specific chemical element. If a calibration method is applied, the intensity variations of the image obtained by mapping indicate the abundance of this element on the surface of the material.

However, a major disadvantage of this technique is that the very high number of laser shots necessary to perform a mapping to high resolution of the surface (resolution between 300 nm and 1 μm), involves analysis times of several hours or even several days, as soon as the analyzed surface exceeds a few square millimeters. This slow acquisition of the image makes LIBS inapplicable as a method of analysis for a large number of applications in biology, and more particularly for genomics and proteomics.

In the field of genomics, biochips represent a major revolution in molecular biology techniques over the past decade. By allowing the simultaneous study of the level of expression of several hundred or even thousands of genes, they make it possible to understand the impact of a disease or a stress (eg resulting from a radiation, a pollution or taking a drug) at the level of the complete genome of an individual. These techniques become more and more used in modern biology. Biochips fall into two broad families, including microfluidic chips and probe matrix chips. The latter are organized into "spot" matrices or measurement points, and are generally obtained by depositing or synthesizing, at precise coordinates on a passive support, molecular probes formed of biopolymers such as DNA, proteins or proteins. antibodies, for example. These probe matrix biochips enable the identification of the targets present in a biological sample when these targets hybridize specifically at each "spot" of probes.

There are, on the one hand, high complexity microarrays (more than 5000 spots) for pan-genomic studies and, on the other hand, low and medium complexity biochips, which are dedicated to a given theme (eg therapeutic tests, biological detector).

The current technology of probe array biochips has a number of major limitations, including: - the high steric hindrance of fluorescent markers, which sporadically alters recognition between probes and probes; targets and thus leads to numerous measurement artifacts that reduce the reproducibility of the experiments;

- the absence of a quantitative measure, which forbids comparison of expression levels between two different targets; and - the high cost of this current technology, both in production and implementation.

This is the reason why several alternatives to this technology have been developed recently, with in particular: - "RT PCR" technologies in microfluidic card

("Reverse Transcriptase Polymerase Chain Reaction", ie a polymerase chain reaction after reverse transcription of a ribonucleic acid into complementary DNA), which makes it possible to amplify up to 386 different targets in parallel, with a simplification of the implementation. improvement of detection, which is penalized by the absence of quantitative measurement for a real comparison between the targets, by the limitation of the number of targets to be analyzed (well below a low complexity biochip) and by a high cost of implementation;

- Nylon film biochips, based on high-volume target hybridization and chemiluminescent labeling, which also provide simplified implementation and improved detection, but are nevertheless penalized by the lack of measurement quantitative, the large required reaction volume (limiting for low concentration sample analyzes) and the very high costs of production and implementation; and

new concepts of non-marking biochips, which are based on the detection of the target by impedance measurement or by surface plasmon resonance (SPR or Surface Plasmon Resonance), and which have been described in particular in David F et al., Bioscience Bioelectron. 2005, in Li CM et al., Front Biosci. 2005 or Macanovic A. et al., Nucleic Acid Research 2004, but which do not allow a quantification of the number of targets, are problematic to achieve high density chips and involve, for both the impedance measurement technique and the SPR technique, measurement artifacts due to the variable size and conformation of the targets.

"ICP" ("Inductively") spectrometric methods

Coupled Plasma ", i.e. induced plasma coupled mass spectrometry) have also been developed, cf. Inchul Yang et al., Analytical Biochemistry (2004), vol.335, 150-161 or Heinrich F. Arlinghaus et al., Analytical Chemistry (1997), vol.69, No. 18, 3747-3753, methods that allow to measure the phosphorus contained in a nucleic acid to estimate for example the rate of hybridization thereof on a "PNA" ("Peptide Nucleic Acid") biochip, ie peptide nucleic acids.

However, it appears that the use of mass spectrometry to measure phosphorus from a plasma generated on the surface of a biochip is a slow method (typically taking several hours for 1 cm ² on the biochip), and that the instrumentation necessary for its implementation is expensive. Moreover, it should be noted that this "ICP" spectrometry technique is not quantitative because it only provides the raw quantity of hybrid nucleotides, without being able to differentiate between the size and the number of biomolecules.

US-A-2006/0105354 discloses a method of real-time quantification of a multitude of labeled nucleic acid targets which have bound to the surface of a probe matrix type biochip, including in particular the emission of an excitation laser beam on the surface of the matrix and the measurement of the light emission of the hybridized targets in response to this excitation beam.

A major disadvantage of this method is that it is not quantitative in the sense indicated above, and it also requires the presence of labeled molecules linked to the target molecules. In the field of proteomics, the problem posed is further complicated. Indeed, in addition to identifying, in a cell extract, the proteins present and their concentrations, it is necessary to _

to identify their post-translational modifications. Indeed, the activity of a protein is very often determined by its post-translational modifications. Of the possible modifications, phosphorylations are probably the most important biologically. Two-dimensional (2D) electrophoresis techniques are the most commonly used parallel analysis techniques for the analysis of protein mixtures. These techniques consist of migrating a mixture of proteins in a gel, successively following two orthogonal directions, according to different physicochemical criteria (e.g., chromatography or electrophoresis). The proteins are then separated according to their affinity to a solvent, their electrical charge, their mass, their shape, their modification, etc., and after having been pigmented by a fluorescent pigment or not, the proteins are identified according to their position or stain on the 2D gels compared to the migrations obtained in a reference gel. To highlight the post-translational modifications, in particular the phosphorylations, it is necessary to carry out the analysis of each spot of the gel. Different methods of analysis are possible, such as mass spectrometry, NMR, immunological labeling, the "ICP" technique, for example. The highlighting of the post-translational modifications is for the moment very heavy to realize.

In recent years, antibody biochips have been available on the market for the analysis of protein extracts. These biochips make it possible to analyze in parallel the expression of several hundred genes in their protein form as long as there are antibodies specific for the desired proteins. However, this technology is limited by the need to label proteins. Unfortunately, chromophores used for labeling randomly alter the affinity of proteins for their antibody. On the other hand, the marking is also random, the results are difficult to interpret and difficult to compare between different experiments. To evaluate by this technique, for example with the rate of post-translational modifications of a protein given between two conditions or two cell types, it is necessary to have two antibodies recognizing each of the forms of the protein. This, in addition to being expensive, is technically very difficult to obtain. The "LIBS" technique represents an alternative for the analysis without prior labeling of segregated biomolecules on all types of supports (eg membrane, gel, plastic support for example in Kapton®, silicon support), these supports being the main types used. in biological analyzes. The method of analysis of a biological product by "LIBS" consists in assaying a constituent chemical element of the target biomolecule previously isolated, segregated or purified on a chromatography, electrophoresis, membrane or biochip support.

As indicated above, the main technological lock of a "LIBS" analysis is the slowness of the process, which greatly limits the use of this technique in biology. Indeed, the analysis supports in biology often make several square centimeters of surface. Imaging, at a resolution of 10 microns, for example, of a 1 cm ² analysis support thus requires 1,000,000 contiguous laser shots to be fired, one shot per position. Of course, this number is to be multiplied by the number of shots to be made per position, if the molecule to be analyzed is in the thickness of the support, such as it produces for the electrophoresis gels for example.

To carry out this analysis in 1 second, it is necessary to have a UV laser of the mega Hertz type with sufficient energy to induce a plasma on the support at each shot (energy typically greater than 60 micro joules per pulse). To perform this analysis in 1 minute, it is necessary to have a UV laser with a frequency equal to 600 kilo Hertz and sufficient energy to induce a plasma on the support at each shot (also greater than 60 micro joules per pulse). This problem can be minimized by performing non-contiguous shots in return for a degradation of the resolution. However, the frequency remains high, even for a very degraded image. It is possible to use lasers with longer wavelengths, for example around 532 nm, because these lasers generally have larger frequencies and higher energies. However, by moving away from the far UV (266 nm), the energy required to obtain an analyzable plasma increases, and for example the lasers at 532 nm - of sufficient energy - have frequencies close to the far UV lasers, which then poses the same problem as for the far UV field.

An object of the present invention is to provide a method for high-speed quantitative measurement of biomolecular targets, in particular proteins, present on or in a biological analysis support, which overcomes all of the aforementioned drawbacks.

For this purpose, the measuring method according to the invention comprises the following steps: a) the measurement points of the support are scanned by moving, focusing and superimposing on each measurement point of this support at least two laser beams by crossings simultaneous of these beams, to extract a hot and confined plasma comprising at least one chemical element to be quantified present in said targets and at least one other chemical element exogenous to these targets and present in known quantity on or in this support, b) detects and analyzes, for each of these measurement points, emission light lines of each plasma that correspond to the or each element to be quantified and to the or each exogenous element, by measuring the respective intensities of these lines, then c) the calibration of the lines of the or each element to be quantified establishing a correlation between the intensities of the lines specific to this element to be quantified and the concentrations of the latter in mixtures in known proportions of the or each element to be quantified and the or each exogenous element, the concentration in each measurement point of the or each element to be quantified or of a group incorporating it within these targets.

The method according to the invention thus makes it possible to "scan" (ie analyze in one sweep) quickly and efficiently all the measuring points of the biochip, while being independent of a variation of the power setting of the lasers and of the sensitivity of the detection of an acquisition to the other, thanks to the presence of an internal reference corresponding to the exogenous element, and to deduce from the above-mentioned step c) the number of atoms of the desired element in each measuring point, to deduce the number of targets at the observed coordinate, all in less than

1/1000 second per reading point on average (read time = number of reads / acquisition time).

It will be noted that this focusing at the same point of the support of several laser beams which is performed in step a) makes it possible to add their respective energies in order to generate a spectrally analyzable plasma.

It will also be noted that the correlation used in step c) between line intensities y and concentration x for the exogenous element or for the element to be quantified, is advantageously of linear type (ie a relation of proportionality, to an affine constant). near, according to the equation y = ax + b).

According to another characteristic of the invention, said biomolecular targets are immobilized before step a) at the surface or in the thickness of the biological analysis support, which is preferably substantially plane and is chosen from the group consisting of biochips, transfer membranes, substrates made of silicon, polyimide (eg Kapton®) and glass, and electrophoresis and chromatography gels.

Advantageously, this support is formed of a biochip comprising on its surface a matrix of native probes, hybridized or complexed by said targets, this matrix comprising a multitude of said measuring points each comprising a plurality of probes, and the or each chemical element to be quantified being present in these targets and, optionally, further in these probes.

Note that when the element to be quantified is both in the probe and in the target, said exogenous element makes it possible to deduce the amount of signal that comes from the probe and the amount of signal that comes from the target. This exogenous element provides an internal calibration that allows the signal to be readjusted to the calibration curves, whatever the signal attenuation due to the setting of the device.

According to another characteristic of the invention, this measurement method further comprises a subsequent step of obtaining one or more images representative of the concentration of the or each element to be quantified or of the or each exogenous element, the intensity each pixel or each of the three colors R, G, B of the latter within the image being representative of the intensity of the line of the corresponding element observed.

The image thus obtained makes it possible to map the abundance of the or each element to be quantified or exogenous on or in the analysis support. The mapping of the thickness of the analysis support is obtained by successive shots in one and the same position, the signals obtained being either accumulated or analyzed separately to carry out a z-mapping of the support, either averaged or sliced. The number of successive shots depends in particular on the desired depth of analysis and the physicochemical properties of the support material.

In the aforementioned case of a probe matrix biochip, the immobilization prior to step a) of the target molecule is obtained, either directly by an interaction with the material constituting the support or a retention in the molecular mesh of the support. or indirectly by an interaction with a ligand (probe) attached to or in the support. As such probe / target interactions, one can for example quote:

hybridization between a target and a probe of nucleic acid or similar type (eg PNA, LNA), protein / protein interactions between a target and a protein probe such as ligand / receptor, antibody / fixed antigen,

- mixed nucleic acid or assimilated / protein interactions, - interactions of nucleic acid / metal, ion or any other free molecule, or

protein / ion interactions or any other free molecule.

Preferably, each laser beam used in step a) is emitted in the infrared-visible-ultraviolet range according to a pulse of duration between 1 fs and 100 ns, with a frequency of between 600 Hz and 1 GHz and energy included. between 0.05 mW and 1 kW.

Still more preferably, each laser beam is emitted in the ultraviolet at a wavelength of 266 nm or 193 nm, using, for example, the harmonics of an Nd: YAG laser (yttrium garnet laser). neodymium doped aluminum), and according to a pulse of less than 10 ns duration preferably equal to 5 ns.

Alternatively, the laser beams used in step a) can be emitted at different wavelengths, for example in the ultraviolet at a wavelength of 266 nm and in the visible at 532 nm, again according to a pulse of less than 10 ns duration and preferably equal to 5 ns.

According to another characteristic of the invention, said beams may advantageously be shaped in step a) in a scanning head, in such a way that:

these beams are reflected tangentially on a pyramid of reference to at least one reflection stage to give collinear beams at the output of this pyramid; these collinear beams pass through an afocal optical system, such as a beam-reducing telescope, which reduces respective diameters of these beams, and the distances separating them mutually, then that

these collinear reduced beams are then focused and crossed on each measuring point by planar, parabolic or ellipsoidal mirrors arranged at the output of said scanning head.

Advantageously, said collinear reduced beams can be focused and crossed on each measuring point of said support by two rotating optical mirrors disks respectively in horizontal and vertical directions and preferably by a deflection periscope coupled to these disks.

It will be noted that the superposition by crossing of the laser beams generates in step a) a power density at each measurement point advantageously greater than 0.5 GW.cm ^-2 , for obtaining by vaporization of a hot plasma which has a lifetime of about 2 μs Advantageously, a single crossing of two laser beams can be used to ablate each measuring point, with a surface area of between 1 μm ² and 10 000 μm ^2. The scanning of the whole measuring points, according to a fixed pitch, can thus be achieved by preferably ellipsoidal mirrors which move, focus and cross the beams on the surface to be analyzed.

Preferably, each confined plasma is associated with at least one activation agent formed of a plasmogenic gas, such as argon, helium, nitrogen or a mixture of these gases.

According to a first preferred embodiment of the invention, the extracted plasmas are respectively confined optically in integration chambers of the light emitted by the corresponding plasma, each of which has a reflecting side wall and which are each provided with at least one optical fiber for acquiring the light accumulated in this chamber, so that each plasma does not interfere with the other measurement points to be analyzed.

According to this first mode, in step a), the biological analysis support is subjected to a relative movement with respect to optical confinement chambers at the same time that said laser beams are crossed, and these chambers are preferably then guided above said support which remains fixed. Alternatively, mutual overlapping of the respective optical apertures of the acquisition optical fibers can be used by staggering them above said support, to cover the entire surface to be analyzed of the latter without relative displacement of these chambers relative to said support. .

According to a second embodiment of the invention, said optical confinement chambers of the extracted plasmas are themselves housed in a closed enclosure which contains the support, which is filled with the plasmogenic gas and whose at least the upper face is transparent to the plasma. or each excitation wavelength of the laser beams and at the wavelengths of acquisition of the light generated by this plasma. According to this second mode, it will be noted that it is possible to move said laser beams simultaneously in relative movement with respect to said support.

According to another characteristic of the invention, the technique of laser-induced optical emission spectroscopy (abbreviated "Laser Induced Breakdown Spectroscopy") for the implementation of steps a) to c is advantageously used. ) and, preferably, the laser-induced fluorescence ("LIF") technique for the same steps is used in parallel.

According to another characteristic of the invention, the plasmas can be generated by X-ray radiation of parallel rays, the source of which is preferably a femtosecond or even nanosecond X-ray laser clocked between 5 Hz and several kHz. The convergence of X-rays is obtained using mirrors (parabolic, ellipsoidal, concave) capable of reflecting X-rays, such as mirrors consisting of a silicon wafer covered by alternating layers of chromium and scandium. The incidence of the radiation is preferably grazing on the surface of the mirror (ie a radiation almost parallel to its surface), in order to allow an important reflection. In addition to the plasma analysis, the device equipped with such an X-ray source allows an analysis of the X-ray fluorescence of the segregated samples on the surface or in the thickness of the biological analysis support. Indeed, by these X-rays, the material - and especially the phosphorus - contained in the samples re-emits energy in the form, among others, of X-rays and other types of electromagnetic radiation; it is X-ray fluorescence or X-ray secondary emission.

The analysis and the mapping of this X-ray fluorescence or of these other radiations make it possible to carry out a rapid and semi-quantitative mapping of the sample on the surface of the support. This rapid analysis may allow for example to define the areas to be analyzed more finely by the technique "LIBS". The acquisition of the X-ray fluorescence can be carried out by using X-ray-doped doped polymer optical fibers in the device. The doping agents used can be x-ray luminescent compounds, and their introduction into the optical fiber transforms the X-ray radiation. in a radiation conductible by the fiber.

This same principle can be used for the selective acquisition of the emission lines of the targeted elements in the plasma, by introducing into the optical fibers of acquisition a chemiluminescent, fluorescent doping element, etc., which is excitable only at the length of waves of the targeted line and which re-emits at a wavelength on the one hand absent from the spectrum of the plasma and on the other hand well conducted by the fiber considered.

Advantageously, said biomolecular targets are chosen from the group consisting of unlabeled nucleic acids, proteins, peptides, polypeptides, polynucleotides such as DNA and sugars. It should be noted that it is generally possible to use as a target any other biological compound that can be characterized by such an element intrinsic to its structure or strongly binding thereto. In the preferred case of a probes matrix biochip support, these probes are chosen from the group consisting of nucleic acids, nucleic acid peptides ("PNA"), acids locked nucleotides ("LNAs"), ribonucleic ethers ("RNAs"), antibodies, hemi-antibodies, hemi-antibodies coupled with nucleic acids and protein receptors. It should be noted that any other biological compound that can specifically bind to a target and can be immobilized on an analysis support can generally be used as a probe.

Advantageously, said or each exogenous element (i.e. does not exist in the target structure) is deposited in known quantity on the analysis support, either homogeneously or specifically with the probes. When they are deposited with the probes, these exogenous elements are either included in compounds that will be grafted onto the support together with the probes in known proportions, or directly implanted in the structure of the probe by atomic substitution or strong interaction. In an even more advantageous mode, the exogenous elements may be included in a molecular matrix facilitating the formation of the plasma (ie substance with high optical absorbance for the excitation wavelength and low ablation energy), this matrix being homogeneously deposited on the surface of the biological analysis support.

It may be advantageous to use "PNA" probes, especially when the concentrations of the targets are very low. Indeed, the very high affinity of the "PNA" for the nucleic acids makes it possible to trap all the oligonucleotide-type targets present in the medium to be analyzed.

In addition, since the "PNA" are capable of spontaneously fixing themselves to a gold surface by their COOH or NH end, these "PNA" molecules are particularly advantageous for probe deposits on a biochip plastic support (eg polyimide Kapton®) covered with a layer of gold a few microns thick.

The fact that the "PNA" do not contain phosphorus, and more generally no atom having an emission line of 253 nm, 194 nm or 203 nm allows to increase the detection sensitivity by completely eliminating the signal of the probe. W

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According to another characteristic of the invention, said or each chemical element to be quantified may be chosen from the group consisting of phosphorus, sulfur, iodine, nitrogen, oxygen and carbon.

Advantageously, nucleic acids or phosphorylated proteins are used as targets, and the phosphorus present in these targets is used as an element to be quantified for detection in the plasma, in step b). Atomic and ionic emission lines of phosphorus. In this case, the phosphorus emission lines are detected in step b) at a value wavelength which is selected from the group consisting of 138 ± 3 nm, 148 ± 3 nm, 154 ± 3 nm, 167 ± 3 nm, 177 ± 3 nm, 190 ± 3 nm, 193 ± 3 nm, 203 ± 3 nm, 213 ± 3 nm and 253 ± 3 nm, and which is preferably 203 ± 3 nm.

Also advantageously, nucleic acids or proteins are used as targets and probes, and sulfur and iodine are used respectively in said targets and probes as an element to be quantified for detection in the plasma, in step b), of atomic and ionic emission lines of sulfur and iodine. In both of these cases, the sulfur emission lines are detected in step b) at a value wavelength which is selected from the group consisting of 167 ± 3 nm, 181 ± 3 nm, 190 ± 3 nm, 199 ± 3 nm, 415 ± 3 nm, 458 ± 3 nm, 922 ± 3 nm, 942 ± 3 nm, 968 ± 3 nm and which is preferably 415 ± 3 nm, and the emission lines of iodine at a wavelength of value which is selected from the group consisting of 150 ± 3 nm, 161 ± 3 nm, 170 ± 3 nm, 178 ± 3 nm, 183 ± 3 nm, 511 ± 3 nm, 661 ± 3 nm, 740 ± 3 nm, 746 ± 3 nm, 804 ± 3 nm, 839 ± 3 nm, 902 ± 3 nm, 905 ± 3 nm, 911 ± 3 nm and 973 ± 3 nm and which is preferably 511 ± 3 nm.

Also in these two latter cases, chlorine or bromine is advantageously used as an exogenous element for said targets and said probes, for the detection in the plasma at step b) of atomic and ionic emission lines of the sulfur and iodine, respectively, to remove the indeterminacy of the signals generated by these probes as well as by these targets. We then detect in step b): the emission lines of the chlorine at a wavelength of value selected from the group consisting of 35 ± 3 nm, 386 ± 3 nm, 413 ± 3 nm, 479 ± 3 nm, 490 ± 3 nm, 499 ± 3 nm, 507 ± 3 nm, 521 ± 3 nm, 539 ± 3 nm, 542 ± 3 nm, 774 ± 3 nm, 725 ± 3 nm, 741 ± 3 nm, 754 ± 3 nm, 822 ± 3 nm, 833 ± 3 nm nm, 837 ± 3 nm, 842 ± 3 nm, 858 ± 3 nm, 868 ± 3 nm, 894 ± 3 nm, 912 ± 3 nm, 919 ± 3 nm, 928 ± 3 nm, 945 ± 3 nm and 959 ± 3 nm. nm; or

the emission lines of bromine at a wavelength of value selected from the group consisting of 154 ± 3 nm, 158 ± 3 nm, 163 ± 3 nm, 614 ± 3 nm, 635 ± 3 nm, 655 ± 3 nm, 663 ± 3 nm, 751 ± 3 nm, 780 ± 3 nm, 793 ± 3 nm, 798 ± 3 nm, 813 ± 3 nm, 827 ± 3 nm, 844 ± 3 nm, 889 ± 3 nm, 916 ± 3 nm nm and 926 ± 3 nm.

DNA is used for said targets and in that the element to be quantified is a cation bound to these targets and selected from the group consisting of sodium, magnesium and potassium for detection. in the plasma, in step b), atomic and ionic emission lines of phosphorus. In this case, it is detected in step b):

the emission lines of sodium at a wavelength of value selected from the group consisting of 268 ± 3 nm, 285 ± 3 nm, 291 ± 3 nm, 292 ± 3 nm, 298 ± 3 nm, 314 ± 3 nm; nm, 321 ± 3 nm, 325 ± 3 nm, 330 ± 3 nm, 353 ± 3 nm, 363 ± 3 nm, 449 ± 3 nm, 466 ± 3 nm, 497 ± 3 nm, 569 ± 3 nm, 589 ± 3 nm nm, 616 ± 3 nm and 819 ± 3 nm; or

the emission lines of magnesium at a wavelength of value selected from the group consisting of 285 ± 3 nm, 880 ± 3 nm, 309 ± 3 nm, 333 ± 3 nm, 383 ± 3 nm, 457 ± 3 nm; nm, 473 ± 3 nm, 518 ± 3 nm, 552 ± 3 nm, 571 ± 3 nm, 925 ± 3 nm, 964 ± 3 nm and 941 ± 3 nm; or

the emission lines of potassium at a wavelength of value selected from the group consisting of 404 ± 3 nm, 535 ± 3 nm, 580 ± 3 nm, 693 ± 3 nm, 766 ± 3 nm, 769 ± 3 nm, 825 ± 3 nm, 850 ± 3 nm, 890 ± 3 nm and 959 ± 3 nm. According to a variant of the invention, the nitrogen and / or the carbon and / or the oxygen present in said targets and said probes are advantageously used as element (s) to be quantified for detection. in the plasma, in step b), atomic emission lines of nitrogen and / or carbon and / or oxygen to evaluate the quantity of targets and probes on said support, which contains neither carbon, neither nitrogen nor oxygen. In this case, it is detected in step b):

the lines of emission of nitrogen at a wavelength of value which is selected from the group consisting of 174 ± 3 nm, 575 ± 3 nm, 744 ± 3 nm, 821 ± 3 nm, 859 ± 3 nm , 865 ± 3 nm, 871 ± 3 nm, 938 ± 3 nm and 870 ± 3 nm, and which is preferably 575 ± 3 nm;

the carbon emission lines at a wavelength of value which is selected from the group consisting of 156 ± 3 nm, 165 ± 3 nm, 175 ± 3 nm, 193 ± 3 nm, 247 ± 3 nm, 538 ± 3 nm, 600 ± 3 nm, 711 ± 3 nm, 833 ± 3 nm, 908 ± 3 nm, 911 ± 3 nm, 965 ± 3 nm and 940 ± 3 nm, and which is preferably 600 ± 3 nm; and or

the oxygen emission lines at a value wavelength which is selected from the group consisting of 615 ± 3 nm, 645 ± 3 nm, 700 ± 3 nm, 725 ± 3 nm, 777 ± 3 nm , 822 ± 3 nm, 844 ± 3 nm and 926 ± 3 nm, and which is preferably 615 ± 3 nm.

It will be noted that the nitrogen and / or the carbon and / or the oxygen present in all the targets and biological probes as element (s) to be quantified for the detection in the plasma, in step b ), atomic emission lines of these elements, thus allows (tent) to evaluate the quantity of global material (ie probes + targets) present on the support, on the aforementioned condition that this support does not contain carbon, d nitrogen or oxygen.

In order to improve the detection limits, it is advantageous: (i) to implement the "LIBS" technique by performing a double laser pulse of the "double LIBS" type, and / or (ii) use this "LIBS" technique in parallel with the "LIF" technique (ie a "LIBS-LIF" combination), and / or

(iii) subjecting the confined plasma and / or the surface of the material to microwave radiation capable of heating the polar molecules that are biomolecules.

By means of these three preferred methods (i), (ii) and (iii), it is notably possible to induce each plasma more easily and to increase the intensity and the lifetime of the atomic emission lines of the targeted elements, thus allowing obtain a better signal-to-noise ratio and thus lower by a factor at least equal to ten the detection threshold of said element to be quantified and therefore the targets from which it is derived.

Specifically concerning this method (i) of double laser pulse, it can be implemented with two other crossed beams for the second pulse (power, frequency, wavelength), of the same nature as those of the first pulses. Indeed, given the lifetime of about 2 μs of the plasma, the latter is optically analyzable from about 100 ns after its formation (end of black body type radiation and emergence of the desired atomic and ionic lines).

This second laser pulse, shortly before its extinction, makes it possible to extend the life of the plasma and to amplify the emission emitted. It is advantageous to choose a wavelength characteristic of the atom for which the target atom (s) have a strong absorption or emission. It is thus possible to increase the light emission of the ions and / or the targeted atoms (in this case phosphorus), by exalting their own fluorescence by this second laser pulse.

According to another characteristic of the method according to the invention, ablation of material on the surface of the biological analysis support and the formation of the plasma can be decoupled. In this case, a first UV laser pulse ablates part of the surface of the biochip and expels it above it, then a second pulse advantageously using a femtosecond laser at a frequency of 600 Hz to 1 GHz creates the plasma and generates the characteristic emission of its constituents. Optionally, a third pulse can then be used to exalt the fluorescence of the targeted compounds.

The method according to the invention thus makes it possible to detect biomolecular targets efficiently, quickly and without marking, in particular from the fluorescence of the constituent atoms of the nucleic acid molecules after the emission of a plasma. In order to have a truly quantitative measurement of these nucleic acid molecules, the aforementioned calibration of the targets is necessarily carried out via standardization of their respective sizes.

A device according to the invention for implementing the aforementioned quantitative measurement method comprises:

a support of biological analysis preferably substantially planar, such as a biochip for example with matrix of probes, a transfer membrane or an electrophoresis gel or of chromatography,

a plasma generation unit which comprises means for focusing and superimposing on each measurement point at least two laser beams by simultaneously crossing these beams on this support in order to extract a hot plasma containing at least one chemical element to be quantified, such as that the phosphorus, which is present in the targets, these means for focusing and superimposing the beams comprising a scanning head adapted to shape collinearly and a mirror system arranged at the output of this head which cooperates with optical discs rotary mirrors for deflecting the beams towards the support so as to scan the measuring points of the latter,

means of optical confinement of each plasma extracted by this plasma generation unit, arranged above this support, and

a spectrography unit which is connected to these confinement means by acquisition optical fibers opening into said confinement means, and which is adapted to detect and analyze the emission light lines of the extracted plasma for each measurement point, such that the concentration in each measuring point of said one or more element (s) to be quantified from one of the intensities of these lines and from calibration curves is determined.

According to another characteristic of the invention, said means for focusing and superimposing the beams can essentially comprise:

said scanning head which comprises:

an inverted return pyramid comprising at least one stage designed to reflect tangentially incident beams emitted by several laser sources so as to make them collinear at the output of this pyramid, and

an afocal optical system arranged below the top of this pyramid, preferably a beam-reducing telescope, designed to receive these collinear beams by reducing their diameters and the distances separating them mutually, and

said system of planar, parabolic or ellipsoidal mirrors which cooperates with said rotating mirror optical disks in horizontal and vertical directions and, preferably, which furthermore cooperates with a deflection periscope coupled to these disks so that the collinear beams reduced by this afocal system are focused and crossed on each measuring point.

According to the aforementioned first embodiment of the invention, said confinement means are able to optically confine each extracted plasma and comprise a plurality of open chambers which are each delimited by a lateral wall arranged perpendicularly to said support, the internal face of this wall being able to reflect the light accumulated in this chamber and in particular the wavelengths of the lines of said chemical elements to be quantified, and at least one of said acquisition optical fibers passing through this wall.

According to a first example of this first embodiment of the invention, said means of optical confinement of each plasma can comprising a plurality of adjacent rings for integrating the light emitted by said plasma, said lateral wall being of substantially circular cross-section, the free end of several of said acquisition fibers opening inside the chamber formed by each ring through openings in said wall.

According to a second example of this first embodiment of the invention, said means of optical confinement of each plasma can comprise a plurality of integration chambers of the light emitted by this plasma, said lateral wall of each chamber being of substantially shaped section. equilateral triangle and these chambers being two by two contiguous by one of their sides each being provided with said acquisition fibers in each of their three vertices.

According to these first and second examples, means for relative displacement of the chambers with respect to the support, such as sliding rails of said chambers equipped with electromagnets, are advantageously provided to cover the entire surface to be analyzed of the support.

According to a third example of this first embodiment of the invention, said means of optical confinement of each plasma can comprise a plurality of integration chambers of the light emitted by this plasma which are equipped with at least two sets of fibers. acquisition arranged staggered, the respective optical apertures are able to cover by mutual overlap the entire surface to be analyzed said carrier.

In this third example, the device according to the invention then does not use relative displacement of the optical confinement chambers with respect to the analysis support.

According to the above-mentioned second embodiment of the invention, said optical confinement chambers of the extracted plasmas are themselves housed in a closed enclosure which contains the support and which is filled with a plasmogenic gas such as argon, helium, nitrogen or a mixture of these gases, at least the upper face of this chamber being transparent to the or each excitation wavelength of the laser beams and to the acquisition wavelengths of the light generated by this plasma, this chamber being equipped with a plasma gas filling valve and an air purge valve.

As indicated above, the biological analysis support used in connection with the device according to the invention is preferably formed of a biochip comprising on its surface a matrix of native probes, hybridized or complexed by said targets, this matrix comprising a multitude said measuring points each comprising a plurality of said probes and said or each chemical element to be quantified being present in these targets and, optionally, further in these probes.

According to another characteristic of the invention, said spectrography unit comprises at least one photomultiplier type spectrograph, and an optical filter that is only transparent at the desired wavelength can be arranged between each optical acquisition fiber and said spectrograph.

According to another preferred feature of the invention, the spectrography unit comprises at least one photomultiplier type detector. Note however that one could also use, for the detection of plasma emission lines, a camera type "CCD" or "CCD" intensified ("Charge Coupled Device", ie device coupled with charge) or a "galette" of micro-channels.

Also preferentially, an optical filter which is only transparent at the desired wavelength can be arranged between each optical acquisition fiber and the spectrograph. In a preferred embodiment, each of the optical acquisition fibers from the optical confinement means (ie each "mother" fiber) is divided into as many fibers as there are wavelengths of elements to be observed. Each "mother" fiber is disposed in a beam opposite a detector which may be a photosensitive cell such as a photomultiplier ("PM" abbreviated), a "Channel Tron", a "slab" of micro-channels, etc. An optical filter selecting only the desired wavelength can be interposed between each optical fiber acquisition and the detector, to inject the light into the latter. In addition, a suitable lens can be glued at the output of each optical fiber.

In general, it will be noted that these filters can advantageously be replaced by diffraction gratings.

The aforementioned characteristics of the present invention, as well as others, will be better understood on reading the following description of several exemplary embodiments of the invention, given by way of illustration and not limitation, said description being made in relation to the attached drawings, of which: Figure 1 is a schematic side view of a plasma generating unit which is included in a quantitative measuring device according to the invention and which is illustrated in connection with a biological analysis medium incorporating the 2 is a schematic view, partly in section and in perspective, of an optical return pyramid included in a laser scanning head of the plasma generating unit of FIG. 1, FIG. is a schematic perspective view of a simplified variant of the deflection pyramid according to FIG. 2, FIG. 4 is a diagrammatic part view 1 of the analysis support of FIG. 1 which is surmounted by means of optical confinement of the plasmas respectively generated at various measurement points, according to an example of a first embodiment of the invention, FIG. schematic view in vertical section along the sectional plane VV of FIG. 4 of one of these confinement means, FIG. 6 is a diagrammatic view in vertical section according to a variant of FIG. 5 of one of these means of optical confinement arranged above the analysis support, FIG. 7 is a schematic view in vertical section according to another variant of FIG. 5 of such optical confinement means, FIG. 8 is a schematic view in vertical section. e according to another variant of FIG. 5 of such optical confinement means, FIG. 9 is a partial schematic view from above of the analysis support of FIG. 1 surmounted by means of optical plasma confinement means according to another example of this first embodiment of the invention, FIG. 10 is a partial schematic view from above. of the analysis support of FIG. 1 surmounted by means of optical confinement of the plasmas according to another example of this first embodiment of the invention, FIG. 11 is a partial schematic view from above of the analysis support of FIG. 1 surmounted optical confinement means according to the example of Figure 9, further illustrating control members of the displacement of these confinement means relative to this support in a given position, Figure 12 is a view similar to Figure 11 showing these control members of the displacement of the confinement means in another operating position, and FIG. 13 is a schematic side view of the analysis support of FIG. 1 which is housed in a Plasma confinement means according to a second embodiment of the invention, which enclosure is illustrated with a portion of the adjacent plasma generating unit.

The quantitative measuring device 1 according to the invention of biomolecular targets comprises:

a preferably substantially planar biological analysis support 2, such as a biochip, for example with a probe array, a transfer membrane or an electrophoresis or chromatography gel, a plasma generation unit 3 (see FIG. 1) which comprises means 4 for focusing and superimposing on each measurement point at least two laser beams by simultaneously crossing these beams on this support 2 to extract a hot plasma P (visible in Figures 5 and 13) containing at least one chemical element to quantify present in these targets, means of confinement 5 of each plasma P extracted by this unit 3, arranged above the support 2 (see FIG. 4 and following), and

a spectrography unit (not shown) which is connected to these confinement means 5, 5 ', 5 "by acquisition optical fibers 6,

6 ', 6 "(visible in FIGS. 4 et seq.) Opening into these confinement means 5, 5', 5", and which is adapted to detect and analyze luminous emission lines of the extracted plasma P for each measurement point , so that the concentration in each measurement point of the element to be quantized is determined from one of the intensities of these lines and from calibration curves.

The focusing means 4 of the laser beams F on each measurement point essentially comprise, with reference to FIG. 1:

a scanning head 7 which is able to shape these bundles F collinearly and which comprises:

an inverted return pyramid 8 comprising at least one stage 8a designed to tangentially reflect incident F beams emitted by several laser sources 9 so as to make them collinear at the output of this pyramid 8 via high quality aluminum mirrors 8b, and

an afocal optical system 10 arranged below the top of this pyramid 8, preferably a beam-reducing telescope, which is designed to receive these collinear beams F 'by reducing their respective diameters and the distances separating them mutually, and

a system of planar, parabolic or ellipsoidal mirrors 11 which is arranged at the output of the scanning head 7 and which cooperates with rotating optical discs 12 and 13 with mirrors 12a and 13a which deviate them in horizontal and vertical directions, and preferably which furthermore cooperates with a deflection periscope 14 coupled to these disks 12 and 13 so that the Collinear bundles F 'reduced by this afocal system 10 are focused and crossed on each measurement point.

This scanning head 7 is used as follows.

The different laser beams F (two in number in the example of FIG. 1) are reflected tangentially on the high-quality mirrors 8b of the return pyramid 8 which ensure their collinearity, for any angle of the direction of incidence. of these bundles F

(preferentially, this angle is equal to 90 °).

As illustrated in FIGS. 2 and 3, the reference pyramid 8, 8 'may have a single stage 8a (FIG. 3) using, for example, four distinct laser sources 9, or several stages 8a that can typically be up to five (FIG. 2) using in this case for example a maximum of one hundred laser sources 9 in parallel.

It should be noted that the increase in the number of sources 9 makes it possible in particular to increase the resolution of the scan, with a greater spatial coverage of the beams on the scanning zone.

On leaving the pyramid 8, the collinear beams F 'pass through the beam reduction telescope 10, consisting of a multiple lens system superimposed reducing the diameter and the mutual distance of these collinear beams F'. For example, bundles F 'with a diameter of 2 mm and spaced 2.5 mm at the exit of the pyramid 8 are reduced, at the output of the telescope 10, to a diameter of 500 microns and a spacing of 500 microns.

These collinear bundles F 'once reduced are then scanned on the analysis support 2 by the two rotating horizontal optical discs 12 and vertical 13 mirrors 12a and 13a ("ORD" for "optical rotating dise") respectively providing deviations horizontal and vertical bundles, and the periscope of deviation 14. These optical disks 12 and 13 can be according to the needs of monopist or multitrack type (ie with one or more concentric rows of mirrors), depending on the number of sources 9, congestion, resolution, etc. The measuring device 1 according to the invention furthermore comprises a system of electronic control (not shown) for precisely controlling and controlling the rotational speeds of these optical discs 12 and 13.

It will be noted that the reduction of the optical pattern of the laser beams F 'by the telescope 10 enables all these beams F' to be reflected on the single mirror 12a of the horizontal rotating disk 12 and the only mirror 13a of the vertical rotary disk 13, in order to converging them to a point identical to the surface of the support 2. Each mirror 12a, 13a is indeed positioned uniquely on the rotating discs 12 and 13 in terms of angle of inclination, so as to converge the different beams F "on a well localized area of support 2.

For reasons of space, the distance between the two rotating discs 12 and 13 is preferably reduced by adding the deflection periscope 14 which adds two additional reflections on the path of the beams. The focusing of the different beams F "on the support 2 can be ensured by the afocal system 10, by playing in particular on the spacing of the optics, by parabolic mirrors which are positioned on the vertical rotating optical disk 13 located at the exit of the head 7, by an auxiliary system composed of lenses and / or focusing mirrors at the exit of the scanning head 7, playing on the inclination of the mirrors of the pyramid to make the beams F 'intended to cross slightly collinear.

In some embodiments, with a configuration using groups of at least two beams, only the beams of each group to be crossed on the medium 2 will be collinear, so that only the collinear beams of each group converge in one. same point of support 2.

At the exit of this scanning head 7, each laser beam F "is focused on the surface to be analyzed of the support 2 by the mirrors of the head

7 and the beams are crossed in at least one measurement point of this surface. The crossing of each beam has a density of power or irradiance at the surface of each measuring point which is greater than 0.5 GW.cm ^'2 which is sufficient for obtaining by vaporisation a hot plasma P with a lifetime of approximately 2 μs, so general to a power threshold per unit area greater than the ablation threshold of the support material.

At these very high energy densities, part of the material of each measuring point is indeed ejected from the surface to be analyzed by vaporization and this hot plasma P, very bright and very short life, is thus generated. This ablated material in the form of plasma P is dissociated into its various atomic and ionic constituents and, at the end of each laser pulse, this plasma P cools rapidly. During this period, the excited atoms and ions emit light radiations that are characteristic of them, because of their return to lower energy levels.

FIGS. 4 to 12 illustrate examples of structures that can be used for the optical confinement means 5, 5 ', 5 "of each generated plasma P, so that it does not interfere with the other measurement points to be analyzed, before simultaneous detection emission lines of the plasma P corresponding to each beam crossing As previously indicated, the optical analysis of the plasma P generated point by point on the surface of the biological analysis support 2 makes it possible to reconstitute an image at each measurement point the quantity of atoms of the element analyzed, allowing to deduce the number of targets according to the abundance of the element in the raw formula of the target.

As illustrated in FIG. 4, the emission lines of the plasma P generated at each measurement point may, for example, be picked up by a plurality of optical acquisition fibers 6 (at least three optical fibers 6), the respective free ends of which are arranged. regularly on a circular ring 5 of 1 to 20 cm in diameter and reduced in height which is designed to optically isolate the plasma P. The inner face of the side wall 5a, 5b, 5c of this ring 5 is a concave mirror (see FIGS. 5 and 6), multi-faceted (see FIG. 7), plane (FIG. 8), ellipsoidal or even parabolic, provided that it is capable of reflecting the wavelengths of the lines of the elements of interest by forming thus a ring of integration of the emitted light. The curvatures or inclinations of the mirrors are preferably designed to converge the light on the opposite inner face of the ring 5.

The ring 5 is pierced at regular intervals with orifices 5d on its side wall 5a, 5b, 5c (eg cylindrical in the example of FIG. 5) to receive the acquisition optical fibers 6. Because of its numerical aperture each acquisition fiber 6 may be capped with a set of suitable lenses, such as a fiber collimator, to correctly inject the radiation emitted by the plasma P into the spectrography unit, the fields respectively seen by the fibers 6 and capped with corrective lenses, or optical apertures, overlapping each other to form a surface comparable to a circle (or to a polygon inscribed in a circle), ie an optical circle 16. Each plasma P is generated in this optical circle 16, which is such that the cumulative optical path of the light of the plasma P to the fibers 6 is constant regardless of the position of the plasma P in this circle 16. In the variant of FIG. 9, the chambers 5 'of optical confinement of the sma P each have an equilateral triangle shape and each have the same type of internal side wall face 5a 'as the aforementioned rings 5. At the three vertices of each triangle 5 'three optical acquisition fibers 6' are respectively arranged, each with an optical opening greater than or equal to 60 °, so as to capture the direct light of the corresponding plasma P and that which is reflected on the walls of the triangle 5 '. It is advantageous to arrange a series of such equilateral triangles 5 'so as to describe a parallelogram encompassing the entire surface of the support 2. Thus, the laser beams, crossed, converged and displaced by the aforementioned mirrors, scan with the desired pitch all each optical circle 16 according to FIG. 4 or each optical triangle 5 'according to the FIG. 9, generating at each point a plasma P whose light is captured by all the acquisition fibers 6, 6 'associated. It will be noted that the cumulative light that is captured by the fibers 6 of each optical circle 16 or those 6 'of each optical triangle 5' has an intensity independent of the position of the plasma P in this circle 16 or optical triangle 5 ', respectively.

This method makes it possible to scan the entire surface to be analyzed by the crossed laser beams with, for example, a pitch of 10 μm which corresponds to the zone ablated at each laser pulse, the optical confinement chambers 5, 5 'allowing the acquisition simultaneous multiple plasmas P.

To allow the analysis of the surfaces directly above the walls 5a, 5b, 5c or 5a 'of these optical confinement chambers 5, 5', it is necessary to carry out a relative displacement of the biological analysis support 2 with respect to these chambers 5, 5 'which, preferably, is made by moving the latter relative to the analysis support 2 which remains fixed.

As illustrated in FIGS. 11 and 12, these optical confinement chambers 5 '(in this triangular example) can slide along guides or rails 17 via support members 17a of the latter arranged on each side of the analysis support 2, under the control of two electromagnets 18 which are arranged outside and on either side of the support 2 and which alternately attract these chambers 5, 5 ', respectively in association with two metal stops 17b for these electromagnets 18.

In the variant of FIG. 10, the surface of the support 2 is analyzed without relative displacement of the optical confinement chambers 5 "with respect to the support 2, staggering the acquisition optical fibers 6" laterally on the one hand. and second of the support 2. By this arrangement, the set of respective optical openings of the acquisition fibers 6 "encompasses the entire surface of the support 2. A slight cover 19 of the optical openings of the optical fibers 6" laterally on either side of the support 2 thus allows the analysis of the surfaces located above the limits of these optical openings. According to another embodiment of the invention illustrated in FIG. 13, the support 2 to be analyzed as well as the above-mentioned means 5, 5 'of optical confinement (in this example constituted by the rings 5) are all arranged in a closed enclosure 20 , at least the upper face 21 of which is provided transparent to the excitation wavelengths of the laser beams and to the acquisition wavelengths of the light generated by this plasma P.

The air contained in this chamber 5 is emptied by means of an air purge valve

22, then the chamber is filled with a plasmogenic gas (e.g. argon / nitrogen, helium) through a filling valve 23, these valves 22 and 23 being mounted in the lower support surface 24 of the enclosure 20.

In conclusion, it will be noted that the optical analysis of the plasma P generated point by point on the surface of the support 2 makes it possible to reconstitute an image of the quantity of atoms of the desired element at each measurement point.

By using more than two lasers, it is also possible to simultaneously generate several P plasmas on the biological support 2 to be analyzed. Under these conditions, an optical confinement unit is provided by generated plasma. The areas of the support 2 masked by this unit can be analyzed after a relative translation of these areas within the optical confinement unit, such as the above-mentioned circle 5 or optical triangle 5 '.

It will be noted that the geometries described for the "LIBS" mapping of the biological analysis supports are also applicable to "LIBS" mapping of relatively flat surfaces comprising non-biological supports (inorganic or organic) to be analyzed.

Claims

1) Method for high-speed quantitative measurement of biomolecular targets, in particular proteins, present on or in a biological analysis support (2), characterized in that it comprises the following steps: a) a scanning of measurement points of the support by moving, focusing and superimposing on each measurement point at least two laser beams (F ") by simultaneous crossings of these beams to extract a hot plasma (P) containing at least one chemical element to quantify present in said targets and at least one other chemical element exogenous to these targets and present in a known quantity on or in this support, b) for detecting and analyzing, for each of these measuring points, light emission lines of each plasma which corresponds to the or each element to be quantified and to the or each exogenous element, by measuring the respective intensities of these lines, then c) is determined, via a calibration preliminaries of the lines of the or each element to be quantified establishing a correlation between the intensities of the lines specific to this element to be quantified and the concentrations of the latter in mixtures in known proportions of the or each element to be quantified and the or each element exogenously, the concentration in each measurement point of the or each element to be quantified or a group incorporating it within these targets.

2) Method according to claim 1, characterized in that it further comprises a subsequent step of obtaining an image representative of the concentration of said or each element to be quantified or said or each exogenous element, the intensity of each pixel or each of the three colors of the latter within this image being representative of the intensity of the line of the corresponding element observed. 3) Method according to claim 1 or 2, characterized in that each of said laser beams (F) used in step a) is emitted in the infrared - visible - ultraviolet range according to a pulse of duration between 1 fs and 100 ns , with a frequency between 600 Hz and 1 GHz and an energy between 0.05 mW and 1 kW.

4) Method according to claim 3, characterized in that said laser beams (F) are emitted in a pulse of less than 10 ns duration and preferably equal to 5 ns, for example in the ultraviolet range to the same length of wave of 266 nm or 193 nm, ie in the ultraviolet at 266 nm and in the visible at 532 nm.

5) Method according to one of the preceding claims, characterized in that said beams (F ") are shaped in step a) in a scanning head (7), so that:

these beams are reflected tangentially on a return pyramid (8) to at least one reflection stage (8a) to give collinear beams (F ') at the output of this pyramid,

these collinear beams pass through an afocal optical system (10), such as a beam reduction telescope, which reduces the respective diameters of these beams and the distances separating them mutually, then that

these collinear reduced beams are then focused and crossed on each measuring point by flat, parabolic or ellipsoidal mirrors (11) arranged at the output of said scanning head.

6) Process according to claim 5, characterized in that said collinear bundles (F ') reduced are focused and crossed on each measuring point of said support (2) by two rotating optical discs (12 and 13) with mirrors (12a and 13a). ) deviating respectively in horizontal and vertical directions and preferably by a deflection periscope (14) coupled to these discs. 7) Method according to claim 5 or 6, characterized in that the superposition by crossing said laser beams (F ") implemented in step a) generates a power density at each measurement point which is greater than 0, 5 GW.cnrf ² , for obtaining by vaporization of each hot plasma (P) which has a lifetime of about 2 μs.

8) Method according to one of the preceding claims, characterized in that is added to each plasma (P) confined at least one activating agent formed of a plasma gas, such as argon, helium, nitrogen or a mixture of these gases.

9) Method according to one of the preceding claims, characterized in that said extracted plasmas (P) are respectively confined optically in chambers (5, 5 ', 5 ") of integration of the light emitted by the corresponding plasma, each of which reflective lateral wall (5a, 5b, 5c, 5a ') and each of which is provided with at least one optical acquisition fiber (6, 6', 6 ") of the light accumulated in this chamber, so that each plasma does not interfere with other measurement points to be analyzed.

10) Method according to claim 9, characterized in that during step a) said biological analysis support (2) is subjected to relative movement with respect to the optical confinement chambers (5, 5 '), which are preferably guided above said support which remains fixed, at the same time that said laser beams (F ") are crossed.

11) A method according to claim 9, characterized in that one uses a mutual overlap (19) of the respective optical openings of said acquisition optical fibers (6 "), arranging the latter staggered above the support (2). ) to cover the entire surface to analyze the latter without relative movement of these chambers (5 ") relative to said support.

12) Process according to claim 9, characterized in that said optical confinement chambers (5 ') of the extracted plasmas (P) are themselves housed in a closed enclosure (20) which contains said support (2), which is filled said plasmogenic gas and at least the upper face (21) is transparent to the or each excitation wavelength of said laser beams and wavelengths of acquisition of the light generated by the plasma.

13) Method according to one of the preceding claims, characterized in that the technique of laser-induced optical emission spectroscopy ("LIBS") is used for the implementation of steps a) to c), preferably with parallel use of the laser induced fluorescence ("LIF") technique.

14) Method according to one of the preceding claims, characterized in that said biomolecular targets are selected from the group consisting of non-labeled nucleic acids, proteins, peptides, polypeptides, polynucleotides such as I ¹ DNA and sugars.

15) Method according to one of the preceding claims, characterized in that it immobilizes prior to step a) said biomolecular targets on the surface or in the thickness of said support (2), which is substantially planar and is selected in the group consisting of biochips, transfer membranes, substrates made of silicon, polyimide and glass, and electrophoresis and chromatography gels.

16) Method according to claim 15, characterized in that said biological analysis support (2) is formed of a biochip comprising at least its surface a matrix of native probes, hybridized or complexed by said targets, this matrix comprising a multitude of said measuring points each comprising a plurality of said probes and said or each chemical element to be quantified being present in said targets and, optionally, furthermore in said probes.

17) The method of claim 16, characterized in that said probes are selected from the group consisting of nucleic acids, nucleic acid peptides ("PNA"), locked nucleic acids ("LNA"), ribonucleic ethers (" ERN "), antibodies, hemi-antibodies, hemi-antibodies coupled with nucleic acids and protein receptors.

18) Method according to claim 16 or 17, characterized in that said biomolecular targets are immobilized before step a) by probe / target interactions which are:

both protein-based, such as ligand / receptor or antibody / fixed antigen interactions, or

- mixed interactions nucleic acid or assimilated / protein, or

interactions of the nucleic acid / metal, ion or other free molecule type, or

protein / ion interactions or any other free molecule.

19) Method according to one of the preceding claims, characterized in that is deposited homogeneously said or each exogenous element in known amount on said analysis medium (2).

20) Method according to one of claims 16 to 18, characterized in that said or each exogenous element is deposited in a known quantity on said analysis support (2) specifically with said probes, this exogenous element being either included in compounds grafted on this support together with the probes in known proportions, or directly implanted in the structure of each probe by atomic substitution or strong interaction.

21) Method according to one of the preceding claims, characterized in that said or each chemical element to be quantified is selected from the group consisting of phosphorus, sulfur, iodine, nitrogen, oxygen and carbon.

22) Method according to claim 21, characterized in that nucleic acids or phosphorylated proteins are used as targets, and in that the phosphorus present in these targets is used as an element to be quantified for the detection in the plasma, in step b), of atomic and ionic emission lines of phosphorus.

23) Method according to claim 22, characterized in that in step b) the phosphorus emission lines are detected at a value wavelength which is selected from the group consisting of 138 + 3 nm, 148 ± 3 nm, 154 ± 3 nm, 167 ± 3 nm, 177 ± 3 nm, 190 ± 3 nm, 193 ± 3 nm, 203 ± 3 nm, 213 ± 3 nm and 253 ± 3 nm, and which is preferably 203 ± 3 nm.

24) Method according to one of claims 16 to 18 and one of claims 21 to 23, characterized in that one uses nucleic acids, for example labeled with sulfur or iodine, or proteins as targets and of probes, and that sulfur and iodine are used respectively in said targets and said probes as an element to be quantified for the detection in the plasma (P), in step b), of lines Atomic and ionic emission of sulfur and iodine.

25) A method according to claim 24, characterized in that it is detected in step b) the emission lines of sulfur at a wavelength which is selected from the group consisting of 167 ± 3 nm, 181 ± 3 nm, 190 ± 3 nm, 199 ± 3 nm, 415 ± 3 nm, 458 ± 3 nm, 922 ± 3 nm, 942 ± 3 nm, 968 ± 3 nm and which is preferably 415 ± 3 nm, and the emission lines of iodine at a value wavelength which is selected from the group consisting of 150 ± 3 nm, 161 ± 3 nm , 170 ± 3 nm, 178 ± 3 nm, 183 ± 3 nm, 511 ± 3 nm, 661 ± 3 nm, 740 ± 3 nm, 746 ± 3 nm, 804 ± 3 nm, 839 ± 3 nm, 902 ± 3 nm , 905 ± 3 nm, 911 ± 3 nm and 973 ± 3 nm and which is preferably 511 ± 3 nm.

26) Method according to claim 24 or 25, characterized in that chlorine or bromine is used as an exogenous element for said targets and said probes for detection in the plasma (P) in step b) Atomic and ionic emission lines of sulfur and iodine, respectively, to remove the indeterminacy of the signals generated by these probes as well as by these targets.

27) Method according to claim 26, characterized in that in step b):

the emission lines of the chlorine at a wavelength of value selected from the group consisting of 35 ± 3 nm, 386 ± 3 nm, 413 ± 3 nm, 479 ± 3 nm, 490 ± 3 nm, 499 ± 3 nm, 507 ± 3 nm, 521 ± 3 nm, 539 ± 3 nm, 542 ± 3 nm, 774 ± 3 nm, 725 ± 3 nm, 741 ± 3 nm, 754 ± 3 nm, 822 ± 3 nm, 833 ± 3 nm nm, 837 ± 3 nm, 842 ± 3 nm, 858 ± 3 nm, 868 ± 3 nm, 894 ± 3 nm, 912 ± 3 nm, 919 ± 3 nm, 928 ± 3 nm, 945 ± 3 nm and 959 ± 3 nm. nm; or else the emission lines of bromine at a wavelength of value selected from the group consisting of 154 ± 3 nm, 158 ± 3 nm, 163 ± 3 nm, 614 ± 3 nm, 635 ± 3 nm, 655 ± 3 nm, 663 ± 3 nm, 751 ± 3 nm, 780 ± 3 nm, 793 ± 3 nm, 798 ± 3 nm, 813 ± 3 nm, 827 ± 3 nm, 844 ± 3 nm, 889 ± 3 nm, 916 ± 3 nm and 926 ± 3 nm.

28) Method according to claim 14 and claim 22 or 23, characterized in that one uses for said targets of DNA and in that a cation bound to these targets is used as the element to be quantified and selected from the group consisting of sodium, magnesium and potassium for detection in the plasma, at the same time as step b), atomic and ionic emission lines of phosphorus.

29) Method according to claim 28, characterized in that it is detected in step b):

the emission lines of sodium at a wavelength of value selected from the group consisting of 268 ± 3 nm, 285 ± 3 nm, 291 ± 3 nm, 292 ± 3 nm, 298 ± 3 nm, 314 ± 3 nm; nm, 321 ± 3 nm, 325 ± 3 nm, 330 ± 3 nm, 353 ± 3 nm, 363 ± 3 nm, 449 ± 3 nm, 466 ± 3 nm, 497 ± 3 nm, 569 ± 3 nm, 589 ± 3 nm nm, 616 ± 3 nm and 819 ± 3 nm; or

the emission lines of magnesium at a wavelength of value selected from the group consisting of 285 ± 3 nm, 880 ± 3 nm, 309 ± 3 nm, 333 ± 3 nm, 383 ± 3 nm, 457 ± 3 nm; nm, 473 ± 3 nm, 518 ± 3 nm, 552 ± 3 nm, 571 ± 3 nm, 925 ± 3 nm, 964 ± 3 nm and 941 ± 3 nm; or

the emission lines of potassium at a wavelength of value selected from the group consisting of 404 ± 3 nm, 535 ± 3 nm, 580 ± 3 nm, 693 ± 3 nm, 766 ± 3 nm, 769 ± 3 nm, 825 ± 3 nm, 850 ± 3 nm, 890 ± 3 nm and 959 ± 3 nm.

30) Method according to one of claims 16 to 18 and according to one of claims 21 to 29, characterized in that one uses the nitrogen and / or carbon and / or oxygen present (s) in said targets and said probes as element (s) to be quantified for the detection in plasma (P), in step b), of the atomic emission lines of nitrogen and / or carbon and / or oxygen for evaluating the quantity of targets and probes on said support (2), which contains no carbon, nitrogen or oxygen.

31) Method according to claim 30, characterized in that in step b): the lines of emission of nitrogen at a wavelength of value which is selected from the group consisting of 174 ± 3 nm, 575 ± 3 nm, 744 ± 3 nm, 821 ± 3 nm, 859 ± 3 nm , 865 ± 3 nm, 871 ± 3 nm, 938 ± 3 nm and 870 ± 3 nm, and which is preferably 575 ± 3 nm; the carbon emission lines at a value wavelength which is selected from the group consisting of 156 + 3 nm, 165 ± 3 nm, 175 ± 3 nm, 193 ± 3 nm, 247 ± 3 nm, 538 ± 3 nm, 600 ± 3 nm, 711 ± 3 nm, 833 ± 3 nm, 908 ± 3 nm, 911 ± 3 nm, 965 ± 3 nm and 940 ± 3 nm, and which is preferably 600 ± 3 nm; and / or - the emission lines of oxygen at a wavelength of value which is selected from the group consisting of 615 ± 3 nm, 645 ± 3 nm, 700 ± 3 nm, 725 ± 3 nm, 777 ± 3 nm, 822 ± 3 nm, 844 ± 3 nm and 926 ± 3 nm, and which is preferably 615 ± 3 nm.

32) Device (1) for implementing a quantitative measurement method according to one of the preceding claims, characterized in that it comprises:

a preferably substantially biological analysis support (2), such as a biochip, for example a probe array, a transfer membrane or an electrophoresis or chromatography gel,

a plasma generation unit (3) comprising means (4) for focusing and superimposing on each measurement point at least two laser beams (F ") by simultaneously crossing these beams on this support to extract a hot plasma therefrom (P) containing at least one chemical element to be quantified, such as phosphorus, which is present in said targets, these means for focusing and superimposing the beams comprising a scanning head (7) able to shape them in a collinear manner and a mirror system (11) arranged at the output of this head which cooperates with rotating optical discs (12 and 13) with mirrors (12a and 13a) to deflect the beams towards the support so as to scan the measuring points of the latter , means for optically confining (5, 5 ', 5 ") each plasma extracted by this plasma generation unit, which are arranged above this support, and

a spectrography unit which is connected to these confinement means by acquisition optical fibers (6, 6 ', 6 ") opening into said confinement means, and which is adapted to detect and analyze emission light lines; extracted plasma for each measuring point, so that the concentration in each measurement point of said one or more element (s) to be quantified from one of the intensities of these lines and from calibration.

33) Device (1) according to claim 32, characterized in that said means (4) for focusing and superimposing by crossing on said support (2) the laser beams (F ") on each measuring point essentially comprise:

said scanning head (7) which comprises:

* a return pyramid (8) returned comprising at least one stage (8a) designed to reflect tangentially incident beams (F) emitted by several laser sources (9) so as to make them collinear output of this pyramid, and

an afocal optical system (10) arranged below the apex of this pyramid, preferably a beam reducing telescope, which is adapted to receive these collinear beams (F ') by reducing their respective diameters and the distances separating them mutually, and

- said system of planar, parabolic or ellipsoidal mirrors (11) which cooperates with said rotating optical discs (12 and 13) with mirrors (12a and 13a) deviating them in horizontal and vertical directions and, preferably, which further cooperates with a deflection periscope (14) coupled to these disks so that collinear beams reduced by this afocal system are focused and crossed on each measurement point. 34) Device (1) according to claim 32 or 33, characterized in that said optical confinement means (5, 5 ', 5 ") are capable of optically confining each extracted plasma (P) and comprise a plurality of open chambers which are each delimited by a lateral wall (5a, 5b, 5c, 5a ') arranged perpendicular to said support (2), the inner face of this wall being able to reflect the accumulated light in this chamber and in particular the wavelengths of the lines of said chemical elements to be quantified, and at least one of said acquisition optical fibers (6, 6 ', 6 ") passing through this wall.

35) Device (1) according to claim 34, characterized in that said means of optical confinement of each plasma (P) comprise a plurality of adjacent rings (5) of integration of the light emitted by said plasma, said side wall (5a, 5b, 5c) being of substantially circular section, the free end of several of said acquisition fibers (6) opening inside the chamber formed by each ring by orifices (5d) formed in said wall, relative displacement means of these chambers with respect to the support (2), such as sliding rails (17) chambers equipped with electromagnets (18), being provided to cover the entire surface to be analyzed said support.

36) Device (1) according to claim 34, characterized in that said optical confinement means of each plasma (P) comprise a plurality of integration chambers (5 ') of the light emitted by said plasma, said side wall ( 5a ¹ ) of each chamber being of substantially equilateral triangle-shaped section and these chambers being two adjacent to each other by one of their sides each being provided with said acquisition fibers (6 ') in each of their three vertices, means for relative displacement of the chambers with respect to the support (2), such as sliding rails (17) of said chambers equipped with electromagnets (18), being provided to cover the entire surface to be analyzed of said support. 37) Device (1) according to claim 34, characterized in that said means of optical confinement of each plasma (P) comprises a plurality of integration chambers (5 ") of the light emitted by this plasma which are equipped with at least two sets of staggered acquisition fibers (6 "), whose respective optical apertures are able to cover by mutual overlap (19) the entire surface to be analyzed of said support (2), without relative displacement of these chambers by report to the latter.

38) Device (1) according to one of claims 32 to 36, characterized in that said optical confinement chambers (5 ¹ ) extracted plasmas (P) are themselves housed in a closed enclosure (20) which contains said support ( 2) and which is filled with a plasmogenic gas such as argon, helium, nitrogen or a mixture of these gases, at least the upper face (21) of this enclosure being transparent to the each excitation wavelength of the laser beams and at the wavelengths of acquisition of the light generated by this plasma, this chamber being equipped with a filling valve (23) with plasmogenic gas and a valve of bleeding air (22).

39) Device (1) according to one of claims 32 to 38, characterized in that said spectrography unit comprises at least one spectrograph of photosensitive cell type and in that a diffraction grating or at least one optical filter transparent only to the The desired wavelength is arranged between each acquisition optical fiber (6, 6 ', 6 ") and said spectrograph.

40) Device (1) according to one of claims 32 to 39, characterized in that said biological analysis support (2) is formed of a biochip comprising on its surface a matrix of native probes, hybridized or complexed by said targets, this matrix comprising a multitude of said measuring points each comprising a plurality of said probes and said or each chemical element to be quantified being present in said targets and, optionally, further in said probes.