FR2874695A1 - Proteomics analyzing method for liquid sample with protein and solvent mixture, involves comparing results of micro-pattern examination with reference data to produce comparison data that is processed to produce sample analyzing information - Google Patents

Abstract

The method involves auto-forming micro-patterns on a support from a sample having a mixture of protein and a solvent. The micro-patterns and/or effects induced during and/or after their formation are examined to produce examination results. The results are compared with reference data to produce comparison data. The comparison data are processed to produce analyzing information of the sample. The reference data are produced by applying previously the sequence of preparation and examination to a mixture of a reference substance and the solvent in predetermined proportions. An independent claim is also included for a system for analyzing a sample comprising a mixture of a protein and a solvent.

Description

The present invention relates to a method of chemical and / or biological analysis of a sample comprising a mixture of at least one protein and a solvent.
The invention also relates to a system for implementing such a method of analysis. The samples to be analyzed may furthermore comprise non-organic or organic molecules, mixtures of proteins or macromolecular biological systems or mixtures of such molecules. It is also conceivable to analyze other products in the form of co-solutes present in the sample.
The invention makes it possible in particular to analyze the proteins, alone or as a mixture, in a sample. This type of analysis, known as proteomic analysis aims, for example, the identification of proteins present in a biological sample, the detection of mutated proteins, the characterization of posttranslational modifications of proteins, such as phosphorylations , identifying protein interactions or detecting proteins that are differentially expressed in at least two biological samples. Proteomic analysis is particularly important in the field of animal and plant biology because it allows the comprehension of biological processes in their entirety. In the field of human and veterinary medicine, it allows, for example, the discovery of markers of diseases that are useful. for the development of early diagnosis methods and effective treatment methods. Proteomic analysis is also used in the development of new drugs, particularly for the characterization of their pharmacological, therapeutic or toxicological properties.
The invention can also be advantageously used in the field of food because it allows, for example, to detect the presence of toxic or allergenic substances as well as in the field of the environment, for example, for the detection of pollutants.
Protein analysis requires several steps, including purification, separation, characterization, and identification steps. The methods used for these different steps are based on the physical, chemical or biological properties of the proteins and require the use of often cumbersome and complex techniques. For example, one- or two-dimensional electrophoresis techniques or liquid chromatography are used to separate the proteins contained in a sample. Classically, different techniques of mass spectrometry or biological recognition techniques, based for example on antigen-antibody interactions, for the identification of proteins are conventionally used.
All these known techniques are generally complex and expensive and require in-depth know-how from the professionals who implement them.
The known analysis techniques also have the disadvantage of providing results that vary significantly depending on the operating conditions. The great variability of the results which characterizes the solutions in the state of the art makes difficult the work in collaboration or the comparisons between works of different origin. The method according to the invention offers the additional advantage of a significantly lower dependence on the operating conditions than in the case of the other known methods and thus offers possibilities for standardization and rationalization of analyzes meeting a need. often expressed by the professionals concerned.
Finally, known analysis techniques often require the proteins to be analyzed to be labeled with probes, for example fluorescent or radioactive probes. The use of such probes increases the duration and costs of the analysis and requires specialized knowledge from the testing laboratory.
The foregoing reasons limit the methods of proteomic analysis known to research applications and practically exclude their large-scale use, especially for diagnostic purposes in the context of effective preventive medicine.
There is therefore an important need for a method and an apparatus which allow a rapid and reliable analysis by inexpensive means which can be used on a large scale for example to diagnose pathologies by the analysis of body fluids or aerosols, of tissues organic or for food or feed control applications. This is the object of the present invention.
The invention is based on the discovery that it is possible to form characteristic micro-motifs in liquid samples comprising at least one protein and deposited on a surface, and that such micro-motifs can be associated in a one-to-one manner with the presence of a protein, a protein complex, a mixture of proteins or a chemical or biochemical co-solution in the sample.
The method according to the invention allows the analysis of a sample comprising a mixture of at least one protein and a solvent. It comprises: a preparatory and examination sequence comprising: a step of depositing the sample on a support having a predetermined surface state; a self-training step, starting from the sample, of a microorganism; patterns on the support, and - a step of examining said micro-patterns and / or effects induced during and / or after their formation, so as to produce examination results, said preparation and examination sequence having has previously been applied to a mixture of a reference substance and said solvent in predetermined proportions, so as to produce reference data, and - a treatment sequence comprising: - a step for comparing said examination results with said data reference, so as to produce comparison data, and - a step for processing said comparison data, so as to produce analysis information of said sample.
The conditions which determine the self-formation of the micro-motifs according to the invention in the first step are preferably dictated by the conditions previously used to obtain the reference results.
It is thus possible, by reproducing specific appearance conditions of characteristic micro-patterns in the liquid sample deposited on the surface of the support, to compare the information produced by an analysis system with similar information associated one-to-one with the presence of a protein, a protein complex, a mixture of proteins or a known co-solute.
Surprisingly, it has been found that such micro-patterns take a form that depends on the constituents of the mixture and the environmental parameters during their formation. It is for this reason that it is necessary to control the environmental parameters, at least those whose influence is preponderant, by giving them the value which prevailed during the reference analysis, to be able to be in the conditions of a one-to-one correspondence between the micro-patterns to be evaluated and those of reference.
The micro-motifs are formed in the deposition on the surface of the support, the mixture of at least one protein in its native form or in its post-translational or mutated forms, said protein preferably consisting of at least 20 amino acids . The sample may optionally also include other non-organic substances or molecules, organic or super-molecular assembly.
The micro-motifs materialize during the liquid / solid separation that takes place within the sample, during a precipitation in a medium which remains liquid or during the elimination of the liquid phase, by example by drying the mixture under conditions of temperature and possibly pressure determined.
The micro-motifs constitute an organized and characteristic self-assembly of the material that precipitates on the deposition surface.
The nature of the micro-patterns according to the invention thus formed is sensitive to many parameters that must be controlled at least in part to successfully implement the process. Indeed, the presence of other substances or molecules in the sample, the chemical and physical nature of the deposition surface as well as the environmental parameters to which the sample is subjected during the self-formation of the micro-patterns, modify the micro-patterns produced.
The constituent characteristic elements of the micro-patterns formed have dimensions of between a few nanometers and several micrometers, preferably between ten nanometers and one micrometer. It is therefore an order of magnitude very far from that of the structures studied by crystallographic techniques which are of the order of Angstremem. The micro-motifs considered in the context of the invention are also very different from those which characterize crystals and quasi-crystals geometrically.
In addition, the appearance of micro-motifs during the implementation of the method according to the invention is of a deterministic nature whereas the crystallization of proteins is a phenomenon whose appearance can be qualified as probabilistic. Unlike crystals and quasicrystals, the structures that make up these micro-motifs are often not made of a homogeneous material on a molecular level. In an advantageous implementation, the self-forming step of the micro-motifs on the support comprises at least partial elimination of the liquid phase of the mixture.
The preferred method of removing the liquid phase from the mixture is drying the sample, in an open medium, at ambient pressure at a controlled temperature. The temperature can be chosen for example between 25 [deg] C and 70 [deg] C. The duration of the drying period may be for example between 30 seconds and 12 hours, and preferably between 1 and 45 minutes. The volume of solvent to be evaporated may be, for example, between 1 and 250 microliters, and preferably between 20 and 50 microliters.
In more elaborate embodiments of the invention, it is possible to measure the ambient atmospheric pressure during the self-training of the micro-patterns to correct the results obtained. It is also advantageous to manage the drying temperature in decreasing steps or in a continuously decreasing law to obtain a faster result without losing resolution. Indeed, the self-training of the micro-patterns according to the invention is a non-linear phenomenon which accelerates strongly at the end of the period. By taking this characteristic into account, it is possible to optimize the process to approach a reverse law. the natural flow of things to save time at the beginning of the period by applying a high temperature and then reducing the temperature as we approach the end of the self-training period micro-patterns . Of course, when the variable temperature forming mode is implemented during analysis of the sample, it must have been previously implemented substantially identically during the step of constitution of the reference information.
In other embodiments, the self-forming step of the micro-patterns comprises a precipitation of solids on the deposition surface within a sample volume that remains liquid during the self-training of the microparticles. reasons.
Whatever the manner of inducing self-formation of the micro-motifs according to the invention, the characteristics of the micro-motifs may be more or less influenced by the presence of non-organic or organic substances or molecules present in the initial mixture. or added before or during the implementation of the analysis method.
The characteristics of the micro-patterns may be more or less influenced by particular environmental conditions applied during their self-formation. Thus subjecting the sample to fields of different natures and intensity can have an influence on the results obtained. For example, by applying electric, magnetic or electromagnetic fields with certain combinations of intensity, waveform and / or frequency, substantial changes in the characteristics of the micromotives have been observed. Likewise, the application of ultrasonic waves, surface waves or vibrations under particular conditions of frequency, intensity, orientation and coupling to the medium to be analyzed can influence the results observed.
The support may be made of different materials, such as synthetic materials, glasses, metals, semiconductor materials, non-organic crystals, organic crystals, metallo-organic crystals, liquid crystals, natural minerals, composite materials, polymers, gels or colloidal substances.
In other embodiments, the surface of the support has been previously treated by superficial chemical and / or biochemical modification of said support, so as to obtain the predetermined surface state of the support, thereby forming a chemically adsorbed surface layer. or physio-adsorbed. Such a chemical or biochemical modification affects the properties, particularly physical, electromagnetic, optical, chemical and / or biochemical, of the surface of the support. Such treatments can be induced by reactions between the surface of the support and a modifying agent, in the solid, liquid, colloidal, gaseous or plasma phase. Products of the silane family are, for example, highly effective surface chemical modifiers.
It is also possible to modify the characteristics of the deposition surfaces according to the invention by the secondary grafting of an appropriate biological system. Such secondary ligands may be for example peptides, proteins, steroids, carbohydrates or lipids. Physical adsorption may further include natural or synthetic amphiphilic molecules. The means used to allow the selective adsorption of proteins of the sample may comprise Langmuir-Blodgett layers, monolayer, bilayer or multilayer complexes, but also supported lipid bilayers as well as self-assembled polyelectolyte layers.
In another embodiment, the determined surface state of the support has been obtained by physical surface modification of the support. The properties, in particular physical, electromagnetic, optical and / or chemical, were then modulated by heat treatment, ion implantation, specific structuring of the surface (etching, stamping, molding, deposits) or by epitaxy.
During the examination of the micro-patterns, the bi-and / or three-dimensional shape of at least a portion of the micro-patterns is detected. Generally, a quadratic or circular portion of area between 400 and 2,500 square microns is sufficient to obtain suitable information. The examination includes the comparison by recognition of the similarities between all or part of the images.
The characteristic micro-motifs can be detected by any microscopy technique, such as optical microscopy, electron microscopy or atomic force microscopy, confocal microscopy, near-field microscopy including any type of coherent or non-coherent light source, polarized or otherwise, as well as all the techniques of illumination in transparency or by reflection, in bright background or in black background. Fluorescent markers can also be used.
The images produced can be used directly by the user by comparison with reference images. The comparison may also include measures of characteristic features, such as the size of repetitive structures, the size of structural elements, measurements of element density, distances between recurring features or characteristic angles between them. elements.
Pattern recognition and possible measurements may advantageously be performed by appropriate image processing software working on the images to be analyzed after capture and digitization in relation to previously determined reference information. The analysis of the new images can thus be fully automated, up to the expected final result such as, for example, the state of phosphorylation of a protein or a diagnosis of a pathology associated with the detection of the presence or the absence of at least one protein or at least one given protein complex in a sample.
It is also possible to examine the micro-patterns through the production of a characteristic image, using diffractive optical techniques. The image produced by these techniques no longer represents the real forms of the micropatterns, but a representative transformation that can be compared to reference images that are also transformed and previously recorded.
According to another embodiment of the invention, the examination of the micro-patterns can be done by measuring the mechanical tensions appearing on the surface of the support during and / or after the formation of the micro-patterns.
The self-formation of micro-patterns, in particular when it relies on the elimination of the liquid phase, causes the appearance of mechanical surface tensions. The measurement of the evolution of these surface tensions over time is a characteristic information source that can be exploited for example to identify a protein in the sample analyzed. The conversion of the mechanical stresses created on the surface of the substrate during the formation of the micro-patterns in a detectable and / or measurable size can be made by multiple techniques, such as micro-shot devices in materials such as silicon by methods used in the semiconductor industry (MEMS). The deposit of samples on micro-levers or microputres thus produced gives rise to deformations, respectively of skews and buckling, able to reveal the self-formation of micro- patterns according to the invention.
The shape changes induced in the microstructures by the surface mechanical stresses resulting from self-forming micro-patterns are easily detectable by optical or electronic means. Depending on the desired applications, the micro-machined devices and the associated reading means may be arranged to work in on-off detection mode or in a mode of measuring the amplitude of the deformations induced by the formation of the micro-patterns.
It is also possible, for example, to use a piezoresistive material, such as an elastomer loaded with conductive particles such as graphite powder, as a substrate for the formation of micromaterials, to measure by means of suitably arranged electrodes variations of resistivity representative of the evolution of mechanical surface tensions resulting from self-training of micro-patterns.
According to another embodiment of the invention, during the examination of the micro-patterns, at least one electrical characteristic of the sample is measured. For example, the evolution during self-formation of the micro-patterns of the conductance of the mixture between electrodes of appropriate shape and arrangement can be measured. The shape and arrangement of the preferred electrodes are respectively circular and concentric. This shape and this arrangement have isotropic characteristics which substantially reduce the noise due to more or less random internal directional effects that arise within the sample such as preferential conductance paths.
In a particularly advantageous embodiment, the comparison is made on a multidimensional basis. It is to add variable elements to enrich the information produced so as to increase the resolution of the system according to the invention in its ability to discriminate certain proteins that can not be distinguished from each other by an analysis. uni-dimensional. For example, it is possible to measure the conductance of the mixture between electrodes of appropriate shape and layout during the self-training of the micro-patterns not only by means of a direct current, but by varying the frequency of the current in a controlled manner. , its intensity, its polarity and more generally its waveform.
Of course, the one-to-one character of the association between a protein, a protein complex or a co-solution product to be detected, with the corresponding reference data, must be maintained, whatever the complexity and the number of dimensions of the protein. representative data space.
The implementation of the method according to the invention is very sensitive to variations of a large number of parameters that must be controlled to remain in the conditions of a one-to-one relationship between the object of interest and its reference signature. . This sensitivity can advantageously be used to increase the resolution of the analysis system, regardless of the acquisition means implemented. The information produced can be enriched by varying, in a controlled manner, the environment of the mixture to be analyzed during the formation of the micro-patterns.
The most critical environmental variable in its ability to influence the result obtained is the temperature at which the sample is subjected during self-formation of the micro-patterns. That is why this quantity must be controlled to obtain reproducible and therefore exploitable results. Temperature can also be a source of data enrichment to improve the resolution of the analysis. Thus, it is possible to envisage comparing the micro-patterns obtained with a plurality of reference substrates on which reference micro-patterns have been formed at different temperatures.
Similarly, when the sample, during the self-training of micro-patterns, is subjected to fields of sufficient magnitude, the result obtained may vary. This is as many means as can be used in the implementation of the method according to the invention to improve the resolution to obtain the desired discrimination capacity. The applicable fields are electrical, magnetic or electromagnetic, but also vibratory, with for example the use of ultrasonic sources or the diffusion of surface waves on the sample support.
The comparison of the micro-patterns obtained with one or more reference micro-patterns can be done in different ways.
The first step is usually to filter to eliminate or reduce any noises that may interfere with the payload. Then, in a second step, a search is made for correspondence between the new data provided by the micro-pattern resulting from the sample to be analyzed and those previously recorded in a database, explicitly or implicitly associated with micro-patterns. reference patterns obtained under similar conditions and analogous to standardized signatures, themselves associated with the proteins or protein complexes to be sought.
In the case of the use of images of the micro-patterns, resulting from a direct visualization system by microscopy followed by an automatic analysis, it will be possible to implement a shape recognition software that will be based on these images. , produced with analogous means, which will have been previously recorded and associated for example with proteins or protein complexes identified under given conditions.
The exact recognition of the data to be analyzed with respect to the reference data can be made difficult in view of the large number of parameters that can influence the results, irrespective of the means of acquisition used.
Therefore, it may be advantageous for the comparison means to provide not an absolute result but a measure of the relative difference between the data from the analysis and the reference data. It is possible to add a threshold device after obtaining a measure of the relative difference between analyzed data and reference data to provide a binary result by comparison between a fixed threshold and the value of the measurement of this difference. . Binary information is sought in many process applications, for example, the presence or absence of an object of interest of a protein nature or a co-solute in the sample. The binary information produced can be arranged to be directly related to the desired end goal, such as, for example, the diagnosis of a pathology.
In addition, the measurement of the difference between the measured information and the recorded information can be exploited in addition to the binary information produced to inform the user of the relevance of the result provided by the system, for example on the probability associated with a diagnosis.
The invention can be applied to the analysis and detection of the proteins included in the sample to be analyzed. In this case, it is deduced from the comparison made in the second step of the method, information on the protein or proteins present in the sample.
In another advantageous use of the invention, the sample comprises, in addition to one or more known proteins, in a mixture in a solvent, a co-solute of unknown nature as an analyte.
Such co-solutes may be co-solution salts, acids, bases or ampholytes, other proteins, antibodies, antigens, organic molecules, non-organic molecules, metallo-organic molecules, nucleic acids such as DNA or RNA, allergens, lipids, polymers, adjuvants causing specific reactions, molecules with specific physical, chemical or pharmacological properties. Some co-solutes may also allow the use of indirect or cross-detection methods based for example on the detection of photons such as fluorescence, on the detection of particles, for example by means of radioactive markers, or on the detection of variations involving electricity or magnetism.
In this case where the protein is known and where the variable element is the co-solute, information on the co-solute as product to be analyzed is deduced from the comparison made in the second step of the process. It may be for example a toxin present in the sample and which should be detected and analyzed.
The subject of the invention is also a system for analyzing samples comprising a mixture of at least one protein and a solvent, for carrying out the process according to the invention. The system comprises: - means for receiving a support having a predetermined surface state, on which a sample has been deposited, - means for inducing a self-formation of micro-patterns on said support, from said sample, - means for examining said micro-patterns and / or effects induced during and / or after their formation, so as to produce examination results, - means for storing and / or accessing reference data obtained by a treatment and examination prerequisites of a reference substance, - means for comparing the examination results to the reference data, so as to produce comparison data, and - means for processing said comparison data so as to produce information of analyzing said sample.
The system further comprises means for deriving from the comparison performed, information relating to the presence or absence of the desired entity of interest, for example a protein, a protein complex or a co-solute, in the sample to be analyzed.
In any case, the system advantageously comprises a set of determined surface state supports corresponding to those used to produce the reference data, for the analysis of different samples. Naturally, it remains within the scope of the invention to multiply the unit processing means in order to increase the productivity of the system or to widen its field of application.
Similarly, all or part of the components of the system can be indifferently physically located at the place of analysis or be externalized without departing from the scope of the present invention. In the case of a physical outsourcing of all or part of the system components, the operating unit of the method according to the invention is based on communications between the elements of the system. Local or remote networking means (LAN or WAN) standard, for example Ethernet or Internet, are ideally suited to an exploded implementation of the invention for purposes of local sharing of resources or access to resources. apart.
There can be many advantages to exploded implementation of the invention, particularly in the economic field. Indeed, the system according to the invention generally becomes less expensive to deploy if the means physically installed at the location of the analysis of the samples are minimized and the reference database is centralized in another location. and / or the means of comparison between the data resulting from the analysis with the reference data and / or the means of generating a result in relation to the expectations of the users such as diagnoses.
In addition, other economic advantages can arise from the fact that access to all or part of the components of the system according to the invention can be conditioned to obtain access rights for example by a financial transaction taking place during each analysis or as part of a subscription to the service or any other form of agreement between the parties concerned.
A centralized reference database also offers advantages in terms of ease of management of the updates which are necessarily frequent in the case of the invention given the range of possible applications and the very large number of protein entities. or co-solution products that may be of interest. In addition, access to the data resulting from the analyzes can allow additional valuations by means of appropriate treatments, for example to extract information that can be reused as part of the continuous improvement of the implementation of the method according to the invention to improve for example its resolution, the scope of its fields of application and its performance, for example in the field of diagnosis.
Of course, any intermediate distribution of the components of the system between the local whole and the exploded whole remains in the object of the invention. For example, the most frequently used reference data may be stored locally in the context of an application of the method, the system being able to consult an external database and possibly specialized for more specific needs.
In the preferred mode of implementation of the system according to the invention, the sample support, which can join a plurality of deposition surfaces, also comprises on-board identification means. These identification means can be physically printed directly or via a label of a bar code, a radio frequency tag or an onboard memory chip. The analysis device according to the invention then includes reading means adapted to the chosen identification solution. In a preferred embodiment, the identification is based on a laser marking on the sample support that the means implemented for the analysis of the micro-patterns can read and decode at no additional cost.The identification means associated with each sample support advantageously comprises a unique code for traceability and ease of management of data from the analysis.
The identification means further comprises a coding which is associated in a one-to-one way with the complete description of all or part of the characteristics of the support: nature of the surfaces of the support, their number, types of treatment they have undergone, process self-training of micro-patterns to be associated with them, batch reference, manufacturer's code, factory number, any calibration data, etc. The coding of a unique number and the encoding of the characteristics of the medium may be treated indifferently as separate objects or as nested objects, for example a unique code which may consist of descriptive sub-codes to which an element is added. unique code.
Thus the analysis system according to the invention can be managed automatically depending on the consumable media that the user chooses and / or depending on the purpose he seeks. Since the system is aware of the operating conditions to be executed by the coding associated with the consumable that is the sample support, it is even possible to ensure that the system guides the user appropriately for the following operations to be performed. The implementation of such a fully automated system offers advantages that are lacking in the state-of-the-art systems, that is to say a use which does not require any particular skill and the elimination of the risks of errors during handling as well as during the management of the results.
One of the preferred embodiments of the system according to the invention consists in associating different detection techniques of the micro-patterns or their induced effects. Indeed, a detection system, for example based entirely on optical microscopy, requires performance and quality of implementation that have a certain cost. The same applies to a detection system based entirely on the analysis of the electrical characteristics of the sample. On the other hand, by combining these two different techniques, the performance requirement for each is significantly lower, which allows a less expensive embodiment using, for example, molded plastic lenses for the optical part and simplified electronics for detection according to the characteristics. electric.
It is also advantageous to provide an analysis system according to the invention in which the set of supports comprises means for retaining one or more determined products in relation to the reference data which are intended to become co-solutes when the The sample will be deposited on the corresponding surfaces.
Furthermore, physical and / or physicochemical means can be added to the analysis system according to the invention, to homogenize the mixing of an additional product with the sample.
The present invention will now be described more precisely from a few embodiments taken as non-limiting examples and illustrated by the appended figures in which: FIGS. 1a and 1b illustrate, with different magnifications, a portion of a reference micro-pattern obtained as described in Example 1; FIGS. 2 to 22 illustrate various reference micro-motifs obtained as described in Examples 2 to 22; FIGS. 23 to 25 illustrate geometric parameters of reference micro-motifs obtained as described in Examples 23 to 25; FIGS. 26a and 26b illustrate the variations of resistivity during the formation of a reference micro-pattern; Fig. 27 illustrates the resistivity versus frequency variations on a reference micro-pattern; Fig. 28 shows a set of images obtained by atomic force microscopy illustrating Example 28; FIGS. 29aECH and 29aREF respectively illustrate an image obtained by simple optical means in the context of experiment n [deg] 1, and the closest reference image obtained with Fibrin + NaO3; FIGS. 29bECH and 29bREF respectively illustrate an image obtained by simple optical means in the context of experiment n [deg] 2, and the closest reference image obtained with BSA + Fibrine + Nac03; FIGS. 29cECH and 29cREF respectively illustrate an image obtained by simple optical means in the context of experiment n [deg] 3, and the closest reference image obtained with BSA + Fibrin + Na03; and 30REF respectively represent an image of a micropod obtained by simple optical means, and the closest reference image obtained with BSA + Na2HPO4; FIG. 31 schematically illustrates the main elements of an analysis system according to the invention; FIG. 32 illustrates a variant of the system of FIG. 31; and FIG. 33 represents a view from above and a side view of an embodiment of an analysis system according to the invention.

Preconditions
In the following examples, the solutions were prepared in milliQ purified water with a Millipore system (resistivity greater than 18 M (cm-1).) Proteins (bovine serum albumin or BSA, fibrin, α-casein, etc.). from the company SIGMA and the salts (Na2CO3, Na2NO3, etc.) of ACROS When the experiments were carried out on optical glass slides (76 × 26 × 1 mm), these were washed beforehand with methanol. and acetone in an ultrasonic bath, for 15 minutes for each solvent, and then dried with optical paper.The droplet deposits of samples were made with Gilson micropipettes.When the measurements were made under a microscope forcibly Atomic Force Microscopy (AFM), a Thermomicroscope Explorer system equipped with a 100 Microm scanner was used in non-contact mode with a scan rate of 1 to 2 Hz and a resolution of 500 x 500 points For each sample, scans at 50 Microm, 25 Microm, 10 Microm and 5 Microm were generally performed. The pens were silicon, the resonance frequency was fo = 260 kHz and the force constant was 45 Newton. The surface was characterized by three-dimensional topographic measurements, based on angular measurement, distance and height measurements, as well as roughness measurements. The average surface roughness was calculated as follows:
Rms = (1 / N) / (Zi-Z) For experiments on surfaces having reactive polar properties, the surface of silicon dioxide pellets was modified with silane derivatives purchased from ABCR Chemicals. To do this, 50 ml solutions were prepared containing 95% ethanol and 5% water by volume to which 1 ml of a silane derivative was added. After 5 minutes, a silanol was formed. The silicon dioxide pellets are then immersed in the solution with slow stirring for 3 to 4 minutes.
The pellets are then washed in ethanol then dried at room temperature and then heated at 130 ° C. for 15 minutes before use.

Example 1
This example illustrates a study of the constitution of micro-motifs of BSA formed in the presence NaNO 3.
A solution of BSA with Fluorescein Iso-ThioCyanate (FITC) (10 g / L) and NaNO 3 (0.154 M) was prepared. Microl drops of this solution were deposited on glass slides and these samples were dried at different temperatures to show the influence of temperature and rate of evaporation on the organization of the protein micromotives. The samples were studied using a white light microscope, a confocal fluorescence microscope and a near field scanning optical microscope (SNOM) operating in transmission. Fluorescence and transmission images were also obtained using the scanning optical microscope. For all measurements, an air-cooled argon laser at 488 nm was used to excite the sample. A combination of an OG515 filter and an OG530 filter was used to separate the fluorescence from the length. excitation wave. A single photon avalanche photodiode (APD) was further used to finally detect the fluorescence emitted by the sample. The topography of the sample is shown in Figure la. Figure 1b illustrates confocal fluorescence with lower magnification. The reference micro-motifs visible in FIGS. 1a and 1b represent long crystalline structures, characteristic of a fluorescent protein within the main branching structures, which shows the importance of the biological element in the organization of the structure. pattern.
This experiment demonstrates the importance of the protein in the organization of patterns: despite a concentrated presence only in small areas included within the patterns, the specific properties of the protein influence the formation and geometry of the general ramifications. this influence is combined with the nature of the salts to determine the exact nature of the patterns. This example also shows the heterogeneous constitution of the material which constitutes the micro-patterns according to the invention.

Example 2
This example illustrates a formation of micro-patterns and a constitution of reference data, in the case of variable cosolute micro-patterns, fixed protein, fixed temperature, and fixed surface, as well as a study of micro-patterns produced by a-casein.
A solution of α-casein (10 g / l) was prepared and drops of 20 μl of this solution were deposited on optical glass slides. After evaporation at 20.degree. C. for 24 hours, the samples were examined under an atomic force microscope and micro-patterns shown in FIG. 2 were observed. The formation of a relatively flat α-casein film with scattered concretions of varying sizes, ranging from 0.540 to 0.853 nm and heights up to 31.85 nm. The average surface roughness is 12.4 nm; pores of about 100 nm are randomly dispersed in the sample.

Example 3
This example illustrates a formation of micro-patterns and a constitution of reference data, in the case of variable cosolute micro-patterns, with fixed protein, fixed temperature, and fixed surface, as well as a study of the micro-patterns produced by acaseine in the presence of Na2CO3.
A solution of α-casein (10 g / l) and Na 2 CO 3 (0.154 M) was prepared and drops of 20 microl of solution were deposited on optical glass slides. After evaporation at 20 ° C. for 24 hours, the surface of the film was subjected to an atomic force microscope under the same conditions as in Example 1. Micro-patterns illustrated in FIG. 3 were observed and characterized. by a cloudy amorphous structure with pores and a slightly inter-fibrillar organization and a surface roughness of 354.2 nm.

Example 4
This example illustrates a formation of variable cosolute micro-patterns, fixed protein, fixed temperature, fixed surface, and constitution of reference data, as well as a study of micro-patterns produced by the BSA.
A solution of BSA (10 g / l) was prepared and drops of 20 microl of this solution were deposited on optical glass slides. After evaporation at 20 ° C. for 24 hours, the surface of the film was subjected to an atomic force microscope under the same conditions as in Example 1. The micro-patterns illustrated in FIG. BSA film is very flat with a roughness of 4.03 nm; concretions of up to 150 nm, as well as randomly dispersed pores are present on the surface.

Example 5
This example illustrates a formation of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, a constitution of reference data, as well as a study of micro-motifs produced by BSA in the presence of Na2CO3.
The procedure was the same as in Example 3 except that BSA was used in place of α-casein. The images of the micro-patterns obtained by atomic force microscopy are illustrated in FIG. 5. Structures in block shapes of variable sizes, ranging between 0.630 microm and 1.54 microm, and having a maximum height of 2, have been observed. 12 Microm; these blocks are made of parallel structures and give a surface average roughness of 365.6 nm.

Example 6
This example illustrates a formation of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, a constitution of reference data, as well as a study of micro-motifs produced by BSA in the presence of Na2HPO4.
The procedure was the same as in Example 5 but Na2HPO4 was used instead of Na2CO3. The images obtained by an atomic force microscope are illustrated in FIG. 6. An amorphous surface having cracks of depth of 1300 nm and concretions of height of 1090 nm conferring a surface average roughness of 374 nm were observed. However, the average surface roughness value for an area with no cracks is 124 nm, showing that the film is relatively flat.

Example 7
This example illustrates a formation of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by BSA in the presence of NaBr.
The procedure was the same as in Example 5 except that NaBr was used instead of Na2CO3. Images of micro-patterns were obtained using the atomic force microscope shown in FIG. 7. Random but often slightly organized aggregates were observed as a linear structure. The conical blocks that form these aggregates, having a relatively large diameter ranging from 1.3 microm to 4.22 microm, can reach a height of 4000 nm. Dendritic structures with an orientation of 90 [deg] are surrounded by relatively deep pores. The average surface roughness is 685.17 nm.

Example 8
This example illustrates the formation and the study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by BSA in the presence of NaBF4.
The procedure was the same as in Example 5, but NaBF4 was used instead of NaCO3. The images obtained are illustrated in FIG. 8. Regular and dendritic structures have been observed which result from concretions organized in an extremely dense manner and having a maximum height of 500 nm. The average surface roughness is 80 nm.

Example 9
This example illustrates the formation and study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by BSA in the presence of NaHCO 3.
The procedure was the same as in Example 5 except that NaHCO 3 was used in place of NaCO 3. The observed images are illustrated in FIG. 9. Dendritic structures with a maximum height of about 600 nm have been observed. The average surface roughness of the surface is 95.23 nm.

Example 10
This example illustrates the formation and study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by a mixture of α-casein and fibrin.
A solution of 10 g / l of α-casein was prepared and 1.5% by volume of a solution of sodium hydroxide saturated with fibrin (3.8 g / l) was added to this solution. Microl drops of this solution were deposited on optical glass slides. After evaporation at 20.degree. C. for 24 hours, the images observed by atomic force microscopy are illustrated in FIG. 10. The micro-patterns have a circular micro-porosity similar to the structure of certain cheeses. Fine concretions are present on the surface and in the pores. The average surface roughness of the surface is 67.5 nm and the maximum difference between the ends is 161.61 nm.

Example 11
This example illustrates the formation and the study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by a mixture of BSA and fibrin. The procedure was the same as in Example 10. that alpha - casein is replaced by bovine serum albumin. The images obtained are illustrated in FIG. 11. Large spherulites with relatively small heights (50 nm) are observed. Concretions with a height of 36 nm are abundantly dispersed on the surface. The average surface roughness is 94.08 nm.

Example 12
This example illustrates the formation and study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by a mixture of α-casein and fibrin with Na 2 CO 3.
A solution of α-casein (10 g / l), fibrin (0.285 g / l) and Na 2 CO 3 (0.154 mol) was prepared. Microl drops of this solution were deposited on optical glass slides. After evaporation at 20.degree. C. for 24 hours, the images obtained in AFM are illustrated in FIG. 12. A cloud layer is observed which covers dense aggregates and pores dispersed on the surface. The average surface roughness is 227.07 nm.

Example 13
This example illustrates the formation and study of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by a mixture of BSA and fibrin with Na 2 CO 3. The procedure was the same as in Example 12 but acaseine has been replaced by BSA. The images obtained in AFM are illustrated in FIG. 13. Fine and dense fibrillar structures of 1.85 microm in length are observed which are grouped into slightly oriented blocks of relatively small size (0.55 microm). The maximum height is 1.29 microm and the average surface roughness is 261.07 nm.

Example 14
This example illustrates the formation and the study of fixed cosolute micro-motifs, fixed protein, variable temperature, fixed surface, produced by BSA in the presence of NaNO 3 obtained at 20 ° C.
A solution of 10 g / l protein and 0.154 M NaNO 3 was prepared and 20 microl drops of this solution were applied to optical glass slides. After evaporation at 20.degree. C. for 24 hours, the images obtained in AFM are illustrated in FIG. 14. A very regular dendritic structure is observed. The dendrites are perfectly organized, branched at right angles always on the same side. The main branches having a width of 3.99 microm and a height of 450 nm, have depth channels of 265 nm. Secondary branches are narrower but slightly larger (474 nm). There is a maximum height at the ends of the dendrites up to 760 nm.

Example 15
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, variable temperature, fixed surface, produced by BSA in the presence of NaNO3 obtained at 37 [deg] C.
The procedure was the same as in Example 14 except that the evaporation was carried out at 37 [deg.] C. The images obtained are shown in Figure 15. We observe a very organized film. The structure is fibrous and pseudodendritic, relatively dense and totally oriented. The main branches are parallel, with channels of about 260 nm. Short lateral branches (7.48 μm) are located along the main branches, always on the same side and at a constant angle of 45 [deg].

Example 16
This example illustrates the formation and the study of fixed cosolute micro-motifs, fixed protein, variable temperature, fixed surface, produced by BSA in the presence of NaN03 obtained at 75 [deg] C.
The procedure was the same as in Example 14 but the evaporation was carried out at 75 [deg.] C. The images obtained are illustrated in FIG. 16. The structures are pseudo-dendritic in appearance. The lateral branches are either long or branched, on the one hand, or short and dense on the other. The width of the main branches varies from 0.971 microm to 4.31 microm, with heights of 700 nm. The branching angles for the main branches as well as for the secondary ones are 45 [deg].

Example 17
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 on a surface treated with aminopropyltrimethoxysilane.
A solution of BSA (10 g / l) and NaNO 3 (0.154 M) was prepared. In order to obtain reactive polar properties, the surface of a silicon dioxide pellet was modified using 3-aminopropyltrimethoxysilane.

layer formed on the surface of the pellet (R = aminopropyl) 20 microl of solution of BSA and NaNO 3 were deposited on the dry modified silicon dioxide pellet. After evaporation at 20.degree. C. for 24 hours, the images obtained in AFM are illustrated in FIG. 17. The film obtained is very organized. The roughness of the surface is high (259 nm) because of the regular crystalline structures. They have the shape of equilateral triangles and have a size of 12 microm. The crystalline blocks are formed of several superimposed planes and are slightly oriented.

Example 18
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 on a surface treated with n-octodecyltrimethoxysilane.
The procedure was the same as in Example 17 except that the surface of the silicon dioxide pellet was treated with n-octodecyltrimethoxysilane, in order to obtain hydrophobic properties, instead of aminopropyltrimethoxysilane.

layer formed on the surface of the pellet (R = octodecyl) Microl of the solution of BSA and NaNO 3 was deposited on the modified silicon dioxide pellet. After evaporation at 20.degree. C. for 24 hours, the images obtained in AFM are illustrated in FIG. 18. Crystalline blocks prolonged by small dendritic structures are observed. The crystals have a regular triangular shape, one of the sides measuring about 10 microm, and apparently formed by superimposed planes. These large blocks give a surface average roughness of 320.2 nm. Fine dendrites without preferential orientation are observed in extension of the crystallitic wedges and having an average height of 50 nm.

Example 19
This example illustrates the formation and the study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 on a surface treated with acetoxypropyltrimethoxysilane.
The procedure was the same as in Example 17, except that the surface of the silicon dioxide pellet with acetoxypropyltrimethoxysilane.

Layer formed on the surface of the pellet (R = acetoxypropyl) The images obtained in AFM are illustrated in FIG. 19. Regular pseudodendritic structures are observed. The development of pseudo-dendrites is fractal and is consistently on the same side. The dendrimers are parallel and their branches are at 90 [deg]. A significant average surface roughness is noted.

Example 20
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 on a surface treated with isocyanatopropyltrimethoxysilane.
The procedure was the same as in Example 17 but the surface of the silicon dioxide pellet was modified with isocyanatopropyltrimethoxysilane to obtain hydrophobic properties.

Layer formed on the surface of the pellet (R = isocyanatopropyl) The images obtained in AFM are illustrated in FIG. 20. Organized structures of the dendritic type are observed. The branches are oriented in two perpendicular directions. The size of the dendrites and their length are random and the average surface roughness is 3 nm.

Example 21
This example illustrates the formation and the study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 on a surface treated with aminopropyltrimethyloxysilane.
The procedure was the same as in Example 17 except that the surface of the silicon dioxide pellet was treated with aminopropyltrimethyloxysilane.
H2N (CH2) 2 CH2 If OMe OMe OMe The images obtained in AFM are illustrated in FIG. 21. A highly organized structure is observed. The average surface roughness of the surface is high (259 nm) because of the regular crystalline structures. They have the shape of equilateral triangles and a size of 12 microm. The crystalline blocks are formed of several superimposed planes and are slightly oriented.

Example 22
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, variable temperature, fixed surface, produced by BSA in the presence of NaNO 3 at 37 ° C., on a surface treated with aminopropyltrimethoxysilane. .
The procedure was the same as in Example 21 with the exception of the evaporation temperature which was set at 37 [deg] C. The images obtained in AFM are illustrated in FIG. 22. A rather amorphous film is observed. Particles in blocks show a slight tendency towards pseudo-dendritic organization. The main branches are parallel and the distances between them are about 15 μm but have a variable size. They are moreover more or less ramified. The average surface roughness value is 489 nm.

Example 23
This example illustrates the formation and the study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 at 20 ° C., on a surface treated with aminopropyltrimethoxysilane. .
FIG. 23 illustrates a structural analysis made on micro-units produced by BSA in the presence of NaNO 3 on a surface treated with aminopropyltrimethoxysilane and obtained at 20 ° C. Surface characteristics were obtained by three-dimensional topographic measurements such as angle, height and distance measurements, as well as roughness measurement using SPMlab software, version 5.01. The raw images were processed with a leveling routine, in which noise points or lines were eliminated. If necessary, the contrast was improved using a masking routine. Surface characteristics as well as surface analysis, particle analysis, critical dimensions and angle measurements are allowed by an analysis sub-program. The structure of micro-patterns is strongly organized. The average surface roughness is high (259 nm) because of the regular crystalline structures. These structures have the shape of individual or stacked equilateral triangles. The detailed analysis gives an average edge length of 8.5 Microm, the edges form an angle of 55 [deg]. The crystalline blocks are formed of several superimposed planes and are slightly oriented.

Example 24
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 at 20 ° C., on a surface treated with n-octyltrimethoxysilane. .
The same analysis protocol was followed as in Example 23. The study is illustrated in FIG. 24. Regular pseudodendritic structures are observed. The development of pseudo-dendrites is fractal and occurs consistently on the same side. The primary, secondary and tertiary branches are perfectly parallel. Lateral branching occurs at angles of 110 [deg]. Distances of 14 microm and 3.5 microm are formed between the main dendrites and the lateral branches, respectively. The average surface roughness is 237.2 nm.

Example 25
This example illustrates the formation and study of fixed cosolute micro-motifs, fixed protein, fixed temperature, variable surface, produced by BSA in the presence of NaNO 3 at 20 ° C. on a surface treated with mercaptopropyltrimethoxysilane.
The same analysis protocol was followed as in Example 23. The study is illustrated in FIG. 25. Regular pseudodendritic structures are observed. The development of pseudo-dendrites occurs on the same side and are parallel, and their length and size are variable. The distance between the main, secondary and tertiary dendrites are 20.6 Microm, 7.4 Microm and 2.4 Microm respectively. The branching angle is 103 [deg]. The dendrites are enlarged by small individual molecules, patterns that are also found on the main branches. The average surface roughness reaches 72 nm.

Example 26
This example illustrates the formation of variable cosolute micro-motifs, fixed protein, fixed temperature, fixed surface, produced by a mixture of CSA and fibrin in the presence of NaCl, as well as a study of the electrical resistivity during the formation of micro -motifs.
A solution of CSA (Chicken Serum Albumin) (10 g / l) and sodium chloride NaCl (0.154 M) as well as fibrin in various proportions was prepared. This solution was deposited between two concentric copper micrometers of thickness Microm having deposited an electrolytic gold on an epoxy glass support. The electrical resistivity during the formation of the micro-patterns was measured using a Fluke Model 189 multimeter, associated with a computer equipped with Flukeview Forms software, the resistance measurements being made every five seconds. Variation in resistivity is shown in the curves of FIG. 26a which further shows a curve corresponding to the CSA alone, a curve corresponding to a mixture of CSA and salt, and two curves corresponding to respective percentages of 2 and 4% of fibrin added to cosolution. It appears that the resistivity varies with time depending on the presence and the concentration of the protein as well as on the presence or absence of the salt.
In the example illustrated in FIG. 26b, CSA was replaced by BSA and fibrin by casein. The curves obtained indicate the evolution of the resistivity of the mixture between the electrodes as a function of different mixing ratios between the proteins.

Example 27
This example illustrates the formation of fixed cosolute micro-patterns, fixed protein, fixed temperature, fixed surface, variable frequency, produced by BSA in the presence of NaCl, as well as a study of the electrical resistivity as a function of frequency during the formation of micro-patterns.
A solution of BSA (10 g / l) and NaCl (0.154 M) was prepared under the same conditions as in Example 26. The solution was deposited as explained in Example 26. It was left for 24 hours at 20.degree. C. to remove the liquid phase before any measurement. The electrical resistivity measurement of the micro-pattern obtained was performed by an HP Network Analyzer. The curve obtained is shown in FIG. 27 which shows the resistivity as a function of frequency and reveals the existence of a first characteristic peak at a frequency of 1.5 MHz and a second characteristic peak at a frequency of 3 MHz. .

Example 28
This example relates to a use of a reference micro-motif database for the identification of proteins and protein complexes. In this example, the implementation of the invention is illustrated in an experiment whose purpose is the detection of a protein complex (MRP-GG-GM2), the presence of which in a body fluid sample indicates the disease of multiple sclerosis. plate.
To do this, a database containing images of the micro-motifs resulting from all the combinations of the proteins concerned, taken in isolation, two by two and in a triple complex, is obtained beforehand on three deposition surfaces having different surface chemical characteristics. (glass, mica and concavalin A). It can be seen that for one or two different deposition surfaces, there are similarities between the micro-motifs obtained which would not allow the identification of all the protein elements, for example (MRP-GG-GM2) on glass very close to ( MRP-GG-GM2) on mica and (GG-MRP) on ConA, or the micro-motifs of (MRP-GG-GM2) on Con A resemble the micro-motifs of (GG-GM2) on mica.
On the other hand, it can be seen that with a number of at least three distinct depositing surfaces judiciously chosen according to the desired objective, the invention makes it possible to definitely discriminate all the possible arrangements of the protein components concerned and in particular the complex of interest sought. Complementary experiments have even demonstrated the capacity of the invention to be able to clearly discriminate proteins almost identical to a mutation.

Example 29
This example illustrates an implementation of the method according to the invention, and a comparison of micro-patterns resulting from an analysis with reference micro-motifs. In this example, cosolute is known and a protein is sought.
The following experiment is carried out: a blind protein is taken, a solution of which is prepared at the rate of 10 g / l, to which is added a salt in cosolution: Na03 (0.154 M). Droplets of 20 microl of solution are deposited on optical glass slides. After evaporation at 20.degree. C. for 24 hours, micro-patterns which have been formed are observed by means of an entry-level optical microscope which has been modified to demonstrate the feasibility of an optical reading solution. low cost. This is a PERL initiation microscope equipped with a x40 lens and an x10 eyepiece. A webcam sensor and electronics were mounted on the eyepiece to capture the image and produce a file in a format compatible with PC-based digital image processing software tools.
The image of Figure 29aECH is obtained. It is compared with all previously stored images from direct imaging equipment giving compatible results such as AFM and optical microscopes. The image found having micro-patterns having sufficient resemblance with those of the sample and obtained under similar conditions (same protein, same deposition surface and same drying temperature) is that illustrated in FIG. 29aREF, which corresponds to to a known protein, fibrin.
The same procedure is used in the context of two other experiments illustrated in FIGS. 29b and 29c, changing the cosolute if necessary.

Example 30
This example illustrates an implementation of the method according to the invention, and a comparison of micro-patterns resulting from an analysis with reference micro-motifs. In this example, the known protein, the unknown cosolute, and a chemical element is sought.
The following experiment is carried out: a known protein, for example BSA, is used, a solution of which is prepared at a rate of 10 g / l, to which is added a salt in cosolution which is selected in a blind manner. Droplets of 20 microl of solution are deposited on optical glass slides. After evaporation at 20.degree. C. for 24 hours, the microparticles which have been formed by means of an AFM atomic atomic force microscope are observed. The image of FIG. 30ECH is obtained. It is compared with all previously stored images from direct imaging equipment giving compatible results such as AFM and optical microscopes. The image found having micromotives with sufficient resemblance to those of the sample and obtained under similar conditions (same protein, same deposition surface and same drying temperature) is that shown in FIG. 30REF, it corresponds to the presence of Na 2 HPO 4 in cosolution. The invention thus makes it possible to identify the nature of a chemical element present in a sample.
Figure 31 schematically illustrates the main elements of a sample analysis system according to the invention for carrying out the analysis method of the invention. As illustrated in FIG. 31, a liquid sample referenced 1 in the form of a mixture of at least one protein and a solvent, or a cosolvent to be analyzed as indicated previously, was deposited on the surface of a support 2 of predetermined surface state. Such a support may for example, as in the embodiment illustrated in FIG. 31, be part of an analysis device referenced 3 as a whole. The device 3 can also comprise means for receiving the support 2 provided with the sample 1. The device 3 further comprises means for causing the self-formation of the micro-patterns on the support 2.
If one chooses the path of self-formation of micro-patterns by at least partial removal of the liquid phase, these means may be for example a drying device. If the self-formation pathway of the micro-motifs is chosen by dephasing in a medium which remains liquid, these means may be chemical means such as the addition of an appropriate ligand or physical means such as a source of current and electrodes for the implementation of an electrolytic solution for example.
The analysis device 3 also comprises means for examining the micromotif during its formation or after its formation, for example, a scanning electron microscope or any other means as described above.
A device 4 is associated with the analysis device 3 so as to control the progress of the process and in particular the elimination of the liquid phase. The device 4 comprises in a memory 5 stored reference data which may for example be constituted by micro-patterns formed beforehand from samples of known composition under substantially identical conditions with those used for the analysis. reference data thus stored in the memory 5 may, for example, comprise reference micro-pattern images such as those illustrated in FIGS. 2 to 22, geometric parameters of reference micro-patterns as illustrated in FIGS. 23 to 25 or electrical resistivity characteristics of reference micro-patterns as illustrated in FIGS. 26 and 27. In general, the data thus stored in the memory 5 constitute a database that makes it possible to detect and analyze by comparison the sample to be analyzed.
For this purpose, the device 4 comprises means of comparison between the result of the examination of the sample 1 carried out in the analysis device 3 and the reference results stored in the memory 5.
An exploitation device of the results, referenced 6 as a whole, can for example allow the display of the result in the form of a probability or a first level information resulting from the presence or absence of a protein, a protein complex or a co-solute in the sample to be analyzed. A device, referenced 7 as a whole, exploits the results of the analysis produced by the device 6 in relation to an adequate knowledge base to provide qualitative and / or quantitative information related to a human problem such as a diagnosis of pathology, recommendations, a level of danger, tests relating to food or feed, or tests relating to the control of the environment.
The device for exploiting the first level results 6 may be virtual in simplified embodiments where means directly providing results related to a human problem from the comparison information are implemented. Devices 6 and 7 can also be fused without departing from the scope of the present invention.
The problem in which the use of the invention is part may also be more directly related to animal or plant biology, for example applications in the veterinary or agricultural field.
The reference database stored in the memory 5 of the device 4 may also be partially or entirely outside the analysis system as in the variant illustrated in Figure 32 in which the identical elements have the same references.
With reference to FIG. 32, the information coming from an external databank can be transmitted, for example via an Internet-type network to the comparison device 4, to supply as previously a signal representative of the result of the analysis and its exploitation by the exploitation device 6.
An exemplary embodiment of an analysis system according to the invention will now be described with reference to FIG. This analysis system comprises: a reading device 10 according to the invention including: an optical microscope subset 20; an image sensor and associated processing electronics subassembly. a setting device 40 ensuring that the microscope is always in sharp focus, by construction, on the micro-patterns which have formed on the deposition surface; a light source 50, for example one or more white electroluminescent diode (s), a subset 60 for driving the sample support (s) ensuring both the transport of the support of sample (s) in the different working areas of the device, the scanning of the associated code in front of a fixed reader if this code is materialized by a barcode, as well as the micro-displacements in the main axis, ahead and back, during the step of observation of micro-patterns; a housing 80 for receiving a sample carrier; an optional connector 70 comprising a set of micro-tip contacts mounted on spring-loaded pistons integral with a support that can be applied by mechanical or electromechanical means to the corresponding contact areas of the sample holder when an extraction is implemented electrical information during the self-training phase of the micro-patterns; a device 90 for driving the sample support (s) to ensure the micro-displacement in an axis perpendicular to the main axis during the step of observation of micro-patterns; a sample support (s) 100; a zone 11 for introducing the sample support and for reading the code associated with the sample support (s); a zone 12 for depositing the sample (s) on the surface (s) provided for this purpose on the sample support (s); a self-forming zone 13 of the micro-patterns, in this example by drying in contact with a thermostat-controlled plate at the set temperature required by the analysis to be carried out; a zone 14 for observing the micro-patterns; and an area 15 for ejection of the sample support (s) 10.
The driving device 9 can for example be made by means of a stepping motor driving a guide laterally bordering the sample support (s) by a screw transforming the rotation of the motor axis in a translational movement. .
The sample support 100 consists here of a glass or translucent plastic plate on which discoidal deposition zones have been delimited by means of a hydrophobic varnish. If the option of measuring electrical quantities during the formation of the micro-patterns is implemented, then the deposition surfaces are provided with concentric annular electrodes. The electrodes, the network of conductors as well as the reception areas of the reader contacts are advantageously made by screen printing by means of conductive and insulating inks commonly used in industry to produce hybrid thick-layer circuits or matrix keyboards. Where appropriate, the deposition zones may receive additional specific surface treatments to provide selectivity effects as described in the invention. Depending on whether the descriptive code of the sample holder (s) will be read by the same means as those used for the examination of micro-patterns or by specific means, the code will be written on the upper face or on the face bottom of the support.
In an alternative embodiment of an analysis system according to the invention, the handling of the samples to be analyzed is simplified by making sure that the consumable delivered to the user contains, in addition to the deposition surfaces and any electrodes, the possible chemical or biochemical products that a given analysis protocol may require in co-solution.
Where appropriate, each support deposition zone, the surface of which has possibly been the subject of a specific treatment, may be provided with a capsule or any other means for retaining a predetermined product which is presented under a specific condition. form such as powder, liquid, paste, or gel. The sample, at the moment it is deposited, contains the product to be analyzed as well as a solvent able to dissolve also the additional product.
It would also be wise to add, in the case of co-solutes included in the consumable, means for mixing the solution comprising the sample and the additional product, to homogenize it before the process of micro self-training. -motifs does not begin.
Such mixing means may be of a physical nature, for example vibrations or physico-chemical, for example by premixing with each additional product contained in the consumable another product having the property of becoming effervescent in the presence of solvent of the sample.
The effervescence of the mixture, which takes place in the first moments following the deposition of the sample, ensures a homogeneous solubilization of the constituents of the mixture. The effervescent product may be either analytically neutral and inert when mixed with the additional products, or may also be added in the same proportions when building reference data to remain in a one-to-one relationship between the reference data and the data produced by the analysis.
Of course, the invention is not limited to the examples that have just been described and many adjustments can be made to these examples without departing from the scope of the invention.

Claims (22)

2. Method according to claim 1, characterized in that the self-forming step of the micro-patterns on the support comprises an at least partial removal of the liquid phase of the mixture. 3. Method according to claim 2 characterized in that the at least partial removal of the liquid phase of the mixture results from a drying operation of the sample under conditions of temperature and possibly controlled pressure. 4. Method according to claim 3, characterized in that the drying operation comprises a decrease of the temperature at which the drying of the sample is carried out, stepwise or according to a law of continuous variation in a manner substantially identical to the manner in which the sample is dried. whose temperature evolved during the step of constitution of the reference data. 5. Method according to claim 1, characterized in that the self-forming step of the micro-patterns comprises a precipitation of solids on the deposition surface within a sample volume which remains liquid during the self-training of said micropatterned. 6. Process according to claim 1, characterized in that the step of self-forming the micro-units is conditioned on the presence of one or more co-solutes determined in the initial mixture. 7. Method according to any one of the preceding claims, characterized in that the self-forming step of the micro-patterns further comprises an application of an electric field and / or magnetic on the sample during the self-training of said micro -motifs. 8. Method according to any one of the preceding claims, characterized in that the self-training step micro-patterns further comprises an application of ultrasonic waves and / or vibration on the sample during the self-training of said micromotives . 9. Method according to any one of the preceding claims, characterized in that the surface of the support has been previously treated by chemical modification and / or biochemical surface of said support, so as to obtain the predetermined surface state. 10. Method according to any one of claims 1 to 8, characterized in that the surface of the support has been previously treated by physical surface modification of said support, so as to obtain the predetermined surface state. 11. Method according to any one of the preceding claims, characterized in that the micro-pattern examination step comprises an examination by optical microscopy, electron, confocal or atomic force. 12. The method of claim 11, characterized in that the micro-pattern examination step comprises a detection of the bi-and / or three-dimensional form of at least a portion of said micro-patterns. 13. The method of claim 11 to 12, characterized in that the micro-pattern examination step further comprises a measurement of the geometrical magnitudes of structural elements of parts of said micro-patterns. 14. Method according to any one of claims 1 to 10, characterized in that the micro-pattern examination step comprises an examination of said micro-patterns by means based on diffractive optics. 15. Method according to any one of the preceding claims, characterized in that the step of micro-patterns comprises a detection and / or measurement of mechanical tensions appearing on the surface of the support, during and / or after the formation. said patterns. 16. The method of claim 15, characterized in that the detection and / or measurement of mechanical stresses appearing on the surface of the support during the formation of micro-patterns implements mechanical or electromechanical microsystems. (MEMS). 17. The method of claim 15, characterized in that the detection and / or measurement of the mechanical stresses appearing on the surface of the support during the formation of the micro-patterns implements a transducer transforming the mechanical voltage variation into a variation of 'an electrical parameter. 18. Method according to any one of the preceding claims, characterized in that the step of examining the micro-patterns during and / or after their formation comprises a measurement of at least one electrical characteristic of the sample. 19. Method according to any one of the preceding claims, characterized in that the micro-patterns obtained during the self-training step have constituent features with dimensions between one thousandth of a micrometer and ten micrometers. 20. Process according to any one of the preceding claims, characterized in that the analysis information obtained at the end of the treatment sequence comprises information relating to a protein or a protein complex of interest. 21. Method according to claim 20, characterized in that the treatment sequence further comprises a step for deriving, from the information on the protein or on the protein complex of interest, qualitative and / or quantitative information about relationship with a human, animal or plant problematic. 22. A method according to any one of claims 1 to 19, wherein the sample may further comprise an analyte, characterized in that the treatment sequence further comprises a step for deriving from the comparison made between the results. examination and reference data, information on the product to be analyzed. 23. The method of claim 22, characterized in that the processing sequence further comprises a step for deriving, from the information on the product to be analyzed, qualitative and / or quantitative information related to a human problem, animal or vegetable. 24. Method according to any one of the preceding claims, characterized in that access to the reference data necessary to perform the comparison with the examination results of the micro-patterns is conditioned by obtaining rights of access from a holder of those rights. 25. Method according to any one of the preceding claims, characterized in that the execution of the step of comparison between the reference data and the examination results of the micro-patterns is conditioned by the obtaining of rights of reproduction. access to a holder of those rights. 26. Method according to one of claims 20 or 21, characterized in that the execution of the processing step to derive information relating to the protein, the protein complex or the product to be analyzed, is conditioned by obtaining rights of access to the holder of those rights. 27. Method according to claim 20 or 22, characterized in that the access to the deduction means of a qualitative and / or quantitative information related to a human, animal or plant problem is conditioned by obtaining rights of access to the holder of the said rights. 28. System for analyzing samples comprising a mixture of at least one protein and a solvent, implementing the method according to any one of the preceding claims, characterized in that it comprises: means for receiving a support (2) having a predetermined surface state on which a sample (1) has been deposited, means for inducing a self-formation of micro-patterns on said support, from said sample, means for examining said micro-motifs and / or effects induced during and / or after their formation, so as to produce examination results, means for storing and / or accessing reference data obtained by prior treatment and examination of a reference substance, - means for comparing the examination results with the reference data, so as to produce comparison data, and means for processing said comparison data so as to produce analysis information of said sample. 29. System according to claim 28, characterized in that the means for processing the comparison data (6) are arranged to deduce from the comparison performed, information on a protein or a protein complex present in the sample to be analyzed. 30. System according to claim 28, for the analysis of samples further comprising an analyte, characterized in that the means for processing the comparison data (6) are arranged to deduce from the comparison carried out, information about the analyte present in the sample. 31. System according to one of claims 28 or 31, for the analysis of samples further comprising an analyte, characterized in that the means (7) for processing the comparison data are arranged to deduce from information on the product to be analyzed, qualitative and / or quantitative information related to a human, animal or plant problem. 32. System according to any one of claims 28 to 31, characterized in that the receiving means are arranged to receive a set of supports comprising at least one deposition surface whose surface state is determined in relation to the data of reference, for the analysis of different samples. 33. System according to claim 32, characterized in that the set of supports whose surface state is determined in relation to the reference data, is provided with associated identification means. 34. System according to claim 33, characterized in that the identification means associated with the set of supports comprise a unique identification code and / or an encoding representative of all or part of characteristics in direct or indirect relation with said support. . 35. System according to one of claims 32 to 34, characterized in that the set of supports, which comprises at least one deposition surface, further comprises means for retaining one or more determined products in relation to the data. reference, said product being intended to become a co-solute when a sample is deposited on the corresponding deposition surface. 36. System according to one of claims 28 to 35, characterized in that it further comprises physical and / or physicochemical means for homogenizing the mixture of an additional product with the sample.