WO2003048757A1

WO2003048757A1 - Method for detecting positioning and assaying of biological molecules separated on a surface, by measuring an electrical property or effect

Info

Publication number: WO2003048757A1
Application number: PCT/FR2002/004136
Authority: WO
Inventors: René BENNES; Dalila Chenivesse; Jacques Bonnet; Cathy Guasch
Original assignee: Centre National De La Recherche Scientifique (Cnrs); Association Francaise Contre Les Myopathies; Universite De Montpellier Ii
Priority date: 2001-12-03
Filing date: 2002-12-02
Publication date: 2003-06-12
Also published as: AU2002364412A1; FR2833082B1; FR2833082A1

Abstract

The invention concerns a method for detecting and quantifying at least an optionally marked biological molecule in a sample, comprising a step of eletrophoretic spatial dispersion of at least a molecule on a support, characterized in that it comprises (i) a step which consists in measuring the surface potential of said support, in each surface point of said support, using at least a probe.

Description

Positioning and dosing detection of separate biological molecules on a surface, using the measurement of an electrical property or effect

The subject of the present invention is a method for detecting and quantifying biological molecules and a device for implementing such a method. PRIOR ART

In recent years, major advances have been made in molecular genetics and in particular in nucleic acid sequencing techniques and those making it possible to reveal mRNA transcripts and their translation products, in a given cell or tissue.

These techniques require the implementation of processes for the spatial dispersion of biological molecules along a line or a plane, according to their physico-chemical properties. This dispersion can for example be carried out by hybridization on the appropriate receptors (DNA chips) or by migration assisted by an electric field (electrophoresis carried out on crosslinked gels and followed or not by transfers to the surface of conductive or insulating, porous or waterproof supports) .

Usually, the molecules thus dispersed are placed in the presence of colored or fluorescent developers or markers which allow their detection by means of optical methods, or radioactive markers detected by radiation sensors. The revealing or marking products are varied, each of them having specific advantages and disadvantages, from the point of view of their effectiveness (sensitivity variable according to the nature of the proteins), their toxicity towards the experimenters or towards the environment, their influence on the subsequent analysis of samples, their adaptation to the performance of quantitative analyzes, or their cost.

Techniques for the visualization of proteins, peptides, or separate DNA fragments are thus known on electrophoresis gels (acrylamide, agarose, etc.) or on other supports (cellulose membranes, after transfer to membrane of nitrocellulose or nylon, on mica, silica or silicon plates, etc.) which constitute visual marking techniques (coloring of proteins or peptides with culvert red, Coomassie blue, amidoblack, silver, gold or by labeling with fluorescent probes or printing of photographic films for proteins labeled with radioactive elements such as ³² P, ³⁵ S and ...). This type of approach is linked to the history of biology, which favored direct observation of the images. However, the implementation of these visualization tools is cumbersome since the majority of the dyes used requires the use of toxic solvents (methanol, butanol, isopropanol, ...) which risk, in the long term, being banned from laboratories because that they involve significant risks to health and the environment. The same is true of certain chemicals used.

In addition, the quality of the final result depends on the staining protocol used, the freshness of the reagents and the expertise of the technician. Currently, electrophoresis gels, in particular 2D gels (= two dimensions, molecular mass / isoelectric point) are used as a basic technique in the study of the proteome. This involves separating, characterizing, and then identifying the proteins of interest initially contained in a sample (biopsy, cell culture, etc.). The technical challenge is considerable, the study of the proteome allowing more direct access to the real physiology of a cell, in particular to the fine metabolic regulations in which multiple protein-protein interactions are involved, after that of the genome (identification genes) and that of the transcriptome (DNA chips, identification of messenger RNAs). The aforementioned 2D gels make it possible to visualize the expression of proteins according to the tissues concerned, the cellular localization (membrane, cytoplasm, etc.), the state of development, the pathology if any, or the therapeutic treatment in progress. In addition, 2D gels could make it possible to determine the level of expression of each protein of interest, if the analysis techniques made it possible to quantify each protein precisely.

In fact, we know that the stage of development or the severity of certain pathologies is related to the rate of production of one or more specific proteins. Current techniques consume time and various chemicals (organic solvents, dyes, detergent salts, etc.). On the other hand, these techniques are all the responsibility of a protein coloring system (amido-blue, Coomassie blue, revelation with silver, colloidal gold, use of fluorochromes) which most often uses l affinity of the dye for aggregated protein-detergent structures (sodium = dodecyl sulfate SDS / protein). In addition, certain proteins escape coloring which itself is almost non-quantifiable.

In view of the prior art, the present inventors have identified the need for a method for detecting and quantifying biological molecules which in particular makes it possible to achieve the following objectives:

- Make the detection of proteins / peptides more quantitative.

- Overcome gels or membranes coloring techniques and therefore reduce the use of solvents.

- Overcome the labeling of peptides (at ³² P for phosphorylated derivatives or using fluorescent probes)

- Detect proteins / peptides which would not have reacted with the dye.

- Eliminate the need to digitize the image by scanner to archive it. - Spot a task on a gel (“spot”) for automatic excision before proteolysis and sequencing.

Consequently, the applicant has endeavored to develop a method for detecting a biological molecule present on a support which has an increased detection sensitivity, compared to the methods of the prior art and which allows quantification. more precise of the biological molecule detected, in particular due to the absence of saturation effect of the detector when said biological molecule is present in large quantity.

The above technical problems have been solved according to the invention, thanks to a method for detecting and quantifying at least one biological molecule, said method using the dipolar properties of this biological molecule.

The carbon skeleton of proteins already presents, because of its chemical structure, a chain of dipoles, those of the peptide bond -CO-NH-. The other charges can be carried by certain side chains such as for example the -OH dipole on the serine, threonine and tyrosine residues; net positive charges are carried by the arginine and lysine residues, in an acid medium; glutamate and aspartate residues carry negative charges in basic medium. The resulting dipole moment can be calculated, according to the method described in the document available on the internet at the following address: http: //bioin-formatics.weizmann. ac.il/dipol/dipint.html. The contribution of these charges or dipoles to the surface potential has sometimes been studied after adsorption or spreading of these proteins or peptides on the surface of a conductive aqueous solution. We can cite studies on melittin (Wacker bauer et al., 1996), gramicidin (Heitz et al., 1989), polyalanine, polyLproline (Isemura et al., 1959), etc.

In these cases, the measurement of the variation of the surface potential at the air-solution interface uses a vibrating electrode of 1 to 2 cm ² which integrates the electrical properties over a large surface (Davies et al., 1963).

Like proteins, nucleic acids also constitute charged biological molecules with a dipole moment.

The present invention relates to a method for detecting and quantifying at least one optionally labeled biological molecule (such as a protein, a peptide, a DNA, an RNA, etc.) in a sample comprising a step of spatial dispersion, of the electrophoresis type, of said at least one molecule on a support, characterized in that it comprises (i) a step of measuring the surface potential of said support, at each surface point of said support, using at least a probe. Advantageously, the support on which the biological molecule (s) are spatially dispersed consists of a support of the acrylamide gel type.

When the molecules can be identified by an electrical effect, the method in accordance with the invention has the particular advantage of reducing the type of nuisance - solvents etc. - mentioned above, and reducing its cost due to its automation, by detecting without revealing physical effects intrinsic to the tasks (spots) of biological molecules (proteins, peptides, nucleic acids on membranes, gels and transfer plates by direct measurement of the surface potential. In addition, it is no longer necessary to mark biological molecules, which avoids the use of radioactive labeling (with ³² P for phosphorylated derivatives) or labeling using fluorescent probes

The method according to the invention makes it possible to avoid the use of intermediate products, such as solvents. The above method makes it possible to directly detect and quantify one or more biological molecules previously dispersed on a support, thanks to the direct measurement of the surface potential, created by each cluster of molecules (spot), by each dipole and each electrical charge of the cluster. It also makes it possible to overcome at least part of using detergents and in some cases avoiding any staining step.

The cost is considerably lowered by simply eliminating consumables and by reducing working time in the least automatable part of the study of the proteome.

At the time of the separation step, or dispersion of the biological molecules or afterwards, it is no longer necessary to identify the clusters or spots by coloring with silver, for example, blue, or, after transfer on membrane, with colloidal gold. On the other hand, the problem of locating a spot cluster of biological molecules for the purpose of automatic excision, before proteolysis and sequencing for example, is completely resolved by the surface potential mapping techniques developed in the context of the invention. The acquisition of an image is carried out according to a matrix, by relative displacement of the sample with respect to the measurement probe. The X and Y coordinates of each surface point of the support for which a measurement of the surface potential is carried out, as well as the measurement of the surface potential at said surface point can be stored, for example in the memory of a digital computer. The memory of the digital computer can be loaded by a program containing the instructions allowing the creation of an image, called 3D, whose X and Y coordinates represent the X and Y coordinates of each surface point of the support for which a measurement surface potential has been performed, and if necessary previously stored, and whose Z coordinates represent the value of the surface potential at said surface point. It is therefore possible to reposition, facing the measuring electrode, or facing a sampling tool, any surface point, of the support for which a detection and quantification measurement has been carried out previously.

Thanks to the method of the invention, the need to digitize the image is also eliminated, for example by scanning in order to archive it, since the mapping technique implemented in the method according to the invention generates a representation of the measurements in a three-dimensional system, for example in the form of a 3D image representation.

Detection using a Kelvin probe is perfectly mastered in a laboratory by normally qualified personnel and the device is capable of withstanding a laboratory atmosphere. In addition to the drawbacks recalled from the techniques of the prior art cited above, these rarely lead to quantifiable results.

Quantification is difficult with staining techniques since, on the one hand, for example in the same gel, the action of a dye varies from one protein to another, and on the other hand, in gels to compare, and for the same protein, the coloring depends on many parameters such as the impregnation or revelation time.

According to a first embodiment of the method, the biological molecule or the complex ion with the same carbon skeleton that it can form in solution is marked with a detectable marker (fluorescent or radioactive).

According to a second embodiment of the method, the biological molecule or the complex ion with the same carbon skeleton that it can form in solution is not labeled.

The value of the surface potential measured at each point on the surface of the support analyzed in accordance with the method of the invention can result from one or more physicochemical conditions, among those indicated below:

- the biological molecule or the complex ion with the same carbon skeleton that it can form in solution is marked and the value of the surface potential results from a different electronic affinity between the molecules of the marker and their environment;

- the biological molecule or the complex ion with the same carbon skeleton that it can form in solution is not marked and the value of the surface potential results from the dipole moment or from the density of electronic charges, respectively.

- the value of the surface potential corresponds to a primary physico-chemical interaction with a marker carried either by the molecule itself, or by the probe.

- The value of the surface potential corresponds to a side effect of the hydrophilic / hydrophobic reaction type of the molecule.

Advantageously, the surface on which the molecule is deposited is functionalized and the probe optimized.

In a particular embodiment of the method, it comprises an additional step of transferring the biological molecule onto a second electrically conductive support prior to step (i) of measuring the surface potential. In this other embodiment, said second support is preferably a membrane wettable with water, such as a cellulose or nitrocellulose membrane impregnated with an electrolyte solution or a graphite membrane. Preferably, the probe used for implementing the method is a Kelvin probe or vibrating capacitor.

Preferably, the vibration of the probe is produced by a motor of the electromagnetic loudspeaker type or by a piezoelectric element, such as embedded vibrating beam or pellet. In another embodiment of the method, it comprises an additional step of transferring the biological molecule onto a second porous non-electrically conductive support, prior to step (i) of measuring the surface potential.

In this other embodiment, said second non-conductive support is preferably a porous nylon membrane.

In a preferred embodiment of the method, the measurement of the surface potential is automated and controlled by computer.

Advantageously, the method of the invention further comprises steps consisting in storing, processing the cartographic data obtained and in comparing these data by computer with profiles obtained from other samples of biological molecules.

According to an advantageous characteristic of the method, the support and the probe are implanted in an enclosure with hygrometry and controlled atmosphere.

Prior to step (i) of measuring the surface potential at any point on the surface of the support, the method of the invention advantageously comprises a measurement calibration step, said calibration step comprising the following steps: (a) depositing a marker, such as colloidal gold, on said support, whereby only the fraction where a deposition of the molecule was carried out fixes said marker, the complementary fraction of the support being naked; (b) place the support opposite the probe so that the surface facing the probe has not undergone any deposition and measure a first surface potential corresponding to the absence of a biological molecule, then place the support vis-à-vis the probe so that the surface facing the probe is fully marked, measure a second surface potential corresponding to the maximum quantity of biological molecules.

The present invention also relates to a device for implementing the method of the invention of the type comprising: - a means for spatial addressing of said at least one biological molecule on said first support,

a mechanical means allowing the automatic approach of the probe and its maintenance at constant distance from the surface of the first support,

- a support block for said first support, moisturizing and conductive - a Kelvin probe, means for electronic control of potential, distance and automatic approach,

a means of zoom effect, a means of measuring said quantity relating to an electrical property carried by the biological molecule, a means of automatic acquisition and processing of the measurements, and a means of coupling to a system direct debit.

The present invention also relates to a device for implementing the method of detection and quantification of a biological molecule described above, characterized in that it comprises:

- a support (24) on the surface of which biological molecules are immobilized;

- a probe (22) located at a distance z (26) from the surface of the support (14);

- a motor (20) causing the sinusoidal variation, of the value of the distance z at the frequency f; - a direct current generator (16) for applying a potential difference V between the probe (21) and the support (14);

- a sinusoidal current generator (24) for applying a sinusoidal potential difference V of frequency f, the frequency f and the frequency f being chosen with sufficiently different values to be able to be easily separated by synchronous detections.

Advantageously, said device also comprises means allowing the displacement of the probe (22) opposite any point on the surface of the support (24).

Thus the present invention relates to the localization and the dosage of biological molecules by measuring an electrical effect carried by these sorted molecules by means of a suitable process (grafting or electric field by example) according to a line, column or plan. In particular, this invention is related to the emerging field of proteomics and transcriptome which involves the identification and characterization of proteins present in biological samples. It therefore finds applications in the fields of characterization of animal or vegetable proteins, diagnosis of diseases or their prevention, medical prognosis, analysis of the effect of therapies, including genetic modifications.

We will now describe in detail the objects, characteristics and advantages of the present invention with the aid of exemplary embodiments and with reference to the accompanying drawings in which

Figure 1 shows a block diagram of the method of measuring surface potential by vibrating capacitor. 10 electric current meter

14 support on the surface of which a surface potential is measured, after possible transfer onto the membrane of the molecule or molecules previously separated, for example onto a gel.

16 DC voltage generator. 22 Measuring probe.

20 Motor causing the sinusoidal variation of the distance z between the surface of the support (14) and the probe (22) at the pulsation ω.

24 Pulse sinusoidal voltage generator ω '. 26 inter-electrode distance, of value z. Figure 2 shows the mechanical diagram of the device. 28 Reference support frames. 30 Support tank, liquid tank

32 Enclosure with controlled humidity 14 Support. 34 Insulation.

36 mechanical connection. 38 Conductive plastic foam.

40 Motor for actuation of table X. 42 Motor for actuation of table Y.

20 Motor causing the sinusoidal variation of the distance z between the surface of the support (14) and the probe (22). 44 mechanical frictionless travel system and drive motor with plunger 46 Z table actuation motor.

48 Stem.

22 Measuring probe.

50 Displacement table along the x-axis X. 52 Displacement table along the y-axis.

26 distance z between the surface of the support (14) and the probe (22).

54 Table for vertical displacement of the mobile unit carrying the probe (22).

Figure 3 shows the block diagram of the electronic assembly

14 support. 40 Table X actuation motor.

42 Table actuation motor Y.

20 Motor causing the sinusoidal variation of the distance Z.

44 mechanical frictionless translation system and translation motor with plunger core. 46 Z table actuation motor.

22 Measuring probe.

Figure 4 shows examples of graphical representation of the results

Fig. 4A: 3-dimensional representation of a protein spot, the two horizontal axes representing the x and y coordinates of the measurement points, and the vertical axis the surface potential detected by the Kelvin method. The protein spot appears in hollow.

Fig. 4B: same image as in 4A, projected in the horizontal plane representing the coordinates of the measurement points. The color code used represents the value of the surface potential. The protein spot appears dark on a light background.

Fig. 4C: section through the same spot of proteins represented in 4-a and 4-b. The amplitude of the variation in surface potential (vertical coordinate) makes it possible to precisely characterize the quantity of proteins present in this spot.

Figure 5: transfer to a nylon type membrane stained with colloidal gold, optically scanned without image processing. The halos are due to residual wetting by PBS.

Figure 6: acrylamide gel stained with Coomassie blue, optically scanned without image processing. Figure 7: image recorded by the vibrating capacitor technique, representing the surface potential of the proteins transferred onto nylon-type support, after coloration with colloidal gold.

FIGS. 8A and 8C: representation of the values of the surface potential during the scanning of lines 40 and 145, resulting from the gel of FIG. 7, also reproduced in FIG. 8B. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detecting a quantitative electrical effect carried by molecules of biological origin previously separated on a surface, as well as on the associated apparatus.

The electrical effects can manifest individually for each biological molecule, or collectively for a set of identical biological molecules.

In what follows, the method according to the invention is detailed in the case of an electrophoresis gel for separating the biological molecules (a), and in the case where said gel has been transferred to a nylon membrane (Western-Blott ) and stained with colloidal gold (b).

EXAMPLES

The illustrative examples which follow are given with reference to the appended figures 1 to 3 and in the following section the results are given with reference to figures 4 to 8. a) Detection of electrical properties or effects carried by biological molecules a.1) supports The supports (14) on which electrical effects can be demonstrated must have a dispersion of the biological molecules on a surface. The position of the biological molecule (s) is representative of its (their) nature. a.2) support support The support (14) is held facing the probe (22) on a support block so that the position of the biological molecules deposited on the surface can be identified, along a line (x coordinate) , along a flat (xy coordinate), or complex (xyz coordinate) surface. The support block must support the support, maintain its hydration and transmit the signals necessary for the operation of the detection system. a.3) Environment and observation environment Hygrometry, temperature, atmosphere and ambient light can be parameters of the experiment which it is necessary to control (32) a.4) Principle of the method The method applies to the detection and determination of electrical effects carried by biological molecules or by markers which have been attached to them. The contrasts observed during a topographic scan of the surface by a measurement probe may be due to a local change in the electronic affinity of the material, a local change in the concentration of electrical charges, or a local change in moments. dipolar.

a.5) Measurement of an electrical effect

Surface potential measurement The surface potential measurement device is based on the principle of the Kelvin vibrating capacitor: the measurement probe (22) and the surface of the support (14) of the molecule placed opposite it constitute a planar capacitor with capacity C. If a potential difference (-E0) is applied externally to the terminals of this capacitor, the capacitor charges under the potential difference Vcp-EO where if q is the charge of the electron, qVcp represents the difference between the energy required to extract an electron from the support and the energy required to extract an electron from the surface of the measurement probe. If the distance z (26) between the measurement electrode and the support is modified, by varying it sinusoidally at the frequency f = ω / 2ïl (20), the capacitance C varies and the current i which flows through the capacitor is given by the relation i = (Vcp-EO) dC / dt where dC / dt represents the variation of C with respect to time (Bonnet et al., 1976). In the experimental set-up in principle of Figure 1, the apparatus for measuring the electric current (10) and the DC voltage generator (16) are also illustrated. The principle of the measurement consists in adjusting the value of E0 so that i =

0. Under these conditions, the measurement of E0 provides the value of Vcp. The method is a method of measuring zero. It does not disturb the support, since under the conditions of the measurement, the latter is crossed by a zero electric current, that the electric field between electrodes is also zero and that the measurement is made without contact between the probe and the support . The electronic assembly (Palau et al., 1988) automatically fulfills the condition i = 0. a.6) Choice of the size and nature of the probe

The size of the probe is adapted to the area covered by the dispersed molecules. a.7) Zoom effect A zoom effect can be obtained by modifying the spacing between the measurement points, up to the maximum resolution allowed by the diameter of the measurement probe. As it is possible to manage the positioning of the support relative to the probe thanks to the scanning system, this zoom effect can be improved by the use of several probes of different diameters. It is thus possible to carry out a rough mapping of the location and to follow it with a finer mapping of one or more selected regions. . a.8) Realization of the probe

The vibrating element (20) of the measurement probe (22) can use different technologies. Microelectronics techniques can be applied to produce the probe when its size is reduced. a.9) Distance from probe to support

The probe-support distance z is kept constant by means of a servo. a.10) Mapping

The mapping of the measurements of the electrical effect carried by the biological molecule (s) is carried out by acquiring the value of the electrical potential when the probe-support distance is maintained at its set value. a.11) Travel

The relative displacement of the probe with respect to the support can be carried out using a cross-motion table (50, 52, 54). a.12) Steering

The control of the cross-motion table is carried out using a computer equipped with suitable cards. The software allows you to choose the coordinates of the starting point of a line, the length of a line, the number of measurement points per line, the number of lines and the spacing between the lines. It also allows to choose the speed of mechanical movement between two points as well as the time allocated to the measurement at each point. The lines can be scanned alternately or always in the same direction. In this last option, the line feed scanning must be fast so as not to excessively increase the acquisition time of an image. a.13) Acquisition

At each measurement point, the analog data supplied by the probe is digitized and then processed. a.14) Display and display of results

When reading a line, the magnitude of the electrical effect is displayed at each measurement point, in the form of a curve whose abscissa axis represents the position of the measurement point, and the ordinate represents the measured quantity.

At the end of the cartographic survey, the display of the results is in the form of a three-dimensional image in shades of gray or in false colors, representing the physical quantity measured at each point of the surface. An example of the visualization of the results is given in Figures 4. This visualization allows a direct comparison with the result of a conventional staining, if necessary. a.15) Processing

The system is equipped with computerized image processing which allows the choice of the image color palette, the choice of a color saturation threshold, the choice of color contrast, as well as the processing of raw data (interpolations or statistical processing ...).

The results are displayed on the screen. This equipment is of course upgradeable. a.16) Saving of measurements

The digitized measurement files are stored in the various usual formats. a.17) Direct debits

The support being mobile, it is possible to manage its positioning in relation to the position of the probe or in relation to any other fixed point. After the cartographic survey and its interpretation, it is thus possible, for example, to position any selected point in front of a sampling system. The molecules removed will be characterized by additional analyzes. b) Use of a vibrating capacitor on a transferred electrophoresis gel b.1) Example of support in the case of the separation of protein molecules In order to apply the method of the present invention, a biological sample is subjected to a preparation in which the proteins are separated along two axes corresponding to two of their physicochemical properties. This is for example the case encountered in the 2D electrophoresis process carried out on acrylamide gels. The proteins can then be partially transferred to the surface of a porous insulating support and their presence revealed by means of the marker constituted by colloidal gold.

Finally, gold atoms are deposited at certain points on the surface of the support, located in places whose relative coordinates are characteristic of the biological molecules present. b.2) the support block

The support block must mechanically maintain the support and allow its hydration. It must also transmit the signals necessary for the electrical operation of the assembly. It is therefore conductive and isolated from the ground. It is made in the form of a tank (30) and constitutes a reserve of hydration liquid. At the bottom of the tank is deposited a conductive plastic foam (38) on which the support thus polarized and hydrated by capillarity rests. In Figure 2, are shown the reference support frame (28), an insulator (34), a mechanical connection (36), actuation motors of the table X, Y, Z respectively (40, 42, 46) and a bracket (48). b.3) Environment

In order to limit losses by evaporation, through the surface of the support, the support and the probe are installed in an enclosure with controlled humidity and atmosphere (32). b.4) Principle of the method

The electrical energy required to collect an electron from the surface of a material depends on the chemical nature of that material. It is therefore different depending on whether it is measured on the surface of the bare support or on the surface of the support covered with a marker, such as gold. The difference in electrical energy results in a difference in electrical potential which can be measured without contact using a probe having an area. In the case where the surface of the support located in front of the measuring electrode is only partially covered with gold, the potential measured takes an intermediate value between that measured on the bare support and that measured on a layer of gold complete, in proportion to the areas concerned (Bonnet et al., 1984). The measured value changes in proportion to the coverage rate. The probe thus performs a quantitative measurement. b.5) Measurement of the surface potential These measurements are detailed in paragraph a.5 above. b.6) Choice of probe

The measurement of the surface potential is carried out relative to the surface potential of the measurement probe. Different materials can be chosen. To guide this choice, we are particularly attentive to the electrical stability of the material. b.7) Realization of the vibrating probe

The vibration is generated by an electromagnetic loudspeaker motor, or by a piezoelectric element (embedded vibrating beam or pellet). b.8) Distance between probe and support The accuracy of the measurement and the reduction of the effect of electrical noise require that the current i detected in the absence of servo-control be as large as possible. For this, the inter-electrode distance z (26) must be as small as possible. Typically, it is of the order of a few hundred micrometers. So that this distance is kept constant during the movement of the support whose surface is to be analyzed in front of the probe, the apparatus is equipped with a servo-control of the distance z between electrodes (Bonnet et al., 1977). To perform this control, the surface potential measuring electrode is also used as a distance sensor. The capacitor of capacitance C is supplied with a sinusoidal voltage V of frequency f = ω '/ 2π (24) different from the vibration frequency f, and a current i = CdV' / dt, of proportional amplitude, is measured at this frequency to the capacitance C and therefore inversely proportional to the distance z.

The values of the frequencies f and f are chosen sufficiently different to be able to be easily separated by synchronous detections. Mechanically, in order to keep the inter-electrode distance constant, the vibrating measurement probe which is guided by a frictionless translation system (44) has its distance smoothly regulated smoothly thanks to the use of a solenoid. and a suspended plunger core. This system provides a translation of 2 mm. Larger displacement amplitudes can be obtained using a rotary motor actuating the micromanipulator tray. This system is activated automatically by the control electronics when the fine compensation system is outside its operating limits. This can happen when the surface of the support is not parallel to that of the reference plane and when the surface of the support is not planar. It is thus possible to work on large supports that are not perfectly flat.

Another effect of the distance control is to allow measurement in noisy surroundings. b.9) Starting a cartography The servo-control of the inter-electrode distance, using the two complementary mechanical control systems, is also used to carry out the automatic approach of the probe during the start-up phase of a cartographic survey. b.10) Displacement of the support In order to carry out a mapping of the variations of the electric potential of the support, it is necessary to carry out measurements on all the points of coordinates X and Y of its surface. Our system therefore performs a relative displacement of the probe (22) relative to the support (14). For example, the probe is kept fixed, and the surface of the support is moved opposite the probe thanks to an XY table with crossed movements (50, 52) whose piloting is managed by a computer. It is thus possible to determine the coordinates of the starting point and the number of measurement points. At each point of determined X and Y coordinates, the computer manages the acquisition of the measurement of the potential of the surface facing the probe. b.11) Computer control

In order to avoid accidental contact between the support and the measuring electrode during this movement, the computer manages the distance of the probe before the return and then its repositioning in ideal measurement conditions when the return has been made. In the event of accidental contact between the measurement probe and the support, the scanning is stopped, the probe is moved away to remove the liquid meniscus which forms between it and the support, then it is again approached in the measurement position before the scanning does not resume. b.12) Acquisition of the measurements Before the measurements are stored, a digital processing of these data is carried out. The stored data actually represents the average of several measurements made on the same point, after deletion of outliers. The experimenter can choose the number of data entering this processing. b.13) Display The screen is divided into multi-windows with drop-down menus. A window shows the potential variations noted during scanning of a line, as a function of the abscissa of the measurement point. This line can be compared to that noted during the previous scan, which was stored and displayed in another window. At the end of the reading, a window displays, in gray tones, a plan view of the potential variations on the surface as a function of the X and Y coordinates of the point. b.14) Processing of measures

A classic quadratic interpolation is used in order to limit the effect of outliers. B.15) Saving and mapping

The files are saved in an ASCII format. The images are then subject to the usual processing using conventional point matrix processing software.

In accordance with the present invention, non-contact measuring devices of the surface electrical potential are used to map the distribution and the quantity of molecules of biological origin separated by an adapted process.

First of all, a one- or two-dimensional dispersion (or network) is produced on the surface of a support. The method according to the invention provides a digital mapping representative of the identity and abundance of the biomolecules detected on the surface of the one- or two-dimensional network. This mapping allows qualitative and quantitative analysis, data storage and processing, including computer comparison of profiles provided by other biological samples. Non-contact measurements of the surface electrical potential can be automated and controlled by computer. They constitute an interesting alternative to conventional optical or nuclear detection methods. They present a complementarity to these methods by highlighting other physical properties, and by allowing the use of other measurement conditions which could be better adapted to the molecules to be detected. They allow quantitative measurements to be made with great sensitivity. The techniques operate at room temperature, and / or in a controlled atmosphere.

The measurement probe can be coupled to a robotic sampling arm. For large supports, the measurement can be made using a suitable Kelvin vibrating capacitor.

Depending on the electrical signals detected, the invention can make it possible to work on different supports, with different developers or markers, or even on undisclosed gels. RESULTS

The results which follow are given with reference to FIGS. 4 to 8.

Tests are carried out using the vibrating capacitor (= Kelvin) on nylon type supports, after coloration with colloidal gold.

We have highlighted variations in surface potentials which correspond to deposits of proteins stained with colloidal gold.

The lateral scale in gray tones translates the value of the potential measured at each point. Passing through a minimum potential value corresponds to the darkest tones.

The curves represented in FIGS. 8A and 8C represent the values of the potential recorded during the scanning of lines 40 and 145 of the electrophoresis gel of FIG. 8B, respectively. As the reminder lines show, the low levels reached, which are different on minima A and B, reveal the quantitative aspect of the characterization.

The data concerning the variations of the surface potential can easily be processed numerically to increase the contrasts and / or to reveal additional information hitherto embedded in a continuous background, for example the region C of FIG. 8A.

This technique can be extended to different supports and different colors, even to non-colored transfers. It could be adapted to measurements on acrylamide gels, which would avoid transfers and allow the automated punching out of detected spots.

Compared to previous coloring techniques, the approach according to the invention gives a more detailed account of the phenomenon, since one can distinguish substructures. The analysis of substructures can usefully provide information on the presence of several proteins under a spot and therefore lead to a higher quality of discrimination between two extremely neighboring proteins,

REFERENCES

Bonnet J., Palau JM, Soonckindt L., Lassabatere L., "On the interest of a current amplifier associated with the vibrating capacitor of the Kelvin method", Apparatus and techniques 213-213 (1976) Bonnet J., Soonckindt and Lassabatere L., "Slave device for the topographic study of output work by the vibrating condenser method of Kelvin, Measurement and Control, MECO 77, Eds. MH Hamza, Acta Press (1977).

Bonnet J., Soonckindt L., Lassabatere L., "The Kelvin probe method for work function topographies: technical problems and solutions", Vacuum 34.7 693- 698 (1984)

Davies J.T. and Rideal E.K., "Interfacial Phenomena" page 64, 1963 Academy Press.

Heitz F., Van Mau N., Bennes R., Daumas P. and Trudelle Y., "Single channels and surface potential of linear gramicidins", Biochemistry 71 (1) 83-8 (1989)

Isemura T. and Ikeda S., "The role of prolyl residue in polypeptide monolayers. I. On the chain configuration deduced from surface pressure and potential measurements", Surface Chemistry of Synthetic Protein Analogs VIII, 32 (2) 178-184 (1959 ) Palau JM, Bonnet J., "Realization of a high-performance Kelvin probe for the topographic study of output work", J. Phys. E: Sci. Instrum. 21,674-679 (1988)

Wackerbauer G., Weis I. and Schwarz G., "Preferential partitioning of melittin into air / water interface: structural and thermodynamic implications", Biophys. J. 71 (3) 1422-7 (1996).

Claims

1. Method for detecting and quantifying at least one biological molecule optionally labeled in a sample comprising a step of spatial dispersion, of the electrophoresis type, of said at least one molecule on a support, characterized in that it comprises (i ) a step of measuring the surface potential of said support, at each surface point of said support, using at least one probe.

2. Method according to claim 1, characterized in that the support is of the acrylamide gel type.

3. Method according to one of claims 1 or 2, wherein the biological molecule or the complex ion of the same carbon skeleton is labeled.

4. Method according to one of claims 1 or 2, wherein the molecule or the complex ion of the same carbon skeleton is not labeled.

5. Method according to any one of claims 1 to 4, comprising an additional step of transfer of the biological molecule to a second support, electrically conductive, before step (i) of measuring the surface potential.

6. Method according to claim 5, characterized in that said second support is a membrane wettable with water such as a cellulose or nitrocellulose membrane impregnated with an electrolyte solution or a graphite membrane.

7. Method according to any one of claims 1 to 4, characterized in that it comprises an additional step of transfer of the biological molecule onto a second porous non-electrically conductive support, prior to step (i) for measuring the surface potential.

8. Method according to claim 9, characterized in that said second support is a nylon membrane.

9. Method according to one of claims 1 to 8, characterized in that the probe is a Kelvin probe or a vibrating capacitor.

10. Method according to claim 9, characterized in that the vibration of the probe is produced by a motor of the electromagnetic loudspeaker type or by a piezoelectric element, such as embedded vibrating beam or pellet.

11. Method according to any one of the preceding claims, characterized in that the measurement of the surface potential is automated and controlled by computer.

12. Method according to any one of the preceding claims, characterized in that it further comprises steps consisting in storing, processing the cartographic data obtained and in comparing by computer these data with profiles obtained from other samples of biological molecules.

13. Method according to any one of the preceding claims, characterized in that the support and the probe are implanted in an enclosure with hygrometry and controlled atmosphere.

14. Method according to any one of claims 1 to 13, characterized in that it comprises, prior to step (i) of measuring the surface potential, an additional step of calibration of the measurement, said step d calibration comprising the following steps:

(a) depositing a marker, such as colloidal gold, on said first support, whereby only the fraction where a deposition of the molecule was carried out fixes said marker, the complementary fraction of the support being naked;

(b) place the support with respect to the probe so that the surface facing the probe has not undergone any deposition and measure a first surface potential then place the support with respect to the probe so that the surface facing the probe is fully marked and measure a second surface potential;

15. Device for implementing the method according to any one of claims 1 to 14, characterized in that it comprises:

- a support (14) on the surface of which biological molecules are immobilized;

- a motor (20) causing the sinusoidal variation, of frequency f of the value of the distance z;

- a direct current generator (16) for applying a potential difference (16) of the probe (21) and the support (14);

- a sinusoidal current generator (24) for applying a potential V / sinusoidal difference of frequency f, the frequency f and the frequency f being chosen with sufficiently different values to be able to be easily separated by synchronous detections.

16. Device according to claim 15, characterized in that it comprises means allowing the displacement of the probe (22) opposite any point on the surface of the support (14).