Patent Publication Number: US-9901934-B2

Title: Method and microsystem for detecting analytes which are present in drops of liquid

Description:
TECHNICAL FIELD 
     The present invention concerns the general field of detection of analytes of interest which are present in a liquid of interest. 
     These analytes of interest can be chemical and/or biological targets, e.g. macromolecules, cells, organelles, pathogens or intercalations. 
     PRIOR ART 
     In numerous fields, attempts are being made to detect analytes of interest of the chemical and/or biological type which may be present in a drop of liquid. 
     This may be the case, for example, to establish a biological or medical diagnosis, or in the fields of genetic engineering or the food industry. Attempts may be made to detect or measure out, in particular, macromolecules, cells, organelles, pathogens or intercalations. 
     Usually, attempts are made to analyse liquid samples of low volume in reduced time, in the simplest and least intrusive possible way. 
     As an illustration, biochips, which form, in the field of molecular biology, microsystems for analysing the hybridization of nucleic acids (DNA and/or RNA), or interactions of the type of antigen/antibody, protein/ligand, protein/protein, enzyme/substrate, etc., may be cited. Attempts may be made to obtain kinetic parameters or equilibrium constants associated with these chemical interactions. 
     In general, analytes of interest which are of the biological and/or chemical type can be detected using a sensor in a microchannel, within which the liquid sample to be analysed circulates. Several detection techniques can be used, such as detection by gravimetry and detection by field effect. 
     Patent application WO2009/141515, which was filed in the name of the applicant, describes a device for gravimetric detection of particles in a fluid medium, in particular biomolecules. The device includes an electromechanical oscillator, the vibration frequency of which depends on the quantity of analytes of interest which are deposited on the surface of the oscillator. 
     More precisely, the device includes a microchannel, in which a liquid including the analytes of interest circulates. Inside the microchannel, a plane electromechanical oscillator is arranged, in the form of, for example, a square plate. One of the faces of the plate defines an analyte detection surface, the functioning of which can be obtained by prior grafting of probes which are capable of binding to the analytes of interest. 
     The oscillator is kept in position, and able to vibrate in its plane, by beams which are arranged at the four apices of the plate, and each connected to the substrate in which the microchannel is formed. 
     The means of actuating the oscillator can include two adjacent electrodes which are arranged near the plate and coplanar therewith. The oscillator is made to vibrate, at its natural frequency of resonance, by electrostatic coupling, via the two actuating electrodes. To do this, the oscillator is brought to a constant electrical potential. 
     The detection means include at least one electrode which is arranged near the plate and facing said actuating electrodes. Modulating the capacitance between the oscillator and the measuring electrode, because of the vibration of the oscillator, generates a capacitive current, called a motional current, at said electrode. 
     By measuring this current, and in particular its spectral response, the vibration frequency of the oscillator, and then the divergence between the effective vibration frequency of the oscillator and the initial frequency, are deduced. The mass of the analytes of interest which are deposited on the detection surface of the oscillator is directly correlated with this frequency divergence. 
     However, this detection device by gravimetry according to the prior art has some disadvantages. 
     Thus the concentration of analytes of interest in the liquid sample is greatly affected by the hydrodynamic forces which are present in the flow of liquid within the microchannel. In fact, the usually micrometric dimensions of the microchannel make the viscosity forces particularly high. The analytes which are present near the walls, and in particular the edges, of the microchannel are then virtually held back by the viscosity forces, which tends to reduce the concentration of analytes which are routed effectively to the sensor. 
     Additionally, the walls of the microchannel are likely to include chemical elements which can contaminate the liquid of interest, and possibly interact with the analytes upstream from the sensor, or with the probe elements of the detection surface, which may interfere with the detection sensitivity of the sensor. 
     Additionally, the plate is immersed in the liquid of interest. Also, the liquid is present, in particular, in the vibration zone of the plate, i.e. between the plate and the lateral electrodes in the case of transduction by capacitive coupling, which results in damping of the vibrations, called “squeeze damping”, to which viscous damping is added, both of which greatly degrade the quality factor of the sensor. The quality factor of such a sensor usually corresponds to the fineness of its resonance peak. Additionally, it is known that the quality factor is correlated with the sensitivity of detection. In other words, the finer a resonance peak is, the more the quality factor will be increased, and the more the sensitivity of detection of the sensor will be increased. The quality factor is commonly determined by the width, at mid-height, of the resonance peak in a graph representing the vibration amplitude as a function of the vibration frequency. However, any other indicator corresponding to the fineness of a resonance peak can be used. 
     Additionally, in the case of gravimetric sensors with capacitive transduction which are immersed in the liquid of interest, it is necessary to cover the faces of the actuating and detecting electrodes with an insulating layer. In fact, in the absence of this layer, there is a risk of electrolysis when the liquid of interest is conductive. On the other hand, the presence of this insulating layer makes it necessary to increase the actuating voltages to obtain the same oscillation amplitude. 
     SUMMARY OF THE INVENTION 
     The object of the invention is to present a method of detecting analytes of interest which are present in a liquid, at least partly overcoming the above-mentioned disadvantages in relation to the implementation of the prior art. 
     For this purpose, the invention relates to a method of detecting analytes of interest which are present in a liquid of interest, including the following steps:
         said liquid of interest is put into contact with a first surface, said surface being parallel to at least one detection surface;   a finger of liquid is formed on said first surface by liquid dielectrophoresis, under the effect of an electrical control, the finger of liquid extending along two approximately coplanar movement electrodes which are arranged on said first surface, said electrodes including at least one drop formation zone facing said at least one detection surface;   the electrical control is stopped, so that the finger of liquid breaks by capillarity, generating at least one drop on one of said drop formation zones, said at least one drop having sufficient thickness to come into contact with said at least one detection surface;   said analytes of interest which are present in said at least one drop are detected by detection means working with said at least one detection surface.       

     Liquid dielectrophoresis (LDEP) is understood to be the application of an electrical force to an electrically insulating or conducting liquid, the force being generated by a non-uniform oscillating electrical field. The formation of a finger of liquid by liquid dielectrophoresis is described, in particular, in the article by Jones entitled “Liquid dielectrophoresis on the microscale”, J. Electrostat., 51-52 (2001), 290-299. When the liquid is in an electrical field, the molecules of the liquid acquire a non-null dipole and are polarised. To the extent that the field is non-uniform, a Coulomb force appears, and induces the movement of the molecules of the liquid, and thus of all the liquid, towards a field maximum. 
     It should be noted that when the electrical control is stopped, the finger of liquid is of unstable form. Capillary instability then develops rapidly, and causes the finger to break into one or more drop(s), which makes it possible to lower the surface energy of the liquid. 
     The method according to the invention thus provides the detection of analytes of interest which are present in a drop of liquid in contact with the detection surface. The method also makes it possible to form multiple drops simultaneously. The drops can come into contact with a single detection surface or distinct detection surfaces. 
     In contrast to the prior art mentioned above, the analytes are no longer carried by a liquid flowing in a microchannel, but by a finger of liquid in contact with the first surface. The influence of viscous forces is thus greatly reduced, to the extent that the total surface of wetted wall is appreciably reduced. The quantity of analytes “trapped” near the walls, here the first surface, is thus appreciably less, which increases the quantity of analytes which are carried effectively to the detection surface. 
     Additionally, by reducing the total surface of wetted wall, the risk of contaminating the liquid of interest by contact with a contaminated surface is greatly reduced. Additionally, the detection surface is in contact with the liquid only when a drop comes to cover it, which appreciably reduces the risk of contaminating the detection surface by interfering chemical elements. 
     Additionally, in the case of an electromechanical oscillator as described above, one face of which forms a detection surface, the absence of liquid in the vibration zone makes it possible to avoid the damping of the vibrations, of the “squeeze damping” type. The quality factor is then preserved. 
     In the case that multiple drops are formed simultaneously from the finger of liquid, and come into contact with multiple detection surfaces at one drop per surface, said detection surfaces can be used to detect different categories of analytes, thus making it possible to detect, precisely and rapidly, a large number of analytes of different categories. 
     It should be noted that when the liquid of interest is surrounded by a gas, actuation of one detection surface does not influence detection at an adjacent detection surface, to the extent that the different corresponding oscillators are not immersed in a liquid. Only the gas is present in the vibration zone of each oscillator, when the latter vibrates in its plane. If it vibrates outside its plane, only the drop on the corresponding detection surface is deformed, without this interfering with the vibrations of an adjacent oscillator. 
     Preferably, said movement electrodes are approximately rectilinear, coplanar and approximately parallel to each other. 
     Said first surface and said at least one detection surface are separated from each other by a height greater than the maximum thickness of the finger of liquid and less than the maximum thickness of said at least one drop of liquid. 
     Thus the finger of liquid is formed on the first surface, without touching said at least one detection surface. When at least one drop is generated by capillary breaking of the fluid finger, it naturally comes into contact with the detection surface, to the extent that the drop has a maximum thickness which is greater than the distance which separates the two surfaces. 
     Advantageously, the probe elements which are capable of binding to the analytes of interest are grafted onto said at least one detection surface, in such a way as to cover it at least partly. 
     These grafted probe elements can be, for example, antibodies, probes for nucleic acids or printed polymers. 
     According to one embodiment, said liquid movement electrodes include multiple drop formation zones, which are each arranged facing a distinct detection surface. When the electrical control stops, the finger of liquid breaks into multiple drops, each situated on one of said drop formation zones, each drop coming into contact with the corresponding detection surface. 
     The drop formation zones can correspond to outgrowths of the coplanar electrodes. Preferably, these outgrowths are in the form of half-discs. 
     Thus the method makes it possible to form multiple drops. The drops are formed simultaneously, and come into contact with the corresponding detection surface simultaneously. 
     Additionally, the placement of the drops is perfectly controlled, to the extent that each drop is formed on the drop formation zone of the movement electrodes. 
     Additionally, the drops all have a calibrated volume. It is possible that each drop has an identical volume. 
     The volume of each drop depends on the size, and in particular the width, of the drop formation zones, the width of the finger of liquid, and the hydrophilic character of the first surface. 
     When the fluid finger is formed on a first surface facing the detection surface, the volume of the drop also depends on the distance which separates the first surface and the detection surface. 
     The width of the fluid finger is approximately equal to the distance 2R between the rectilinear parts of the outer edges of the movement electrodes. 
     Each detection surface can include probe elements which are capable of binding to different analytes of interest according to the detection surfaces being considered. It is then possible to proceed with detection of analytes of different categories, according to the type of probe elements. 
     According to another embodiment, said liquid movement electrodes include multiple drop formation zones, which are arranged facing the same detection surface. When the electrical control stops, the finger of liquid breaks into multiple drops, each situated on one of said drop formation zones, each drop coming into contact with said corresponding detection surface. 
     As before, the drops are formed simultaneously, and have a calibrated volume. The volume of each drop can also be identical. 
     Advantageously, said movement electrodes each include inner and outer edges, the inner edges being arranged approximately facing each other, and the outer edges having approximately rectilinear parts. 
     Said rectilinear parts are separated from each other by a distance 2R, and the drop formation zones are separated from each other by a distance which advantageously is between eight and ten times the distance R, and preferably of the order of nine times the distance R, and preferably 9.016R. 
     This distance is approximately equal to the most unstable wavelength of the finger of liquid. 
     According to one embodiment, said liquid movement electrodes include a single drop formation zone which directly faces a single detection surface. When the electrical control stops, the finger of liquid breaks into a single drop, situated on said drop formation zone, said drop coming into contact with said detection surface. 
     Preferably, said movement electrodes are covered with a dielectric layer. 
     Preferably, said first surface is hydrophobic, and said at least one detection surface is at least partly hydrophilic. 
     According to a first preferred embodiment of the invention, said detection surface is a face of a plane electromechanical oscillator which is capable of vibrating. 
     Said detection step can then include the following substeps:
         the oscillator is set to vibrate at a predetermined frequency and according to a predetermined vibration mode;   the effective vibration frequency of the oscillator is measured;   a divergence between the measured vibration frequency and the predetermined vibration frequency is calculated.       

     This divergence is due to the mass of the drop which is deposited on the detection surface. When the detection surface is made functional with specific probes, the divergence is also due to the interactions between the targets which are present in the liquid of interest and the probes. The term “gravimetric detection” can also be used. 
     Preferably, at least one actuating electrode is arranged facing the edge of said oscillator, preferably parallel to the latter, and advantageously coplanar with the latter. Said setting of the oscillator to vibrate is implemented by electrostatic coupling between the oscillator and said at least one actuating electrode, by generating an alternating electrical field between said oscillator and said at least one actuating electrode. 
     Said oscillator can thus be brought to a constant electrical potential, and an alternating electrical voltage can be applied to said at least one actuating electrode. 
     A measuring electrode is arranged facing the edge of said oscillator, preferably parallel to the latter, and advantageously coplanar with the latter. Said step of measuring the vibration frequency of the oscillator includes measuring an electric current circulating from said measuring electrode, said electric current being generated by capacitive coupling between the oscillator and said measuring electrode. Several measuring electrodes can be arranged, these measuring electrodes then being coupled capacitively to the oscillator. 
     Alternatively, analytes of interest are detected by piezoelectricity. Said at least one detection surface includes a layer of an electrically conducting material which forms a reference electrode, and is covered with a layer of a dielectric piezoelectric material, the latter being covered at least partly by at least one measuring electrode. Said step of measuring the vibration frequency of the oscillator includes measuring an electric current circulating from said measuring electrode, said electric current being generated by capacitive coupling between the reference electrode and the measuring electrode, the latter being brought to a given electrical potential by polarisation of the piezoelectric layer because of the vibration of the oscillator. 
     According to a variant, said piezoelectric layer is covered at least partly by two measuring electrodes, each formed of a metallic track and arranged approximately parallel to each other. Said step of measuring the vibration frequency of the oscillator also includes measuring a second electric current from at least one of said measuring electrodes, said second electric current being generated by capacitive coupling between said measuring electrodes. 
     Alternatively, analytes of interest are detected by a technique according to which the oscillator forms a resonant electrical grid. An electrode forming a channel is arranged facing the edge of said oscillator, preferably parallel to the latter, and advantageously coplanar with the latter, said electrode forming a channel being connected to an electrode forming a source, which is brought to a first constant electrical potential, and to an electrode forming a drain, which is brought to a second electrical potential. Said step of measuring the vibration frequency of the oscillator includes measuring the variations of the electric current which circulates in the electrode forming a channel, said variations being induced by field effect between the oscillator and the electrode forming a channel. 
     Alternatively, analytes of interest are detected by a detection technique by field effect, according to which the oscillator forms a resonant electrical channel. Said oscillator is an electrode forming a channel, and is connected to an electrode forming a source, which is brought to a first constant electrical potential, and to an electrode forming a drain, which is brought to a second electrical potential. Said step of measuring the vibration frequency of the oscillator includes measuring the variations of the electric current which circulates in the electrode forming a channel, said variations being induced, by field effect, by analytes of interest being deposited on the detection surface of the oscillator. 
     Advantageously, said at least one detection surface has a hydrophilic zone which is intended to be covered by said at least one drop, the outline of the hydrophilic zone coinciding approximately with the nodal lines of the oscillator according to the vibration mode in which it is stressed. 
     According to a preferred second embodiment of the invention, said detection surface includes multiple nanowires, each connected to an electrode forming a source, to which a direct voltage is applied, and to an electrode forming a drain, to which a direct voltage is applied. Said step of detecting analytes of interest includes measuring the variations of the electric current which circulates in said nanowires, said variations being induced, by field effect, by analytes of interest being deposited on said detection surface. 
     The invention also concerns a method of detecting analytes of interest which are present in a liquid of interest, including the following steps:
         said liquid is put into contact with a principal surface formed of a surface of a substrate, a surface of a plane detector forming a detection surface and a surface of means of supporting the oscillator relative to said substrate;   a finger of liquid is formed on said principal surface by liquid dielectrophoresis, under the effect of an electrical control, the finger of liquid extending along two movement electrodes which are arranged on said principal surface, said electrodes including at least one drop formation zone, each located on said detection surface of the detector;   the electrical control is stopped, so that the finger of liquid breaks by capillarity, generating at least one drop on one of said drop formation zones;   said analytes of interest which are present in said at least one drop are detected by electrical detection means working with said at least one detection surface.       

     In contrast to the previously described method, here the surface on which the finger of liquid is formed and the at least one detection surface are coplanar. 
     Advantageously, the probe elements which are capable of binding to the analytes of interest are grafted onto said at least one detection surface, in such a way as to cover it at least partly. 
     Said detection surface is a face of a plane electromechanical oscillator which is capable of vibrating. Said detection step can then include the following substeps:
         the oscillator is set to vibrate at a predetermined frequency and according to a predetermined vibration mode;   the effective vibration frequency of the oscillator is measured;   a divergence between the measured vibration frequency and the predetermined vibration frequency is calculated.       

     This divergence is due to the mass of the drop which is deposited on the detection surface. When the detection surface is made functional with specific probes, the divergence is also due to the interactions between the targets which are present in the liquid of interest and the probes. The term “gravimetric detection” can also be used. 
     Preferably, at least one actuating electrode is arranged facing the edge of said oscillator, preferably parallel to the latter, and advantageously coplanar with the latter. Said setting of the oscillator to vibrate is implemented by electrostatic coupling between the oscillator and said at least one actuating electrode, by generating an alternating electrical field between said oscillator and said at least one actuating electrode. For example, the oscillator is brought to a constant electrical potential, and an alternating electrical voltage is applied to said at least one actuating electrode. 
     A measuring electrode is arranged facing the edge of said oscillator, preferably parallel to the latter, and advantageously coplanar with the latter. Said step of measuring the vibration frequency of the oscillator includes measuring an electric current circulating from said measuring electrode, said electric current being generated by capacitive coupling between the carried oscillator and said measuring electrode. 
     The oscillators described above (piezoelectric oscillators, oscillators with a resonant grid or resonant channel) can also be used in this embodiment. 
     The invention also concerns a device for detecting analytes of interest, to implement the detection method with a non-coplanar drop formation surface and detection surface, according to one of the above characteristics. The detection device includes:
         a first surface and at least one detection surface, said first surface being parallel to said at least one detection surface and arranged at a determined distance from the latter;   a tank of liquid of interest, arranged so that said liquid can be put into contact with said first surface;   electrical means of forming, by liquid dielectrophoresis, a finger of liquid from said tank on the first surface, said electrical means including two approximately coplanar movement electrodes which are arranged on said first surface and include at least one drop formation zone facing said at least one detection surface;   means of detecting analytes of interest in a drop of said liquid in contact with said at least one detection surface, said detection means working with said at least one detection surface.       

     Finally, the invention also concerns a device for detecting analytes of interest, to implement the detection method with a coplanar drop formation surface and detection surface, according to one of the above characteristics. The detection device includes:
         a substrate, at least one plane electromechanical oscillator, and means of supporting each oscillator relative to said substrate, a principal surface being formed of a surface of said substrate, a surface of said oscillator forming a detection surface and a surface of said means of support;   a tank of liquid of interest, arranged so that said liquid can be put into contact with said principal surface;   electrical means of forming, by liquid dielectrophoresis, a finger of liquid from said tank on the principal surface, said electrical means including two approximately coplanar movement electrodes which are arranged on said principal surface and include at least one drop formation zone each located on said detection surface;   means of detecting analytes of interest in a drop of said liquid in contact with said at least one detection surface, said detection means working with said at least one detection surface.       

     Other advantages and characteristics of the invention will appear in the non-limiting detailed description below. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments of the invention will now be described, as non-limiting examples, referring to the attached drawings, in which: 
         FIG. 1  is a schematic view in longitudinal cross-section of a detection device according to the first preferred embodiment of the invention, in which the detection technique is gravimetric; 
         FIG. 2A  is a schematic view from below of the substrate forming a cover of the device shown in  FIG. 1 , the cover being equipped with two movement electrodes; 
         FIG. 2B  is a detailed view of part of the movement electrodes shown in  FIG. 2A ; 
         FIG. 3  is a detailed schematic view in transverse cross-section of part of the detection device shown in  FIG. 1 ; 
         FIG. 4A  is a schematic perspective view of part of the detection device shown in  FIG. 1 ; 
         FIG. 4B  is a schematic plan view of the plane electromechanical oscillator, which is surrounded by actuating electrodes and measuring electrodes, of the part of the detection device shown in  FIG. 4A ; 
         FIGS. 5A to 5C  are schematic views in longitudinal cross-section of the detection device shown in  FIG. 1 , showing the formation of drops of liquid; 
         FIGS. 6A and 6B  are views in transverse cross-section ( FIG. 6A ) and plan views ( FIG. 6B ) of part of the detection device according to a variant of the first preferred embodiment, in which the detection technique is piezoelectric; 
         FIG. 7  is a schematic perspective view of part of the detection device according to a variant of the first preferred embodiment, in which the oscillator forms a resonant electrical grid; 
         FIG. 8  is a schematic perspective view of part of the detection device according to a variant of the first preferred embodiment, in which the oscillator forms a resonant electrical channel; 
         FIG. 9  is a schematic view of part of the detection device according to the second preferred embodiment, in which the detection surface includes multiple nanowires; 
         FIG. 10  is a schematic perspective view of part of the detection device according to the third embodiment of the invention, in which the drop formation surface and the detection surfaces are coplanar. 
     
    
    
     DETAILED PRESENTATION OF A PREFERRED EMBODIMENT 
       FIG. 1  shows a device for detecting analytes of interest which are present in a liquid, according to a first embodiment of the invention. 
     The detection device  1  includes a lower substrate  10  and an upper substrate  20  forming a cover, arranged facing each other. 
     The cover  20  has a lower face formed of a dielectric layer  22  and a hydrophobic layer  23 . The free surface of said hydrophobic layer is called the first surface  24 . 
     The lower substrate  10  includes multiple electromechanical oscillators  30 , which are capable of being set to vibrate. Said oscillators  30  are described in detail below. The upper face  31  of each oscillator is called the detection surface  31 , and faces the first surface  24  of the cover  20 . 
     In all the description below, by convention a direct orthonormal frame in Cartesian co-ordinates (X, Y, Z) is used, as shown in  FIG. 1 . The plane (X, Y) is parallel to said surfaces, and the direction Z is oriented from the detection surfaces  31  to the first surface  24  of the cover. 
     The terms “upper” and “lower” should be understood here in terms of orientation following the direction Z of said frame. 
     Said detection surfaces  31  are coplanar, and separated from the first surface  24  by a determined distance H. 
     The cover  20  includes an aperture  25  which passes through and opens into the first surface  24 . The aperture  25  can be filled with liquid, in which analytes of interest may be present, thus forming a liquid tank  25 . 
     The liquid has an electrical conductivity of the order of a few μS·cm −1  to a few mS·cm −1 , e.g. between 1 μS·cm −1  and 100 mS·cm −1 , preferably of the order of 10 mS·cm −1 . 
     The detection device  1  includes electrical means of forming a finger of liquid by liquid dielectrophoresis on the first surface  24  of the cover  20 . 
     These means are similar to those which are presented in the article by Ahmed and Jones entitled “Optimized liquid DEP droplet dispensing”, J. Micromech. Microeng., 17 (2007), 1052-1058. 
     Thus, as  FIGS. 2A and 2B  show, two movement electrodes  40 ,  41  are arranged on the first surface  24 , and include multiple drop formation zones  42 , each facing a different detection surface. 
     The electrodes  40 ,  41  are each formed of a metallic track. They are parallel to each other, coplanar and approximately rectilinear. 
     As  FIG. 2B  shows more precisely, each track  40 ,  41  includes an inner edge  40 I,  41 I and an outer edge  40 E,  41 E. The inner edges  40 I,  41 I are arranged facing each other. 
     Said drop formation zones  42  are formed of plane protuberances or plane bumps  42 - 0  and  42 - 1 , which extend to the outside of each movement electrode  40 ,  41 . The bumps  42 - 0  and  42 - 1  are part of the electrodes  40 ,  41  and are coplanar with them. 
     The bumps  42 - 0  and  42 - 1  here are arranged symmetrically in relation to each other, and each belong to a different movement electrode  40 ,  41 . 
     Thus the movement electrodes  40 ,  41  include rectilinear parts  43  and drop formation zones  42 , which are connected to each other by said rectilinear parts  43 . 
     The inner edges  40 I,  41 I of the movement electrodes  40 ,  41  are separated from each other by a distance g. The rectilinear parts  43  have a width w, and each bump  42 - 0 ,  42 - 1  is a half-disc of radius Rb, the centre of which is located in the continuation of the outer edge  40 E,  41 E of the rectilinear parts  43 . The notation of these various distances is similar to what is used in the article by Ahmed and Jones cited above. 
     2R is the distance separating the outer edges  40 E,  41 E of the rectilinear parts  43  of the movement electrodes  40 ,  41 . 
     The drop formation zones  42  are arranged equidistantly from each other, the distance preferably being between 8R and 10R, and preferably 9.016R. 
     As is explained in detail below, the distance which separates the drop formation zones  42  is approximately equal to the most unstable wavelength λ max  of the finger of liquid which extends along the movement electrodes  40 ,  41 . 
     The movement electrodes  40 ,  41  are connected to a voltage generator  44  ( FIG. 2A ), which makes it possible to apply a potential difference between the electrodes  40 ,  41 . 
     The voltage which is applied is an alternating voltage, the frequency of which is, for example, between a few kilohertz and a few megahertz, e.g. between 10 kHz and 10 MHz, and between 10 kHz and 100 kHz, and of a preferred voltage of a few RMS volts to a few hundred RMS volts. 
     Finally, as mentioned above with reference to  FIG. 1 , a dielectric layer  22  is arranged in such a way as to cover the lower face  21  of the cover  20  and the movement electrodes  40 ,  41 . A hydrophobic layer  23  covers the dielectric layer  22 . Advantageously, the dielectric layer and the hydrophobic layer can be a single layer of the same material. 
     The lower substrate  10  includes multiple detectors, in the form of plane electromechanical oscillators  30  which are capable of being set to vibrate ( FIG. 1 ). Each oscillator  30  has an upper face called a detection surface  31 . 
     The detection surfaces  31  are coplanar with and separated by a distance H from the first surface  24  of the cover  20 . 
     The oscillators  30  can be similar or identical to those described in the international application WO2009/141515 cited above, or any other gravimetric detector known to the person skilled in the art (beams, cantilevers etc.). 
     As  FIG. 3  shows, each oscillator  30  here is a square plate which is arranged directly facing a drop formation zone  42  of the movement electrodes  40 ,  41 . However, it can be in other forms, e.g. a disc, a ring or a polygon. 
     The plate  30  is arranged above a cavity  11 , which enables it to vibrate in and out of its plane. 
     As  FIGS. 4A and 4B  show, the plate  30  is mounted on the lower substrate by support means  50 , here beams, which are distributed at the four apices of the oscillator and oriented following the diagonals of the latter. These beams can be, for example, of silicon, polysilicon, tungsten, nickel or any other material which is used in the field of micro-electromechanical or nano-electromechanical systems (MEMS, NEMS). 
     Actuating means are provided to set each oscillator to vibrate. 
     At least one actuating electrode  60  is arranged facing the edge of said oscillator  30 , preferably parallel to the latter, and advantageously coplanar with the latter. 
       FIG. 4B  shows two adjacent actuating electrodes  60 ,  61  which are arranged near the oscillator  30 . 
     The actuating electrodes  60 ,  61  are separated from the oscillator  30  by a distance of the order of a few hundred nanometers, e.g. 100 nm or 300 nm. 
     A voltage generator (not shown) is connected to the actuating electrodes  60 ,  61 , to apply to each of them an alternating electrical voltage of determined frequency, and the oscillator  30  is brought to a constant electrical potential. Control means (not shown) are connected to the voltage generator, for choosing the parameters of the voltage to be set. The frequency of the applied voltage is advantageously equal to the natural resonant frequency of the oscillator. 
     It should be noted that the oscillator  30  can vibrate, preferably in its plane, according to a predetermined vibration mode chosen from Lamé mode, volume extension mode or the mode called “wine glass”, or any other mode of outline. 
     As described in detail below, the oscillator  30  is set to vibrate by electrostatic coupling between the oscillator  30 , which is brought to a constant electrical potential, and said actuating electrodes  60 ,  61 , to which an alternating electrical voltage of predetermined frequency is applied. 
     The analytes of interest are detected here by gravimetry. 
     As  FIG. 4B  shows, two adjacent measuring electrodes  70 ,  71  are arranged facing the edge of said oscillator  30 , preferably parallel to the latter, and advantageously coplanar with the latter. They have the same distance separating them from the oscillator  30  as the actuating electrodes  60 ,  61 . 
     As described in detail below, said step of measuring the vibration frequency of the oscillator includes measuring an electric current which circulates from said measuring electrodes  70 ,  71 . This electric current is generated by capacitive coupling between the oscillator  30  and the measuring electrodes  70 ,  71 . 
     Finally, the means (not shown) of storing and analysing the measured electrical signals are connected to the means of measuring the generated electric current and the means of controlling the actuating electrodes. They make it possible to calculate the effective vibration frequency of the oscillator on the one hand, and to detect the analytes of interest from a divergence between the measured vibration frequency and the initially set predetermined vibration frequency. 
     It should be noted that the detection surface  31  of the oscillator  30  advantageously has a hydrophilic zone, which is intended to be covered by said drop. The outline of the hydrophilic zone can advantageously coincide approximately with the nodal lines of the oscillator according to the vibration mode in which it is stressed. This makes it possible to attenuate the energy dissipation caused by the vibration of the triple line of the drop, this vibration then being of negligible amplitude. 
     Additionally, the probe elements which are capable of binding to the analytes of interest can be grafted onto said detection surface, in such a way as to cover it at least partly. These grafted probe elements can be, for example, antibodies, probes for nucleic acids or printed polymers. 
     The probe elements can be different according to the detection surfaces. Thus each detection surface is intended to receive a different category of analytes of interest. 
     The lower substrate  10  can be implemented in a material such as monocrystalline silicon, polycrystalline silicon, diamond, silicon nitride, silicon oxide, nickel, tungsten or platinum. The material of the upper substrate  20  can be chosen from among the above-mentioned materials, but glass, pyrex or an organic material such as polycarbonate or PEEK will be preferred. The upper substrate will advantageously be transparent. The thickness of the upper substrate can be between a few hundred microns and a few millimeters. 
     The movement electrodes  40 ,  41  are implemented in a metallic material, e.g. gold or aluminium. The electrodes  40 ,  41  can have a width w of the order of 20 μm, and be separated from each other by a distance g of the order of 20 μm. The width of the finger of liquid will thus be of the order of R=w+g/2=30 μm. The bumps can be half-discs of radius Rb=0.98R. 
     The dielectric layer  22  which covers the movement electrodes  40 ,  41  can be, for example, of SiO 2 , Al 2 O 3 , HfO 2 , SiN, and have a thickness between 100 nm and a few microns. It makes it possible to avoid the electrolysis of the liquid if the latter was in direct contact with the movement electrodes  40 ,  41 . 
     The hydrophobic layer  23  which forms the first surface  24  can be of SiOC, PTFE (polytetrafluoroethylene) or parylene, and have a thickness of a few microns. 
     The oscillator  30  is a square plate of width between 5 μm and a few hundred microns. Its thickness is typically less than or equal to a tenth of its width. It is implemented in a material which is chosen from monocrystalline silicon, polycrystalline silicon, diamond, silicon nitride, silicon oxide, nickel, tungsten or platinum. 
     The distance H which separates the first surface  24  and the detection surfaces  31  can be of the order of a few tens of microns, e.g. 50 μm. 
     Each detection surface  31  has a hydrophilic zone, corresponding to the zone which is intended to receive the formed drop. This hydrophilic zone can be formed by structuring a hydrophobic layer which has previously been deposited on the detection surface. Alternatively, the hydrophilic zone can be formed by chemical treatment, starting with hydrophobic silanes and hydrophilic silanes. 
     The detection device  1  according to the first preferred embodiment of the invention operates as follows, referring to  FIGS. 5A to 5C . 
     According to a first step ( FIG. 5A ), the liquid of interest is put into contact with the first surface  24 , from the liquid tank  25 . 
     An oscillating, non-uniform electrical field is generated under the effect of an electrical control, by applying a suitable voltage to the two movement electrodes  40 ,  41 . 
     An electrostatic force is then applied to the liquid, and causes the formation of a finger of liquid on said first surface  24  by liquid dielectrophoresis ( FIG. 5B ). 
     The finger of liquid extends along the two movement electrodes  40 ,  41 . It should be noted that the speed at which the liquid moves is high, of the order of 10 cm/s. Thus for a length of movement electrodes  40 ,  41  of the order of 5 mm, 50 ms are sufficient to form the finger of liquid. 
     The finger of liquid approximately covers the movement electrodes  40 ,  41  throughout their length, and its width is approximately equal to the distance 2R defined above, corresponding to the distance which separates the outer edges of the electrodes  40 ,  41  in their rectilinear part. 
     Next, when the electrical control stops ( FIG. 5C ), the finger of liquid breaks by capillarity into multiple drops, each on a drop formation zone. 
     In fact, the finger of liquid, in the absence of electrostatic force, is naturally unstable. The finger breaks under the effect of hydrodynamic instability of Rayleigh-Plateau type. In fact, this breaking of the finger into multiple drops makes it possible to reduce the surface energy of the liquid. 
     The instability is a competition between capillarity and inertia, and the most unstable wavelength is such that k max ·R=1/√{square root over (2)}, where k max  is the wave number. The most unstable wavelength is therefore written λ max =9.016R. 
     Additionally, the drop formation zones are separated from each other by a distance which approximately equals λ max . The drop formation zones make it possible to deform the interface of the finger of liquid at the λ max  wavelength, and thus to “preselect” the desired wavelength. 
     Thus the drops are formed simultaneously, and are each located in a drop formation zone. 
     Each drop has a calibrated volume. The volume depends on the width 2R of the finger of liquid and the distance λ max  between the drop formation zones. 
     The drops which are formed have sufficient thickness to come into contact with the corresponding detection surface  31 . 
     Additionally, the distance H which separates the first surface  24  and each detection surface  31 , and the lateral dimensions g and w of the movement electrodes  40 ,  41 , are adapted so that the finger of liquid has a maximum thickness which is less than the distance H, and each drop which is formed has a maximum thickness which is greater than this distance H. 
     The finger of liquid wets only the first surface  24 , without being in contact with the detection surfaces  31 . When the finger breaks, the drops which are formed come naturally into contact with said detection surfaces  31 . 
     Each plane electromechanical oscillator  30  is set to vibrate by electrostatic coupling with the actuating electrodes, according to a predetermined frequency and a predetermined vibration mode. 
     Said set predetermined frequency is preferably the resonant frequency of the oscillator  30 . 
     For this, an alternating voltage of frequency equal to the resonant frequency of the oscillator  30  is applied to the actuating electrodes, with a phase difference of π relative to each other. Lamé&#39;s vibration mode is thus obtained. 
     Volume extension mode or “wine glass” mode can also be obtained, with different polarisations of actuating electrodes, as is shown in detail by international application WO2009/141515. 
     The effective vibration frequency of the oscillator  30  is then measured. The frequency actually depends on the mass of the drop, and if appropriate on the quantity of analytes of interest which are grafted onto the detection surface of the oscillator when the latter is made functional. 
     The modulation of the capacitance between the oscillator and the two measuring electrodes generates an electric current which circulates from these two electrodes. 
     The storage and analysis means make it possible, starting from the measurement of the electric current measured at the measuring electrodes, to determine the effective vibration frequency of the oscillator. 
     They calculate the divergence between the set initial frequency and the measured frequency, and deduce from it the presence of analytes of interest which are deposited on the surface of the oscillator. 
     Thus the method according to the invention makes it possible to detect, precisely and rapidly, the analytes of interest which may be present in the liquid. 
     It should be noted that the liquid is brought rapidly onto the detection surfaces. 
     The formation of the finger of liquid is actually very rapid, with a speed of movement of the liquid of the order of 10 cm/s. Only 50 ms are necessary to form a 5 mm finger of liquid. Additionally, the drops are formed even more rapidly, to the extent that the characteristic time of a capillarity/inertia instability is √{square root over (ρR 3 /σ)}, or 0.05 ms for a liquid density ρ=1000 kg/m 3 , a half-width of finger R=50 μm and a liquid/air surface tension σ=0.072 Nm. 
     Additionally, the drops are formed simultaneously and perfectly arranged on the detection surfaces. They can be of identical, calibrated volume, approximately equal to πR 2 λ max /2. 
     Additionally, the liquid has only been in contact with the first surface, thus limiting to a large extent the risks of contaminating the liquid while it is routed to the detection surfaces. 
     In the case of the gravimetric detection of the first preferred embodiment of the invention, the liquid is in the form of drops which are arranged on the detection surfaces. In contrast to the example of the prior art described above, the oscillators are no longer immersed in the liquid. Additionally, the oscillations are no longer damped by the liquid, which preserves the intrinsic quality factor of the oscillators from all degradation of this type. 
     Additionally, the triple line of the drop coincides with the outline of the hydrophilic zone of the detection surface, and with the nodal lines of the vibration mode of the oscillator. It is thus on a zero movement line of the oscillator. This makes it possible to minimise the interactions between the vibrating oscillator and the liquid, the effect of said interactions being, in particular, calorific dissipation, which degrades the quality factor of the oscillator. Thus the degradation of the quality factor of the oscillator because of the presence of the drop is minimised. 
     It should be noted that each electromechanical oscillator can alternatively be in the form of a beam. The beam can be doubly fixed, i.e. mounted on support means at its two ends. It can also be fixed at the centre, and thus be mounted on two support means in its middle, or fixed at four points by being mounted on four support means, each arranged between the middle and an end of the beam. In the latter case, the support means are lateral beams, which are connected to the oscillator at a quarter of the vibration wavelength, and arranged at the nodes of the determined vibration mode. 
     According to a first variant of the first preferred embodiment of the invention, the gravimetric detection is not implemented by capacitive coupling but in a piezoelectric manner. 
     It should be noted that the formation of drops by breaking a finger of liquid which is formed by liquid dielectrophoresis is here identical to what is described above. 
     In the same way, the actuation of the electromechanical oscillators can be identical to the first preferred embodiment. 
     As  FIGS. 6A and 6B  show, said detection surfaces  31  each include a layer  32  of an electrically conductive material, which forms a reference electrode, and is implemented in molybdenum, for example. 
     The reference electrode  32  is covered with a layer of a dielectric piezoelectric material, e.g. aluminium nitride (AlN). This material has a crystallographic orientation &lt;002&gt; on the molybdenum layer. Additionally, because of this crystallographic orientation, the intensity of the electrical field generated by polarisation of the AlN layer  33  is greater for the same intensity of mechanical stress. 
     The AlN layer  33  is covered at least partly by two measuring electrodes  72 ,  73  which are brought to a constant, opposite electrical potential. The electrodes  72 ,  73  are metallic tracks which cross the detection surface in a zigzag, and extend on two support girders  50 . They are parallel to each other and separated by a constant distance. 
     The measuring electrodes  72 ,  73  are covered with a dielectric layer. 
     The detection surface can have a hydrophilic zone, to make it possible to check the location of the drop on the detection surface. 
     When the oscillator vibrates, the AlN layer  33  is deformed and polarised. The reference electrode  32  is then brought to a determined potential via the AlN layer  33 . 
     The variations of capacitance between the reference electrode  32  and each measuring electrode  72 ,  73  because of the mechanical vibrations of the oscillator cause the appearance of an electric current which circulates in the measuring electrodes  72 ,  73 . 
     By measuring the electric current, the effective vibration frequency of the oscillator  30  is deduced. 
     It is then possible to calculate the divergence between the measured frequency and the set initial frequency, and to deduce from it the presence of analytes of interest which are deposited on the detection surface  31 . 
     Additionally, the mechanical vibrations of the oscillator  30  induce a variation of the distance which separates the two measuring electrodes  72 ,  73  from each other. This variation causes a variation of the capacitance between these two electrodes  72 ,  73 , and causes the appearance of a second electric current. 
     Measurement and analysis of this second electric current, in addition to those of the first current, make it possible to deduce even more precisely the effective vibration frequency of the oscillator, which makes the detection of analytes of interest even more efficient. 
     According to a second variant of the first preferred embodiment of the invention, each oscillator  30  of the detection device  1  forms a resonant grid, by analogy with field effect transistors. 
     It should be noted that the formation of drops by breaking a finger of liquid which is formed by liquid dielectrophoresis is here identical to what is described above. 
     In the same way, the actuation of the electromechanical oscillators is identical to the first preferred embodiment. 
     As  FIG. 7  shows, a measuring electrode  74  forming a channel is arranged facing the edge of said oscillator  30 , preferably parallel to the latter, and advantageously coplanar with the latter, at a distance from the oscillator equal to the separation between the actuating electrodes  60 ,  61  and the oscillator  30 , that is a few hundred nanometers. 
     The electrode forming a channel  74  is connected at one end to an electrode forming a source  74 S, which is brought to a first constant electrical potential, and at the opposite end to an electrode forming a drain  74 D, which is brought to a second electrical potential. The two electrical potentials are different. The electrode forming a channel  74  is thus subjected to a direct voltage. 
     The oscillator  30  can also be brought to a constant electrical potential. 
     When the oscillator  30  is set to vibrate at its resonant frequency, it forms a resonant grid. 
     The source-drain current which is generated by field effect at the resonant frequency of the oscillator which is set to vibrate, and more precisely the variations of the current, are measured. These electric current variations are induced by capacitive coupling between the oscillator  30  and the electrode forming a channel  74 . 
     From the measurement of these variations, the presence of analytes of interest deposited on the detection surface is deduced. 
     According to a third variant of the first preferred embodiment of the invention, each oscillator of the detection device forms a resonant channel, by analogy with field effect transistors. 
     It should be noted that the formation of drops by breaking a finger of liquid which is formed by liquid dielectrophoresis is here identical to what is described above. 
     In the same way, the actuation of the electromechanical oscillators is identical to the first preferred embodiment. 
     As  FIG. 8  shows, each oscillator  30  is an electrode forming a channel, and is connected at one end to an electrode forming a source  75 S, which is brought to a first constant electrical potential, and at the opposite end to an electrode forming a drain  75 D, which is brought to a second electrical potential. The two electrical potentials are different. The oscillator is thus subjected to a direct voltage. 
     The source electrode  75 S and drain electrode  75 D can be arranged on the substrate  10  and connected to the oscillator  30  by electrically conducting support beams  50 . 
     The source-drain current which circulates in the oscillator  30 , and in particular the current variations which are induced by field effect by analytes of interest being deposited on the detection surface  31  of the oscillator  30 , are measured. 
     From the measurement of these variations, the presence of analytes of interest deposited on the detection surface  31  is deduced. 
     According to a second preferred embodiment of the invention, the detection surface is no longer a face of an electromechanical oscillator, but a determined zone of the upper face of the lower substrate  10 . 
     The detection surface  31  then includes multiple nanowires  76 , which cover it at least partly. 
     The nanowires  76  are implemented in a semiconductor material, e.g. silicon or carbon in the form of nanotubes. 
     The nanowires  76  are each connected at one end to an electrode forming a source  75 S, which is brought to a first constant electrical potential, and at the other end to an electrode forming a drain  75 D, which is brought to a second constant electrical potential. Each nanowire is thus subjected to a direct electrical voltage. 
     By analogy with field effect transistors, the nanowires form a channel through which free carriers (electrons or holes according to the nature and the type of doping of the channel) pass. 
     Thus, at a given fall of source-drain potential, the current which circulates through the nanowires, and more particularly its variations induced by field effect by the presence of analytes of interest deposited on its surface, are measured. These analytes, by their charge, actually modulate the grid potential of the transistor. 
     Detection of the analytes of interest is thus deduced from the variations of the measured current. 
     The method of forming drops from a finger of liquid which is formed by liquid dielectrophoresis is identical to what is described above, and is not repeated here. 
     According to a third embodiment of the invention, the detection device includes a coplanar drop formation surface and detection surface. 
     The detection device includes a substrate  10 , which includes at least one detector  30 . A detector can thus be a plane electromechanical oscillator. Support means ensure that each oscillator  30  is maintained relative to the substrate  10 . 
       FIG. 10  shows part of such a detection device, including a single oscillator  30 . 
     Electromechanical oscillators  10 , setting them to vibrate from actuating electrodes  60 ,  61 , and detection by gravimetry from measuring electrodes  70 ,  71  here are identical or similar to what has been described with reference to the first preferred embodiment of the invention. 
     A surface, called the principal, for forming the finger of liquid and for detection is formed of a surface of said substrate  10 , a surface of said oscillator  30  forming the detection surface  31 , and a surface of said support means  50 . 
     As for the first preferred embodiment, a tank of liquid of interest (not shown) is arranged so that it can put said liquid into contact with said principal surface. The tank can be formed from an aperture which passes through the substrate and opens into the principal surface. 
     Two movement electrodes  40 ,  41  extend from said tank at the level of the principal surface. They include drop formation zones  42 . 
     They extend on the surface of the substrate, and continue on the face of the oscillators  30  each of which forms a detection surface  31 , via the support beams  50 . 
     The method of forming the finger of liquid is identical to what is described above. The finger of liquid is formed by liquid dielectrophoresis, and extends on the substrate  10  and oscillators  30  via the corresponding support beams  50 . 
     The drop formation zones  42  are arranged on each detection surface  31 . 
     Thus when the electrical control stops, the finger of liquid breaks by capillarity into multiple drops, each being arranged on a drop formation zone  42 , and thus on a detection surface  31  of the corresponding oscillator  30 . 
     Each oscillator  30  is set to vibrate, preferably at its resonant frequency, by capacitive coupling with the actuating electrodes  60 ,  61 , which are arranged facing the edge of the oscillator  30 . 
     Analytes are detected in the drops as described with reference to the first preferred embodiment, by capacitive coupling between the oscillator  30  and two measuring electrodes  70 ,  71 , which are arranged facing the edge of the oscillator  30 . 
     From the measured electric current, the frequency divergence between the effective vibration frequency and the set initial frequency is deduced. 
     The presence of analytes of interest is detected from this calculated divergence, or as being said calculated divergence. 
     Of course, various modifications can be made by the person skilled in the art to the invention which has just been described, as non-limiting examples only. 
     As a variant of the various embodiments described above, analytes of interest can be detected by optical means which work with the detection surface. 
     The detection surface can be one face of a lower substrate, and include a hydrophilic part which is intended to be in contact with the drop to be analysed. This substrate, at this detection surface, can be implemented in a transparent material. The part of the substrate facing this surface is also implemented in a transparent material. This detection surface can be illuminated by a light source, and coupled to a photodetector. 
     The detection surface can also be similar to the surface of an electrophysiological recording sensor for ionic currents passing through cellular membranes. 
     The detection surface is then a porous membrane, of diameter between 100 nm and a few millimeters, the diameter of the pores being between a few nanometers and a few microns. 
     Such a membrane can count from one to about a hundred pores or more. 
     The membrane is implemented using an insulating material, e.g. silicon nitride, silicon oxide, parylene. 
     Because of a pressure difference between the upper face of the membrane, i.e. that which is in contact with the collected drop, and the lower face of the membrane, this face is opposite the upper face. 
     The coplanar substrate of the detection surface has, on said surface, an opening which acts as a fluid chamber, one of the walls of which is the lower face of the membrane. 
     Thus the membrane separates the collected drop from the microfluid chamber. 
     This microfluid chamber can be filled with a saline buffer. 
     A potential difference is usually applied on one side and the other of the membrane. 
     Preferably, pressure control means make it possible to apply, on one side and the other of the membrane, a pressure difference such that the drop is kept supported against the membrane, according to an analogous configuration to a pipette with a plane surface. 
     In this way, when the drop which is formed on the detection surface contains cells, the latter are agglutinated and invaginated on the membrane under the effect of the suction exerted by the pressure difference, when one exists, and under the effect of the potential difference which exists on one side and the other of the membrane, the latter effect being known by the name of attraction by electrophoresis. 
     Means of measuring the potential difference between two measurement points which are arranged on one side and the other of the membrane are also available. 
     It is known that the external envelope of the cells consists of a lipidic bilayer, which can be represented by two charged surfaces, the two surfaces being separated by a layer of insulant. 
     This results from the hydrophilic character of the polar heads of the lipids which form the two layers. Thus each surface of a cell can be modelled by a capacitor. 
     When the liquid medium in which the cells are bathed includes molecules with which the membranous proteins which are contained in the lipidic bilayer are likely to interact, the lipidic bilayer can be modified, and in particular be partly opened, and then allow ionic species to pass through the membrane between the interior of the cells and the fluid chamber, because of the potential difference which is applied on one side and the other of the membrane. 
     This ionic current can be quantified by the means described above for measuring the potential difference.