EP1703987A1 - Planar electronebulization sources modeled on a calligraphy pen and the production thereof. - Google Patents

Planar electronebulization sources modeled on a calligraphy pen and the production thereof.

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Publication number
EP1703987A1
EP1703987A1 EP04805823A EP04805823A EP1703987A1 EP 1703987 A1 EP1703987 A1 EP 1703987A1 EP 04805823 A EP04805823 A EP 04805823A EP 04805823 A EP04805823 A EP 04805823A EP 1703987 A1 EP1703987 A1 EP 1703987A1
Authority
EP
European Patent Office
Prior art keywords
electrospray
source
liquid
support
plate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP04805823A
Other languages
German (de)
French (fr)
Other versions
EP1703987B1 (en
Inventor
Steve Arscott
Séverine LE GAC
Christian Druon
Christian Rolando
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Universite de Lille 1 Sciences et Technologies
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite de Lille 1 Sciences et Technologies
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Publication of EP1703987A1 publication Critical patent/EP1703987A1/en
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Publication of EP1703987B1 publication Critical patent/EP1703987B1/en
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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/165—Electrospray ionisation
    • H01J49/167—Capillaries and nozzles specially adapted therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
    • B05B5/00—Electrostatic spraying apparatus; Spraying apparatus with means for charging the spray electrically; Apparatus for spraying liquids or other fluent materials by other electric means
    • B05B5/025—Discharge apparatus, e.g. electrostatic spray guns
    • B05B5/0255—Discharge apparatus, e.g. electrostatic spray guns spraying and depositing by electrostatic forces only
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0013—Miniaturised spectrometers, e.g. having smaller than usual scale, integrated conventional components
    • H01J49/0018—Microminiaturised spectrometers, e.g. chip-integrated devices, Micro-Electro-Mechanical Systems [MEMS]

Definitions

  • the present invention relates to original electrospray sources, their manufacturing process and their applications.
  • Electrospray is the phenomenon which transforms a liquid into a nebuliser under the action of a high voltage (M. CLOUPEAU “Electrohydrodynamic spraying functioning modes: a critical revie. Journal of Aérosol Science (1994), 25 (6), 1021-1036 ").
  • the liquid is brought into a capillary and is subjected to a direct or alternating high voltage or to a superposition of the two (Z. HUNEITI et al., "The study of AC coupled DC fields on conducting liquid jets", Journal of Electrostatics (1997), 40 & 41 97-102).
  • the liquid is nebulized under the action of tension.
  • the surface of the meniscus formed by the liquid is elongated to form one or more Taylor cones from which are ejected charged liquid droplets which evolve to give a gas containing charged particles.
  • the formation of the nebulisate is observed when the electrical forces due to the application of the voltage compensate for and exceed the surface tension forces of the liquid. on the section of the capillary at the end of said capillary.
  • the size of the capillary, and more precisely its outlet orifice, is in direct relation with the flow of liquid leaving the capillary and the voltage to be applied to observe the nebulization phenomenon.
  • electrospray regimes There are two distinct electrospray regimes which are distinguished by their establishment characteristics: • the so-called classical regime which corresponds to capillary outlet sizes of 100 ⁇ m, fluid flow rates in the range of 1-20 ⁇ L / min and high voltages of 3-4 kV; • the so-called nanoelectronisation regime where the liquid flow rates are less than 1 ⁇ L / min, the high voltage of approximately 1 kV and the internal diameter of the capillaries of 1-10 ⁇ m (M. WILM et al, "Analytical Properties of the Nanoelectrospray Ion Source ", Analytical Chemistry (1996), 68 (1), 1-8.).
  • a voltage comprising an AC component allows the stabilization of the electrospray process by synchronization on its natural frequency (F. CHARBONNIER et al., "Differentiating between Capillary and Counter Electrode Processes during Electrospray lonization by Opening the Short Circuit at the Collector.
  • Electrochemically ionizable derivatives Analytical Chemistry (1998), 70 (8), 1544-1554; F. ZHOU and al. "Electrochemistry Combined Online with Electrospray Mass Spectrometry", Analytical Chemistry (1995), 67 (20), 3643-3649).
  • the fields of application of electrospray are as follows: • First, the ionization of molecules (M. DOLE et al., "Molecular beams of macroions", Journal of Chemical Physics (1968), 49 (5) , 2240-2249; LL MACK et al., "Molecular beams of macroions. II", Journal of Chemical Physics (1970), 52 (10), 4977-4986; US Patent 4,209,696; M. YAMASHITA et al.
  • Such drops can be deposited on a support (CJ McNEAL et al., "Thin film deposition by the electrospray method for californium-252 plasma desorption studies of involatile molecules", Analytical Chemistry (1979), 51 (12), 2036-2039 ; RC MURPHY et al., “Electrospray loading of field desorption emitters and desorption chemical lonization probes", Analytical Chemistry (1982), 54 (2), 336-338) for example a plate for either the production of analysis chips like DNA or peptide chips, dedicated to high-throughput analysis (VN MOROZOV et al., "Electrospray Deposition as a Method for Mass Fabrication of Mono- and Multicomponent Microarrays of Biological and Biologically Active Substances", Analytical Chemistry (1999 ), 71 (15), 3110-3117; R.
  • MOERMAN et al. "Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots", Analytical Chemistry (2001 May 15), 73 (10) , 2183-2189; NV AVSEENKO et al., "Imm unoassay with Multicomponent Protein Microarrays Fabricated by Electrospray Déposition ", Analytical Chemistry (2002), 74 (5), 927-933), i.e. the deposit of solutions on a MALDI plate (for" Matrix Assisted Laser Desorption lonization ”) before an analysis by mass spectrometry (J.
  • a third application is the deposition of particles of controlled size contained within the liquid (IW LENGGORO et al., "Sizing of Colloidal Nanoparticles by Electrospray and Differential Mobility Analyzer Methods", Langmuir (2002), 18 (12), 4584- 4591). The particles can also be replaced by cells for the preparation of cell chips.
  • a fourth application is the injection of the drops formed by electrospray into a liquid leading to well defined size emulsions (RJ PFEIFER et al., "Charge-to-mass relation for electrohydrodynamically sprayed liquid droplets", Physics of Fluids (1958 -1988) (1967), 10 (10), 2149-54; C.
  • the sources used for nanoelectronebulization are in the form of capillaries made of glass or fused silica. They are manufactured by hot stretching or by acid attack of the material in order to give an outlet of 1 to 10 ⁇ m (M. WILM et al., "Electrospray and Taylor-Cone theory, Dole 's beam of macromolecules at last?", International Journal of Mass Spectrometry and Ion Processes (1994), 136 (2-3), 167-180).
  • the electrospray voltage can be applied via an appropriate conductive external coating: a metallic coating such as gold or an Au / Pd alloy (GA VALASKOVIC et al., “Long-lived metalized tips for nanoliter electrospray mass spectrometry", Journal of the American Society for Mass Spectrometry (1996), 7 (12), 1270-1272), argent (Y.-R CHEN et al., "A simple method for fabrication of silver-coated sheathless electrospray emitters", Rapid Coiximunications in Mass Spectrometry (2003), 17 (5), 437-441), a carbon-based material (X.
  • a metallic coating such as gold or an Au / Pd alloy
  • GA VALASKOVIC et al. “Long-lived metalized tips for nanoliter electrospray mass spectrometry", Journal of the American Society for Mass Spectrometry (1996), 7 (12), 1270-1272
  • argent Y.-R CHEN et al., "
  • the electrospray voltage can also be applied via the liquid with the introduction of a metallic wire into the source (KWY FONG et al., "A novel nonmetallized tip for electrospray mass spectrometry at nanoliter flow rate", Journal of the American Society for Mass Spectrometry (1999), 10 (1), 72-75).
  • KWY FONG et al. "A novel nonmetallized tip for electrospray mass spectrometry at nanoliter flow rate", Journal of the American Society for Mass Spectrometry (1999), 10 (1), 72-75.
  • the devices of the prior art dedicated to nanoelectronebulization suffer from several weaknesses (B. FENG et al., "A Simple Nanoelectrospray Arrangement With Controllable Flowrate for Mass Analysis of Submicroliter Protein Samples", Journal of the American Society for Mass Spectrometry (2000), 11, 94-99): • First of all, these capillaries are not very robust.
  • Standard commercial sources are therefore unsuitable, firstly for controlled, reproducible and quality nebulization, secondly for the use of robots due to the entirely manual nature of their mode of use, and thirdly, for integration on a fluid microsystem, as discussed below.
  • These faults hamper certain fields of application of electrospray which currently require robotization and process automation. This is the case of the fields of application listed above: analysis by mass spectrometry, the deposition of drops of calibrated size and writing on a scale less than a micrometer using a tip.
  • microtechnology techniques are used for the manufacture of integrated microsystems of characteristic size of the order of a micrometer and which bring together a series of reaction and / or analytical, chemical and / or biochemical / biological processes.
  • the rise of microfluidics in the fields of chemistry and biology, where speed and process automation are required today, can be explained by: • the gain in speed of the processes, due to the fact that the speed mainly depends on the size of the devices; this gain in speed is particularly important for fields of application such as medical diagnosis or environmental analysis, where an instant response is often expected, • the possibility of parallelization of processes; microtechnology allows the simultaneous manufacture of a large number of identical devices, • the compatibility of microfabricated objects with a robotic interface for the automation of processes, • the adequacy of the volumes handled with those available to the experimenter in the case, inter alia, biological or environmental analyzes, • the limitation going until the suppression of the human intervention, which is often source of error and contamination, • a gain in sensitivity, for certain analysis techniques, including mass spectrometry with
  • Microfluidic devices are manufactured using microtechnology techniques.
  • Today a wide range of materials is available for these microfabrications, ranging from silicon and quartz (common materials in microtechnology) to glasses, ceramics and polymer-type materials, such as elastomers or plastics.
  • microfluidics benefits from both: • the heritage of materials and manufacturing techniques developed and used for microelectronic applications and, • new manufacturing processes, developed in parallel and adapted to others emerging materials of great interest for microfluidic applications, such as plastic-type materials, the main attraction of which is their low cost. More specifically, the possible materials for technological manufacturing applicable to chemistry and biology are (T.
  • McCREEDY "Fabrication techniques and materials co monly used for the production of microreactors and micro total analytical Systems", TrAC, Trends in Analytical Chemistry (2000), 19 (6), 396-401): • materials of the semiconductor type such as silicon, traditional materials in microtechnology which benefit from robust and proven manufacturing techniques; among these manufacturing techniques are lithography, physical and chemical engravings among others (PJ FRENCH et al., “Surface versus bulk micromachining: the contest for suitable applications", Journal of Micromechanics and Microengineering (1998), 8 (2 ), 45-53). Therefore, silicon in particular is the most interesting material in terms of manufacturing small structures at scales of ten nanometers.
  • glass a less expensive material than quartz and silicon, which is widely used because of its surface properties suitable for establishing an electroosmotic flux (K. SATO et al., "Integration of chemical and biochemical analysis Systems into a glass microchip", Analytical Sciences (2003), 19 (1), 15-22).
  • silanol groups line the surface of the glass. They suggest a subsequent chemical modification of the glass surfaces.
  • its transparency properties make it a material of choice in the case of optical detection.
  • microfabrication techniques have been applied to the production of electrospray or advanced needle-type sources with a view to: • improving the overall quality of the capillaries in terms of control of the manufacturing processes, reproducibility of the sources and their dimensions, • to produce a large number of identical or differing devices by one or several dimensions, on the same material plate, like microcomponents in microelectronics, in order to promote the automation and robotization of electrospray.
  • the manufacture using microtechnology techniques of electrospray tips obeys two trends: • the manufacture of an electrospray tip which reproduces classic geometry, that is to say a microfabricated capillary and, most often of circular section. Also included in this class are microfabricated needles intended for another application, such as injecting chemical substances or measuring biological potential.
  • electrospray source such as a microchannel or capillary outlet manufactured using microtechnology techniques and having a tapered profile.
  • microfabricated electrospray devices are based, like fluidic microsystems, on the use of different types of materials and different types of processes. According to the first trend, which aims to produce a capillary type geometry, the following descriptions are listed: • According to this approach, electrospray sources made of silicon nitride have been manufactured using conventional photolithography techniques and engraving (A. DESAI et al., "MEMS Electrospray Nozzle for Mass Spectrometry", Int. Conf. on Solid-State Sensors and Actuators, Transducers '97, (1997)).
  • the dimensions of said devices are a length of 40 ⁇ m and an internal diameter of the outlet orifice of 1 to 3 ⁇ m. Said sources were tested in mass spectrometry at nebulization voltages close to 4 kV and a liquid flow rate of
  • TANG et al. "Generation of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometrie sensitivity", Analytical Chemistry (2001), 73 (8), 1658-1663).
  • Their dimensions are as follows: 30 ⁇ m in internal diameter at their outlet orifice and 250 ⁇ m in height.
  • the dimensions of said devices are too large for a nanoelectronebulization regime since the voltage required for the observation of a nebulisate is 7 kV and the fluid flow rate is estimated at 30 ⁇ L / min.
  • the manufacturing process is also complex. These sources are in the form of a series of nine sources arranged in a 3 x 3 square. They operate simultaneously and nebulize the same solution.
  • the second tendency is to machine a point at the exit of a microchannel or to create a point structure which acts as a source of electrospray.
  • the angle of the pointed structure does not seem to have any influence on the nebulization phenomenon.
  • the voltage to be applied is very high and, under these conditions, the liquid tends to spread over the exit surface, on the edge of the microsystem (R. RAMSEY et al., "Generating Electrospray from Microchip Devices Using Electroosmotic Pumping” , Analytical Chemistry (1997), 69 (6), 1174-1178; Q.
  • the peak effect can be achieved by inserting a triangular planar structure between the two plates of materials defining a microchannel (the support in which the microchannel is machined and the cover). consists of a parylene sheet 5 ⁇ m thick (J. KAMEOKA et al., "An electrospray lonization source for integration with microfluidics", Analytical Chemistry (2002), 74 (22), 5897-590 1).
  • the system incorporates four identical electrospray devices placed in parallel.
  • the nebulization voltage required is 2.5-3 kV for a fluid flow of 300 nL / min. No inter-source interference was observed.
  • An eight-pointed star-shaped device was made of polymethylmethacrylate (PMMA) (C.-H. YUAN et al., "Sequential Electrospray
  • Each of the branches of the star constitutes an independent microfluidic system and the tip of each branch is a source of nebulization.
  • Each branch thus integrates a microchannel with a section of 300 x 376 ⁇ m, the pointed structure forms an angle of 90 ° and the eight liquid reservoirs are grouped in the center of the star.
  • the voltage applied for the establishment of a Taylor cone is high and equal to 3.8 kV, which is explained by the very large dimensions of the section of the microchannel at its end.
  • the manufacturing process described is based on the machining of channels using a knife, a technique which does not allow channels and nebulization devices of small dimensions to be produced.
  • PDMS polydimethylsiloxane
  • the nebulization orifice is rectangular and of variable dimensions ranging from 30 x 100 ⁇ m to 30 x 50 ⁇ m according to the microtechnology process used for their manufacture.
  • nebulization voltage ranged from 2.5 kV to 3.7 kV for solutions at 1 to 10 ⁇ M and high flow rates from a few 100 nL / min to several ⁇ L / min.
  • polyimide another material of relatively hydrophobic polymer type, was used for the manufacture of nebulization sources (GB-A-2 379 554; V.
  • GOBRY et al. "Microfabricated polymer injector for direct mass spectrometry coupling", Proteomics (2002), 2 (4), 405-412; JS ROSSIER et al., "Thin-chip microspray System for high-performance Fourier-transform ion-cyclotron resonance mass spectrometry of biopolymers", Angewandte Chemie, International Edition (2003 ), 42 (1), 54-58) integrated on a microsystem, or at least, connected to a microchannel of section 120 x 45 ⁇ m.
  • the system, the microchannel and the point structure are produced by plasma etching of the polyimide.
  • the system cover is made of polyethylene / polyethylene terephthalate.
  • the type of structure chosen for these different devices is practically inseparable from the material used for their production.
  • the nebulization voltage is most often applied at the reservoir of the device, if the system includes a reservoir, or, if not, at the level of the liquid supply which is carried out using a capillary connected to the device.
  • the capillary is conductive (in stainless steel for example), or the connection rests on a metal fitting.
  • These beam-type structures which have a width of 210 ⁇ m at their point, are manufactured in parallel on the same system. They allow the ejection of drops having a volume in the range from femtoliter to picolitre, the volume deposited depends linearly on the contact time between the tip and the surface, with a flow rate of up to 100 deposits per minute.
  • the AFM microscopy technique has the advantage of high resolution and very high writing precision. Three operating modes are possible, and depending on the mode chosen, the surface condition can be checked before and after passing the chemical molecular writing solution. However, this technique requires the use of heavy, bulky, expensive and complex equipment. Two molecular writing devices described in the literature can also be cited. They derive from the technique using an AFM microscopy tip but are based on the use of a microfabricated tip. The first device (A. LEWIS et al., "Fountain pen nanochemistry: Atomic force control of chrome etching", Applied Physics Letters (1999), 75 (17), 2689-2691; H.
  • TAHA et al. "Protein printing with an atomic force sensing nanofountainpen”, Applied Physics Letters (2003), 83 (5), 1041-1043), is in the form of a micropipette manufactured using microtechnology techniques and the tip of which can have dimensions as small as 3 and 10 nm for its internal and external diameters respectively.
  • This micropipette is nevertheless integrated into an AFM apparatus for its use.
  • the ejection of the solution is here caused not by contacting but by exerting pressure on the liquid column.
  • This device has been tested for its ability to deliver solutions for etching a layer of chromium deposited on a glass plate.
  • the second device (IW RANGELOW et al., "" NANOJET “: Tool for the nanofabrication", Journal of Vacuum Science & Technology, B: Microelectronics and Nanometer Structures (2001), 19 (6), 2723-2726; J. VOIGT et al., "Nanofabrication with scanning nanonozzle 'Nanojet'", Microelectronic Engineering (2001), 57-58 1035-1042) consists of spikes made of silicon covered with Cr / Au, having a pyramidal shape and an outlet for size less than .100 nm.
  • This device does not deliver a chemical solution as in the previous example, but free radicals in the gas phase produced by a plasma discharge which attack the material placed opposite the tip.
  • the device does not only consist of a microfabricated tip but it also includes machinery for producing highly reactive species, such as a radiofrequency or microwave plasma discharge, which can attack the substrate.
  • highly reactive species such as a radiofrequency or microwave plasma discharge
  • these two examples certainly present a microfabricated tip which replaces the conventional AFM microscopy tip, but they do not make it possible to dispense with the heavy and expensive peripheral machinery necessary for their operation.
  • this technique is based on bringing into contact or almost bringing into contact the tip and the substrate. As a result, the parameters of operation must be very carefully checked to avoid any deterioration of the surface finish due to excessive force exerted on the tip.
  • the present invention relates to a two-dimensional electrospray device having a geometry of the calligraphy feather type, the tip of which acts as a seat for nebulization.
  • the subject of the invention is therefore a source of electrospray comprising a structure comprising at least one flat and thin point in cantilever with respect to the rest of the structure, said point being provided with a capillary slot practiced throughout the thickness of the tip and which terminates at the end of the tip to form the ejection orifice of the electrospray source, the source comprising means for supplying the capillary slit with liquid to be nebulized and means for application of an electrospray voltage on said liquid.
  • the supply means comprise at least one reservoir in fluid communication with the capillary slot.
  • the structure comprises a support and a plate integral with the support and a part of which constitutes said point.
  • the supply means may comprise a reservoir constituted by a recess formed in said plate and in fluid communication with the capillary slot.
  • the means for applying an electrospray voltage may comprise at least one electrode arranged so as to be in contact with said liquid to be nebulized.
  • the means for applying an electrospray voltage may comprise the support, at least partially electrically conductive, and / or the plate at least partially electrically conductive.
  • the plate has a surface hydrophobic to the liquid to be nebulized.
  • the means for applying an electrospray voltage may comprise an electrically conductive wire arranged to be able to be in contact with said liquid to be nebulized.
  • the supply means may include a capillary tube. They can comprise a channel produced in a microsystem supporting said structure and in fluid communication with the capillary slit.
  • the means for applying the voltage also allow the application of the voltages necessary for any device placed upstream in fluid continuity with the object of the present invention.
  • the subject of the invention is also a method of manufacturing a structure being a source of electrospray, comprising: - The production of a support from a substrate, the production of a plate comprising a part constituting a flat and thin tip, said tip being provided with a capillary slot, for conveying a liquid to be nebulized, practiced throughout the thickness of the tip and which ends at the end of the tip, - the solidarisatiori of said plate on the support, the tip being in cantilever with respect to the support.
  • This process can include the following stages: - the supply of a substrate to make the support, - the delimitation of the support by means of trenches engraved in the substrate, the deposition, on an area of the substrate corresponding to the future point of the structure , of sacrificial material according to a determined thickness, - the deposition of the plate on the support delimited in the substrate, the point of the plate being located on the sacrificial material, - the elimination of the sacrificial material, - the detachment of the support relative to to the substrate by cleavage at said trenches.
  • the plate deposition step can be a deposition of a plate comprising in recess in fluid communication with the capillary slot in order to constitute a reservoir.
  • the method may further comprise a step of depositing at least one electrode intended to ensure electrical contact with the liquid to be nebulized.
  • the source of electrospray according to the invention can be used to obtain ionization of a liquid by electronebulization before its analysis in mass spectrometry. It can also be used to obtain a production of drops of liquid of calibrated size or the ejection of particles of fixed size. It can also be applied to the production of molecular writing using chemical compounds. It can also be applied to the definition of the electrical junction potential of a device in fluid continuity.
  • FIGS. 1A and 1B are top and side views respectively of a source of electrospray according to the present invention
  • - Figure 2 is a perspective view of the tip end of a source of electrospray according to the present invention
  • FIGS. 3A to 3H are top views illustrating a method of manufacturing the electrospray source shown in FIGS. 1A and 1B
  • FIGS. 4A and 4B illustrate a cleavage technique that can be used for the implementation of the manufacturing process illustrated by FIGS. 3A to 3H
  • FIG. 1A and 1B are top and side views respectively of a source of electrospray according to the present invention
  • FIGS. 3A to 3H are top views illustrating a method of manufacturing the electrospray source shown in FIGS. 1A and 1B
  • FIGS. 4A and 4B illustrate a cleavage technique that can be used for the implementation of the manufacturing process illustrated by FIGS. 3A to 3H
  • FIG. 5 represents an assembly used during a test during which a source of electrospray according to the invention is associated with a mass spectrometer
  • - Figure 6 is a graph representing the total ion current obtained during the test using a source of electrospray according to the invention
  • Figure 7 is a mass spectrum obtained during the test using a source of electrospray according to the invention in the assembly of FIG. 5
  • - FIG. 8 represents another assembly used during a test during which a source of electrospray according to the invention is associated with a mass spectrometer
  • - Figure 9 is a graph representing the total ion current obtained during the test using a source of electrospray according to the invention ion, in the assembly of FIG. 8, - FIG.
  • FIG. 10 is a mass spectrum obtained during the test using a source of electrospray according to the invention in the assembly of FIG. 8, - FIG. 11 represents a mass spectrum fragmentation of the glu-fibrinopeptide obtained with a source of electrospray according to the present invention, - Figure 12 represents a mass spectrum obtained for a digest of Cytochrome C via a source of electrospray according to the present invention, - Figure 13 is a graph representing the total ion current obtained during a test using a source of electrospray according to the invention, - Figure 14 represents a mass spectrum obtained during a test using a source of electrospray according to the present invention, - Figure 15 is a graph representing the total ion current recorded on a mass spectrometer of the ion trap type during a coupling test using an electrospray source according to the present invention, - Figure 16 represents the corresponding mass spectrum to the graph in Figure 15.
  • the present invention is inspired by the structure and the mode of operation of a calligraphy pen.
  • the planar sources which are the subject of the present invention consist of the same elements as a calligraphy pen: a liquid reservoir and a two-dimensional capillary slot formed in a point.
  • the present invention may include, if necessary, an electrical contact zone to which the voltage necessary to apply is applied. the establishment of a nebulisate.
  • This contact zone can be structured with multiple and independent contacts and in particular three contacts corresponding to a working electrode, also making it possible to apply the electrospray voltage, a reference electrode and a measurement electrode to allow chemical modification. by electrochemistry in order to favor the process of electrospray or to study it.
  • FIG. 1A and 1B A source of electrospray according to the present invention is shown in Figures 1A and 1B, Figure 1A being a top view and Figure 1B a side view.
  • This source of electrospray comprises a support 1 and a plate 2 integral with the support 1. A part of the plate 2 forms a point 3 in cantilever with respect to the support 1.
  • the plate 2 has in its center a recess 4 revealing the surface of the support 1 and constituting a reservoir.
  • the operation of the device is based on the following principles.
  • the liquid reservoir 4 contains the liquid or serves as a transit for the supply of liquid.
  • the liquid is then guided by the capillary slot 5 upstream of which is located the reservoir 4 of liquid.
  • the tip of the structure allows the establishment of an electrospray. This results in the following operating mode.
  • the liquid of interest is deposited or conveyed in the liquid reservoir 4 by an appropriate method. It is guided towards the end 6 of the structure by capillarity.
  • FIG. 2 represents a three-dimensional view of the capillary slot at the end 6 of the tip 3.
  • the role of the reservoir 4 is to contain the liquid to be nebulized and to gradually supply the capillary slot 5.
  • the topology of the structure is two-dimensional.
  • the plate 2 is made of a material of hydrophobic nature, and even more hydrophobic than that constituting the support 1 supporting the plate 2, material which lines the bottom of the tank.
  • the liquids envisaged for the nebulization will a priori be of rather hydrophilic character, such as purely aqueous solutions or semi-aqueous semi-alcoholic, for example mixtures methanol / water 50/50.
  • the capillary slot 5 and the end 6 of the tip 3 are made of the material forming the plate 2 and their dimensions are determined during the manufacturing process. In FIG. 2 are indicated dimensions to be considered for the operation of the electrospray source: the width w of the slit, its height h and its length 1. It is assumed that liquid is present in the capillary slit 5.
  • Equation 1 governing the capillarity effect of a liquid in a capillary tube, the cosine of the contact angle ⁇ must be positive to observe the capillary effect, and this, independently of the effect of gravity.
  • Equation 1 where (r) is the internal radius of the capillary, (h r ) the height by which the liquid rises in the capillary tube, (p) the density of the liquid, ( ⁇ ) is the contact angle of the liquid on the internal walls of the capillary tube and (g) is the acceleration of gravity.
  • r C S a r sv - ⁇ SL (Eq u ati on 2) where ⁇ sv is the surface tension at the solid-vapor interface and ⁇ SL is the surface tension at the solid-liquid interface.
  • Young's equation (equation 2) implies that Ysv> YS L and therefore that the solid-liquid interaction is favored compared to that solid- steam.
  • the term r appears in equation 1. Whether or not the capillary effect is observed depends on its value.
  • the term r corresponds to the radius of the capillary tube and, in the case of the device which is the subject of the present invention, to the dimension of the capillary slot 5.
  • the nebulization device may or may not include conductive zones (see Figure 3H). These conductive zones if they are located at the level of the liquid reservoir 4 serve as electrodes for bringing the nebulization voltage.
  • these electrodes will be used to modify the species present in the liquid.
  • electrochemical processes take place during the ionization of the molecules.
  • the conductive areas located on either side of the capillary slot 5 at the end 6 of the tip 3 would allow them to be studied. Furthermore, these phenomena lead to an increase in the ionization yield and, as a result, to an improvement in the analysis conditions.
  • the presence of a greater quantity of radical species increases the speed of etching of the substrate.
  • these conductive zones in particular if their role is to bring the nebulization voltage, may not be necessary. Indeed, if a conductive material (metal, Si ..) is used to make the support 1 or the plate 2, the voltage will be directly applied to this conductive material. Finally, a device which does not include conductive zones and for which the materials are not conductive can * be used in electronebulization provided that the electrical contact is made via the liquid. A metal wire immersed in the solution to be nebulized, at the level of the reservoir 4 or any other conductive contact will thus ensure the role of application of the nebulization voltage.
  • a conductive material metal, Si ..
  • the device can also be connected to a source of liquid supply upstream of the reservoir 4, like a capillary supplying a solution coming from another device, from another structure.
  • the capillary can correspond to an outlet of the separation column.
  • this capillary brings the liquid to the nebulization device from its initial location.
  • Said capillary can be a conventional commercial capillary made of fused silica. It can also be a microfabricated capillary, that is to say a microchannel integrated on the system supporting the source.
  • the capillary can be a hydrophilic track materialized on the support 1.
  • the plate 2 is integrated on a fluid microsystem and plays the role of interface between said microsystem and the outside world where the solution leaving the microsystem is used .
  • the conductive properties of the device or of one of its elements can be used to electrically supply any system in fluid relationship with the device.
  • said feather type plates can be used in isolation or be integrated in large numbers on the same support, and this, for the parallelization of the nebulization.
  • said feather-type plates are independent or not from each other and the nebulized solutions are either the same in order to increase the nebulization of said solution, or different and, in this case, the feathers operate sequentially in nebulization.
  • the integration of said feather-type plates can be carried out linearly with an alignment of said plates on one side of the support or in a circular fashion on a round support.
  • the passage from one source to the other then takes place respectively by translation or by rotation of the support.
  • a wide range of materials can now be envisaged for microtechnological manufacturing and in particular of fluidic microsystems: glass, silicon-based materials (Si, Si0 2 silicon nitride ...), quartz, ceramics as well as a large number macromolecular, plastic or elastomeric materials.
  • the geometry chosen for the present invention is compatible with manufacturing using any type of material, and this, for the different parts making up the electrospray source: the support 1, the feather-type plate 2 and the conductive areas.
  • the technological manufacturing process also involves one or more other material (s), the choice of which is suitable. depending on the materials selected for elements 1, 2 and 3.
  • a generic process for manufacturing electrospray sources according to the invention is shown in FIGS. 3A to 3H. This manufacturing process can be divided into seven major steps which are detailed below, so as to be applicable to any type of material.
  • the first step in this manufacturing process is the choice of the substrate intended to constitute the support for the electrospray source.
  • This substrate 10 (see FIG. 3A) can be made of macromolecular material, glass or else silicon or even metal. In the case of this exemplary embodiment, it is a silicon substrate 250 ⁇ m thick.
  • the start of the process conditions the end of the manufacturing of the electrospray devices.
  • a layer 11 of so-called protective material is deposited on part of the substrate 10.
  • the material of the layer 11 is chosen according to the nature of the material of the substrate 10 so that an attack on the layer 11 does not affect the substrate 10.
  • the layer of protective material is a layer of silicon oxide 20 nm thick.
  • the layer 11 is of variable thickness according to the nature of the materials of the substrate 10 and layer 11.
  • Layer 11 is subjected to a lithography step intended to reveal the areas of the substrate to be attacked in order to define. cleavage lines delimiting the support of the structure.
  • FIG. 3C shows the result obtained: the lines 13, consisting of V-shaped trenches, delimiting the support of the structure to be obtained.
  • a layer of sacrificial material is deposited on the substrate 10. This layer of sacrificial material 14 will allow, at the end of manufacture at the tip of the structure, to overhang its support before the cleavage operation.
  • the substrate 10 is covered with a thin layer of sacrificial material of sufficient thickness so that, after its removal, the tip is sufficiently separated from the substrate 10, but nevertheless sufficiently fine to be able to overcome any problem of stress and curvature of the point overhanging the support.
  • the layer of sacrificial material is a layer of nickel 150 nm thick.
  • the layer of sacrificial material is then subjected to an appropriate lithography and etching step in order to keep only a zone of this material. 14 corresponding to the tip of the structure (see Figure 3D).
  • the fourth step can be implemented.
  • the substrate 10 is then covered with a layer of a material intended to constitute the plate of the structure.
  • the material of this layer may be silicon or based on silicon, a metal or even a material of the polymer or ceramic type.
  • the layer of material intended to constitute the plate is a layer 35 ⁇ m thick in polymer SU-8 2035 purchased in pre-poly erect form from Microchem and polymerized by a photolithographic process.
  • the thickness of this layer is chosen appropriately. On this thickness indeed depend the performance in ionization of the nebulization device, as it was explained previously.
  • the thickness of this layer directly influences the height h of the capillary slit and, according to the above, the greater h, the greater w must be so as not to modify the ratio R.
  • the challenge is to reduce w as much as possible in order to increase performance.
  • the overhanging point can bend once detached from the support due to the stresses exerted on the material.
  • Those skilled in the art are able to adapt the present specifications according to the nature of the material of this layer and thus to define the optimal thickness of material to be deposited.
  • This layer then undergoes a lithography step and an attack in order to form the feather-type plate 2, that is to say in addition to its bulk, the reservoir 4, the capillary slot 5 and the tip 3 (see the figure 3E). This attack is adapted according to the material of the plate.
  • the fifth step can then be undertaken.
  • the zone 14 of sacrificial material under the tip 3 can be removed.
  • the sacrificial material is removed by an appropriate chemical attack.
  • the solution for this chemical attack must be chosen judiciously so that all the sacrificial material is removed without neither the support nor the plate being affected. The materials of these elements must therefore not be sensitive to this chemical solution.
  • the structure shown in Figure 3F is obtained.
  • the sixth step concerns the implantation of conductive zones on the structure.
  • this step is only included in the manufacturing process if such conductive zones are provided. Whether these zones are located at the level of the reservoir 4 (application of the nebulization voltage) or at the level of the tip (physicochemical study electrodes), the manufacturing process is the same.
  • the realization of the conductive zones 3 at the level of the tank alone will be detailed here. These conductive zones can be made of metal or carbon.
  • the structure is first subjected to a masking step so that only the zones corresponding to the formation of the conductive zones are exposed.
  • the conductive material chosen is then deposited by a PECVD technique (vapor deposition by chemical plasma techniques) on the structure.
  • the conductive zones are made of palladium and have a thickness of 400 nm.
  • Figure 3G shows the structure obtained.
  • Two conductive zones 7 and 8 surround the reservoir 4 and allow an electrical potential to be applied to it.
  • the seventh step of this method of manufacturing the nebulization source is the detachment of the support 1 relative to the substrate 10 and in particular, the overhanging of the tip 3 relative to the support 1 using the cleavage lines 13 materialized at the second step in this manufacturing process.
  • the structure obtained is shown in Figure 3H.
  • An advantageous cleavage technique is illustrated by FIGS. 4A and 4B in the case of the overhang of the point.
  • a fixed metal wire 20 is placed under the support 1 at the level of the cleavage trenches 13 formed on either side of the point. Jointly, two forces are exerted on the substrate at the locations indicated in Figure 4A by arrows.
  • FIG. 4B shows the cleavage in progress.
  • This generic manufacturing process is then adapted according to the materials chosen for each element of the electrospray source.
  • the first field of applications targeted by the present invention is the electrospraying of biological or chemical solutions to be analyzed by mass spectrometry. Mass spectrometry is currently the technique of choice for the analysis, characterization and identification of proteins. However, since the end of the genome decryption, biologists in particular are increasingly interested in proteomics, a science which aims to study and characterize all the proteins of an individual. These proteins, in every human being, are present in more than 10 6 different molecules including post-translational modifications.
  • the second type of targeted applications by the present invention is the deposit of calibrated drops on a smooth or rough surface. This is of prime interest for the preparation of DNA chips, peptides, PNA or any other type of molecules.
  • This type of application requires a device capable of delivering fluid in discrete form, drops of liquid of calibrated size, the size depending most often on the resolution expected in the preparation of the analysis plates. The smaller the drops, the closer their deposition can be on the plate and the greater the density of deposits and therefore of substances to be analyzed.
  • the device forming the subject of the present invention can be re used for this purpose.
  • the width of the capillary slot 5, as well as the value of the voltage applied for the ejection of the drops conditions the size of the drops ejected by said nebulization device.
  • the resolution of the analysis plates can be adjusted according to the width of the slot of the device.
  • the nebulization voltage can be alternating and thus give a deposition rate in drops / minute directly dependent on the frequency of the alternating voltage.
  • the deposit of calibrated drops as presented above can be used for the preparation of analysis plates such as DNA chips.
  • the present nebulization device having a feather type geometry can it for example be connected at the outlet of the separation column and allow coupling between a separation technique and an online analysis by mass spectrometry of the MALDI type.
  • the drops of liquid can be replaced by cells. In this case, the cells are likewise discreetly ejected and deposited, for example, on a plate for the preparation of cell chips.
  • the third application targeted by the present invention is molecular writing at scales of the order of a hundred nanometers.
  • this type of operation is carried out at using AFM microscopy tips, operating using heavy and bulky equipment.
  • the ejection of the liquid is based on bringing the tip and the deposition substrate into contact or quasi-contact in the case of AFM or on the application of pressure on the liquid.
  • An adaptation of this technique is to eject the liquid under the action of a tension and not using pressure or contacting. Indeed, in both cases, the ejection is caused when the tensioning forces of the liquid at the tip of the pipette are "exceeded" by another force applied to the column of liquid.
  • the formation of reactive species is intrinsic to the process of electrospraying.
  • This fluid ejection technique eliminates any complex apparatus for producing reactive species such as free radicals, such as a plasma or microwave discharge, upstream of the structure which delivers the liquid.
  • the present invention can therefore be used for such molecular writing purposes on a smooth or rough substrate, the release of the writing solution (pseudo-ink) here being controlled by applying a voltage.
  • a major stake is to minimize the size of the tip of the tip, this dimension conditioning the size of the ejections by nebulization and consequently the resolution expected in writing on the final substrate.
  • the width of the tip is less than or equal to a micrometer.
  • Another factor influencing the size of the ejections and the fluid flow rate is the nebulization voltage applied to the liquid.
  • Example 1 Design of microfabricated nanoelectronebulization sources according to the present invention.
  • a first example concerns the dimensions and the shapes chosen to produce a nebulization device as described in the present invention.
  • This first device has small dimensions at its tip due to the field of applications targeted, that is to say a nanoelectronebulization for the ionization of solutions before their analysis by mass spectrometry.
  • the device is produced in accordance with FIGS. 1A and 1B.
  • the reservoir 4 of the device has the dimensions 2.5 mm x 2.5 mm xe ( ⁇ m) where e is the thickness of the layer of material used to make the plate 2.
  • the value of e is close to that of h, considered below, the thickness of sacrificial material being the order of a hundred nanometers.
  • Example 2 Manufacture of the design sources described in Example 1 using silicon and SU-8 materials.
  • the second example relates to the fabrication by microtechnology of the nebulization sources, as described in example 1.
  • the materials used are silicon for the support 1 and the negative photolithographic resin SU-8 for the feather-type plate 2.
  • the method of manufacturing follows from the process described above. It is suitable for the materials chosen.
  • a 3 inch oriented n-doped silicon substrate (100) is covered with a 200 nm layer of silicon oxide (Si0 2 ), then masked by lithography.
  • the Si0 2 layer is attacked by a acid solution of HF: H 2 0 on the unmasked areas.
  • the exposed silicon is then attacked by a sodium hydroxide solution (KOH) so as to materialize the cleavage lines.
  • KOH sodium hydroxide solution
  • a 150 nm layer of nickel is then deposited on the silicon surface by spraying technique under argon (Plassys MP 450S).
  • the nickel layer is attacked locally by UV photolithography (positive photosensitive resin AZ1518 [l, 2 ⁇ m], etching solution HN0 3 / H 2 0 (1: 3)) so that only nickel remains under the tip of the pen.
  • UV photolithography positive photosensitive resin AZ1518 [l, 2 ⁇ m], etching solution HN0 3 / H 2 0 (1: 3)
  • the silicon wafer is dehydrated at 170 ° C. for 30 min, so as to optimize the adhesion of the SU-8 resin to the silicon surface.
  • a 35 ⁇ m layer of SU-8 resin is spread on the silicon substrate using a spinner to homogenize the thickness thereof before the next photolithography step.
  • the feather-type plate 2 is produced in this layer of resin SU-8 using conventional UV photolithography techniques.
  • the nickel layer is attacked with the acid solution (HN0 3 / H 2 0) described above. This nickel etching step does not affect the SU-8 resin even if this process can take several hours.
  • the silicon substrate 1 is sawn according to the technique illustrated in FIGS. 4A and 4B.
  • the technique used here preserves the structure of the pen, as it was previously detached from its support.
  • a photograph of scanning electron microscopy (Hitachi S4700) of the feather-type nebulization source manufactured according to this process confirms the correct detachment of the point relative to its support.
  • the manufacturing process described above does not include the production of electrodes.
  • Example 3 Design of a particle ejection device of a hundred micrometers.
  • a third example relates to the dimensions and the shapes chosen to make a particle ejection device having a size of a hundred micrometers, as described in the present invention.
  • This device has larger dimensions than that described in Example 1.
  • the dimensions of the capillary slot 5 and of the reservoir 4 must be compatible with the handling of objects of a hundred micrometers. Because of this range of dimensions, the device described in Example 3 also applies to the handling of cells of size approaching 100 ⁇ m in diameter, for the preparation of cell chips for example.
  • the reservoir 4 of said device has the dimensions 1 cm x 1 cm xe ( ⁇ m) where e is the thickness of the plate 2.
  • the value of e is defined as a function of the width of the capillary slot 5 so as to have a form factor R at the end 6 of the plate which is greater than 1.
  • the particles handled by this device have a size of a hundred micrometers, therefore the capillary slot 5 must have a width greater than 100 ⁇ m. However, since the particles may tend to aggregate, this width should not be chosen too large. It is preferably close to twice the size of the particles handled. Therefore, the width of the slot is fixed at 150 ⁇ m, and the thickness of the plate at 200 ⁇ m.
  • the material retained for the manufacture of the feather-type plate 2 is here again the negative photolithographic resin SU-8 and the material chosen for the support 1 is glass.
  • the SU-8 resin is interesting here for handling particles such as cells, because these cells do not adhere to this material. Therefore, the glass support 1 is also covered with a thin layer of SU-8 resin in order to prevent any unwanted adhesion of the cells to the device.
  • Example 4 Test of the nebulization sources manufactured according to Example 2 in mass spectrometry. I: Application of the voltage using a platinum wire.
  • Example 4 is the test of the nebulization sources manufactured as described in Example 2 for an analysis in mass spectrometry.
  • the nebulization voltage is applied to the liquid to be nebulized using a platinum wire immersed in the liquid at the level of the reservoir as illustrated in FIG. 5.
  • the nebulization device is placed on a part.
  • mobile 30 can be moved in xyz. This moving part 30 comprises a metal part 31 on which the ionization voltage is applied in the mass spectrometer 25.
  • the silicon support 1 is carefully insulated from this metal part 31 when the device is fixed to said moving part 30 because semiconductor properties of this material.
  • the electrical contact between the metal part 31 and the reservoir of the device is ensured using a platinum wire 32 introduced into the reservoir and which plunges into the solution to be analyzed 33.
  • the solution used for the nebulization tests a standard peptide solution (Gramicidine S), is deposited in the device reservoir and the moving part 30 is introduced into the inlet of the mass spectrometer 25.
  • the tests are carried out on a mass spectrometer of the ion trap type from Thermo Finnigan (LCQ DE ⁇ A XP +). Voltage is then applied to the liquid.
  • FIG. 6 is a graph representing the total ion current recorded by the mass spectrometer for an experiment carried out for 2 minutes with a solution of Gramicidine S at 5 ⁇ M and an ionization voltage at 0.8 kV.
  • the ordinate axis represents the relative intensity I R.
  • the abscissa axis represents time.
  • FIG. 7 corresponds to the mass spectrum obtained with a solution of Gramicidine S at 5 ⁇ M and a voltage of 1.2 kV. The mass spectrum was average over 2 minutes of signal acquisition, ie 80 scans.
  • Example 5 Test of the nebulization sources manufactured according to Example 2 in mass spectrometry. II: Application of the voltage on the silicon support Example 5 is close to Example 4, but here the voltage is not applied using a platinum wire but by exploiting the semiconductor properties silicon. Example 5 is therefore the mass spectrometry test of nebulization sources manufactured according to Example 2 with an application of the ionization voltage on the material constituting the support 1 of the nebulization device.
  • the nebulization device is fixed on a movable part 40 which can be moved in xyz and comprising a metallic part 41.
  • the silicon support 1 is brought into electrical contact with the metallic part 41 of the movable part 40 on which is applied the ionization voltage in the mass spectrometer 25.
  • the device is fixed to the movable part 40 using a teflon tape which surrounds the device upstream of the tank.
  • the test is carried out as previously after introduction of the moving part 40 into the ion trap 25 and application of the voltage.
  • the capillary slot has a width of 8 ⁇ m.
  • the tests were carried out with another standard peptide, Glu-Fibrinopeptide B.
  • the ionization voltages, here, are in the same range as before, from 1 to 1.4 kV for concentrations in peptide less than 1 ⁇ M.
  • FIG. 9 represents the total ion current measured during 3 minutes of signal acquisition with a 0.1 ⁇ M solution and a voltage of 1.1 kV.
  • I R is the relative intensity and t the time.
  • Figure 10 is the mass spectrum obtained for this acquisition and average over the period of 3 minutes, ie 120 scans. I R is the relative intensity.
  • Example 6 Test of the nebulization sources produced according to Example 2 in mass spectrometry. III: Fragmentation experience (MS / MS).
  • Example 6 is identical to Example 5 on how to conduct the test.
  • the test setup is identical to that of the previous example, the nebulization device corresponds to that described in Example 1 and produced according to the manufacturing process described in Example 2.
  • the voltage is applied directly to the material of the support 1, silicon, via the metallic zone 41 included on the moving part 40 introduced into the mass spectrometer 25 (see FIG. 8).
  • the capillary slot has a width of 8 ⁇ m.
  • the solution is the same as above, a standard peptide solution, Glu-Fibrinopeptide B at concentrations less than or equal to 1 ⁇ M.
  • FIG. 11 represents the fragmentation spectrum obtained during this experiment with a 0.1 ⁇ M solution and a voltage of 1.1 kV.
  • I R is the relative intensity. The spectrum was average over 2-3 minutes of acquisition of the nebulization signal. The different MS / MS fragments are annotated with their sequence.
  • Example 7 Test of the nebulization sources produced according to Example 2 in mass spectrometry. IV: Application to the analysis of a biological mixture.
  • Example 7 is identical to Example 5 (same device manufactured according to the same process and tested under the same conditions with application of the voltage on the silicon support 1) except that the sample analyzed here is no longer a peptide standard but a complex mixture of peptides obtained by digestion of a protein, Cytochrome C. This digestate is made up of 13 peptides of different lengths and physicochemical properties. This digestate is tested at a concentration of 1 ⁇ M and with an ionization voltage of 1.1-1.2 kV. The width of the capillary slot is 8 ⁇ m.
  • Example 12 represents the mass spectrum obtained for the digest of Cytochrome C at 1 ⁇ M with a voltage of 1.2 kV.
  • I R is the relative intensity. The peaks are annotated with the sequence of the fragment and its state of charge. Of the 15 peptides, 11 are clearly identified during this experiment.
  • Example 8 Test of the nebulization sources produced according to Example 2 in mass spectrometry.
  • V Continuous supply of said device using a syringe pump or a nanoLC chain placed upstream.
  • Example 8 is identical to Example 5 (same device manufactured according to the same process and tested under the same conditions with application of the voltage on the silicon support 1) except that the sample analyzed here is brought to said device in continuous by a capillary connected to a syringe pump or a chain of nanoLC upstream.
  • the liquid flow rate was set at 500 nL / min.
  • the solution for this test is identical to that of Example 5, except that the concentration of the Glu-Fibrinopeptide B peptide is here 1 ⁇ M and the nebulization voltage has been set at 1.2 kV.
  • the width of the capillary slot is 8 ⁇ m.
  • FIG. 13 shows the total ion current recorded during a nebulization test carried out over a period of 6 minutes under said conditions.
  • I R is the relative intensity and t the time.
  • FIG. 14 represents the corresponding and average mass spectrum over this 6-minute acquisition period, ie 240 scans.
  • I R is the relative intensity.
  • the coupling to a nanoLC chain liquid chromatography at a flow rate of 1 to 1000 nL / min
  • the fluid flow rate is 100 nL / min, the ionization voltage of 1.5 kV.
  • the separation experiment is carried out on a Cytochrome C digestate at 800 fmol / ⁇ L and 800 fmol of this digestate are injected onto the separation column.
  • the width of the capillary slot is 10 ⁇ m.
  • Figure 15 shows the total ion current detected on the mass spectrometer during the separation experiment. I R is the relative intensity and t the time.
  • FIG. 16 is the mass spectrum obtained for the peak indicated in FIG. 15 at the retention time of 23.8 min. It corresponds to the elution and analysis of the 92-99 fragment of Cytochrome C.
  • I R is the relative intensity.

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Abstract

Pen nib structure, for electro-spray ionization (ESI), has at least one thin and flat point (3) in relation to the remainder of the body (1) on a supporting plate (2), in the structure of a pen nib. The point has a capillary slot (5) through the whole thickness, leading to the tip (6) forming an opening to eject the sample vapor. Pen nib structure, for electro-spray ionization (ESI), has at least one thin and flat point (3) in relation to the remainder of the body (1) on a supporting plate (2), in the structure of a pen nib. The point has a capillary slot (5) through the whole thickness, leading to the tip (6) forming an opening to eject the sample vapor. The body has a well (4) to hold the sample, and is connected to an electrode to deliver a voltage for vaporizing.

Description

SOURCES DΕLECTRONEBULISATION PLANAIRES SUR LE MODELE D'UNE PLUME DE CALLIGRAPHIE ET LEUR FABRICATION SOURCES OF PLANAR ELECTRONEBULIZATION ON THE MODEL OF A CALLIGRAPHY FEATHER AND THEIR MANUFACTURE
DESCRIPTIONDESCRIPTION
DOMAINE TECHNIQUE La présente invention concerne des sources d'électronébulisation originales, leur procédé de fabrication et leurs applications .TECHNICAL FIELD The present invention relates to original electrospray sources, their manufacturing process and their applications.
ETAT DE LA TECHNIQUE ANTERIEURE L' électronébulisation est le phénomène qui transforme un liquide en un nebulisat sous l'action d'une haute tension (M. CLOUPEAU "Electrohydrodynamic spraying functioning modes: a critical revie . Journal of Aérosol Science (1994), 25(6), 1021-1036"). Pour ce faire, le liquide est amené dans un capillaire et est soumis à une haute tension continue ou alternative ou à une superposition des deux (Z. HUNEITI et al., "The study of AC coupled DC fields on conducting liquid jets", Journal of Electrostatics (1997), 40 & 41 97- 102) . En sortie de capillaire, le liquide est nébulisé sous l'action de la tension. La surface du ménisque formé par le liquide est allongée pour former un ou des cônes de Taylor d'où sont éjectées des gouttelettes de liquide chargées qui évoluent pour donner un gaz contenant des particules chargées . La formation du nebulisat est observée lorsque les forces électriques dues à l'application de la tension compensent et dépassent les forces de tension de surface du liquide sur la section du capillaire en l'extrémité dudit capillaire. La taille du capillaire, et plus précisément son orifice de sortie, est en relation directe avec le débit de liquide sortant du capillaire et la tension à appliquer pour observer le phénomène de nébulisation. Il existe deux régimes distincts d'électronébulisation qui se distinguent de par leurs caractéristiques d'établissement : • le régime dit classique qui correspond à des tailles de sortie de capillaire de 100 μm, des débits de fluide dans la gamme de 1-20 μL/min et des hautes tensions de 3-4 kV ; • le régime dit de nanoélectronébulisation où les débits de liquide sont inférieurs à 1 μL/min, la haute tension d'environ 1 kV et les diamètre internes des capillaires de 1-10 μm (M. WILM et al, "Analytical Properties of the Nanoelectrospray Ion Source", Analytical Chemistry (1996), 68(1), 1-8.). L'application d'une tension comportant une composante alternative permet la stabilisation du processus d'électronébulisation par synchronisation sur sa fréquence propre (F. CHARBONNIER et al., "Differentiating between Capillary and Counter Electrode Processes during Electrospray lonization by Opening the Short Circuit at the Collector. Analytical Chemistry (1999), 71(8), 1585-1591). La composition chimique des gouttes produites par le phénomène d'électronébulisation peut être améliorée en vue de ses applications par l'application de tensions multiples et indépendantes qui permettent la modification chimique des espèces présentes dans le liquide par électrochimie (voir la demande de brevet US 2003/0015656/ G. J. VAN BERKEL, "Enhanced Study and Control of Analyte Oxidation in Electrospray Using a Thin-Channel, Planar Electrode Emitter", Analytical Chemistry (2002), 74(19), 5047-5056; G.J. VAN BERKEL et al., "Derivatization for electrospray lonization mass spectrometry. 3. Electrochemically ionizable derivatives", Analytical Chemistry (1998), 70(8), 1544- 1554; F. ZHOU et al. "Electrochemistry Combined Online with Electrospray Mass Spectrometry", Analytical Chemistry (1995), 67(20), 3643-3649). Les domaines d' applications de 1' électronébulisation sont les suivants : • En premier lieu, l'ionisation de molécules (M. DOLE et al., "Molecular beams of macroions", Journal of Chemical Physics (1968), 49(5), 2240-2249 ; L. L. MACK et al., "Molecular beams of macroions. II", Journal of Chemical Physics (1970), 52(10), 4977-4986 ; le brevet US 4 209 696; M. YAMASHITA et al., "Electrospray ion source. Another variation on the free-jet thème", Journal of Physical Chemistry (1984), 88(20), 4451-4459 ; M. YAMASHITA et al . , "Négative ion production with the electrospray ion source", Journal of Physical Chemistry (1984), 88(20), 4671-4675) avant leur analyse par spectrométrie de masse en fonction du rapport m/z où m est la masse de l' analyte et z sa charge. Dans ce cas, le débit de liquide est continu. • Une deuxième application des dispositifs d'électronébulisation est la production de gouttes de taille calibrée. De telles gouttes peuvent être déposées sur un support (C. J. McNEAL et al., "Thin film déposition by the electrospray method for californium-252 plasma desorption studies of involatile molécules", Analytical Chemistry (1979), 51(12), 2036- 2039 ; R. C. MURPHY et al., "Electrospray loading of field desorption emitters and desorption chemical lonization probes", Analytical Chemistry (1982), 54(2), 336-338) par exemple une plaque pour, soit la production de puces d'analyse comme les puces à ADN ou à peptides, dédiées à une analyse à haut débit (V. N. MOROZOV et al., "Electrospray Déposition as a Method for Mass Fabrication of Mono- and Multicomponent Microarrays of Biological and Biologically Active Substances", Analytical Chemistry (1999), 71(15), 3110- 3117; R. MOERMAN et al., "Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots", Analytical Chemistry (2001 May 15), 73(10), 2183-2189 ; N. V. AVSEENKO et al., "Immunoassay with Multicomponent Protein Microarrays Fabricated by Electrospray Déposition", Analytical Chemistry (2002), 74(5), 927- 933) , soit le dépôt de solutions sur une plaque MALDI (pour "Matrix Assisted Laser Desorption lonization") avant une analyse par spectrometrie de masse (J. AXELSSON et al., "Improved reproducibility and increased signal intensity in matrix-assisted laser desorption/ionization as a resuit of electrospray sample préparation", Rapid Communications in Mass Spectrometry (1997), 11(2), 209-213). Ces gouttes peuvent aussi être manipulées, soit pour l'injection de liquide dans une balance hydrodynamique pour la manipulation de gouttes uniques (M. J. BOGAN et al., "MALDI-TOF-MS analysis of droplets prepared in an electrodynamic balance: "wall-less" sample préparation", Analytical Chemistry (2002), 74(3), 489- 496) , soit pour leur collecte pour conduire à des molécules encapsulées ou présentant un état cristallin métastable (I. G. LOSCERTALES et al., "Micro/nano encapsulation via electrified coaxial liquid jets", Science (Washington, DC, United States) (2002), 295(5560), 1695-1698). Ici, l'éjection a lieu de manière discrète les dimensions des sources dépendent grandement de la taille des dépôts à réaliser. • Une troisième application est le dépôt de particules de taille contrôlée contenues au sein du liquide (I. W. LENGGORO et al., "Sizing of Colloidal Nanoparticles by Electrospray and Differential Mobility Analyzer Methods", Langmuir (2002), 18(12), 4584-4591). Les particules peuvent également être remplacées pas des cellules pour la préparation de puces à cellules. • Une quatrième application est l'injection des gouttes formées par électronébulisation dans un liquide conduisant à des émulsions de taille bien définies (R. J. PFEIFER et al., "Charge-to-mass relation for electrohydrodynamically sprayed liquid droplets", Physics of Fluids (1958-1988) (1967), 10(10), 2149-54; C. TSOURIS et al., "Expérimental Investigation of Electrostatic Dispersion of Nonconductive Fluids into Conductive Fluids", Industrial & Engineering Chemistry Research (1995) , 34(4), 1394-1403 ; R. HENGELMOLEN et al., "Emulsions from aérosol sprays", Journal of Colloid and Interface Science (1997), 196(1), 12-22). • Une cinquième application est l'écriture moléculaire sur une plaque à l'aide de molécules ou de solutions chimiques (S. N. JAYASINGHE et al., "A novel process for simulataneous printing of multiple tracks from concentrated suspensions", Materials Research Innovations (2003), 7(2), 62-64.), en vue de la fonctionnalisation du matériau ou d'un traitement chimique localisé, à une échelle pouvant être inférieure au micromètre. Ces diverses applications peuvent être également combinées entre elles . Usuellement, les sources utilisées pour la nanoelectronebulisation se présentent sous forme de capillaires en verre ou en silice fondue. Elles sont fabriquées par étirement à chaud ou par attaque acide du matériau afin de donner un orifice de sortie de 1 à 10 μm (M. WILM et al., "Electrospray and Taylor-Cone theory, Dole' s beam of macromolecules at last?", International Journal of Mass Spectrometry and Ion Processes (1994), 136(2-3), 167-180). La tension d'électronébulisation peut être appliquée via un revêtement extérieur conducteur approprié : un revêtement métallique comme l'or ou un alliage Au/Pd (G. A. VALASKOVIC et al., "Long-lived metalized tips for nanoliter electrospray mass spectrometry", Journal of the American Society for Mass Spectrometry (1996) , 7(12), 1270-1272), l'argent (Y.-R CHEN et al., "A simple method for fabrication of silver-coated sheathless electrospray emitters", Rapid Coiximunications in Mass Spectrometry (2003), 17(5), 437-441), un matériau à base de carbone (X. ZHU et al., "A Colloidal Graphite-Coated Emitter for Sheathless Capillary Electrophoresis/Nanoelectrospray lonization Mass Spectrometry", Analytical Chemistry (2002), 74(20), 5405-5409) ou un polymère conducteur comme le polyaniline (P. A. BIGWARFE et al., "Polyaniline-coated nanoelectrospray emitters: performance characteristics in the négative ion mode", Rapid Communications in Mass Spectrometry (2002), 16(24), 2266-2272). La tension d'électronébulisation peut aussi être appliquée via le liquide avec l'introduction d'un fil métallique dans la source (K. W. Y. FONG et al., "A novel nonmetallized tip for electrospray mass spectrometry at nanoliter flow rate", Journal of the American Society for Mass Spectrometry (1999), 10(1), 72-75). Néanmoins, les dispositifs de l'art antérieur dédiés à la nanoelectronebulisation souffrent de plusieurs faiblesses (B. FENG et al., "A Simple Nanoelectrospray Arrangement With Controllable Flowrate for Mass Analysis of Submicroliter Protein Samples", Journal of the American Society for Mass Spectrometry (2000), 11, 94-99) : • Tout d'abord, ces capillaires sont peu robustes. Leur procédé de fabrication est mal contrôlé et fournit des sources de dimensions peu reproductibles ; • Le revêtement conducteur externe se détériore rapidement ; • Leur mode d'utilisation est peu commode du fait de leur géométrie de type aiguille : le liquide à nébuliser doit être introduit manuellement dans l'aiguille à l'aide d'une micropipette et d'un embout adapté de forme effilée ; • Le chargement de la solution conduit à l'introduction de bulles d'air dans l'aiguille qui peuvent perturber ultérieurement la stabilité du nebulisat, elles doivent donc être chassées ; • Enfin, le plus souvent, l'orifice de sortie est trop petit pour permettre le passage du liquide ; de ce fait, les capillaires doivent d'abord être cassés doucement le long d'une paroi, ce qui accroît encore le caractère aléatoire de leurs dimensions . Ainsi, les sources standard commerciales sont-elles peu adaptées, premièrement à une nébulisation contrôlée, reproductible et de qualité, deuxièmement à l'utilisation de robots du fait du caractère entièrement manuel de leur mode d'utilisation, et, troisièmement, à une intégration sur un microsystème fluidique, comme discuté dans la suite. Ces défauts entravent certains domaines d'applications de l' éléctronébulisation qui nécessitent à l'heure actuelle une robotisation et une automatisation des processus. Ceci est le cas des domaines d'applications recensés ci-dessus : l'analyse par spectrometrie de masse, le dépôt de gouttes de taille calibrée et l'écriture à une échelle inférieure au micromètre à l'aide d'une pointe. Ces deux dernières décennies ont vu l'avènement de la microfluidique dans les domaines de la chimie et de la biologie. Ce secteur résulte en partie de la miniaturisation des outils de laboratoire et donc du mariage entre microtechnologie et biologie ou microtechnologie et analyse chimique. Ainsi, les techniques de microtechnologie sont-elles mises à profit pour la fabrication de microsystèmes intégrés de taille caractéristique de l'ordre du micromètre et qui rassemblent une série de processus réactionnels et/ou analytiques, chimiques et/ou biochimiques/biologiques. L'essor de la microfluidique dans les domaines de la chimie et de la biologie, où la rapidité et l'automatisation des processus sont aujourd'hui requises, s'explique par : • le gain en vitesse des processus, du fait que la vitesse dépend principalement de la taille des dispositifs ; ce gain en vitesse est particulièrement important pour des champs d' pplications de type diagnostic médical ou analyse environnementale, où une réponse instantanée est souvent attendue, • la possibilité de parallélisation des processus ; la microtechnologie permet la fabrication simultanée d'un grand nombre de dispositifs identiques, • la compatibilité des objets microfabriqués avec une interface robotique en vue de l'automatisation des processus, • l'adéquation des volumes manipulés avec ceux dont l'expérimentateur dispose dans le cas, entre autres, des analyses biologiques ou environnementales, • la limitation allant jusqu'à la suppression de l'intervention humaine, qui est souvent source d'erreur et de contamination, • un gain en sensibilité, pour certaines techniques d'analyse, dont la spectrometrie de masse avec une ionisation par électronébulisation, • globalement, de nouvelles performances qui ne correspondent pas seulement à une diminution d'échelle des outils et des techniques bien établis. Les dispositifs microfluidiques sont fabriqués à l'aide des techniques de microtechnologie. Une large gamme de matériaux est aujourd'hui disponible pour ces microfabrications, gamme qui va du silicium et du quartz (matériaux usuels en microtechnologie) aux verres, céramiques et matériaux de type polymère, comme les élastomères ou les plastiques. Ainsi, la microfluidique bénéficie-t-elle à la fois : • de l'héritage des matériaux et des techniques de fabrication développés et utilisés pour des applications microélectronique et, • de nouveaux procédés de fabrication, développés en parallèle et adaptés à d' autres matériaux émergents et de grand intérêt pour des applications microfluidiques, comme les matériaux de type plastique, dont l'attrait principal réside dans leur faible coût. Plus précisément, les matériaux envisageables pour des fabrications technologiques applicables à la chimie et à la biologie sont (T. McCREEDY, "Fabrication techniques and materials co monly used for the production of microreactors and micro total analytical Systems", TrAC, Trends in Analytical Chemistry (2000), 19(6), 396-401) : • les matériaux de type semi-conducteurs comme le silicium, matériaux traditionnels en microtechnologie qui bénéficient de techniques de fabrication robustes et éprouvées ; parmi ces techniques de fabrication, on compte la lithographie, les gravures physiques et chimiques entre autres (P. J. FRENCH et al., "Surface versus bulk micromachining: the contest for suitable applications", Journal of Micromechanics and Microengineering (1998), 8(2), 45- 53) . De ce fait, le silicium notamment est le matériau le plus intéressant en termes de fabrication de petites structures à des échelles de la dizaine de nanomètres. De plus, sa chimie de surface est maîtrisée, les traitements mettant en jeu les fonctions silanols présentes à sa surface. Mais ses propriétés semi- conductrices ne sont pas toujours adaptées en fonction des applications visées. Il n'est pas transparent ce qui empêche toute technique de détection optique (absorbance UV, fluorescence, luminescence) . Le coût du matériau lui-même le rend impropre pour certaines fabrications de masse (objets à usage unique notamment) . • le quartz, utilisé pour le développement des premiers microsystèmes (J. S. DANEL et al., "Quartz: a material for microdevices", Journal of Micromechanics and Microengineering (1991), 1(4), 187- 98) , qui est devenu peu attrayant du fait de son coût fortement élevé ; il est donc progressivement abandonné en dépit de ses propriétés physico-chimiques . • le verre, matériau moins cher que le quartz et le silicium, qui est beaucoup utilisé du fait de ses propriétés de surface adaptées à l'établissement d'un flux électroosmotique (K. SATO et al., "Intégration of chemical and biochemical analysis Systems into a glass microchip", Analytical Sciences (2003), 19(1), 15-22). De même que pour le silicium, des groupements silanols tapissent la surface du verre. Ils laissent envisager une modification chimique ultérieure des surfaces de verre. De plus, ses propriétés de transparence en font un matériau de choix dans le cas d'une détection optique. Cependant, les techniques de fabrication ne sont pas aussi bien maîtrisées que pour le silicium; les profils de gravure sont moins propres et le rapport de forme est fort médiocre (T. R. DIETRICH et al., "Fabrication technologies for microsystems utilizing photoetchable glass", Microelectronic Engineering (1996), 30(1-4), 497-504) . D'autre part, c'est un matériau fragile et cassant. • les matériaux de type polymère, qui regroupent les plastiques les élastomères . Leur avantage principal est leur faible coût qui est compatible avec des productions de masse à bas prix de revient. La multiplicité de ces matériaux conduit à une large gamme de propriétés physico-chimiques . Leur inconvénient majeur est leur faible résistance aux hautes températures et leur sensibilité aux conditions de solvant utilisées classiquement en chimie et en biologie, milieu organique, acide, basique, qui peuvent entraîner une dégradation du matériau voire même sa dissolution. Par ailleurs, la chimie de surface de ces matériaux est mal connue, ce qui rend difficile tout traitement ultérieur des surfaces engendrées afin d'en modifier les propriétés . Les techniques de fabrication sont tout autres et sont basées sur des techniques de moulage/injection, d'ablation laser, de LIGA (acronyme allemand pour "Lithographie, Galvanoformung, Abformung") (J. HRUBY, " Overview of LIGA microfabrication", AIP Conférence Proceedings (2002), 625(High Energy Density and High Power RF) , 55-61), de photolithographie, de gravure plasma. • les matériaux de types céramiques (W. BAUER, "Ceramic materials in the microsystem technology", Keramische Zeitschrift (2003), 55(4), 266- 270), qui sont des substrats inorganiques de faible coût de fabrication à l'image des matériaux plastiques. Un avantage majeur est que leur fabrication ne nécessite pas d'équipements dédiés d'entretien onéreux comme des salles blanches mais repose sur des processus simples et rapides (ablation laser, laminage, moulage, procédé sol-gel) , réduisant encore le prix de revient des structures icrofabriquées . Leur état de surface est comparable à celui du verre ou du silicium et enfin, le capotage est plus facile que pour d'autres matériaux, comme le verre. En particulier, les techniques de microfabrication ont été appliquées à la réalisation de sources d'électronébulisation ou de pointe type aiguille en vue : • d' améliorer la qualité globale des capillaires en termes de contrôle des procédés de fabrication, de reproductibilité des sources et de leurs dimensions, • de produire un grand nombre de dispositifs identiques ou différant entre eux par une ou plusieurs dimensions, sur une même plaque de matériau, à l'image des microcomposants en microélectronique, afin de promouvoir l'automatisation et la robotisation de l'électronébulisation. Les fabrications à l'aide des techniques de microtechnologie de pointes d'électronébulisation obéissent à deux tendances : • la fabrication d'une pointe d'électronébulisation qui reproduit la géométrie classique, c'est-à-dire un capillaire microfabriqué et, le plus souvent, de section circulaire. Dans cette classe peuvent être inclues également les aiguilles microfabriquées destinées à une autre application, comme celle d'injection de substances chimiques ou de mesure de potentiel biologique. • la conception d'une source d'électronébulisation comme une sortie de microcanal ou capillaire fabriqué à l'aide de techniques de microtechnologie et ayant un profil effilé. Ces dispositifs d'électronébulisation microfabriqués reposent, à l'image des microsystèmes fluidiques, sur l'utilisation de différents types de matériaux et différents types de procédés . Selon la première tendance, qui vise à produire par voie technologique une géométrie de type capillaire, on recense les descriptions suivantes : • Selon cette approche, des sources d'électronébulisation en nitrure de silicium ont été fabriquées à l'aide de techniques classiques de photolithographie et de gravure (A. DESAI et al., "MEMS Electrospray Nozzle for Mass Spectrometry", Int. Conf. on Solid-State Sensors and Actuators, Transducers '97, (1997)). Les dimensions desdits dispositifs sont une longueur de 40 μm et un diamètre interne de l'orifice de sortie de 1 à 3 μm. Lesdites sources ont été testées en spectrometrie de masse à des tensions de nebulisation voisines de 4 kV et un débit de liquide deSTATE OF THE PRIOR ART Electrospray is the phenomenon which transforms a liquid into a nebuliser under the action of a high voltage (M. CLOUPEAU "Electrohydrodynamic spraying functioning modes: a critical revie. Journal of Aérosol Science (1994), 25 (6), 1021-1036 "). To do this, the liquid is brought into a capillary and is subjected to a direct or alternating high voltage or to a superposition of the two (Z. HUNEITI et al., "The study of AC coupled DC fields on conducting liquid jets", Journal of Electrostatics (1997), 40 & 41 97-102). At the capillary outlet, the liquid is nebulized under the action of tension. The surface of the meniscus formed by the liquid is elongated to form one or more Taylor cones from which are ejected charged liquid droplets which evolve to give a gas containing charged particles. The formation of the nebulisate is observed when the electrical forces due to the application of the voltage compensate for and exceed the surface tension forces of the liquid. on the section of the capillary at the end of said capillary. The size of the capillary, and more precisely its outlet orifice, is in direct relation with the flow of liquid leaving the capillary and the voltage to be applied to observe the nebulization phenomenon. There are two distinct electrospray regimes which are distinguished by their establishment characteristics: • the so-called classical regime which corresponds to capillary outlet sizes of 100 μm, fluid flow rates in the range of 1-20 μL / min and high voltages of 3-4 kV; • the so-called nanoelectronisation regime where the liquid flow rates are less than 1 μL / min, the high voltage of approximately 1 kV and the internal diameter of the capillaries of 1-10 μm (M. WILM et al, "Analytical Properties of the Nanoelectrospray Ion Source ", Analytical Chemistry (1996), 68 (1), 1-8.). The application of a voltage comprising an AC component allows the stabilization of the electrospray process by synchronization on its natural frequency (F. CHARBONNIER et al., "Differentiating between Capillary and Counter Electrode Processes during Electrospray lonization by Opening the Short Circuit at the Collector. Analytical Chemistry (1999), 71 (8), 1585-1591) .The chemical composition of the drops produced by the electrospray phenomenon can be improved with a view to its applications by the application of multiple and independent voltages which allow chemical modification species present in the liquid by electrochemistry (see patent application US 2003/0015656 / GJ VAN BERKEL, "Enhanced Study and Control of Analyte Oxidation in Electrospray Using a Thin-Channel, Planar Electrode Emitter", Analytical Chemistry (2002), 74 (19), 5047-5056; GJ VAN BERKEL et al., "Derivatization for electrospray lonization mass spectrometry. 3. Electrochemically ionizable derivatives", Analytical Chemistry (1998), 70 (8), 1544-1554; F. ZHOU and al. "Electrochemistry Combined Online with Electrospray Mass Spectrometry", Analytical Chemistry (1995), 67 (20), 3643-3649). The fields of application of electrospray are as follows: • First, the ionization of molecules (M. DOLE et al., "Molecular beams of macroions", Journal of Chemical Physics (1968), 49 (5) , 2240-2249; LL MACK et al., "Molecular beams of macroions. II", Journal of Chemical Physics (1970), 52 (10), 4977-4986; US Patent 4,209,696; M. YAMASHITA et al. , "Electrospray ion source. Another variation on the free-jet theme", Journal of Physical Chemistry (1984), 88 (20), 4451-4459; M. YAMASHITA et al., "Negative ion production with the electrospray ion source" , Journal of Physical Chemistry (1984), 88 (20), 4671-4675) before their analysis by mass spectrometry as a function of the ratio m / z where m is the mass of the analyte and z its charge. In this case, the liquid flow is continuous. • A second application of electrospray devices is the production of drops of calibrated size. Such drops can be deposited on a support (CJ McNEAL et al., "Thin film deposition by the electrospray method for californium-252 plasma desorption studies of involatile molecules", Analytical Chemistry (1979), 51 (12), 2036-2039 ; RC MURPHY et al., "Electrospray loading of field desorption emitters and desorption chemical lonization probes", Analytical Chemistry (1982), 54 (2), 336-338) for example a plate for either the production of analysis chips like DNA or peptide chips, dedicated to high-throughput analysis (VN MOROZOV et al., "Electrospray Deposition as a Method for Mass Fabrication of Mono- and Multicomponent Microarrays of Biological and Biologically Active Substances", Analytical Chemistry (1999 ), 71 (15), 3110-3117; R. MOERMAN et al., "Miniaturized electrospraying as a technique for the production of microarrays of reproducible micrometer-sized protein spots", Analytical Chemistry (2001 May 15), 73 (10) , 2183-2189; NV AVSEENKO et al., "Imm unoassay with Multicomponent Protein Microarrays Fabricated by Electrospray Déposition ", Analytical Chemistry (2002), 74 (5), 927-933), i.e. the deposit of solutions on a MALDI plate (for" Matrix Assisted Laser Desorption lonization ") before an analysis by mass spectrometry (J. AXELSSON et al., "Improved reproducibility and increased signal intensity in matrix-assisted laser desorption / ionization as a resuit of electrospray sample preparation", Rapid Communications in Mass Spectrometry (1997), 11 (2), 209-213). These drops can also be handled, either for the injection of liquid in a hydrodynamic balance for handling single drops (MJ BOGAN et al., "MALDI-TOF-MS analysis of droplets prepared in an electrodynamic balance:" wall-less "sample preparation", Analytical Chemistry (2002), 74 ( 3), 489-496), either for their collection to lead to molecules encapsulated or having a metastable crystalline state (IG LOSCERTALES et al., "Micro / nano encapsulation via electrified coaxial liquid jets", Science (Washington, DC, United States) (2002), 295 (5560), 1695-1698). Here, the ejection takes place in a discreet manner the dimensions of the sources depend greatly on the size of the deposits to be produced. • A third application is the deposition of particles of controlled size contained within the liquid (IW LENGGORO et al., "Sizing of Colloidal Nanoparticles by Electrospray and Differential Mobility Analyzer Methods", Langmuir (2002), 18 (12), 4584- 4591). The particles can also be replaced by cells for the preparation of cell chips. • A fourth application is the injection of the drops formed by electrospray into a liquid leading to well defined size emulsions (RJ PFEIFER et al., "Charge-to-mass relation for electrohydrodynamically sprayed liquid droplets", Physics of Fluids (1958 -1988) (1967), 10 (10), 2149-54; C. TSOURIS et al., "Experimental Investigation of Electrostatic Dispersion of Nonconductive Fluids into Conductive Fluids", Industrial & Engineering Chemistry Research (1995), 34 (4) , 1394-1403; R. HENGELMOLEN et al., "Emulsions from aerosol sprays ", Journal of Colloid and Interface Science (1997), 196 (1), 12-22). • A fifth application is molecular writing on a plate using molecules or chemical solutions (SN JAYASINGHE et al., "A novel process for simulataneous printing of multiple tracks from concentrated suspensions", Materials Research Innovations (2003), 7 (2), 62-64.), for the functionalization of the material or a chemical treatment localized, on a scale which can be less than a micrometer. These various applications can also be combined with one another. Usually, the sources used for nanoelectronebulization are in the form of capillaries made of glass or fused silica. They are manufactured by hot stretching or by acid attack of the material in order to give an outlet of 1 to 10 μm (M. WILM et al., "Electrospray and Taylor-Cone theory, Dole 's beam of macromolecules at last?", International Journal of Mass Spectrometry and Ion Processes (1994), 136 (2-3), 167-180). The electrospray voltage can be applied via an appropriate conductive external coating: a metallic coating such as gold or an Au / Pd alloy (GA VALASKOVIC et al., "Long-lived metalized tips for nanoliter electrospray mass spectrometry", Journal of the American Society for Mass Spectrometry (1996), 7 (12), 1270-1272), argent (Y.-R CHEN et al., "A simple method for fabrication of silver-coated sheathless electrospray emitters", Rapid Coiximunications in Mass Spectrometry (2003), 17 (5), 437-441), a carbon-based material (X. ZHU et al., "A Colloidal Graphite-Coated Emitter for Sheathless Capillary Electrophoresis / Nanoelectrospray lonization Mass Spectrometry", Analytical Chemistry (2002), 74 (20), 5405-5409) or a conductive polymer such as polyaniline (PA BIGWARFE et al., "Polyaniline-coated nanoelectrospray emitters: performance characteristics in the negative ion mode", Rapid Communications in Mass Spectrometry ( 2002), 16 (24), 2266-2272). The electrospray voltage can also be applied via the liquid with the introduction of a metallic wire into the source (KWY FONG et al., "A novel nonmetallized tip for electrospray mass spectrometry at nanoliter flow rate", Journal of the American Society for Mass Spectrometry (1999), 10 (1), 72-75). Nevertheless, the devices of the prior art dedicated to nanoelectronebulization suffer from several weaknesses (B. FENG et al., "A Simple Nanoelectrospray Arrangement With Controllable Flowrate for Mass Analysis of Submicroliter Protein Samples", Journal of the American Society for Mass Spectrometry (2000), 11, 94-99): • First of all, these capillaries are not very robust. Their manufacturing process is poorly controlled and provides sources of dimensions that are difficult to reproduce; • The external conductive coating deteriorates rapidly; • Their mode of use is inconvenient due to their needle type geometry: the liquid to be nebulized must be introduced manually into the needle using a micropipette and a suitable tip of tapered shape; • The loading of the solution leads to the introduction of air bubbles in the needle which can later disturb the stability of the nebulisat, they must therefore be removed; • Finally, most often, the outlet orifice is too small to allow the passage of the liquid; therefore, the capillaries must first be gently broken along a wall, which further increases the randomness of their dimensions. Standard commercial sources are therefore unsuitable, firstly for controlled, reproducible and quality nebulization, secondly for the use of robots due to the entirely manual nature of their mode of use, and thirdly, for integration on a fluid microsystem, as discussed below. These faults hamper certain fields of application of electrospray which currently require robotization and process automation. This is the case of the fields of application listed above: analysis by mass spectrometry, the deposition of drops of calibrated size and writing on a scale less than a micrometer using a tip. These last two decades have seen the advent of microfluidics in the fields of chemistry and biology. This sector results in part of the miniaturization of laboratory tools and therefore of the marriage between microtechnology and biology or microtechnology and chemical analysis. Thus, microtechnology techniques are used for the manufacture of integrated microsystems of characteristic size of the order of a micrometer and which bring together a series of reaction and / or analytical, chemical and / or biochemical / biological processes. The rise of microfluidics in the fields of chemistry and biology, where speed and process automation are required today, can be explained by: • the gain in speed of the processes, due to the fact that the speed mainly depends on the size of the devices; this gain in speed is particularly important for fields of application such as medical diagnosis or environmental analysis, where an instant response is often expected, • the possibility of parallelization of processes; microtechnology allows the simultaneous manufacture of a large number of identical devices, • the compatibility of microfabricated objects with a robotic interface for the automation of processes, • the adequacy of the volumes handled with those available to the experimenter in the case, inter alia, biological or environmental analyzes, • the limitation going until the suppression of the human intervention, which is often source of error and contamination, • a gain in sensitivity, for certain analysis techniques, including mass spectrometry with ionization by electrospray, • overall, new performances which do not correspond only to a reduction in scale of well-established tools and techniques. Microfluidic devices are manufactured using microtechnology techniques. Today, a wide range of materials is available for these microfabrications, ranging from silicon and quartz (common materials in microtechnology) to glasses, ceramics and polymer-type materials, such as elastomers or plastics. Thus, microfluidics benefits from both: • the heritage of materials and manufacturing techniques developed and used for microelectronic applications and, • new manufacturing processes, developed in parallel and adapted to others emerging materials of great interest for microfluidic applications, such as plastic-type materials, the main attraction of which is their low cost. More specifically, the possible materials for technological manufacturing applicable to chemistry and biology are (T. McCREEDY, "Fabrication techniques and materials co monly used for the production of microreactors and micro total analytical Systems", TrAC, Trends in Analytical Chemistry (2000), 19 (6), 396-401): • materials of the semiconductor type such as silicon, traditional materials in microtechnology which benefit from robust and proven manufacturing techniques; among these manufacturing techniques are lithography, physical and chemical engravings among others (PJ FRENCH et al., "Surface versus bulk micromachining: the contest for suitable applications", Journal of Micromechanics and Microengineering (1998), 8 (2 ), 45-53). Therefore, silicon in particular is the most interesting material in terms of manufacturing small structures at scales of ten nanometers. In addition, its surface chemistry is under control, the treatments bringing into play the silanol functions present on its surface. However, its semiconductor properties are not always adapted according to the intended applications. It is not transparent which prevents any optical detection technique (UV absorbance, fluorescence, luminescence). The cost of the material itself makes it unsuitable for certain mass manufacturing (objects for single use in particular). • quartz, used for the development of the first microsystems (JS DANEL et al., "Quartz: a material for microdevices", Journal of Micromechanics and Microengineering (1991), 1 (4), 187-98), which has become little attractive due to its high cost; it is therefore gradually abandoned despite its physico-chemical properties. • glass, a less expensive material than quartz and silicon, which is widely used because of its surface properties suitable for establishing an electroosmotic flux (K. SATO et al., "Integration of chemical and biochemical analysis Systems into a glass microchip", Analytical Sciences (2003), 19 (1), 15-22). As with silicon, silanol groups line the surface of the glass. They suggest a subsequent chemical modification of the glass surfaces. In addition, its transparency properties make it a material of choice in the case of optical detection. However, the manufacturing techniques are not as well mastered as for silicon; the etching profiles are less clean and the aspect ratio is very poor (TR DIETRICH et al., "Fabrication technologies for microsystems utilizing photoetchable glass", Microelectronic Engineering (1996), 30 (1-4), 497-504). On the other hand, it is a fragile and brittle material. • polymer-type materials, which include plastics and elastomers. Their main advantage is their low cost which is compatible with mass production at low cost. The multiplicity of these materials leads to a wide range of physicochemical properties. Their major drawback is their low resistance to high temperatures and their sensitivity to the solvent conditions conventionally used in chemistry and biology, organic, acidic, basic medium, which can cause degradation of the material or even its dissolution. Furthermore, the surface chemistry of these materials is poorly understood, which makes any subsequent treatment of the surfaces generated difficult in order to modify their properties. Manufacturing techniques are quite different and are based on molding / injection techniques, laser ablation, LIGA (German acronym for "Lithography, Galvanoformung, Abformung") (J. HRUBY, "Overview of LIGA microfabrication", AIP Conférence Proceedings (2002 ), 625 (High Energy Density and High Power RF), 55-61), photolithography, plasma etching. • ceramic type materials (W. BAUER, "Ceramic materials in the microsystem technology", Keramische Zeitschrift (2003), 55 (4), 266-270), which are inorganic substrates of low manufacturing cost in the image plastic materials. A major advantage is that their manufacture does not require dedicated expensive maintenance equipment such as clean rooms but is based on simple and rapid processes (laser ablation, rolling, molding, sol-gel process), further reducing the cost price. icrofabricated structures. Their surface condition is comparable to that of glass or silicon and finally, the rollover is easier than for other materials, such as glass. In particular, microfabrication techniques have been applied to the production of electrospray or advanced needle-type sources with a view to: • improving the overall quality of the capillaries in terms of control of the manufacturing processes, reproducibility of the sources and their dimensions, • to produce a large number of identical or differing devices by one or several dimensions, on the same material plate, like microcomponents in microelectronics, in order to promote the automation and robotization of electrospray. The manufacture using microtechnology techniques of electrospray tips obeys two trends: • the manufacture of an electrospray tip which reproduces classic geometry, that is to say a microfabricated capillary and, most often of circular section. Also included in this class are microfabricated needles intended for another application, such as injecting chemical substances or measuring biological potential. • the design of an electrospray source such as a microchannel or capillary outlet manufactured using microtechnology techniques and having a tapered profile. These microfabricated electrospray devices are based, like fluidic microsystems, on the use of different types of materials and different types of processes. According to the first trend, which aims to produce a capillary type geometry, the following descriptions are listed: • According to this approach, electrospray sources made of silicon nitride have been manufactured using conventional photolithography techniques and engraving (A. DESAI et al., "MEMS Electrospray Nozzle for Mass Spectrometry", Int. Conf. on Solid-State Sensors and Actuators, Transducers '97, (1997)). The dimensions of said devices are a length of 40 μm and an internal diameter of the outlet orifice of 1 to 3 μm. Said sources were tested in mass spectrometry at nebulization voltages close to 4 kV and a liquid flow rate of
50 nL/min avec des peptides standard à une concentration de quelques micromolaires. La tension de nebulisation est appliquée en amont dudit dispositif, au niveau de la jonction avec un capillaire d'alimentation en liquide, et ce, sur une connexion métallique en platine . • Des sources d'électronébulisation fabriquées en matériau de type polymère, le parylène, matériau photolithographiable ont également été décrites (demande interntionale WO-A-00/30167; L. LICKLIDER et al., "A Micromachined Chip-Based Electrospray Source for Mass Spectrometry", Analytical Chemistry (2000), 72(2), 367-375). Ces sources ont un orifice de sortie de 5 x 10 μm et ont été présentées comme partie intégrante d'un microsystème fluidique en silicium. Elles sont connectées à des microcanaux de 100 μm de largeur et de 5 μm de hauteur. La tension requise pour la nebulisation est ici plus faible, de l'ordre de 1,2 à 1,8 kV dans des conditions de concentration et de débit de fluide équivalentes ; la tension est appliquée sur un fil métallique mis en contact avec la solution à nébuliser. • Le silicium a aussi été utilisé pour la microfabrication de structures de type aiguille. La demande internationale WO-A-00/15321 décrit un dispositif d'électronébulisation ressemblant à une cheminée, de diamètre interne de 10 μm pour un diamètre externe de 20 μm et une hauteur de 50 μm. On peut se référer également à l'article de G. A. SCHULTZ et al., intitulé "A Fully Integrated Monolithic Microchip Electrospray Device for Mass Spectrometry", Analytical Chemistry (2000), 72(17), 4058-4063. Ces sources résultent d'une gravure physique dite profonde du matériau. Leur fonctionnement en électronébulisation est décrit avec des hautes tensions de 1,25 kV, qui sont appliquées sur le capillaire d' alimentation en fluide situé à l'arrière de la source et qui est en matériau conducteur. Le prototype a été présenté intégré sur une plaque comprenant 100 sources de ce type, identiques et fonctionnant indépendamment les unes des autres. Le silicium et un procédé de fabrication similaire ont également été utilisés pour former des structures de type aiguille qui sont employées soit comme sources d'électronébulisation (P. GRISS et al., "Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry", Journal of Micromechanics and Microengineering (2002), 12(5), 682- 687; J. SJODAHL et al., "Characterization of micromachined hollow tips for two-dimensional nanoelectrospray mass spectrometry", Rapid Communications in Mass Spectrometry (2003), 17(4), 337- 341) , soit comme aiguilles de mesure de potentiels biologiques (demande internationale WO-A-03/15860; P. GRISS et al., "Micromachnied électrodes for biopotential measurements", IEEE/ASME Journal of Microelectromechanical Systems, 2001, 10, 10-16) . Leur forme varie quelques peu en fonction de leur application; les dispositifs d'électronébulisation ressemblent aux dispositifs en silicium décrits ci- dessus, avec néanmoins, un profil qui se rétrécit en leur pointe conduisant à un plus petit orifice de sortie, alors que les aiguilles destinées à des mesures de potentiels biologiques ont une pointe très effilée. Le procédé de fabrication desdits dispositifs en silicium à l'aide de techniques de gravure profonde est fort complexe et nécessite un appareillage coûteux, encombrant et les performances, en termes de tension de nebulisation entre autres, des structures obtenues sont médiocres comparées à celles de sources standard commerciales. Par ailleurs, leur géométrie se prête mal à une intégration sur un microsystème fluidique. • L'article de L. LIN et al., intitulé "Silicon processed microneedles", IEEE Journal of Mictroelectromechanical Systems (1999) , 8, 78-84) décrit des micro-aiguilles qui sont connectées à un réseau microfluidique. Ces aiguilles ont été développées pour l'injection de substances chimiques in situ et non pour de la nebulisation, mais la géométrie de type aiguille de ces dispositifs est proche de celle des sources de nanonébulisation. Ces aiguilles sont fabriquées en nitrure de silicium et présentent un orifice de sortie rectangulaire de 9 x 30-50 μm et une hauteur de 1 à 6 mm. • Des structures de type aiguille ont enfin été fabriquées en un autre matériau polymère, le polycarbonate, à l'aide d'un procédé d'ablation laser (K. TANG et al., "Génération of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometrie sensitivity", Analytical Chemistry (2001), 73(8), 1658-1663). Leurs dimensions sont les suivantes : 30 μm de diamètre interne en leur orifice de sortie et 250 μm de hauteur. Pour cet exemple encore, les dimensions desdits dispositifs sont trop grandes pour un régime en nanoelectronebulisation puisque la tension requise pour l'observation d'un nebulisat est de 7 kV et le débit de fluide est estimé à 30 μL/min. Le procédé de fabrication est par ailleurs complexe. Ces sources se présentent sous forme d'une série de neuf sources arrangées selon un carré 3 x 3. Elles opèrent simultanément et nebulisent la même solution. La deuxième tendance est d'usiner une pointe à la sortie d'un microcanal ou de créer une structure en pointe qui tient lieu de source d'électronébulisation. L'angle de la structure en pointe ne semble pas avoir d'influence sur le phénomène de nebulisation. Selon cette deuxième tendance: • Les tentatives de nebulisation à la sortie d'un microcanal, sur la tranche d'un microsystème se sont révélées peu concluantes . La tension à appliquer est très élevée et, dans ces conditions, le liquide a tendance à s'étaler sur la surface de sortie, sur la tranche du microsystème (R. RAMSEY et al., "Generating Electrospray from Microchip Devices Using Electroosmotic Pumping", Analytical Chemistry (1997), 69(6), 1174-1178; Q. XUE et al., "Multichannel Microchip Electrospray Mass Spectrometry", Analytical Chemistry (1997), 69(3), 426- 430; B. ZHANG et al., "Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry", Analytical Chemistry (1999), 71(15), 3258-3264) . Ces essais ont été améliorés par un traitement chimique approprié de la surface de sortie ou en assistant de façon pneumatique la formation du nebulisat. Ceci démontre l'importance de travailler avec une structure en pointe qui conduit à une concentration du champ électrique et qui permet ainsi la nebulisation. • L'effet de pointe peut être réalisé par insertion d'une structure plane triangulaire entre les deux plaques de matériaux définissant un microcanal (le support dans lequel le microcanal est usiné et le couvercle) . Cette structure plane triangulaire est constituée d'une feuille de parylène de 5 μm d'épaisseur (J. KAMEOKA et al., "An electrospray lonization source for intégration with microfluidics", Analytical Chemistry (2002), 74(22), 5897-5901). Le système intègre quatre dispositifs d'électronébulisation identiques placés en parallèle. La tension de nebulisation requise est de 2,5-3 kV pour un débit de fluide de 300 nL/min. Aucune interférence inter-sources n'a été observée. • Un dispositif en forme d'étoile à huit branches a été fabriqué en polyméthylméthacrylate (PMMA) (C.-H. YUAN et al., "Sequential Electrospray50 nL / min with standard peptides at a concentration of a few micromolars. The nebulization voltage is applied upstream of said device, at the junction with a liquid supply capillary, and this, on a metallic platinum connection. • Sources of electrospray made of polymer type material, parylene, photolithographic material have also been described (international application WO-A-00/30167; L. LICKLIDER et al., "A Micromachined Chip-Based Electrospray Source for Mass Spectrometry ", Analytical Chemistry (2000), 72 (2), 367-375). These sources have an outlet of 5 x 10 μm and have been presented as an integral part of a silicon fluid microsystem. They are connected to microchannels 100 μm wide and 5 μm high. The voltage required for nebulization is lower here, of the order of 1.2 to 1.8 kV under equivalent concentration and fluid flow conditions; the voltage is applied to a metal wire brought into contact with the solution to be nebulized. • Silicon has also been used for microfabrication of needle-like structures. International application WO-A-00/15321 describes a electrospray device resembling a chimney, with an internal diameter of 10 μm for an external diameter of 20 μm and a height of 50 μm. We can also refer to the article by GA SCHULTZ et al., Entitled "A Fully Integrated Monolithic Microchip Electrospray Device for Mass Spectrometry", Analytical Chemistry (2000), 72 (17), 4058-4063. These sources result from a so-called deep physical etching of the material. Their operation in electrospray is described with high voltages of 1.25 kV, which are applied to the fluid supply capillary located at the rear of the source and which is made of conductive material. The prototype was presented integrated on a plate comprising 100 sources of this type, identical and operating independently of each other. Silicon and a similar manufacturing process have also been used to form needle-like structures that are used either as sources of electrospray (P. GRISS et al., "Development of micromachined hollow tips for protein analysis based on nanoelectrospray ionization mass spectrometry ", Journal of Micromechanics and Microengineering (2002), 12 (5), 682- 687; J. SJODAHL et al.," Characterization of micromachined hollow tips for two-dimensional nanoelectrospray mass spectrometry ", Rapid Communications in Mass Spectrometry (2003 ), 17 (4), 337-341), or as needles for measuring biological potentials (international application WO-A-03/15860; P. GRISS et al., "Micromachnied electrodes for biopotential measurements", IEEE / ASME Journal of Microelectromechanical Systems, 2001, 10, 10-16). Their shape varies somewhat depending on their application; electrospray devices resemble the silicon devices described above, with, however, a profile which narrows at their tip leading to a smaller outlet, while the needles intended for biological potential measurements have a very sharp tip. tapered. The process for manufacturing said silicon devices using deep etching techniques is very complex and requires expensive, bulky equipment and the performance, in terms of nebulization voltage among others, of the structures obtained are poor compared to those of sources. commercial standards. Furthermore, their geometry does not lend itself well to integration on a fluid microsystem. • The article by L. LIN et al., Entitled "Silicon processed microneedles", IEEE Journal of Mictroelectromechanical Systems (1999), 8, 78-84) describes micro-needles which are connected to a microfluidic network. These needles were developed for the injection of chemical substances in situ and not for nebulization, but the needle-like geometry of these devices is close to that of the sources of nanonebulisation. These needles are made of silicon nitride and have a rectangular outlet of 9 x 30-50 μm and a height of 1 to 6 mm. • Needle-like structures were finally made from another polymer material, polycarbonate, using a laser ablation process (K. TANG et al., "Generation of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometrie sensitivity", Analytical Chemistry (2001), 73 (8), 1658-1663). Their dimensions are as follows: 30 μm in internal diameter at their outlet orifice and 250 μm in height. For this example again, the dimensions of said devices are too large for a nanoelectronebulization regime since the voltage required for the observation of a nebulisate is 7 kV and the fluid flow rate is estimated at 30 μL / min. The manufacturing process is also complex. These sources are in the form of a series of nine sources arranged in a 3 x 3 square. They operate simultaneously and nebulize the same solution. The second tendency is to machine a point at the exit of a microchannel or to create a point structure which acts as a source of electrospray. The angle of the pointed structure does not seem to have any influence on the nebulization phenomenon. According to this second trend: • Attempts to nebulize at the exit of a microchannel, on the edge of a microsystem have proved inconclusive. The voltage to be applied is very high and, under these conditions, the liquid tends to spread over the exit surface, on the edge of the microsystem (R. RAMSEY et al., "Generating Electrospray from Microchip Devices Using Electroosmotic Pumping" , Analytical Chemistry (1997), 69 (6), 1174-1178; Q. XUE et al., "Multichannel Microchip Electrospray Mass Spectrometry ", Analytical Chemistry (1997), 69 (3), 426-430; B. ZHANG et al.," Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry ", Analytical Chemistry (1999), 71 (15), 3258- These tests have been improved by appropriate chemical treatment of the outlet surface or by pneumatically assisting the formation of the nebulisate. This demonstrates the importance of working with a pointed structure which leads to a concentration of the electric field and which allows nebulization. • The peak effect can be achieved by inserting a triangular planar structure between the two plates of materials defining a microchannel (the support in which the microchannel is machined and the cover). consists of a parylene sheet 5 μm thick (J. KAMEOKA et al., "An electrospray lonization source for integration with microfluidics", Analytical Chemistry (2002), 74 (22), 5897-590 1). The system incorporates four identical electrospray devices placed in parallel. The nebulization voltage required is 2.5-3 kV for a fluid flow of 300 nL / min. No inter-source interference was observed. • An eight-pointed star-shaped device was made of polymethylmethacrylate (PMMA) (C.-H. YUAN et al., "Sequential Electrospray
Analysis Using Sharp-Tip Channels Fabricated on a Plastic Chip", Analytical Chemistry (2001), 73(6),Analysis Using Sharp-Tip Channels Fabricated on a Plastic Chip ", Analytical Chemistry (2001), 73 (6),
1080-1083) . Chacune des branches de l'étoile constitue un système microfluidique indépendant et la pointe de chaque branche est une source de nebulisation. Chaque branche intègre ainsi un microcanal de section 300 x 376 μm, la structure en pointe forme un angle de 90° et les huit réservoirs de liquide sont regroupés au centre de l'étoile. La tension appliquée pour l'établissement d'un cône de Taylor est élevée et égale à 3,8 kV, ce qui s'explique par les dimensions fort larges de la section du microcanal en son extrémité. Par ailleurs, le procédé de fabrication décrit repose sur l'usinage de canaux à l'aide d'un couteau, technique qui ne permet pas de réaliser des canaux et des dispositifs de nebulisation de petites dimensions . • Un autre matériau de type polymère, le polydiméthylsiloxane (PDMS) , a servi à la réalisation de structures en pointe destinées à l'électronébulisation suivant trois voies de fabrication microtechnologiques différentes, une méthode basée sur l'ablation de matériau, un procédé utilisant une double couche de résine photolithographiable et un procédé de moulage de la résine (demande internationale WO-A-02/55990; J. S. KIM et al., "Microfabrication of polydiméthylsiloxane electrospray ionization emitter", Journal of Chromatography, A (2001), 924(1-2), 137-145; J.-S. KIM et al . , "Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry", Journal of the American Society for Mass Spectrometry (2001) , 12(4), 463-469; J.-S. KIM et al., "Miniaturized multichannel electrospray ionization emitters on poly (dimethylsiloxane) microfluidic devices", Electrophoresis (2001), 22(18), 3993-3999). L'orifice de nebulisation est rectangulaire et de dimensions variables allant de 30 x 100 μm à 30 x 50 μm selon le procédé de microtechnologie utilisé pour leur fabrication. Dans les différents cas, la tension de nebulisation allait de 2,5 kV à 3,7 kV pour des solutions à 1 à 10 μM et des débits élevés de quelques 100 nL/min à plusieurs μL/min. • Enfin, le polyimide, autre matériau de type polymère relativement hydrophobe a été utilisé pour la fabrication de sources de nebulisation (GB-A-2 379 554; V. GOBRY et al., "Microfabricated polymer injector for direct mass spectrometry coupling", Proteomics (2002), 2(4), 405-412; J. S. ROSSIER et al., "Thin-chip microspray System for high-performance Fourier-transform ion-cyclotron résonance mass spectrometry of biopolymers", Angewandte Chemie, International Edition (2003), 42(1), 54-58) intégrées sur un microsystème, ou tout du moins, connectées à un microcanal de section 120 x 45 μm. Le système, le microcanal et la structure en pointe sont fabriqués par gravure plasma du polyimide. Le couvercle du système est en polyéthylène/polyéthylène téréphtalate. Le fonctionnement desdites sources en électronébulisation a été validé pour des échantillons de peptides standard à 5 μM, s'écoulant à 140 nL/min et pour des tensions de nebulisation de 1,6 à 1,8 kV. Un autre dispositif fabriqué dans le même matériau a été présenté, différant du précédent de par sa topologie ouverte et la finesse de l'épaisseur (50 μm) de matériau utilisée pour sa fabrication. Cette structure dite mince a été testée pour des tensions d'ionisation de 1 à 2,3 kV appliquées ici sur une électrode de carbone intégrée sur le dispositif. Globalement, les dispositifs de nebulisation recensés ci-dessus présentent des conditions de fonctionnement non conformes pour une nebulisation à petite échelle (dimensions trop grandes, tensions de nebulisation trop élevées) et résultent le plus souvent de procédés de fabrication fort complexes. De plus, le type de structure choisi pour ces différents dispositifs est pratiquement indissociable du matériau utilisé pour leur réalisation. Pour les différents dispositifs présentés ci-dessus, la tension de nebulisation est le plus souvent appliquée au niveau du réservoir du dispositif, si le système inclut un réservoir, ou, dans le cas contraire, au niveau de l'alimentation en liquide qui est effectuée à l'aide d'un capillaire connecté au dispositif. Dans ce cas, soit ' le capillaire est conducteur (en acier inoxydable par exemple) , soit la connexion repose sur un raccord métallique. Cependant, il a été proposé d'intégrer, sur le dispositif de nebulisation, une électrode ou zone conductrice sur laquelle est appliquée la tension de nebulisation (T. C. ROHNER et al., "Polymer microspray with an integrated thick-film microelectrode", Analytical Chemistry (2001), 73(22), 5353-5357). Cette zone conductrice est réalisée à base d'encre de carbone dans l'exemple cité. Enfin, l'application de ces dispositifs est ciblée pour de l'électronébulisation précédant une analyse par spectrometrie de masse et ne se prête pas à un autre type d'application. Par ailleurs, les dispositifs de dépôt de gouttes calibrées issus de la microtechnologie ne reposent pas sur la nebulisation de la solution mais sur un effet mécanique avec la mise en contact de la pointe microfabriquée sur la surface de dépôt. Ainsi : • Une structure mimant celle d'un stylo plume a été décrite pour l'élaboration de plaques de type des puces à ADN avec la déposition régulière de gouttes calibrées sur une surface lisse (voir la demande internationale WO-A-03/53583) . Le dispositif comprend une tranchée gravée dans le matériau se terminant sur une pointe par laquelle le liquide sort . Cette structure est dite flexible et le liquide à déposer sort par mise en contact de la pointe flexible avec le substrat de dépôt, l'angle de contact étant de 20-30° par rapport à la verticale. L'application majeure ciblée par cette invention est la préparation de puces à ADNs ou autres composés à analyser. • P. BELAUBRE et al. dans l'article "Fabrication of biological microarrays using microcantilevers", Applied Physics Letters (2003) , 82 (18) , 3122-3124, proposent une structure de type poutre ouverte pour le dépôt de gouttes de taille reproductible. L'application du dispositif est la préparation de puces à ADN ou à protéines de façon automatisée. La structure de type poutre est tout d'abord plongée dans la solution à déposer, puis est mise en contact avec la surface de dépôt. L'éjection du liquide est provoquée par la mise en contact entre la pointe et ladite surface. Une particularité de ce dispositif est l'intégration dans la structure de type poutre d' électrodes en aluminium qui permettent d'accroître le chargement en liquide de la pointe lorsque cette dernière est trempée dans la solution à déposer, par effet électrostatique. Ces structures de type poutre, qui ont une largeur de 210 μm en leur pointe, sont fabriquées en parallèle sur un même système. Elles permettent l'éjection de gouttes ayant un volume dans la gamme du femtolitre jusqu'au picolitre, le volume déposé dépendant linéairement du temps de contact entre la pointe et la surface, avec un débit pouvant atteindre 100 dépôts par minute. Enfin, l'écriture moléculaire à des échelles de l'ordre du nanometre est principalement décrite avec une pointe de microscopie AFM (Microscopie à Force Atomique) qui est trempée dans une solution chimique, à l'image d'une plume de stylo (G. AGARWAL et al., "Dip-Pen Nanolithography in Tapping Mode", Journal of the American Chemical Society (2003), 125(2), 580- 583; les demandes internationales WO-A-03/48314 et WO- A-03/52514; H. ZHANG et al., "Direct-write dip-pen nanolithography of proteins on modified silicon oxide surfaces", Angewandte Chemie, International Edition (2003), 42(20), 2309-2312; L. FU et al., "Nanopatterning of "Hard" Magnetic Nanostructures via Dip-Pen Nanolithography and a Sol-Based Ink", Nano Letters (2003), 3(6), 757-760; H. ZHANG et al., "Fabrication of sub-50-nm solid-state nanostructures on the basis of dip-pen nanolithography", Nano Letters (2003), 3(1), 43-45). L'écriture a ensuite lieu par mise en contact ou après rapprochement, suivant le mode d'utilisation de l'AFM sélectionné, de la pointe et d'une surface lisse. La solution chimique peut aussi être une solution qui attaque le matériau sur lequel elle est déposée et servir ainsi à la gravure de canaux ou d'autres structures. La technique de microscopie AFM présente l'avantage d'une forte résolution et d'une très grande précision d'écriture. Trois modes de fonctionnement sont possibles, et suivant le mode choisi, l'état de surface peut être contrôlé avant et après passage de la solution chimique d'écriture moléculaire. Néanmoins, cette technique impose l'utilisation d'un appareillage lourd, encombrant, onéreux et complexe . Deux dispositifs d'écriture moléculaire décrits dans la littérature peuvent également être cités. Ils dérivent de la technique utilisant une pointe de microscopie AFM mais reposent sur l'utilisation d'une pointe microfabriquée. Le premier dispositif (A. LEWIS et al., "Fountain pen nanochemistry: Atomic force control of chrome etching", Applied Physics Letters (1999), 75(17), 2689-2691 ; H. TAHA et al., "Protein printing with an atomic force sensing nanofountainpen", Applied Physics Letters (2003), 83(5), 1041-1043), se présente sous forme d'une micropipette fabriquée à l'aide de techniques de microtechnologie et dont la pointe peut avoir des dimensions aussi petites que 3 et 10 nm pour ses diamètres internes et externes respectivement . Cette micropipette est néanmoins intégrée dans un appareillage AFM pour son utilisation. L'éjection de la solution est ici provoquée non pas par une mise en contact mais en exerçant une pression sur la colonne de liquide. Ce dispositif a été testé pour son aptitude à délivrer des solutions de gravure d'une couche de chrome déposée sur une plaque de verre. Le deuxième dispositif (I. W. RANGELOW et al., ""NANOJET": Tool for the nanofabrication", Journal of Vacuum Science & Technology, B: Microelectronics and Nanometer Structures (2001), 19(6), 2723-2726; J. VOIGT et al., "Nanofabrication with scanning nanonozzle 'Nanojet'", Microelectronic Engineering (2001) , 57-58 1035-1042) consiste en des pointes réalisées en silicium couvert de Cr/Au, ayant une forme pyramidale et un orifice de sortie de taille inférieure à .100 nm. Ce dispositif délivre non pas une solution chimique comme dans l'exemple précédent, mais des radicaux libres en phase gazeuse produits par une décharge plasma qui viennent attaquer le matériau mis en regard de la pointe. Ainsi, le dispositif ne consiste-t-il pas uniquement en une pointe microfabriquée mais inclut-il également une machinerie de production d'espèces très réactives, comme une décharge plasma radiofréquence ou microonde, qui peuvent attaquer le substrat. Ces deux exemples présentent certes une pointe microfabriquée qui remplace la pointe conventionnelle de microscopie AFM, mais ils ne permettent pas de s'affranchir de la machinerie périphérique lourde et onéreuse nécessaire à leur fonctionnement. D'autre part, cette technique repose sur une mise en contact ou quasi-mise en contact de la pointe et du substrat. De ce fait, les paramètres de fonctionnement doivent être très minutieusement contrôlés pour éviter toute détérioration de l'état de surface due à une trop grande force exercée au niveau de la pointe.1080-1083). Each of the branches of the star constitutes an independent microfluidic system and the tip of each branch is a source of nebulization. Each branch thus integrates a microchannel with a section of 300 x 376 μm, the pointed structure forms an angle of 90 ° and the eight liquid reservoirs are grouped in the center of the star. The voltage applied for the establishment of a Taylor cone is high and equal to 3.8 kV, which is explained by the very large dimensions of the section of the microchannel at its end. Furthermore, the manufacturing process described is based on the machining of channels using a knife, a technique which does not allow channels and nebulization devices of small dimensions to be produced. • Another polymer-type material, polydimethylsiloxane (PDMS), was used to produce advanced structures intended for electrospray according to three different microtechnological manufacturing routes, a method based on the ablation of material, a process using a double layer of photolithographic resin and a process for molding the resin (international application WO-A-02/55990; JS KIM et al., "Microfabrication of polydimethylsiloxane electrospray ionization emitter", Journal of Chromatography, A (2001), 924 ( 1-2), 137-145; J.-S. KIM et al., "Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry", Journal of the American Society for Mass Spectrometry (2001), 12 (4), 463- 469; J.-S. KIM et al., "Miniaturized multichannel electrospray ionization emitters on poly (dimethylsiloxane) microfluidic devices", Electrophoresis (2001), 22 (18), 3993-3999). The nebulization orifice is rectangular and of variable dimensions ranging from 30 x 100 μm to 30 x 50 μm according to the microtechnology process used for their manufacture. In the different cases, the nebulization voltage ranged from 2.5 kV to 3.7 kV for solutions at 1 to 10 μM and high flow rates from a few 100 nL / min to several μL / min. Finally, polyimide, another material of relatively hydrophobic polymer type, was used for the manufacture of nebulization sources (GB-A-2 379 554; V. GOBRY et al., "Microfabricated polymer injector for direct mass spectrometry coupling", Proteomics (2002), 2 (4), 405-412; JS ROSSIER et al., "Thin-chip microspray System for high-performance Fourier-transform ion-cyclotron resonance mass spectrometry of biopolymers", Angewandte Chemie, International Edition (2003 ), 42 (1), 54-58) integrated on a microsystem, or at least, connected to a microchannel of section 120 x 45 μm. The system, the microchannel and the point structure are produced by plasma etching of the polyimide. The system cover is made of polyethylene / polyethylene terephthalate. The operation of said electrospray sources has been validated for samples of standard peptides at 5 μM, flowing at 140 nL / min and for nebulization voltages of 1.6 to 1.8 kV. Another device made of the same material was presented, differing from the previous one by its open topology and the thinness of the thickness (50 μm) of material used for its manufacture. This so-called thin structure was tested for ionization voltages from 1 to 2.3 kV applied here on a carbon electrode integrated on the device. Overall, the nebulization devices listed above have non-compliant operating conditions for nebulization on a small scale (too large dimensions, too high nebulization voltages) and most often result from very complex manufacturing processes. In addition, the type of structure chosen for these different devices is practically inseparable from the material used for their production. For the various devices presented above, the nebulization voltage is most often applied at the reservoir of the device, if the system includes a reservoir, or, if not, at the level of the liquid supply which is carried out using a capillary connected to the device. In this case, either ' the capillary is conductive (in stainless steel for example), or the connection rests on a metal fitting. However, it has been proposed to integrate, on the nebulization device, an electrode or conductive area to which the nebulization voltage is applied (TC ROHNER et al., "Polymer microspray with an integrated thick-film microelectrode", Analytical Chemistry (2001), 73 (22), 5353-5357). This conductive zone is produced based on carbon ink in the example cited. Finally, the application of these devices is targeted for electrospray preceding a mass spectrometry analysis and does not lend itself to another type of application. Furthermore, the devices for depositing calibrated drops originating from microtechnology do not rely on the nebulization of the solution but on a mechanical effect with the contacting of the microfabricated tip on the deposition surface. Thus: • A structure mimicking that of a fountain pen has been described for the production of DNA chip type plates with the regular deposition of calibrated drops on a smooth surface (see international application WO-A-03/53583 ). The device comprises a trench engraved in the material ending on a point through which the liquid leaves. This structure is said to be flexible and the liquid to be deposited leaves by bringing the flexible tip into contact with the deposition substrate, the contact angle being 20-30 ° relative to the vertical. The major application targeted by this invention is the preparation of DNA chips or other compounds to be analyzed. • P. BELAUBRE et al. in the article "Fabrication of biological microarrays using microcantilevers", Applied Physics Letters (2003), 82 (18), 3122-3124, propose an open beam type structure for depositing drops of reproducible size. The application of the device is the preparation of DNA or protein chips in an automated manner. The beam-type structure is first immersed in the solution to be deposited, then is brought into contact with the deposition surface. The ejection of the liquid is caused by the contact between the tip and said surface. A special feature of this device is the integration into the beam-type structure of aluminum electrodes which make it possible to increase the liquid loading of the tip when the latter is soaked in the solution to be deposited, by electrostatic effect. These beam-type structures, which have a width of 210 μm at their point, are manufactured in parallel on the same system. They allow the ejection of drops having a volume in the range from femtoliter to picolitre, the volume deposited depends linearly on the contact time between the tip and the surface, with a flow rate of up to 100 deposits per minute. Finally, molecular writing on scales of the order of a nanometer is mainly described with a tip of AFM microscopy (Atomic Force Microscopy) which is dipped in a chemical solution, like a pen nib (G AGARWAL et al., "Dip-Pen Nanolithography in Tapping Mode", Journal of the American Chemical Society (2003), 125 (2), 580-583; international applications WO-A-03/48314 and WO-A- 03/52514; H. ZHANG et al., "Direct-write dip-pen nanolithography of proteins on modified silicon oxide surfaces", Angewandte Chemie, International Edition (2003), 42 (20), 2309-2312; L. FU and al., "Nanopatterning of" Hard "Magnetic Nanostructures via Dip-Pen Nanolithography and a Sol-Based Ink", Nano Letters (2003), 3 (6), 757-760; H. ZHANG et al., "Fabrication of sub -50-nm solid-state nanostructures on the basis of dip-pen nanolithography ", Nano Letters (2003), 3 (1), 43-45). The writing then takes place by contacting or after approximation, depending on the mode of use of the selected AFM, the tip and a smooth surface. The chemical solution can also be a solution which attacks the material on which it is deposited and thus serve for the etching of channels or other structures. The AFM microscopy technique has the advantage of high resolution and very high writing precision. Three operating modes are possible, and depending on the mode chosen, the surface condition can be checked before and after passing the chemical molecular writing solution. However, this technique requires the use of heavy, bulky, expensive and complex equipment. Two molecular writing devices described in the literature can also be cited. They derive from the technique using an AFM microscopy tip but are based on the use of a microfabricated tip. The first device (A. LEWIS et al., "Fountain pen nanochemistry: Atomic force control of chrome etching", Applied Physics Letters (1999), 75 (17), 2689-2691; H. TAHA et al., "Protein printing with an atomic force sensing nanofountainpen ", Applied Physics Letters (2003), 83 (5), 1041-1043), is in the form of a micropipette manufactured using microtechnology techniques and the tip of which can have dimensions as small as 3 and 10 nm for its internal and external diameters respectively. This micropipette is nevertheless integrated into an AFM apparatus for its use. The ejection of the solution is here caused not by contacting but by exerting pressure on the liquid column. This device has been tested for its ability to deliver solutions for etching a layer of chromium deposited on a glass plate. The second device (IW RANGELOW et al., "" NANOJET ": Tool for the nanofabrication", Journal of Vacuum Science & Technology, B: Microelectronics and Nanometer Structures (2001), 19 (6), 2723-2726; J. VOIGT et al., "Nanofabrication with scanning nanonozzle 'Nanojet'", Microelectronic Engineering (2001), 57-58 1035-1042) consists of spikes made of silicon covered with Cr / Au, having a pyramidal shape and an outlet for size less than .100 nm. This device does not deliver a chemical solution as in the previous example, but free radicals in the gas phase produced by a plasma discharge which attack the material placed opposite the tip. Thus, the device does not only consist of a microfabricated tip but it also includes machinery for producing highly reactive species, such as a radiofrequency or microwave plasma discharge, which can attack the substrate. These two examples certainly present a microfabricated tip which replaces the conventional AFM microscopy tip, but they do not make it possible to dispense with the heavy and expensive peripheral machinery necessary for their operation. On the other hand, this technique is based on bringing into contact or almost bringing into contact the tip and the substrate. As a result, the parameters of operation must be very carefully checked to avoid any deterioration of the surface finish due to excessive force exerted on the tip.
EXPOSE DE L'INVENTION La présente invention concerne un dispositif d'électronébulisation bidimensionnel ayant une géométrie de type plume de calligraphie, dont la pointe tient lieu de siège pour la nebulisation. L'invention a donc pour objet une source d'électronébulisation comportant une structure comprenant au moins une pointe plate et mince en porte- à-faux par rapport au reste de la structure, ladite pointe étant pourvue d'une fente capillaire pratiquée dans toute l'épaisseur de la pointe et qui aboutit à l'extrémité de la pointe pour former l'orifice d'éjection de la source d'électronébulisation, la source comprenant des moyens d'approvisionnement de la fente capillaire en liquide à nébuliser et des moyens d'application d'une tension d'électronébulisation sur ledit liquide. Selon un mode avantageux, les moyens d'approvisionnement comprennent au moins un réservoir en communication fluidique avec la fente capillaire. De préférence, la structure comprend un support et une plaque solidaire du support et dont une partie constitue ladite pointe. Les moyens d'approvisionnement peuvent comprendre un réservoir constitué par un evidement formé dans ladite plaque et en communication fluidique avec la fente capillaire. Les moyens d'application d'une tension d'électronébulisation peuvent comprendre au moins une électrode disposée de façon à être en contact avec ledit liquide à nébuliser. Dans le cas où la structure comprend un support et une plaque solidaire du support, les moyens d'application d'une tension d'électronébulisation peuvent comprendre le support, au moins partiellement électriquement conducteur, et/ou la plaque au moins partiellement électriquement conductrice. Avantageusement, la plaque présente une surface hydrophobe au liquide à nébuliser. Les moyens d'application d'une tension d'électronébulisation peuvent comprendre un fil électriquement conducteur disposé pour pouvoir être en contact avec ledit liquide à nébuliser. Les moyens d'approvisionnement peuvent comprendre un tube capillaire. Ils peuvent comprendre un canal réalisé dans un microsystème supportant ladite structure et en communication fluidique avec la fente capillaire. Selon un mode avantageux, les moyens d'application de la tension (électrode, support, plaque, fil) permettent également l'application des tensions nécessaires pour tout dispositif placé en amont en continuité fluidique avec l'objet de la présente invention. L'invention a aussi pour objet un procédé de fabrication d'une structure étant une source d'électronébulisation, comprenant : - la réalisation d'un support à partir d'un substrat, la réalisation d'une plaque comportant une partie constituant une pointe plate et mince, ladite pointe étant pourvue d'une fente capillaire, pour véhiculer un liquide à nébuliser, pratiquée dans toute l'épaisseur de la pointe et qui aboutit à l'extrémité de la pointe, - la solidarisatiori de ladite plaque sur le support, la pointe étant en porte-à-faux par rapport au support . Ce procédé peut comprendre les étapes suivantes : - la fourniture d'un substrat pour réaliser le support, - la délimitation du support au moyen de tranchées gravées dans le substrat, le dépôt, sur une zone du substrat correspondant à la future pointe de la structure, de matériau sacrificiel selon une épaisseur déterminée, - le dépôt de la plaque sur le support délimité dans le substrat, la pointe de la plaque étant située sur le matériau sacrificiel, - l'élimination du matériau sacrificiel, - le détachement du support par rapport au substrat par clivage au niveau desdites tranchées . L ' étape de dépôt de la plaque peut être un dépôt d'une plaque comprenant en evidement en communication fluidique avec la fente capillaire afin de constituer un réservoir. Le procédé peut comprendre en outre une étape de dépôt d'au moins une électrode destinée à assurer un contact électrique avec le liquide à nébuliser. La source d'électronébulisation selon l'invention peut être utilisée pour obtenir une ionisation d'un liquide par electronebulisation avant son analyse en spectrometrie de masse. Elle peut aussi être utilisée pour obtenir une production de gouttes de liquide de taille calibrée ou l'éjection de particules de taille fixée. Elle peut encore s'appliquer à la réalisation d'une écriture moléculaire à l'aide de composés chimiques. Elle peut encore s'appliquer à la définition du potentiel électrique de jonction d'un dispositif en continuité fluidique.PRESENTATION OF THE INVENTION The present invention relates to a two-dimensional electrospray device having a geometry of the calligraphy feather type, the tip of which acts as a seat for nebulization. The subject of the invention is therefore a source of electrospray comprising a structure comprising at least one flat and thin point in cantilever with respect to the rest of the structure, said point being provided with a capillary slot practiced throughout the thickness of the tip and which terminates at the end of the tip to form the ejection orifice of the electrospray source, the source comprising means for supplying the capillary slit with liquid to be nebulized and means for application of an electrospray voltage on said liquid. According to an advantageous mode, the supply means comprise at least one reservoir in fluid communication with the capillary slot. Preferably, the structure comprises a support and a plate integral with the support and a part of which constitutes said point. The supply means may comprise a reservoir constituted by a recess formed in said plate and in fluid communication with the capillary slot. The means for applying an electrospray voltage may comprise at least one electrode arranged so as to be in contact with said liquid to be nebulized. In the case where the structure comprises a support and a plate integral with the support, the means for applying an electrospray voltage may comprise the support, at least partially electrically conductive, and / or the plate at least partially electrically conductive. Advantageously, the plate has a surface hydrophobic to the liquid to be nebulized. The means for applying an electrospray voltage may comprise an electrically conductive wire arranged to be able to be in contact with said liquid to be nebulized. The supply means may include a capillary tube. They can comprise a channel produced in a microsystem supporting said structure and in fluid communication with the capillary slit. According to an advantageous mode, the means for applying the voltage (electrode, support, plate, wire) also allow the application of the voltages necessary for any device placed upstream in fluid continuity with the object of the present invention. The subject of the invention is also a method of manufacturing a structure being a source of electrospray, comprising: - The production of a support from a substrate, the production of a plate comprising a part constituting a flat and thin tip, said tip being provided with a capillary slot, for conveying a liquid to be nebulized, practiced throughout the thickness of the tip and which ends at the end of the tip, - the solidarisatiori of said plate on the support, the tip being in cantilever with respect to the support. This process can include the following stages: - the supply of a substrate to make the support, - the delimitation of the support by means of trenches engraved in the substrate, the deposition, on an area of the substrate corresponding to the future point of the structure , of sacrificial material according to a determined thickness, - the deposition of the plate on the support delimited in the substrate, the point of the plate being located on the sacrificial material, - the elimination of the sacrificial material, - the detachment of the support relative to to the substrate by cleavage at said trenches. The plate deposition step can be a deposition of a plate comprising in recess in fluid communication with the capillary slot in order to constitute a reservoir. The method may further comprise a step of depositing at least one electrode intended to ensure electrical contact with the liquid to be nebulized. The source of electrospray according to the invention can be used to obtain ionization of a liquid by electronebulization before its analysis in mass spectrometry. It can also be used to obtain a production of drops of liquid of calibrated size or the ejection of particles of fixed size. It can also be applied to the production of molecular writing using chemical compounds. It can also be applied to the definition of the electrical junction potential of a device in fluid continuity.
BRÈVE DESCRIPTION DES DESSINS L'invention sera mieux comprise et d'autres avantages et particularités apparaîtront à la lecture de la description qui va suivre, donnée à titre d'exemple non limitatif, accompagnée des dessins annexés parmi lesquels : les figures 1A et 1B sont des vues respectivement de dessus et de côté d'une source d'életronébulisation selon la présente invention, - la figure 2 est une vue en perspective de l'extrémité de la pointe d'une source d'électronébulisation selon la présente invention, - les figures 3A à 3H sont des vues de dessus illustrant un procédé de fabrication de la source d'électronébulisation représentée aux figures 1A et 1B, les figures 4A et 4B illustrent une technique de clivage utilisable pour la mise en œuvre du procédé de fabrication illustré par les figures 3A à 3H, - la figure 5 représente un montage utilisé lors d'un test au cours duquel une source d'électronébulisation selon l'invention est associée à un spectromètre de masse, - la figure 6 est un graphe représentant le courant ionique total obtenu au cours du test utilisant une source d'électronébulisation selon l'invention, dans le montage de la figure 5, la figure 7 est un spectre de masse obtenu au cours du test utilisant une source d'électronébulisation selon l'invention dans le montage de la figure 5, - la figure 8 représente un autre montage utilisé lors d'un test au cours duquel une source d'életronébulisation selon l'invention est associée à un spectromètre de masse, - la figure 9 est un graphe représentant le courant ionique total obtenu au cours du test utilisant une source d'électronébulisation selon l'invention, dans le montage de la figure 8, - la figure 10 est un spectre de masse obtenu cours du test utilisant une source d'électronébulisation selon l'invention dans le montage de la figure 8, - la figure 11 représente un spectre de masse de fragmentation du glu-fibrinopeptide obtenu avec une source d'électronébulisation selon la présente invention, - la figure 12 représente un spectre de masse obtenu pour un digestat de Cytochrome C par l'intermédiaire d'une source d'électronébulisation selon la présente invention, - la figure 13 est un graphe représentant le courant ionique total obtenu au cours d'un test utilisant une source d'électronébulisation selon l'invention, - la figure 14 représente un spectre de masse obtenu au cours d'un test utilisant une source d'électronébulisation selon la présente invention, - la figure 15 est un graphe représentant le courant ionique total enregistré sur un spectromètre de masse de type trappe ionique lors d'un test en couplage utilisant une source d'électronébulisation selon la présente invention, - la figure 16 représente le spectre de masse correspondant au graphe de la figure 15.BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood and other advantages and features will appear on reading the description which follows, given by way of nonlimiting example, accompanied by the appended drawings among which: FIGS. 1A and 1B are top and side views respectively of a source of electrospray according to the present invention, - Figure 2 is a perspective view of the tip end of a source of electrospray according to the present invention, - FIGS. 3A to 3H are top views illustrating a method of manufacturing the electrospray source shown in FIGS. 1A and 1B, FIGS. 4A and 4B illustrate a cleavage technique that can be used for the implementation of the manufacturing process illustrated by FIGS. 3A to 3H, - FIG. 5 represents an assembly used during a test during which a source of electrospray according to the invention is associated with a mass spectrometer, - Figure 6 is a graph representing the total ion current obtained during the test using a source of electrospray according to the invention, in the assembly of Figure 5, Figure 7 is a mass spectrum obtained during the test using a source of electrospray according to the invention in the assembly of FIG. 5, - FIG. 8 represents another assembly used during a test during which a source of electrospray according to the invention is associated with a mass spectrometer, - Figure 9 is a graph representing the total ion current obtained during the test using a source of electrospray according to the invention ion, in the assembly of FIG. 8, - FIG. 10 is a mass spectrum obtained during the test using a source of electrospray according to the invention in the assembly of FIG. 8, - FIG. 11 represents a mass spectrum fragmentation of the glu-fibrinopeptide obtained with a source of electrospray according to the present invention, - Figure 12 represents a mass spectrum obtained for a digest of Cytochrome C via a source of electrospray according to the present invention, - Figure 13 is a graph representing the total ion current obtained during a test using a source of electrospray according to the invention, - Figure 14 represents a mass spectrum obtained during a test using a source of electrospray according to the present invention, - Figure 15 is a graph representing the total ion current recorded on a mass spectrometer of the ion trap type during a coupling test using an electrospray source according to the present invention, - Figure 16 represents the corresponding mass spectrum to the graph in Figure 15.
EXPOSÉ DÉTAILLÉ DE MODES DE RÉALISATION PARTICULIERSDETAILED PRESENTATION OF PARTICULAR EMBODIMENTS
La présente invention s'inspire de la structure et du mode de fonctionnement d'une plume de calligraphie. Les sources planaires qui font l'objet de la présente invention sont constituées des mêmes éléments qu'une plume de calligraphie : un réservoir à liquide et une fente capillaire bidimensionnelle formée dans une pointe. La présente invention peut comporter, si cela est nécessaire, une zone de contact électrique sur laquelle est appliquée la tension nécessaire à l'établissement d'un nebulisat. Cette zone de contact peut être structurée avec des contacts multiples et indépendants et en particulier trois contacts correspondant à une électrode de travail, permettant également d'appliquer la tension d'électronébulisation, une électrode de référence et une électrode de mesure pour permettre la modification chimique par électrochimie en vue de favoriser le processus d'électronébulisation ou de l'étudier. Ces électrodes permettent également le contrôle du processus d'électronébulisation par synchronisation sur sa fréquence propre. De même que dans la plume de calligraphie, le liquide est amené par capillarité dans la fente vers l'extrémité de la pointe de la structure de type plume où il est éjecté. L'éjection a lieu non pas par action mécanique, mais sous forme de nebulisation par application d'une haute tension sur le liquide . Une source d'électronébulisation selon la présente invention est représentée aux figures 1A et 1B, la figure 1A étant une vue de dessus et la figure 1B une vue de côté . Cette source d'électronébulisation comprend un support 1 et une plaque 2 solidaire du support 1. Une partie de la plaque 2 forme une pointe 3 en porte- à-faux par rapport au support 1. La plaque 2 comporte en son centre un evidement 4 révélant la surface du support 1 et constituant un réservoir. Une fente capillaire 5, révélant également le support 1, relie le réservoir 4 à l'extrémité 6 de la pointe 3 qui forme un orifice d'éjection pour la source d'électronébulisation. Le fonctionnement du dispositif repose sur les principes énoncés suivants . Le réservoir de liquide 4 contient le liquide ou sert de transit pour l'alimentation en liquide. Le liquide est ensuite guidé par la fente capillaire 5 en amont de laquelle est situé le réservoir 4 de liquide. La pointe de la structure permet l'établissement d'un électronébulisat . II en découle le mode de fonctionnement suivant. Le liquide d'intérêt est déposé ou acheminé dans le réservoir de liquide 4 par une méthode adéquate. Il est guidé vers l'extrémité 6 de la structure par capillarité. La source est amenée sur son site d'utilisation (par exemple devant un spectromètre de masse) . Un potentiel est appliqué au liquide de façon à observer le nebulisat à l'extrémité 6 de la pointe. La physique de la source ayant une géométrie de type plume repose sur les propriétés des matériaux qui la constituent et sur les dimensions de ses différents éléments. La figure 2 représente une vue tridimensionnelle de la fente capillaire au niveau de 1 ' extrémité 6 de la pointe 3. Le rôle du réservoir 4 est de contenir le liquide à nébuliser et d'alimenter progressivement la fente capillaire 5. La topologie de la structure est bidimensionnelle. La plaque 2 est en un matériau à caractère hydrophobe, et même plus hydrophobe que celui constituant le support 1 supportant la plaque 2, matériau qui tapisse le fond du réservoir. Ceci permet de limiter les pertes de liquide hors du réservoir. Il est intéressant de noter à ce point que les liquides envisagés pour la nebulisation seront a priori à caractère plutôt hydrophile, tels que des solutions purement aqueuses ou mi-aqueuses mi-alcooliques, par exemple des mélanges méthanol/eau 50/50. La fente capillaire 5 et l'extrémité 6 de la pointe 3 sont constituées dans le matériau formant la plaque 2 et leurs dimensions sont déterminées lors du procédé de fabrication. Sur la figure 2 sont indiquées des dimensions à considérer pour le fonctionnement de la source d'électronébulisation : la largeur w de la fente, sa hauteur h et sa longueur 1. On suppose que du liquide est présent dans la fente capillaire 5. Lorsque la source d'électronébulisation est présenté en regard de la zone où la nebulisation est souhaitée, l'effet de gravité sur ce liquide est négligeable. Les facteurs qui vont intervenir pour le remplissage de la fente capillaire par le liquide sont : l'angle de contact (α) du liquide sur le matériau constituant la plaque 2, la tension de surfaceThe present invention is inspired by the structure and the mode of operation of a calligraphy pen. The planar sources which are the subject of the present invention consist of the same elements as a calligraphy pen: a liquid reservoir and a two-dimensional capillary slot formed in a point. The present invention may include, if necessary, an electrical contact zone to which the voltage necessary to apply is applied. the establishment of a nebulisate. This contact zone can be structured with multiple and independent contacts and in particular three contacts corresponding to a working electrode, also making it possible to apply the electrospray voltage, a reference electrode and a measurement electrode to allow chemical modification. by electrochemistry in order to favor the process of electrospray or to study it. These electrodes also allow the control of the electrospray process by synchronization on its natural frequency. As in the calligraphy pen, the liquid is brought by capillarity into the slit towards the end of the tip of the pen-like structure where it is ejected. The ejection takes place not by mechanical action, but in the form of nebulization by application of a high voltage to the liquid. A source of electrospray according to the present invention is shown in Figures 1A and 1B, Figure 1A being a top view and Figure 1B a side view. This source of electrospray comprises a support 1 and a plate 2 integral with the support 1. A part of the plate 2 forms a point 3 in cantilever with respect to the support 1. The plate 2 has in its center a recess 4 revealing the surface of the support 1 and constituting a reservoir. A capillary slot 5, also revealing the support 1, connects the reservoir 4 to the end 6 of the tip 3 which forms a ejection port for the source of electrospray. The operation of the device is based on the following principles. The liquid reservoir 4 contains the liquid or serves as a transit for the supply of liquid. The liquid is then guided by the capillary slot 5 upstream of which is located the reservoir 4 of liquid. The tip of the structure allows the establishment of an electrospray. This results in the following operating mode. The liquid of interest is deposited or conveyed in the liquid reservoir 4 by an appropriate method. It is guided towards the end 6 of the structure by capillarity. The source is brought to its site of use (for example in front of a mass spectrometer). A potential is applied to the liquid so as to observe the nebulisate at the end 6 of the tip. The physics of the source having a feather type geometry is based on the properties of the materials which constitute it and on the dimensions of its different elements. FIG. 2 represents a three-dimensional view of the capillary slot at the end 6 of the tip 3. The role of the reservoir 4 is to contain the liquid to be nebulized and to gradually supply the capillary slot 5. The topology of the structure is two-dimensional. The plate 2 is made of a material of hydrophobic nature, and even more hydrophobic than that constituting the support 1 supporting the plate 2, material which lines the bottom of the tank. this allows limit liquid loss from the tank. It is interesting to note at this point that the liquids envisaged for the nebulization will a priori be of rather hydrophilic character, such as purely aqueous solutions or semi-aqueous semi-alcoholic, for example mixtures methanol / water 50/50. The capillary slot 5 and the end 6 of the tip 3 are made of the material forming the plate 2 and their dimensions are determined during the manufacturing process. In FIG. 2 are indicated dimensions to be considered for the operation of the electrospray source: the width w of the slit, its height h and its length 1. It is assumed that liquid is present in the capillary slit 5. When the source of electrospray is presented next to the area where nebulization is desired, the effect of gravity on this liquid is negligible. The factors which will intervene for the filling of the capillary slit with the liquid are: the contact angle (α) of the liquid on the material constituting the plate 2, the surface tension
(γ) du liquide et les dimensions (1 et h) de la fente capillaire 5. D'après l'équation 1, régissant l'effet de capillarité d'un liquide dans un tube capillaire, le cosinus de l'angle de contact α doit être positif pour observer l'effet de capillarité, et ceci, indépendamment de l'effet de gravité. (Equation 1) où (r) est le rayon interne du capillaire, (hr) la hauteur dont monte le liquide dans le tube capillaire, (p) la densité du liquide, (α) est l'angle de contact du liquide sur les parois internes du tube capillaire et (g) est l'accélération de la pesanteur. rC Sa = rsv -χSL (Equation 2) où γsv est la tension de surface à l'interface solide-vapeur et γSL est la tension de surface à l'interface solide-liquide. Tout d'abord, dans le cas où α < 90° (cos > 0), l'équation de Young (équation 2) implique que Ysv > YSL et donc que l'interaction solide-liquide soit favorisée comparée à celle solide-vapeur. Le terme r apparaît dans l'équation 1. De sa valeur dépend l'observation ou non de l'effet de capillarité. Le terme r correspond au rayon du tube capillaire et, dans le cas du dispositif faisant l'objet de la présente invention, à la dimension de la fente capillaire 5. Si le liquide pénètre dans la fente capillaire, il se forme un pont-liquide entre les deux parois de la fente capillaire. On peut ainsi définir un rapport de forme R pour la fente capillaire 5, correspondant au rapport h/w. Il résulte de ce qui précède que R doit être supérieur à une valeur critique pour observer un effet de capillarité dans la fente capillaire 5 et pour que la formation du pont-liquide dans la fente capillaire 5 soit favorisée du point de vue énergétique. Le dispositif de nebulisation peut ou non inclure des zones conductrices (voir la figure 3H) . Ces zones conductrices si elles sont situées au niveau de réservoir de liquide 4 servent d'électrodes pour amener la tension de nebulisation. Par contre, si elles se situent au niveau de la fente capillaire 5, ces électrodes serviront à modifier les espèces présentes dans le liquide. Dans le cas d'une application de type electronebulisation avant analyse par spectrometrie de masse, des processus électrochimiques interviennent lors de l'ionisation des molécules. Les zones conductrices implantées de part et d' autre de la fente capillaire 5 au niveau de l'extrémité 6 de la pointe 3 permettraient de les étudier. Par ailleurs, ces phénomènes conduisent à une augmentation du rendement d'ionisation et, de ce fait, à une amélioration des conditions d'analyse. Dans le cas d'une application de type écriture moléculaire, la présence d'une quantité plus importante d'espèces radicalaires accroît la vitesse de gravure du substrat. Néanmoins, suivant la nature du matériau choisi pour réaliser le support 1 de la source d'électronébulisation, ces zones conductrices, en particulier si leur rôle est d'amener la tension de nebulisation, peuvent ne pas être nécessaires. En effet, si un matériau conducteur (métal, Si..) est utilisé pour réaliser le support 1 ou la plaque 2, la tension sera directement appliquée sur ce matériau conducteur. Enfin, un dispositif ne comprenant pas de zones conductrices et pour lequel les matériaux ne sont pas conducteurs peut* être utilisé en electronebulisation pourvu que le contact électrique soit réalisé via le liquide. Un fil métallique plongeant dans la solution à nébuliser, au niveau du réservoir 4 ou tout autre contact conducteur assurera ainsi le rôle d'application de la tension de nebulisation. Le dispositif peut être également connecté à une source d' alimentation en liquide en amont du réservoir 4, comme un capillaire amenant une solution provenant d'un autre appareil, d'une autre structure. Par exemple, pour une application de type spectrometrie de masse, le capillaire peut correspondre à une sortie de colonne de séparation. Pour une application de type dépôt de gouttes de taille calibrée ou écriture moléculaire, ce capillaire amène le liquide vers le dispositif de nebulisation depuis sa localisation initiale. Ledit capillaire peut être un capillaire classique commercial en silice fondue. Il peut également être un capillaire microfabriqué, c'est-à- dire un microcanal intégré sur le système supportant la source. Le capillaire peut être une piste hydrophile matérialisée sur le support 1. Dans ces deux derniers cas, la plaque 2 est intégrée sur un microsystème fluidique et joue le rôle d'interface entre ledit microsystème et le monde extérieur où la solution sortant du microsystème est utilisée. Enfin, les propriétés conductrices du dispositif ou d'un de ses éléments peuvent être utilisées pour alimenter électriquement tout système en relation fluidique avec le dispositif. De surcroît, lesdites plaques de type plume peuvent être utilisées de façon isolée ou être intégrées en grand nombre sur un même support, et ce, en vue de la parallélisation de la nebulisation. Dans ce cas, lesdites plaques de type plumes sont indépendantes ou non les unes des autres et les solutions nébulisées sont, soit les mêmes afin d'accroître la nebulisation de ladite solution, soit différentes et, dans ce cas, les plumes fonctionnent de façon séquentielle en nebulisation. L'intégration desdites plaques de type plume peut être réalisée de façon linéaire avec un alignement desdites plaques sur un côté du support ou de façon circulaire sur un support rond. Le passage d'une source à l'autre s'effectue alors respectivement par translation ou par rotation du support. Une large gamme de matériaux est aujourd'hui envisageable pour des fabrications microtechnologiques et en particulier de microsystèmes fluidiques : verre, matériaux à base de silicium (Si, Si02 nitrure de silicium...) , quartz, céramiques ainsi qu'un grande nombre de matériaux macromoléculaires, plastiques ou élastomères . La géométrie retenue pour la présente invention est compatible avec des fabrications utilisant tout type de matériaux, et ce, pour les différentes parties composant la source d'électronébulisation : le support 1, la plaque de type plume 2 et les zones conductrices . Le procédé de fabrication technologique fait de plus intervenir un ou plusieurs autre (s) matériau (x) dont le choix est adapté en fonction des matériaux retenus pour les éléments 1, 2 et 3. Un procédé générique de fabrication de sources d'électronébulisation selon l'invention est représenté aux figures 3A à 3H. Ce procédé de fabrication peut être découpé en sept étapes majeures qui sont détaillées ci-dessous, de façon à être applicable à n'importe quel type de matériau. La première étape de ce procédé de fabrication est le choix du substrat destiné à constituer le support de la source d'électronébulisation. Ce substrat 10 (voir la figure 3A) peut être en matériau macromoléculaire, en verre ou bien en silicium ou encore en métal. Dans le cas de cet exemple de réalisation, c'est un substrat de silicium de 250 μm d'épaisseur. Le début du procédé conditionne la fin de la fabrication des dispositifs d'électronébulisation. Il s'agit de la matérialisation sur le support du dispositif de lignes qui aideront au clivage du substrat afin de libérer la pointe de la source et permettre la nebulisation. Selon la deuxième étape, une couche 11 de matériau dit de protection est déposée sur une partie du substrat 10. Le matériau de la couche 11 est choisi en fonction de la nature du matériau du substrat 10 de façon qu'une attaque de la couche 11 n'affecte pas le substrat 10. Dans cet exemple de réalisation, la couche de matériau de protection est une couche d'oxyde de silicium de 20 nm d'épaisseur. La couche 11 est d'épaisseur variable suivant la nature des matériaux du substrat 10 et de la couche 11. La couche 11 est soumise à une étape de lithographie destinée à révéler les zones du substrat à attaquer pour définir . des lignes de clivage délimitant le support de la structure. Les zones correspondantes de la couche 11 sont attaquées afin de fournir des fenêtres 12 révélant le substrat 10 (voir la figure 3B) . Une fois ces zones du substrat révélées, elles sont soumises à une attaque appropriée de façon à matérialiser les lignes de clivage 13. Enfin, la couche 11 restante est éliminée. La figure 3C montre le résultat obtenu : les lignes 13, constituées de tranchées à section en V, délimitant le support de la structure à obtenir. Au cours d'une troisième étape, une couche de matériau sacrificiel est déposée sur le substrat 10. Cette couche de matériau sacrificiel 14 permettra en fin de fabrication à la pointe de la structure de surplomber son support avant 1 ' opération de clivage . Le substrat 10 est recouvert d'une fine couche de matériau sacrificiel d'épaisseur suffisante pour que, après sa suppression, la pointe soit suffisamment séparée du substrat 10, mais néanmoins suffisamment fine pour pouvoir s'affranchir de tout problème de contrainte et de courbure de la pointe en surplomb du support. Dans cet exemple de réalisation, la couche de matériau sacrificiel est une couche de nickel de 150 nm d'épaisseur. La couche de matériau sacrificiel est alors soumise à une étape de lithographie et d'attaque appropriée afin de ne garder de ce matériau qu'une zone 14 correspondant à la pointe de la structure (voir la figure 3D) . La quatrième étape peut être mise en œuvre. Le substrat 10 est alors recouvert d'une couche d'un matériau destinée à constituer la plaque de la structure. En fonction du matériau du substrat, le matériau de cette couche peut être du silicium ou à base de silicium, un métal ou même un matériau de type polymère ou céramique. Dans cet exemple de réalisation, la couche de matériau destinée à constituer la plaque est une couche de 35 μm d'épaisseur en polymère SU-8 2035 acheté sous forme pré-poly érisée chez Microchem et polymérisé par un procédé photolithographique. L'épaisseur de cette couche est choisie de façon appropriée. De cette épaisseur dépendent en effet les performances en ionisation du dispositif de nebulisation, comme il a été expliqué précédemment. L'épaisseur de cette couche influence directement la hauteur h de la fente capillaire et, d'après ce qui précède, plus h est grand, plus w doit être grand afin de ne pas modifier le rapport R. Or, en fonction de l'application finale de la source de nebulisation, l'enjeu est de diminuer au maximum w afin d'accroître les performances. En revanche, si l'épaisseur de la couche destinée à constituer la plaque est trop fine, la pointe en surplomb peut se courber une fois décollée du support du fait des contraintes exercées sur le matériau. L'homme de l'art est en mesure d'adapter le présent cahier des charges en fonction de la nature du matériau de cette couche et ainsi de définir l'épaisseur optimale de matériau à déposer. Cette couche subit alors une étape de lithographie et une attaque afin de former la plaque de type plume 2, c'est-à-dire en plus de son encombrement, le réservoir 4, la fente capillaire 5 et la pointe 3 (voir la figure 3E) . Cette attaque est adaptée en fonction du matériau de la plaque. Il peut s'agir d'une technique de gravure chimique, d'une attaque physique dans le cas d'un matériau à base de silicium ou d'un métal, d'une attaque physique ou d'une photolithographie suivie d'une révélation dans le cas d'un polymère photolithographiable. La cinquième étape peut alors être entreprise. Une fois la plaque 2 formée, la zone 14 de matériau sacrificiel sous la pointe 3 peut être ôtée. Le matériau sacrificiel est ôté par une attaque chimique appropriée. La solution pour cette attaque chimique doit être choisie judicieusement de façon à ce que tout le matériau sacrificiel soit supprimé sans que ni le support ni plaque ne soient affectés . Les matériaux de ces éléments ne doivent donc pas être sensibles à cette solution chimique. On obtient la structure montrée à la figure 3F. La sixième étape concerne l'implantation de zones conductrices sur la structure. Comme mentionné précédemment, cette étape n'est incluse dans le procédé de fabrication que s'il est prévu de telles zones conductrices. Que ces zones se situent au niveau du réservoir 4 (application de la tension de nebulisation) ou au niveau de la pointe (électrodes d'études physicochimiques), le procédé le fabrication est le même. La réalisation des zones conductrices 3 au niveau du réservoir seule sera détaillée ici. Ces zones conductrices peuvent être en métal ou en carbone. La structure est d'abord soumise à une étape de masquage afin que seules les zones correspondant à la formation des zones conductrices soient dégagées. Le matériau conducteur choisi est alors déposé par une technique de PECVD (déposition en phase vapeur par techniques de plasma chimique) sur la structure. Dans cet exemple de réalisation, les zones conductrices sont en palladium et ont une épaisseur de 400 nm. La figure 3G montre la structure obtenue. Deux zones conductrices 7 et 8 encadrent le réservoir 4 et permettent d'y appliquer un potentiel électrique. La septième étape de ce procédé de fabrication de la source de nebulisation est le détachement du support 1 par rapport au substrat 10 et notamment, la mise en surplomb de la pointe 3 par rapport au support 1 en utilisant les lignes de clivage 13 matérialisées à la deuxième étape de ce procédé de fabrication. La structure obtenue est représentée à la figure 3H. Une technique de clivage avantageuse est illustrée par les figures 4A et 4B dans le cas de la mise en surplomb de la pointe. Un fil métallique fixe 20 est placé sous le support 1 au niveau des tranchées de clivage 13 réalisées de part et d'autre de la pointe. Conjointement, deux forces sont exercées sur le substrat aux endroits indiqués sur la figure 4A par des flèches. La séparation préalablement effectuée de la pointe 3 par rapport au support 1 assure ainsi de ne pas endommager la pointe lors de l'étape de clivage. La figure 4B montre le clivage en cours de réalisation. Ce procédé de fabrication générique est ensuite adapté en fonction des matériaux choisis pour chaque élément de la source d'électronébulisation. Le premier champ d' applications ciblé par la présente invention est l'électronébulisation de solutions biologiques ou chimiques à analyser par spectrometrie de masse. La spectrometrie de masse est à l'heure actuelle la technique de choix pour l'analyse, la caractérisation et l'identification des protéines. Or, depuis la fin du décryptage du génome, les biologistes notamment s'intéressent de plus en plus à la protéomique, science qui vise à étudier et à caractériser l'ensemble des protéines d'un individu. Ces protéines, chez tout être humain, sont présentes à raison de plus de 106 molécules différentes en incluant les modifications post-traductionnelles . Ce point justifie le besoin à l'heure actuelle, de techniques et d'outils d'analyse compatibles avec une automatisation en vue d'une analyse à haut débit, et ce, notamment pour la spectrometrie de masse du fait de sa pertinence dans le cadre de l'étude des protéines. Les échantillons (ou solutions à analyser) dont dispose le biologiste sont souvent de taille restreinte (inférieure ou égale au 1 μL) et contiennent peu de matériel biologique, ce qui impose de travailler avec une technique d'analyse très sensible et consommant peu d'échantillon. Ceci fait de la spectrometrie de masse avec une ionisation par nanoelectronebulisation une des techniques d'analyse les plus utilisées pour la caractérisation des protéines. Dans ce contexte, l'enjeu majeur est la diminution au maximum des dimensions de l'extrémité de la pointe de la source. En effet, comme mentionné dans l'introduction, il existe deux régimes d'électronébulisation pour ce type d'application, le plus intéressant en termes d'automatisation et de gain en sensibilité étant le régime de nanoelectronebulisation. Cependant, à l'heure actuelle, la vitesse d'analyse est limitée, le débit d'échantillons restreint du fait que la nanoESI-MS (pour "nano ElectroSpray Ionization - Mass(γ) of the liquid and the dimensions (1 and h) of the capillary slot 5. According to equation 1, governing the capillarity effect of a liquid in a capillary tube, the cosine of the contact angle α must be positive to observe the capillary effect, and this, independently of the effect of gravity. ( Equation 1 ) where (r) is the internal radius of the capillary, (h r ) the height by which the liquid rises in the capillary tube, (p) the density of the liquid, (α) is the contact angle of the liquid on the internal walls of the capillary tube and (g) is the acceleration of gravity. r C S a = r sv -χ SL (Eq u ati on 2) where γ sv is the surface tension at the solid-vapor interface and γ SL is the surface tension at the solid-liquid interface. First of all, in the case where α <90 ° (cos> 0), Young's equation (equation 2) implies that Ysv> YS L and therefore that the solid-liquid interaction is favored compared to that solid- steam. The term r appears in equation 1. Whether or not the capillary effect is observed depends on its value. The term r corresponds to the radius of the capillary tube and, in the case of the device which is the subject of the present invention, to the dimension of the capillary slot 5. If the liquid enters the capillary slot, a liquid bridge is formed between the two walls of the capillary slot. It is thus possible to define an aspect ratio R for the capillary slot 5, corresponding to the ratio h / w. It follows from the above that R must be greater than a critical value to observe a capillary effect in the capillary slot 5 and for the formation of the liquid bridge in the capillary slot 5 to be favored from the energy point of view. The nebulization device may or may not include conductive zones (see Figure 3H). These conductive zones if they are located at the level of the liquid reservoir 4 serve as electrodes for bringing the nebulization voltage. By cons, if they are located at the capillary slot 5, these electrodes will be used to modify the species present in the liquid. In the case of an electronebulization type application before analysis by mass spectrometry, electrochemical processes take place during the ionization of the molecules. The conductive areas located on either side of the capillary slot 5 at the end 6 of the tip 3 would allow them to be studied. Furthermore, these phenomena lead to an increase in the ionization yield and, as a result, to an improvement in the analysis conditions. In the case of an application of the molecular writing type, the presence of a greater quantity of radical species increases the speed of etching of the substrate. However, depending on the nature of the material chosen to make the support 1 for the electrospray source, these conductive zones, in particular if their role is to bring the nebulization voltage, may not be necessary. Indeed, if a conductive material (metal, Si ..) is used to make the support 1 or the plate 2, the voltage will be directly applied to this conductive material. Finally, a device which does not include conductive zones and for which the materials are not conductive can * be used in electronebulization provided that the electrical contact is made via the liquid. A metal wire immersed in the solution to be nebulized, at the level of the reservoir 4 or any other conductive contact will thus ensure the role of application of the nebulization voltage. The device can also be connected to a source of liquid supply upstream of the reservoir 4, like a capillary supplying a solution coming from another device, from another structure. For example, for an application of mass spectrometry type, the capillary can correspond to an outlet of the separation column. For an application such as a droplet of calibrated size or molecular writing, this capillary brings the liquid to the nebulization device from its initial location. Said capillary can be a conventional commercial capillary made of fused silica. It can also be a microfabricated capillary, that is to say a microchannel integrated on the system supporting the source. The capillary can be a hydrophilic track materialized on the support 1. In the latter two cases, the plate 2 is integrated on a fluid microsystem and plays the role of interface between said microsystem and the outside world where the solution leaving the microsystem is used . Finally, the conductive properties of the device or of one of its elements can be used to electrically supply any system in fluid relationship with the device. In addition, said feather type plates can be used in isolation or be integrated in large numbers on the same support, and this, for the parallelization of the nebulization. In this case, said feather-type plates are independent or not from each other and the nebulized solutions are either the same in order to increase the nebulization of said solution, or different and, in this case, the feathers operate sequentially in nebulization. The integration of said feather-type plates can be carried out linearly with an alignment of said plates on one side of the support or in a circular fashion on a round support. The passage from one source to the other then takes place respectively by translation or by rotation of the support. A wide range of materials can now be envisaged for microtechnological manufacturing and in particular of fluidic microsystems: glass, silicon-based materials (Si, Si0 2 silicon nitride ...), quartz, ceramics as well as a large number macromolecular, plastic or elastomeric materials. The geometry chosen for the present invention is compatible with manufacturing using any type of material, and this, for the different parts making up the electrospray source: the support 1, the feather-type plate 2 and the conductive areas. The technological manufacturing process also involves one or more other material (s), the choice of which is suitable. depending on the materials selected for elements 1, 2 and 3. A generic process for manufacturing electrospray sources according to the invention is shown in FIGS. 3A to 3H. This manufacturing process can be divided into seven major steps which are detailed below, so as to be applicable to any type of material. The first step in this manufacturing process is the choice of the substrate intended to constitute the support for the electrospray source. This substrate 10 (see FIG. 3A) can be made of macromolecular material, glass or else silicon or even metal. In the case of this exemplary embodiment, it is a silicon substrate 250 μm thick. The start of the process conditions the end of the manufacturing of the electrospray devices. This is the materialization on the support of the line device which will help the cleavage of the substrate in order to free the tip of the source and allow nebulization. According to the second step, a layer 11 of so-called protective material is deposited on part of the substrate 10. The material of the layer 11 is chosen according to the nature of the material of the substrate 10 so that an attack on the layer 11 does not affect the substrate 10. In this exemplary embodiment, the layer of protective material is a layer of silicon oxide 20 nm thick. The layer 11 is of variable thickness according to the nature of the materials of the substrate 10 and layer 11. Layer 11 is subjected to a lithography step intended to reveal the areas of the substrate to be attacked in order to define. cleavage lines delimiting the support of the structure. The corresponding zones of the layer 11 are attacked in order to provide windows 12 revealing the substrate 10 (see FIG. 3B). Once these areas of the substrate are revealed, they are subjected to an appropriate attack so as to materialize the cleavage lines 13. Finally, the remaining layer 11 is eliminated. FIG. 3C shows the result obtained: the lines 13, consisting of V-shaped trenches, delimiting the support of the structure to be obtained. During a third step, a layer of sacrificial material is deposited on the substrate 10. This layer of sacrificial material 14 will allow, at the end of manufacture at the tip of the structure, to overhang its support before the cleavage operation. The substrate 10 is covered with a thin layer of sacrificial material of sufficient thickness so that, after its removal, the tip is sufficiently separated from the substrate 10, but nevertheless sufficiently fine to be able to overcome any problem of stress and curvature of the point overhanging the support. In this exemplary embodiment, the layer of sacrificial material is a layer of nickel 150 nm thick. The layer of sacrificial material is then subjected to an appropriate lithography and etching step in order to keep only a zone of this material. 14 corresponding to the tip of the structure (see Figure 3D). The fourth step can be implemented. The substrate 10 is then covered with a layer of a material intended to constitute the plate of the structure. Depending on the material of the substrate, the material of this layer may be silicon or based on silicon, a metal or even a material of the polymer or ceramic type. In this embodiment, the layer of material intended to constitute the plate is a layer 35 μm thick in polymer SU-8 2035 purchased in pre-poly erect form from Microchem and polymerized by a photolithographic process. The thickness of this layer is chosen appropriately. On this thickness indeed depend the performance in ionization of the nebulization device, as it was explained previously. The thickness of this layer directly influences the height h of the capillary slit and, according to the above, the greater h, the greater w must be so as not to modify the ratio R. Now, as a function of the final application of the nebulization source, the challenge is to reduce w as much as possible in order to increase performance. On the other hand, if the thickness of the layer intended to constitute the plate is too thin, the overhanging point can bend once detached from the support due to the stresses exerted on the material. Those skilled in the art are able to adapt the present specifications according to the nature of the material of this layer and thus to define the optimal thickness of material to be deposited. This layer then undergoes a lithography step and an attack in order to form the feather-type plate 2, that is to say in addition to its bulk, the reservoir 4, the capillary slot 5 and the tip 3 (see the figure 3E). This attack is adapted according to the material of the plate. It can be a chemical etching technique, a physical attack in the case of a material based on silicon or a metal, a physical attack or a photolithography followed by a revelation in the case of a photolithographic polymer. The fifth step can then be undertaken. Once the plate 2 has been formed, the zone 14 of sacrificial material under the tip 3 can be removed. The sacrificial material is removed by an appropriate chemical attack. The solution for this chemical attack must be chosen judiciously so that all the sacrificial material is removed without neither the support nor the plate being affected. The materials of these elements must therefore not be sensitive to this chemical solution. The structure shown in Figure 3F is obtained. The sixth step concerns the implantation of conductive zones on the structure. As mentioned previously, this step is only included in the manufacturing process if such conductive zones are provided. Whether these zones are located at the level of the reservoir 4 (application of the nebulization voltage) or at the level of the tip (physicochemical study electrodes), the manufacturing process is the same. The realization of the conductive zones 3 at the level of the tank alone will be detailed here. These conductive zones can be made of metal or carbon. The structure is first subjected to a masking step so that only the zones corresponding to the formation of the conductive zones are exposed. The conductive material chosen is then deposited by a PECVD technique (vapor deposition by chemical plasma techniques) on the structure. In this exemplary embodiment, the conductive zones are made of palladium and have a thickness of 400 nm. Figure 3G shows the structure obtained. Two conductive zones 7 and 8 surround the reservoir 4 and allow an electrical potential to be applied to it. The seventh step of this method of manufacturing the nebulization source is the detachment of the support 1 relative to the substrate 10 and in particular, the overhanging of the tip 3 relative to the support 1 using the cleavage lines 13 materialized at the second step in this manufacturing process. The structure obtained is shown in Figure 3H. An advantageous cleavage technique is illustrated by FIGS. 4A and 4B in the case of the overhang of the point. A fixed metal wire 20 is placed under the support 1 at the level of the cleavage trenches 13 formed on either side of the point. Jointly, two forces are exerted on the substrate at the locations indicated in Figure 4A by arrows. The separation performed beforehand of the tip 3 with respect to the support 1 thus ensures that not damage the tip during the cleavage step. Figure 4B shows the cleavage in progress. This generic manufacturing process is then adapted according to the materials chosen for each element of the electrospray source. The first field of applications targeted by the present invention is the electrospraying of biological or chemical solutions to be analyzed by mass spectrometry. Mass spectrometry is currently the technique of choice for the analysis, characterization and identification of proteins. However, since the end of the genome decryption, biologists in particular are increasingly interested in proteomics, a science which aims to study and characterize all the proteins of an individual. These proteins, in every human being, are present in more than 10 6 different molecules including post-translational modifications. This point justifies the need at present for analysis techniques and tools compatible with automation for high-throughput analysis, and this, in particular for mass spectrometry because of its relevance in protein study. The samples (or solutions to be analyzed) available to the biologist are often of limited size (less than or equal to 1 μL) and contain little biological material, which requires working with a very sensitive analysis technique and consuming little sample. This makes mass spectrometry with nanoelectronebulization ionization one of the most used analysis techniques for protein characterization. In this context, the major stake is the reduction to the maximum of the dimensions of the end of the point of the source. Indeed, as mentioned in the introduction, there are two electrospray regimes for this type of application, the most interesting in terms of automation and gain in sensitivity being the nanoelectronebulization regime. However, at present, the analysis speed is limited, the sample throughput restricted by the fact that the nanoESI-MS (for "nano ElectroSpray Ionization - Mass
Spectrometry") repose entièrement sur des processus manuels. Les outils actuels ne se prêtent pas à une analyse robotisée et automatisée. Ce contexte explique les motivations pour le développement de la présente invention pour ce type d' applications . Le deuxième type d' applications ciblé par la présente invention est le dépôt de gouttes calibrées sur une surface lisse ou rugueuse. Ceci est de prime intérêt pour la préparation de puces à ADN, à peptides, à PNA ou tout autre type de molécules . Ce type d'applications requiert un dispositif capable de délivrer du fluide sous forme discrète, des gouttes de liquide de taille calibrée, la taille dépendant le plus souvent de la résolution espérée dans la préparation des plaques d'analyse. Plus les gouttes sont petites, plus leur dépôt peut être rapproché sur la plaque et plus la densité en dépôts et donc en substances à analyser est grande. Le dispositif faisant l'objet de la présente invention peut être utilisé à cette fin. La largeur de la fente capillaire 5, ainsi que la valeur de la tension appliquée pour l'éjection des gouttes conditionne la taille des gouttes éjectées par ledit dispositif de nebulisation. Ainsi la résolution des plaques d'analyse peut-elle être ajustée en fonction de la largeur de la fente du dispositif. Enfin, la tension de nebulisation peut être alternative et ainsi donner une vitesse de dépôt en gouttes/minute dépendant directement de la fréquence de la tension alternative. Le dépôt de gouttes calibrées comme présenté ci-dessus peut être utilisé pour la préparation de plaques d'analyse comme les puces à ADN. Il peut aussi être appliqué à la préparation de cibles MALDI (pour "Matrix-Assisted Laser Desorption/lonization") sur lesquelles les échantillons à analyser par spectrometrie de masse avec une ionisation MALDI ici, sont déposés de façon discrète avant leur cristallisation et leur introduction dans le spectromètre de masse. Ainsi, le présent dispositif de nebulisation ayant une géométrie de type plume peut-il être par exemple connecté en sortie de colonne de séparation et permettre un couplage entre une technique séparative et une analyse en ligne par spectrometrie de masse de type MALDI. Les gouttes de liquide enfin peuvent être remplacées par des cellules. Dans ce cas, les cellules sont de même éjectées de façon discrète et déposées par exemple sur une plaque en vue de l'élaboration de puces à cellules. La troisième application ciblée par la présente invention est l'écriture moléculaire à des échelles de l'ordre de la centaine de nanomètres . A l'heure actuelle, ce type d'opérations est réalisé à l'aide de pointes de microscopie AFM, fonctionnant à l'aide d'un appareillage lourd et encombrant. L'éjection du liquide repose sur une mise en contact ou quasi-contact de la pointe et du substrat de dépôt dans le cas de l'AFM ou sur l'application d'une pression sur le liquide. Une adaptation de cette technique est d'éjecter le liquide sous l'action d'une tension et non à l'aide d'une pression ou d'une mise en contact. En effet, dans les deux cas, l'éjection est provoquée lorsque les forces de tension du liquide au niveau de la pointe de la pipette sont « dépassées » par une autre force appliquée à la colonne de liquide. Ceci est envisageable avec un dispositif d'électronébulisation où la force électrique vient surpasser celle de tension du liquide et ainsi engendrer la formation de gouttelettes. D'autre part, la formation d'espèces réactives est intrinsèque au processus d'électronébulisation. Cette technique d'éjection du fluide supprime tout appareillage complexe de production d'espèces réactives comme des radicaux libres, tel qu'une décharge plasma ou micro-onde, en amont de la structure qui délivre le liquide. La présente invention peut donc être utilisée à de telles fins d'écriture moléculaire sur un substrat lisse ou rugueux, la libération de la solution d'écriture (pseudo-encre) étant ici régie par application d'une tension. De même que pour le premier champ d'applications, un enjeu majeur est de minimiser la taille de l'extrémité de la pointe, cette dimension conditionnant la taille des éjections par nebulisation et par conséquent la résolution espérée en écriture sur le substrat final. La largeur de la pointe est inférieure ou égale au micromètre. Un autre facteur influençant la taille des éjections et le débit de fluide est la tension de nebulisation appliquée au liquide. Enfin, la production d'espèces réactives, si le dispositif est utilisé pour dispenser une solution d'attaque du substrat, peut être accrue avec l'implantation d'électrodes au sein de la structure de type plume qui délivre le fluide. Ces électrodes sont alors le siège de réactions électrochimiques conduisant à la formation d' espèces réactives .Spectrometry ") relies entirely on manual processes. Current tools do not lend themselves to robotic and automated analysis. This context explains the motivations for the development of the present invention for this type of applications. The second type of targeted applications by the present invention is the deposit of calibrated drops on a smooth or rough surface. This is of prime interest for the preparation of DNA chips, peptides, PNA or any other type of molecules. This type of application requires a device capable of delivering fluid in discrete form, drops of liquid of calibrated size, the size depending most often on the resolution expected in the preparation of the analysis plates. The smaller the drops, the closer their deposition can be on the plate and the greater the density of deposits and therefore of substances to be analyzed. The device forming the subject of the present invention can be re used for this purpose. The width of the capillary slot 5, as well as the value of the voltage applied for the ejection of the drops conditions the size of the drops ejected by said nebulization device. Thus the resolution of the analysis plates can be adjusted according to the width of the slot of the device. Finally, the nebulization voltage can be alternating and thus give a deposition rate in drops / minute directly dependent on the frequency of the alternating voltage. The deposit of calibrated drops as presented above can be used for the preparation of analysis plates such as DNA chips. It can also be applied to the preparation of MALDI targets (for "Matrix-Assisted Laser Desorption / lonization") on which the samples to be analyzed by mass spectrometry with a MALDI ionization here, are deposited discretely before their crystallization and their introduction in the mass spectrometer. Thus, the present nebulization device having a feather type geometry can it for example be connected at the outlet of the separation column and allow coupling between a separation technique and an online analysis by mass spectrometry of the MALDI type. Finally, the drops of liquid can be replaced by cells. In this case, the cells are likewise discreetly ejected and deposited, for example, on a plate for the preparation of cell chips. The third application targeted by the present invention is molecular writing at scales of the order of a hundred nanometers. Currently, this type of operation is carried out at using AFM microscopy tips, operating using heavy and bulky equipment. The ejection of the liquid is based on bringing the tip and the deposition substrate into contact or quasi-contact in the case of AFM or on the application of pressure on the liquid. An adaptation of this technique is to eject the liquid under the action of a tension and not using pressure or contacting. Indeed, in both cases, the ejection is caused when the tensioning forces of the liquid at the tip of the pipette are "exceeded" by another force applied to the column of liquid. This can be envisaged with an electrospray device where the electric force comes to exceed that of tension of the liquid and thus generate the formation of droplets. On the other hand, the formation of reactive species is intrinsic to the process of electrospraying. This fluid ejection technique eliminates any complex apparatus for producing reactive species such as free radicals, such as a plasma or microwave discharge, upstream of the structure which delivers the liquid. The present invention can therefore be used for such molecular writing purposes on a smooth or rough substrate, the release of the writing solution (pseudo-ink) here being controlled by applying a voltage. As for the first field of applications, a major stake is to minimize the size of the tip of the tip, this dimension conditioning the size of the ejections by nebulization and consequently the resolution expected in writing on the final substrate. The width of the tip is less than or equal to a micrometer. Another factor influencing the size of the ejections and the fluid flow rate is the nebulization voltage applied to the liquid. Finally, the production of reactive species, if the device is used to dispense a solution for attacking the substrate, can be increased with the implantation of electrodes within the feather-like structure which delivers the fluid. These electrodes are then the site of electrochemical reactions leading to the formation of reactive species.
On va maintenant s'intéresser aux exemple suivants . Exemple 1 : Design de sources de nanoelectronebulisation microfabriquées selon la présente invention. Un premier exemple concerne les dimensions et les formes choisies pour réaliser un dispositif de nebulisation comme décrit dans la présente invention. Ce premier dispositif présente de petites dimensions en sa pointe du fait du domaine d'applications visé, c'est-à-dire une nanoelectronebulisation pour l'ionisation de solutions avant leur analyse par spectrometrie de masse. Le dispositif est réalisé conformément aux figures 1A et 1B. Le réservoir 4 du dispositif a pour dimensions 2,5 mm x 2,5 mm x e (μm) où e est l'épaisseur de la couche de matériau utilisée pour réaliser la plaque 2. La valeur de e est proche de celle de h, considérée ci- après, l'épaisseur de matériau sacrificiel étant de l'ordre de la centaine de nanomètres. La largeur de la fente capillaire 5 est de 8 μm à l'extrémité 6 de la pointe 3. De la valeur de cette largeur de fente découle l'épaisseur de la plaque 2 de façon à observer l'effet de capillarité et la pénétration effective du liquide dans la fente capillaire 5. Ceci est régi par la valeur du paramètre R défini comme le rapport entre la hauteur h et la largeur w de la fente, R = h/w. Il apparaît que ce rapport doit être supérieur à 1 pour que l'effet de capillarité soit observé. Ainsi, l'épaisseur de la plaque doit-elle être supérieure à une dizaine de micromètres. Par ailleurs, pour s'affranchir des problèmes de contraintes mécaniques qui se traduisent par un recourbement de la structure en extrémité 6, cette épaisseur a été fixée à 35 μm.We will now focus on the following examples. Example 1: Design of microfabricated nanoelectronebulization sources according to the present invention. A first example concerns the dimensions and the shapes chosen to produce a nebulization device as described in the present invention. This first device has small dimensions at its tip due to the field of applications targeted, that is to say a nanoelectronebulization for the ionization of solutions before their analysis by mass spectrometry. The device is produced in accordance with FIGS. 1A and 1B. The reservoir 4 of the device has the dimensions 2.5 mm x 2.5 mm xe (μm) where e is the thickness of the layer of material used to make the plate 2. The value of e is close to that of h, considered below, the thickness of sacrificial material being the order of a hundred nanometers. The width of the capillary slit 5 is 8 μm at the end 6 of the tip 3. From the value of this slit width follows the thickness of the plate 2 so as to observe the capillarity effect and the effective penetration of liquid in the capillary slot 5. This is governed by the value of the parameter R defined as the ratio between the height h and the width w of the slot, R = h / w. It appears that this ratio must be greater than 1 for the capillary effect to be observed. Thus, the thickness of the plate must be greater than ten micrometers. Furthermore, to overcome the problems of mechanical stresses which result in a bending of the structure at the end 6, this thickness has been set at 35 μm.
Exemple 2 : Fabrication des sources de design décrit dans l'exemple 1 à l'aide des matériaux silicium et SU-8. Le deuxième exemple concerne la fabrication par microtechnologie des sources de nebulisation, comme décrit dans l'exemple 1. Les matériaux utilisés sont le silicium pour le support 1 et la résine photolithographiable négative SU-8 pour la plaque de type plume 2. Le procédé de fabrication découle du procédé décrit ci-dessus. Il est adapté aux matériaux choisis . Un substrat de silicium orienté (100) et dopé n, de 3 pouces, est recouvert d'une couche de 200 nm d'oxyde de silicium (Si02) , puis masqué par lithographie. La couche de Si02 est attaquée par une solution acide de HF:H20 sur les zones non masquées. Le silicium exposé est ensuite attaqué par une solution de soude (KOH) de façon à matérialiser les lignes de clivage. Une couche de 150 nm de nickel est ensuite déposée sur la surface de silicium par technique de pulvérisation sous argon (Plassys MP 450S) . La couche de nickel est attaquée de façon locale par photolithographie UV (résine positive photosensible AZ1518 [l,2μm], solution de gravure HN03/H20 (1:3)) de façon à ce qu'il ne reste du nickel que sous la pointe de la plume. Après suppression de toute trace de résine photolithographiable, la plaque de silicium est déshydratée à 170 °C pendant 30 min, de façon à optimiser l'adhésion de la résine SU-8 sur la surface de silicium. Une couche de 35 μm de résine SU-8 est étalée sur le substrat de silicium à l'aide d'une tournette pour en homogénéiser l'épaisseur avant l'étape suivante de photolithographie. La plaque de type plume 2 est réalisée dans cette couche de résine SU-8 à l'aide de techniques classiques de photolithographie UV. Après développement de la résine SU-8 avec le réactif approprié (acétate de l-méthoxy-2- propanol, PGMEA) , la couche de nickel est attaquée avec la solution acide (HN03/H20) décrite ci-dessus. Cette étape d'attaque chimique du nickel n'affecte pas la résine SU-8 même si ce procédé peut prendre plusieurs heures. Enfin, après séchage du dispositif, le substrat 1 de silicium est scié selon la technique illustrée aux figures 4A et 4B. La technique utilisée ici préserve la structure de la plume, comme cette dernière a été auparavant décollée de son support. Une photographie de microscopie électronique à balayage (Hitachi S4700) de la source de nebulisation de type plume fabriquée selon ce procédé confirme le décollement correct de la pointe par rapport à son support. Le procédé de fabrication décrit ci-dessus n'inclut pas la réalisation d'électrodes.Example 2: Manufacture of the design sources described in Example 1 using silicon and SU-8 materials. The second example relates to the fabrication by microtechnology of the nebulization sources, as described in example 1. The materials used are silicon for the support 1 and the negative photolithographic resin SU-8 for the feather-type plate 2. The method of manufacturing follows from the process described above. It is suitable for the materials chosen. A 3 inch oriented n-doped silicon substrate (100) is covered with a 200 nm layer of silicon oxide (Si0 2 ), then masked by lithography. The Si0 2 layer is attacked by a acid solution of HF: H 2 0 on the unmasked areas. The exposed silicon is then attacked by a sodium hydroxide solution (KOH) so as to materialize the cleavage lines. A 150 nm layer of nickel is then deposited on the silicon surface by spraying technique under argon (Plassys MP 450S). The nickel layer is attacked locally by UV photolithography (positive photosensitive resin AZ1518 [l, 2μm], etching solution HN0 3 / H 2 0 (1: 3)) so that only nickel remains under the tip of the pen. After removing all traces of photolithographic resin, the silicon wafer is dehydrated at 170 ° C. for 30 min, so as to optimize the adhesion of the SU-8 resin to the silicon surface. A 35 μm layer of SU-8 resin is spread on the silicon substrate using a spinner to homogenize the thickness thereof before the next photolithography step. The feather-type plate 2 is produced in this layer of resin SU-8 using conventional UV photolithography techniques. After development of the SU-8 resin with the appropriate reagent (1-methoxy-2-propanol acetate, PGMEA), the nickel layer is attacked with the acid solution (HN0 3 / H 2 0) described above. This nickel etching step does not affect the SU-8 resin even if this process can take several hours. Finally, after drying of the device, the silicon substrate 1 is sawn according to the technique illustrated in FIGS. 4A and 4B. The technique used here preserves the structure of the pen, as it was previously detached from its support. A photograph of scanning electron microscopy (Hitachi S4700) of the feather-type nebulization source manufactured according to this process confirms the correct detachment of the point relative to its support. The manufacturing process described above does not include the production of electrodes.
Exemple 3 : Design de dispositif d'éjection de particules d'une centaine de micromètres. Un troisième exemple concerne les dimensions et les formes choisies pour réaliser un dispositif d'éjection de particules ayant une taille d'une centaine de micromètres, comme décrit dans la présente invention. Ce dispositif présente des dimensions plus larges que celui décrit dans l'exemple 1. Ici, les dimensions de la fente de capillaire 5 et du réservoir 4 doivent être compatibles avec la manipulation d'objets d'une centaine de micromètres. Du fait de cette gamme de dimensions, le dispositif décrit dans l'exemple 3 s'applique également à la manipulation de cellules de taille avoisinant 100 μm de diamètre, pour la préparation de puces à cellules par exemple. Le réservoir 4 dudit dispositif a pour dimensions 1 cm x 1 cm x e (μm) où e est l'épaisseur de la plaque 2. De même que dans l'exemple 1, la valeur de e est définie en fonction de la largeur de la fente capillaire 5 de façon à avoir un facteur de forme R en l'extrémité 6 de la plaque qui soit supérieur à 1. Les particules manipulées par ce dispositif ont une taille de la centaine de micromètres, donc la fente capillaire 5 doit avoir une largeur supérieure à 100 μm. Cependant, les particules pouvant avoir tendance à s'agréger, cette largeur ne doit pas être choisie trop grande. Elle est de préférence voisine du double de la taille des particules manipulées. De ce fait, la largeur de la fente est fixée à 150 μm, et l'épaisseur de la plaque à 200 μm. Le matériau retenu pour la fabrication de la plaque de type plume 2 est ici encore la résine photolithographiable négative SU-8 et le matériau choisi pour le support 1 est le verre. La résine SU-8 est intéressante ici pour la manipulation de particules comme les cellules, car ces cellules n'adhèrent pas sur ce matériau. De ce fait, le support 1 en verre est lui aussi couvert d'une fine couche de résine SU-8 afin de prévenir toute adhésion non désirée des cellules sur le dispositif.Example 3: Design of a particle ejection device of a hundred micrometers. A third example relates to the dimensions and the shapes chosen to make a particle ejection device having a size of a hundred micrometers, as described in the present invention. This device has larger dimensions than that described in Example 1. Here, the dimensions of the capillary slot 5 and of the reservoir 4 must be compatible with the handling of objects of a hundred micrometers. Because of this range of dimensions, the device described in Example 3 also applies to the handling of cells of size approaching 100 μm in diameter, for the preparation of cell chips for example. The reservoir 4 of said device has the dimensions 1 cm x 1 cm xe (μm) where e is the thickness of the plate 2. As in example 1, the value of e is defined as a function of the width of the capillary slot 5 so as to have a form factor R at the end 6 of the plate which is greater than 1. The particles handled by this device have a size of a hundred micrometers, therefore the capillary slot 5 must have a width greater than 100 μm. However, since the particles may tend to aggregate, this width should not be chosen too large. It is preferably close to twice the size of the particles handled. Therefore, the width of the slot is fixed at 150 μm, and the thickness of the plate at 200 μm. The material retained for the manufacture of the feather-type plate 2 is here again the negative photolithographic resin SU-8 and the material chosen for the support 1 is glass. The SU-8 resin is interesting here for handling particles such as cells, because these cells do not adhere to this material. Therefore, the glass support 1 is also covered with a thin layer of SU-8 resin in order to prevent any unwanted adhesion of the cells to the device.
Exemple 4 : Test des sources de nebulisation fabriquées selon l'exemple 2 en spectrometrie de masse. I : Application de la tension à l'aide d'un fil de platine. L'exemple 4 est le test des sources de nebulisation fabriquées comme décrit dans l'exemple 2 pour une analyse en spectrometrie de masse. Dans ce premier exemple, la tension de nebulisation est appliquée à du liquide à nébuliser à l'aide d'un fil de platine plongé dans le liquide au niveau du réservoir comme illustré sur la figure 5. Le dispositif de nebulisation est placé sur une pièce mobile 30 pouvant être déplacée en xyz . Cette pièce mobile 30 comporte une partie métallique 31 sur laquelle est appliquée la tension d'ionisation dans le spectromètre de masse 25. Le support 1 de silicium est précautionneusement isolé de cette partie métallique 31 lors de la fixation du dispositif sur ladite pièce mobile 30 du fait des propriétés semi-conductrices de ce matériau. Le contact électrique entre la partie métallique 31 et le réservoir du dispositif est assuré à l'aide d'un fil de platine 32 introduit dans le réservoir et qui plonge dans la solution à analyser 33. La solution utilisée pour les tests de nebulisation, une solution de peptide standard (Gramicidine S) , est déposée dans le réservoir du dispositif et la pièce mobile 30 est introduite dans l'entrée du spectromètre de masse 25. Les tests sont effectués sur un spectromètre de masse de type trappe ionique de chez Thermo Finnigan (LCQ DEÇA XP+) . La tension est alors appliquée au liquide. Une caméra installée sur la trappe ionique permet de visualiser la formation du cône de Taylor, une fois la tension appliquée. La fente capillaire à une largeur de 8 μm. La figure .6 est un graphe représentant le courant ionique total enregistré par le spectromètre de masse pour une expérience menée pendant 2 minutes avec une solution de Gramicidine S à 5 μM et une tension d'ionisation à 0,8 kV. L'axe des ordonnées représente l'intensité relative IR. L'axe des abscisses représente le temps. La figure 7 correspond au spectre de masse obtenu avec une solution de Gramicidine S à 5 μM et une tension de 1,2 kV. Le spectre de masse a été moyenne sur 2 minutes d'acquisition du signal soit 80 scans. Exemple 5 : Test des sources de nebulisation fabriquées selon l'exemple 2 en spectrometrie de masse. II : Application de la tension sur le support en silicium L'exemple 5 est proche de l'exemple 4, mais ici la tension n'est pas appliquée à l'aide d'un fil de platine mais en exploitant les propriétés semi- conductrices du silicium. L'exemple 5 est donc le test en spectrometrie de masse de sources de nebulisation fabriquées selon l'exemple 2 avec une application de la tension d'ionisation sur le matériau constituant le support 1 du dispositif de nebulisation. De même que précédemment, le dispositif de nebulisation est fixé sur une pièce mobile 40 pouvant être déplacée en xyz et comportant une partie métallique 41. Ici, le support 1 de silicium est mis en contact électrique avec la partie métallique 41 de la pièce mobile 40 sur laquelle est appliquée la tension d'ionisation dans le spectromètre de masse 25. Le dispositif est fixé sur la partie mobile 40 à l'aide d'un ruban de téflon qui entoure le dispositif en amont du réservoir. Le test est conduit comme précédemment après introduction de la pièce mobile 40 dans la trappe ionique 25 et application de la tension. La fente capillaire possède une largeur de 8 μm. Les tests ont été menés avec un autre peptide standard le Glu-Fibrinopeptide B. Les tensions d'ionisation, ici, sont dans la même gamme que précédemment, de 1 à 1,4 kV pour des concentrations en peptide inférieures à 1 μM. La figure 9 représente le courant ionique total mesuré pendant 3 minutes d'acquisition du signal avec une solution à 0,1 μM et une tension de 1,1 kV. IR est l'intensité relative et t le temps. La figure 10 est le spectre de masse obtenu pour cette acquisition et moyenne sur la période de 3 minutes soit 120 scans. IR est l'intensité relative.Example 4: Test of the nebulization sources manufactured according to Example 2 in mass spectrometry. I: Application of the voltage using a platinum wire. Example 4 is the test of the nebulization sources manufactured as described in Example 2 for an analysis in mass spectrometry. In this first example, the nebulization voltage is applied to the liquid to be nebulized using a platinum wire immersed in the liquid at the level of the reservoir as illustrated in FIG. 5. The nebulization device is placed on a part. mobile 30 can be moved in xyz. This moving part 30 comprises a metal part 31 on which the ionization voltage is applied in the mass spectrometer 25. The silicon support 1 is carefully insulated from this metal part 31 when the device is fixed to said moving part 30 because semiconductor properties of this material. The electrical contact between the metal part 31 and the reservoir of the device is ensured using a platinum wire 32 introduced into the reservoir and which plunges into the solution to be analyzed 33. The solution used for the nebulization tests, a standard peptide solution (Gramicidine S), is deposited in the device reservoir and the moving part 30 is introduced into the inlet of the mass spectrometer 25. The tests are carried out on a mass spectrometer of the ion trap type from Thermo Finnigan (LCQ DEÇA XP +). Voltage is then applied to the liquid. A camera installed on the ion trap allows you to view the formation of the Taylor cone, once the voltage is applied. The capillary slot has a width of 8 μm. Figure .6 is a graph representing the total ion current recorded by the mass spectrometer for an experiment carried out for 2 minutes with a solution of Gramicidine S at 5 μM and an ionization voltage at 0.8 kV. The ordinate axis represents the relative intensity I R. The abscissa axis represents time. FIG. 7 corresponds to the mass spectrum obtained with a solution of Gramicidine S at 5 μM and a voltage of 1.2 kV. The mass spectrum was average over 2 minutes of signal acquisition, ie 80 scans. Example 5: Test of the nebulization sources manufactured according to Example 2 in mass spectrometry. II: Application of the voltage on the silicon support Example 5 is close to Example 4, but here the voltage is not applied using a platinum wire but by exploiting the semiconductor properties silicon. Example 5 is therefore the mass spectrometry test of nebulization sources manufactured according to Example 2 with an application of the ionization voltage on the material constituting the support 1 of the nebulization device. As previously, the nebulization device is fixed on a movable part 40 which can be moved in xyz and comprising a metallic part 41. Here, the silicon support 1 is brought into electrical contact with the metallic part 41 of the movable part 40 on which is applied the ionization voltage in the mass spectrometer 25. The device is fixed to the movable part 40 using a teflon tape which surrounds the device upstream of the tank. The test is carried out as previously after introduction of the moving part 40 into the ion trap 25 and application of the voltage. The capillary slot has a width of 8 μm. The tests were carried out with another standard peptide, Glu-Fibrinopeptide B. The ionization voltages, here, are in the same range as before, from 1 to 1.4 kV for concentrations in peptide less than 1 μM. FIG. 9 represents the total ion current measured during 3 minutes of signal acquisition with a 0.1 μM solution and a voltage of 1.1 kV. I R is the relative intensity and t the time. Figure 10 is the mass spectrum obtained for this acquisition and average over the period of 3 minutes, ie 120 scans. I R is the relative intensity.
Exemple 6 : Test des sources de nebulisation fabriquées selon l'exemple 2 en spectrometrie de masse. III : Expérience de fragmentation (MS/MS) . L'exemple 6 est identique à l'exemple 5 sur la façon de conduire le test . Le montage de test est identique à celui de l'exemple précédent, le dispositif de nebulisation correspond à celui décrit dans l'exemple 1 et réalisé selon le procédé de fabrication décrit dans l'exemple 2. La tension est appliquée directement sur le matériau du support 1, le silicium, via la zone métallique 41 incluse sur la pièce mobile 40 introduite dans le spectromètre de masse 25 (voir la figure 8) . La fente capillaire a une largeur de 8 μm. La solution est la même que précédemment, une solution de peptide standard, le Glu-Fibrinopeptide B à des concentrations inférieures ou égales à 1 μM. Ici, le peptide est soumis à une expérience de fragmentation. Le peptide sous forme dichargée (M+2H)2+ est spécifiquement isolé dans la trappe ionique et est fragmenté (paramètre d'énergie de collision normalisée de 30%, facteur d'activation de radiofréquence fixé à 0,25) . La figure 11 représente le spectre de fragmentation obtenu lors de cette expérience avec une solution à 0,1 μM et une tension de 1,1 kV. IR est l'intensité relative. Le spectre a été moyenne sur 2-3 minutes d'acquisition du signal de nebulisation. Les différents fragments de MS/MS sont annotés avec leur séquence .Example 6: Test of the nebulization sources produced according to Example 2 in mass spectrometry. III: Fragmentation experience (MS / MS). Example 6 is identical to Example 5 on how to conduct the test. The test setup is identical to that of the previous example, the nebulization device corresponds to that described in Example 1 and produced according to the manufacturing process described in Example 2. The voltage is applied directly to the material of the support 1, silicon, via the metallic zone 41 included on the moving part 40 introduced into the mass spectrometer 25 (see FIG. 8). The capillary slot has a width of 8 μm. The solution is the same as above, a standard peptide solution, Glu-Fibrinopeptide B at concentrations less than or equal to 1 μM. Here, the peptide is subjected to a fragmentation experiment. The peptide in dicharged form (M + 2H) 2+ is specifically isolated in the ion trap and is fragmented (normalized collision energy parameter of 30%, radio frequency activation factor fixed at 0.25). FIG. 11 represents the fragmentation spectrum obtained during this experiment with a 0.1 μM solution and a voltage of 1.1 kV. I R is the relative intensity. The spectrum was average over 2-3 minutes of acquisition of the nebulization signal. The different MS / MS fragments are annotated with their sequence.
Exemple 7 : Test des sources de nebulisation fabriquées selon l'exemple 2 en spectrometrie de masse. IV : Application à l'analyse d'un mélange biologique. L'exemple 7 est identique à l'exemple 5 (même dispositif fabriqué selon le même procédé et testé dans les même conditions avec application de la tension sur le support 1 en silicium) sauf que l'échantillon analysé ici n'est plus un peptide standard mais un mélange complexe de peptides obtenu par digestion d'une protéine, le Cytochrome C. Ce digestat se compose de 13 peptides de longueurs et de propriétés physico-chimiques différentes . Ce digestat est testé à une concentration de 1 μM et avec une tension d'ionisation de 1,1-1,2 kV. La largeur de la fente capillaire est de 8 μm. La figure 12 représente le spectre de masse obtenu pour le digestat de Cytochrome C à 1 μM avec une tension de 1,2 kV. IR est l'intensité relative. Les pics sont annotés avec la séquence du fragment ainsi que son état de charge. Sur les 15 peptides, 11 sont clairement identifiés lors de cette expérience. Exemple 8 : Test des sources de nebulisation fabriquées selon l'exemple 2 en spectrometrie de masse. V : Alimentation dudit dispositif en continu à l'aide d'un pousse-seringue ou d'une chaîne de nanoLC placé en amont. L'exemple 8 est identique à l'exemple 5 (même dispositif fabriqué selon le même procédé et testé dans les même conditions avec application de la tension sur le support 1 en silicium) sauf que l'échantillon analysé ici est amené sur ledit dispositif en continu par un capillaire connecté à un pousse-seringue ou une chaîne de nanoLC en amont. Pour le couplage à un pousse-seringue, le débit de liquide a été fixé à 500 nL/min. La solution pour ce test est identique à celle de l'exemple 5, sauf que la concentration du peptide Glu-Fibrinopeptide B est ici de 1 μM et la tension de nebulisation a été fixée à 1,2 kV. La largeur de la fente capillaire est de 8 μm. La figure 13 présente le courant ionique total enregistré lors d'un test de nebulisation mené sur une période de 6 minutes dans lesdites conditions . IR est l'intensité relative et t le temps. La figure 14 représente le spectre de masse correspondant et moyenne sur cette période d'acquisition de 6 minutes soit 240 scans. IR est l'intensité relative. Le couplage à une chaîne de nanoLC (chromatographie liquide à un débit de 1 à 1000 nL/min) a été effectué avec des conditions classiques de couplage entre une séparation sur nanoLC et une analyse en ligne par spectrometrie de masse sur une trappe ionique. Le débit de fluide est de 100 nL/min, la tension d'ionisation de 1,5 kV. L'expérience de séparation est effectuée sur un digestat de Cytochrome C à 800 fmol/μL et 800 fmol de ce digestat sont injectés sur la colonne de séparation. La largeur de la fente capillaire est de 10 μm. La figure 15 représente le courant ionique total détecté sur le spectromètre de masse lors de l'expérience de séparation. IR est l'intensité relative et t le temps. La figure 16 est le spectre de masse obtenu pour le pic indiqué sur la figure 15 au temps de rétention de 23,8 min. Il correspond à l'élution et à l'analyse du fragment 92-99 du Cytochrome C. IR est l'intensité relative. Example 7: Test of the nebulization sources produced according to Example 2 in mass spectrometry. IV: Application to the analysis of a biological mixture. Example 7 is identical to Example 5 (same device manufactured according to the same process and tested under the same conditions with application of the voltage on the silicon support 1) except that the sample analyzed here is no longer a peptide standard but a complex mixture of peptides obtained by digestion of a protein, Cytochrome C. This digestate is made up of 13 peptides of different lengths and physicochemical properties. This digestate is tested at a concentration of 1 μM and with an ionization voltage of 1.1-1.2 kV. The width of the capillary slot is 8 μm. FIG. 12 represents the mass spectrum obtained for the digest of Cytochrome C at 1 μM with a voltage of 1.2 kV. I R is the relative intensity. The peaks are annotated with the sequence of the fragment and its state of charge. Of the 15 peptides, 11 are clearly identified during this experiment. Example 8: Test of the nebulization sources produced according to Example 2 in mass spectrometry. V: Continuous supply of said device using a syringe pump or a nanoLC chain placed upstream. Example 8 is identical to Example 5 (same device manufactured according to the same process and tested under the same conditions with application of the voltage on the silicon support 1) except that the sample analyzed here is brought to said device in continuous by a capillary connected to a syringe pump or a chain of nanoLC upstream. For coupling to a syringe pump, the liquid flow rate was set at 500 nL / min. The solution for this test is identical to that of Example 5, except that the concentration of the Glu-Fibrinopeptide B peptide is here 1 μM and the nebulization voltage has been set at 1.2 kV. The width of the capillary slot is 8 μm. FIG. 13 shows the total ion current recorded during a nebulization test carried out over a period of 6 minutes under said conditions. I R is the relative intensity and t the time. FIG. 14 represents the corresponding and average mass spectrum over this 6-minute acquisition period, ie 240 scans. I R is the relative intensity. The coupling to a nanoLC chain (liquid chromatography at a flow rate of 1 to 1000 nL / min) was carried out with conventional coupling conditions between a separation on nanoLC and an online analysis by mass spectrometry on a hatch ionic. The fluid flow rate is 100 nL / min, the ionization voltage of 1.5 kV. The separation experiment is carried out on a Cytochrome C digestate at 800 fmol / μL and 800 fmol of this digestate are injected onto the separation column. The width of the capillary slot is 10 μm. Figure 15 shows the total ion current detected on the mass spectrometer during the separation experiment. I R is the relative intensity and t the time. FIG. 16 is the mass spectrum obtained for the peak indicated in FIG. 15 at the retention time of 23.8 min. It corresponds to the elution and analysis of the 92-99 fragment of Cytochrome C. I R is the relative intensity.

Claims

REVENDICATIONS
1. Source d'électronébulisation comportant une structure comprenant au moins une pointe plate et mince (3) en porte-à-faux par rapport au reste de la structure, ladite pointe (3) étant pourvue d'une fente capillaire (5) pratiquée dans toute l'épaisseur de la pointe et qui aboutit à l'extrémité (6) de la pointe (3) pour former l'orifice d'éjection de la source d'électronébulisation, la source comprenant des moyens d'approvisionnement (4) de la fente capillaire (5) en liquide à nébuliser et des moyens d'application d'une tension d'électronébulisation sur ledit liquide. 1. Source of electrospray comprising a structure comprising at least one flat and thin point (3) in cantilever with respect to the rest of the structure, said point (3) being provided with a capillary slot (5) formed throughout the thickness of the tip and which ends at the end (6) of the tip (3) to form the ejection orifice of the electrospray source, the source comprising supply means (4) capillary slit (5) of liquid to be nebulized and means for applying an electrospray voltage to said liquid.
2. Source d'électronébulisation selon la revendication 1, caractérisée en ce que les moyens d'approvisionnement comprennent au moins un réservoir (4) en communication fluidique avec la fente capillaire (5) .2. Source of electrospray according to claim 1, characterized in that the supply means comprise at least one reservoir (4) in fluid communication with the capillary slot (5).
3. Source d'électronébulisation selon la revendication 1, caractérisée en ce que la structure comprend un support (1) et une plaque (2) solidaire du support et dont une partie constitue ladite pointe (3) .3. Source of electrospray according to claim 1, characterized in that the structure comprises a support (1) and a plate (2) integral with the support and a part of which constitutes said point (3).
4. Source d'électronébulisation selon la revendication 3, caractérisée en ce que les moyens d'approvisionnement comprennent un réservoir (4) constitué par un evidement formé dans ladite plaque (2) et en communication fluidique avec la fente capillaire (5). 4. Source of electrospray according to claim 3, characterized in that the supply means comprise a reservoir (4) constituted by a recess formed in said plate (2) and in fluid communication with the capillary slot (5).
5. Source d'électronébulisation selon l'une quelconque des revendications 1 à 4, caractérisée en ce que les moyens d'application d'une tension d'électronébulisation comprennent au moins une électrode (7, 8) disposée de façon à être en contact avec ledit liquide à nébuliser.5. Electrospray source according to any one of claims 1 to 4, characterized in that the means for applying an electrospray voltage comprise at least one electrode (7, 8) arranged so as to be in contact with said liquid to be nebulized.
6. Source d'électronébulisation selon l'une quelconque des revendications 3 ou 4, caractérisée en ce que les moyens d'application d'une tension d'électronébulisation comprennent le support, au moins partiellement électriquement conducteur, et/ou la plaque au moins partiellement électriquement conductrice.6. Electrospray source according to any one of claims 3 or 4, characterized in that the means for applying an electrospray voltage comprise the support, at least partially electrically conductive, and / or the plate at least partially electrically conductive.
7. Source d'électronébulisation selon l'une quelconque des revendications 1 à 4, caractérisée en ce que les moyens d'application d'une tension d'électronébulisation comprennent un fil électriquement conducteur (32) disposé pour pouvoir être en contact avec ledit liquide à nébuliser.7. Electrospray source according to any one of claims 1 to 4, characterized in that the means for applying an electrospray voltage comprise an electrically conductive wire (32) arranged to be able to be in contact with said liquid to nebulize.
8. Source d'électronébulisation selon l'une quelconque des revendications 1 à 7, caractérisée en ce que les moyens d'approvisionnement comprennent un tube capillaire.8. A source of electrospray according to any one of claims 1 to 7, characterized in that the supply means comprise a capillary tube.
9. Source d'électronébulisation selon l'une quelconque des revendications 1 à 7, caractérisée en ce que les moyens d'approvisionnement comprennent un canal réalisé dans un microsystème supportant ladite structure et en communication fluidique avec la fente capillaire. 9. Source of electrospray according to any one of claims 1 to 7, characterized in that the supply means comprise a channel produced in a microsystem supporting said structure and in fluid communication with the capillary slot.
10. Source d'électronébulisation selon l'une des revendications 3 ou 4, caractérisée en ce que la plaque (2) présente une surface hydrophobe au liquide à nébuliser. 10. Source of electrospray according to one of claims 3 or 4, characterized in that the plate (2) has a hydrophobic surface to the liquid to be nebulized.
11. Procédé de fabrication d'une structure étant une source d'électronébulisation, comprenant : - la réalisation d'un support (1) à partir d'un substrat (10), la réalisation d'une plaque (2) comportant une partie constituant une pointe plate et mince (3), ladite pointe étant pourvue d'une fente capillaire (5) , pour véhiculer un liquide à nébuliser, pratiquée dans toute l'épaisseur de la pointe et qui aboutit à l'extrémité de la pointe, - la solidarisation de ladite plaque (2) sur le support (1) , la pointe (3) étant en porte-à-faux par rapport au support.11. A method of manufacturing a structure being a source of electrospray, comprising: - producing a support (1) from a substrate (10), producing a plate (2) comprising a part constituting a flat and thin point (3), said point being provided with a capillary slot (5), for conveying a liquid to be nebulized, practiced throughout the thickness of the point and which terminates at the end of the point, - The joining of said plate (2) on the support (1), the tip (3) being cantilevered relative to the support.
12. Procédé selon la revendication 11, caractérisé en ce qu'il comprend les étapes suivantes : - la fourniture d'un substrat (10) pour réaliser le support (1) , - la délimitation du support (1) au moyen de tranchées (13) gravées dans le substrat (10) , - le dépôt, sur une zone du substrat correspondant à la future pointe de la structure, de matériau sacrificiel (14) selon une épaisseur déterminée, - le dépôt de la plaque (2) sur le support (1) délimité dans le substrat (10) , la pointe (3) de la plaque (2) étant située sur le matériau sacrificiel (14), l'élimination du matériau sacrificiel (14), - le détachement du support (1) par rapport au substrat (10) par clivage au niveau desdites tranchées (13) .12. Method according to claim 11, characterized in that it comprises the following steps: - the supply of a substrate (10) for producing the support (1), - the delimitation of the support (1) by means of trenches ( 13) etched in the substrate (10), - the deposition, on an area of the substrate corresponding to the future point of the structure, of sacrificial material (14) according to a determined thickness, - the deposition of the plate (2) on the support (1) delimited in the substrate (10), the point (3) of the plate (2) being located on the sacrificial material (14), the elimination of the sacrificial material (14), - the detachment of the support (1) relative to the substrate (10) by cleavage at the level of said trenches (13).
13. Procédé selon la revendication 12, caractérisé en ce que l'étape de dépôt de la plaque (2) est un dépôt d'une plaque comprenant en evidement en communication fluidique avec la fente capillaire (5) afin de constituer un réservoir (4) .13. Method according to claim 12, characterized in that the step of depositing the plate (2) is depositing a plate comprising in recess in fluid communication with the capillary slot (5) in order to constitute a reservoir (4 ).
14. Procédé selon l'une des revendications 12 ou 13, caractérisé en ce qu'il comprend en outre une étape de dépôt d'au moins une électrode (7, 8) destinée à assurer un contact électrique avec le liquide à nébuliser. 14. Method according to one of claims 12 or 13, characterized in that it further comprises a step of depositing at least one electrode (7, 8) intended to ensure electrical contact with the liquid to be nebulized.
15. Application de la source d'électronébulisation selon l'une quelconque des revendications 1 à 10 pour obtenir une ionisation d'un liquide par electronebulisation avant son analyse en spectrometrie de masse. 15. Application of the electrospray source according to any one of claims 1 to 10 to obtain an ionization of a liquid by electronebulization before its analysis in mass spectrometry.
16. Application de la source d'électronébulisation selon l'une quelconque des revendications 1 à 10 pour obtenir une production de gouttes de liquide de taille calibrée ou l'éjection de particules de taille fixée.16. Application of the electrospray source according to any one of claims 1 to 10 to obtain a production of drops of liquid of calibrated size or the ejection of particles of fixed size.
17. Application de la source d'électronébulisation selon l'une quelconque des revendications 1 à 10 à la réalisation d'une écriture moléculaire à l'aide de composés chimiques.17. Application of the electrospray source according to any one of claims 1 to 10 to the production of molecular writing using chemical compounds.
18. Application de la source d'électronébulisation selon l'une quelconque des revendications 1 à 10 à la définition du potentiel électrique de jonction d'un dispositif en continuité fluidique. 18. Application of the electrospray source according to any one of claims 1 to 10 to the definition of the electrical junction potential of a device in fluid continuity.
EP04805823A 2003-11-12 2004-11-10 Planar electronebulization sources modeled on a calligraphy pen and the production thereof. Not-in-force EP1703987B1 (en)

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FR0350820A FR2862006B1 (en) 2003-11-12 2003-11-12 PLANAR ELECTRONEBULATING SOURCES ON THE MODEL OF A CALLIGRAPHIC FEATHER AND THEIR MANUFACTURE.
PCT/FR2004/050580 WO2005046881A1 (en) 2003-11-12 2004-11-10 Planar electronebulization sources modeled on a calligraphy pen and the production thereof.

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US20070252083A1 (en) 2007-11-01
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DE602004013195T2 (en) 2009-06-25
CA2545213A1 (en) 2005-05-26
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CA2545213C (en) 2012-02-21
JP4800218B2 (en) 2011-10-26
US8294119B2 (en) 2012-10-23
DE602004013195D1 (en) 2008-05-29
WO2005046881A1 (en) 2005-05-26
FR2862006A1 (en) 2005-05-13

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