EP1703987B1 - Planar electronebulization sources modeled on a calligraphy pen and the production thereof. - Google Patents

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

TECHNICAL AREA

The present invention relates to original electrospray sources, their method of manufacture and their applications.

STATE OF THE PRIOR ART

Electrospray is the phenomenon that transforms a liquid into a nebulisat under the action of a high voltage ( M. CLOUPEAU "Electrohydrodynamic Spraying Operating Modes: A Critical Review." Journal of Aerosol Science (1994), 25 (6), 1021-1036 To do this, the liquid is fed into a capillary and is subjected to a continuous or alternating high voltage or 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 outlet of the capillary, the liquid is nebulized under the action of the tension. The surface of the meniscus formed by the liquid is elongated to form a Taylor cone (s) from which charged liquid droplets are ejected, evolving to form a charged particle-containing gas. The formation of the nebulisate is observed when the electrical forces due to the application of the tension compensate for and exceed the surface tension forces of the liquid on the section of the capillary at the end of said capillary.

• 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 .).

The size of the capillary, and more precisely its outlet orifice, is in direct relationship 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 distinguished by their establishment characteristics:

• the so-called conventional regime which corresponds to capillary output 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 nano-electro-depressurization 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 own frequency ( F. CHARBONNIER et al., "Differentiating between Capillary and Counter Electrode Processes during Electrospray Ionization 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 for its applications by the application of multiple and independent voltages that allow the chemical modification species present in the liquid by electrochemistry (see patent application US 2003/0015656 ; GJ VAN BERKEL, "Enhanced Study and Control of Analyzing 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 ionization 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 ).

• 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 theme", 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 deposition by the electrospray method for californium-252 plasma desorption studies of involatile molecules", Analytical Chemistry (1979), 51(12), 2036-2039 ; R. C. MURPHY et al., "Electrospray loading of field desorption emitters and desorption chemical ionization 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 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 ; N. V. AVSEENKO et al., "Immunoassay with Multicomponent Protein Microarrays Fabricated by Electrospray Deposition", Analytical Chemistry (2002), 74(5), 927-933 ), soit le dépôt de solutions sur une plaque MALDI (pour "Matrix Assisted Laser Desorption Ionization") avant une analyse par spectrométrie de masse ( J. AXELSSON et al., "Improved reproducibility and increased signal intensity in matrix-assisted laser desorption/ionization as a result of electrospray sample preparation", 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., "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 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.

The fields of application of electrospray are:

• 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. ; the patent US 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 source ion electrospray", Journal of Physical Chemistry (1984), 88 (20), 4671-4675 ) before their analysis by mass spectrometry as a function of the m / z ratio where m is the mass of the analyte and 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 may 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 ionization probes", Analytical Chemistry (1982), 54 (2), 336-338 ) for example a plate for, or the production of analysis chips such as DNA or peptide chips, dedicated to a high-throughput analysis ( VN MOROZOV et al., "Electrospray Deposition as a Method for Mass Production 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., "Immunoassay with Multicomponent Protein Microarrays Fabricated by Electrospray Deposition", Analytical Chemistry (2002), 74 (5), 927-933 ), or the deposit of solutions on a MALDI plate (for "Matrix Assisted Laser Desorption Ionization") before mass spectrometry analysis ( J. AXELSSON et al., "Improved Reproducibility and Enhanced Signal Intensity in Matrix-assisted Laser Desorption / Ionization as a Result of Electrospray Sample Preparation", Rapid Communications in Mass Spectrometry (1997), 11 (2), 209-213 ). These drops can also be manipulated, either for the injection of fluid 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 encapsulated molecules 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 discrete manner the dimensions of the sources depend greatly on the size of the deposits to be made.
• A third application is the deposition of controlled size particles 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. ). Particles can also be replaced by cells for the preparation of cell chips.
• A fourth application is the injection of drops formed by electrospray in 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 the molecular writing on a plate using molecules or chemical solutions ( SN JAYASINGHE et al., "A novel process for simulataneous printing of multiple tracks of concentrated suspensions", Materials Research Innovations (2003), 7 (2), 62-64. .), in view of the functionalization of the material or a localized chemical treatment, on a scale that may be less than one micrometer.

These various applications can also be combined with each other.

Usually, the sources used for nanoelectrospray are in the form of glass or fused silica capillaries. They are manufactured by hot stretching or acid etching of the material 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 a suitable conductive outer coating: a metal 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 ), money ( Y.-R CHEN et al., "A simple method for the manufacture of silver-coated sheathless electrospray emitters", Rapid Communications 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 Ionization 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 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. ).

• 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 nébulisat, 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.

Nevertheless, the devices of the prior art dedicated to nanoelectro-deposition 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 not very reproducible;
• The outer conductive coating deteriorates rapidly;
• Their mode of use is inconvenient because of their needle-like geometry: the liquid to be sprayed 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 into the needle which can subsequently disturb the stability of the nebulisate, they must therefore be driven out;
• Finally, most often, the outlet orifice is too small to allow the passage of the liquid; as a result, the capillaries must first be gently broken along a wall, which further increases the randomness of their dimensions.

Thus, commercial standard sources are unsuited, firstly to a controlled, reproducible and quality nebulization, secondly to the use of robots because of the entirely manual nature of their mode of use, and, third, to an integration. on a fluidic microsystem, as discussed in the following.

These defects hinder certain areas of applications of electro-fogging that currently require robotization and automation of processes. This is the case of the application areas identified above: mass spectrometry analysis, the deposition of calibrated sized drops and the writing at a scale below 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 thus the marriage between microtechnology and biology or microtechnology and chemical analysis. Thus, microtechnology techniques are used for the production of integrated microsystems of characteristic size of the order of one micrometer and which bring together a series of reaction and / or analytical, chemical and / or biochemical / biological processes.

• 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'applications 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 spectrométrie 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.

The rise of microfluidics in the fields of chemistry and biology, where speed and automation of processes are now required, is explained by:

• the speed gain of the processes, because the speed depends mainly on the size of the devices; this gain in speed is particularly important for fields of applications 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 volumes handled with those available to the experimenter in the case of, inter alia, biological or environmental analyzes,
• the limitation to the removal of human intervention, which is often a source of error and contamination,
• a gain in sensitivity, for certain analytical techniques, including mass spectrometry with electrospray ionization,
• overall, new performances that do not only correspond to a downscaling of well-established tools and techniques.

• 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.

Microfluidic devices are manufactured using microtechnology techniques. A wide range of materials is now available for these microfabrications, ranging from silicon and quartz (materials used in microtechnology) to glasses, ceramics and polymer-type materials, such as elastomers or plastics. So, does microfluidics benefit at the same time:

• the legacy of materials and manufacturing techniques developed and used for microelectronics applications and,
• new manufacturing processes, developed in parallel and adapted to other emerging materials of great interest for microfluidic applications, such as plastic type materials, whose main attraction is their low cost.

• 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., "Integration 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 Conference 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 microfabriquées. 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.

More specifically, the possible materials for technological productions applicable to chemistry and biology are ( T. McCREEDY, "Manufacturing techniques and materials commonly used for the production of microreactors and micro total analytical systems", TrAC, Trends in Analytical Chemistry (2000), 19 (6), 396-401 ):

• semiconductor type materials such as silicon, traditional materials in microtechnology that benefit from robust and proven manufacturing techniques; these manufacturing techniques include lithography, physical and chemical etchings, 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 about ten nanometers. In addition, its surface chemistry is controlled, the treatments involving the silanol functions present on its surface. But its semiconducting properties are not always adapted according to the targeted 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-produced products (single-use objects 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 unattractive because of its high cost; it is therefore gradually abandoned despite its physicochemical properties.
• glass, a cheaper material than quartz and silicon, which is widely used because of its surface properties suitable for the establishment of an electroosmotic flow ( K. SATO et al. "Integration of chemical and biochemical analysis systems into a microchip", Analytical Sciences (2003), 19 (1), 15-22 ). As with silicon, silanol groups lurk 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, manufacturing techniques are not as well controlled as for silicon; the engraving profiles are less clean and the aspect ratio is very poor ( TR DIETRICH et al., "Manufacturing technologies for microsystems utilizing photo etchable glass", Microelectronic Engineering (1996), 30 (1-4), 497-504. ). On the other hand, it is a fragile and brittle material.
• polymeric materials, which include plastics and elastomers. Their main advantage is their low cost which is compatible with low-cost mass production. The multiplicity of these materials leads to a wide range of physicochemical properties. Their major disadvantage is their low resistance to high temperatures and their sensitivity to solvent conditions conventionally used in chemistry and biology, organic medium, acidic, basic, which can lead to degradation of the material or even its dissolution. Moreover, the surface chemistry of these materials is poorly known, which makes it difficult to further treat the surfaces generated 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 Conference 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 inexpensive inorganic substrates of manufacture in the image of plastic materials. A major advantage is that their manufacture does not require dedicated expensive maintenance equipment such as clean rooms but relies on simple and fast processes (laser ablation, rolling, molding, sol-gel process), further reducing the cost price. microfabricated structures. Their surface condition is comparable to that of glass or silicon and finally, the cowling is easier than for other materials, such as glass.

• 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.

In particular, the microfabrication techniques have been applied to the production of electrospray sources or needle tip type in order to:

• to improve the overall quality of capillaries in terms of control of manufacturing processes, reproducibility of sources and their dimensions,
• to produce a large number of devices identical or different from each other by a or several dimensions, on the same plate of material, like microcomponents in microelectronics, to promote the automation and robotization of electrospray.

• 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.

The fabrications using microtechnology techniques of electrospray spikes obey two trends:

• the manufacture of an electrospray tip that reproduces the conventional geometry, that is to say a microfabricated capillary and, most often, circular section. In this class may also be included microfabricated needles for another application, such as injection of chemical substances or measurement of biological potential.
• the design of an electrospray source as a microchannel or capillary outlet manufactured using microtechnology techniques and having a tapered profile.

These microfabricated electrospray devices rely, like fluidic microsystems, on the use of different types of materials and different types of processes.

• 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 spectrométrie de masse à des tensions de nébulisation voisines de 4 kV et un débit de liquide de 50 nL/min avec des peptides standard à une concentration de quelques micromolaires. La tension de nébulisation 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 nébulisation 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 nébulisation 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 nébulisation, 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., "Generation of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometric 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 nanoélectronébulisation puisque la tension requise pour l'observation d'un nébulisat 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 nébulisent la même solution.

According to the first trend, which aims to technologically produce a capillary-type geometry, the following descriptions are listed:

• According to this approach, sources of silicon nitride electrospray have been produced using conventional photolithography and etching techniques ( 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 microns and an internal diameter of the outlet orifice of 1 to 3 microns. Said sources were tested by mass spectrometry at nebulization voltages of about 4 kV and a liquid flow rate of 50 nL / min with standard peptides at a concentration of a few micromolar. The nebulization voltage is applied upstream of said device, at the junction with a liquid supply capillary, and this, on a platinum metal connection.
• Sources of electrospray made of polymer-type material, parylene, photolithographable 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 part of a silicon microsystem. They are connected to microchannels 100 μm wide and 5 μm high. The required voltage for nebulization is here lower, of the order of 1.2 to 1.8 kV under conditions of concentration and equivalent fluid flow; the voltage is applied to a metal wire brought into contact with the solution to be sprayed.
• Silicon has also been used for microfabrication of needle-like structures. International demand WO-A-00/15321 describes a chimney-like electrospray device 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 of 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 a 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 measuring needles for biological potential (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 a little according to their application; the electrospray devices resemble the silicon devices described above, with, nevertheless, a profile that narrows at their tip leading to a smaller exit orifice, while the needles for biological potential measurements have a very sharp point. tapered. The method of manufacturing said silicon devices using deep etching techniques is very complex and requires expensive equipment, cumbersome and performance, in terms of nebulization voltage among others, the structures obtained are poor compared to those of sources commercial standard. Moreover, their geometry is poorly suited to integration on a fluidic microsystem.
• The article of L. LIN et al., Entitled "Silicon processed microneedles", IEEE Journal of Microelectromechanical Systems (1999), 8, 78-84 ) describes micro-needles that are connected to a microfluidic network. These needles have been developed for the injection of chemicals in situ and not for nebulization, but the needle-like geometry of these devices is close to that of nanonebulization sources. These needles are made of silicon nitride and have a rectangular exit orifice of 9 x 30-50 μm and a height of 1 to 6 mm.
• Needle-type structures have finally been made of another polymeric material, polycarbonate, using a laser ablation method ( K. TANG et al., "Generation of multiple electrosprays using microfabricated emitter arrays for mass spectrometric sensitivity", Analytical Chemistry (2001), 73 (8), 1658-1663 ). Their dimensions are as follows: 30 μm internal diameter at their outlet orifice and 250 μm height. For this example again, the dimensions of said devices are too large for a nanoelectro-evaporation regime since the voltage required for the observation of a nebulisat is 7 kV and the fluid flow rate is estimated at 30 μL / min. The manufacturing process is otherwise 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.

• Les tentatives de nébulisation 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 nébulisat. Ceci démontre l'importance de travailler avec une structure en pointe qui conduit à une concentration du champ électrique et qui permet ainsi la nébulisation.
• 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 ionization source for integration with microfluidics", Analytical Chemistry (2002), 74(22), 5897-5901 ). Le système intègre quatre dispositifs d'électronébulisation identiques placés en parallèle. La tension de nébulisation 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 Electrospray 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 nébulisation. 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 nébulisation 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 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 ). L'orifice de nébulisation 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 nébulisation 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 nébulisation ( 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 resonance 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 nébulisation 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.

The second trend is to machine a tip at the output of a microchannel or create a peak structure that acts as a source of electrospray. The angle of the pointed structure does not seem to have any influence on the phenomenon of nebulization. According to this second trend:

• The attempts of nebulization at the exit of a microchannel on the edge of a microsystem 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), 3258-3264. ). These tests have been improved by appropriate chemical treatment of the exit surface or by pneumatically assisting the formation of the nebulisate. This demonstrates the importance of working with a peak structure that leads to a concentration of the electric field and thus allows nebulization.
• The peak effect can be achieved by inserting a triangular plane structure between the two material plates defining a microchannel (the support in which the microchannel is machined and the cover). This triangular flat structure consists of a 5 μm thick parylene sheet ( J. KAMEOKA et al., "An electrospray ionization source for integration with microfluidics", Analytical Chemistry (2002), 74 (22), 5897-5901. ). The system incorporates four identical electrospray devices in parallel. The required nebulization voltage is 2.5-3 kV for a fluid flow rate of 300 nL / min. No inter-source interference was observed.
• An eight-pointed star-shaped device was made of polymethyl methacrylate (PMMA) ( C.-H. YUAN et al., "Sequential Electrospray Analysis Using Sharp-Tip Channels Fabricated on a Plastic Chip," Analytical Chemistry (2001), 73 (6), 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 integrates a microchannel 300 x 376 microns section, the tip structure forms a 90 ° angle and the eight liquid tanks 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 microchannel section at its end. Furthermore, the manufacturing method described is based on the machining of channels with a knife, a technique that does not allow for the production of channels and nebulizing devices of small dimensions.
• Another polymer-type material, polydimethylsiloxane (PDMS), has been used for the realization of electrospray-based structures in three different microtechnological manufacturing processes, a method based on ablation of material, a process using a double photolithographable resin layer and a resin molding process (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 microns to 30 x 50 microns 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 of some 100 nL / min to several μL / min.
• Finally, polyimide, another relatively hydrophobic polymeric material material has been 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 section 120 x 45 microns. The system, the microchannel and the point structure are made by plasma etching of the polyimide. The system lid is made of polyethylene / polyethylene terephthalate. The operation of said sources in electrospray 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 by its open topology and the fineness of the thickness (50 microns) of material used for its manufacture. This so-called thin structure has been tested for ionization voltages of 1 to 2.3 kV applied here on a carbon electrode integrated on the device.

Overall, the nebulizing devices identified above have non-compliant operating conditions for small-scale nebulization (too large dimensions, too high nebulization voltages) and most often result from highly complex manufacturing processes. In addition, the type of structure chosen for these different devices is virtually indissociable material used for their realization.

For the various devices presented above, the nebulization voltage is most often applied at the level of the reservoir of the device, if the system includes a reservoir, or, in the opposite case, at the level of the supply of liquid which is carried out using a capillary connected to the device. In this case, either the capillary is conductive (stainless steel for example), or the connection is based on a metal connector. However, it has been proposed to integrate, on the nebulization device, an electrode or conductive zone on 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 made based on carbon ink in the example cited.

Finally, the application of these devices is targeted for electrospray prior to a mass spectrometry analysis and does not lend itself to another type of application.

• 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.

In addition, the devices for depositing calibrated drops from microtechnology are not based on the nebulization of the solution but on a mechanical effect with the contact of the microfabricated tip on the deposition surface. So :

• A structure mimicking that of a fountain pen has been described for the development of DNA microarray plates with the regular deposition of calibrated drops on a smooth surface (see international application WO-A-03/53583 ). The device includes a trench etched into the material terminating at a point through which the liquid exits. This structure is said to be flexible and the liquid to be deposited comes out by contacting the flexible tip with the deposition substrate, the contact angle being 20-30 ° with respect to the vertical. The major application targeted by this invention is the preparation of microarrays or other compounds to be analyzed.
• P. BELAUBRE et al. in the article "Manufacturing of biological microarrays using microcantilevers", Applied Physics Letters (2003), 82 (18), 3122-3124 , propose an open beam type structure for the deposition of 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 particularity of this device is the integration in the beam type structure of aluminum electrodes which increase the liquid loading of the tip when the latter is soaked in the solution to be deposited, by electrostatic effect. These beams-type structures, which have a width of 210 microns at their peak, are manufactured in parallel on the same system. They allow the ejection of drops having a volume in the range of femtolitre to picolitre, the deposited volume linearly depending on the contact time between the tip and the surface, with a flow rate of up to 100 deposits per minute.

Finally, molecular writing at nanometer scales is mainly described with a tip of AFM microscopy (Atomic Force Microscopy) which is soaked in a chemical solution, like a pen pen ( 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 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., "Manufacturing 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 bringing into contact 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 that attacks the material on which it is deposited and thus serve to etch channels or other structures. The AFM microscopy technique has the advantage of a high resolution and a very high writing accuracy. Three modes of operation are possible, and depending on the mode chosen, the surface condition can be controlled before and after passing the chemical molecular writing solution. Nevertheless, this technique requires the use of heavy equipment, bulky, expensive and complex.

Two molecular writing devices described in the literature can also be cited. They derive from the technique using a tip of AFM microscopy but rely 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 whose tip can have dimensions as small as 3 and 10 nm for its internal and external diameters respectively. This micropipette is nevertheless integrated in an AFM apparatus for its use. The ejection of solution here is caused not by contacting but by exerting pressure on the liquid column. This device has been tested for its ability to deliver etching solutions of a chromium layer deposited on a glass plate. The second device ( IW RANGELOW et al., "" NANOJET ": Tool for 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 Cr / Au-covered silicon, having a pyramidal shape and an outlet orifice smaller than 100 nm. This device delivers not a chemical solution as in the previous example, but free radicals in the gas phase produced by a plasma discharge that attack the material placed opposite the tip. Thus, the device does not consist not only in a microfabricated tip but 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 have a microfabricated tip that replaces the conventional tip of AFM microscopy, but they do not allow to overcome the heavy and expensive peripheral machinery necessary for their operation. On the other hand, this technique relies on contacting or quasi-contacting the tip and the substrate. As a result, the parameters of must be very carefully checked to avoid any deterioration of the surface condition due to excessive force exerted on the tip.

Another electrospray device is described in the application US 5,165,601 .

STATEMENT OF THE INVENTION

The present invention relates to a two-dimensional electrospray device having a calligraphy feather type geometry, the tip of which acts as a seat for nebulization.

The subject of the invention is therefore an electrospray source comprising a structure comprising at least one flat and thin tip cantilevered with respect to the remainder of the structure, said tip being provided with a capillary slot practiced throughout the body. thickness of the tip and which ends at the end of the tip to form the ejection orifice of the source of electrospray, the source comprising means for supplying the capillary slit with liquid to be sprayed and means for applying an electrospray voltage to said liquid.

According to an advantageous embodiment, 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 tip. The supply means may comprise a reservoir consisting of 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 to be in contact with said liquid to be sprayed.

In the case where the structure comprises a support and a plate secured to the support, the means for applying an electrospray voltage may comprise the support, at least partially electrically conductive, and / or the at least partially electrically conductive plate. Advantageously, the plate has a hydrophobic surface to the liquid to be sprayed.

The means for applying an electrospray voltage may comprise an electrically conductive wire arranged to be in contact with said liquid to be sprayed.

The supply means may comprise a capillary tube. They may comprise a channel made in a microsystem supporting said structure and in fluid communication with the capillary slot.

According to an advantageous embodiment, 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.

• 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 solidarisation de ladite plaque sur le support, la pointe étant en porte-à-faux par rapport au support.

The invention also relates to 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 portion constituting a flat and thin tip, said tip being provided with a capillary slot, for conveying a liquid to be sprayed, made throughout the thickness of the tip and which ends at the end of The point,
• the securing of said plate on the support, the tip being cantilever with respect to the support.

• 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.

This method may comprise the following steps:

• providing a substrate for carrying out the support,
• the delimitation of the support by means of trenches etched into the substrate,
• depositing, on an area of the substrate corresponding to the future point of the structure, sacrificial material according to a determined thickness,
• depositing the plate on the support delimited in the substrate, the tip of the plate being located on the sacrificial material,
• the elimination of the sacrificial material,
• detaching the support from the substrate by cleavage at said trenches.

The step of depositing the plate may be a deposit of a plate comprising a recess in fluid communication with the capillary slot to form a reservoir. The method may further comprise a step of depositing at least one electrode intended to provide electrical contact with the liquid to be sprayed.

The source of electrospray according to the invention can be used to obtain ionization of a liquid by electrospray before its analysis by mass spectrometry. It can also be used to obtain a production of liquid drops of calibrated size or the ejection of particles of fixed size. It can still be applied to the realization of a molecular writing with the help of chemical compounds. It can still be applied to the definition of the electric potential of junction of a device in fluidic continuity.

BRIEF DESCRIPTION OF THE DRAWINGS

• 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 oeuvre 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.

The invention will be better understood and other advantages and particularities will appear on reading the following description, given by way of non-limiting example, accompanied by the appended drawings among which:

• the Figures 1A and 1B are views respectively from above and from the side of an electrospray source according to the present invention,
• the figure 2 is a perspective view of the end of the tip of an electrospray source according to the present invention,
• the Figures 3A to 3H are top views illustrating a method of manufacturing the electrospray source shown in FIGS. Figures 1A and 1B ,
• the Figures 4A and 4B illustrate a cleavage technique usable for the implementation of the manufacturing process illustrated by the Figures 3A to 3H ,
• the figure 5 represents an assembly used during a test in which an electrospray source according to the invention is associated with a mass spectrometer,
• the figure 6 is a graph representing the total ionic current obtained during the test using an electrospray source according to the invention, in the assembly of the figure 5 ,
• the figure 7 is a mass spectrum obtained during the test using an electrospray source according to the invention in the assembly of the figure 5 ,
• the figure 8 represents another assembly used during a test in which a source of electrospray according to the invention is associated with a mass spectrometer,
• the figure 9 is a graph representing the total ionic current obtained during the test using an electrospray source according to the invention, in the assembly of the figure 8 ,
• the figure 10 is a mass spectrum obtained during the test using an electrospray source according to the invention in the assembly of the figure 8 ,
• the figure 11 represents a fragmentation mass spectrum of the glu-fibrinopeptide obtained with an electrospray source according to the present invention,
• the figure 12 represents a mass spectrum obtained for a Cytochrome C digestate via an electrospray source according to the present invention,
• the figure 13 is a graph representing the total ionic current obtained during a test using an electrospray source according to the invention,
• the figure 14 represents a mass spectrum obtained during a test using an electrospray source according to the present invention,
• the figure 15 is a graph representing the total ion current recorded on an ion trap type mass spectrometer during a coupling test using an electrospray source according to the present invention,
• the figure 16 represents the mass spectrum corresponding to the graph of the figure 15 .

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

The present invention is inspired by the structure and 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 tip. The present invention may comprise, if necessary, an electrical contact zone on which is applied the voltage necessary to the establishment of a nebulisat. 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 to promote or study the electrospray process. These electrodes also allow the control of the electrospray process by synchronization on its own frequency. As in the pen of calligraphy, the liquid is brought by capillarity into the slot towards the end of the tip of the feather-type structure where it is ejected. The ejection takes place not by mechanical action, but in the form of nebulization by applying a high voltage on the liquid.

An electrospray source according to the present invention is shown in FIGS. Figures 1A and 1B , the Figure 1A being a view from above and the Figure 1B a side view.

This source of electrospray comprises a support 1 and a plate 2 integral with the support 1. A portion of the plate 2 forms a tip 3 cantilevered with respect to the support 1. The plate 2 comprises at 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 electrospray source.

The operation of the device is based on the following stated 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 mode of operation. The liquid of interest is deposited or conveyed into the liquid reservoir 4 by a suitable 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 nebulisat at the tip 6 of the tip.

The physics of the source having a feather-type geometry rests on the properties of the materials that constitute it and on the dimensions of its different elements. The figure 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 gradually feed the capillary slot 5. The topology of the structure is two-dimensional. The plate 2 is a hydrophobic material, and even more hydrophobic than that constituting the support 1 supporting the plate 2, material lining the bottom of the tank. this allows to limit the losses of liquid out of the tank. It is interesting to note at this point that the liquids envisaged for nebulization are a priori rather hydrophilic character, such as purely aqueous or half-aqueous solutions half-alcoholic, for example 50/50 methanol / water mixtures.

où (r) est le rayon interne du capillaire, (h_r) la hauteur dont monte le liquide dans le tube capillaire, (ρ) 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. $$\mathit{\gamma }\mathrm{cos\alpha }={\mathit{\gamma }}_{\mathit{SV}}-{\mathit{\gamma }}_{\mathit{SL}}$$

où γ_SV est la tension de surface à l'interface solide-vapeur et γ_SL est la tension de surface à l'interface solide-liquide.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. On the figure 2 indicated dimensions for the operation of the source of electrospray: the width w of the slot, its height h and its length 1. It is assumed that liquid is present in the capillary slot 5. When the source of electrospray is presented next to the area where the nebulization is desired, the effect of gravity on this liquid is negligible. The factors that will intervene for the filling of the capillary gap by the liquid are: the contact angle (α) of the liquid on the material constituting the plate 2, the surface tension (γ) of the liquid and the dimensions (1 and h) of the capillary slot 5. According to equation 1, governing the capillary effect of a liquid in a capillary tube, the cosine of the contact angle α must be positive to observe the capillarity effect , and this, regardless of the effect of gravity. $$h_r=\frac{2\mathit{\gamma }\cos \alpha }{\mathit{\rho gr}}$$

where (r) is the internal radius of the capillary, (h _r ) the height from which the liquid rises in the capillary tube, (ρ) 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. $$\mathit{\gamma }\cos ={\mathit{\gamma }}_{\mathit{SV}}-{\mathit{\gamma }}_{\mathit{SL}}$$

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, in the case where α <90 ° (cos α> 0), the Young equation (equation 2) implies that γ _SV > γ _SL and thus that the solid-liquid interaction is favored compared to that solid-vapor. The term r appears in Equation 1. Its value depends on the observation or not of the capillarity effect. The term r corresponds to the radius of the capillary tube and, in the case of the device forming the subject of the present invention, to the size of the capillary slot 5. If the liquid penetrates into the capillary slot, a liquid bridge is formed. between the two walls of the capillary cleft. It is thus possible to define a ratio of shape 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 that the formation of the liquid bridge in the capillary slot 5 is favored from the energy point of view.

The nebulizing device may or may not include conductive areas (see figure 3H ). These conductive areas if they are located at the level of the liquid reservoir 4 serve as electrodes to bring the nebulization voltage. On the other hand, if they are located at the level of the capillary slot 5, these electrodes will be used to modify the species present in the liquid. In the case of an electrospray-type application before analysis by mass spectrometry, electrochemical processes occur during the ionization of the molecules. The conductive zones implanted on either side of the capillary slot 5 at the end 6 of the tip 3 would make it possible to study them. Moreover, these phenomena lead to an increase in the ionization efficiency and, as a result, to an improvement in the analysis conditions. In the case of a molecular writing type application, the presence of a larger amount of radical species increases the etching rate of the substrate.

Nevertheless, depending on the nature of the material chosen to produce the support 1 of 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 that does not include conductive zones and for which the materials are not conductive can be used in Electro-fogging provided that the electrical contact is made via the liquid. A metal wire immersed in the solution to be sprayed, at the reservoir 4 or any other conductive contact and ensure the role of application of the nebulization voltage.

The device may also be connected to a liquid supply source upstream of the tank 4, such as a capillary delivering a solution from another apparatus of another structure. For example, for a mass spectrometry type application, the capillary may correspond to a separation column output. For an application of the type of droplets of calibrated size or molecular writing, this capillary brings the liquid to the nebulization device from its initial location. Said capillary may be a conventional commercial fused silica capillary. It can also be a microfabricated capillary, that is to say a microchannel integrated on the system supporting the source. The capillary may be a hydrophilic track materialized on the support 1. In the latter two cases, the plate 2 is integrated on a fluidic microsystem and acts as an interface between said microsystem and the outside world where the solution leaving the microsystem is used . Finally, the conductive properties of the device or one of its elements can be used to electrically power any system in fluidic relation with the device.

In addition, said feather plates can be used in isolation or be integrated in large numbers on the same support, and for the parallelization of the nebulization. In this case, said feather-type plates are independent or not of 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 pens function 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 circularly on a round support. The passage from one source to another is then performed respectively by translation or by rotation of the support.

A wide range of materials is nowadays conceivable for microtechnological manufacturing and in particular for fluidic microsystems: glass, silicon-based materials (Si, SiO ₂ , silicon nitride, etc.), quartz, ceramics and a large number of materials. number of macromolecular materials, plastics or elastomers.

The geometry selected for the present invention is compatible with fabrications using any type of materials, and this, for the different parts of the source of electrospray: the support 1, the feather plate 2 and the conductive areas. The technological manufacturing process also involves one or more other material (s) whose choice is adapted depending on the materials selected for elements 1, 2 and 3.

A generic method for manufacturing electrospray sources according to the invention is shown in Figures 3A to 3H . This manufacturing process can be cut into seven major steps which are detailed below, so as to be applicable to any type of material.

The first step of this manufacturing process is the choice of the substrate intended to constitute the support of the source of electrospray. This substrate 10 (see figure 3A ) can be of macromolecular material, glass or silicon or metal. In the case of this embodiment, it is a silicon substrate of 250 .mu.m thick.

The beginning of the process conditions the end of the manufacture of electrospray devices. It is the materialization on the support of the device of lines which will help the cleavage of the substrate in order to release the point of the source and to allow nebulization.

According to the second step, a layer 11 of protective material is deposited on a portion 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 of the layer 11 does not affect the substrate 10. In this embodiment, the layer of protective material is a 20 nm thick layer of silicon oxide. The layer 11 is of variable thickness depending on the nature of the materials of the substrate 10 and the layer 11. The layer 11 is subjected to a lithography step intended to reveal the areas of the substrate to be attacked to define cleavage lines delimiting the support of the structure. The corresponding areas of the layer 11 are etched to provide windows 12 revealing the substrate 10 (see FIG. figure 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. The figure 3C shows the result obtained: the lines 13, consisting of trenches V section, defining the support of the structure to obtain.

During a third step, a layer of sacrificial material is deposited on the substrate 10. This layer of sacrificial material 14 will ultimately enable 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 thin enough to be able to overcome any problem of stress and curvature from the point above 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 one area of the material 14 corresponding to the tip of the structure (see 3D figure ).

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 silicon-based, a metal or even a material of polymer or ceramic type. In this embodiment, the layer of material intended to constitute the plate is a 35 μm thick layer of polymer SU-8 2035 purchased in pre-polymerized form from Microchem and polymerized by a photolithographic process. The thickness of this layer is chosen appropriately. This thickness indeed depend on the ionization performance of the nebulization device, as explained above. The thickness of this layer directly influences the height h of the capillary slit and, according to the foregoing, plus h is large, plus w must be large in order not to modify the ratio R. Or, depending on the final application of the source of nebulization, the stake is to decrease to the maximum w in order to increase the performances. On the other hand, if the thickness of the layer intended to constitute the plate is too thin, the overhanging tip may 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 specification depending on the nature of the material of this layer and thus to define the optimum thickness of material to be deposited.

This layer then undergoes a lithography step and an attack 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 FIG. 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 silicon-based material or a metal, a physical attack or a photolithography followed by a revelation in the case of a photolithographable 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 appropriate chemical attack. The solution for this chemical attack must be carefully chosen so that all the sacrificial material is removed without the support or plate being affected. The materials of these elements must not be sensitive to this chemical solution. We obtain the structure shown in figure 3F .

The sixth step concerns the implantation of conductive zones on the structure. As mentioned above, this step is included in the manufacturing process only if such conductive areas are provided.

Whether these areas are at the level of the reservoir 4 (application of the nebulization voltage) or at the tip (electrodes of physico-chemical studies), the manufacturing process is the same. The realization of the conductive zones 3 at the reservoir alone will be detailed here.

These conductive zones may be 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 disengaged. The conductive material chosen is then deposited by a PECVD (chemical vapor deposition technique) technique on the structure. In this embodiment, the conductive zones are made of palladium and have a thickness of 400 nm. The figure 3G shows the structure obtained. Two conductive areas 7 and 8 surround the tank 4 and allow to apply an electric potential.

The seventh step of this method of manufacturing the source of nebulization is the detachment of the support 1 with respect to the substrate 10 and in particular, the overhang of the tip 3 relative to the support 1 by using the cleavage lines 13 materialized in FIG. second step of this manufacturing process. The structure obtained is represented in figure 3H .

An advantageous cleavage technique is illustrated by the Figures 4A and 4B in the case of overhanging the tip. A fixed wire 20 is placed under the support 1 at the cleavage trenches 13 made on either side of the tip. Together, two forces are exerted on the substrate at the locations indicated on the Figure 4A by arrows. The separation previously made of the tip 3 relative to the support 1 thus ensures that not damage the tip during the cleavage step. The 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 application targeted by the present invention is the electrospray 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 decoding of the genome, biologists in particular are increasingly interested in proteomics, a science that aims to study and characterize all the proteins of an individual. These proteins, in any human being, are present in more than 10 ⁶ different molecules including post-translational modifications. This point justifies the need at the present time for analytical techniques and tools that are compatible with automation for high-throughput analysis, especially for mass spectrometry because of its relevance in the field of mass spectrometry. framework of the study of proteins. The samples (or solutions to be analyzed) available to the biologist are often small (less than or equal to 1 μL) and contain little biological material, which requires working with a very sensitive analytical technique and consuming few sample. This makes mass spectrometry with nanoelectrospray ionization one of the most widely used analytical techniques for characterization of proteins. In this context, the major issue is the reduction to the maximum dimensions of the tip of the source tip. Indeed, as mentioned in the introduction, there are two modes of electrospray for this type of application, the most interesting in terms of automation and gain in sensitivity being the nanoelectro-boiling regime. However, at present, the analysis speed is limited, the flow of samples restricted because the nanoESI-MS (for "nano ElectroSpray Ionization - Mass 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 application targeted by the present invention is the deposition 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 most often depending on the resolution expected in the preparation of the analysis plates. The smaller the drops, the closer their deposit can be on the plate and the higher the density in deposits and therefore in analytes. The device which is the subject of the present invention can be used for this purpose. The width of the capillary slot 5, as well as the value the voltage applied for the ejection of the drops conditions the size of the drops ejected by said nebulizing 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 may be alternating and thus give a deposition rate in drops / minute directly dependent on the frequency of the AC voltage. The deposition of calibrated drops as presented above can be used for the preparation of assay plates such as DNA chips. It can also be applied to the preparation of MALDI targets (for "Matrix-Assisted Laser Desorption / Ionization") on which the samples to be analyzed by mass spectrometry with a MALDI ionization here, are deposited in a discrete manner before their crystallization and their introduction. in the mass spectrometer. Thus, the present nebulizing device having a feather-type geometry can it be for example connected to the output of the separation column and allow coupling between a separation technique and an online analysis by mass spectrometry MALDI type. The drops of liquid finally can be replaced by cells. In this case, the cells are likewise ejected discretely and deposited for example on a plate for the development of cell chips.

The third application targeted by the present invention is the molecular writing at scales of the order of one hundred nanometers. At present, this type of operation is carried out at using AFM microscopy tips, operating with heavy and bulky equipment. The ejection of the liquid is based on a contact or quasi-contact of the tip and the deposition substrate in the case of the AFM or the application of a pressure on the liquid. An adaptation of this technique is to eject the liquid under the action of a voltage and not with the help of pressure or contact. Indeed, in both cases, the ejection is caused when the liquid tension forces at the tip of the pipette are "exceeded" by another force applied to the liquid column. This is possible with an electrospray device where the electric force exceeds that of voltage of the liquid and thus cause the formation of droplets. On the other hand, the formation of reactive species is intrinsic to the electrospray process. This technique of ejection of the fluid eliminates any complex apparatus for producing reactive species such as free radicals, such as a plasma or microwave discharge, upstream of the structure that delivers the liquid.

The present invention can therefore be used for such purposes of molecular writing on a smooth or rough substrate, the release of the writing solution (pseudo-ink) being governed here by applying a voltage. As for the first field of applications, a major stake is to minimize the size of the end of the tip, this dimension conditioning the size of the ejections by nebulization and consequently the expected resolution write on the final substrate. The width of the tip is less than or equal to one micrometer. Another factor influencing the size of the ejections and the fluid flow is the nebulization voltage applied to the liquid. Finally, the production of reactive species, if the device is used to dispense an etching solution from the substrate, can be increased with the implantation of electrodes within the feather-like structure that delivers the fluid. These electrodes are then the seat of electrochemical reactions leading to the formation of reactive species.

We will now focus on the following examples.

Example 1: Design of microfabricated nanoelectrospray sources according to the present invention.

A first example concerns the dimensions and shapes chosen for producing a nebulization device as described in the present invention.

This first device has small dimensions in its tip because of the intended field of application, that is to say a nanoelectro-debulization for the ionization of solutions before their analysis by mass spectrometry. The device is made in accordance with Figures 1A and 1B . The reservoir 4 of the device has 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 slot 5 is 8 μm at the end 6 of the tip 3. From the value of this slot width results the thickness of the plate 2 so as to observe the capillarity effect and the effective penetration 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 capillarity effect to be observed. Thus, the thickness of the plate must be greater than ten micrometers. Moreover, to overcome the problems of mechanical stresses resulting in bending of the structure at the end 6, this thickness was set at 35 microns.

Example 2: Manufacture of design sources described in Example 1 using silicon and SU-8 materials.

The second example relates to the fabrication by microtechnology of nebulization sources, as described in Example 1. The materials used are silicon for support 1 and photolithographable negative resin SU-8 for the feather plate 2. The method of The manufacture derives from the process described above. It is adapted to the chosen materials.

An oriented silicon substrate (100) and n-doped, 3 inches, is coated with a 200 nm layer of silicon oxide (SiO ₂ ), and masked by lithography. The SiO ₂ layer is attacked by a acid solution of HF: H ₂ O on unmasked areas. The exposed silicon is then attacked with a sodium hydroxide solution (KOH) so as to materialize the cleavage lines. A layer of 150 nm of nickel is then deposited on the silicon surface by argon sputtering technique (Plassys MP 450S). The nickel layer is etched locally by UV photolithography (positive photoresist AZ1518 [1.2μm], etching solution HNO ₃ / H ₂ O (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 spinning wheel to homogenize the thickness before the next photolithography step. The feather plate 2 is made in this SU-8 resin layer using conventional UV photolithography techniques. After developing the SU-8 resin with the appropriate reagent (1-methoxy-2-propanol acetate, PGMEA), the nickel layer is etched with the acid solution (HNO ₃ / H ₂ O) described above. This nickel etching step does not affect the SU-8 resin even though this process may take several hours. Finally, after drying of the device, the silicon substrate 1 is sawn according to the technique illustrated in FIGS. Figures 4A and 4B . The technique used here preserves the structure of the feather, as the latter was previously peeled off its support. A photograph of Scanning electron microscopy (Hitachi S4700) of the feather-type fogging source manufactured according to this method confirms the correct detachment of the tip from its support.

The manufacturing method described above does not include the production of electrodes.

Example 3 Design of particle ejection device of a hundred micrometers.

A third example concerns the dimensions and shapes chosen for producing a device for ejecting particles 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 the tank 4 must be compatible with the handling of objects of a hundred microns. Because of this range of dimensions, the device described in Example 3 also applies to the manipulation of cells of size close to 100 μm in diameter, for the preparation of cell chips, for example.

The reservoir 4 of said device has the dimensions 1 cm × 1 cm × (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 that is greater than 1. The particles handled by this device have a size of one hundred micrometers, so the capillary slot 5 must have a width greater than 100 μm. However, the particles may tend to aggregate, this width should not be chosen too large. It is preferably close to double the size of the particles handled. As a result, the width of the slot is set 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 photolithographable 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. As a result, the glass support 1 is also covered with a thin layer of SU-8 resin to prevent unwanted adhesion of the cells to the device.

Example 4 Test Nebulization Sources Manufactured According to Example 2 in Mass Spectrometry. I: Application of the tension using a platinum wire.

Example 4 is the test of nebulization sources manufactured as described in Example 2 for mass spectrometry analysis. In this first example, the nebulizing voltage is applied to the liquid to be sprayed using a platinum wire immersed in the liquid at the reservoir as shown in FIG. figure 5 .

The nebulizing device is placed on a moving part 30 that can be moved in xyz. This mobile 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 isolated from this metal part 31 when the device is fixed on 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 by means of a platinum wire 32 introduced into the reservoir and which is immersed in the solution to be analyzed 33. The solution used for the nebulization tests, a standard peptide solution (Gramicidin S) is deposited in the reservoir of the device and the moving part 30 is introduced into the input of the mass spectrometer 25. The tests are carried out on an ion trap type mass spectrometer from Thermo Finnigan (LCQ DECA XP +). The voltage is then applied to the liquid. A camera installed on the ion trap allows to visualize the formation of the Taylor cone, once the applied voltage. The capillary slit at a width of 8 μm.

The figure 6 is a graph representing the total ion current recorded by the mass spectrometer for an experiment conducted for 2 minutes with a solution of Gramicidin S at 5 μM and a ionization voltage at 0.8 kV. The y-axis represents the relative intensity I _R. The x-axis represents time. The figure 7 corresponds to the mass spectrum obtained with a solution of Gramicidin S at 5 μM and a voltage of 1.2 kV. The mass spectrum was averaged over 2 minutes of signal acquisition, ie 80 scans.

Example 5 Test 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 exploiting the semiconductor properties of 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 before, the nebulizing device is fixed on a moving part 40 that can be moved in xyz and having a metal part 41. Here, the support 1 of silicon is placed in electrical contact with the metal part 41 of the moving part 40 on which the ionization voltage is applied in the mass spectrometer 25. The device is fixed on the mobile part 40 by means of a Teflon tape which surrounds the device upstream of the tank. The test is conducted as previously after introduction of the moving part 40 into the ion trap and application of the voltage. The capillary gap has a width of 8 μm.

The tests were conducted with another standard peptide Glu-Fibrinopeptide B. The ionization voltages here are in the same range as above, from 1 to 1.4 kV for peptide less than 1 μM. The figure 9 represents the total ionic current measured during 3 minutes of signal acquisition with a solution of 0.1 μM and a voltage of 1.1 kV. I _R is the relative intensity and t is the time. The figure 10 is the mass spectrum obtained for this acquisition and averaged over the period of 3 minutes, ie 120 scans. I _R is the relative intensity.

Example 6 Test Nebulization Sources Manufactured 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 assembly is identical to that of the previous example, the nebulization device corresponds to that described in Example 1 and carried out according to the manufacturing method described in Example 2. The voltage is applied directly to the material of the invention. support 1, the silicon, via the metal zone 41 included on the moving part 40 introduced into the mass spectrometer 25 (see FIG. figure 8 ). The capillary gap has a width of 8 μm.

The solution is the same as above, a standard peptide solution, Glu-Fibrinopeptide B at concentrations of less than or equal to 1 μM. Here, the peptide is subjected to a fragmentation experiment. Peptide in dicharged form (M + 2H) ²⁺ is specifically isolated in the ion trap and is fragmented (standardized collision energy parameter of 30%, radiofrequency activation factor set at 0.25).

The figure 11 represents the fragmentation spectrum obtained during this experiment with a solution of 0.1 μM and a voltage of 1.1 kV. I _R is the relative intensity. The spectrum was averaged over 2-3 minutes of acquisition of the nebulization signal. The different fragments of MS / MS are annotated with their sequence.

Example 7 Test Nebulization Sources Manufactured 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 composed 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 slit is 8 μm.

The figure 12 represents the mass spectrum obtained for the digestate 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 as well as its state of charge. Of the 15 peptides, 11 are clearly identified in this experiment.

Example 8 Test Nebulization Sources Manufactured According to Example 2 in Mass Spectrometry V: Feeding said device continuously using a syringe pump or a chain of nanoLC placed upstream.

Example 8 is identical to Example 5 (same device manufactured according to the same method and tested under the same conditions with application of the voltage on the silicon support 1) except that the sample analyzed here is fed 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 of 1 μM and the nebulization voltage was set at 1.2 kV. The width of the capillary slit is 8 μm.

The figure 13 shows the total ion current recorded during a nebulization test conducted over a period of 6 minutes under said conditions. I _R is the relative intensity and t is the time. The figure 14 represents the corresponding mass spectrum averaged over this acquisition period of 6 minutes, 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 nanoLC separation and an on-line mass spectrometry analysis on a hatch. ionic. The fluid flow rate is 100 nL / min, the ionization voltage is 1.5 kV. The separation experiment is performed on a Cytochrome C digestate at 800 fmol / μL and 800 μmol of this digestate are injected onto the separation column. The width of the capillary slit is 10 μm. The figure 15 represents the total ionic current detected on the mass spectrometer during the separation experiment. I _R is the relative intensity and t is the time. The figure 16 is the mass spectrum obtained for the peak indicated on the figure 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 (18)

1. Electrospray source having a structure comprising at least one flat and thin tip (3) in cantilever in relation to the rest of the structure,
   characterized in that said tip (3) is provided with a capillary slot (5) formed through the complete thickness of the tip and which ends up at the end (6) of the tip (3) to form the ejection orifice of the electrospray source, the source comprising means of supplying (4) the capillary slot (5) with liquid to be nebulised and means of applying an electrospray voltage to said liquid.
2. Electrospray source according to claim 1, characterized in that the supply means comprise at least one reservoir (4) in fluidic communication with the capillary slot (5).
3. Electrospray source according to claim 1, characterized in that the structure comprises a support (1) and a wafer (2) integral with the support and in which a part constitutes said tip (3).
4. Electrospray source according to claim 3, characterized in that the supply means comprise a reservoir (4) constituted by a recess formed in said wafer (2) and in fluidic communication with the capillary slot (5).
5. Electrospray source according to any of claims 1 to 4, characterized in that the means of applying an electrospray voltage comprise at least one electrode (7, 8) arranged so as to be in contact with said liquid to be nebulised.
6. Electrospray source according to any of claims 3 or 4, characterised in that the means of applying an electrospray voltage comprise the support, at least partially electrically conductive, and/or the wafer at least partially electrically conductive.
7. Electrospray source according to any of claims 1 to 4, characterised in that the means of applying an electrospray voltage comprise an electrically conductive wire (32) arranged in order to be able to be in contact with said liquid to be nebulised.
8. Electrospray source according to any of claims 1 to 7, characterised in that the supply means comprise a capillary tube.
9. Electrospray source according to any of claims 1 to 7, characterised in that the supply means comprise a channel formed in a microsystem supporting said structure and in fluidic communication with the capillary slot.
10. Electrospray source according to one of claims 3 or 4, characterised in that the wafer (2) has a surface hydrophobic to the liquid to be nebulised.
11. Method of manufacturing a structure being an electrospray source, comprising:

    - the formation of a support (1) from a substrate (10),

    - the formation of a wafer (2) having a part constituting a flat and thin tip (3), said tip being provided with a capillary slot (5), to convey a liquid to be nebulised, formed in the complete thickness of the tip and which ends up the end of the tip,

    - making said wafer (2) integral on the support (1), the tip (3) being in cantilever in relation to the support.
12. Method according to claim 11, characterised in that it comprises the following steps:

    - the provision of a substrate (10) to form the support (1),

    - the delimitation of the support (1) by means of trenches (13) etched in the substrate (10),

    - the deposition, on a zone of the substrate corresponding to the future tip of the structure, of sacrificial material (14) according to a determined thickness,

    - the deposition of the wafer (2) on the support (1) delimited in the substrate (10), the tip (3) of the wafer (2) being situated on the sacrificial material (14),

    - the elimination of the sacrificial material (14),

    - the detachment of the support (1) in relation to the substrate (10) by cleavage at the level of said trenches (13).
13. Method according to claim 12, characterised in that the step of deposition of the wafer (2) is a deposition of a wafer comprising a recess in fluidic communication with the capillary slot (5) in order to constitute a reservoir (4).
14. Method according to one of claims 12 or 13, characterised in that it further comprises a step of depositing at least one electrode (7, 8) intended to assure an electrical contact with the liquid to be nebulised.
15. Application of the electrospray source according to any of claims 1 to 10 to obtain an ionisation of a liquid by electrospraying before its analysis by mass spectrometry.
16. Application of the electrospray source according to any 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 of the electrospray source according to any of claims 1 to 10 to the carrying out of a molecular writing by means of chemical compounds.
18. Application of the electrospray source according to any of claims 1 to 10 to the definition of the electrical junction potential of a device in fluidic continuity.

Images