Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution

Definitions

• the invention relates to a laboratory on a chip comprising a microfluidic network and a coplanar electrospray nose. It relates in particular to the coupling of a laboratory on a chip with a mass spectrometer.
• mass spectrometer For almost ten years, many avenues have been explored to couple different microfluidic devices to mass spectrometers. Indeed, optical detection methods such as spectrophotometry or fluorescence are not suitable for the detection of biomolecules such as proteins or peptides, detection which is of particular interest in the field of proteomics.
• the limits are either the sensitivity or the need to prepare the sample (fluorescent labeling), which, in the case of the identification of proteins after enzymatic digestion for example, presents a problem since the peptides obtained are a priori not known.
• Mass spectrometry is therefore often used since it gives information on the nature of the samples analyzed (intensity spectrum according to the mass / charge ratio) with very good sensitivity (femtomole / ⁇ l), and that it allows analyze complex mixtures of molecules.
• a pre-treatment of the sample is carried out before the analysis. Through example, this pre-treatment consists of a separation of the chemical and / or biological compounds, preceded and / or followed by a concentration of the species.
• micro-fluidic devices for enzymatic digestion Lian Ji Jin, "A microchip-based proteolytic digestion system driven by electroosmotic pumping", Lab Chip, 2003, 3, 11-18
• capillary electrophoresis B. Zhang et al., "Microfabricated Devices for Capillary Electrophoresis-Electrospray Mass Spectrometry", Anal. Chem., Vol.
• microfluidic / mass spectrometry coupling can be based on a technique of ionization of the sample by electronebulization or electrospray (or ESI for ElectroSpray Ionization).
• the pre-treated liquid sample leaving the micro-fluidic chip is nebulized in an ion gas or in a multitude of charged droplets which can enter the mass spectrometer (MS) for analysis.
• MS mass spectrometer
• This nebulization passes through the deformation of the interface formed between the outgoing liquid and the surrounding gas (liquid meniscus / gas) and the "drop" of liquid takes a conical shape called "Taylor cone".
• the volume of this cone constitutes a dead volume for the outgoing liquid (geometric space in which the chemical compounds can mix), which is undesirable, especially when the last stage of the pretreatment consists precisely in a separation of the compounds sample chemicals.
• the sample is pre-treated "outside the ESI device” then placed manually (with a pipette) in a hollow needle whose end is electrically conductive ("PicoTip emitter” of New Objective for example).
• An electric field is imposed between the conductive part of the PicoTip and the entry of the MS, which allows the formation of a Taylor cone at the exit of the PicoTip and the nebulization of the sample.
• the “pointed” cylindrical geometry of PicoTip is ideal for the formation of a small Taylor cone, but the limits on the minimization of their size (conventionally with external diameter 360 ⁇ m and internal diameter 10 ⁇ m), those on obtaining good reproducibility by the manufacturing techniques used (stretching) and their fragility in use are the main reasons for seek to make other types of nebulization devices.
• micro-technologies such as plane silicon technologies for example (etching, machining, deposition in thin layers and photolithography of various materials on substrates having very large lateral dimensions compared to their thickness), we often speak of "electrospray nose” (Tai et al., "MEMS electrospray nozzle for mass spectroscopy", WO-A-98/35376).
• electrospray nose Ti et al., "MEMS electrospray nozzle for mass spectroscopy", WO-A-98/35376.
• micro-technologies can allow ESI interfaces to be created by defining tip-like structures (such as PicoTips) but smaller (to limit the volume of the Taylor cone), more reproducible and less fragile, which is of interest in itself (see document WO-A- 00/30167).
• micro-technologies can make it possible to produce devices integrating a fluidic network making it possible to ensure the pretreatment of the sample and an ESI type interface.
• advantages reduction in dead output volumes, reproducibility, robustness of the ESI interface
• an integrated preprocessing device continuous preprocessing protocol with analysis, reduction overall analysis times, minimization of reagent volumes.
• PDMS poly (dimethylsiloxane) chip also showing through channels intended to be placed opposite an MS for nebulization of the sample.
• the authors take advantage of the hydrophobicity of PDMS to obtain a small Taylor cone, hence the limitation of the dead output volume.
• the proposed device does not incorporate a nebulization electrode.
• the tests are carried out using a platinum wire immersed in the inlet tank of the ⁇ SI channel, which cannot be a good solution, that is to say without adding dead volume, for a possible integration into a fluid pre-treatment network.
• PDMS technology remains a limited technology which does not yet allow the design of complex microfluidic networks of characteristic size of the order of a micrometer.
• PicoTip is only 10 ⁇ m.
• the use of polymeric materials imposes strong limits on possible chemical functionalizations or of the internal walls of the outlet channel or of a possible fluidic pre-treatment network of the sample. Indeed, until now, most of these functionalizations have been developed on silicon or on glass.
• the manufacturing technology proposed is not collective and the nebulization electrode is produced on the outside of the ESI tip. V. Gobry et al. ("Microfabricated polymer injector for direct mass spectrometry coupling",
• the technologies claimed for producing an electrospray nose fitted with an upstream filter are surface technologies making it possible to produce hollow structures in silicon nitride in the first case and in parylene in the second. These surface technologies are based on the use of a sacrificial layer (in phosphosilicate glass PSG), which as its name suggests, is not preserved until the end of the technological sequence. The removal of this layer, produced by chemical etching, determines the hollow structures.
• the present invention provides a microfluidic device allowing various sample treatments and having a good interface with an ESI type mass spectrometer, which requires: - A production technology compatible with that of '' a fluid pre-treatment network (reservoirs, micro-channels, reactors ...) and an ESI interface at the output (tip geometry, minimum output dimensions ...), and this, to allow the realization of the complete device on the same support or the same set of supports seeing a technological sequence common to the two integrated entities. - An integration design without dead volumes. The integration of a nebulization electrode inside the outlet channel and near the device outlet.
• the object of the invention is therefore a laboratory on a chip comprising a support, at least one fluid network, at least one fluid inlet orifice connected to the fluid network and at least one fluid outlet orifice connected to the fluid network, a thin layer integral with the support and in which the fluid network and an electrospray nose are made , the electrospray nose being overhanging with respect to the support and comprising a channel, one end of which is connected to the fluid network and the other end of which constitutes said fluid outlet orifice, the channel being equipped with electrical conduction means forming at minus an electrode, characterized in that the thin layer is a layer fixed by direct sealing on the support.
• the rear face of the support that is to say that which does not support the thin layer, can advantageously be of an inert nature. It therefore does not participate in the operation of the device. In particular, it then has no electrical connection.
• the support is made of semiconductor material
• the electrical conduction means can be a doped part of said support.
• the support can be of conductive material.
• This laboratory may include a cover hermetically covering the fluid network, this cover being provided with a means of fluid access to the fluid inlet orifice.
• the laboratory on a chip may comprise a cover hermetically covering the fluidic network, this cover being provided with a means of fluid access to the orifice fluid inlet and being provided with said electrical conduction means.
• the cover may be of conductive material.
• the electrical conduction means can therefore be located both in the support and in the cover and can be produced either by the support or the cover made of conductive material, or by metal tracks deposited on the support or the insulating cover, or be parts doped with support or cover in semiconductor material.
• FIG. 1 is a diagram of a laboratory on chip according to the present invention
• FIG. 2 shows the structure
• FIG. 3 represents the COMOSS structure of a pre-concentration reactor used in the laboratory on chip of FIG. 1
• FIG. 3A shows a detail of FIG. 3
• FIG. 4 represents the COMOSS structure of a chromatography reactor used in the lab on chip of figure 1
• figure 4A shows a detail of figure 4
• - figure 5 is an enlarged view of a detail of figure 1 showing the ESI interface
• - figures 6A to 6D illustrate a first embodiment of a laboratory on chip according to the present invention
• FIGS. 7A and 7B illustrate a second embodiment of a laboratory on chip according to the present invention
• FIGS. 7A and 7B illustrate a second embodiment of a laboratory on chip according to the present invention
• FIGS. 8A to 8D illustrate a third embodiment of realization of a laboratory on chip according to the present invention
• FIGS. 9A to 9H illustrate a fourth embodiment of a laboratory on chip according to the present invention
• - FIGS. 10A and 10E illustrate a fifth mode of r realization of a laboratory on chip according to the present invention
• FIGS. 11A to 11F illustrate a sixth embodiment of a laboratory on chip according to the present invention
• - Figure 12 illustrates a top view of a substrate comprising a plurality of devices according to the present invention.
• FIG. 1 is a diagram of a laboratory on a chip 1 according to the present invention. This device can be 18 mm long by 5 mm wide.
• the fluidic network We first describe the fluidic network intended to prepare a complex biological sample in order to identify its protein content.
• This fluid network consists of a set of reservoirs and channels, a digestion, enzymatic reactor, a pre-concentration reactor and a separation reactor by liquid electro-chromatography.
• the basic structure of all these reactors is a deep cavity provided with a large number of studs of square or hexagonal section ...
• This kind of structure is known under the name of COMOSS (for "Collocated MOnolith Support Structures”).
• COMOSS for "Collocated MOnolith Support Structures”
• R2, R3 and R4 contain a mixture of water / acetonitrile ACN / formic acid TFA (95%; 5%; 0.1 %)
• R5 contains a water / acetonitrile / formic acid mixture (20%; 80%; 0.1%).
• the digest collected in the R2 tank must be concentrated before separation. For this, it is pumped by electro-osmosis to the R3 tank (trash can). All of the peptides resulting from the enzymatic digestion are then "captured" by the pre-concentration reactor 3 of small volume, hence the concentration.
• a gradient of acetonitrile produced by mixing the buffer of R4 and that of R5 in structure 4 of the "serpentine" type (2 cm in length), then selectively detaches the peptides according to their affinity with the stationary phase (C18 for example ) of the pre-concentration reactor 3. These are again “captured” by the chromatography column 5, which is denser than the pre-concentration reactor 3.
• the enrichment of the mixture with ACN again makes it possible to selectively unhook these peptides from the chromatography column 5, and take them, separated, to the outlet 6 of the chip 1 where the liquid is nebulized towards the inlet of a mass spectrometer not shown.
• An affinity reactor for a given protein (not shown) can be used to capture the latter in a multi-protein mixture conveyed through this reactor.
• the affinity reactor can be functionalized with antibodies and the elution buffer can be made up of competing proteins (vis-à-vis the antibody) with that which one wishes to "capture" in the multi-protein complex.
• the upstream affinity reactor COMOSS structure it is intended to specifically capture a protein, a family of proteins, or a multi-protein complex in the complex biological sample.
• the tools used for this step can be antibodies, but also for example small molecules which have specificity of interaction with the protein (s) sought.
• the COMOSS structure of the enzymatic digestion reactor is made from a set of section studs hexagonal of 10 ⁇ m allowing to define a network of channels of approximately 5 ⁇ m. Its useful width a is constant (640 ⁇ m), but its real width b is 892 ⁇ m. The length c of the active part of the reactor is 15 mm. Its other geometric characteristics, to be read in conjunction with Figure 2, are described in the following table:
• This structure optionally makes it possible to organize silica "beads" of a few micrometers (Beads Bangs Laboratories distributed by Serotec France for example) functionalized (Trypsin for example) in order to bring to the reactor its enzymatic properties or to increase them.
• the enzyme grafted onto the studs can be trypsin.
• the protocol used is that described in document FR-A-2 818 662.
• FIG. 2A shows a detail of the zone of the reactor referenced 11 in FIG. 2.
• the studs 12 of hexagonal section are recognized, making it possible to define the network of channels 13.
• the reference 14 designates possibly used silica beads.
• FIG. 3A shows a detail of the zone of the reactor referenced 21 in FIG. 3.
• the studs 22 of square section are recognized so as to define the network of channels 23.
• the separation reactor by liquid electro-chromatography The COMOSS structure of the separation reactor, shown in fig. 4, is made from a set of pads with a square section of 10 ⁇ m making it possible to define a network of channels of approximately 2 .mu.m. Its useful width g is constant (160 ⁇ m), but its actual width h is 310 ⁇ m.
• the length i of the active part d ⁇ _ ⁇ reactor is 12 mm. Its other geometric characteristics, to be read in parallel with Figure 4, are described in the following table:
• the reactor can be made in three parts of 12 mm in length each as shown in Figure 1.
• This structure can optionally organize functionalized silica beads to provide to the reactor or increase its affinity properties (C18 grafting for example).
• FIG. 4A shows a detail of the zone of the reactor referenced 31 in FIG. 4.
• the studs 32 of square section are recognized making it possible to define the network of channels 33.
• FIG. 5 is an enlarged view of the output of the chip, referenced 6 in FIG. 1.
• the outlet channel 40 is planar and of rectilinear axis with respect to the fluid network. In other words, the outlet channel 40 remains parallel to the planes of the different substrates used for the production. This configuration avoids the dead volumes that the partial or total travel of the thickness of one or more of these substrates could cause, after having traversed a portion parallel to the planes of these substrates. This avoids any turn, which as it was underlined above is essential, in particular for transporting previously separated samples.
• the section of the outlet channel 40 can be adapted by working preferentially on the transverse dimensions (in the plane of the substrate) of the latter, which gives the possibility of achieving “soft restrictions” avoiding dead volumes.
• these remarks are illustrated by the existence of a “connection” 41 between the outlet of the channel of the chromatography reactor 5 and the outlet channel 40.
• Such a restriction is essential for connecting fluid structures of “large »Dimensions (" large "volumes,” large “affinity capacity for example ”) to a structure of the ESI interface type for which, as previously pointed out, it is desirable to minimize the output surface by reaching typically dimensions on the order of a micrometer to a few micrometers.
• the outlet channel 40 opens into a point-like structure 42, a structure with variable external section making it possible to limit the surface of the liquid / gas and liquid / solid interfaces presented by the liquid leaving with its environment, thanks to its end of small interior and exterior sections, while retaining robustness during use thanks to its wide section end.
• the interior of the outlet channel 40 is provided with an electrode 43 making it possible to impose an electrical potential on the liquid present at the outlet of the device, which is necessary to nebulize the sample (stability of the Taylor cone) and / or participate in its electroosmotic pumping. All of these elements provide a complete flat ESI interface, since robust, without dead volumes of connection to fluidic networks and allowing the birth of a Taylor cone of good stability.
• the fluidic network is simplified and reduced to an inlet tank, an inlet channel, a microreactor and an outlet channel with constant section opening into the tip type structure.
• FIGS. 6A to 6D This embodiment is illustrated by FIGS. 6A to 6D. It uses only one SOI substrate. Such substrates are sold by the company "Soitec". The electrodes, the conductive tracks and the electrical contact pickups are produced in a single localized doping step of the silicon.
• FIG. 6A shows an SOI substrate 50 consisting of a support 51 made of silicon 500 ⁇ m thick, successively supporting a layer of silicon oxide 52 4 ⁇ m thick and a thin layer 53 of silicon 25 ⁇ m thick. The thin layer 53 is locally doped to provide a first electrically conductive circuit formed from zones 54 and 55 and a second electrically conductive circuit formed from zones 56 and 57.
• FIG. 6B illustrates the production of the fluid network in the thin layer 53.
• the fluid network is obtained by DRIE etching (for “Deep Reactive Ion Etching”). Etching the thin layer of silicon
• the fluid network produced comprises an inlet tank 61, an inlet channel 62, a microreactor 63 and an outlet channel 64.
• the outlet channel defined here then has two side walls and a horizontal wall ("the ground"). Note that one end 58 of the doped area 55 is located at the bottom of the inlet tank 61 and that one end 59 of the doped area 57 is located at the bottom of part of the outlet channel 64.
• FIG. 6C illustrates the tip clearance. This is obtained by chemical etching of the part of the oxide layer 52 located on the right part of the figure. After this etching, the tip-type structure 65 is released and forms an overhang above the support 51. It should be noted that the outlet channel 64 always comprises the ground 66. An electrical isolation step is then carried out on the fluidic network . This is obtained by a thermal oxidation of 3 ⁇ m in thickness of the silicon of the thin layer 53.
• the support 51 of silicon must not be oxidized otherwise the tip-type structure 65 would no longer be overhanging.
• This thermal oxidation step is necessary to electrically isolate the liquid present in the fluid network from the outside. This electrical isolation is necessary, for example, when electroosmotic pumping is used or when separation by electrophoresis or an electrochemical reaction is present in the fluid network.
• the next step is to clear the electrical contacts. To release the electrodes (the ends 58 and 59) and the contact pickups (the zones 54 and 56), it is necessary to locally etch the layer of thermal Si0 2 (3 ⁇ m) produced previously. This step can be carried out by a laser engraving technique offered by the NovaLase Company in Pessac (Gironde, France). To obtain the on-chip laboratory according to the invention, the support 51 is cleaved as shown in FIG. 6D to release the structure of the tip type 65.
• Second embodiment closure of the device described in the first embodiment by a structured pyrex cover. According to this embodiment, the device
• the cover plate 71 has an end portion 72 overhanging so that the plate 71 does not cover the point-like structure 65. It also includes a through hole 73 intended to ensure fluid communication with the inlet tank 61 of the device 70.
• the cover plate 71 can be a pyrex substrate, for example that available under the reference Corning 7740.
• FIGS. 8A to 8D This embodiment is illustrated by FIGS. 8A to 8D. It uses only one SOI substrate.
• FIG. 8A shows an SOI substrate 80 consisting of a support 81 of 500 ⁇ m thick silicon, successively supporting a layer of silicon oxide 82 1 ⁇ m thick and a thin layer 83 of 25 ⁇ m silicon d 'thickness.
• FIG. 8B illustrates the construction of the fluid network in the thin layer 83.
• the fluid network is obtained by DRIE etching.
• the etching of the upper silicon layer 83 is: either partial in order to preserve a
• the fluid network produced comprises an inlet tank 91, an inlet channel 92, a microreactor 93 and an outlet channel 94.
• the etching of the thin layer 83 also defines the tip-like structure 95.
• the tip-like structure 95 is then released by total chemical etching of the part of the oxide layer 82 which has been revealed by the etching of the layer 83 and also of that which is under the structure of type tip 95 (see FIG. 8C).
• An electrical isolation step is then carried out on the fluid network. This is obtained by thermal oxidation of 3 ⁇ m in thickness of the silicon of the thin layer 83. Then, by "lifting-off" of metal, the contact pick-ups 84 and 86 are made, the electrodes
• the support 81 can then be cleaved to release the pointed structure 95.
• the substrate 100 having a face 102 on which are made, by localized doping, two electrically conductive circuits.
• the first conductive circuit is formed zones 104 and 105 and the second conductive circuit is formed by zones 106 and 107.
• the substrate 100 then undergoes, from the face 102, an RIE etching (for "Reactive Ion Etching") or a chemical etching by means of KOH to obtain a recess 101 in anticipation of the tip-like structure and of the cleavage of the substrate (see FIG. 9B).
• Another silicon substrate 110 is then fixed by direct sealing on the face 102 of the substrate 100 (see FIG. 9C).
• the substrate 110 is then thinned until a thin layer 111 is obtained (see FIG. 9D).
• the fluid network is then produced as shown in FIG.
• the thin layer 111 partially or totally undergoes DRIE etching.
• the fluid network includes an inlet reservoir 121, an inlet channel 122, a microreactor 123 and an outlet channel 124.
• the etching of the thin layer 111 also defines the tip-like structure 125.
• a step of electrical isolation of the fluid network is then carried out. This is achieved by thermal oxidation.
• the purpose of the next step is to clear the contact pickups 104 and 106 (see Figure 9F). For this it is necessary to locally etch the thin layer 111 and the thermal oxide. This step can be carried out by a laser engraving technique.
• Step 9G represents the direct sealing of a cover plate 131 on the thin layer 111.
• the cover plate 131 comprises a part d end 132 overhanging so that the plate 131 does not cover the point-like structure 125. It also has a through hole 133 intended to ensure fluid communication with the inlet reservoir 121.
• the covering plate 131 may be a substrate pyrex.
• This embodiment uses an SOI substrate and a pyrex substrate (“Corning” 7740) as a cover.
• the electrodes, the conductive tracks and the electrical contact pick-ups are produced by depositing metal (aluminum, platinum, gold, etc.) and photolithography on the underside of the pyrex cover, in which they are "embedded”.
• FIG. 10A shows an SOI substrate 140 consisting of a support 141 made of silicon 500 ⁇ m thick, successively supporting a layer of silicon oxide 142 1 ⁇ m thick and a thin layer 143 of silicon 25 ⁇ m thick.
• FIG. 10B shows the device obtained after a step of DRIE etching of the thin layer 143. The etching makes it possible to produce the fluid network.
• This comprises an inlet reservoir 151, an inlet channel 152, a microreactor 153 and an outlet channel 154.
• the etching of the thin layer 143 is also carried out on two edges of the substrate 140 until the layer is revealed of oxide 142. It makes it possible to define the structure of the tip type 155.
• This etching step is conventional in microtechnology. She uses a 5000 ⁇ thick silicon oxide mask produced in an oven at 1050 ° C in a humid atmosphere. A 1.3 ⁇ m layer of “Shipley S 1813 SP15” photosensitive resin is then spread on “SVG” track (adhesion promoter: HMDS vapor). The IX motifs are exposed, then developed with “Shipley MIF 310” on the “SVG” track. The oxide mask can then be engraved in RIE
• FIG. 10C which shows the point-like structure 155 overhanging. This figure also shows that the oxide, previously revealed on the other edge of the substrate 140, was eliminated during the etching to reveal the edge 144 of the support 141.
• electrical isolation from the network fluidics is obtained by thermal oxidation. This oxidation takes place in an oven at 1150 ° C in a humid atmosphere.
• FIG. 10D represents the direct sealing of a covering plate 161 on the thin layer 143.
• the covering plate 161 has an end portion 162 overhanging so that the plate 161 does not cover the point-like structure 155. It also includes a through hole 163 intended to ensure fluid communication with the inlet reservoir 151.
• the cover plate 161 may be a pyrex substrate.
• the plate 161 comprises, on the face intended to come into contact with the thin layer 143, a metal track 164 arranged so that its internal end 165 serves as an electrode for the outlet channel 154 and that its external end 166 serves as an electrical contact resumption.
• This direct sealing step is carried out at 400 ° C.
• the structure of the pyrex cover (engraving and "inlaying" of the metal track) is done according to the following technological stages: • Realization of the engravings for obviously cutting and "box for metal track”: - Cr / Au / Cr / Au deposit (50 A / 3000 ⁇ / 50 ⁇ / 3000 ⁇ ), spreading of photosensitive resin "Shipley S 1813 SP15" on track “SVG", thickness 1, 3 ⁇ m, - exposure of IX patterns and cutout recess, - development on track "SVG” with the developer "Shipley MIF 319", - KI / I 2 etching, - Cr etching with the solution called “Cr Etch”, - removal of the resin by “stripping” using "Posistrip” or HNO 3 fuming, spreading of photosensitive resin "Shipley S 1813 SP15” on track “SVG", thickness 1.3 ⁇ m, exposure of patterns IX ⁇ recess cutout and metal track box ', - development on “S
• FIG. 11A shows a first silicon substrate 170 having a recess 171 in anticipation of the tip type structure and of the cleavage of this substrate.
• the recess is obtained by RIE, DRIE or KOH engraving.
• FIG. 11B shows that a second silicon substrate 180 has been fixed to the etched face of the substrate 170. This fixing has been obtained by direct sealing.
• FIG. 11C shows that the second substrate has been thinned to give a thin layer 181 of silicon.
• the fluid network is then produced as shown in FIG. 11D. During this step, the thin layer 181 undergoes DRIE etching.
• the fluid network includes an inlet tank 191, an inlet channel 192, a microreactor 193 and an outlet channel 194.
• the etching of the thin layer 181 also defines the tip-like structure 195 and makes it possible to reveal the edge 184 of the substrate 170.
• an electrical isolation step is then carried out on the fluid network. This is achieved by thermal oxidation.
• FIG. 11E represents the direct sealing of a covering plate 201 on the thin layer 181.
• the covering plate 201 has an end portion 202 overhanging so that the plate 201 does not cover the point-like structure 195. It also includes a through hole 203 intended to ensure fluid communication with the inlet tank 191.
• the cover plate 201 may be a pyrex substrate.
• the plate 201 comprises, on the face intended to come into contact with the thin layer 181, a metal track 204 arranged so that its internal end 205 serves as an electrode for the outlet channel 194 and that its external end 206 serves as an electrical contact resumption.
• FIG. 12 shows how all of the “fluid network and electrospray nose” devices 211 can be distributed on a circular substrate 210 in order to have a single object with N microfluidic devices, thus facilitating use "high speed” analyzes.
• the fluidic networks are drawn radially, along the radii of the circular substrate 210.
• N nose of electrospray are then distributed according to the circumference of the substrate, and it suffices to rotate it manually or automatically to carry out a sequence in mass spectrometer series 212.
• the substrate support can be mounted on a rotary axis. The preparation of the samples can be carried out beforehand in parallel on the N devices.
• ElectroSpray Ionization As an example, we can cite the analysis of samples in the biomedical and pharmaceutical industries: - genetic, proteomic analyzes (identification of proteins, etc.), - drug development.

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