Classifications

• • 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
  • B01L3/02—Burettes; Pipettes
  • B01L3/0241—Drop counters; Drop formers
  • B01L3/0244—Drop counters; Drop formers using pins
• • 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
  • B01L3/02—Burettes; Pipettes
  • B01L3/0241—Drop counters; Drop formers
  • B01L3/0244—Drop counters; Drop formers using pins
  • B01L3/0248—Prongs, quill pen type dispenser
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/12—Specific details about manufacturing devices
• • 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
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
• • 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
  • B01L2300/16—Surface properties and coatings
  • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0433—Moving fluids with specific forces or mechanical means specific forces vibrational forces
  • B01L2400/0439—Moving fluids with specific forces or mechanical means specific forces vibrational forces ultrasonic vibrations, vibrating piezo elements
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T29/00—Metal working
  • Y10T29/49—Method of mechanical manufacture
  • Y10T29/49995—Shaping one-piece blank by removing material
  • Y10T29/49996—Successive distinct removal operations

Definitions

• the present invention relates to a device for localized and actively controlled deposition of at least one biological solution in the form of micro-drops.
• dippen pen lithography is a technique derived from atomic force microscopy and which makes it possible to form patterns on a surface using a diffusion effect by molecular transport at the level meniscus of water that forms between the tip of an atomic force microscope and the surface on which the deposit is made.
• the operating principle is based on the difference in hydrophilicity or wettability properties of the tip and the surface.
• the surface must indeed be more hydrophilic than the tip to generate a molecular diffusion from the tip to the surface.
• the resolution obtained can be less than a micron and it is also possible to envisage the deposition of different biological molecules but this supposes carrying out a change of the tip (which will have been previously immersed in the solution to be deposited) for each solution.
• microsystems have also been proposed for making deposits for the manufacture of biochips. These are generally microfluidic structures, for example the one described in the following article:
• micromachined silicon structures with microfabricated channels are micromachined silicon structures with microfabricated channels, and their use is in all respects comparable to that of an inkjet system.
• These “closed” tube-shaped structures are very difficult to clean, which constitutes an obstacle to the use of the same device for depositing droplets of different liquids.
• electrospray for depositing controlled by an electric field adjustable very small amounts of organic molecules.
• electrospray consists of applying an electric field high enough to ionize and atomize the liquid to be deposited.
• the droplets thus produced have sub-micrometric dimensions and evaporate before waiting for the deposition surface; in this way, thin films are produced. It is therefore a problem different from that targeted by the present invention, that is to say the deposition of droplets of a volume of the order of a picolitre or ferntolitre.
• the devices for electrospray consist of micropipettes containing a needle-shaped electrode, so they cannot be washed effectively and must be replaced each time the liquid is changed.
• the present invention achieves these objectives by the use, as a deposition system, of one or more silicon micro-levers comprising at least one electrode allowing the liquid to be deposited to be manipulated by electrostatic effects.
• An object of the invention is a deposition device allowing precise localized deposition and actively controlled microdrops, in particular of diameter less than 10 microns, and more particularly of diameter of the order of 1 micron.
• Another object of the invention is a deposition device allowing precise and actively controlled localized deposition of microdrops on microstructures such as bridges, beams or membranes.
• Another object of the invention is a deposition device making it possible to deposit different biological molecules.
• Another object of the invention is a deposition device for depositing microdrops without contact with the structure or microstructure on which the deposition takes place.
• Another object of the invention is a deposition device making it possible to deposit microdrops by contact with a structure or a microstructure, under conditions which preserve the integrity of the structure or of the microstructure.
• At least one of the abovementioned objectives is achieved using a device for depositing biological solutions comprising at least one planar silicon lever having a central body and an end region forming a point in which a slot or a groove, characterized in that it has at least one metal track formed on one face of the central body and at least partially skirting a said slot or groove.
• Said slot or groove advantageously extends from said tip to a reservoir formed in the central body.
• said metal track or tracks at least partially run along said tank.
• the reservoir is a non-opening cavity formed from a main face of the central body.
• the tank is constituted by a through opening formed between two opposite main faces of the central body.
• a said slot or groove and / or a said reservoir and / or a said metal track is optionally coated with Si0 2 .
• the lever advantageously has at least one hydrophobic region made of silicon or else of silicon oxide coated with hydrophobic silane.
• the device has at least one implanted piezoresistor.
• the or each lever has at least one integrated actuator making it possible to control its bending.
• said actuator comprises a piezoelectric layer deposited on a surface of said lever.
• said actuator comprises a metal bimetallic strip and a heating resistor deposited on a surface of said lever.
• the invention also relates to a method of manufacturing the device as defined above, characterized in that it implements: a) at least one deposit of silicon oxide on a front face of a silicon on insulator substrate having a buried insulating layer, b) producing for each lever at least one metal track. c) at least one chemical attack or ion etching by the front face of the silicon substrate to define the contour of the levers, and at least one slot or groove, the contour of the levers being defined by chemical attack or ion etching up to the insulating layer buried, d) chemical attack or ion etching by the rear face of the substrate to remove including the buried insulating layer and release at least one lever.
• the method can be characterized in that b also comprises: b1) a second oxide deposit on the front face to isolate at least one metal track.
• the method can be characterized in that c comprises a chemical attack or ion etching up to the buried insulating layer to define, in addition to the contour of the levers, a slot and / or a through opening constituting a reservoir for at least one lever.
• the method can be characterized in that c comprises a first chemical attack or ion etching of the substrate which is stopped before the buried insulating layer to define at least one groove and / or one non-emerging cavity forming a reservoir, for at least one lever and a second chemical attack or ion etching of the substrate, up to the buried insulating layer to define at least the contour of the levers.
• the first chemical attack or ion etching can be carried out so that the contour of the levers is defined over part of their thickness.
• a step of implanting at least one piezoresistor is provided.
• the method also includes a step of depositing an integrated actuator.
• said step of depositing an integrated actuator comprises depositing by cathode sputtering a piezoelectric film of PbZr0 3 / PbTi0 3 .
• Said piezoelectric film is advantageously isolated from the liquid by a layer made of a material chosen from: silicon oxide, PTFE called "Teflon", a polymer.
• said step of depositing an integrated actuator comprises chemical deposition at low pressure
• LPCVD LPCVD of a layer of Si 3 N followed by an evaporative deposition of a layer of Cr and a layer of Au to achieve a heating resistance, thus forming a metallic bimetallic strip.
• the invention also relates to a method for sampling at least one biological solution using a device as defined above, characterized in that the sampling and the retention of said biological solution are assisted by electric field effect by applying a difference potential between said metal tracks.
• a measurement of the variation of the electrical resistance of said piezoresistor is carried out after the sampling to determine the amount of biological solution sampled.
• the invention also relates to a method for depositing at least one biological solution using a device as defined above, characterized in that the deposition of said biological solution is assisted by electric field effect by applying a potential difference between said metal tracks, which are maintained at the same potential, and a deposition surface comprising at least one conductive layer.
• a device comprising a piezoresistor advantageously a measurement of the variation in the electrical resistance of said piezoresistor is carried out after deposition to determine the amount of biological solution deposited.
• the invention also relates to a method for depositing at least one biological solution using a row of devices as defined above, each comprising a piezoresistor and an integrated actuator, characterized in that the contact force of each lever with the deposition area is determined by measuring the variation of the electrical resistance of each piezoresistor implanted and actively monitored by each integrated actuator.
• FIG. 5 illustrates a sectional view VI-VI of a variant lever having an integrated piezoresistor
• FIG. 6A and 6B illustrates a sectional view VI-VI of two other lever variants having an integrated actuator
• FIGS. 7A and 7B illustrate a device consisting of a set of identical levers forming a row.
• FIG. 8A to 8J illustrate a method of manufacturing levers according to the invention.
• FIG. 9A-9D illustrate the different methods of loading and depositing a liquid.
• the levers are preferably of rectangular shape (central body 1) terminated by a triangular end 2 forming a point 3.
• a rectangular tank 6 or 7 can be inserted at the upper end of channel 4 or 5.
• Two metal tracks 8 and 9 run along channel 4 or 5 and / or tank 6 or 7.
• the geometric dimensions of the levers can be as follows:
• Lever length 1 to 2 mm
• Width 100 ⁇ to 300 ⁇ , for example 210 ⁇ m
• Thickness 1 to 20 ⁇ m (depending on the thickness of the starting SOI substrate)
• Channel width 2 to 20 ⁇ m, for example 5 ⁇ m
• Tank length 200 to 600 ⁇ m, for example 250 ⁇ m
• Width of the tank 50 to 150 ⁇ m, and for example 80 ⁇ m
• Width of the conductive tracks 1 to 40 ⁇ m, and for example
• the channel can be a groove 4 formed over a part of the thickness of the lever from a surface 11 or a through slot 5 which extends between the faces 1 1 and 12.
• the channel can communicate with a non-opening reservoir constituted by a cavity 6 formed from a main face 1 1 of the central body 1 of the lever, or else with a through reservoir 7 constituted by an opening 7 formed between the main faces 11 and 12 of the central body 1.
• FIGS. 1A and 1B illustrate the case of a slot 5, FIGS. 2A and 2B, of a slot 5 and of an opening tank 7, FIGS. 3A and 3B illustrate the case of a groove 4 and of a non-opening tank 6, and finally FIGS. 4A and 4B illustrate the case of a slot 5 and a non-opening tank 6.
• the case (not shown) of a lever having a groove 4 and a open tank 6 can also be implemented.
• the metal tracks 8 and / or 9 run along the reservoir 6 or 7 (FIGS. 2A, 2B, 3A, 3B, 4A and 4B) and / or the groove 4 (FIGS. 3A, 3B) and / or the slot 5 (FIGS. 1A, 1 B, 2A, 2B, 3A and 3B).
• a single metal track 8 or 9 may be present.
• an actuator constituted by a piezoelectric layer 38 (figure 6A) or a metallic bimetallic strip comprising a layer of Si 3 N 33, a layer of Chromium 35 and a layer of Gold 37 (figure 6B ).
• a piezoresistor 31 Figure 5
• Both the piezoresistor 31 and the actuator 33-35-37 or 38 are isolated from the liquid by a passivation layer 32.
• the device according to the invention makes it possible in particular: a) A reduction in the volumes deposited: the deposits made with the present system have, for example, a diameter of the order of 10 microns (picoliter), this characteristic being moreover configurable; obtaining microdrops of the order of 1 ⁇ m in diameter (femtoliter) is possible and makes the device compatible with nanotechnology type approaches which are currently emerging (depositing drops on nanosensors in particular); and b) the possibility of actively controlling the loading and deposition of the liquid via the metal tracks 8 and / or 9, used as electrodes to exploit the effects of electrowetting, dielectrophoresis and electrospray; and / or c) the possibility of depositing a wide variety of organic biological materials (DNA, proteins, cells, etc.) or inorganic (polymers, photosensitive resins, etc.) and / or d) Possible use of very small volumes.
• the deposits made with the present system have, for example, a diameter of the order of 10 microns (picoliter), this characteristic
• FIG. 7B shows, for example, a row in which the first lever is flexed towards the deposition surface by the action of said integrated actuator, the second is flexed in the direction opposite to said surface to avoid contact and the third is left in its rest position.
• the arrows F1 and F2 indicate the direction of movement of the tip induced by the integrated actuator in the case of the first and second lever respectively.
• thermomechanical actuation (bimetal ).
• the process for manufacturing levers for deposition is based on the collective manufacturing techniques of microelectronics. A series of technological steps is carried out on a silicon on insulator substrate (SOI: Silicon On Insulator).
• the first part of the process includes a succession of thin layer processing ( Figures 8A and 8C), and the second part consists of a series of micro-machining operations in order to define the levers.
• the first step is a deposition of silicon oxide 22 by LPCVD (chemical vapor deposition at low pressure), on the front face 21 of a silicon substrate 20 having a buried oxide layer 30.
• the oxide layer 22 serves as an insulator between the substrate and the following metallizations.
• an under-stripping makes it possible to produce the metal tracks 25, namely a photolithography followed by a metallic deposit 25 by evaporation then a removal of the resin (which was used for masking the metallized regions) in acetone and with the application of ultrasound, and finally an annealing of the metallization.
• the last step of the thin film part is a second localized deposit 26 of silicon oxide (FIG. 8C) by LPCVD to isolate the metallizations from the liquid when using the levers, followed by photolithography to access the contact pads. metallizations by chemical attack of silicon oxide.
• photolithography on the front side in the silicon layer 27 makes it possible to define the contours of the levers.
• a first plasma etching (reactive ion etching or RIE) is then carried out for silicon oxide and then a second plasma etching is carried out for monocrystalline silicon (FIG. 8D).
• levers with through channel slot 5 crossing the entire thickness of the lever
• a single step is sufficient (as shown in FIG. 8D) by stopping the etching of the silicon on the oxide layer of the substrate on oxide SOI.
• this oxide (FIG. 8A) is deposited then opened by chemical attack at the level of the contacts of the piezoresistor and a metallic deposit is produced (FIG. 8B) by a pickling, which takes into account the tracks serving as electrodes and the tracks for the piezoresistors.
• a pickling which takes into account the tracks serving as electrodes and the tracks for the piezoresistors.
• One or more piezoresistors installed on at least some of the levers make it possible to have one or more strain gauges whose variation in resistance makes it possible to detect in particular the contact of the lever with a surface. This makes it possible in particular to ensure adjustment of the coplanarity of the levers during a collective filing.
• a piezoelectric film 30, for example consisting of a mixture of PbZr0 3 and PbTi0 3 in a ratio 54/46 can be deposited by sputtering, as described in:
• the deposit can be made for example on the rear face of the lever, as illustrated in FIG. 8F. Alternatively, it can be carried out on the oxide layer 26 which covers the metal tracks 25 as illustrated in FIG. 8G.
• the piezoelectric actuator must be isolated from the liquid by a layer 32 of oxide or of any material making it possible to ensure effective insulation: PTFE called “Teflon”, polymer (PDMS, resin, etc.).
• Teflon polymer
• the actuator can consist of a metal bimetallic strip.
• Figures 8H-8L show the different stages of making such a device. First, a layer 33 of Si 3 N is deposited by a chemical low pressure vapor deposition process
• LPCVD chromium
• FIG. 8H a layer 35 of chromium (figure 81) and a layer 37 of gold to constitute the heating resistance (figure 8L), thus forming a bimetallic strip, are deposited by thermal evaporation.
• a layer of doped polycrystalline silicon can also be used as a heating resistor. There follows a lithography step to define the contours of these elements, the deposition of an insulating oxide layer and making the electrical contacts of the heating resistor.
• the metal tracks constitute the heart of the invention, because they make it possible to control the rise of the liquid in the slot or groove during filling of the device, and its fall during the deposition by field effect.
• a first technique consists in using an alternating electric field to confine a polarizable liquid (water for example) in zones of strong electric field (the use of a continuous field is possible, but can induce annoying effects, such as liquid electrolysis or damage to biomolecules).
• a polarizable liquid water for example
• the liquid literally "plates” on the electrodes.
• a completely similar effect but whose physical origin is different, occurs for conductive liquids.
• a liquid can be “conductive” or “dielectric” depending on the frequency of the electric field applied to it. If, in the frequency range considered, the liquid constitutes a dielectric, the electrodes may not be coated with insulation.
• electrowetting makes it possible to modify the wettability properties of a surface (contact angle between the surface and the liquid) by applying a potential difference between said surface and the liquid. , and thus control the capillary effects. If a potential difference of a few Volts at 10 V is applied between the electrodes and a conductive surface, the field effect can induce contactless deposition. A higher potential difference (beyond kV) can induce electrospray.
• Such a treatment consists, for example, of a hydrophobic silane bond, for example a silane having a methyl or fluorinated group as a termination, which is deposited on silicon oxide.
• This compound is deposited on silicon oxide in the form of self-assembled monolayers and has the advantage of being highly hydrophobic.
• the techniques for creating residual charges in the oxide by implantation or irradiation technique can be envisaged to increase the wettability or hydrophilicity properties of the passivation layer (layer of cold oxide for example).
• the surface of the device is made highly hydrophobic and the loading of the liquid is carried out by virtue of the dielectrophoresis and electrowetting effects mentioned above. In this way, cleaning of the device is facilitated and the deposition of several different liquids without contamination is made possible.
• a three-axis micro-robot (X, Y, Z) makes it possible to use the microlevers according to the invention for the filling and deposition phases.
• this involves immersing the microstructures in a tank containing the solution to be deposited and filling the micro-channels by field effect, possibly assisted by capillarity.
• the micro-robot makes it possible to position the microstructures very precisely relative to a surface intended to receive the deposit.
• the deposition is then carried out by direct contact with the surface or by non-contact field effect.
• the electrospray deposition technique is also conceivable insofar as the applied field is large enough to generate a nebulization and an atomization of the biomolecules.
• the robot is for example a commercially available three-axis robot X, Y, and Z, with a pitch of 50 nanometers, largely compatible with a diameter of deposits to be produced of the order of 10 to 20 microns. This precision allows fine control of the lever-deposition surface contact, thus giving better volume uniformity of the spots produced.
• a further improvement in contact control is obtained by the use of an actuator, for example piezoelectric or thermomechanical, integrated in the microstructure.
• an actuator for example piezoelectric or thermomechanical
• the integrated actuators make it possible to individually control the contact of each device with the surface.
• the integrated piezoresistors allow the robot and said actuators to be controlled.
• each axis The movement along each axis is ensured by a stepping motor.
• Each motor supplied with alternating current, is associated with a linear position sensor allowing a closed-loop position control.
• the angle of incidence that is to say the angle of contact between the lever and the surface on which the deposition is carried out, has a notable influence on the size of the drops deposited. The most satisfactory results are obtained with an angle close to 60 °. It should be noted that, during the contact phase, this angle varies from 60 ° to 45 ° for a descent of the lever after contact of 50 microns (for the value of the descent distance of the lever after contact, we will adopt for the following the term "contact depth"). The greater or lesser bearing force thus varies the volume of liquid deposited.
• the angle is made variable thanks to a moving part fixed on the Z axis and in rotation relative to the Y axis. It is possible to control this angle directly from microcontrollers connected to the control system.
• the deposition can be carried out in the following manner, as illustrated by FIGS. 9A-9D.
• the first step ( Figure 9A) consists of filling the channel and the reservoir (where it exists) machined in the axis of the levers.
• the control software makes it possible to position the levers above the tank containing the liquid to be deposited and to immerse them in this liquid.
• An electric field is then created by applying a voltage between the electrodes machined on the levers and the liquid; the levers are then moved outside the liquid and the robot positions them above the location of the first deposit to be made.
• the volume deposited depends on the depth, the angle and the contact time.
• the field effect can also be used to control the volume of the deposit: a decrease in the electric field between the conductive tracks increases the quantity of liquid deposited, and vice versa.
• the deposition is controlled individually for each lever thanks to the integrated actuators, which act on the characteristics of the contact, and to the electrodes.
• a potential difference of some volts at 10 V is applied between the metal tracks and the deposit surface, which must be conductive, or include a conductive coating; the field effect (dielectrophoresis) thus induced sucks up the liquid.
• a higher potential difference (beyond kV) can induce electrospray.
• This process is repeated for each set of drop-off points, according to a programming established by the user, until the number of points that can be made without recharging is reached. If this happens, the robot interrupts the deposit task and resumes that of loading with liquid.

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• Clinical Laboratory Science (AREA)
• Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Health & Medical Sciences (AREA)
• Micromachines (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Automatic Analysis And Handling Materials Therefor (AREA)
• Steroid Compounds (AREA)
• Peptides Or Proteins (AREA)
• Physical Or Chemical Processes And Apparatus (AREA)
• Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
• Media Introduction/Drainage Providing Device (AREA)
• Infusion, Injection, And Reservoir Apparatuses (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

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