Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1065—Multiple transfer devices
• • 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
  • B01L3/0262—Drop counters; Drop formers using touch-off at substrate or container
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1034—Transferring microquantities of liquid
  • G01N2035/1037—Using surface tension, e.g. pins or wires
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N35/1065—Multiple transfer devices
  • G01N35/1074—Multiple transfer devices arranged in a two-dimensional array
• • 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
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/2575—Volumetric liquid transfer

Definitions

• the present invention relates to the field of controlled sampling and deposition of substances by a similar device or electromechanical system, in particular for the deposition on a surface of a large number of molecules of different chemical or biological products (DNA solutions, proteins, chemical reagents or others), and relates to a device for sampling and depositing on a surface of solutions in very small quantities, in the form of a network (s) of high density microdrops.
• the known devices carrying out the deposition by projection do not make it possible to control the shape of the projected drop; nor precisely the quantity of substance deposited per drop.
• such a projection can, in certain cases, lead to the bursting of the projected drop and therefore to the contamination of neighboring drops and / or of the surface extending between said deposited drops. This technique is therefore not suitable for a quantitatively controlled high density deposition.
• the subject of the invention is an apparatus for sampling and depositing on a surface, in the form of microdrops, of solutions, in particular chemical or biological, comprising at least one micropipette or deposition point mounted on a movable head according to at least one direction, at least between one or more sampling site (s) and one or more deposit site (s), characterized in that the micropipette (s) are hollow and are mounted in the body of said head, with the ability to relative translation with respect to the latter, by means of a device for controlling and limiting the application forces of the micropipette (s) on the deposition surface, associated with a device for guiding the latter (s) with respect to to said head, during or with a view to bringing them into contact with said surface.
• sampling and deposit sites will advantageously be arranged in a flat matrix arrangement on plates or in respective respective receptacles.
• the movable head could, for example, be movable in two perpendicular directions, by being mounted on a fixed support structure, and the plates or receptacles receiving said sites can then be independently movable in directions parallel to each other and perpendicular in plane containing the two directions for moving the movable head or the micropipettes mounted on the latter.
• the head may be supported by a structure which is itself movable in a direction perpendicular to the plane. containing the two directions of movement of the movable head (for example an arm or a gantry displaceable in translation), the plates or receptacles then being fixed.
• said plates or receptacles When the head is only movable in one direction, provision may be made for said plates or receptacles to be movable in two directions perpendicular in a plane orthogonal to the direction of movement of the head or for said plates or receptacles to be movable in one direction and that the support structure of the head is also movable in another direction, the three directions of movement being perpendicular two by two.
• the movable head will have three degrees of freedom relative to the collection site (s) and / or the storage site (s).
• each micropipette has a body, cylindrical or not, traversed longitudinally by a channel and having a tapered end, for example of substantially conical shape, ending in a terminal, annular and planar contact surface.
• the tapered end has, before opening on the terminal contact surface, a connection surface providing a continuous passage between the conical surface of the end tapered and the planar contact surface, advantageously generated by a portion of parabolic curve.
• the micropipette could, for example, have a cylinder body which tapers conically at its free or deposit end.
• each micropipette can be provided, at its free end or deposit, with a substantially annular protuberant formation, circumferentially continuous or not, surrounding the tapered end and separated from the latter by an annular recess zone formed or formed in said end of the body of the micropipette in question, said protruding annular formation ending in a plane terminal contact surface, situated in a parallel plane, and if necessary coinciding, with the plane comprising the contact surface of the tapered end .
• These contact surfaces can be offset from one another in the longitudinal direction of the body of each micropipette and, in this case, the deposition sites are located in corresponding recesses or recesses.
• the guide device specific to each micropipette or common to all the micropipettes, can provide only simple guidance at a single guide surface but, to obtain better precision of movement and positioning, it will advantageously ensure double guiding of the body of each micropipette, at the level of two guiding zones in translation spaced apart relative to the movable head in the body of which it is mounted (clearance of an intermediate space for sensors and / or actuator elements).
• each control and force limitation device consists of a passive compliant device, such as an elastically flexible or compressible intermediate element, ensuring controlled transmission of the thrust forces and of the translational movement between the body of the movable head and the micropipette concerned.
• each control and force limitation device consists of an active device in the form of an actuator secured to the movable head, acting directly on the body of the micropipette considered or on a force transmission part formed or fixed on the latter with a view to bringing it into contact with the surface and controlled by means of a regulation and control loop also incorporating a means of direct or indirect measurement or determination of the force of application of said micropipette on the deposition surface.
• each control and force limitation device consists of a mixed device integrating an actuator controlled by a regulation and servo control loop integrating a means of direct measurement or determination or indirect and acting on the associated micropipette via a passive compliant device.
• the determination of the application force can, for example, be carried out by an indirect measurement, by observation and / or estimation.
• the quantity of liquid withdrawn and deposited by each micropipette is controlled by means carrying out a variation in pressure of a gas at the end of the micropipette opposite its free or deposition end.
• FIG. 1 is a schematic perspective view of an embodiment of the apparatus according to the invention
• Figure 2 is a view by transparency and on a different scale of a part of a micropipette forming part of the apparatus shown in Figure 1
• Figure 3 is a side elevational view in section of a portion of the movable head forming part of the apparatus shown in Figure 1 and in which a micropipette is mounted, in connection with a passive compliant device
• Figure 4 is a side elevational view in section of a portion of the movable head forming part of the apparatus shown in Figure 1 and in which a micropipette is mounted, in connection with a device for controlling and limiting d 'mixed effort
• FIG. 5 is a block diagram representing in the form of function blocks a possible structure of a regulation and force control loop which can be implemented in the device according to the invention
• Figure 6 is a schematic representation of the fluid circuit associated with the movable head forming part of the device according to the invention
• Figure 7 is a view, on a different scale, of the detail A of Figure 2 (the outline of the drop deposited is shown in broken lines)
• Figure 8 is a view similar to that of Figure 2 of an alternative embodiment of the end of a micropipette forming part of the apparatus shown in Figure 1
• Figures 9 A and 9B are representations diagrams showing the deposition of a drop on two supports presenting sites of deposit with different spacings, with a micropipette according to FIG. 8.
• the apparatus 1 for sampling and depositing on a surface 2 in the form of microdrops 2 ′ of solutions, in particular chemical or biological comprises at least one micropipette or deposition tip 3 mounted on a head 4 movable in a direction Y or Z or two different directions Y and Z, at least between one or more sampling site (s) 5 and one or more deposit site (s) 6.
• the head 4 is mounted mobile in two directions Y and Z on a support structure of the fixed gantry type 12 and the plates or receptacles 5 ′ and 6 ′ receiving the sites 5 and 6 respectively are movable independently in parallel directions X and XI , the assembly being installed on a fixed support plane 13.
• micropipette (s) 3 are mounted in the body of said head 4, with the ability to translate relative to the latter, by means of a device 7 for controlling and limiting the application forces of the micropipette (s) 3 on the deposition surface 2, associated with a device 8, 8 ′ for guiding in translation this or these latter with respect to said head 4, when or with a view to bringing them into contact with said surface 2.
• the apparatus 1 further comprises, on the one hand, an arm or a gantry 12 carrying the movable head 4 and allowing at least its displacement along an axis or the displacement of the micropipettes 3 in a plane, preferably in two orthogonal directions Y and Z and, on the other hand, a support plane 13 (of a table or of a similar piece of furniture) on which the site or sites 5 for sampling the solutions are placed, for example in the form of plates, wells or similar containers, the sites 6 for depositing microdrops in the microdrops 2 ′ of solutions withdrawn by the micropipettes 3 and a site or station 14 for emptying and washing said micropipettes 3, the arm or gantry 12 which can be moved in translation in a direction X substantially perpendicular to the plane Y, Z of movement of the movable head 4 or of the micropipettes 3 mounted on the latter.
• the apparatus 1 consists of a programmable robot, the gantry-shaped arm 12 supporting the movable head 4 moves
• Figure 1 defines the different possible axes of movement for the movable head 4, namely a longitudinal movement (axes X, XI) via the gantry 12 or plates 5 'and 6', a transverse movement (axis Y) by translation of the head on the support bar of said gantry 12, and a vertical movement (axis Z) for removal or deposition.
• the movement for the purpose of deposition can result from the only movement of the mobile head 4 (passive device 7) or from the combination of a first coarse movement of the mobile head 4 and a final fine movement of each micropipette or deposition tip 3 by an actuator 10 (active or mixed device 7).
• the deposition head 4 is advantageously made up of four sub-assemblies: the deposition points 3, a device 8, 8 ′ for precise guidance and positioning, a device 7 for controlling the contact forces and a set of capillaries and chamber pressure (not shown specifically) allowing the control of the pressure inside the deposition tips 3.
• the deposition head 4 makes it possible to deposit drops 2 ′ of predefined shape (circular in most cases) with great regularity and very good repeatability and is provided with one or more micropipettes or tips 3.
• the movable head 4 comprises a plurality of micropipettes 3 arranged in rows and columns according to a matrix structure of two dimensions. The number of points 3 must be compatible with the size and density of drops 2 * of the microarray to be produced.
• the table above gives, by way of non-limiting indication, possibilities of using heads with multiple points according to the invention.
• the deposition head 4 makes it possible to control the exact volume withdrawn from each well and to carry out an effective washing (contamination rate less than
• the deposition points 3 used are, as shown in FIGS. 1, 3 and 4, of hollow cylindrical shape (cylindrical body 3 'traversed longitudinally by a channel 3 ").
• the outside diameter D' of the body 3 '(without effect on the size of the drops) must allow a good grip and precise guidance of the tip, the latter being cut into a cone at one 3 ′′ from its ends.
• FIG. 2 shows a sketch of the realization of a deposit tip 3.
• the conical part (its specific shape and its slope do not matter) allows to reduce the diameter and to go from the outside diameter D 'to the diameter terminal D (external diameter of the terminal contact surface 3 ""), the latter being of the same order of magnitude as the diameter of the drops 2 'to be deposited (a diameter of 150 ⁇ m leads to drops of diameter of 180 ⁇ m, by example).
• the connection surface 3 '"" part of the external surface of the micropipette wetted during the contact of the latter with the deposition surface) between the tapered part 3 "" and the terminal contact surface 3 “” can be conical or have a different shape. For example, we may want to optimize the size of the droplets according to the variations in viscosity or surface tension of the solutions, which will require a particular shape and configuration of the 3 '"tapered part and the connection surface. 3 "".
• connection surface 3 "'” makes a continuous passage between the conical surface of the tapered end 3' "and the planar contact surface 3" ", advantageously generated by a portion of parabolic curve.
• connection surface 3 "'" should advantageously allow the gradual passage from the terminal 3 "" plane and cylindrical surface to the conical 3 "surface. It is therefore necessary to have a tangent connection and a gradual variation in the slope.
• the internal diameter influences two essential parameters of the deposition process, namely the contact pressure of the tip with the deposition surface and the speed of the washing process.
• the value of the internal diameter d is preferably between 0.2 and 0.8 times the value of the terminal diameter D.
• the guide device 8, 8 ′ allows the deposition tips 3 to slide freely along an axis perpendicular to the deposition surface 2. Those skilled in the art will note that only the final movement of arrival in contact with the sampling or deposition surface must be perpendicular, the prior movement possibly being a movement pivoting or a series of translational movements in different directions.
• the drop 2 ' is deposited by contact between the tip 3 and the surface 2.
• the reserve of solution for the deposit of several drops 2' is constituted by the interior volume (channel 3 ") of the tip 3 and by any additional volume (larger diameter cylinder for example) behind the tip 3.
• the guide device 8, 8 ′ may be common to all the points 3 or specific to each.
• the approach and contacting movement is obtained by controlling the Z axis of the gantry 12, by an actuator 10 specially dedicated to this task or by the combination of the two aforementioned actions (device 7 mixed).
• the device 7 for controlling the contact forces between the deposition tip 3 and the surface 2 is an essential element for obtaining quality microarrays.
• the terminal surface 3 "" of the deposition tip is flat and makes it possible to guarantee a maximum value of contact pressure (and therefore sealing) between the tip 3 and the surface 2 by controlling the interaction force tip- area.
• This force is controlled by a passive compliant device 9 or by an active device 10 (actuator), which guarantees the quality of the deposit of the drops as well as the attachment of the molecules (of DNA for example) on the surface.
• This device 7 as well as the shape of the tips 3 prevent liquid splashes on the surface 3, as well as the bursting of the drops 2 '.
• Figure 3 schematically shows a passive compliant device.
• This device 9 is constituted by a flexible element (spring blade type) or compressible element (tension-compression spring type) which exerts a force on the tip 3 or on a part 9 ′ secured to the tip, this force being regulated by an element calibration (for example screw or washer forming an adjustment shim), placed at the rear of the compliant device 9 opposite the stop 9 ′ secured to the micropipette 3 or on the same side as the latter.
• the interaction force with the deposition surface 2 is therefore a result of the initial setting force and the displacement value along the Z axis during deposition.
• Figure 4 shows an embodiment of the device 7 in the form of a mixed active device.
• This device consists of an actuator 10 which exerts a force on the tip 3 or on a piece 9 'integral with the tip 3. This force is exerted either directly (not shown), or by means of a compliant device 9 (spring type) mounted between a force transmission part 10 'on which the actuator 10 and the part 9' act.
• the force for movement and contacting is created by an actuator 10 (electric, pneumatic or hydraulic).
• an electromagnetic actuator for example an electromagnet or a linear motor
• an electronic control device (not shown) makes it possible to control the force exerted.
• the force is measured either directly by a sensor 11 inserted between the compliant device 9 and the tip 3, or indirectly by an observer model and the measurement of deformation of the compliant device.
• the force control makes it possible to control the force of interaction with the surface 2 and in particular its maximum value. This action guarantees that the surface, its coating or any other product that may have been deposited on it, or the tips 3 themselves, is not damaged.
• the control is performed either by a purely electronic analog device, or by a digital microprocessor system or by both at the same time.
• the instruction is drawn up as a function of the task to be carried out (microarray) and of the material or materials deposited on the deposit surface 2 or constituting the latter.
• the compliant device 9 makes it possible to absorb the shock upon contact. In the absence of a compliant device 9, the shock is absorbed by an appropriate choice of the control setpoint.
• FIG. 5 details the various functions relating to the assembly for regulating and controlling the contact forces.
• the value of the contact force is obtained either by the direct measurement of this one, or indirectly by the determination using an equation of mechanical behavior and the measurement of a quantity of the same type or of different nature . It is for example possible to determine the contact force by measuring the displacement of the tip and the dynamic equation of behavior of the mobile part (the determination technique can be based on an estimator or on an observer).
• the stage supplying the actuator is for example a power amplifier (for example a linear stage known as "push-pull", a stage known under the designation PWM, etc.).
• the regulation and control loops are composed of the functions of measurement and / or observation / prediction, filtering, comparison, correction and generation of setpoint signals (command).
• the functions of observation / prediction, filtering, comparison, correction and generation of setpoint signals can be performed in a known manner by a digital microprocessor device, or by an analog solution.
• One solution may consist in separating the two functions, that is to say providing a first surface 3 "" carrying out the deposition and a second surface 17 'carrying out the limitation of the normal movement of the micropipette 3 considered.
• FIGS. 8, 9A and 9B illustrate an embodiment of a micropipette corresponding to the solution indicated above, implemented with plates comprising sites or deposition zones 6, 19 and sites or support zones 20 .
• Zone 19 is the surface for depositing chemical or biological products.
• Zone 19 is therefore covered with the same adherent products (polylysine, silane, etc.) as usual in the production of microarrays.
• Zone 20 receives no deposit of product and has the function only to stop the movement along the axis perpendicular to the deposit surface.
• the micropipette 3 has in this case a particular shape adapted to the deposition operation. It has an additional contact or connection surface 17 ′ which comes into connection with zone 20.
• the contact pressure between the micropipette and zone 20 is limited by the same devices 5 to 7 as those described above.
• the terminal surface 3 "" of the micropipette 3 is designed so that it is then close to or just in contact with the deposition zone 19. It is of course necessary to provide a level difference between the surfaces 3 "" and 17 'compatible with the difference in level of the receiving surfaces 19 and 20.
• the central part of the micropipette 3 has the same characteristics as those described in relation to FIG. 2, namely a terminal surface 3 "", a conical surface 3 "", a substantially cylindrical surface 3 'and possibly a connecting surface 3' "". It is also always crossed by a 3 "circulation channel leading to the 3" "surface.
• the zones 19 and 20 can be located in mutually offset planes, as can the surfaces 17 'and 3 "".
• the zone 18 forms a hollow clearly separating the surface 3 '"from the surface 17' and has the essential role of preventing the liquid wetting the surface 3" "from wetting the surface 17 '.
• FIG. 9A shows the position of the micropipette 3 during a deposition near a previous deposition. In this case, the hollow is chosen with a depth greater than the height of the deposits previously formed.
• FIG. 9B shows the case of a deposit with partial overlap of the previous deposit by the micropipette 3.
• a particular device is developed to allow the cleaning of the micropipettes 3. Another device is used to remove the liquid wetting the surfaces 17 ' and 18 after a sample.
• a capillary / associated pressure chamber assembly makes it possible to connect the deposition tip (s) to a pump (peristaltic or other) and makes it possible to circulate fluids (water, solvent, air, gas) inside the tips. It thus performs a double function: effective cleaning during washing (described below) and precise control of the quantities of liquid handled (which is particularly important when sampling solutions of biological or chemical compounds), thus allowing save the solutions.
• FIG. 6 shows a possibility of making a fluid circuit associated with the movable head 4.
• the pressure chamber 15 makes it possible to balance the pressures when several micropipettes or tips 3 are controlled by the same pump channel.
• the deposit points 3 are connected to the pressure chamber 15 by flexible connection pipes 15 ′ and can optionally be connected to one another by connection tees 15 ".
• the pressure chamber 15 can be a specific connection element or be made up of the connecting pipes themselves.
• the role of the pressure chamber is to make the pipes connected to the tips 3 communicate with the pumps 16 or the fluid storage tanks 16 ′ (under pressure or not).
• the control unit (computer) are opened depending on the washing or deposit processes.
• the electro-valve EV1 controls the pressure or vacuum of the chamber and the connecting pipes via a gaseous fluid.
• the solenoid valve EN2 controls via a liquid fluid the pressure or vacuum of the chamber and the connecting pipes. The liquid fluid is mainly used during the washing process.
• the solenoid valve EN3 allows to connect the chamber and the connecting pipes at atmospheric pressure and thus to balance the pressures on either side of the column of solution contained in the tips
• the subject of the invention is also a method of depositing high density networks of microdrops of solutions on a surface by means of the apparatus described above, characterized in that it consists in bringing the mobile head 4 to a sampling site 5 , to collect, by soaking the ends of the micropipettes 3 in wells or similar containers of a sampling site 5, a determined quantity of the solutions present in these containers, to transfer the mobile head 4 to a deposit site 6, deposit, in the form of a network of microdrops 2 ', a determined quantity of the solutions taken from the deposition surface 2 by pressure-controlled support of said pipettes 3 on said surface 2, possibly repeating this deposition operation one or more times by successively moving the movable head 4 towards one or more other deposition sites 6, at displacing the movable head 4 to the emptying and washing station 14 and to clean the micropipettes 3 and the other elements which have been in contact with the sampled solutions, with a view to their decontamination, and to repeat all of the above operations until exhaustion solutions to be sampled and / or
• the washing or cleaning of the tips 3 can be broken down into several stages: - the rejection of the solutions contained in the tips,

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Clinical Laboratory Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Sampling And Sample Adjustment (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Devices For Use In Laboratory Experiments (AREA)
• Automatic Analysis And Handling Materials Therefor (AREA)

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• 2001
  • 2001-02-23 FR FR0102509A patent/FR2821284B1/fr not_active Expired - Fee Related
• 2002
  • 2002-02-22 EP EP02706891A patent/EP1361925A2/de not_active Withdrawn
  • 2002-02-22 WO PCT/FR2002/000669 patent/WO2002068121A2/fr not_active Ceased
  • 2002-02-22 CA CA002438859A patent/CA2438859A1/fr not_active Abandoned
  • 2002-02-22 AU AU2002241057A patent/AU2002241057A1/en not_active Abandoned
  • 2002-02-22 US US10/468,871 patent/US20040141883A1/en not_active Abandoned

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