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
  • B01L3/50—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
  • B01L3/502738—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 integrated valves
• • 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
  • B01L3/502715—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 interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • 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
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0621—Control of the sequence of chambers filled or emptied
• • 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
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
• • 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
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
  • B01L2200/0668—Trapping microscopic beads
• • 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
  • B01L2300/04—Closures and closing means
  • B01L2300/041—Connecting closures to device or container
  • B01L2300/045—Connecting closures to device or container whereby the whole cover is slidable
• • 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
  • B01L2300/087—Multiple sequential chambers
• • 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
  • B01L2300/18—Means for temperature control
• • 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
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • 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
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
  • B01L2400/065—Valves, specific forms thereof with moving parts sliding valves
• • 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
  • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
  • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
• • 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
  • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
  • G01N2035/00099—Characterised by type of test elements
  • G01N2035/00158—Elements containing microarrays, i.e. "biochip"

Definitions

• the present invention relates to a device for preparing and / or treating an organic sample.
• Such a device is intended in particular for use in the context of the automation of biological protocols, particularly complex biological protocols.
• such a device can be applied to the detection of pathogens or molecules, nucleic acids or proteins, of a pathogen.
• Such a biological protocol should preferably be performed in a low cost consumable device, related to the detection module, and which is changed between each test.
• This consumable can be inserted into a treatment apparatus containing the expensive components, for example mechanical or optical components.
• a first known device used in particular by the company Genpoint and implementing a preparation robot marketed by the company Tecan, comprises three-dimensional displacement means of a and a plate having a plurality of wells, the wells containing either a reagent or a sample.
• the pipette is moved over the plate so as to be positioned in a well to collect a quantity of reagent, then positioned in the well containing the sample to deliver the amount of reagent in this well, this successively for reactive shell.
• Such a device has the disadvantage of using mechanical means of precision for the displacement of the pipette which have a complex structure and are difficult to transport.
• US-B-6,734,684 for its part also describes a single-use cartridge comprising a set of chambers and reservoirs.
• a single process chamber is used which can be fluidically communicated with other chambers or tanks selectively through channels in a rotatable member.
• US-B-6, 964, 862 discloses a device comprising a disposable member having walls-separated chambers for fluid communication above a predetermined pressure. Each chamber is filled with a specific fluid before closing. The communication of the fluids contained in two adjacent chambers is carried out by mechanical pressure on one of the two chambers, which causes the appearance of an opening in the separating wall.
• This last device makes it possible to simplify the realization of the communication between the rooms, and also makes it possible to limit the contamination between tests.
• the quantities of liquids used have smaller and smaller volumes. These quantities become so small that the use of single - use containers with integrated reagents is difficult. Indeed, for cost reasons, the materials used for making chambers or tanks are produced from basic and cheap plastics, such as polyolefins. These materials do not provide a long-lasting seal and have poor barrier properties without adequate treatment. Thus, a diffusion can take place through the walls. This results in particular changes in the concentration of reagents due to the evaporation of the solvent. Such evaporation may be neglected in the case of quantities of several hundred ⁇ , but can not be neglected in the case of reagent volumes less than 50 ⁇ , especially during high incubations and for significant periods (several hours) .
• the size of the devices themselves is reduced. It can be estimated that the size limit to allow easy handling is of the order of 10 mm. An operator can not assemble or manipulate elements of this size given the time that would be required for each operation and the risk of loss of parts of the device. It is more practical to use sets of disposable devices comprising up to several hundred devices. An automatic device will handle disposable disposable devices.
• the devices are assembled from several elements from different manufacturers. It is therefore appropriate that the structure of these devices is adapted for an assembly which must be automated given the tolerances imposed which are of the order of ten] im.
• this application relates to a device for preparing, processing and / or analyzing a biological sample
• a device for preparing, processing and / or analyzing a biological sample comprising a base and a slide, movable in translation relative to the base, comprising a set of storage chambers and / or reaction means for receiving a fluid, the chambers being separated by walls so as to constitute a set of adjacent chambers, the slide further comprising a contact surface on which open first fluidic communication means connected to the volume internal chamber, the contact surface of the slide being positioned opposite a contact surface of the base comprising at least one position at the lake lie are arranged second fluid communication means connected to detection means.
• connection between the drawer, so-called mobile part, and the base, so-called fixed part is effected by means of at least two particularly sophisticated seals since in a different material than the base or the drawer and positioned from concentrically with respect to the opening for the passage of the sample.
• the present invention aims to solve all or part of the disadvantages mentioned above.
• the present invention relates to a device for preparing, treating and / or analyzing at least one liquid, prefer! a biological sample, comprising:
• reaction support having an assembly of at least two chambers, this assembly comprising:
• At least one central channel comprising:
• an internal channel for connecting the chambers to one another so as to form a set of adjacent chambers
• an external pipe for connecting the internal pipe opening outwards, and at least one channel opening through a chamber, located substantially upstream and downstream of each chamber, said upstream channel and downstream channel,
• a slide movable in translation relative to the reaction medium, between at least two positions, and having, for each position, at least three control means adapted to cooperate in closing and / or opening the opening channels, for put into fluid communication (open control means) or not (closed control means) the internal volume of the chambers with the outside of the device.
• the reaction support consists of two parts:
• a base having means allowing the translation of the slide in translation
• reaction element comprising the chambers and channels.
• the reaction element consists of two parts:
• each drawer control means comprises a sealing means adapted to cooperate with the contact surface of the reaction medium at each position in translation.
• all the chambers, channels and means of control in fluid relation are aligned with each other for each translational position.
• the device has:
• a first means vis-à-vis the first channel
• a second means vis-à-vis the second channel
• a third means vis-à-vis the third channel.
• At least one of the chambers comprises a zone of 1 ectu re.
• the drawer has in at least one position a magnet capable of acting on at least one of the chambers for allow the separation of magnetic particles present in the liquid.
• the base comprises in at least one position a magnet capable of acting on at least one of the chambers to allow the separation of magnetic particles present in the liquid.
• the present invention also relates to an analysis apparatus adapted to use a device, as described above, which comprises:
• driving means allowing the relative movement of the slide with respect to the reaction medium, transfer means (addition, withdrawal or movement within the device) of all or part of a liquid, such as a biological sample to be treated or a liquid (s) necessary (washing liquid, elution) to the implementation of the method below, and mixing thereof, and
• control means adapted to allow smooth operation and good synchronization of the drive means, transfer means.
• control means adapted to allow smooth operation and good synchronization of the drive means, transfer means.
• control means when it is intended to allow the incubation of the biological sample further comprising heating means themselves operating under the control of the control means.
• the apparatus comprises, at the level of the transfer means, a pipette tip or a needle adapted to cooperate with each of the control means at each position.
• the invention also proposes a method of using a device, previously described, or implementing an apparatus, as disclosed above, in which:
• a liquid is introduced into the first chamber by injecting it into the second control means, opening the first control means and closing the third control means, and / or
• a liquid is introduced into the second chamber by injecting it into the second control means, opening the third control means and closing the first control means, and / or
• a liquid is introduced into the first and second chambers by injecting it into the first control means, opening the third control means and closing the second control means, and / or
• a liquid is introduced into the first and second chambers by injecting it into the third control means, opening the first control means and closing the second control means, and / or purging a liquid in the first chamber by injecting or aspirating a fluid into the first control means, opening the second control means and closing the third control means, and / or
• a liquid present in the second chamber is purged by injecting or aspirating a fluid in the second control means, opening the third control means and closing the first control means, and / or
• a liquid present in the chamber is purged by injecting or aspirating a fluid into the third control means, by opening the first means of control and closing the second control means, and / or
• a liquid is incubated in the first chamber by closing the first and second control means and applying a heat source to said first chamber, and / or
• a precise volume of a liquid is sampled at the first chamber, firstly by opening the first and second control means, closing the third control means and pushing with a fluid the liquid, via the first control means, until it overflows at the second means, finally by translating the drawer relative to the support, and / or
• a precise volume of a liquid is sampled at the first and second chambers, firstly by opening the first and third control means, closing the second control means and pushing, with the aid of a fluid, the liquid, via the first control means, until it overflows at the third control means, finally by translating the drawer relative to the support, and / or is sampled a specific volume of a liquid at the level of the first and second chambers, firstly, by opening the first and third control means, closing.
• the second control means and pushing, with a fluid, the liquid, via the third control means, until it overflows at the first control means, finally by translating the drawer by relative to the support, and / or a reciprocating movement of a liquid at the first chamber, firstly, by injecting it into the first control means, by opening the second means of controlling and closing the third control means, then, translating the slide relative to the support in a position that closes the second control means, and finally pushing, with the aid of a fluid, by the first means of at least once controlling the liquid present in the first chamber towards or in the second chamber by compressing the air trapped in said second chamber, and / or
• this other control means when purging, a liquid present in one or both of the first and second chambers by injecting a fluid by one of the control means and by evacuating it by one of the other control means, this other control means is connected to a liquid surplus collection tank via a channel.
• the present invention finally relates to a method of separation and incubation within a device, as described above, or implementing an apparatus, according to the characteristics described above, comprising the following steps:
• this liquid is incubated by keeping the control means closed and by applying a second heat source to said second chamber, allowing hybridization of the nucleic acids on the reaction chip while incubating at 40 to 80 ° C, preferably at substantially 65 ° C. C C,
• Figure 1 is a perspective view of a first device according to the invention seen from above.
• Figure 2 is a view of the device identical to that of Figure 1 but further comprising an incubator.
• Figure 3 is a partial view of the device of Figure 1, in which the drawer has been removed; there is therefore only the base of the device carrying a reported microfluidic reaction chip is present.
• Figure 4 is a sectional view along A-A of Figure 1 on an enlarged scale with respect to this Figure 1.
• Figure 5 is an enlarged view of a portion of the section along A-A of Figure 1 or the reaction chip of Figure 4 on an enlarged scale with respect to these Figures 1 and 4.
• Figure 6 is a sectional view along B-B of Figure 5.
• Figure 7 is a bottom view of the drawer constituting a part of a device according to the invention.
• Figure 8 is a sectional view along C-C of Figure 7 on an enlarged scale with respect to Figure 7.
• Figure 9 is a sectional view of a device according to the invention, combining:
• reaction chip carried by a base, not shown in this figure, as shown in FIG. 4, and
• Figure 10 is a sectional view similar to Figure 9 of said device which is in a configuration in relation to a second process step.
• Figure 11 is a sectional view similar to Figures 9 and 10 of the device which is in a configuration in connection with a third process step.
• Figure 12 is a sectional view similar to Figures 9 to 11 of said device which is in a configuration in relation to a fourth step of the method.
• Figure 13 is a sectional view identical to
• Figures 9 to 12 of the device which is in a configuration in connection with a fifth process step.
• Figure 14 is a sectional view similar to Figures 9 to 13, of said device which is in a configuration in relation to a sixth stage of the orocédé.
• Figure 15 is a representation of the denaturation curve of nucleic acids (including RNA strands) at a temperature of 90 ° C of the device according to the invention, before incubation at 65 ° C.
• Figure 16 is a representation of the temperature setting of the same device but for 3 hours, still to demonstrate the temperature stability of n.ot. invention.
• FIG. 17 is a representation of the heating of said device according to the invention for 20 minutes.
• the present invention relates to a device 1 intended to allow the treatment of a biological sample 2.
• a device 1 is well represented, for example, in FIG. 1.
• This processing device 1 comprises, on the one hand, a reaction medium 3 and, on the other hand, a drawer 4.
• the drawer 4 can slide in the support 3 according to the arrow Fl.
• a reverse movement may also be possible, or a joint movement of the support 3 and drawer 4.
• the support 3 consists of three parts, non-referenced, a lower part and two upper parts are facing. Each of the two upper parts constitutes with the lower part a shoulder which creates a slide 23.
• the drawer 4 has various channels which allow to connect non-visible chambers in this figure, as will be explained later.
• the support 3 comprises a certain number of elements on the upper face of its lower plate consisting in particular of grooves 25 which facilitate sliding along F1 and contact between support 3 and slide 4.
• Figure 2 is identical to Figure 1, but in this a reported element a. been added. This is a incubator 17 which allows the device to incubate the biological sample 2 when it is present within the device 1.
• other elements are also referenced in this FIG. 2. These include the upstream reservoir 19 and the downstream reservoir 20 for collecting the liquid surplus, the vent 21 present between the tanks 19 and 20 and the outside. There are therefore in this figure two vents, one on the left and one on the right each associated with one of the two tanks 19 and 20.
• Figure 2 has indexing holes 18 which are positioned between two channels namely the channel. upstream 10 and the downstream channel 11, which will be. better defined u 1 later.
• indexing holes 18 could be positioned elsewhere on the map or be useless because of the use of a stepping motor whose advanced corresponds to the distance between two channels for example 10 acai acents.
• indexing holes 18 may also be replaced by magnets which have a certain function in the context of the method of use of the device 1.
• FIG. 3 shows only a part of the device 1, namely the reaction support 3.
• the reaction support 3 we better see the three-element structure of said support 3, namely the lower plate and the two upper, non-referenced elements, which thanks to shoulders constitute with this lower plate the two slides 23.
• the reaction element or reaction chip 15 which is placed i) in place within the support 3 on the upper part of the lower plate of it. -this .
• This chip 15 is advantageously fixed by gluing, or even simply clipped or set on the upper face 15 of the base 14 of the support 3.
• the manufacture of the support 3 may be made by plastic injection, in particular from a single material. Different materials can be used for the support 3 which should preferably have the following properties:
• the position of the injection point must be chosen so as to allow satisfactory flatness and filling.
• the design of the base must take into account the deformation under the stress due to the mounting of the slide 4.
• the support 3 may be made for example of polycarbonate (PC), polystyrene (PS), polymethyl methacrylate (PMMA), polyetherimide (PEI), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), acrylonitrile-butadiene-styrene (ABS), styrene-acrylonitrile (SAN).
• PC polycarbonate
• PS polystyrene
• PMMA polymethyl methacrylate
• PEI polyetherimide
• PET polyethylene terephthalate
• PEN polyethylene naphthalate
• ABS acrylonitrile-butadiene-styrene
• SAN styrene-acrylonitrile
• the drawer 4 is also an injected plastic part that can be made in two different ways, with a single injection or a double injection of two different materials.
• the simple infection allows an easier and less expensive realization, the co-injection makes it possible to improve the robustness of the assembly.
• the drawer 4 made of a single material can be made from different polymers, such as thermoplastic polymers or thermoplastic elastomers.
• the drawer 4 is made of a single material, which allows of course to simplify its manufacture.
• the latter 4 comprises two parts constituted by two different materials, the part which constitutes the body of said drawer comprising a more rigid material than that constituting the control means.
• the base 14 of the support 3 is particularly smooth to allow the proper translation along the Fl of the drawer 4. It is also noted on this surface 14 of the support 3 the presence of grooves 25 on said contact surface 13 allowing the passage of the control means 12, 22 and 32.
• Figure 4 is a sectional representation along ⁇ - ⁇ of Figure 1.
• the reaction medium 3 is monoblock in this embodiment.
• the reaction element 15 is an insert. This insert is thus glued within the support 3, however it is visible from the outside via a read window 30 which corresponds to a reading zone 16 of the reaction element 15.
• the slide 4 in FIG. this sectional view thus comprises three control means referenced from left to right 12, 22 and 32. These means of control 12, 22 and 32 cooperate with the reaction chip 15.
• the lower surface of the slide 4 comprises three protrusions which extend downwards at the control means 12, 22 and 32.
• the control means 12, 22 and 32 correspond directly to the pipes 9 and the upstream channels 10 and 11 of the repositioning chip 15, and can be moved in translation along Fl at the grooves 25.
• This reaction chip 15 is for example well described in Figure 5, which represents a magnification of said chip 15 in this Figure 4. In this case, there is the presence of three channels connecting the internal cavity not referenced in this figure towards the upper part of said chip 15. This is the external pipe 9 of the upstream channel 10 and the downstream channel 11.
• reading window 30, well represented in FIG. 4 corresponds to a reading window 31 of the element 15, as is well represented in FIG. 5.
• this chip 15 is better understood according to a sectional view along BB of FIG. 5.
• the external pipe 9 makes it possible to connect an internal pipe 8 and opening towards the outside.
• This internal channel 8 connects two chambers 5 on the left and 6 on the right.
• the chamber 5 also comprises on its left an upstream channel 10 which connects said chamber 5 to the outside.
• the chamber 6 comprises a downstream channel 11 which connects said chamber 6 to the outside. It is therefore these channels 9, 10 and 11 of the chip 15, and therefore of the support 3, which correspond to the control means 12, 22 and 32 of the spool 4.
• the internal and external piping assembly 8 situated between the two chambers 5 and 6 of the reaction element 15 constitute, the central channel 7, as shown in Figure 6.
• the reactive chip 29 is present which makes it possible to have a contact which is only optically with the interior of the reaction chamber 6.
• This reaction chip 29 therefore comprises a reading zone 16.
• Figure 7 shows a more detailed view of the drawer 4. It has five vertical rows of three control means 12, 22 and 32. Each of these groups of three control means corresponds to a process step that is desired to achieve. It is already noted in this Figure 7 that the control means 12, 22 and 32 may have three different configurations depending on the symbol used, namely a white circle, a white circle with a small white circle inside. or finally a white circle with a small black circle inside. These uses will be better explained in relation to Figure 8 a little later.
• indexing holes 18 are at each vertical row of control means. Again these indexing holes 18 can also be used for magnetization by enclosing each a small magnet.
• tanks 19 and 20 All the channels that connect the control means 12, 22 and 32 with the upstream or downstream tanks 19 are referenced 24, they are liquid surplus discharge channels to these reservoirs 19 and 20. Of course for this be effective, tanks 19 and 20 also communicate outwards by means of a vent 21 whether for the tank 19 or for the tank 20 to the outside.
• Cut C-C of Figure 8 shows the nature of the control means 12, 22 and 32 at the third line of the control means of Figure 7, which corresponds to a third step.
• control means 12 is pierced with a through hole (a white circle with a small inner black circle) allowing the fluidic continuity as will be better described in Figures 9 to 14.
• the control means 22 meanwhile is in the case of Ligure presented completely sealed and allows no passage of liquid 2 (a white circle).
• the third control means 32 meanwhile, comprises a blind hole (a white circle with a small inner white circle) which increases the volume but without allowing the evacuation of liquid 2.
• Figure 9 shows a way in which the slide 4 and the reaction chip 15 can cooperate together to manage the fluidics of the device 1. This is the first position.
• the control means will take the references 12P1, 22P1 and 32P1.
• This position Pl corresponds in Figure 7 to the vertical alignment of the three control means 12, 22 and 32 located rightmost of this figure.
• control means 12P1 is a through hole corresponding to the upstream channel 10 of the chamber 5, which allows the biological sample 2 to penetrate, according to F2, into said reaction chip 15
• the liquid 2 will be able to continue its path within said chip 15 via the internal pipe 8 and the external pipe 9; external channel 9 which cooperates with the control means 22P1 which allows the liquid outlet F3 1on.
• sample volume 2 is not important: the liquid containing magnetic beads passes control means 12 to 22. All the balls are captured in the chip 15 during the passage of the liquid
• reaction chip 15 is as smooth as possible so as not to damage the control means 12, 22 and 32 when the sliding according to Fl is carried out.
• FIG. 10 corresponds to the same configuration as FIG. 9, that is to say that the control means 12P1 and 22P1 are extensions of the channels 10 and 9, the control means 32P1 being an element which partitions the downstream channel 11.
• the thrust on F4 which is. similar to F2 is performed by a fluid that has a tendency to push the liquid outside according to F5.
• This fluid is a gas, preferably air, or a liquid, preferably oil, immiscible with the liquid 2, whatever it may be (biological sample, elution buffer or others)
• the liquid 2 consists of magnetic particles comprising nucleic acids which have been fixed according to the technique described in patent EP-B-0.389.063, an agglomerate 27 of magnetic particles and nucleic acids is present at the level of the 26.
• the chamber 6 remains empty.
• the slide 4 is translated according to F1 with respect to the support 3.
• this movement can be either the movement of the slide 4 relative to the fixed support 3 or by moving the support 3 movable relative to the drawer 4 fixed, or by a relative movement of the slide 4 relative to the support 3, both being movable.
• control means are modified and become control means 12P2, 22P2 and 32P2.
• the control means 22P2 is substantially identical to the 22P1 previously described, that is to say that it is a through hole that corresponds to the pipe 9.
• the control means 12P2 is a control means which is liquid-tight
• the control means 32P2 is an element which is identical to the control means 22P2, namely that it allows the passage of a fluid along F6 inside the channel downstream 11 and the reaction chamber 6.
• the liquid 2 can then continue its migration to the inner pipe 8 and the outer pipe 9 to emerge according to F7 at the control means 22P2.
• the agglomerate 27 of magnetic particles and nucleic acids is always present in front of a magnet 26 which is located under the chamber 5.
• This liquid 2 is preferably an elution solution which makes it possible to extract the nucleic acids present on magnetic particles coated with silica, which is the case here and as described in particular in EP-B-0,389,063.
• Figure 12 shows a third position and thus configuration of the control means 12P3, 22P3 and 32P3.
• the central control means 22P3 is closed while the third means 32P3 is provided with a through hole.
• the first control means 12P3 is equipped with a blind hole which is connected to the upstream channel 10, which makes it possible to increase the volume of air at the level of the chamber 5.
• the liquid With respect to the reciprocating movement, when the air in the control means 32P3 returns to atmospheric pressure, the liquid automatically returns to its position in the chamber 6 allowing the presence of the acids.
• the nuclei and the reaction chip 29 This will be facilitated by the presence of air within the first control means 12 P3 which will be compressed and expanded depending on the force applied by the fluid according to F8 or F9.
• This configuration allows the passage and the return a number of times of the elution liquid 2 at the agglomerate of magnetic particles and nucleic acids 27, the purpose being to allow the detachment of the nucleic acids and the recovery of those -this
• the three control means 12P4, 22P4 and 32P4, according to this fourth mode of configuration are all three sealed.
• the liquid 2 is returned to its initial position, that is to say that the air trapped in the chamber 5 is returned to its initial position since there is no more pressure according to F8 or F9. It is in this position that the incubator 17, described in FIG. 2, will make it possible to heat the chamber 6, and possibly the. chamber 5, in order to incubate said liquid 2.
• a first incubation is carried out at 40 ° C. for 5 minutes
• the second incubation is carried out at 65 ° C. for 16 hours preceded by denaturation at 90 ° C. for a few minutes.
• the agglomerate present at the level of the chamber 5 in front of the magnet 26 is an agglomerate 28, which consists only of magnetic particles, the nucleic acids having been in large part, even completely, reprized by the elution liquid 2.
• FIG. 14 shows that, in this fifth configuration, the control means 12P5 is closed, the control means 22P5 is open and allows the arrival of a fluid according to F12. And finally the third control means 32P5 is also open to allow the evacuation of the elution liquid 2 according to F13.
• the nucleic acids have been captured by capture nucleic acids, called detection probes, fixed on the reaction chip 29. There will therefore be hybridization between the capture chip 29 and nucleic acids, which were present in the starting biological fluid 2. As a result, may, using the window 31, perform a reading within the reaction chip 15 to know whether or not there has been hybridization.
• this reaction chip 29 may comprise one or more spots for analyzing the presence of one or more types of nucleic acids of interest.
• the principle of the device 1, according to the invention, is to slide control means, said joints, 12, 22 and 32, on the hybridization reaction chip 15 during the protocol.
• the injection by the open joints is by elastic sealing between the injection means (a pipette cone associated with a pipette for example) and the elastic seal.
• the joints are distributed over a plastic drawer 4 so as to reproduce the steps of the protocol.
• the part of the joints that presses the chip is thicker than the plastic card to ensure a good seal.
• the liquids 2 are injected using a cone present at a pipette (manual operation) or an instrument (automatic operation).
• the movable slide 4 slides in the support 3 between several successive positions (position tolerance is: +/- 0.4 mm).
• the seals are in contact only with the chip 15, but not with the rest of said support 3.
• the volume of this chip 15 is of the order of 1 pL.
• the device In another embodiment of the device 1, it carries a hybridization reaction chip 29, included in a Micronit® 15 plastic chip or glass chip, and an incubator 17.
• the magnets 26 have a diameter of 1.5 mm, a length of 3 mm and are integrated in a fixed part under the chip 15. They consist of neodymium-iron-boron with a remanence of about 1.2 Tesla.
• the complete dimensions of said device 1 are 10 cm in length by 4.5 cm in width and 2 cm in height and respectively 5.5 cm by 3.5 cm and 0.5 mm in height for the drawer 4. These values were chosen to facilitate manufacturing and use but could easily be modified up or down.
• the volume of the hybridization and elution liquid must allow a complete filling of the chamber 6.
• the volume of each chamber is between 0.1 and 100 ⁇ , preferably between 0.1 and 10 ⁇ and even more preferably between 0 and 10 ⁇ . , 1 and 1 ⁇ .
• a chamber is of a volume of 0.25 ⁇ L, 0.5 to 0.8 ⁇ L of hybridization buffer is introduced.
• the exact volume is defined during the translation according to Fl.
• the air is injected with a pipette tip and the necessary pressure can be controlled manually or automatically to allow the transfer of liquid 2.
• the pressure is constant throughout the elution, ie 5 minutes at 40 ° C.
• Plastic chip Chip 15 is machined in PMMA or PTFE.
• the channels are 500 ⁇ wide and 200 ⁇ high.
• the hybridization volume is of the order of 1 ⁇ L.
• the length, width and thickness of said chip 29 is 18 mm, 4 mm and 450 ⁇ m. More specifically as regards the thickness, the body (upper part) and the cover (lower part) have a thickness of between 30 to 400 ⁇ m, preferably a thickness of 170 to 400 ⁇ m at the reading zones.
• the channels are closed by an adhesive film ARseal TM (Ref .: DEV-90404 from the company Adhesive Reseach - Glen Rock, PA, USA) of 150-pin thick.
• ARseal TM Ref .: DEV-90404 from the company Adhesive Reseach - Glen Rock, PA, USA
• This chip of identical size to the previous one has two chambers 5 and 6.
• the chamber 5 allows the capture of the magnetic beads and has dimensions that are comparable to and compatible with those of the magnets 26 (D: 1.5 mm, L: 3 mm).
• This chip 29 was manufactured by the company Micronit (Enschede, the Netherlands).
• the chip 29 has been described above in PMMA plastic or glass, but it could just as easily be made of silicon, metal or any type of hard plastic that is impermeable to gases and liquids, such as Teflon for example. It is preferable to chamfer the chip 15, in particular made of glass, for example on an abrasive surface type sandpaper, to promote sliding according to Fl without tearing joints on the edge of the chip.
• the device 1 is machined in PolyMethyl Methacrylate, called PMMA.
• the sealing joints are molded in B.A.U. silicone. from Plastiform (Ref .: 310 120 15N, Thise, France) or butyl rubber or polyisobutylene, called Butyl Rubber. These protrude from the contact surface by a height of between 100-300 ⁇ m.
• the objective is to control the position of the liquid segment during a pressurization of 10 minutes.
• the indicator is the good return to the initial position of the liquid.
• the device 1 is set in the configuration of FIG. 11, that is to say control means 12P2 closed and control means 22 P2 and 32P2 open.
• the hybridization chamber 6 is filled in excess with a liquid 2.
• Said device 1 is then translated according to Fl in the configuration of FIG. 12, that is to say open control means 32P3 and control means 12P3 closed by blind hole and 22P3 closed.
• the chip 15 is pressurized by a pump connected to a cone inserted into the open seal. Pressure is set to 1 bar (relative to Patm) and monitored by pressure gauge (Keller 70621) at regular intervals.
• the liquid migrates from the hybridization chamber to the middle of the capture chamber.
• the limit pressure is determined by regularly increasing the pressure " until a leak appears Device 1 has a limit pressure greater than 4 bar, ie without leakage during pressurization on the network The results are the same whether the chip is plastic or glass.
• EXAMPLE 2 Management of fluids (plastic chip or glass):
• the objective is to test the basic functions of the device 1, namely and especially the tightness of the joints, the flow, the ease of use, etc.
• the filling of a channel is very easy.
• the cone fits easily and creates a perfectly sealed connection.
• the liquid exits through the other open valve.
• the liquid segment does not move (visual control) when the movable drawer 4 changes position. It is thus possible to fill the second channel without changing the position of the first liquid segment.
• the emptying of the channels is done by circulating air,
• an EasyQ incubator set to obtain a temperature of 65 ° C at the chip level for plastic chips.
• the incubator integrated in the device according to the invention for Micronit ⁇ chips.
• the chip may be Teflon (PTFE) to limit absorption and evaporation through the chip.
• PTFE Teflon
• the holes through the chip are 200-300 ⁇ to limit the liquid-seal contact.
• Material properties for joints The tables below describe the permeability and water absorption properties of different polymers.
• Table 2 Gas permeability for polymers in the medical sector (1) H 2 0 in cm '. mni / nf. day. ATM
• the experimental protocol is an incubation for several hours at 65 ° C. in a device whose joints of the incubation stage have been modified. A part, not shown in the Figures, which compresses the seals can be screwed on the fixed part of the device, which improves the sealing of the system.
• the first step is an incubation of colored water throughout the component. If successful, the second step is an incubation of hybridization buffer slightly colored in half of the chip (same protocol).
• the liquid loss is close to 1 ⁇ l, on the initial 2 ⁇ L.
• the seal has taken the form of the chip; (holes and surface roughness).
• the loss is estimated at 46% in 16 hours of incubation.
• the loss is estimated at 35% in 21:00, or 27% in 16 hours.
• Two glass capillaries (inner diameter 500 ⁇ m, ie equivalent to the hole diameter of the plastic chip) are filled with dye and sealed at the ends with a B.A.D silicone nipple and an epoxy adhesive, which serves as a reference.
• the evaporated volume in the capillary is around 2 ⁇ L. This result represents an average evaporation of 0.1 ⁇ L per hole and by incubation for 16 hours.
• the loss due to the seal should not exceed 0.3 ⁇ L during incubations on plastic chips, because there are three holes in the reaction chip.
• the loss is estimated at 19% in 16h00.
• the lower seal is faulty, perhaps following a tearing when moving the movable part.
• the leak observed may be the consequence.
• This material is sensitive to shearing (tearing).
• This material is more waterproof than the previously tested silicone.
• the loss in liquid is estimated at 24% over 22 hours. That is 17% over 16 hours of incubation.
• the holes in the chip are 200 ⁇ m in diameter instead of 500 ⁇ m previously.
• the first step is an idea.
• a temperature probe was added under the chip (piercing the fixed part of the device) to control the temperature throughout the incubation.
• This one is colored in order to be able to visualize the progression of the liquid segment.
• Butyl rubber is a suitable material for the incubation stage.
• the joints of the device are therefore of two different materials namely: silicone for the fluidic steps and butyl rubber for the incubation step.
• the incubation step in the Micronit® chip is critical because the hybridization volume is 250 nL (half-chamber volume).
• the incubator is integrated into the base of the device.
• the temperature rise time is of the order of 15 minutes.
• the transition from plastic chip incubation to a glass chip is not a major problem. Reducing the incubation volume from 1 ⁇ L to 0.25 ⁇ L does not affect the results.
• a rise in temperature is provided at 90-95 ° C before the actual incubation at 65 ° C. This step followed by a 24 hour incubation at 65 ° C. was performed (FIG. 15).
• Half of the chip is filled with hybridization buffer according to the protocol (step shown in Figures 11 and 12).
• the chip is closed (step shown in Figure 13) for incubation.
• the incubator is pre-set to heat the chip to 90 ° C. Once the temperature is reached, the incubator is set at 65 ° C. The chip therefore comes down gently at 65 ° C for incubation.
• FIG. 16 and 17 show: Figure 16 stabilization in emperature with a standard deviation of 0.1 C (mean 63.5 ° C) over 3 hours, and
• Figure 17 the temperature setting of the test device in 20 minutes s.
• the aim is to characterize the efficiency of flux capture of magnetic beads in the microfluidic component.
• the theory and practice show that magnets with dimensions of the order of a millimeter are large enough to capture small magnetic particles of a size or diameter of 0.1 to several ⁇ , present in a microfluidic chamber, as long as the distance between magnet; and room is. less than 400 ⁇ .
• the reaction chip 15 has a 170 ⁇ m thick glass lid and the magnet is 3 mm long and 1.5 mm in diameter. Nevertheless, the flow velocity must be less than 10 pL / min to allow effective capture of at least 90% of the magnetic particles.
• Magnetic capture particles have demonstrated with the device according to the invention an effective capture. With a flow rate of 5 ⁇ L / min, and for three different experiments, the capture efficiency was 95, 96 and 97%, for magnetic particles of 200 nm. Adembeads Amino Diameter [Ref. 200 nm AMINO-ADEMBEADS, Ademtech, Pessac, France] (1.6 ⁇ L in 50 ⁇ l of capture buffer). In order to compare, a yield greater than 95% was obtained with a manual protocol.
• T. / ultimate objective is to be able to carry out agitation during ultrasound hybridization.
• the first segment of a plastic chip 1 ⁇ L is filled with colored water (1 ⁇ L) and the second with water without dye (1 ⁇ L).
• the assembly is sealed in a silicone seal identical to the joints of the device.
• a first assembly is. placed in the ultrasound bath. The mixture is visually performed in 1 to 2 minutes.
• a glass capillary with a diameter of 520 ⁇ is coupled to an ultrasonic transducer controlled by a frequency generator. The dispersion of magnetic beads previously attracted by a magnet is observed. The frequency is 50-150 kHz.
• the redispersion of a pellet of magnetic beads in a glass capillary by means of ultrasound is therefore possible.
• the redispersion time is less than 5 seconds.
• Half of the device is filled with colored water and the second with water. Slow staining of the staining is observed. This technique is not very effective.
• a - Validation of the device pure use Fluid management of the device is by sealing between a cone, or pipette tip, and a flexible seal. Most types of cones are compatible with the current device. All the hybridization steps can be done by hand by a trained technician.
• the most critical step is the capture of magnetic particles. If the capture is in flux, a simple syringe-type device of the Agitate BioAnalyser (Bioanalyzer 2100, Santa Clara, CA, USA) is required: based on a defined fluidic resistance coupled to a volume of air under pressure). allows to circulate S 'starting sample at about 5 pL / min in the device and whatever the volume of the sample. Hybridization can be done in an oven.
• the incubator, agitator and reader are external to the system.
• the instrument is based on a two - axis Y / Z piper arm that picks up and deposits cones, draws reagents and ejects them into the device. After each step, an arm or table an axis shifts the device and the reagents one notch.
• the pipetting device thus has access to new cones and reagents.
• the technician inserts racks of samples, reagents, cones and devices according to the invention into the machine. If the fluid handling time is one hour per test, there is approximately 15 tests to allow the machine to run all night without the need for recharging.
• the device is based on an existing platform.
• Control means cooperating with the channel opening 10 Support contact surface 3 where the channels 9, 10 and 11 open

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