CHIP FOR MICRO-ORGANISM LYSIS WITH VALVE, GRINDER AND USE

THEREOF

FIELD OF INVENTION

[0001] The present invention relates to a chip for micro-organism lysis comprising valves, a mechanical system operating with such chip and methods of micro-organism lysing.

BACKGROUND OF INVENTION

[0002] Micro-organism lysis is the phenomenon of destruction - partially or totally - of the external protection of the micro-organism. In particular, cellular lysis is the destruction of the cellular membrane. After lysis, the internal content of the microorganism is released and can be extracted or identified by various methods. Especially, nucleic acid material - DNA or RNA - can be released and used as starting material for Polymerase Chain Reaction, leading to identification of the type of micro-organism. Protein material may be also retrieved and used for micro-organism identification. Such methods are now very popular in numerous domains, including diagnostic - human health and veterinary - and quality control in food industry, hygiene or cosmetics.

[0003] Various methods for micro-organism lysis are known, and may be classified under three categories. Chemical lysis uses chemicals to degrade the external protection of micro-organism. However, these chemicals may also degrade the valuable internal content of micro-organism. Thermal lysis uses heat to degrade the external protection of micro-organism. But heating is usually leading to degradation of nucleic acid material and proteins. Finally, mechanical lysis aims at disrupting the external protection of microorganisms by a mechanical stress.

[0004] International patent application WO 2015181743 describes in particular a device for mechanical lysis. In said device, the mechanical lysis is carried out by shearing a suspension of micro-organisms between two walls, one of the two walls having a rough glass bearing surface. However, results obtained, especially on Gram-positive bacteria, are poor and use of glass is not allowed in food and health domains.

[0005] European patent application EP 3222989 discloses a mechanical lysis device in which a suspension of micro-organism is grinded on an abrasive surface. However, use of a grinding surface leads to complex procedures and sensitivity of identification is not sufficient.

[0006] Moreover, several known mechanical lysis devices comprise one inlet port and one outlet port. This implies that injection of the sample and injection of the washing solution are performed by the same inlet port. In the same manner, the collection of the content of the cell and the removal of the waste solution is performed by the same outlet port. In an automatized system, where a single device - syringe typically - is used for several successive lysis devices, this would lead to cross-contamination.

[0007] It is thus desirable for a device for lysis of micro-organism to be easily transportable, efficient and highly reliable with low volume of sample, and allowing for separation of internal material of micro-organism from external protection without crosscontamination.

[0008] A purpose of this invention is therefore to provide a chip for micro-organism lysing comprising several superimposed layers and membranes forming a lysing chamber and four ports fluidically connected to the lysing chamber by several channels, each channel comprising a valve. The mechanical lysis achieved in the lysing chamber allows the separation of the internal material of microorganism which has been injected. After rinsing, the internal material will be collected through specific channels which can be closed using the valves, thus avoiding cross -contamination.

SUMMARY

[0009] This invention thus relates to a chip for micro-organism lysing comprising a. a multilayer stack comprising i. a first double-sided adhesive layer comprising a chamber hole; ii. a flexible filter layer in direct contact with the first double-sided adhesive layer and covering the chamber hole; iii. a second double-sided adhesive layer in direct contact with the flexible filter layer, said second double-sided adhesive layer comprising a chamber hole; iv. a first flexible membrane on the first double-sided adhesive layer, opposite the flexible filter; v. a second flexible membrane on the second double-sided adhesive layer, opposite the flexible filter; wherein all chamber holes are superimposed to form a lysing chamber; and b. a lower support on the second flexible membrane of the multilayer stack, opposite the second double- sided adhesive layer; c. an upper support on the first flexible membrane of the multilayer stack, opposite the first double- sided adhesive layer; wherein a sample inlet port, a rinsing inlet port, an elution outlet port, a waste outlet port, a pestle hole and four valve apertures are distributed on the lower support and/or the upper support; wherein the sample inlet port and the rinsing inlet port are in fluidic communication with the lysing chamber via inlet holes in layers of the multilayer stack and respectively a sample inlet channel and a rinsing inlet channel; wherein the elution outlet port and the waste outlet port are in fluidic communication with the lysing chamber via outlet holes in layers of the multilayer stack and respectively an elution outlet channel and a waste outlet channel; wherein the pestle hole is aligned with the lysing chamber; wherein at least part of each channel is comprised in a double- sided adhesive layer between the first flexible membrane and the lower support, or comprised in the lower support wherein the chip is divided in two sides separated by the plane of the flexible filter layer and wherein at least part of the sample inlet channel is comprised in one of said sides and at least part of the elution outlet channel is comprised in the other side; wherein each channel comprises a valve hole; wherein the four valve holes are respectively aligned with one of the four valve apertures, for each channel, at least one of the flexible membranes and/or at least one of the doublesided adhesive layers between said channel and the valve aperture aligned with the valve hole of said channel being devoid of openings aligned with one of the four valve apertures; and d. fastening means to maintain the upper support and lower support in contact with the multilayer stack.

[0010] Indeed, this chip allows mechanical lysis of micro-organisms avoiding degradation of its internal content, nucleic acid material and proteins.

[0011] Moreover, using a filter allows the lysis of micro-organisms for analysis in food and health domains.

[0012] Furthermore, the chip of the present invention allows a simple mechanical lysis procedure and an increase of the sensitivity of identification.

[0013] Moreover, the use of the lower and upper support allows to increase the rigidity of the multilayer stack and membranes all over the chip except in the lysis chamber allowing the applied mechanical action to be gathered on the lysis chamber and in the four valve apertures allowing the deformation of the flexible membranes and/or doublesided adhesives.

[0014] Finally, the sample, the rinsing liquid, the waste liquid and the internal content of the micro-organisms are provided to or extracted from the lysing chamber via a specific channel. The channels being selectively closed by valves, this allows to avoid any crosscontamination.

[0015] According to other advantageous aspects of the invention, the fastening means comprise a third double- sided adhesive layer in direct contact between the upper support and the multilayer stack, said third double-sided adhesive layer comprising a pestle hole superimposed with the lysing chamber, inlet/outlet holes aligned with the inlet/outlet ports. [0016] According to other advantageous aspects of the invention, the sample inlet port, the rinsing inlet port, the elution outlet port, the waste outlet port, the pestle hole and the four valve apertures are comprised in the upper support.

[0017] Indeed, this allows an easier access of the ports and apertures all on the same side of the chip. The lower support may thus lie on a flat surface during the use of the chip.

[0018] According to other advantageous aspects of the invention, the sample inlet port, the rinsing inlet port, the elution outlet port and the waste outlet port are comprised in the upper support and the pestle hole and the four valve apertures are comprised in the lower support.

[0019] Indeed, this allows a more compact grinding system since the two faces of the chip are used allowing to use the rods, the pipettes or syringes and the grinder on each side of the chip.

[0020] According to other advantageous aspects of the invention, the fastening means comprise a fourth double- sided adhesive layer in direct contact between the lower support and the multilayer stack, said fourth double-sided adhesive layer comprising a pestle hole superimposed with the lysing chamber, and inlet/outlet holes aligned with the inlet/outlet ports.

[0021] According to other advantageous aspects of the invention, at least part of the elution outlet channel and the waste channel is comprised in the fourth double-sided adhesive layer and each of the fourth double-sided adhesive layer and the second flexible membrane comprises a communication hole.

[0022] According to other advantageous aspects of the invention, at least one opening is spread over the flexible membranes and/or the double- sided adhesive layers, each opening being aligned with one of the four valve apertures.

[0023] According to other advantageous aspects of the invention, flexible membranes are polyethylene terephthalate membranes, preferably flexible membranes have a thickness ranging from 15 pm to 150 pm. [0024] Indeed, using polyethylene terephthalate allows to obtain membranes which are biologically compatible.

[0025] Moreover, the thickness range is optimized: below 15 pm the membranes are subject to tearing while above 150 pm there is a loss of efficiency.

[0026] According to other advantageous aspects of the invention, the double-sided adhesive layers are flexible and have a thickness ranging from 90 pm to 350 pm.

[0027] According to other advantageous aspects of the invention, the flexible filter layer is a polycarbonate layer, preferably flexible filter layer has a porosity ranging from 0.2 pm to 1 pm.

[0028] According to other advantageous aspects of the invention, for each channel, the flexible membranes and the double- sided adhesive layers devoid of said channel comprised between the support comprising the valve aperture aligned with the valve hole comprised in said channel and the double-sided adhesive layer comprising said channel are the upper layers and the flexible membranes and the double-sided adhesive layers devoid of said channel comprised between the double-sided adhesive layer comprising said channel and the support devoid of the valve aperture aligned with the valve hole comprised in said channel are the lower layers, and for at least one of the four valve apertures, each layer comprised between the flexible membrane of the upper layers and the flexible membrane of the lower layers or the support not comprising said valve aperture comprises an opening aligned with said valve aperture so that the closing of the channel is reversible.

[0029] Indeed, this allows for reversibly opening and closing the channel according to the step of the lysing which is performed.

[0030] According to other advantageous aspects of the invention, for each channel, the flexible membranes and the double- sided adhesive layers devoid of said channel comprised between the support comprising the valve aperture aligned with the valve hole comprised in said channel and the double-sided adhesive layer comprising said channel are the upper layers and the flexible membranes and the double-sided adhesive layers devoid of said channel comprised between the double-sided adhesive layer comprising said channel and the support devoid of the valve aperture aligned with the valve hole comprised in said channel are the lower layers, and for at least one of the valve apertures aligned, either each layer comprised between the flexible membrane comprised in the upper layers and the double-sided adhesive layer comprised in the lower layers, or each layer comprised between the double-sided adhesive layer comprised in the upper layers and the flexible membrane comprised in the lower layers or the support not comprising said valve aperture comprises an opening aligned with said valve aperture so that the closing of the channel by contact between the double-sided adhesive layer and the flexible membrane or the support not comprising said valve aperture is reversible under pressure higher than 0.1 MPa.

[0031] Indeed, this allows for closing the channel and opening it only if a pressure higher than the predetermined pressure is achieved in the channel. The channel may thus stay closed without need of applying the actuator.

[0032] According to other advantageous aspects of the invention, for each channel, the flexible membranes and the double- sided adhesive layers devoid of said channel comprised between the support comprising the valve aperture aligned with the valve hole comprised in said channel and the double-sided adhesive layer comprising said channel are the upper layers and the flexible membranes and the double-sided adhesive layers devoid of said channel comprised between the double- sided adhesive layer (111) comprising said channel and the support devoid of the valve aperture aligned with the valve hole comprised in said channel are the lower layers, and for at least one of the four valve apertures, each layer comprised between the double-sided adhesive layer comprised in the upper layers and the double-sided adhesive layer comprised in the lower layers comprises an opening aligned with said valve aperture so that the closing of the channel by contact between the double- sided adhesive layers is permanent. [0033] Indeed, this allows for irreversibly closing the channel. Therefore, applying a rod on said valve will definitely close the channel.

[0034] The present invention also relates to a multi-chip comprising at least two chips as described above, preferably four or eight chips, wherein the upper supports of all chips form a single upper support and the lower supports of all chips form a single lower support.

[0035] According to other advantageous aspects of the invention, the at least two chips are distributed in a row.

[0036] The present invention also relates to a system for micro-organism lysing comprising a. a chip for micro-organism lysing or a multi-chip as disclosed hereabove, b. a grinder comprising i. a platform with a recess configured to receive the chip for microorganism lysing or the multi-chip; ii. a pestle configured to be aligned with the lysing chamber; iii. a motor to rotate said pestle; iv. pressuring means to press said pestle against the lysing chamber; and c. a set of four actuators, each actuator comprising a rod configured to be independently inserted in the valve apertures and pressed against the support not comprising the valve apertures into which said actuator is inserted, thereby closing independently one of the four channels.

[0037] Indeed, this system is compact and the compatibility of the elements is optimized to increase the efficiency of the mechanical lysis.

[0038] Moreover, the application of a pressure and the rotation of the pestle allow to increase the efficiency of the cell lysis by efficiently deforming the cell membrane.

[0039] According to other advantageous aspects of the invention, the system for microorganism lysing further comprises automated fluidic devices - such as syringes, pumps and tubes, pipettes ... - to supply sample in the sample inlet port, to supply rinsing solution in the rinsing inlet port, to collect sample in the elution outlet port and to collect waste in the waste outlet port.

[0040] The invention also relates to a method for micro-organism lysing comprising a. Introducing a sample comprising micro-organisms via the sample inlet import in a chip for micro-organism lysing or in a multi-chip according to one aspect of the invention; b. Pressing the lysing chamber with a rotating pestle; and c. Recovering the content of cells via the elution outlet port.

DEFINITIONS

[0041] In the present invention, the following terms have the following meanings:

[0042] “Direct contact” between two layers means that both layers are not separated by another layer.

[0043] “Flexible” refers to elements of the chip, in particular filter and membranes, that are able to deform under pressure in the direction normal to their surface (bending), and are able to deform within their surface and form pleats (stretching and folding) when sheared.

[0044] “Micro-organism” refers to biological entities such as viruses, archaea, bacteria, fungi and cells.

[0045] “On” in relationship with two layers, means that one layer is laid on or under - there is no privileged vertical direction - the other layer, either in direct contact or separated by one or several intermediate layers.

[0046] “Valve” refers to a superposition of chip layers, comprising openings or not, the openings being aligned with a hole comprised on the path of a channel. The closing of the valve is performed by the deformation of the layers devoid of openings through the hole of the channel. The deformed layer is thus in contact with another flexible layer, an adhesive layer or a support, this contact allowing the closing of the valve.

DETAILED DESCRIPTION

[0047] This disclosure relates to a chip 100 for micro-organism lysing.

[0048] Functionally, the chip comprises a support, an inlet port and a lysing chamber comprising a flexible filter layer. The chip allows to inject a sample, through the inlet port, in the lysing chamber able to be pressed and sheared strongly against the support by an external mechanical device. By this way, micro-organisms in the sample are efficiently grinded, leading to mechanical lysis. Thanks to the porosity of the flexible filter layer, separation of the internal material of micro-organisms from their outer envelope - cell membrane, capsid... - is achieved, the former flowing through the flexible filter layer whereas the latter are blocked by the flexible filter layer. Thus, the internal material of micro-organisms is accumulated - downstream - in the side of the lysing chamber opposite the side - upstream - of lysing chamber connected to the inlet port. Thus, the internal material may be collected for further analysis, such as PCR amplification. The complete structure of chips is detailed hereafter.

First basic configuration

[0049] In a first basic configuration represented in figures 1, 2 A and 3, the chip 100 comprises a multilayer stack 110 comprising a first double-sided adhesive layer 111 comprising a chamber hole. A flexible filter layer 112 is in direct contact with the first double-sided adhesive layer 111. The flexible filter layer 112 covers the chamber hole. A second double- sided adhesive layer 111 is in direct contact with the flexible filter layer 112. The second double-sided adhesive layer 111 comprises a chamber hole. The first and second double-sided adhesive layer 111 thus sandwich the flexible filter layer 112. In other words, the chip 100 is divided in two sides separated by the plane of the flexible filter layer 112. Indeed, the flexible filter layer 112 is, by definition of the layer, flat, its length and width are large compared to its height. The sides of the flexible filter layer 112 are thus the surfaces of the filter separated by its height. The flexible filter layer 112 may thus be seen as lying in a plane at the interface between the first double- sided adhesive layer 111 and the second double- sided adhesive layer 111.

[0050] All chamber holes are superimposed to form the lysing chamber 120. The thickness of the double- sided adhesive layers 111 and the chamber holes define a hollow space in which the sample can be introduced.

[0051] In the first basic configuration, a flexible membrane 113 is on the first doublesided adhesive layer 111, opposite the flexible filter layer 112. A second flexible membrane 113 is on the second double-sided adhesive layer 111, opposite the flexible filter layer 112. The second flexible membrane 113 allows to increase the number of possibilities, in term of position and reversibility, to close the valves as it is explained hereafter. The second flexible membrane 113 may comprise a communication hole 118 which is preferably located so as to be superimposed on the periphery of the lysing chamber 120. The communication hole 118 may be advantageously smaller than the chamber hole, so that the second flexible membrane 113 covers almost completely the lysing chamber 120, except for a small exit through the communication hole 118.

[0052] The separation of the chip in two sides thus implies that the flexible membranes 113 and the adhesive layers 111 are distributed into a first stack and a second stack of layers, the two stacks being separated by the flexible filter layer 112. The first stack is thus on one side of the flexible filter layer 112 and the second stack is on the other side of the flexible filter layer 112.

[0053] In this disclosure, the filter layer 112 and the membranes 113 are flexible. By flexible, it is meant that these flat elements are able to deform under pressure in the direction normal to their surface (bending) as represented in figures 4A (before the deformation) and 4B (during the deformation), and are able to deform within their surface and form pleats (stretching and folding) when sheared. Although flexible, filter layer 112 and membranes 113 are not torn under constraint considered and they have a stable shape in absence of pressure and/or shear. Preferably, the second flexible membrane 113 is similar to the first flexible membrane 113 - same material and same flexibility properties.

The double-sided adhesive layers 111 may also be flexible.

[0054] As listed above, the chip 100 further comprises a lower support 115. This lower support 115 is on the second flexible membrane 113 of the multilayer stack 110, opposite the second double-sided adhesive layer 111. Preferably, the lower support 115 is rigid (z.e., not flexible).

[0055] The chip 100 further comprises an upper support 114 on the first flexible membrane 113 of the multilayer stack 110, opposite the first double-sided adhesive layer 111. A sample inlet port 130, a rinsing inlet port 135, an elution outlet port 140, a waste outlet port 145, a pestle hole and four valve apertures 180 are distributed on the lower support 115 and/or the upper support 114. For example, the upper support 114 comprises the sample inlet port 130, the rinsing inlet port 135, the elution outlet port 140, the waste outlet port 145, the pestle hole aligned with the lysing chamber 120 and the four valve apertures 180 - represented in a horse-shoe shaped area in figures 1 and 3. In another example, the sample inlet port 130, the rinsing inlet port 135, the elution outlet port 140 and the waste outlet port 145 are comprised in the upper support 114 (figure 6) and the pestle hole and the four valve apertures 180 are comprised in the lower support 114 (figure 7). The upper support 114 and the lower support 115 are thus not defined according to their position above or below the flexible filter layer 112. Indeed, the arrangement of the layers may be changed in mirror with respect to the flexible filter layer 112. In the following, in order to simplify the description, the support comprising the ports is considered as the upper support 114. However, the whole description may be read with the upper support 114 and the lower support 115 exchanged.

[0056] Preferably, the upper support 114 is rigid. The lower support 115 and upper support 114 provide with mechanical stability of the chip 100, allowing easier handling. Moreover, the ports on the upper support 114 allows to introduce in or collect the sample from the chip 100, with a fluidic device (a micropipette for instance), where the ports help positioning the tip of the fluidic devices correctly. Figure 2B represents the assembled chip. The multilayer stack 110 is comprised between the lower support 115 and upper support 114. [0057] In the first basic configuration, the chip 100 further comprises fastening means to maintain the upper support 114 and lower support 115 in contact with the multilayer stack 110.

[0058] The sample inlet port 130, rinsing inlet port 135, elution outlet port 140 and waste outlet port 145 are in fluidic communication with the lysing chamber 120.

[0059] As explained above, the sample inlet port 130 allows to inject a sample in the lysing chamber 120. The rinsing inlet port 135 allows to inject rinsing liquid in the lysing chamber 120 whereas the waste outlet port 145 allows to collect the soiled rinsing liquid out of the lysing chamber 120. The rinsing liquid thus has a viscosity which allows to easily pass through the flexible filter layer 112. Finally, the elution outlet port 140 - in other words a sample outlet port - allows, after mechanical lysis, to collect the internal material of a micro-organism present in the sample that has flown through the flexible filter layer 112.

[0060] More precisely, the sample inlet port 130 is in fluidic communication with the lysing chamber 120 via a sample inlet channel 170 while the rinsing inlet port 135 is in fluidic communication with the lysing chamber 120 via a rinsing inlet channel 170. Several inlet holes are present in the layers of the multilayer stack 110. The inlet holes form two small inlet pits respectively connecting the sample inlet port 130 and the rinsing inlet port 135 to the sample inlet channel 170 and the rinsing inlet channel 170.

[0061] The elution outlet port 140 is in fluidic communication with the lysing chamber 120 via a elution outlet channel 170 - in other words a sample outlet channel - while the waste outlet port 145 is in fluidic communication with the lysing chamber 120 via a waste outlet channel 170. Outlet holes are also present in the layers of the multilayer stack 110. The outlet holes form two small outlet pits respectively connecting the elution outlet port 140 and the waste outlet port 145 to the elution outlet channel 170 and the waste outlet channel 170.

[0062] At least part of the sample inlet channel 170 is comprised in one side of the flexible filter layer 112 and at least part of the elution outlet channel 170 is comprised in the other side. In other words, at least part of the sample inlet channel 170 is comprised in a layer of the first stack and at least part of the elution outlet channel 170 is comprised in a layer of the second stack. Therefore, the overall length of all the channels 170 cannot be comprised in a single layer 113 or in a single support (114, 115). Indeed, since the liquid passes through the flexible filter layer 112 during the grinding, the extremity of the sample inlet channel 170 connecting the lysing chamber 120 must be on a side of the flexible filter layer 112 opposite the extremity of the elution outlet channel 170 connecting the lysing chamber 120.

[0063] The inlet holes and outlet holes are preferably present in each layer or membrane between the upper support 114 and the lower support 115. Indeed, although inlet and outlet holes are not required on all layers and membranes to ensure fluidic communication between inlet ports or outlet ports and the channels 170, they are advantageous. Indeed, when sample is deposited in the sample inlet port 130 with a micropipette for instance, the tip of the micropipette may enter inside the multilayer stack 110 and become stuck by an adhesive, thus plugging the micropipette or degrading sample delivery. Inlet and outlet holes in all double- sided adhesive layers 111 and flexible membranes 113 prevent this drawback.

[0064] At least part of each channel 170 (sample inlet channel, rinsing inlet channel, elution outlet channel and/or waste outlet channel) is either comprised in a double-sided adhesive layer 111 (for example the first or the second double- sided adhesive layer 111) between the first flexible membrane 113 and the lower support 115, or in the lower support 115. However, the four channels 170 shall not intersect to avoid any contamination between the rinsing liquid, the sample and the extracted internal material. When the channel 170 is comprised in a double-sided adhesive layer 111, the channel may correspond to a furrow passing through the overall thickness of the double-sided adhesive layer 111. When the channel 170 is comprised in the lower support 115, the channel is grooved in said support but does not pass through the overall thickness of the support. Grooving at least part of the channel 170 in the lower support 115 has several advantages: grooving a channel in a rigid element is easier than designing it within an adhesive layer. And pressure drop through a channel grooved in a rigid element is much lower than within an adhesive layer: thus, pressure required to collect the sample from the lysing chamber 120 is reduced.

[0065] Each channel 170 comprises a valve hole 150 so that the valve hole 150 is on the path of the channel 170. The size (diameter or longer dimension) of the valve hole 150 may be equal to or larger than the width of the corresponding channel 170. The four valve holes 150 are respectively aligned with one of the four valve apertures 180. Moreover, for each channel 170, at least one of the flexible membranes 113 and/or at least one of the double-sided adhesive layers 111 between said channel 170 and the valve aperture 180 aligned with the valve hole 150 of said channel 170 is devoid of openings aligned with said valve apertures 180 aligned with the valve hole 150 of said channel 170. Therefore, when a rod is inserted in one of the four valve apertures 180 and pressed against the support (114, 115) opposite the valve apertures 180, the flexible membranes 113 and/or double-sided adhesives 111 devoid of openings are deformed through the valve holes 150 and close the corresponding channel 170 as represented in figure 4B. Therefore, the closing of the valve - and thus of the channel 170 - is not carried out by the rod itself but by the deformed layer (111, 113) which forms a barrage over the whole width and height of the channel, the deformed layer (111, 113) being in contact with a flexible membrane 113, a double-sided adhesive layer 111 or a support (114, 115). In other words, the rod is never in contact with the liquid inside the channel 170. This advantageously prevents any cross -contamination between the rod 550 and the liquid. Therefore, the same rod 550 may be used to close several valves. Moreover, the presence of the layers (111, 113) devoid of openings allows to increase the number of possibilities, in term of position and reversibility, to close the channels as it is explained hereafter. Preferably, the doublesided adhesive layer 111 in direct contact with the support (114, 115) comprising the valve apertures 180 is devoid of valve hole 150. Indeed, the closing of the valve being performed by the deformation of a layer, a layer must be present between the valve aperture 180 and the valve hole 150.

[0066] The valve apertures 180 are thus distinct from the ports (130, 135, 140, 145). Indeed, the presence of the layer (111, 113) devoid of opening between the valve apertures 180 and the channels 170 does not allow to inject or collect liquid in the channel 170 through the valve apertures 180.

[0067] One channel 170 may be comprised in a plurality of double-sided adhesive layers 111 or in at least one double-sided adhesive layer 111 and the lower support 115, so that the channel 170 is divided into several parts. Therefore, said channel 170 comprises one valve hole 150 in each part of the channel and these valve holes 150 are aligned. Moreover, openings may be comprised in the layers comprised between two layers comprising said channel so that to ensure fluidic communication along the overall channel. For example, in figure 2A, the rinsing inlet channel 170 connecting the rinsing inlet port 135 to the lysing chamber 120 comprises a first part included in the first doublesided adhesive layer 111 and a second part comprised in the second double-sided adhesive layer 111. The two parts of this channel 170 are in fluid communication through the valve hole 150 comprised in the downstream end of the first part of the channel 170 and in the upstream end of the second part of the channel 170.

[0068] The valve holes 150 of at least two channels comprised in two non-adjacent layers may be aligned. For example, considering a sample inlet channel 170 entirely comprised in the first flexible membrane 113 and a waste inlet channel 170 completely comprised in the second double-sided adhesive layer 111, the valve holes 150 of these channels may be aligned. This advantageously allows to simultaneously close these two channels 170. However, this does not allow to selectively close only one of these two channels 170.

[0069] Moreover, additional communication holes in the layers of the multilayer stack 110 may form, with the communication hole 118 comprised in the second flexible membrane 113, a communication pit connecting the chamber holes comprised between the flexible filter layer 112 and the lower support 115 with a channel 170. The channels 170 may thus be in fluidic communication with the lysing chamber 120 through the communication hole 118.

[0070] At least one opening 160 may be spread over the flexible membranes 113 and/or the double-sided adhesive layers 111. The openings 160 are located in a horse-shoe shaped area or oval area in figures 1, 2A and 3. Each opening 160 is aligned with one of the four valve apertures 180 of the upper or lower support (114, 115). Thus, since the valve apertures 180 are aligned with the valve holes 150 comprised in each channel 170, the openings 160 are also aligned with the valve holes 150 on the path of the channels 170. These openings advantageously allow to select the flexible membranes 113 and/or the double-sided adhesive layers 111 that will be deformed to close the corresponding channel 170. Indeed, as described hereafter, the reversibility of the closure of the channel depends on the contact between the flexible membranes 113 and/or the double-sided adhesive layers 111 when the channel is closed. The reversibility of the closure will be described in details hereafter.

[0071] In this disclosure, flexible membranes 113 may be selected in any material suitable to resist mechanical stress and shear required for micro-organism lysing. In particular flexible membranes 113 made of polyethylene terephthalate (PET) are suitable, especially PET flexible membranes having a thickness ranging from 15 pm to 150 pm.

[0072] In this disclosure, double-sided adhesive layers 111 may be selected in any material suitable, for example any flexible material suitable to resist mechanical stress and shear required for micro-organism lysing. The double-sided adhesive layers 111 preferably have a thickness ranging from 90 pm to 350 pm.

[0073] In this disclosure, flexible filter layer 112 may be selected in any material suitable to resist mechanical stress and shear required for micro-organism lysing. In particular flexible filter layer 112 made of polycarbonate (PC) are suitable, especially PC flexible filters a porosity ranging from 0.2 pm to 1 pm. Flexible filter layer 112 preferably have a thickness ranging from 5 pm to 50 pm.

Second basic configuration

[0074] In a second basic configuration, a second flexible filter layer 112 is introduced in the multilayer stack 110 of the chip 100 in order to maximize the volume of the injected sample. In this configuration, the second flexible filter 112 is aligned on the first flexible filter layer 112 and is in direct contact with the second double-sided adhesive 111, opposite with the second flexible membrane 113. In this configuration, a separating layer comprising a chamber hole aligned with the flexible filter layers 112 is inserted between the two flexible filter layers 112. The separating layer may be a double- sided adhesive layer 111. In this second basic configuration, the separating layer thus divides the chip 100 into two sides: the first stack of layers (111, 113) and the second stack of layers (111, 113).

[0075] In this second basic configuration, the sample inlet channel 170 and the rinsing inlet channel 170 must be positioned in the separating layer in order to inject the sample between the two flexible filter layers 112 whereas the elution outlet channel 170 and the waste outlet channel 170 must be positioned downstream the two flexible filter layers 112.

[0076] In the following, first and second basic configurations may be independently used in all configurations and variants. Indeed, a single flexible filter layer 112 may be replaced by a pair of flexible filter layers 112 in all configurations and variants disclosed hereafter.

Variants

[0077] In a first variant compatible with both configurations, the fastening means comprise a third double- sided adhesive layer 111 in direct contact between the upper support 114 and the multilayer stack 110, said third double-sided adhesive layer 111 comprising a pestle hole superimposed with the lysing chamber 120. The third doublesided adhesive layer 111 further comprises two inlet holes respectively aligned with the sample inlet port 130 and rinsing inlet port 135 thereby contributing to the inlet pits and two outlet holes respectively aligned with the elution outlet port 140 and waste outlet port 145 thereby contributing to the outlet pits. As explained above, when the valve apertures 180 are comprised in the upper support 114, this third double-sided adhesive layer 111 preferably not comprise the part of a channel 170 comprising the valve hole 150.

[0078] In a second variant compatible with both configurations, the fastening means comprise a fourth double- sided adhesive layer 111 in direct contact between the lower support 115 and the second flexible membrane 113. The fourth double- sided adhesive layer 111 further comprises two inlet holes respectively aligned with the sample inlet port 130 and rinsing inlet port 135 thereby contributing to the inlet pits and two outlet holes respectively aligned with the elution outlet port 140 and waste outlet port 145 thereby contributing to the outlet pits. The fourth double-sided adhesive layer 111 may comprise a channel 170. For example, at least part of the elution outlet channel 170 and the waste channel 170 is comprised in the fourth double-sided adhesive layer 111. In this last embodiment, each of the fourth double- sided adhesive layer 111 and the second flexible membrane 113 comprises a communication hole 118. Indeed, to reach the fourth doublesided adhesive layer 111, the liquid must pass though the second flexible membrane 113. The second flexible membrane 113 thus comprises a communication hole 118 preferably aligned with the communication hole 118 of the fourth double- sided adhesive layer 111. If the whole length of the elution outlet channel and the waste channel 170 are comprised in the fourth double-sided adhesive layer 111, one extremity of each of the elution outlet channel and the waste channel 170 is in fluidic communication with the communication hole 118. As explained above, when the valve apertures 180 are comprised in the lower support 114, this fourth double-sided adhesive layer 111 preferably not comprise the part of a channel 170 comprising the valve hole 150.

[0079] The first and second variant are compatible so that the fastening means may comprise both the third double-sided adhesive layer 111 and the fourth double-sided adhesive layer 111. In this specific configuration, the cohesion of the chip 100 is advantageously ensured by adhesives over the whole surface of the chip 100, leading to a very good resistance of the chip 100 during handling, sample introduction and collection and grinding. The combination of the first and second variant in the first configuration is represented in figures 1, 2A and 3.

[0080] Additional layers may be introduced in the chip 100. These additional layers may be metallic film providing with electric conductivity and heat generation - by Joule effect - or heat conductivity. Other additional layers may be rubber films allowing to define valves on the fluidic paths. Other additional layers may be hydrophobic layers - like PTFE or surface modified plastics - to improve hydrodynamic conditions like sliding on the walls/boundaries leading to a reduction of pressure required to generate flows in the fluidic paths. Other additional layers may be spacer layers, intended to increase the volume of the lysing chamber 120 for instance, made of any material. Such additional layers shall comprise inlet holes, outlet holes or pestle holes to maintain the fluidic connections disclosed hereabove.

Reversibility of the closure of the channels

[0081] For each channel 170, a set of upper layers and a set of lower layers is defined. Considering a particular channel 170, the upper layers comprise the flexible membranes 113 and the double-sided adhesive layers 111 devoid of said channel 170 and comprised between the support (114, 115) comprising the valve aperture 180 aligned with the valve hole 150 comprised in said channel 170 and the double-sided adhesive layer 111 comprising said channel 170. The lower layers for said channel are the flexible membranes 113 and the double-sided adhesive layers 111 devoid of said channel 170 comprised between the double-sided adhesive layer 111 comprising said channel 170 and the support (115, 114) devoid of the valve aperture 180 aligned with the valve hole 150 comprised in said channel 170.

[0082] For example, in the embodiment wherein the upper support 114 comprises the four valve apertures 180 and one of the channels 170 is comprised in the second doublesided adhesive layer 111, the upper layers for said channel 170 comprise the flexible membranes 113 and the double-sided adhesive layers 111 comprised between the upper support 114 and the second double- sided adhesive layer 111. The lower layers are the flexible membranes 113 and the double-sided adhesive layers 111 comprised between the second double- sided adhesive layer 111 and the lower support 115.

[0083] In the example wherein the four valve apertures 180 are comprised in the lower support 114 and one of the channels 170 comprised in the second double-sided adhesive layer 111, the upper layers for said channel 170 comprise the flexible membranes 113 and the double-sided adhesive layers 111 comprised between the lower support 115 and the second double-sided adhesive layer 11. The lower layers are the flexible membranes 113 and the double-sided adhesive layers 111 comprised between the second double-sided adhesive layer 111 and the upper support 114. [0084] In the first basic configuration, considering a channel 170 comprised in the first double- sided adhesive layer 111, the upper layers only comprise the first flexible membrane 113 whereas the lower layers comprise the second double-sided adhesive layer 111 and the second flexible membrane 113.

[0085] As recited above, the reversibility of the closure of the channel depends on the contact between the flexible membranes 113 and/or the double- sided adhesive layers 111 when the corresponding valve is closed.

[0086] In a reversible configuration represented in figures 4A and 4B, the closure of a channel 170 is reversible meaning that, when a closed valve (figure 4B, rod 550 inserted in the corresponding valve aperture 180 for closing the channel 170) is opened (rod 550 removed), the flexible layers substantially recover their undeformed shape so that said channel 170 is open again (figure 4A). To do so, each layer comprised between the flexible membrane 113 of the upper layers and the flexible membrane 113 of the lower layers or the support (114, 115) not comprising said valve aperture 180 comprises an opening 160 aligned with said valve aperture 180. By this way, the flexible membrane 113 of the upper layers is in direct contact with the flexible membrane 113 of the lower layers when the rod 550 is pressed against the support (114, 115) opposite the valve aperture 180. For example, in figure 4B, the valve apertures 180 are in the upper support 114. The flexible membranes 113 are deformed by the insertion of the rods 550 and are pressed against the lower support 115. The flexible membrane 113 being not adhesive, the contact between these layers does not lead to any sticking and the flexible membranes 113 are free to recover their undeformed shape when the pressure by the rod 550 ceases. This reversible configuration is particularly advantageous for the waste outlet channel 170 which has to be closed and then opened during the lysing process as described hereafter. This reversible configuration is represented in figures 1, 2A and 3.

[0087] In a semi-reversible configuration, the closure of a channel 170 is semi-reversible meaning that, when a rod 550 inserted in the corresponding valve aperture 180 for closing the channel 170 is not pressed anymore against the support (114, 115) opposite the valve aperture 180, the flexible layers in contact when the rod 550 applied the pressure remain stick together. The flexible layers may come unstuck when the pressure in the closed channel 170 becomes higher than 0.1 MPa. This semi-reversible configuration may be reached by two manners: either, for at least one of the valve apertures 180, each layer comprised between the flexible membrane 113 comprised in the upper layers and the double-sided adhesive layer 111 comprised in the lower layers, or for at least one of the valve apertures 180, each layer comprised between the double- sided adhesive layer 111 comprised in the upper layers and the flexible membrane 113 comprised in the lower layers or the support (114, 115) not comprising said valve aperture 180 comprises an opening 160 aligned with said valve aperture 180.

By this way, the closing of the channel 170 is performed by contact between a doublesided adhesive layer 111 and a flexible membrane 113 or the support (114, 115) opposite the valve aperture 180. For example, when the valve apertures 180 are in the upper support 114, the double-sided adhesive layer 111 of the upper layers may be deformed by the insertion of a rod 550 in one of the valve apertures 180 and is pressed against the lower support 115 or a flexible membrane 113 of the lower layers. In another example, when the valve apertures 180 are in the upper support 114, the flexible membrane 113 of the upper layers may be deformed by the insertion of a rod 550 in one of the valve apertures 180 and is pressed against the double-sided adhesive layer 111 of the lower layers. The double- sided adhesive layer 111 being adhesive while the flexible membrane 113 or the supports (114, 115) being not adhesive, this leads to a contact which is sticky enough to leave the channel 170 closed when the pressure of the rod 550 ceased but not sticky enough to maintain a pressure higher than 0.1 Mpa. The double-sided adhesive layer 111 is preferably flexible allowing to be deformed when closing the channel 170. This semi-reversible configuration is particularly advantageous for the elution outlet channel 170 which has to be closed during the injection of the sample then opened when an elution liquid is provided with pressure in the lysing chamber 120 to allow the internal material to reach the elution outlet port 140 and then closed again to avoid any way back of the internal material.

[0088] In a permanent configuration, the closure of a channel 170 is permanent meaning that, when a rod 550 inserted in the corresponding valve aperture 180 for closing the channel 170 is not pressed anymore against the support (114, 115) not comprising said valve aperture 180, the flexible layers in contact when the rod 550 applied the pressure remain permanently (definitely) stuck together. To do so, each layer comprised between the double-sided adhesive layer 111 comprised in the upper layers and the double-sided adhesive layer 111 comprised in the lower layers comprises an opening aligned with said valve aperture 180. By this way, the double-sided adhesive layer 111 of the upper layers is in direct contact with the double- sided adhesive layer 111 of the lower layers when the rod 550 is pressed against the support (114, 115) not comprising said valve aperture 180. The double-sided adhesive layers 111 being both adhesive, the contact between these layers leads to a strong sticking. The double-sided adhesive layers 111 are preferably flexible allowing to be deformed when closing the channel. This permanent configuration is particularly advantageous for the sample inlet channel 130 which has to be opened only when injecting the sample into the lysing chamber 120 and then definitely closed.

System for a micro-organism lysing

[0089] This disclosure also relates to a system for micro-organism lysing represented in figure 5. The system comprises a chip 100 for micro-organism lysing as disclosed hereabove. The system further comprises a grinder 500 (figure 5) comprising a platform (not shown) with a recess (not shown) configured to receive the chip 100 for microorganism lysing, a pestle 520 configured to be aligned with the lysing chamber 120, a motor 530 to rotate said pestle 520, and pressuring means (not shown) to press said pestle 520 against the lysing chamber 120. Pressuring means may be spring, a hydraulic or pneumatic pressure source, hydraulic or pneumatic cylinders, motor with screw, or a heavy mass. The system further comprises a set of four actuators, each actuator comprising a rod 550 configured to be independently inserted in the valve apertures 180 of the upper support 114 and pressed against the support (114, 115) opposite the valve aperture 180, thereby closing independently one of the four channels 170.

[0090] Chip 100 and grinder 500 are designed to co-operate in such a way that the chip 100 is immobilized in the grinder 500 while the pestle 520 is pressed through the pestle hole and rotated. The chip 100 is immobilized in the platform by any usual means, such as a hood or slides arranged on covers. [0091] The pestle 520 may have any structure on its grinding part. It may be smooth or irregular.

[0092] Advantageously, the grinding part of the pestle 520 is irregular, with a step shape. By step shape, it is meant that the grinding part comprises a discontinuity of thickness. During lysing, pestle 520 is rotated so that pressure applied on the lysing chamber 120 is not uniform: some parts are heavily pressed, while other parts are almost not pressed. Thus, shearing is not uniform and flexible membranes 113 or the double-sided adhesive layers 111 are sheared, distorted, bent in an irregular manner, leading to improved lysis.

[0093] The system may further comprise automated fluidic devices 560 - such as syringes, pumps and tubes, pipettes... - to supply sample in the sample inlet port 130, to supply rinsing solution in the rinsing inlet port 135, to collect sample in the elution outlet port 140 and to collect waste in the waste outlet port 145.

Multi-chip

[0094] This disclosure also relates to a multi-chip comprising at least two chips 100 for micro-organism mechanical lysing as described above. The upper support 114 of all chips 100 forms a single upper support. The lower support 115 of all chips 100 forms a single lower support. The chips 100 are thus maintained together by the single upper and/or lower surfaces. Preferably, the multi-chip comprises four or eight chips 100. An example of multi-chip is represented in figure 8.

[0095] In the multi-chip, the ports (130, 135, 140, 145) may be arranged in an array corresponding to a standard multichannel pipette design. For example, a standard multichannel pipette design comprises 4, 8 or 12 aligned channels spaced of about 9 to 14 mm. The multi-chip may thus comprise an array of at least one row of 4, 8 or 12 waste outlet ports 145, at least one row of 4, 8 or 12 elution outlet ports 140, at least one row of 4, 8 or 12 rinsing inlet ports 135 and/or at least one row of 4, 8 or 12 sample inlet ports 130. [0096] For example, the at least two chips 100 are distributed in a row. It is preferable that the inlet channels and outlet channels of the individual chips 100 do not intersect so as to avoid any cross -contamination.

[0097] The multi-chip is advantageous because the same sample may undergo different lysing protocols in the same multi-chip, allowing for detection of several microorganisms. This is particularly advantageous when using a multichannel pipette since the introduction to and extraction from the chips is parallelized which leads to time saving.

Method for micro-organism lysing

[0098] This disclosure also relates to a method for micro-organism lysing. In this method, a sample comprising micro-organisms is introduced in a chip 100. Then, the lysing chamber 120 of the chip 100 is pressed with a rotating pestle 520 and the internal material contained in the micro-organisms is recovered via the elution outlet port 140.

[0099] In particular, the method for micro-organism lysing may comprise the following steps. A sample comprising micro-organisms is introduced in a chip 100 as disclosed hereabove through the sample inlet port 130, preferably using a first syringe 560. Then, the sample is washed using rinsing solution injected in the lysing chamber 120 through the rinsing inlet port 135, preferably using a pump or a tube. The dirty waste liquid is then collected through the waste outlet port 145, preferably using a pump or a tube. The lysing chamber 120 of the chip 100 is then pressed with a rotating pestle 520 and the internal material contained in the micro-organisms is recovered through the elution outlet port 140, preferably using a pipette tip. Using different ports (130, 135, 140 and 145) connected to the lysing chamber via non-intersecting channels 170 advantageously allows to avoid contamination between the sample, the collected material and the rinsing liquids.

[0100] Advantageously, the chip 100 is placed in the recess of the platform of a grinder 500, said grinder 500 comprising a pestle 520 configured to be aligned with the lysing chamber 120 of the chip 100 for micro-organism lysing, a motor 530 to rotate said pestle 520, and pressuring means to press said pestle 520 against the lysing chamber 120. The grinder 500 disclosed hereabove is especially suitable for this method. [0101] In an embodiment, the pressure exerted by pestle 520 is ranging from 0.02 Mpa to 1.5 Mpa, preferably from 0.1 Mpa to 1 Mpa. The pressure may be selected according to the exact chip geometry, the type of flexible membranes 113 and filter(s) 112, and also to the type of micro-organisms to be lysed. The pressure exerted by pestle 520 is the force applied by pestle 520 divided by the surface of pestle grinding part. The force applied is ranging from I N to 24 N, for a surface of pestle grinding part ranging from 15 mm ² to 50 mm ² .

[0102] In an embodiment, the rotational speed of pestle 520 is ranging from lO rpm to 250 rpm, preferably from 30 rpm to l80 rpm. Rotational speeds of pestle 520 of 30 rpm, 60 rpm, 90 rpm, 120 rpm and 150 rpm are especially adequate. The rotational speed may be selected according to the exact chip geometry, the type of flexible membranes 113 and filter(s) 112, and also to the type of micro-organisms to be lysed.

[0103] In an embodiment, the duration of grinding is ranging from 30 s to 10 min. A short grinding duration is preferable, to improve output of system for micro-organism lysing. Duration of grinding or 1 min, 2 min, 3 min are especially adequate.

[0104] Appropriate conditions for lysing Gram-negative salmonella enterica are a grinding time of 1 min at 60 rpm with a pestle force of 12 N, with chip designed as in the first configuration with both the first and second variant (presence of the third and fourth double- sided adhesive layers 111).

[0105] Appropriate conditions for lysing Gram-positive listeria monocytogenes are a grinding time of 2 min at 60 rpm with a pestle force of 12 N, with chip designed as in the first configuration with both the first and second variant.

[0106] Appropriate conditions for lysing saccharomyces cerevisiae yeasts are a grinding time of 1 min at 120 rpm with a pestle force of 20 N, with chip designed as in the first configuration with both the first and second variant.

BRIEF DESCRIPTION OF THE DRAWINGS [0107] Figure 1 is a view of the structure of a chip - elements shown next to each other instead of superimposed, upper support on the left, lower support on the right - according to the combination of the first and the second variant of the first configuration of the disclosure.

[0108] Figure 2A is a 3-dimensional exploded view of the structure of a chip according to the combination of the first and the second variant of the first configuration of the disclosure. Figure 2B is a 3-dimensional view of the chip.

[0109] Figure 3 is a view similar to figure 1 at four different steps of the lysing method.

[0110] Figure 4 is a sectional side view of a chip according to the combination of the first and the second variant of the first configuration of the disclosure, highlighting valves in open configuration (figure 4A) and closed configuration (figure 4B). The section plane of Figure 4 follows the sample inlet channel and waste outlet channel.

[0111] Figure 5 shows the association of a grinder, four actuators and a chip forming a system for micro-organism lysing.

[0112] Figure 6 shows an upper view of another embodiment of a chip wherein the ports are on the upper support while the valve apertures are on the lower support as observed in figure 7.

[0113] Figure ? shows a side view of the chip of figure 6. The valve apertures are visible on the lower support.

[0114] Figure 8 shows a multi-chip according to one embodiment of the disclosure.

EXAMPLES

[0115] The present invention is further illustrated by the following examples. Method for micro-organism lysing detailed steps

[0116] This example illustrates the use of the valves during the lysing process. This example may be applied for both the first and second configurations. [0117] This example is illustrated in figure 3 for a chip in the first configuration with both the first and the second variant (presence of the third and fourth double- sided adhesive layers 111). The ports (130, 135, 140 and 145) and valve apertures 180 are distributed on the upper support 114.

[0118] In this example, the sample inlet channel 170 is entirely comprised in the first double-sided adhesive layer 111. This sample inlet channel 170 thus connects the sample inlet port 130 to the lysing chamber 120 via the first double-sided adhesive layer 111 and comprises a valve hole 150.

[0119] The rinsing inlet channel 170 is also entirely comprised in the first double- sided adhesive layer 111 without intersecting the sample inlet channel 170. This rinsing inlet channel 170 thus connects the rinsing inlet port 135 to the lysing chamber 120 via the first double-sided adhesive layer 111 and comprises a valve hole 150.

[0120] The elution outlet channel 170 and the waste outlet channel 170 both comprise a first part included in the fourth double- sided adhesive layer 111 and a second part included in the lower support 115 without intersecting each other. The elution outlet channel 170 thus connects the elution outlet port 140 to the lysing chamber 120 while the waste outlet channel 170 connects the waste outlet port 145 to the lysing chamber 120. The two parts of the elution outlet channel 170 are in fluid communication through the valve hole 150 comprised in the upstream end of the first part of the channel 170 and in the downstream end of the second part of the channel 170. In the same manner, the two parts of the waste outlet channel 170 are in fluid communication through the valve hole 150 comprised in the upstream end of the first part of the channel 170 and in the downstream end of the second part of the channel 170. In this example, the downstream end of the first part of the elution outlet channel 170 and waste outlet channel 170 are in fluidic communication with the lysing chamber through the communication hole 118.

[0121] The closure of the valves - and thus the channels 170 - of this example is reversible. Indeed, openings 160 are comprised in all the layers but the two flexible membranes 113 (one in the upper layers and one in the lower layers). This implies that only the flexible membranes 113 are deformed when a rod 550 inserted in one of the ports (130, 135, 140, 145) is pressed against the lower support 115 (figure 4B). Thus, only the non-adhesive flexible membranes 113 are in contact and recover their undeformed shape when the pressure applied by the rod 550 ceases (rod 550 removed, figure 4A).

[0122] In figure 3, each of the four rows represents a view of the same chip at a different step of the lysing method - elements shown next to each other instead of superimposed, upper support on the left, lower support on the right. The first raw is the sample injection into the lysing chamber 120, the second raw is the washing, drying and grinding of the sample, the third raw is the elution of the internal material and the fourth raw is the collection of the internal material.

[0123] In figure 3, the crosses on the valve holes 150 show the valve holes 150 wherein a rod 550 is inserted in order to close the corresponding channel 170.

[0124] During the injection of the sample (first raw), the elution outlet channel 170 and the rinsing inlet channel 170 are reversibly closed. Indeed, the elution outlet channel 170 and the rinsing inlet channel 170 must remain as clean as possible during the whole lysing to avoid any contamination. Therefore, to prevent any flow of the sample in these two channels 170, a rod 550 is inserted in the corresponding valve apertures 180 and pressed against the lower support 115. In this example, 1 milliliter of matrix and 1 milliliter of air are injected.

[0125] During the washing and drying of the sample (second raw), the sample inlet channel 170 is closed while the elution outlet channel 170 is maintained closed by insertion of rods 550 in the corresponding valve apertures 180. A rinsing solution comprising 1 milliliter of wash buffer and 1 milliliter of air are injected through the rinsing inlet port 135. The dirty rinsing solution is sucked through the flexible filter layer 112 and is collected via the waste outlet port 145.

[0126] The washed sample is then mechanically lysed - or grinded - by applying, with the pestle 520 a pressure on the lysing chamber 120. The internal material is flowing through the flexible filter layer 112. After the lysing, the internal material is contained in the chamber hole of the second double-sided adhesive layer 111 on both sides of the filter. [0127] During the elution (third raw), the waste outlet channel 170 is closed by insertion of a rod 550 in the corresponding valve aperture 180 and the elution outlet channel 170 is opened by removal of the rod 550 in the corresponding valve aperture 180. 30 microliters of elution buffer mixed with air is injected in the lysing chamber 120 via the rinsing inlet port 135. The elution buffer passes through the flexible filter layer 112 and pushes the internal material out of the lysing chamber 120 through the elution outlet channel 170.

[0128] The elution outlet channel 170 is then closed (fourth raw) by insertion of a rod 550 in the corresponding valve aperture 180 in order to avoid any return of the elution towards the lysing chamber 120. The elution (containing the internal material) is then collected through the elution outlet port 140.

[0129] Example 2: Chip performance

[0130] Two chips are used, with the following specifications:

[0131] Chip design I according to first configuration: elements are laid in this order - direct contact - from top to bottom: upper support 114 with sample inlet port 130, rinsing inlet port 135, elution outlet port 140, waste outlet port 145 and valve apertures 180; third double-sided adhesive 111; first flexible membrane 113; first doublesided adhesive 111 with sample and rinsing inlet channels 170; flexible filter 112; second double-sided adhesive 111; second flexible membrane 113; fourth double-sided adhesive 111 with part of the waste and elution outlet channels 170 and lower support 115 comprising the rest of the waste and elution outlet channels 170 grooved. All doublesided adhesive layers 111 and flexible membrane 113 have inlet hole and outlet hole. In design I; the volume of the lysing chamber 120 upstream of the filter 112 is about 30 pL.

[0132] Chip design II according to first configuration: elements are laid in this order - direct contact - from top to bottom: upper support 114 with sample inlet port 130, rinsing inlet port 135, elution outlet port 140, waste outlet port 145 and valve apertures 180; third double-sided adhesive 111; first flexible membrane 113; additional double-sided adhesive 111; spacer layer; first double-sided adhesive 111 with sample and rinsing inlet channels 170; flexible filter 112; second double-sided adhesive 111 with waste and elution outlet channels 170; second flexible membrane 113; fourth doublesided adhesive 111 and lower support 115. All double- sided adhesive layers 111; spacer layer and flexible membrane 113 have inlet hole and outlet hole. In design II; the spacer layer and additional double-sided adhesive layer define a thicker lysing chamber: volume of lysing chamber 120 upstream of the filter 112 about 45 pL.

[0133] The two chips have a chamber hole of diameter 10 mm, inlet holes and outlet holes of diameter 4 mm. Double-sided adhesive thickness is 150 pm. Flexible membrane 113 thickness is 50 pm. Flexible filter 112 is a disc of diameter 13 mm and thickness 10 pm. Upper support 114 and lower support 115 are in polycarbonate, with a thickness of 2 mm, resp. 1 mm.

[0134] Grinding protocol used in this example has the following specifications. Chip is inserted in the grinder 500 as shown on figure 5. Then pestle 520 - step shaped surface of grinding part about 25 mm ² - is brought in rotation at rotational speed of 60 rpm and pressed against the lysing chamber 120 with a force of 12 N for 2 min - pressure applied by the pestle 520 is about 0.5 Mpa.

[0135] Two types of protocols have been used. In both protocols, the channels are opened and closed following the steps described in Example 1.

PCR protocol

[0136] A micro-organism is cultured overnight in suitable conditions of temperature, humidity and medium. Then, micro-organisms are transferred into Ringer’s solution and diluted to reach a colony forming unit (CFU) of 10 ⁴ CFU/mL. Then 25 mL of said solution is added to 225 mL of BPW culture medium - buffered peptone water - and 25 g of a mixture of frozen vegetables into a culture bag, yielding a 10 ³ CFU/mL sample. The sample is homogenized using a Stomacher - BagMixer® 400 from Interscience, 8 coups/s, 400W power - during 1 min. This process aims at preparing a sample representative of a food product containing 10 ³ CFU/mL of a known micro-organism.

[0137] 1 mL of said sample is introduced in a chip through the sample inlet port 130.

Then, 1 mL of rinsing buffer is introduced in the chip through the rinsing inlet port 135. The dirty rinsing solution is collected via the waste outlet port 145. The chip is placed in the grinder 500 for 1 min, with determined pestle 520, pressure and rotational speed. A 20 pL sample is collected from the lysing chamber 120 through the elution outlet port 140 and tested by Polymerase Chain Reaction (PCR) in conditions suitable for the nucleic acid of interest, allowing for detection of the micro-organisms. Cycle threshold (Ct) is then reported. The lowest the Ct value, the best the lysis performance.

DNA dosing protocol

[0138] A micro-organism is cultured overnight in suitable conditions of temperature, humidity and medium. Then, micro-organisms are transferred into Ringer’s solution and diluted to reach a colony forming unit (CFU) of 10 ⁷ CFU/mL. 1 mL of said sample is introduced in a chip through the sample inlet port 130. Then, 1 mL of rinsing buffer is introduced in the chip through the rinsing inlet port 135. The dirty rinsing solution is collected via the waste outlet port 145. The chip is placed in the grinder 500 for 1 min, with determined pestle 520, pressure and rotational speed. A 20 pL sample is collected from the lysing chamber 120 through the elution outlet port 140. DNA content is then measured according to state-of-the-art methods (in ng/pL), as well as Cycle threshold (Ct). Note that sample is much more concentrated in DNA protocol (10 ⁷ CFU/mL) as compared to PCR protocol (10 ³ CFU/mL): Ct in DNA protocol is thus much lower than Ct in PCR protocol.

[0139] The following tables show experimental results. For each experiment, measurements were reproduced three or five times, result is an average. When results are highly dispersed, an indication of dispersion is given.

[0140] Various designs and parameters have been used in PCR protocol (10 ³ CFU/mL), showing overall a very good performance for lysing and detection of micro-organisms. It appears that polycarbonate-based filters (F2 and F5) give better results, as well as PET membranes (M2 and M3). Chip design I also shows the best results, with Ct 31 and 32, when filter F5 and membrane M3 are used.

[0141] Comparative experiments have been run, using the DNA protocol (10 ⁷ CFU/mL).

Chip I in association with filter F5 and membrane M3 shows a much better sensibility as compared to prior art methods. DNA retrieved after lysis is at least 5 times more concentrated.

Table 1

NUMERICAL REFERENCES 100 - Chip / 110 - Multilayer stack / 111 - Double sided adhesive layer / 112 - Flexible filter layer / 113 - Flexible membrane / 114 - Upper support / 115 - Lower support / 118 - Communication hole / 120 - Lysing chamber / 130 - Sample inlet port / 135 - Rinsing inlet port / 140 - Elution outlet port / 145 - Waste outlet port / 150 - Valve holes / 160 - Openings / 170 - Channels / 180 - Valve apertures / 500 - Grinder / 520 - Pestle / 530 - Motor / 550 - Rod / 560 - Fluidic devices