Chips, grinders and their uses for lysing microorganisms

Abstract

The present invention relates to a chip for mechanical lysis of microorganisms comprising a lysis chamber, a mechanical system operating with such a chip and a method for mechanical lysis of microorganisms.

Description

[Technical field]

[0001] The present invention relates to a chip for microbial lysis, a mechanical system operating with such a chip and a method for microbial lysis. [Background technology]

[0002] Microbial lysis is a phenomenon that destroys - partially or completely - the external protection of a microorganism. In particular, cell lysis is the destruction of the cell membrane. After lysis, the internal contents of the microorganism are released and can be extracted or identified by various methods. In particular, nucleic acid material - DNA or RNA - is released and can be used as starting material for the polymerase chain reaction that leads to the identification of the microorganism type. Protein material can also be extracted and used for the identification of the microorganism. Such methods are now very common in many fields, including diagnostics and quality control in the food industry, hygiene or cosmetics.

[0003] Various methods for microbial lysis are known and can be divided into three categories: Chemical lysis uses chemical agents that degrade the external protection of the microorganism. However, these chemical agents may also degrade the useful internal contents of the microorganism. Thermal lysis uses heat to degrade the external protection of the microorganism. However, heating usually leads to the degradation of nucleic acid material and proteins. Finally, mechanical lysis aims at the destruction of the external protection of the microorganism by mechanical stress.

[0004] In addition, it is desirable for an apparatus for lysis of microorganisms to be easily portable, efficient, reliable with small sample sizes, and capable of separating the internal material of the microorganism from its external protective material.

[0005] WO2015181743 describes, inter alia, an apparatus for mechanical lysis. In said apparatus, mechanical lysis is carried out by shearing action of a microbial suspension between two walls, one of which has a rough glass-containing surface. However, the results obtained are not good, especially for gram-positive bacteria, and the use of glass is not acceptable for the food and health sector.

[0006] European patent application EP 3222989 discloses a mechanical lysis device in which a suspension of microorganisms is ground with an abrasive surface, but the use of a grinding surface is a complicated procedure and is not sufficiently sensitive for identification.

[0007] The subject of this disclosure is a particular chip structure, in which various layers are used to define chambers that allow for the separation of the internal material of microorganisms while mechanical lysis is achieved, which overcomes some or all of the above mentioned drawbacks. Summary of the Invention

[0008] The present invention relates to a chip for mechanical lysis of microorganisms, said chip comprising: a) a multi-layer stack, i. a first double-sided adhesive layer containing a chamber hole; ii. a flexible filter layer in direct contact with the first double-sided adhesive layer and covering the chamber aperture; iii. a second double-sided adhesive layer in direct contact with the flexible filter layer and containing the chamber holes; and iv. a flexible membrane on the first double-sided adhesive layer opposite the flexible filter; a multi-layer stack with all chamber holes overlapping to form a lysis chamber; and b) a closure surface on a second double-sided adhesive layer of the multi-layer stack opposite the flexible membrane, the closure surface being the second flexible membrane; c) an inlet in fluid communication with the lysis chamber.

[0009] Indeed, this tip allows the mechanical lysis of microorganisms while avoiding the degradation of their internal contents, nucleic acid material and proteins.

[0010] Furthermore, the use of filters allows the lysis of microorganisms for analysis in the food and health sector.

[0011] Furthermore, the chip of the present invention allows for a simple mechanical lysis procedure and increased sensitivity for identification.

[0012] Finally, the use of a second flexible membrane can increase the efficiency of lysis and reduce variability in the reproducibility of lysis efficiency.

[0013] In other advantageous embodiments of the invention, the chip for microbial lysis comprises one or more of the following characteristics, taken alone or in any technically possible combination:

[0014] Chip for mechanical lysis of microorganisms a) a rear cover on the membrane side of the multi-layer stack, containing a pestle hole aligned with the lysis chamber; b) a rigid cover on the second flexible membrane opposite the rear cover; and c) fastening means for holding the rigid cover and the rear cover in contact with the sealed multi-layer stack; The inlet is on the rigid cover and fluid communication is arranged by overlapping inlet holes in the layers of the multi-layer stack and in the second flexible membrane to form an inlet chamber and an inlet channel between the chamber hole and an inlet hole in one of the double-sided adhesive layers.

[0015] Additionally, the rear and rigid cover provide mechanical stability to the chip, allowing for easier handling.

[0016] Additionally, an inlet may be located on the rigid cover to allow connection of a cone or syringe for injection of liquid components.

[0017] Furthermore, the pestle holes can increase the efficiency of the mechanical lysis of microorganisms by defining an area for the application of mechanical action that corresponds precisely to the size of the lysis chamber.

[0018] Finally, the use of a rigid cover and fixing means allows for increased rigidity of the multilayer stack and membranes of the entire chip except for the lysis chamber, allowing the applied mechanical action to be concentrated in the lysis chamber.

[0019] The chip for mechanical lysis of microorganisms further comprises an outlet in fluid communication with the lysis chamber, the outlet being on the rigid cover; The fluid communication noted as i.A1 is arranged by overlapping an outlet hole in a layer of the multilayer stack and in the second flexible membrane to form an outlet chamber; and an outlet channel between the chamber hole and the outlet hole in the double-sided adhesive layer that does not include an inlet channel. The fluid communication noted as ii.B1 is provided by a hole in the second flexible membrane that is superimposed on the periphery of the lysis chamber; and an outlet channel between the hole and the outlet of the rigid cover.

[0020] Indeed, the inlet and outlet on the same side of the same cover can facilitate the manipulations performed on the microorganisms since simultaneous use of tools at the inlet and outlet can be performed on the same side of the chip.

[0021] Additionally, locating an outlet on the rigid cover allows for connection of a cone or syringe for collection of the liquid component.

[0022] The fastening means includes a third double-sided adhesive layer in direct contact between the rear cover and the multi-layer stack, said third double-sided adhesive layer including a pestle hole aligned with the lysis chamber.

[0023] The fastening means includes a third double-sided adhesive layer in direct contact between the rear cover and the multi-layer stack, and including a pestle hole superimposed with the lysis chamber; and a fourth double-sided adhesive layer in direct contact between the rigid cover and the second flexible membrane, the layer including an inlet hole superimposed with the inlet chamber; and i. an outlet hole overlapping another outlet hole for fluid communication A1; or ii. A chamber hole superimposed with the lysis chamber for fluid communication B1.

[0024] At least one of the flexible films is a polyethylene terephthalate film, preferably at least one of the flexible films has a thickness in the range of 15 μm to 150 μm, more preferably all of the flexible films are polyethylene terephthalate films having a thickness in the range of 15 μm to 150 μm.

[0025] Indeed, the use of polyethylene terephthalate makes it possible to obtain a membrane that is biologically compatible.

[0026] Furthermore, below 15 μm the membrane is susceptible to tearing, and above 150 μm the efficiency decreases, so this thickness range is optimal.

[0027] The flexible filter layer is a polycarbonate layer, and preferably the flexible filter layer has pores in the range of 0.2 μm to 1 μm and / or a thickness in the range of 5 μm to 50 μm.

[0028] The present invention also relates to a multi-chip comprising at least two tips for mechanical lysis of microorganisms, preferably in one aspect of the invention, four or eight tips, the closed surfaces of all the tips forming a single closed surface.

[0029] In other advantageous aspects of the invention, the multichip comprises one or more of the following features, either alone or in combination: The outlets or inlets are arranged in an array corresponding to standard multi-channel pipette designs. At least two chips are arranged in a row.

[0030] The present invention also relates to a system for mechanical lysis of microorganisms, the system comprising: a. a chip or multi-tip for mechanical lysis of microorganisms according to one embodiment of the present invention; b. A grinding machine, i. a platform having a recess configured to receive a tip or multiple tips for mechanical lysis; ii. a pestle configured to be aligned with the lysis chamber; iii. a motor for rotating the pestle; and and iv. a pressurizing means for pressing said pestle against a dissolution chamber.

[0031] Indeed, the system is compact and the compatibility of its components is optimized to enhance the efficiency of the mechanical lysis.

[0032] Furthermore, the application of pressure and rotation allows for efficient deformation of the cell membrane, increasing the efficiency of cell lysis.

[0033] In another advantageous embodiment of the invention, the system for mechanical lysis of microorganisms comprises the following features: the pestle grinding section comprises one step.

[0034] In effect, this can create a pinch-smash area in the lysis chamber, allowing for the disruption of cells.

[0035] The present invention also relates to a method for mechanical lysis of microorganisms, the method comprising the steps of: a. introducing a sample containing microorganisms through an inlet of a chip or multichip for mechanical lysis of microorganisms according to one aspect of the present invention; b. pressurizing the lysis chamber with a rotating pestle; and c. Recovering the contents of the cells.

[0036] Indeed, the simultaneous application of pressure and rotation makes it possible to increase the efficiency of cell lysis by efficient deformation of the cell membrane.

[0037] In other advantageous embodiments of the invention, the method for mechanical lysis of microorganisms comprises one or more of the following characteristics, taken alone or in any technically possible combination:

[0038] A tip or multi-tip for mechanical lysis of microorganisms is placed in a recess in the platform of a grinder, said grinder comprising: i. a pestle configured to be aligned with a lysis chamber of a tip or multi-tip for mechanical lysis of microorganisms; ii. a motor for rotating the pestle; and iii. a pressurizing means for pressurizing said pestle against the lysis chamber.

[0039] Indeed, the alignment of the pestle with the lysis chamber allows for efficient distribution of pressure to the lysis chamber.

[0040] The force of the pestle is in the range of 1N to 24N, and the rotation speed of the pestle is in the range of 30 rpm to 150 rpm.

[0041] This range of forces and rotational speeds increases the efficiency of cell lysis without damaging the cell contents.

[0042] definition For the purposes of the present invention, the following terms have the following meanings:

[0043] "Direct contact" between two layers means that both layers are not separated by another layer.

[0044] "Flexible" means chip components, particularly filters and membranes, that can deform under pressure in a direction perpendicular to their surface (bending) and can deform and crease (stretch and fold) in their plane when subjected to shear forces.

[0045] "Microorganism" means biological material such as viruses, archaea, bacteria, fungi and cells.

[0046] In the context of two layers, "over" means that one layer is positioned above the other layer, either in direct contact or separated by one or several intermediate layers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The present disclosure relates to a chip 100 for mechanical lysis of microorganisms. In a first basic configuration, the chip 100 includes a multi-layer stack 110 including a first double-sided adhesive layer 111 that includes 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 includes a chamber hole. Finally, a flexible membrane 113 is on the first double-sided adhesive layer 111, opposite the flexible filter layer 112. Furthermore, all the chamber holes are overlapped to form a lysis chamber 120.

[0048] The chip 100 further includes a closing surface on the second double-sided adhesive layer 111 of the multi-layer stack 110 opposite the flexible membrane 113, the closing surface being the second flexible membrane 113. and the inlet 130 are in fluid communication with the lysis chamber 120. In this configuration, the chip 100 is entirely flexible, with a well-defined chamber bounded by the two flexible membranes 113, separated into two parts by a flexible filter layer 112 in the first basic configuration, and separated into three parts by two flexible filter layers 112 in the second basic configuration. Preferably, the second flexible membrane 113 is similar - of the same material and the same flexibility properties - to the first flexible membrane 113.

[0049] In this first basic configuration, the chip 100 includes two exterior surfaces - a flexible membrane 113 on one side and a closed side on the opposite side - inside of which two adhesive layers 111 sandwich a flexible filter layer 112. The thickness of the double-sided adhesive layers 111 and the chamber holes define a cavity into which a sample can be introduced through an inlet 130. The combination of the multilayer stack 110 and the closed side then becomes a sealed multilayer stack 110.

[0050] In this disclosure, the filter layer 112 and the membrane 113 are flexible. By flexible, we mean that these flat components can deform in a direction perpendicular to their surface under pressure (bending) and can deform and crease in their plane when subjected to shear forces (stretching and folding). Although the filter layer 112 and the membrane 113 are flexible, they do not tear under the constraints considered, and they have a stable shape in the absence of pressure and / or shear forces.

[0051] Finally, the chip 100 can facilitate the injection of a sample into a chamber that can be strongly deformed and sheared by an external mechanical device: the microorganisms in the sample can be efficiently crushed, leading to mechanical lysis. Due to the pores of the filter 112, separation of the internal material of the microorganisms from their outer membranes - cell membrane, capsid, ... - is achieved, and the internal material of the microorganisms passes through the filter, while the outer membrane is prevented from passing through by the filter. Thus, the internal material of the microorganisms is accumulated on the side of the lysis chamber 120 - downstream - opposite to the side of the lysis chamber 120 - upstream - connected to the inlet 130, where it can be collected for further analysis, such as PCR amplification.

[0052] In the second basic configuration, a second flexible filter layer 112 is introduced into the multi-layer stack 110 of the chip 100 to maximize the amount of sample injected. In this configuration, a second double-sided adhesive layer 112 is aligned to the first flexible filter layer 112 and is in direct contact with the second double-sided adhesive layer opposite the closure surface. In this configuration, a separation layer containing chamber holes aligned to the flexible filter layer 112 is inserted between the two flexible filter layers 112. The separation layer may be a double-sided adhesive layer.

[0053] In the following, the first and second basic configurations can be used independently in all configurations and variants, and in fact the single flexible filter layer 112 can be replaced by a pair of flexible filter layers 112 in all configurations and variants disclosed hereinafter.

[0054] In this first configuration embodiment, the tip 100 further includes a rear cover 115 on the membrane side of the multi-layer stack 110 that includes a pestle hole aligned with the lysis chamber 120. This rear cover 115 provides mechanical stability to the tip 100 and allows for easier handling. The pestle hole is aligned with the lysis chamber 120 so that an external component, such as the pestle 520 of the mechanical grinder 500, can engage the pestle hole to compress and apply shear forces to the flexible membrane 113 and flexible filter layer 112 of the tip 100.

[0055] In this embodiment, the chip 100 also includes a rigid cover 114 on the second flexible membrane 113 opposite the rear cover 115. This rear cover 114 also provides mechanical stability to the chip 100, allowing for easier handling. In addition, the rigid cover 114 is the surface against which the flexible membrane 113 and the flexible filter layer 112 are pressed by external components to increase pressure and shear forces. Both covers - the rear cover 115 and the rigid cover 114 - are held in contact with the sealed multi-layer stack 110 using fastening means.

[0056] Finally, in this embodiment, the layers of the multilayer stack 110 and the second flexible membrane 113 contain superimposed inlet holes, forming a small inlet chamber. In one of the double-sided adhesive layers 111 of the multilayer stack 110 of the first basic configuration, the inlet channel 116 achieves a fluid connection between the chamber hole and the inlet hole. In the case of the second basic configuration, the inlet channel 116 needs to be placed in the separation layer between the two flexible filter layers 112 to inject the sample. Finally, the inlet 130 of the chip is placed on the rigid cover 114 above the inlet chamber, and is therefore in fluid communication with the lysis chamber 120 via the inlet channel 116.

[0057] Finally, in this embodiment, the chip 100 is reinforced by both covers such that a liquid sample deposited in the inlet 130 can flow through the inlet chamber and then through the inlet channel 116 to the interior of the lysis chamber 120. This is particularly interesting for introducing a sample into the chip 100 using, for example, a micropipette, as the inlet 130 and both covers aid in precisely positioning the tip of the micropipette.

[0058] An outlet 140 may be on the rigid cover 114 and in fluid communication with the lysis chamber 120 for collecting the internal material of the microorganisms after lysis.

[0059] In a first variant configuration shown in a non-limiting manner in FIG. 1, the fluid communication between the outlet 140 and the lysis chamber 120 is noted as A1 and is designed as follows: the layer of the multilayer stack 110 and the second flexible membrane 113 contain superimposed outlet holes to form a small outlet chamber. In the first basic configuration, the double-sided adhesive layer 111 of the multilayer stack 110 that does not contain the inlet channel 116 contains an outlet channel 117 that establishes a fluid connection between the chamber hole and the outlet hole. In the case of the second basic configuration, both double-sided adhesive layers 111 of the multilayer stack 110 that does not contain the inlet channel 116 contain an outlet channel 117 between the respective chamber hole and the outlet hole. Finally, the outlet 140 of the chip is disposed on the rigid cover 114 above the outlet chamber, and is thus in fluid communication with the lysis chamber 120 via the outlet channel 117. In this variant configuration, the sample can flow from the inlet 130 to the outlet 140 via the lysis chamber 120 and the flexible filter layer 112. Thus, only the internal material of the microorganisms can be collected at the outlet 140 .

[0060] In a second variant configuration shown in a non-limiting manner in FIG. 2, the fluid communication between the outlet 140 and the lysis chamber 120 is noted as B1 and is designed as follows: The second flexible membrane 113 includes a communication hole 118 arranged to overlap the periphery of the lysis chamber 120. The communication hole 118 is advantageously smaller than the chamber hole, so that the second flexible membrane 113 almost completely closes the lysis chamber 120, except for a small outlet via the communication hole 118. A side view of the chip 100 with the communication hole 118 is shown in FIG. 3, and an enlarged view is shown in FIG. 4. In the first basic configuration, the rigid cover 114 on the side of the second flexible membrane 113 includes an outlet channel 117 between the communication hole 118 and the outlet 140. In the second basic configuration, the second flexible membrane 113 includes a through hole 118 arranged to overlap at the periphery of the lysis chamber 120, and the rigid cover 114 includes an outlet channel 117 between the through hole 118 and the outlet 140. Furthermore, in this configuration, the layers of the multi-layer stack 110 and the second flexible membrane 113 include overlapping outlet holes to form a small outlet chamber, and the first double-sided adhesive layer 111 includes an outlet channel 117 between the chamber hole and the outlet hole. Advantageously, the outlet channel 117 is formed as a groove in the surface of the rigid cover 114, as shown in Figures 2 and 3. Advantageously, a recess 510 is bored in the rigid cover 114 in alignment with the lysis chamber 120, the through hole 118 allows fluid flow between the lysis chamber 120 and said recess 510, and the outlet channel 117 is formed as a groove between said recess 510 and the outlet 140. In this variant, the internal material of the microorganisms can be collected almost directly in the lysis chamber 120. This second variant has several advantages: the groove formation of the outlet channel 117 in the rigid component is easier than designing the outlet channel 117 in the adhesive layer. Furthermore, the pressure drop through the outlet channel 117 of the rigid component is much smaller than in the adhesive layer: therefore, the pressure required for the introduction of the sample into the lysis chamber 120 is smaller in this second variant.

[0061] In a particular aspect of this embodiment - applicable to both variant configurations - the fastening means intended to hold both covers in contact with the sealed multi-layer stack 110 comprises a third double-sided adhesive layer 111 in direct contact between the rear cover 115 and the multi-layer stack 110, said third double-sided adhesive layer 111 comprising a pestle hole superimposed with the melting chamber 120.

[0062] In a particular embodiment of the first variant configuration, the fastening means intended to hold both covers in contact with the sealed multi-layer stack 110 comprises a third double-sided adhesive layer 111 in direct contact between the rear cover 115 and the multi-layer stack 110, and also comprises a pestle hole superimposed with the lysis chamber 120; and a fourth double-sided adhesive layer 111 in direct contact between the rigid cover 114 and the second flexible membrane 113. The fourth double-sided adhesive layer 111 further comprises an inlet hole superimposed with the inlet chamber and an outlet hole superimposed with the other outlet hole. In this particular configuration, the bonding force of the chip 100 is ensured by the adhesive over the entire surface of the chip 100, leading to a very good resistance of the chip 100 during handling, sample introduction and crushing.

[0063] In a particular embodiment of the second variant configuration, the fastening means intended to hold both covers in contact with the closed multi-layer stack 110 comprises a third double-sided adhesive layer 111 in direct contact between the rear cover 115 and the multi-layer stack 110, and also comprises a pestle hole superimposed with the lysis chamber 120; and a fourth double-sided adhesive layer 111 in direct contact between the rigid cover 114 and the second flexible membrane 113. The fourth double-sided adhesive layer 111 further comprises an inlet hole superimposed with the inlet chamber and a chamber hole superimposed with the lysis chamber 120. In this particular configuration, the bonding force of the chip 100 is ensured by the adhesive, leading to a very good balance between the adhesive surface - for the stability of the chip during handling, sample introduction and grinding - and the retention of the flexible filter layer 112 free of any adhesive on the filtering area.

[0064] In the first configuration, the double-sided adhesive layer 111 and the flexible membrane 113 may all include inlet and outlet holes. These holes are not all necessary to ensure fluid communication between the inlet 130 and the lysis chamber 120, and between the lysis chamber 120 and the outlet 140, as required, but it is advantageous. Indeed, when placing a sample in the inlet 130, for example, with a micropipette, the tip of the micropipette will enter the multi-layer stack 110 and become stuck by the adhesive, thus clogging the micropipette or degrading the sample delivery function. The inlet and outlet holes in all the double-sided adhesive layers 111 and the flexible membrane 113 prevent this drawback.

[0065] In the second configuration, the closing surface is a rigid cover 114. In this configuration, the chip 100 is entirely rigid, with a well-defined chamber bounded by a flexible membrane 113 and a rigid cover 114, and separated into two parts by a flexible filter layer 112. The rear cover 114 provides mechanical stability to the chip 100 and allows for easier handling.

[0066] In an embodiment of the second configuration, the tip 100 further includes a rear cover 115 on the side of the multi-layer stack 110 opposite the rigid cover 114, which includes a pestle hole aligned with the lysis chamber 120. The pestle hole is aligned with the lysis chamber 120 so that an external component, such as the pestle 520 of the mechanical grinder 500, can engage the pestle hole to press the flexible membrane 113 and flexible filter layer 112 of the tip 100 against the rigid cover 114, applying a shear force. Both covers, the rear cover 115 and the rigid cover 114, are held in contact with the sealed multi-layer stack 110 using fastening means.

[0067] An outlet 140 may be on the rigid cover 114 and in fluid communication with the lysis chamber 120 for collecting the internal material of the microorganisms after lysis.

[0068] In a first variant configuration of this embodiment, the fluid communication between the outlet 140 and the lysis chamber 120 is noted as A2 and is designed as follows: the layers of the multilayer stack 110 contain overlapping outlet holes to form a small outlet chamber. In the first basic configuration, one double-sided adhesive layer 111 of the multilayer stack 110 contains an outlet channel 117 that establishes a fluid connection between the chamber hole and the outlet hole. In the case of the second basic configuration, both double-sided adhesive layers 111 of the multilayer stack 110 that are not disposed between the two flexible filter layers 112 contain an outlet channel 117 between their respective chamber holes and the outlet hole. Finally, the outlet 140 of the chip 100 is disposed on the rigid cover 114 above the outlet chamber, and is therefore in fluid communication with the lysis chamber 120 via the outlet channel 117.

[0069] In a second variant configuration of this embodiment, the fluid communication between the outlet 140 and the lysis chamber 120 is noted as B2 and is designed as follows: In a first basic configuration, the rigid cover 114 on the side of the lysis chamber includes an outlet channel 117 between the chamber hole and the outlet 140. In the case of a second basic configuration, the layers of the multilayer stack 110 include superimposed outlet holes to form a small outlet chamber. In this configuration, the first double-sided adhesive layer 111 includes an outlet channel 117 between the chamber hole and the outlet hole, and the outlet hole and the rigid cover 114 include an outlet channel 117 between the chamber hole and the outlet 140. Advantageously, the outlet channel 117 is formed as a groove in the surface of the rigid cover 114. Advantageously, a recess 510 is bored in the rigid cover 114 in alignment with the lysis chamber 120, whereby the outlet channel 117 is formed as a groove between said recess 510 and the outlet 140.

[0070] Finally, in this embodiment - for both variant configurations - the layers of the multilayer stack 110 contain superimposed inlet holes, forming a small inlet chamber. In the first basic configuration, one of the double-sided adhesive layers 111 of the multilayer stack 110 that does not contain an outlet channel 117 contains an inlet channel 116 that establishes a fluid connection between the chamber hole and the inlet hole. In the case of the second basic configuration, the inlet channel 116 must be contained in a separation layer that is placed between the two flexible filter layers 112. Finally, the inlet 130 of the chip 100 is placed on the rigid cover 114 above the inlet chamber and is therefore in fluid communication with the lysis chamber 120 via the inlet channel 116.

[0071] In this embodiment, the sample introduced through the inlet 130 flows into the lysis chamber -upstream- and through the flexible filter layer 112. Thus, only the internal material of the microorganisms can be collected at the outlet 140 -downstream.

[0072] In a particular aspect of this embodiment - applicable to both variant configurations - the fastening means intended to hold both covers in contact with the sealed multi-layer stack 110 comprises a third double-sided adhesive layer 111 in direct contact between the rear cover 115 and the multi-layer stack 110, said third double-sided adhesive layer 111 comprising a pestle hole superimposed with the melting chamber 120.

[0073] In the second configuration, the double-sided adhesive layer 111 and the flexible membrane 113 may all include inlet and outlet holes. These holes are not all necessary to ensure fluid communication between the inlet 130 and the lysis chamber 120, and between the lysis chamber 120 and the outlet 140, as required, but they are advantageous. Indeed, when placing a sample in the inlet 130, for example, with a micropipette, the tip of the micropipette will enter the multi-layer stack 110 and become stuck by the adhesive, thus clogging the micropipette or degrading the sample delivery function. Inlet and outlet holes in all the double-sided adhesive layers 111 and the flexible membrane 113 prevent this drawback.

[0074] Additional layers may be introduced into the chip 100 to increase the thickness of the chamber. Other additional layers may be metal films that provide electrical conductivity and - by Joule effect - heat generation or thermal conductivity. Other additional layers may be rubber membranes that can define valves in the fluidic pathway. Other additional layers may be hydrophobic layers - like PTFE or surface-modified plastics - to improve the hydrodynamic conditions such as sliding on the walls / boundaries, leading to a reduction in the pressure required to generate flow in the fluidic pathway. Other additional layers may be spacer layers, for example, aimed at increasing the volume of the lysis chamber made from any material. Such additional layers should include inlet holes, outlet holes or pestle holes to maintain the fluidic connection disclosed above.

[0075] In the present disclosure, the flexible membrane 113 may be selected from any material suitable for withstanding the mechanical and shear stresses required for lysis of microorganisms. In particular, flexible membranes 113 made of polyethylene terephthalate (PET), particularly PET flexible membranes having a thickness in the range of 15 μm to 150 μm, are suitable.

[0076] In the present disclosure, the flexible filter layer 112 may be selected from any material suitable for withstanding the mechanical and shear stresses required for lysis of microorganisms. In particular, flexible filter layers 112 made of polycarbonate (PC) are preferred, particularly PC flexible filters having pores in the range of 0.2 μm to 1 μm. The flexible filter layer 112 has a thickness in the range of 5 μm to 50 μm.

[0077] The present invention also relates to a multichip comprising at least two chips 100, preferably four or eight (FIG. 8A) of the above disclosed chips 100. The closing surfaces of all the chips form a single closing surface, and the chips 100 are held together by the single closing surface.

[0078] In a multi-tip, the inlets 130 and / or outlets 140 may be arranged in an array corresponding to a standard multi-channel pipette design. For example, a standard multi-channel pipette design may include 4, 8 or 12 aligned channels spaced approximately 9-14 mm apart. A multi-tip may thus include an array of at least one row of 4, 8 or 12 outlets 140 and / or at least one row of 4, 8 or 12 inlets 130.

[0079] At least two chips 100 are preferably arranged in a row, as shown in Figures 8 A and 8 B. The inlet and outlet channels of the individual chips 100 preferably do not intersect to avoid any cross-contamination.

[0080] Multi-tips are advantageous because in the same multi-tips the same sample can be subjected to different lysis protocols, allowing the detection of several microorganisms. This is particularly advantageous when using multi-channel pipettes, since the introduction into and extraction from the chip can be done in parallel, saving time.

[0081] The present invention also relates to a system for lysis of microorganisms. The system includes the tip for lysis of microorganisms 100 disclosed above. The system further includes a grinder 500 (FIG. 5) including a platform having a recess 510 configured to receive the tip for lysis of microorganisms 100, a pestle 520 configured to align with the lysis chamber 120, a motor 530 for rotating said pestle 520, and a pressure means 540 for applying pressure to said pestle 520 against the lysis chamber 120. The pressure means 540 may be a spring, a hydraulic or pneumatic pressure source, a hydraulic or pneumatic cylinder, a screw motor, or a heavy weight.

[0082] The tip 100 and grinder 500 are designed to cooperate such that the tip 100 is secured in the grinder 500 while compressing and rotating the pestle 520 - through the pestle hole if the tip 100 includes a rigid cover 114 - against the lysis chamber 120. A diagram of the tip 100 inserted into the grinder 500 is shown in FIG.

[0083] The grinder 500 may be designed to receive the tip 100 including the cover, in which case the pestle 520 is engaged through the pestle aperture to apply pressure to the lysis chamber 120. The tip 100 is secured to the platform by conventional means such as a hood or slide placed over the cover.

[0084] Alternatively, the grinder 500 can be designed to receive the tip 100 without a cover, i.e., a flexible tip. In this case, the shape of the platform is configured to fit the exact shape of the flexible tip 100, and a hood is used to secure the tip 100. A pestle 520 then pressurizes the lysis chamber 120 against the hood. With this uncovered tip 100, which exhibits a symmetrical configuration with respect to the flexible filter layer 112, pressure can be applied to both sides of the tip 100.

[0085] The pestle 520 may have any structure for its grinding portion: it may be a smooth structure, as shown in Figure 7 (left), or an irregular structure.

[0086] The crushed portion of the pestle 520 is irregular and advantageously has a step shape, as shown in FIG. 7 (right). By step shape it is meant that the crushed portion comprises a discontinuous thickness. During melting, the pestle 520 is rotated, whereby the pressure applied to the melting chamber 120 is not uniform: some parts are highly pressurized while other parts are hardly pressurized. Thus, the shear action is not uniform and the membrane 113 is irregularly sheared, distorted and bent, leading to improved melting.

[0087] The present disclosure also relates to a method for lysis of microorganisms, in which a sample containing microorganisms is introduced into the above-disclosed chip 100 via the inlet 130. The lysis chamber 120 of the chip 100 is then pressurized with a rotating pestle 520 and internal materials contained in the microorganisms are collected.

[0088] Advantageously, the chip 100 is placed in a recess 510 of the platform of a grinder 500, said grinder 500 comprising a pestle 520 configured to align with the lysis chamber 120 of the chip 100 for lysis of microorganisms, a motor 530 for rotating said pestle 520, and a pressure means 540 for pressing said pestle 520 against the lysis chamber 120. The pestle 520 may have any structure for its grinding portion. It may be smooth or irregular in structure. The grinder 500 disclosed above is particularly suitable for this method.

[0089] In an embodiment, the pressure applied by the pestle 520 ranges from 0.02 MPa to 1.5 MPa, preferably from 0.1 MPa to 1 MPa. The pressure can be selected depending on the exact tip geometry, the type of flexible membrane 113 and filter 112, and also the type of microorganism to be lysed. The pressure applied by the pestle 520 is the force applied by the pestle 520 divided by the surface area of ​​the pestle crushing part. The applied force ranges from 1 N to 24 N, and the surface area of ​​the pestle crushing part is 15 mm 2 ~50mm 2 The range is.

[0090] In one embodiment, the rotation speed of the pestle 520 is in the range of 30 rpm to 200 rpm, preferably 50 rpm to 150 rpm. Rotation speeds of the pestle 520 of 60 rpm, 90 rpm and 120 rpm are particularly suitable. The rotation speed can be selected depending on the exact tip geometry, the type of flexible membrane 113 and filter 112, and also the type of microorganism to be lysed.

[0091] In one embodiment, the grinding time is in the range of 30 seconds to 10 minutes. Shorter grinding times are preferred to improve the output of the system for microbial lysis. Grinding times of 1 minute, 2 minutes, and 3 minutes are particularly suitable.

[0092] Suitable conditions for lysing Gram-negative Salmonella are 1 minute grinding time at 60 rpm, pestle force of 12 N, and tip design V, as described below.

[0093] Suitable conditions for lysing Gram-positive Listeria are 2 minutes grinding time at 60 rpm, pestle force of 12 N, and tip design V, as described below.

[0094] Suitable conditions for lysing Saccharomyces cerevisiae yeast cells are a grinding time of 1 minute at 120 rpm, a pestle force of 20 N, and a tip design of V, as described below. [Brief description of the drawings]

[0095] [Figure 1] 1 is an exploded view of a tip structure according to a first modified configuration of the first configuration of the present disclosure. [Diagram 2] 1 is an exploded view of a tip structure according to a second modified configuration of the first configuration of the present disclosure. FIG. [Diagram 3] 1 is a side cross-sectional view of a chip according to a second modified configuration of the first configuration of the present disclosure. [Figure 4] FIG. 4 is an enlarged view of a portion of FIG. [Diagram 5] FIG. 1 is a diagram of a grinding machine, with an external view on the left and a cross-sectional view on the right in order to visualize the motor and the pressing means. [Figure 6] 1 shows the relationship of the grinder and chips forming a system for lysis of microorganisms. [Figure 7] FIG. 1 shows a pestle with a smooth grinding portion (left) and an irregular - stepped shaped - grinding portion (right). [Figure 8]Figure 8A is a simplified diagram of a multi-chip containing eight chips, and Figure 8B is a simplified diagram of a multi-chip containing two chips. These figures show only the inlets 130 and outlets 140 on the rigid cover 114 aligned with the rear cover 115 to accurately position each chip in the multi-chip. Some components (117, 118) are shown, but the internal multi-layer structure is omitted.

[0096] Working Example The present invention is further illustrated by the following examples. Various chips are used with the following specifications:

[0097] Chip design I according to the first configuration: The components are placed from top to bottom - in direct contact - in the following order as shown in Figure 1: rigid cover 114 with inlet 130 and outlet 140; double-sided adhesive layer 111 (fixing means); flexible membrane 113; double-sided adhesive layer 111 with outlet channel 117; flexible filter 112; double-sided adhesive layer 111 with inlet channel 116; flexible membrane 113; double-sided adhesive layer 111 (fixing means) and rear cover 115. All double-sided adhesive layers 111 and flexible membrane 113 have inlet and outlet holes. In design I, the volume of the lysis chamber 120 upstream of the filter 112 is about 30 μL.

[0098] Chip design II according to the first configuration: The components are placed from top to bottom - in direct contact - in the following order: rigid cover 114 (closing surface) with inlet 130 and outlet 140; double-sided adhesive layer 111 (fixing means); flexible membrane 113; double-sided adhesive layer 111; spacer layer; double-sided adhesive layer 111 with outlet channel 117; flexible filter 112; double-sided adhesive layer 111 with inlet channel 116; flexible membrane 113; double-sided adhesive layer 111 (fixing means) and rear cover 115. All double-sided adhesive layers 111; spacer layer and flexible membrane 113 have inlet and outlet holes. In design II, the spacer layer and additional double-sided adhesive layer define a thicker lysis chamber: the volume of the lysis chamber 120 upstream of the filter 112 is about 40 μL.

[0099] Chip design III according to the second configuration: The components are placed from top to bottom - in direct contact - in the following order: rigid cover 114 (closing surface) with inlet 130 and outlet 140; double-sided adhesive layer 111 (fixing means); spacer layer; double-sided adhesive layer 111 with outlet channel 117; flexible filter 112; double-sided adhesive layer 111 with inlet channel 116; flexible membrane 113; double-sided adhesive layer 111 (fixing means) and rear cover 115. All double-sided adhesive layers 111; spacer layer and flexible membrane 113 have inlet and outlet holes. In design III, the volume of the lysis chamber upstream of filter 112 is similar to design II; however, lysis chamber 120 is sealed by rigid cover 114 instead of flexible membrane 113.

[0100] Chip design IV according to the second configuration: The components are placed from top to bottom - in direct contact - in the following order: rigid cover 114 (closing surface) with inlet 130, outlet 140 and outlet channel 117 formed as a groove; double-sided adhesive layer 111; flexible filter 112; double-sided adhesive layer 111 with inlet channel 116; flexible membrane 113; double-sided adhesive layer 111 (fixing means) and rear cover 115. All double-sided adhesive layers 111 and flexible membrane 113 have inlet and outlet holes. In design IV, the volume of the lysis chamber 120 upstream of the filter 112 is similar to design I; however, the lysis chamber 120 is sealed by the rigid cover 114 instead of the flexible membrane 113.

[0101] Chip design V according to the first configuration: The components are placed from top to bottom - in direct contact - in the following order: rigid cover 114 with inlet 130, outlet 140 and outlet channel 117 formed as a groove; double-sided adhesive layer 111 (fixing means); flexible membrane 113 with communicating hole 118; double-sided adhesive layer 111; flexible filter 112; double-sided adhesive layer 111 with inlet channel 116; flexible membrane 113; double-sided adhesive layer 111 (fixing means) and rear cover 115 (Figure 2). All double-sided adhesive layers 111 and flexible membrane 113 have inlet and outlet holes. In design V, the volume of the lysis chamber 120 upstream of the filter 112 is similar to design I; however, the lysis chamber 120 is fluidly connected to the outlet 140 via a hole in the flexible membrane 113 and the outlet channel 117 formed as a groove on the rigid cover 114. FIG. 3 is a side cross-sectional view of chip design V, with an enlarged view in FIG.

[0102] All chips have a chamber hole with a diameter of 10 mm, and inlet and outlet holes with a diameter of 4 mm. The thickness of the double-sided adhesive layer is 150 μm. The thickness of the flexible membrane 113 is 50 μm. The flexible filter 112 is a disk with a diameter of 13 mm and a thickness of 10 μm. The rigid cover 114 and the rear cover 115 are made of polycarbonate and are 2 mm and 1 mm thick, respectively.

[0103] The grinding protocol used in these examples has the following specifications: The tips were inserted into the grinder 500 as shown in Figure 6. The pestle 520 - the stepped surface of the grinding portion - was then inserted into the grinder 500 at a distance of approximately 25 mm. 2 - is rotated at a rotation speed of 60 rpm and pressurized with a force of 12 N against the lysis chamber 120 for 2 minutes, the pressure exerted by the pestle 520 being about 0.5 MPa.

[0104] Two types of protocols were used.

[0105] PCR protocol The microorganisms are cultured overnight under suitable conditions of temperature, humidity and medium. The microorganisms are then transferred to Ringer's solution and incubated for 10 min. 4 The solution is then diluted to reach a colony forming unit (CFU) of 0.001 CFU / mL. Then, 25 mL of the solution is added to 225 mL of BPW medium - buffered peptone water - and 25 g of the frozen vegetable mixture is added to the culture bag, and 10 3 A sample of CFU / mL is obtained. The sample is homogenized using a Stomacher-Interscience BagMixer® 400, 8 strokes / sec, 400W power for 1 minute. This process removes 10 of the known microorganisms. 3 The goal is to prepare a representative sample of the food containing CFU / mL.

[0106] 1 mL of the sample is introduced into the tip. Then, 1 mL of rinse buffer is introduced into the tip. The tip is placed in the grinder 500 for 1 minute using a given pestle 520, pressure and rotation speed. A 20 μL sample is collected from the lysis chamber 120 and tested by polymerase chain reaction (PCR) under conditions suitable for the nucleic acid of interest, allowing detection of the microorganism. The cycle threshold (Ct) is then reported. The lowest Ct value indicates the best lysis performance.

[0107] DNA Administration Protocol The microorganisms are cultured overnight under suitable conditions of temperature, humidity and medium. The microorganisms are then transferred to Ringer's solution and incubated for 10 min. 7 The sample is diluted to reach colony forming units (CFU) of CFU / mL. 1 mL of the sample is introduced into the tip. Then, 1 mL of rinse buffer is introduced into the tip. The tip is placed in the grinder 500 for 1 minute with a given pestle 520, pressure and rotation speed. 20 μL of the sample is collected from the lysis chamber 120. The DNA content (in ng / μL) is then measured by state of the art methods, as well as the cycle threshold (Ct). The DNA protocol (10 7 CFU / mL samples were analyzed using the PCR protocol (10 3Note that the Ct for the DNA protocol is much more highly concentrated than the CFU / mL; therefore, the Ct for the PCR protocol is much smaller.

[0108] The following table shows the experimental results. For each experiment, measurements were repeated three or five times and the results were averaged. In cases where the results were highly scattered, a measure of the variance is given.

[0109] PCR protocol (10 3 In terms of CFU / mL, different designs and parameters were used, showing an overall very good performance in terms of lysis and detection of microorganisms. Polycarbonate-based filters (F2 and F5), as well as PET membranes (M2 and M3) appear to give better results. Chip designs I and V also show the best results, with Ct 31 and 32, when filter F5 and membrane M3 are used. [Table 1]

[0110] DNA Protocol (10 7 A comparative experiment was carried out using 100% ELISA kits (100% CFU / mL). Chip V, which combines filter F5 and membrane M3, shows a much better sensitivity compared to the prior art method. The DNA extracted after lysis is at least 5 times more concentrated. [Table 2] Filter: F1-Sartorius Cellulose Acetate 0.45μm (SCA) Reference Number: 11106 13N F2-Polycarbonate Isopore Membrane Filter 0.4μm (IMF4) Reference Number: HTBP01300 F3-Mixed Cellulose Ester Membrane 0.65μm (CEM) Reference Number: DAWP01300 F4-Mixed cellulose ester membrane 0.45μm (MF) Reference number: HAWP02500 F5-Polycarbonate Isopore Membrane Filter 0.6μm (IMF6) Reference Number: DTTP01300 film: M1-Microamp film (PMMA 35μm thick with one adhesive side) M2 - 23μm thick PET film. M3 - 50μm thick PET film.

[0111] For the microorganism Listeria monocytogenes ATCC13932 annotated as μO1, PCR tests were performed with the Surectect kit provided by ThermoFisher (reference number: PT0300A). DNA administration was performed using the Invitrogen™ Qubit™ dsDNA HS Assay Kit after fluorescence measurement with the Invitrogen™ Fluorometre Qubit™ 4 reader.

[0112] Effect of pestle type Using the chip design I with DNA protocol, the types of pestle 520 were compared. With the smooth pestle 520 in Figure 7 (left), the total DNA concentration is measured at 0.78 (±0.02) ng / μL. With the step-shaped pestle 520 in Figure 7 (right), the total DNA concentration is measured at 5.2 (±0.4) ng / μL. The irregular shape of the crushed part of the pestle 520 significantly increases the amount of DNA released, showing evidence of a more quantitative lysis of the microorganisms.

[0113] numeric reference 100-chip / 110-multilayer stack / 111-double-sided adhesive layer / 112-flexible filter layer / 113-flexible membrane / 114-rigid cover / 115-rear cover / 116-inlet channel / 117-outlet channel / 118-connecting hole / 120-melting chamber / 130-inlet / 140-outlet / 500-crusher / 510-recess / 520-pestle / 530-motor / 540-pressure means

Claims

1. A chip (100) for mechanical lysis of microorganisms, comprising: a) a multi-layer stack (110), i. a first double-sided adhesive layer (111) containing a chamber hole; ii. a flexible filter layer (112) in direct contact with the first double-sided adhesive layer (111) and covering the chamber aperture; iii. a second double-sided adhesive layer (111) in direct contact with the flexible filter layer (112) and containing chamber holes; and iv. a flexible membrane (113) on the first double-sided adhesive layer (111) opposite the flexible filter layer (112); A multi-layer stack (110) where all the chamber holes overlap to form a lysis chamber (120); and b) a closing surface on a second double-sided adhesive layer (111) of said multi-layer stack (110) opposite the flexible membrane (113), said closing surface being the second flexible membrane (113); c) a chip (100) for mechanical lysis of microorganisms, comprising an inlet (130) in fluid communication with said lysis chamber (120).

2. a) a rear cover (115) on the flexible membrane side of the multi-layer stack (110) that includes a pestle hole aligned with the lysis chamber (120); b) a rigid cover (114) on the second flexible membrane (113) opposite said rear cover (120); and c) fastening means (110) for holding a rigid cover (114) and a rear cover (115) in contact with said sealed multi-layer stack (110); 2. The chip for mechanical lysis of microorganisms (100) of claim 1, wherein the inlet (130) is on the rigid cover (114) and the fluid communication is arranged by overlapping inlet holes in a layer of the multilayer stack (110) and in the second flexible membrane (113) to form an inlet chamber and an inlet channel (116) between the chamber hole and the inlet hole of one of the double-sided adhesive layers (111).

3. further comprising an outlet (140) in fluid communication with said lysis chamber (120), said outlet (140) being on said rigid cover (114); and i. said fluid communication noted as A1 is arranged by overlapping an outlet hole in a layer of said multi-layer stack (110) and in said second flexible membrane (113) to form an outlet chamber; and an outlet channel (117) between said chamber hole and an outlet hole in said double-sided adhesive layer (111) that does not include an inlet channel (116); or ii. The chip (100) for mechanical lysis of microorganisms according to claim 2, wherein the fluid communication noted as B1 is arranged by a communication hole (118) in the second flexible membrane (113), the communication hole (118) being superimposed on the periphery of the lysis chamber (120); and an outlet channel (117) between the communication hole and an outlet (140) of the rigid cover (114).

4. 4. The chip (100) for mechanical lysis of microorganisms according to claim 2 or 3, wherein the fixing means comprises a third double-sided adhesive layer (111) in direct contact between the rear cover (115) and the multi-layer stack (110), the third double-sided adhesive layer (111) comprising a pestle hole superimposed with the lysis chamber (120).

5. The fastening means comprises a third double-sided adhesive layer (111) in direct contact between said rear cover (115) and said multi-layer stack (110) and comprising a pestle hole superimposed with said lysis chamber (120); and a fourth double-sided adhesive layer (111) in direct contact between said rigid cover (114) and said second flexible membrane (113) and comprising an inlet hole superimposed with said inlet chamber; and i. an outlet hole overlapping another outlet hole for fluid communication A1; or ii. a chamber hole superimposed with said lysis chamber for fluid communication B1.

6. The chip (100) for mechanical lysis of microorganisms according to any one of claims 1 to 5, wherein the flexible membrane (113) is a polyethylene terephthalate membrane (113), preferably having a thickness in the range of 15 μm to 150 μm.

7. The chip (100) for mechanical lysis of microorganisms according to any one of claims 1 to 6, wherein the flexible filter layer (112) is a polycarbonate layer, preferably the flexible filter layer (112) has pores in the range of 0.2 μm to 1 μm.

8. A multi-tip comprising at least two tips, preferably four or eight tips, for mechanical lysis of microorganisms according to any one of claims 1 to 7, wherein the closing surfaces of all tips form a single closing surface.

9. The multi-chip of claim 8 , wherein the at least two chips are arranged in a row.

10. 1. A system for mechanical lysis of microorganisms, comprising: a. a chip (100) for mechanical lysis of microorganisms according to any one of claims 1 to 7 or a multichip according to claims 8 or 9, b. A grinder (500), i. a platform having a recess (510) configured to receive the tip (100) or the multi-tips for mechanical lysis; ii. a pestle (520) configured to be aligned with the lysis chamber (120); iii. a motor (530) for rotating said pestle (520); and iv. a grinding machine comprising a pressurizing means (540) for pressing said pestle (520) against said dissolution chamber (120).

11. 11. The system for mechanical lysis of microorganisms of claim 10, wherein the crushing portion of the pestle comprises one step.

12. 1. A method for mechanical lysis of microorganisms comprising: a. introducing a sample containing microorganisms through the inlet (130) of the chip for mechanical lysis of microorganisms (100) according to any one of claims 1 to 7 or the multichip according to claims 8 or 9; b. pressurizing the lysis chamber (120) with a rotating pestle (520); and c. harvesting the contents of the cells.

13. The tip (100) or the multi-tip for mechanical lysis of microorganisms is placed in a recess in a platform (510) of a grinder (500), the grinder (500) comprising: i. a pestle (520) configured to be aligned with the lysis chamber (120) of the tip (100) for lysis of microorganisms; ii. a motor (530) for rotating said pestle (520); and iii. A method for mechanical lysis of microorganisms according to claim 12, comprising a pressurizing means (540) for pressurizing said pestle (520) against said lysis chamber (120).

14. 14. The method for mechanical lysis of microorganisms according to claim 13, wherein the force of the pestle (520) is in the range of 1N to 24N and / or the rotation speed of the pestle (520) is in the range of 30 rpm to 150 rpm.

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