WO2012146458A2

WO2012146458A2 - A method for producing a polymer-based microfluidics system for bioanalytics using biological membranes

Info

Publication number: WO2012146458A2
Application number: PCT/EP2012/055591
Authority: WO
Inventors: Ingrid IMHOF; Eugen Müller; Helmut Schift; Louis Tiefenauer
Original assignee: Paul Scherrer Institut
Priority date: 2011-04-26
Filing date: 2012-03-29
Publication date: 2012-11-01
Also published as: WO2012146458A3

Abstract

Simple fabrication procedures have been developed for a fully polymer-based microfluidic system containing a thin PEEK polymer sheet in which one pore or arrays of pores of 1 to 20 micrometer diameter and a low aspect ratio of about 1:1 allow spontaneous formation of lipid bilayers. Such stable lipid bilayers, separating two fluidic compartments of small volumes (0.1 to 100 microliters), are used to investigate processes across lipid bilayers such as perfusion, or peptide and protein-mediated translocation of ions or molecules. The device allows the simple preparation of bilayers and electrochemical and optical detection of signals related to membrane-associated protein activities and perfusion. Applications are seen for bioanalytical investigations, medical diagnosis, environmental analysis and toxicology. Further applications are in processes where diffusion across pores is a determining factor, such as in chemical synthesis, crystallization and nanoparticle investigations. Said method for producing a microfluidic system (10) comprises the steps of: a) providing a first high performance thermoplastic polymer foil (2) having a thickness in the range of 1 to 100 micrometer, preferably 6 to 50 micrometer; b) generating a plurality of pores (6) in said first foil (2); said pores (6) having an aspect ratio in the range of 0.25 to 15, preferably in the range of 0.5 to 5; c) providing at least one second high performance thermoplastic polymer foil (4) having an embossed channel structure (8) for guiding a liquid; d) aligning the first foil (2) and at least one second foil (4) in order to enable an access to the channel structure (8) via the pores (6); and e) bonding the first foil (2) to at least one second foil (4) by thermal pressing with a pressure power of up to 2.5 MN and a predetermined course of the temperature in the range of 20 to 160°C.

Description

A method for producing a polymer-based microfluidics system for bioanalytics using biological membranes

The present invention relates to a method for producing a polymer- based microfluidics system for bioanalytics with biological membranes .

Microfluidics systems (MFS) have been developed for decades, especially for applications in biosciences. In principle an aqueous or organic fluid is transported by capillary forces, centrifugal forces or pumps trough microchannels . Connected to these channels are tubes containing the fluids of interest and detectors. Optional mixing structures or heating devices can also be integrated into the MFS. Generally, the channels are formed by fixing a cover sheet above a line structured bottom slice by so called lamination or bonding processes.

Various materials have been used in MFS fabrication: glass, silicon, polymers and metals. Polymers are widely used as

materials in MFS fabrication because they have specific

advantages: they are not brittle, transparent, flexible, easily to be structured, resistant to organic solvent but suitable for chemical derivatization and cheap. The development of polymeric- based MFS has become an important area in the field of (bio) analytics, specifically also for compact remote systems such as biosensing. Biosensors are defined as analytical devices in which specific recognition elements such as enzymes, antibodies, receptors, ion channels or transporters are in an intimate contact with the detectors, providing a signal related to the

concentration of the compound of interest (analyte) . Sensitivity is directly related to binding avidity, amplification and sample volume. MFS offer the advantage to reduce the sample volume to very low values i.e below that of a water drop of about 20 microliters. Furthermore, the two compartments allow easy exchange of solutions on both sides without pressure fluctuation and mechanical vibrations. By integrating the recognition element and the detector into the MFS, a compact device can be constructed, ideally produced using established fabrication processes.

In the European Patent EP 1 697 752 Bl, an assay chip for the investigation of the functionality of non-lipid molecules and their interactions with molecules is disclosed, comprising:

a) a nanopore substrate having a plurality of nanopores;

b) a suitable substantially planar support layer deposited on said nanopore substrate having a plurality of nanopores corresponding with said nanopores of said nanopore substrate; c) a biologically effective layer being capable to host at least a non-lipid molecule or functional molecule, deposited on said support layer and covering the plurality of nanopores, resulting in accessible nanopores from both sides of the biologically effective layer for measurements.

This assay chip offers an array of nanopores of macroscopic lateral dimension therefore providing both, supporting area to stabilize the biological effective layers (defined as a layer that preserves the full functionality of the non-lipid molecule hosted therein) , such as a lipid bilayer membrane, and pores in a

predefined density in which the biological effective layer remains fully fluid. This assay chip therefore offers a versatile system for various applications, like drug screening, functional protein analysis, toxicity analysis and the like. Due to applied

technology for generating Si₃N₄-layers on silicon chips the resulting Si₃N-membranes with the nanopores after etching are mechanically stable. The small aspect ratio of the pore diameter to the thickness of Si₃N₄-membrabanes with the nanopores allows an un-impeded diffusion of macromolecules across both, the lipid layer membrane and across the non-lipid molecule, such as membrane proteins, integrated therein. Further, the mechanically stabilized biologically effective layer (that means the solid support layer being the Si₃N-membrane with the nanopores and the biologically effective layer immobilized thereupon) offers free access from both sides of the biologically effective layer what allows the investigation of a variety of interactions of molecules, such as natural ligands (e.g. hormones) or the interaction with artificial effector molecules (such as drugs) with functional integrated membrane proteins and to elucidate the mechanism of signal

transduction. Due to the accessibility from both sides, the transport of ions, molecules and particles across the biological effective layer by transporter proteins can be investigated in a micro-chamber system, i.e. in a two-compartment system. Surface patterning and microspotting technologies will allow to address specific nanopore arrays. Furthermore, the integrated membrane proteins are in a lipidic environment and mobile and can therefore directly be investigated on their response to allosteric effects which is crucial for the development of new drugs targeting e.g. GCPRs . The total surface area being nanostructured is in a range that on the one hand a sufficient number of membrane protein molecules are present in order to enable conventional methods to detect distinct molecular interactions by means of fluorescence or other sensitive methods. On the other hand the amount of precious membrane proteins and/or binding compounds needed is comparably low for the achievement of the desired screening process.

This assay chip with the nanostructured silicon nitride membrane supports the biologically effective layer and thus bio-mimicks the stabilizing cytoskeleton . Materials for a suitable support layer are silicon nitride (S1₃N₄) or silicon oxide (S1O₂) and the nanopore substrate is potentially of silicon and carbon containing materials, but also can be a metal, a dielectrica, a glass or a ceramic. Suitable is insofar meant as a definition that the properties of the support material do allow adhesion of the lipid layer that is supposed to be supported by the support layer. Additionally, it should be pointed out that already the support layer may have chemical and

topographical properties that promote the fusion of the lipid layer on the support layer.

In order to improve or induce the formation and/or fusion and/or immobilization of the fluidic lipid bilayers on the support layer to a desired extent, the surface of the support layer may be modified resulting in a promotion layer, i.e. using chemically activated hydrophobic or hydrophilic silanes or other components as well as modifications of physical nature such as topographical or electrical modifications. This promotion layer may be designed according to the properties of the lipid bilayer to be supported and according to the mechanism responsible for the formation of the lipid bilayer. Besides all the afore-mentioned features and advantages, the formation of the assay chip according to the EP 1 697 752 is unfortunately quiet expensive and requires lithographic processes in order to generate the pores in the nanosize range. Apparently, polymers are also the preferential materials for MFS fabrication. Polymers are linear or branched organic compounds with a relatively high molecular mass which form a solid material at room temperature. Polymers are characterized by: density, transparency, glass transition temperature, organic solvents resistivity, electrical insulation and dielectric properties, elastic modulus (stiffness), robustness against wear and

radiations and crystallinity . For MFSs PDMS, PMMA, Dyneon THV and other thermoplastic polymers have been used. Thus, the micrometer sized fluidic channels in the thin foil sheets or microstructured PDMS-slabs have to be packed together by a "holder". Such

mechanically stable holding devices are often fabricated from PMMA because it is transparent, stable and can be easily mechanically processed. However, PMMA is not resistant against some organic solvents such as acetone. PMMA should therefore only be used to provide mechanical stability of the device and for the connecting tubes and should not come into contact with the channel fluids.

It is therefore an object of the present invention to provide a method for the fabrication of polymer-based microfluidic system for bioanalytics with biological membranes which is inexpensive and results in a MFS that offers the major advantages incurred with the assay chip according to EP 1 697 752 Bl, in particular it should be easy to be produced, simple in handling and robust to permit longevity.

These objectives are achieved according to the present invention by a method for producing a microfluidic system, comprising the steps of:

a) providing a first high performance thermoplastic polymer foil having a thickness in the range of 1 to 100 micrometer, preferably 6 to 50 micrometer;

b) generating a plurality of pores in said first foil; said pores having an aspect ratio in the range of 0.25 to 15, preferably in the range of 0.5 to 5;

c) providing at least one second high performance thermoplastic polymer foil having an embossed channel structure for guiding aqueous or organic liquids;

d) aligning the first foil and the at least one second foil in order to enable an access to the channel structure via the pores; and

e) bonding the first foil to at least one second foil by thermal pressing with a pressure power of up to 2.5 MN and a predetermined course of the temperature in the range of 20 to 160°C.

High performance thermoplastic polymers are known as dense polymer which have a high transition point and an excellent chemical resistance. Typical materials of this group are PEEK

(polyetheretherketone ) , PEK (polyetherketone ) , PSU (polysulfone) , PEI (polyetherimide ) , PES (polyethersulfone) and PPSU

(polyphenylsulfone) . Due to their high density the present

invention takes advantage of these materials that are also

relatively stable in very thin (< 50 micrometer) sheets. For freestanding planar lipid bilayer formation small uniform pores have been generated offering an advantageous aspect ratio in order to host a biological lipid bilayer, wherein the mechanical stability of such perforated thin foils has to be observed as a limiting factor of the entire device. The final MFS is easy to handle and robust. In the context of the present invention, PEEK is

considered a preferred material but the other materials can be used as well. The other polymer has to be choosen under the circumstances that this polymer also meets the required material properties (mechanical stability, electric capacitance, inertness, transparency, biocompatibility) as good as PEEK does.

With respect to the robustness of the MFS, it is useful when at least one second foil has a thickness higher than that one of the first foil, preferably in the range of 100 to 500 micrometer. Of course, it is possible to sandwich the first foil between two second foils that both offer accessibility to the pores via the channel system in these second foils.

A suitable way to generate the pores may consist in lasering the pores into the first foil using picosecond-pulsed laser. The laser pulses therefore provide regular pores having rather even rims which allow for the reproducible deposition of the lipids and formation of stable lipid bilayers in the pores.

For the investigation of exchange processes with rather low process rates, it can be very helpful, when the plurality of pores is assembled as an array of pores. Thereby, larger areas of the assay can be used to observe the biological transport phenomena. Translocation rates by some membrane proteins are high, the total area of free-standing lipid membrane needed is small and

consequently only a few pores required (even a single pore is sufficient to monitor most ion channels) .

In order to achieve separated assay systems for the

investigations, it is useful when a number of pores out of the plurality of pores is aligned with separated channel structures. Therefore, the transport phenomena at a single pore can be

observed under the determined supply of reactive substances using a detection method for translocated substances in the target volume. In order to achieve a parallel detection it is useful when individual pores or arrays of pores can be addressed by separate channel structures. A suitable method for generating the channel structure can be achieved when the channel structure is generated by hot-embossing the at least one second foil. Polymers covered with

photolithographic resists can also be structured by silicon- related technologies, but it is much easier to do it directly by hot embossing using a metallic stamp (cast) . In most cases the cast is produced by using mechanical tools. Such casts are durable and suitable for multiple uses. The hot embossing technique is well established and can be used for microchannel imprinting in polymer sheets, in particular in PEEK foils.

A further preferred embodiment of the present invention provides for a combination of different materials. For example, for the first foil and the second foil different high performance

thermoplastic polymers are used. The selection can be made due to their different properties, i.e. related to the electrical properties, the chemical resistance, the heat distortion

temperature, the hydrolysis resistance, the melt viscosity, the mechanical strength and rigidity.

A further preferred embodiment of the present invention provides for two different options of assembling the microfluidic system. According to the first option, the at least one second foil can be gluelessly bonded to a micro titer plate and the first foil is subsequently bonded to the second foil. According to the second option, the first foil can be bonded to the second foil and the compound comprising the first and the second foil is subsequently gluelessly bonded to a micro titer plate. Preferred embodiments of the present invention are explained hereinafter with more detail, in particular with reference to the attached drawings which depict in Figure 1 a schematic view on a first PEEK foil 2 and a second PEEK foil 4. The applications described in the following paragraph require that the thin first PEEK foil 2 comprises multiple pores (array) 6. Since the diameter of the individual pores 6 should be as small as possible,

mechanical drilling is excluded. Structuring by etching or light is favored. Etching requires several protection layers which first have to be added and structured using photolithography. Such a procedure leads to very small pores (down to 500 nm) , but includes several steps. The so-generated smallest pores will have a high aspect ratio (diameter / thickness) of about 12, since the

thinnest polymer foils available are 6 micrometers.

Presently, the pores 6 are directly generated by pulsed lasers, i.e. by locally melting the PEEK polymer. Some parameters of the laser are kept fixed and the others are varied. Preferentially, the number of applied pulses is varied. In order to achieve regular pores, the energy has to be high enough to penetrate the foil, but not too high in order to not enlarge the pore or

generate surface roughness. Soft materials need less energy than stiff ones. However, the quality of the pores (diameter,

uniformity, smooth etches) can not be exactly predicted.

Nevertheless, the pore quality is a critical factor for the lipid bilayer formation and stability. In particular, the rims should be rather smooth in order to generate a suitable site for the

formation of the lipid bilayer.

A MFS 10 should be leak-proof and appearance of bubbles should be avoided and unwanted bubbles in the device easily removable. The second PEEK foil 4 shows a channel structure 8 with channels 8a to 8e for the supply of liquids to and/or from the pores 6. The channels 8a to 8e must also be leak-proof at an elevated fluid pressure. This can be achieved by pressing ultraflat channel forming polymer slabs together at low forces, but only for some specific polymers such as PDMS (polydimethylsiloxane, silicone) and PEEK. To achieve a long-term stable MFS, the foils 2, 4 are bonded together to build the MFS 10. Glue should be avoided since it can fill the microchannels 8a to 8e and can be dissolved by organic solvents. In the present invention, the two PEEK foils 2 and 4 are bonded using optionally a chemically activation of the foils 2, 4 and an application of pressure and/or heat to form a stable connection. Such lamination processes are widely used also for PEEK

(polyetheretherketone ) that is FDA-approved as a biocompatible material for different applications. Chemical activation can on one hand permanently modify surfaces also within the channels 8a to 8e resulting in unwanted reactivities. On the other hand, the required good wetability of surfaces for aqueous solutions can be achieved by plasma treatment or by chemical modification methods. Bonding by applying pressure and/or heat in combination with localized surface activation processes before or afterwards will result in waterproofed devices with predefined wetability of channels. However, pressing two sheets together at an elevated temperature increases the risk of channel deformation. Thus, the bonding process has to be optimized in order to achieve tight connections of the foils 2, 4 and to retain the microstructure (channels 8a to 8e and pores 6) previously generated in the foils 2, 4.

Lipid bilayers 12 are formed by self-assembly processes. Most commonly, lipids are dissolved in an organic solvent such as decane and the lipid solution applied to the pores 6. This so called painting is simple and quite reproducible. Within the pore 6 the lipids self-assemble to a bilayer and this solvent separtes from the bilayer formed by rising, since its density is lower than that of water. However, there is always a residual ring of organic solvent (annulus) at the edges of the pore 6. This reservoir can be an advantage for the stability of bilayers, as surplus lipids favor a self-regeneration of bilyers after rupture. The extension of the annulus depends on the geometry of the pore and surface properties. Bilayers are more stable in pores of smaller

diameters, because the probability of self-healing is higher. It has been shown that a pore diameter of 1 micrometer or less results in long-term stable lipid bilayers 12. However, the stability of a painted bilayer also depends on the quality of the rims of the pores 6. The roughness of the edges may result in smaller or larger annuli and thus influence the long-term

stability of the suspended bilayer. Such stable free-standing bilayers resemble natural biological membranes. The formation of peptidic channels with simple

structures is usually not affected by the residual organic

solvent. However, this may affect the more complex structure of integral membrane proteins and thus impede their activity.

Therefore, other techniques are required to achieve planar lipid bilayers 12 with integrated membrane proteins 14 therein. The research on new techniques to create stable planar lipid bilayers 12 with integrated membrane proteins 14 in nanopores is presently actively further pursued.

Quantitative measurements of spontaneous perfusion of ions and molecules across lipid membranes are of interest in research and many applications in biosciences. Diffusion rates of ions and molecules across such artificial lipid bilayers will strongly depend on the following conditions: the ion gradient, the

lipophilicity of the transported molecule, the temperature and the lipid composition of the planar lipid bilayer 12. The MFS 10 consisting of two compartments has the major advantage that on both sides of the bilayers 12 are comparable volumes, avoiding a fast saturation and thus making possible the monitoring of diffusion processes across the bilayer. For example, the diffusion of ions or molecules across lipid bilayers 12 of liposomes of atto-liter volumes (10^~18 liter) occurs in less than one second, making accurate measurements nearly impossible even when using stopped-flow devices. This invention allows to address both side of the free-standing lipid bilayer generated in the micropore by fluidic microchannels and/or to monitor locally translocated species .

Furthermore, protein-mediated translocation of ions and molecules across lipid bilayers 12 can hardly be discriminated from passive diffusion. Thus, stable planar lipid bilayers 12 integrated in the MFS 10 will offer a unique tool to quantify both, spontaneous diffusion and protein-meditated translocations across biological membranes at predefined conditions. Furthermore, uptake or

accumulation of drug candidates into cells can be mimicked. The most important application of the MFS 10 is to investigate and measure the activity of integral membrane proteins 14. About one third of all proteins of the human genome are membrane proteins. Unlike from soluble proteins, only little is known about membrane proteins which regulate information exchange and mass transport into cells. Major classes of membrane proteins include ion

channels, receptors, transporters and so-call GPCRs (G protein- coupled receptors) . The structures of only a limited number of MPs have been solved to atomic resolution. Detailed knowledge of the structure of a protein is essential to fully understand their working mechanism. Therefore, quantitative assays for protein activity measurements are highly welcome in the field of membrane protein research.

MPs are also exceptionally important in medical research. About half of all drug targets are membrane proteins. A drug activates, inhibits or modulates specifically the activity of the target MP resulting in cellular responses. Obviously, assays for MPs are highly welcome in drug discovery as a major tool to assess the potential of candidate compounds on a pre-given target molecule. Such membrane protein activity assays may also be established to monitor the toxicity of fluids. Activity measurements of membrane proteins are based on detecting a physical signal. For ion channels, the ion flow across the membrane is measured using electrochemical methods. Electrical impedance spectroscopy is widely used to determine changes of resistance and capacitance resulting from such membrane protein activities. More directly, ion flows are measured at an applied voltage as an increase in current. Such a V-clamp measurement needs to be well protected from environmental electromagnetic noise and the capacitance of the carrier material should be kept as low as possible. PEEK and in general polymers are very good isolators because they have a very low inherent capacitance due to the low ε (epsilon) value. It is assessed that below a thickness of about 10 micrometer the inherent capacitance will be too high for sensitive measurements. Thus, foils of about 10 micrometer thickness balance the contradicting requirements to increase stability of bilayers by reducing pores sizes with low aspect ratio on one hand, and to achieve an acceptable capacitance by not too thin capacitors on the other hand. Most important, such thin PEEK-foils remain mechanically stable; this is a prerequisite for handling and they are suitable for microstructuring by lasers as described above. Alternatively, translocated molecules can also be detected using optical methods, specifically fluorescence. Since amorphous PEEK is transparent, direct optical measurement is possible with the lower limit of about 380 nm. The present invention therefore provides a method for a

fabrication of the polymer-based micro fluidics system 10 used to generate stable planar lipid bilayers for the investigation of transmembrane processes, such as signal transduction, ion or molecule transports or translocation/passive diffusion, membrane permeability, wherein the combination of material properties, such as mechanical stability, electrochemical capacitance, elasticity, required for a successful fabrication is fulfilled by the use of PEEK foils. Of course, other polymer materials with similar properties may also be used.

According to the present invention, in the first PEEK foil 2 with a thickness in the range of 5 to 50 micrometer, pores 6a in the range of 5 to 30 micrometer diameter are generated at a

preferential aspect ratio of 1:1 (thickness to diameter) . Small pores are crucial for the long-term stability of the suspended lipid bilayers therein. The aspect ratio is important for many classes of membrane proteins. Ion channel measurements are not affected by a high aspect ratio. However, carriers and

transporters (co-transporters, symporters, and antiporters) have considerably lower transport rates than ion channels, resulting in low concentrations of translocated compounds in the trans-side compartment. Therefore, high aspect ratios of pores slow down diffusion which is not acceptable. Therefore, thin foils are potentially useful with the formation of arrays of micropores.

The generation of uniform and precisely arranged micropores 6a is achieved by using a picosecond pulsed laser. The optimal

structuring parameters (number of pulses and time) for the

material PEEK have been determined. In foils of 12 micrometer thickness, pore diameters of 10 to 20 micrometers and smooth edges have been reproducibly generated. Thus, the intended aspect ratio of about 1 can be achieved by this laser technique. The same pulse laser device is used to generate the larger pores (6b to 6e) needed for connecting the fluidic channels by burning a circle in the thin foil resulting in a pore of a diameter of channel width. The channel structure 8 in the second thicker PEEK foil 4 (200 to 500 micrometer) are made by hot embossing processes. A

mechanically defined stamp carrying the channel structure is first made in brass. The embossing process in the relatively hard/stiff PEEK requires elevated pressure and temperature. The edges of the channels in the second PEEK foil 4 are sharp as they should form a rectangular cross section of the channels 8a to 8e.

The bonding of the first thin micropore PEEK foil 2 to the second thicker channel PEEK foil 4 is done by thermal bonding. Pressure and temperature are critical factors to achieve a stable bonding. It is not obvious that the geometry of the microchannels is retained, i.e. not deformed by this treatment. The organic solvent stable bonding of PEEK to PEEK results in leak-proof channels 8a to 8e, which can be filled with various fluids including organic solvents. Pressure and subpressure can be applied to the closed fluidic system; a prerequisite to fully control fluid flow.

Due to the high density of PEEK, arrays of regularly arranged and well separated micropores 6 can be generated by laser pulses. The pores 6a relevant for bilayer formation are uniform in size and exhibit a smooth rim. The array format can be adapted to the intended use. The distance between individual pores in arrays has to be larger than one pore diameter. For ion channels one or a few pores are required whereas transporter measurements require an array of many pores. Applications such as nanoparticle separation or diffusion controlled chemical reactions demand even larger pore areas .

The fully PEEK-based MFS 10 is of highest stability and inertness and transports fluids to a micro- or nanopore which is in contact with the second (trans-) compartment. Controllable transport of nanoliter volumes of fluids to this trans-compartment is used to trigger chemical reactions for analysis and synthesis or physical reaction such as (protein) crystallization. Such MFS with a low aspect ratio are also potentially useful devices for separation processes of nanoparticles at applied pressures and electrical potentials.

The generation of the lipid bilayer in the pores 6a is done by using microfluidics via the inlet pore 6b and the outlet pore 6e. The pore 6d is used as an access to a service channel 8d. The measurement of the activity of the membrane protein 14 can be measured at pore 6c. This example represents the specific example for a separate channel system 8 that is exclusively shared among the pores 6a to 6e.

Claims

Patent Claims

1. A method for producing a microfluidic system (10), comprising the steps of:

a) providing a first high performance thermoplastic polymer foil (2) having a thickness in the range of 1 to 100 micrometer, preferably 6 to 50 micrometer;

b) generating a plurality of pores (6) in said first foil (2); said pores (6) having an aspect ratio in the range of 0.25 to 15, preferably in the range of 0.5 to 5;

c) providing at least one second highperformance thermoplastic polymer foil (4) having an embossed channel structure (8) for guiding a liquid;

d) aligning the first foil (2) and at least one second foil (4) in order to enable an access to the channel structure (8) via the pores ( 6 ) ; and

e) bonding the first foil (2) to at least one second foil (4) by thermal pressing with a pressure power of up to 2.5 MN and a predetermined course of the temperature in the range of 20 to 160°C.

2. The method according to claim 1 wherein the at least one second foil (4) has a thickness higher than that one of the first foil (2), preferably in the range of 100 to 500 micrometer.

3. The method according to claim 1 or 2 wherein the generation of the plurality of pores (6) is achieved by lasering the pores (6) into the first foil (2) using picosecond-pulsed laser.

4. The method according to any of the preceding claims, wherein the plurality of pores (6) is assembled as an array of pores.

5. The method according to any of the preceding claims, wherein a number of pores out of the plurality of pores are aligned with a separated channel system (8, 8a to 8e) ) .

6. The method according to any of the preceding claims, wherein the channel structure (8) is generated by hot-embossing the at least one second foil.

7. The method according to any of the preceding claims, wherein PEEK is used as a high performance thermoplastic polymer.

8. The method according to any of the preceding claims, wherein the high performance thermoplastic polymer is selected from a group consisting of PEK, PSU, PEI, PES and PPSU.

9. The method according to claim 7 or 8, wherein for the first foil (2) and the second foil (4) different high performance thermoplastic polymers are used.

10. The method according to any of the preceding claims, wherein the at least one second foil (4) is glueless bonded to a micro titer plate and the first foil (2) is subsequently bonded to the second foil ( 4 ) .

11. The method according to any of the preceding claims 1 to 9, wherein the first foil (2) is bonded to the second foil (4) and the compound comprising the first and the second foil is subsequently gluelessly bonded to a micro titer plate.