WO2014009484A1

WO2014009484A1 - Multifunctional sensing membrane-based platform for tissue or cell culturing and monitoring

Info

Publication number: WO2014009484A1
Application number: PCT/EP2013/064705
Authority: WO
Inventors: Luigi Sasso; Karsten Brandt ANDERSEN; Jaime CASTILLO-LEÓN; Jan Bert GRAMSBERGEN; Winnie Edith Svendsen
Original assignee: Danmarks Tekniske Universitet
Priority date: 2012-07-11
Filing date: 2013-07-11
Publication date: 2014-01-16
Also published as: WO2014009486A1

Abstract

The present application discloses a water-permeable sensor membrane comprising i) a first layer of a conductive material defining at least one electrode and having a thickness of 0.1-,000 µm; ii) a second layer of a nanostructure material build on the first layer; and iii) a third, topmost, layer of a conducting polymer material defining at least one electrode and having a thickness of 0.001-1.0 µm. The application also discloses a tissue or cell culture sample monitoring assembly comprising a sensor assembly and a tissue sample or a cell culture sample arranged on top of the third layer of the sensor membrane, and a method of monitoring the concentration or presence of a tissue analyte in the proximity of a tissue sample or cell culture sample.

Description

MULTIFUNCTIONAL SENSING MEMBRANE-BASED PLATFORM FOR TISSUE OR CELL

CULTURING AND MONITORING

FIELD OF THE INVENTION

The present invention relates to a sensor membrane particularly useful as a platform for tissue or cell culturing and monitoring.

BACKGROUND OF THE INVENTION

Organotypic brain slice cultures, typically prepared from immature animals, can grow and survive for several weeks or even months in vitro, and allow long-term drug treatments, as well as long-term monitoring of biological processes, as is often necessary in studies of neurotoxicity, neuro-protection and neuro-repair. Therefore these cultures are an often used model in research on neurodegenerative diseases.

The concentration of energy substrates in the growth medium such as glucose, lactate or glutamine, neurotransmitters such as glutamate, GABA (gamma-aminobutyric acid) or dopamine, or cell death markers such as choline, glycerol or lactate dehydrogenase (LDH), is today determined by offline methods including electrochemical techniques and high pressure liquid chromatography systems. However, this approach is both time-consuming, labor- intensive and only the accumulative concentration of the analytes released since last medium change can be measured.

For the determination of the physical release site of different analytes in a brain slice, it is currently necessary to cut the brain slices and apply analyte specific dyes to these thinner slices, which is finally analysed using fluorescent microscopy. Alternatively, in order to provide information about e.g. dopamine release from a tissue sample, HPLC analysis of the medium collected after incubation for e.g. a few days is typically conducted. However, in order to measure dopamine in a culture medium using HPLC with electrochemical detection it is necessary to change serum-containing or Neurobasal medium with a more simple physiological medium (e.g. Ringer's solution or Hanks balanced salt solution). In organotypic mesencephalic slice cultures dopamine levels above detection levels (about 20 femtomoles in a 50 μΙ sample) are reached after about 1 hour incubation in the presence of a dopamine reuptake blocker (See ref. Larsen et al. 2008 Eur. J.Neurosci. 28, 569-576). Alternatively, the dopamine release can be directly measured by placing a tissue sample on an electrode. Sasso et al. (J. Nanosci. Nanotechnolo. 2012, Vol. 12, 3027-3083) recently described self-assembled diphenylalanine peptide nanowires (PNW) used for gold sensor modifications. The peptide nanowires could be further modified by functionalization with polypyrrole (PNW/PPy) and such a modified sensor could be used as a dopamine sensor. Moreover, the PNW modified gold surfaces were found useful for growing cells, i.e. cell growth was not hindered by the peptide nanostructure. However, conventional electrodes are not particularly suited for long-term studies of tissue samples or cell cultures due to localised cell necrosis and/or inhomogeneous growth.

WO 2008/054611 A2 discloses engineered conductive polymer films to mediate biochemcial interactions, in particular a functionalized conductive polymer film comprising a conductive polymer and at least one receptor embedded in or absorbed to the conductive polymer. The polymer film is typically coated onto a device or an electrode.

Michalska et al., Journal of Solid State Electrochemistry, Springer Verlag, Germany, Vol. 8, No. 6, May 2004, pages 381-389, disclose PEDOT films for multifunctional membranes for electrochemical ion sensing. Marti et al., Journal of Materials Chemistry, Royal Society of Chemistry, UK, Vol. 20, No. 47, 2010, pages 10652-10660, discloses nanostructured conducting poly(N-methylpyrrole) polymers for dopamine detection.

Hence, there is a need for sensors and methods that are able to overcome the above- mentioned short-comings. SUMMARY OF THE INVENTION

In their continuing work to develop membrane-based sensors, the present inventors have realised that the previous constructions for analysis of tissue samples and/or cell culture samples do not effectively allow for continuous measurement of e.g. dopamine release and for suitable conditions for tissue and cell survival, such as stable gas and nutrients medium concentrations. For example, previous HPLC methods require sampling (thereby eliminating a truly continuous monitoring of e.g. dopamine release) and a limited choice for growth media. On the other hand, the arrangement of tissue samples and/or cell culture samples directly on an electrode does not allow for reliable growth of the tissue sample or cell culture samples.

So, in a first aspect the present invention relates to a water-permeable sensor membrane, cf. claim 1. The invention further provides a sensor assembly, cf. claim 12, a tissue sample or cell culture sample monitoring assembly, cf. claim 13, and a method of monitoring the concentration or presence of a cell tissue analyte, cf. claim 14.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates a schematic for the sensor membrane assembly, and the superimposition of each individual layer in the order described, i.e. a first layer of conductive material at the bottom, a second layer of nanostructure material in the middle and a third layer of conducting polymer material at the top.

Figure 2 illustrates the tissue sample or cell culture sample monitoring assembly, with the biological sample (tissue or cell culture) directly on the 3-layer sensor assembly connected with the external equipment via an electrical connection to the first layer.

Figure 3 illustrates the tissue sample or cell culture sample monitoring assembly including examples of mark up lines included in the 3^rd layer assisting in the positioning of the cultures on the proper location of the membrane. DETAILED DISCLOSURE OF THE INVENTION

The present inventors have now realised that a particular sensor membrane construction allows for continuous monitoring of release of analytes while at the same time providing excellent conditions for tissue samples and cell culture samples. Moreover, the possibly patterned based sensor membrane will be able to provide researchers with this knowledge on tissue cultures that are still alive and are thereby able to monitor how the production of specific analytes such as dopamine changes over time in different parts of the brain slice. This will especially be valuable in stem cell research for the monitoring of incorporation of stem cells in specific parts of the brain. In this way it is possible to monitor the properties and behaviour of the live implanted cells rather than investigating whether they have been integrated after prolonged culturing, for example the validation of the differentiation of neuronal stem cells into dopaminergic neurons and subsequent maturation until the stage that these neurons are able to release dopamine. The sensor membrane may also preferably allow visualisation of the morphological integration of new-born neurons into a brain slice culture using fluorescent microscopy. Release of dopamine and integration of new-born neurons into an existing neuronal network may occur at different time scales. A patterned multi-electrode sensor membrane furthermore makes it possible to monitor the concentration of different analytes simultaneously during the culture at different positions providing even more information to the researcher.

Such a patterned sensor membrane can be realized using a photosensitive conductive polymer and standard micro fabrication techniques. In this way specific electrodes can be defined both in the top layer of conducting material and in the conducting membrane itself.

Sensor membrane

As described above, the present invention i.a. provides a water-permeable sensor membrane comprising a) a first layer of a conductive material defining at least one electrode and having a thickness of from 0.1-1,000 μιη; b) a second layer of a nanostructure material build on the first layer and e.g. having a thickness of 0.01-100 μιη; and c) a third, topmost, layer of a conducting polymer material defining at least one electrode and having a thickness of 0.001-1.0 μιη.

It should be understood that even though the sensor membrane is defined as having three layers, it may be possible to include even further layers so as to improve the sensor with respect to e.g. water-permeability, structural stability, biocompatibility, conductivity, etc.

In one embodiment, however, the sensor membrane only includes the three layers as defined above and as further described below.

In another embodiment, the sensor membrane includes the three layers as defined above and as further described below, but where the first layer is arranged on a porous material. In one variant, the sensor membrane is arranged on a membrane insert conventionally used for tissue or cell culturing, e.g. a Biopore™ inserts from Milipore (e.g. a PTFE membrane with a pore size of about 0.4 μιη).

The sensor membrane should be overall water-permeable so as to allow an aqueous medium to penetrate the membrane from the bottom face (first layer) and reach the upper surface (third layer) where a tissue sample or cell culture sample is intended to be arranged, as well as allowing the same aqueous medium to penetrate the membrane from the top face to the underlying surface for efficient medium exchange, needed to guarantee optimal cell or tissue growth conditions.

In the present context, the term "water-permeable" is intended to mean having the ability to allow aqueous solutions, holding relevant biological components needed for the tissue or cell culturing, to pass through the material.

Typically, a suitable water-permeability is present when the lowest pore size of either one of the 3 layers is in the range of 0.05-100 μιη, e.g. 0.08-50 μιη, or 0.1-10 μιη, such as 0.2-3 μιη. Moreover, the sensor membrane should also be conductive so as to allow for electron transfer between the redox components in the analyte and external equipment (e.g. a potentiostat or other electrical components).

The overall thickness of the sensor membrane is typically 0.1-1,100 μιη, e.g. 0.2-1.0 μιη, 10- 50 μιη or 100-500 μιη. The first layer

The first layer (also referred to as the bottom layer) consists of a conductive material.

In view of the overall water-permeability of the sensor membrane, it should be understood that the conductive material should allow water (an aqueous medium) to penetrate there through. Typically, the porosity of the first layer is at least 40 %, such as at least 50 %, e.g. between 60 % and 90 %, or between 65 % and 85 %.

The first layer defines at least one electrode, such as one of a set of electrodes for measuring an analyte.

In one embodiment, the first layer defines a single electrode.

In another interesting embodiment, the first layer is patterned, e.g. such that two or more electrodes are defined. In one variant, the patterning defines two or more individually addressable electrodes of the conductive material. Moreover, the individually addressable electrodes may consist of at least two sub-groups of electrodes for measuring different analytes. In this case, it will be necessary to pattern or modify the third uppermost layer so that areas of this third layer are sensitive to different analytes. The first layer typically has a thickness of 0.1-1,000 μιη, such as 1.0-500 μιη, e.g. 1.0-20 μιη, 10-80 μιη, 50-120 μιη or 100-400 μιη.

In one embodiment, the first layer is a porous metal material. Examples of suitable materials are silver, gold and platinum. When the first layer is a porous metal material, it typically has a thickness of 0.1-1,000 μιη, such as 1.0-500 μιη, e.g. 1.0-20 μιη, 10-80 μιη, 50-120 μιη or 100-400 μιη. Moreover, a first layer of a porous metal material typically has a porosity of at least 40 %, such as at least 50 %, e.g. between 60 % and 90 %, or between 65 % and 85 %.

When the first layer is a porous metal material, it can be prepared through a sintering process of metallic nanoparticles or nanowires into the desired membrane size. When the first layer is a patterned porous metal material, it can be prepared by standard micro and nanofabrication processes in which a polymer layer patterned by UV and deep UV lithography, e-beam lithography, nano-imprint lithography, block co-polymer lithography or by other means are utilized as a masking material.

In another embodiment, the first layer is a porous intrinsically conducting polymer material. Examples of suitable materials are PEDOT (such as PEDOT-based polymers) . When the first layer is a porous intrinsically conducting polymer material, it typically has a thickness of 0.1- 1,000 μιη, such as 1.0-500 μιη, e.g. 1.0-20 μιη, 10-80 μιη, 50-120 μιη or 100-400 μιη. Moreover, a first layer of a porous intrinsically conducting polymer material typically has a porosity of at least 40 %, such as at least 50 %, e.g. between 60 % and 90 %, or between 65 % and 85 %.

When the first layer is a porous intrinsically conducting polymer material, it can be prepared by chemical polymerization casting methods [H. Allcock, M. Hofmann, S. Lvov, X.Y. Zhou, D. Macdonald. Proton Conducting Polymer Membranes, US Patent 6,759,157 (2004).] .

The porosity of a layer of an intrinsically conducting polymer material can be controlled in a track etching process where a thin film of the polymer material is subjected to a high energy ion (for instance Argon) bombardment in which process the material around individual ion impacts becomes soluble whereas the non disturbed area remains insoluble. In this way the porosity of the layer can be controlled by the ion dose and by standard photolithography the porosity can be confined into smaller areas if desired, thereby providing a suitable patterning. The individual electrodes can be defined by another photolithography patterning process where the conductivity of the polymer is increased locally to define the electrode and corresponding connections.

In still another embodiment, the first layer is a porous polymer electrode with incorporated metallic nanoparticles. Illustrative examples hereof are any conductive polymers like conductive SU-8 [M. Marelli, G. Divitini, C. Collini, L. Ravagnan, G. Corbelli, C. Chisleri, A. Gianfelice, C. Lenardi, P. Milani, L. Lorenzelli. Flexible and biocompatible microelectrode arrays fabricated by supersonic cluster beam deposition on SU-8. J. of Micromech. Microeng. 21(4), 045013 (2011)] (with doped Pt or Au particles). When the first layer is a porous polymer electrode with incorporated metallic nanoparticles, it typically has a thickness of 0.1- 1,000 μιη, such as 1.0-500 μιη, e.g. 1.0-20 μιη, 10-80 μιη, 50-120 μιη or 100-400 μιη.

Moreover, a first layer of a porous polymer electrode with incorporated metallic nanoparticles typically has a porosity of at least 40 %, such as at least 50 %, e.g. between 60 % and 90 %, or between 65 % and 85 %.

When the first layer is a porous polymer electrode with incorporated metallic nanoparticles, it can be prepared by standard microfabrication technique like lithography, in particular for the purpose of patterning. In a currently most preferred embodiment, the first layer is porous gold having a thickness of 10-500 μιη.

In another currently preferred embodiment, the first layer is a porous layer of a conducting polymer, e.g. PEDOT, having a thickness of 10-500 μιη.

The second layer The second layer is not a uniform, covering layer in the conventional sense, but is rather a three-dimensional structure build on top of the first (membrane) layer, and on which the third layer is coated. Hence, the second layer consisting of the nanostructure material can be said to separate the first layer (see above) and the third layer (see below).

In the present context, the term "nanostructure material" is intended to mean solid state structures having at least one dimension in the nanometer range, such as nanotubes, nanowires or nanofibers, present in order to optimize the biocompatibility properties or the electrochemical properties of the sensor membrane.

In some embodiments, the second layer consists of extruding nanostructures attached to the first layer and covered by the third layer. Due to the nanostructure (and a suitable degree of hydrophilicity) of the second layer, it not be limiting for the penetration of water (e.g. an aqueous medium) there through.

The nanostructure material on the first layer typically has a thickness (i.e. height) of 0.01- 100 μιη, such as 2.0-8.0 μιη, e.g. 2.0-4.0 μιη, or 6.0-8.0 μιη.

Illustrative examples of the materials useful as the second layer are nanostructures, such as peptide nanowires, e.g. diphenylalanine nanowires, and EAK16, K24, and MAX 1 amyloid nanostructures. Examples of "EAK16" are the peptides EAK16-I, EAK16-II, and EAK16-IV (each of the formula C₇0H₁2₁N2₁O2₅, 1657 g/mol) available from Invitrogen (Huntsville, AL), having the amino acid sequences AEAKAEAKAEAKAEAK (EAK16-I, - + - + - + - +), AEAEAKAKAEAEAKAK (EAK16-II, - - + + - - + +), and AEAEAEAEAKAKAKAK (EAK16-IV, - 1- + + +), respectively, where A=Ala, E=Glu, and K=Lys. K24 is a 24-residue peptide

(NH2-KLEALYVLGFFGFFTLGIMLSYIR-COOH). MAX 1 is a peptide of the structure (VK)₄-VDPPT- (KV)₄-NH₂.

Alternatively, non-biological three-dimensional materials like black silicon nanograss, carbon nanotubes or other inorganic nanowires or nanotubes may also be used. Preferably, the nanostructure material (the second layer) is capable of promoting the viability of tissue or cell cultures.

In one embodiment, the second layer is prepared by an aniline vapour aging, high temperature treatment of a diphenylalanine film for the self-assembly of peptide nanowires [J. Ryu and C.B. Park. High-Temperature Self-Assembly of Peptides into Vertically Well- Aligned Nanowires by Aniline Vapor. Advanced Materials 20(19), 3754 (2008)] .

In another embodiment, the second layer is prepared by standard microfabrication techniques for the creation of titanium oxide nanotubes [S. Jin and S. Oh. Biocompatible vertically aligned nanotube array structure on biocompatible substrate, useful for in vitro testing of drugs, chemicals, toxins, orthopedic or dental bone repair, comprises laterally separated nanotube arrangement. See also WO 2006/116752 A2; EP 1 879 522 A2; US 2009/220561 Al] . In a currently most preferred embodiment, the second layer comprises peptide nanowires (PNW), preferably diphenylalanine nanowires, and has a thickness (i.e. height) of 2-10 μιη, e.g. 3-8 μιη.

The third layer The third, topmost, layer consists of a conducting polymer material.

Typically, the polymer material of the third layer holds functionalizations specific to a certain cell tissue analyte. The cell tissue analyte for which the polymer material provides selectivity is typically selected from neurotransmitters, e.g. selected from dopamine, glutamate, GABA, DOPAC, HVA and norepinephrine, as well as other analytes and metabolites like glycerol, choline (cell death markers), glucose, lactate, and pyruvate. Particularly interesting are dopamine, GABA and norepinephrine.

In order to adapt the sensor membrane to exhibit selectivity for dopamine, the third layer may be composed of an electropolymerized polypyrrole film doped with polystyrene sulfonate counter-ions [Sasso et al., Analyst, 2013, Vol. 138, 3651-3659] . In order to adapt the sensor membrane to exhibit selectivity for glutamate, enzymes such as glutamate oxidase, horseradish peroxidase and ascorbate oxidase wired via

poly(ethyleneglycol) diglycidyl ether (PEDGE) to an Os(bpy)₂CI-complexed,ethylamine- quaternized, redox polymer (POs-EA) may be incorporated in the third layer. Reference is also made to the papers by Oldenziel and Westerink in Anal. Chem. 2005 In order to adapt the sensor membrane to exhibit selectivity for GABA, enzymes such as gabase and glutamate oxidase can be incorporated in to the third layer (M. Zhang and L. Mao, Frontiers in Bioscience, 10, 345-352, 2005).

Illustrative examples of suitable polymers for establishing the third layer are e.g. polymers selected from polypyrroles (PPY), polyanilines (PANI), and polythiophenes such as poly(3,4- ethylenedioxythiophene) (PEDOT).

In some embodiments, it is preferred that the third layer comprises a polymer selected from conducting polymers having the ability to be electro-polymerized, i.e. to be polymerized under the influence of an electric current. Synthesizing the third layer by electro- polymerization at individually addressable electrodes (present on the first layer) allows for the different localized functionalization of the electrodes, meaning that the sensor membrane gains more than one specificity, one for each of the functionalized individually addressable electrodes.

The third layer defines at least one electrode, such as one of a set of electrodes for measuring an analyte. In one embodiment, the third layer defines a single electrode.

In another interesting embodiment, the third layer is patterned, e.g. such that two or more electrodes are defined. In one variant, the patterning of the first and the third layer defines two or more individually addressable electrodes of the conducting polymer material.

Moreover, the individually addressable electrodes may consist of at least two sub-groups of electrodes having selectivity for different analytes.

With respect to the measuring of an analyte (or different analytes) at different regions of e.g. a brain slice, it may even be advantageous that the patterning of the third layer includes mark-up lines (or dots or dashed lines) for facilitating the proper arrangement and orientation of e.g. a brain slice. Such a mark-up may alternatively be provided by means of a pigment layer or a dye or the like as illustrated in Figure 3.

It should be understood, that in order to obtain two or more individually addressable electrodes, the first layer must be patterned. The third layer will then be patterned simply because the first layer is patterned due to the nature of the polymer deposition methods.

The third layer typically has a thickness of 0.001-1.0 μιη, such as 0.01-0.08 μιη, e.g. 0.01- 0.02 μιη, or 0.06-0.08 μιη.

The third layer is typically designed with such a thickness that it does not affect the permeability of the overall sensor membrane.

In one embodiment, the third layer can be prepared by coating the first two layers with an intrinsic conducting polymer, or a polymer made conducting by metal nanoparticle doping, and then using microfabrication techniques (such as mask-assisted lithography) to pattern the third layer, for example by defining conductive and non-conductive sections of the third layer.

In another embodiment, the third layer can be prepared by electro-polymerization, by catalyzing the polymer formation onto the individually addressable electrodes present in the first layer. In this way, several functionalization of the third layer can be implemented into the sensor membrane, by using different materials for the third layer onto each different electrode present on the first layer.

In a currently most preferred embodiment, the third layer is prepared from poly(3,4- ethylenedioxythiophene) (PEDOT), which can easily be patterned by microfabrication techniques like mask-assisted lithography.

Specific embodiments of the invention

One currently preferred embodiment of the invention relates to a water-permeable sensor membrane comprising : a) a first layer of a conductive material defining a porous metal electrode and having a thickness of from 0.1-1,000 μιη; b) a second layer build on the first layer, said second layer comprising peptide nanowires (PNW), preferably diphenylalanine nanowires, and having a thickness of 0.01-100 μιη; and c) a third, topmost, layer of a poly(3,4-ethylenedioxythiophene) (PEDOT) defining an electrode and having a thickness of 0.001-1.0 μιη.

Such a sensor membrane is especially suited for measuring dopamine.

Another currently preferred embodiment of the invention relates to a water-permeable sensor membrane comprising : a) a first layer of PEDOT defining a porous polymer electrode and having a thickness of from 0.1-1,000 m; b) a second layer build on the first layer, said second layer comprising peptide nanowires (PNW), preferably diphenylalanine nanowires, and having a thickness of 0.01-100 μιη; and c) a third, topmost, layer of a polypyrrole defining an electrode and having a thickness of 0.001-1.0 m. Such a sensor membrane is especially suited for measuring dopamine if the third layer e.g. is an electropolymerized polypyrrole film doped with polystyrene sulfonate counter-ions. Selectivity for other analytes can be obtained if the third layer is an electropolymerized film doped with enzymes such as e.g. gabase and glutamate oxidase for GABA detection or glutamate oxidase for glutamate detection (M. Zhang and L. Mao, Frontiers in Bioscience, 10, 345-352, 2005) . A sensor assembly

The present invention also provides a sensor assembly comprising a sensor membrane as defined hereinabove (either the three-layer membrane or the single layer membrane), a metering device, and one or more conductors electrically connecting the electrode(s) of the sensor membrane with the metering device. Examples of suitable metering devices are any commercially available potentiostat (electronic hardware required to control a two- or three-electrode cell and run most electroanalytical experiments), or simpler versions of such a device, like an amperometric detector (a detector designed to apply a potential to a channel and measures the current response).

The individual electrodes of the sensor membrane (the electrode(s) of the third layer) are electrically connected with the metering device, e.g. by use of, e.g., conducting glue, silver paste or conducting tape.

The metering device may further be electrically connected to a reference electrode and to a counter electrode arranged in a conventional manner.

A tissue sample or cell culture sample monitoring assembly The present invention further provides a tissue sample or cell culture sample monitoring assembly comprising a sensor assembly as defined hereinabove, and a tissue sample or a cell culture sample arranged on top of the third layer of the sensor membrane of the sensor assembly (or simply on top of the single layer sensor membrane).

The end user will typically be able to arrange the tissue sample or cell culture sample, e.g. an organ tissue sample, on the present sensor membrane in the same way as for conventional tissue culture membranes. Examples of typical tissue samples are organ tissue samples from the brain or spine of rats, mice or other test animals along with neuronal cell lines, e.g. PC- 12 cells. Furthermore the sensor assembly could be used in the study of differentiation of neuronal stem cells into for instance dopaminergic, noradrenergic or serotonergic neurons. As mentioned above, if the sensor electrode is adapted for measurement of an analyte (or different analytes) at different regions of e.g. a brain slice, the patterning of the third layer may include mark-up lines (or dots or dashed lines) for facilitating the proper arrangement and orientation of e.g. a brain slice, or such a mark-up may be provided by means of a pigment layer or a dye or the like.

Advantageously, the monitoring assembly further comprises a receptacle wherein the sensor membrane and the tissue sample or cell culture sample are arranged, said receptacle further comprising a liquid medium for said tissue sample or cell culture sample.

The receptacle may be in the form of conventional trays capable of holding a plurality (e.g. 6 or 12) of individual sensor membranes. Each well holds a sensor membrane with the tissue sample or cell culture sample and a liquid medium for the tissue sample or cell culture sample. Such a tray is typically equipped with a lid.

A method

Still further, the present invention provides a method of continuously or intermittently monitoring the concentration or presence of a cell tissue analyte in the proximity of a tissue sample or cell culture sample, said method comprising the step of a) providing a monitoring assembly as defined hereinabove (either the three-layer membrane or the single layer membrane), and b) measuring the concentration or presence of the cell tissue analyte by electrochemical techniques.

In one interesting embodiment, the monitoring is conducted continuously, hence the accurate development of the concentration (or presence) of the cell tissue analyte can be monitored.

Measurement can be done either by amperometry, where then the current response can be related to the amount of analyte (e.g. dopamine) released. As an alternative to a continuous measurement, it is of course also possible to obtain several point measurements over a period of time (i.e. virtually continuous in the sense of achieving a data point e.g. every 10 min. for a period of e.g. several weeks), using other

electrochemical techniques like cyclic voltammetry or linear sweep voltametry or electrochemical impedance spectroscopy, or even several amperometric measurements e.g. every 10 min. or the like.

EXAMPLES

Example 1 The first layer in the membrane assembly can consist of either metal or an intrinsically conducting polymer. In the case of intrinsically conducting polymers the membrane can be prepared by a chemical polymerization casting methods [H. Allcock, M. Hofmann, S. Lvov, X.Y. Zhou, D. Macdonald. Proton Conducting Polymer Membranes, US Patent 6,759,157 (2004).] . In another approach the conducting polymer membrane can be prepared from a track etching procedure of a polymer film. In the case of metal membranes these can be prepared by a sintering process of metal nanoparticles.

The second layer in the membrane assembly can consist of diphenylanaline peptide nanowires. In which case the layer is prepared by first preparing a solution of diphenylalanine dipeptide monomers dissolved in hexaflouro-2-isopropanol (HFP) at a concentration ranging from 20 mg/mL to 200 mg/mL. This solution is deposited on the first layer and the HFP is evaporated. When aged at 100°C in aniline vapour the diphenylalanine peptide nanowires will form. The orientation of the formed nanowires is controlled by the gradient of the aniline vapour concentration therefore to insure a vertical orientation a vertical gradient of the vapour must be insured. The density and the length of the formed wires are governed by the concentration of the monomers and the growth time respectively.

The third conducting polymer layer can be prepared by electropolymerization from a solution containing the monomer precursor, by applying a constant potential to the sensor membrane relative to the polymerization potential, e.g. 0.7 V for 10 seconds in the case of polypyrrole. The monomer solution can contain counter-ion dopants or enzymes needed for the specificity and sensitivity increase with respect to a specific analyte, e.g. polystyrene sulfonate ions in the case of dopamine or gabase and glutamate oxidase for GABA detection.

The characterization of the sensor membrane can be achieved by standard electrochemical techniques. The sensor membrane can be used as working electrode in a standard 3- electrode setup electrochemical cell, having a Pt counter electrode and a Ag/AgCI reference electrode immerged in an electrolyte solution and with all electrodes connected to an external potentiostat. Cyclic voltammetry can be used to study the sensor membrane response to the specific analytes intended for the detection as well as standard redox couples, e.g. potassium ferri/ferrocyanide, by swiping the potential in a window relevant to the oxidation and reduction of each analyte, e.g. 0.2-0.8 V, and by observing how the anodic and cathodic current responses obtained change with respect to potential sweep rate, e.g. ranges of 10- 250 mV/s, and concentration of the analyte, e.g. ranges of 0.01-10 mM for standard redox couples and 0.05-800 nM for analyte solutions. Amperometry can be used to investigate the sensor membrane current response to subsequent additions of analytes with varying concentrations at constant potentials close to the oxidation/reduction potentials resulting from the cyclic voltammetry investigation. An amperometric calibration can be achieved by constructing a plot of current response vs. analyte concentration, yielding statistical values such as minimum detection limit and the concentration range of linear response.

The membrane assembly can be utilized as standard tissue or cell culture membrane.

Therefore the preparation of the experiments is similar to the preparation of normal culture experiments. First the membrane is placed in a 6 well plate, which is filled with medium so that the bottom part of the membrane is in contact with the culture medium and the top part with a thin layer of the medium withdrawn from below due to capillary forces. The tissue sample or cell culture sample is placed on the membrane and the 6 well plate moved to an incubator that controls the temperature and humidity. All of the preparation steps must be performed in a (sterile) laminar flow bench.

The analyte detection from cells or tissue can be obtained by amperometric measurements at potentials close to the oxidation/reduction potential peaks of specific analytes, e.g. -0.1 V, 0.3 V or 0.6 V, by connecting the sensor membrane to a potentiostat in the setup discussed above, with the electrolyte solution being the culture medium. Upon the release of analyte from the cells or tissue, a change will appear in the current trace that can yield information about the analyte and the mechanics of the release. Example 2

The first layer in the membrane assembly can consist of either metal or an intrinsically conducting polymer. In the case of intrinsically conducting polymers the membrane can be prepared by a chemical polymerization casting methods [H. Allcock, M. Hofmann, S. Lvov, X.Y. Zhou, D. Macdonald. Proton Conducting Polymer Membranes, US Patent 6,759,157 (2004).] . In another approach the conducting polymer membrane can be prepared from a track etching procedure of a polymer film. In the case of metal membranes these can be prepared by a sintering process of metal nanoparticles. The second layer in the membrane assembly can consist of diphenylanaline peptide nanowires. In which case the layer is prepared by first preparing a solution of diphenylalanine dipeptide monomers dissolved in hexaflouro-2-isopropanol (HFP) at a concentration ranging from 20 mg/mL to 200 mg/mL. This solution is deposited on the first layer and the HFP is evaporated. When aged at 100°C in aniline vapour the diphenylalanine peptide nanowires will form. The orientation of the formed nanowires is controlled by the gradient of the aniline vapour concentration therefore to insure a vertical orientation a vertical gradient of the vapour must be insured. The density and the length of the formed wires are governed by the concentration of the monomers and the growth time respectively. The peptide nanowires can be functionalized to contain counter-ion dopants or enzymes needed for the specificity and sensitivity increase with respect to a specific analyte, e.g. polystyrene sulfonate ions in the case of dopamine or gabase and glutamate oxidase for GABA detection.

The third conducting polymer layer can be prepared by electropolymerization from a solution containing the monomer precursor, by applying a constant potential to the sensor membrane relative to the polymerization potential, e.g. 0.7 V for 10 seconds in the case of polypyrrole. In this example, the third layer is passive in terms of sensitivity but is needed to guarantee an additional stability in the entrapment of the functional components anchored to the peptide nanowires.

The characterization of the sensor membrane can be achieved by standard electrochemical techniques. The sensor membrane can be used as working electrode in a standard 3- electrode setup electrochemical cell, having a Pt counter electrode and a Ag/AgCI reference electrode immerged in an electrolyte solution and with all electrodes connected to an external potentiostat. Cyclic voltammetry can be used to study the sensor membrane response to the specific analytes intended for the detection as well as standard redox couples, e.g. potassium ferri/ferrocyanide, by swiping the potential in a window relevant to the oxidation and reduction of each analyte, e.g. 0.2-0.8 V, and by observing how the anodic and cathodic current responses obtained change with respect to potential sweep rate, e.g. ranges of 10- 250 mV/s, and concentration of the analyte, e.g. ranges of 0.01-10 mM for standard redox couples and 0.05-800 nM for analyte solutions. Amperometry can be used to investigate the sensor membrane current response to subsequent additions of analytes with varying concentrations at constant potentials close to the oxidation/reduction potentials resulting from the cyclic voltammetry investigation. An amperometric calibration can be achieved by constructing a plot of current response vs. analyte concentration, yielding statistical values such as minimum detection limit and the concentration range of linear response. The membrane assembly can be utilized as standard tissue or cell culture membrane.

Therefore the preparation of the experiments is similar to the preparation of normal culture experiments. First the membrane is placed in a 6 well plate, which is filled with medium so that the bottom part of the membrane is in contact with the culture medium and the top part with air. The tissue sample or cell culture sample is placed on the membrane and the 6 well plate moved to an incubator that controls the temperature and humidity. All of the preparation steps must be performed in a laminar flow bench.

The analyte detection from cells or tissue can be obtained by amperometric measurements at potentials close to the oxidation/reduction potential peaks of specific analytes, e.g. -0.1 V, 0.3 V or 0.6 V, by connecting the sensor membrane to a potentiostat in the setup discussed above, with the electrolyte solution being the culture medium. Upon the release of analyte from the cells or tissue, a change will appear in the current trace that can yield information about the analyte and the mechanics of the release.

Claims

1. A water-permeable sensor membrane comprising a. a first layer of a conductive material defining at least one electrode and having a thickness of 0.1-1,000 μιη; b. a second layer of a nanostructure material build on the first layer; and c. a third, topmost, layer of a conducting polymer material defining at least one electrode and having a thickness of 0.001-1.0 μιη.

2. The sensor membrane according to any one of the preceding claims, wherein said polymer material of the third layer holds functionalizations specific to a certain cell tissue analyte.

3. The sensor membrane according to any one of the preceding claims, wherein the cell tissue analyte for which the polymer material provides selectivity is selected from

neurotransmitters, e.g. selected from dopamine, GABA and norepinephrine.

4. The sensor membrane according to any one of the preceding claims, wherein the third layer comprises a polymer selected from conducting polymers having the ability to be electropolymerized .

5. The sensor membrane according to any one of the preceding claims, wherein the third layer comprises a polymer selected from polypyrroles (PPY), polyanilines (PANI), and polythiophenes like poly(3,4-ethylenedioxythiophene) (PEDOT).

6. The sensor membrane according to any one of the preceding claims, wherein the second layer consists of nanostructures, such as peptide nanowires, e.g. diphenylalanine nanowires, and EAK16, K24, and MAX 1 amyloid nanostructures.

7. The sensor membrane according to any one of the preceding claims, wherein the second layer is capable of promoting the viability of tissue or cell cultures.

8. The sensor membrane according to any one of the preceding claims, wherein the first layer is a porous metal material, or the first layer is a porous intrinsically conducting polymer material, or the first layer is a porous polymer material with incorporated metallic

nanoparticles.

9. The sensor membrane according to any one of the preceding claims, wherein the first layer is patterned, e.g. such that the patterning defines two or more individually addressable electrodes of the conductive material.

10. The sensor membrane according to any one of the preceding claims, wherein the third layer is patterned, e.g. such that the individually addressable electrodes consist of at least two sub-groups of electrodes for measuring different analytes.

11. The sensor membrane according to any one of the preceding claims, wherein the second layer comprises peptide nanowires, e.g. diphenylalanine nanowires, and wherein the third, topmost, layer comprises of poly(3,4-ethylenedioxythiophene) (PEDOT) and holds functionalizations specific to dopamine.

12. A sensor assembly comprising a sensor membrane according to any one of the preceding claims, a metering device, and one or more conductors electrically connecting the electrode(s) of the third layer of the sensor membrane with the metering device.

13. A tissue sample or cell culture sample monitoring assembly comprising a sensor assembly according to claim 12 and a tissue sample or a cell culture sample arranged on top of the third layer of the sensor membrane of the sensor assembly, e.g. further comprising a receptacle wherein the sensor membrane and the tissue sample or the cell culture sample are arranged, said receptacle further comprising a liquid medium for said tissue sample or cell culture sample.

14. A method of continuously or intermittently monitoring the concentration or presence of a tissue analyte in the proximity of a tissue sample or cell culture sample, said method comprising the step of: a. providing a monitoring assembly as defined in claim 13, b. measuring the concentration or presence of the cell tissue analyte by electrochemical techniques.