Optically transparent microelectrode arrays for electrochemical and electrophysiological measurements or stimulation

Abstract

The present invention relates to a microelectrode array device and method for biological imaging comprising a well for containing a liquid medium, an optically transparent substrate, and a microelectrode array comprising a plurality of electrode pads, each electrode pad comprising a conductive film having a thickness between 5 nm and 1000 nm, comprising a carbon nanotube network and / or graphene. The microelectrode array further comprises at least one counter electrode and a conductive trace connecting the at least one counter electrode to a voltage source such that the electrode pads can be connected to a voltage source and a potential difference can be provided between the electrode pads and the counter electrode.

Description

[Technical field]

[0001] The present invention relates to a microelectrode array device for biological imaging and a method for cell imaging. [Background technology]

[0002] The plasma membrane of living cells contains ion channels that can open or close to regulate the electrical potential between the inner and outer plasma membrane surfaces. This membrane potential can be sensed with electrodes, and the information can be used in assessing the state or activity of the cell. Sensing the membrane potential is particularly important in the study of neurons, but a variety of other cell types also undergo membrane potential changes that may be desired to be measured. In addition, the state or activity of a cell can be determined by the release of a neurotransmitter, Ca. 2+ The biomolecular release can also be evaluated based on the release of ions, or other biomolecules. Therefore, it is desirable to measure both the electrical signal and the release of the biomolecules. The electrical signal is measured using electrodes, and the release of the biomolecules is detected by either electrochemical electrodes or fluorescent signals. For this reason, transparent electrodes that can measure both electrical and electrochemical signals are highly needed, as they can be used to multiplex the electrical signal with the biochemical signal.

[0003] Currently available microelectrode array structures are often made of opaque materials, typically metals (Patent Document 1), making it impossible to image cells / organoids / tissues before, during, or after measurements with the disclosed microelectrode array (MEA) structures. When considering neurotransmitter measurements, it is very important to visually see the cell or cell part on the measurement pad, since it is not possible to be sure whether there is a synapse or an electrically active ion channel region on top of the measurement pad. Opaque electrodes and wiring also prevent the detection of proteins, lipids, sugars, or Ca. 2+ This makes it impossible to measure fluorescently labeled biomolecules such as ion concentration fluctuations. It is therefore advantageous for the MEA structure to be transparent for use in both academic environments and industries such as the pharmaceutical industry.

[0004] Wiring has previously been made from transparent materials, but to the inventor's knowledge, there are no transparent pad materials or products available. There are some devices that use indium tin oxide or conductive polymers, but their electrical, electrochemical, or optical properties could still be improved. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US RE38,323 E

[0006] It is an object of the present invention to provide a microelectrode array device which offers the described improvements over known microelectrode array devices. Summary of the Invention [Means for solving the problem]

[0007] This object is achieved by means of the features of the independent claims. The dependent claims refer to preferred embodiments.

[0008] The microelectrode array (MEA) devices described herein can be used as electrochemical sensors or as electrophysiological instruments to measure and / or stimulate explanted tissue samples such as cells, organoids, or brain slices. Because the proposed MEA structure is transparent, cells, organoids, or tissue samples can be imaged under a microscope or optically stimulated through the MEA structure. This allows the use of microscopes in which either the objective lens or the excitation source or both are located below the MEA. This allows the combined use of electrochemical / electrophysiological measurements with fluorescence-based techniques or other techniques that require optical observation of the sample, e.g. patch clamping.

[0009] According to a first aspect, the present invention relates to a microelectrode array device and method for bioimaging, comprising a well for containing a liquid medium, an optically transparent substrate, and a microelectrode array comprising a plurality of electrode pads. Each electrode pad comprises a conductive film, preferably having a thickness between 5 nm and 1000 nm, comprising a carbon nanotube network and / or graphene. The microelectrode array further comprises at least one counter electrode and conductive traces connecting the at least one counter electrode to a voltage source such that the electrode pads can be connected to a voltage source and a potential difference can be provided between the electrode pads and the counter electrode. Such a microelectrode array device has improved electrical, electrochemical, and / or optical properties. Furthermore, the electrode pads can perform both electrical and electrochemical measurements, thus improving the functionality of the microelectrode array structure. Furthermore, the porous conductive film can be modified with metal particles or conductive polymers to further improve the electrical conductivity and improve the chemical sensor performance.

[0010] Preferably, the substrate is made of at least one of glass, quartz, a transparent polymer, a transparent metal oxide, and a cellulose film.

[0011] Preferably, each electrode pad is 1 μm 2 to 1000μm 2 Between 1 μm and 2 μm, preferably 1 μm 2 to 500 μm 2 More preferably, between 10 μm 2 to 100μm 2 Alternatively, each electrode pad includes a surface area of ​​1 mm 2 From 100mm 2 Between 1 mm and 2 mm, preferably 2 From 10mm 2 Includes the surface area between.

[0012] Preferably, each electrode pad has a light transmittance of at least 70%, more preferably at least 80%, and most preferably at least 90% within the wavelength spectrum of 250 nm to 900 nm, or each electrode pad has a light transmittance of at least 70%, more preferably at least 80%, and most preferably at least 90% within the wavelength spectrum of 250 nm to 3000 nm. Preferably, these light transmittances are achieved over at least 50%, more preferably at least 70%, and even more preferably at least 90% of the wavelengths in the above-mentioned wavelength spectrum, or for each wavelength in the above-mentioned wavelength spectrum. In some cases, light transmittance at specific wavelengths (e.g., wavelengths where fluorescence is expected may be sufficient).

[0013] In some embodiments, the conductive film is deposited using press transfer, spin coating, dip coating, liquid phase adsorption, spray coating, inkjet printing, screen printing, electrochemical deposition, arc discharge, evaporation, sputtering, physical vapor deposition, chemical vapor deposition (CVD), or plasma enhanced CVD, preferably the film is deposited using aerosol chemical vapor deposition (CVD) dry deposited by press transfer.

[0014] Preferably, the carbon nanotube network film has a thickness between 10 nm and 400 nm, preferably between 30 nm and 200 nm, more preferably between 30 nm and 100 nm.

[0015] Preferably, the conductive traces are formed of the same material as the electrode pads.Preferably, the plurality of electrode pads are arranged in a grid or repeating pattern.

[0016] Preferably, the microelectrode array device further comprises contact pads disposed on the optically transparent substrate adjacent to the grid of electrode pads, one contact pad for each electrode pad, and conductive traces connecting each pair of the electrode pads and the contact pads.

[0017] Preferably, the plurality of electrode pads are further coated with a cell growth promoter, preferably the growth promoter comprises collagen, laminin, polyornithine (poly-L-ornithine) and / or an extracellular matrix factor.

[0018] According to a further aspect, the present invention also provides a method of cell imaging using a microelectrode array device, comprising the steps of: i. depositing cells or tissue onto the microelectrode array in the well; ii. measuring the voltage and / or current from each electrode pad; iii. imaging the cell or tissue using a light microscope and / or a fluorescent microscope.

[0019] Preferably, steps ii and iii are carried out simultaneously and / or step iii comprises fluorescent imaging of fluorescently labelled cells or tissues.

[0020] Preferably, the method comprises the further step of introducing a biochemical reagent into the well during step ii, preferably the biochemical reagent comprises a neurotransmitter, a pharmacological agent, and / or a cell signalling agent.

[0021] Preferably, the method comprises the further step of stimulating the cells or tissue by establishing a voltage difference between the electrode pad and at least one counter electrode.

[0022] Preferably, step ii comprises performing chronoamperometric, voltammetric and / or impedance measurements of the electrode pads. [Brief description of the drawings]

[0023] The subject matter of the present invention will be described in more detail in the following text with reference to preferred exemplary and non-limiting embodiments as illustrated in the accompanying drawings, which disclose embodiments of the present invention for purposes of illustration only, and in particular, the disclosure provided by the drawings and the description is not meant to limit the scope of protection afforded by the present invention.

[0024] [Figure 1] FIG. 1 is a schematic top view of an example of a microelectrode array design. [Diagram 2] 1 is a schematic cross-sectional view of a portion of an exemplary microelectrode array. [Diagram 3] FIG. 1 shows an example of a combination of microscopy and electrical and / or electrochemical measurements performed by the method. [Figure 4] FIG. 13 is an exemplary immunofluorescence image of primary cortical astrocytes imaged through an optically transparent electrode according to the invention, staining cells for nuclei (DAPI, blue), focal adhesions (vinculin, green) and actin cytoskeleton (phalloidin, red), with commonly used filters (DAPI, FITC, TXRED) providing data derived along the full wavelength range of fluorescence microscopy, which are indicated above the figure. [Diagram 5] FIG. 13 is a graph of experimental results of chronoamperometric performance of a transparent CNT network sheet sample measured during a dopamine injection series (0 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM dopamine) in cell culture medium, with each line representing an individual electrode pad. [Figure 6] FIG. 13 is a graph of experimental results of the chronoamperometric performance of another transparent CNT network sheet sample measured by a dopamine injection series (0 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM dopamine) in cell culture medium, with each line representing an individual electrode pad. [Figure 7]1 is a graph of experimental results of light transmittance through a glass reference slide and a glass reference slide (lower line) deposited with a carbon nanotube network (CNT) for use in an electrode pad according to the present invention. [Figure 8] FIG. 1 shows two examples of chronoamperometric data from one electrode pad with a high iron (AC) SWCNT conductive film and a second electrode pad with a low iron (DF) SWCNT conductive film. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Although the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description should be considered as illustrative or exemplary and non-limiting. Therefore, the present invention is not limited to the disclosed embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art from a study of the drawings, the disclosure, and the appended claims, and by practicing the claimed invention. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality and may mean "at least one".

[0026] Figure 1 shows an example of a microelectrode array 100 that can be implemented in a microelectrode array device. The microelectrode array 100 is formed on an optically transparent substrate 10. The microelectrode array 100 comprises a plurality of electrode pads 20, which may be arranged in a grid or patterned array on the surface of the substrate 10, as in Figure 1. The substrate 10 also supports at least one counter electrode 30, which together with the electrode pads 20 can close an electrical circuit related to an external voltage source. Conductive traces 40 are also supported on the substrate 10 and connect the electrode pads 20 to a provided voltage source.

[0027] The substrate 10 is made of an optically transparent material, including but not limited to glass, quartz, transparent polymers, metal oxides, cellulose films, or combinations of the aforementioned materials. If the substrate 10 is conductive, an insulating material 70 molded as a layer can be placed between the substrate 10 and the electrode pads. In some embodiments, the substrate 10 forms the entire bottom surface of the well 200. Alternatively, the optically transparent substrate 10 may support only the electrode pads 20, with other features, such as the counter electrode 30, being placed on an adjacent non-transparent substrate in the well. In the example shown in FIG. 1, the optically transparent substrate 10 underlies the electrode pads 20, the counter electrode 30, and any reference electrode 60, if present (dashed square in FIG. 1).

[0028] The conductive traces 40 can be formed of the same material as the electrode pads 20. For electrode pads formed from carbon nanotubes, a CNT network layer can also be deposited simultaneously as the conductive traces 40, and the conductivity of the conductive traces 40 can be improved by the addition of a conductive polymer. Alternatively, the conductive traces 40 can be made of a transparent material, such as a conductive polymer, indium tin oxide (ITO), graphene, graphene nanoparticles, or a combination of the aforementioned materials. The electrical signal resulting from the electrode pads 20 can be sent to an oscilloscope, potentiostat, or any other device capable of generating or reading an electrical signal, including but not limited to potential, current, or frequency.

[0029] The electrode pad 20 comprises a conductive film 22 that includes or is made from a carbon nanotube network (CNT) and / or graphene layer. In some particular embodiments, the conductive film 22 may be formed from single-walled carbon nanotubes (SWCNT). The CNT network and / or graphene can be disposed on the substrate 10 by techniques including, but not limited to, press transfer, spin coating, dip coating, liquid adsorption, spray coating, inkjet printing, screen printing, electrochemical deposition, arc discharge, evaporation, sputtering, physical vapor deposition, chemical vapor deposition (CVD), or plasma enhanced CVD. The conductive film 22 layer can be patterned into the desired surface pattern by top-down techniques such as laser patterning, dry etching, oxygen plasma etching, mechanical removal from unprotected areas, wet etching, etc. Alternatively, the conductive film 22 can be directly fabricated into the desired pattern by techniques such as screen printing, inkjet printing, three-dimensional printing, lift-off, or techniques based on the modification of surface chemistry to obtain selective deposition by spin coating, dip coating, spray coating, or liquid adsorption. Advantageously, the microelectrode array 100 structures described herein can be manufactured on a large scale without necessarily requiring the use of ultra-clean cleanroom environments and tooling. Thus, the microelectrode array 100 structures can be manufactured directly on large substrates 10, and the structures can also be manufactured in a roll-to-roll fashion.

[0030] For conductive films 22 comprising or made from graphene, preferably chemical vapor deposited (CVD) graphene, the growth and transfer are commonly known processes. Single or multi-layer graphene is most conveniently grown by CVD on a metal catalyst foil, most commonly copper. The graphene side of the foil is then coated with a polymer (usually PMMA) or polymer tape, and the Cu foil is then etched away in an etching bath. The film is then transferred to a substrate (usually Si / SiO 2) and immersed in a cleaning solution to remove the polymer. After cleaning, the film can be further transferred onto other substrates or processed into devices by patterning (e.g. laser patterning or lithography + oxygen plasma etching). Such transfer films (e.g. Trivial Transfer Graphene available from ACS Material) and transfer films on polymer (Easy Transfer: Monolayer Graphene on Polymer Film available from Graphenea) are commercially available. Alternatively, an ink with graphene flakes, preferably a conductive polymer, can be inkjet printed.

[0031] The conductive film 22 also uses an enzyme to modify the analyte of interest into a separate molecule and by-product that is detected, for example, the enzyme L-glutamate oxidase to convert L-glutamate (the analyte of interest) into α-ketoglutaric acid + ammonia + H (which is detected electrochemically). 2 O 2 Alternatively, the conductive layer can be modified with antibodies or aptamers to bind to specific biomolecules, changing the state of the electrode (typically impedance), which can be measured and the information used to estimate the concentration of that specific biomolecule. Specific binding of a target / analyte / antigen can also block the active area of ​​the electrode or result in less efficient diffusion of electroactive species from the culture medium (a redox reporter may be added). Conformational changes upon binding of the analyte can have the same effect. Thus, changes in the voltameric or chronoamperometric signal can be correlated with changes in target / analyte / antigen concentration. The conductive film 22 can also be modified with metal films and / or metal-containing nanoparticles and / or microparticles to improve sensitivity and / or selectivity to specific analytes of interest, as well as electrical conductivity. Additionally, the conductive film 22 can be coated with growth promoters that can improve cell viability.

[0032] The conductive film 22 may have a conductive polymer coating (e.g., polypyrrole or PEDOT:PSS) added to it. This may help provide the conductive film 22 with a larger surface area, higher conductivity and / or electrochemical activity. The polymer coating may further improve the conductivity, for example by doping the CNT layer and partially filling any gaps between the CNT nanowires in the film, which may be particularly useful when the wires have very small line widths. Such a polymer coating may also protect the electrode from biofouling, making it more biocompatible. However, the CNT conductive film 22 alone (with an appropriate protein coating) is sufficiently biocompatible and sensitive even after fouling that occurs in cell culture medium.

[0033] Depending on the needs of the particular experiment being used, the impedance of the conductive film 22 may be controlled by adjusting the thickness or density of the network. The diameter of the electrode pads may be easily adapted, for example, between 10,000 μm, depending on whether it is desired to use the electrodes for the measurement of individual cells or cell populations. Each electrode pad may be as small as 1 μm 2 to 1000μm 2 Between 1 μm and 2 μm, preferably 1 μm 2 to 500 μm 2 More preferably, between 10 μm 2 to 100μm 2 may provide a surface area between

[0034] The microelectrode array devices described herein are advantageous because they allow for multiplexing of information obtained from stimulation with electrical or electrochemical recordings and / or fluorescence microscopy techniques. The transparent microelectrode array devices described herein, potentially with the exception of the external contact pads 50, include or are made from optically transparent materials and allow imaging of a cell or organoid or tissue sample before and during electrical or electrochemical measurements of that same cell / organoid / tissue. Furthermore, the same electrode pad 20 can perform electrophysiological measurement techniques and electrochemical techniques, thus providing multiple functions of the same electrode pad 20.

[0035] The microelectrode array device described herein allows for automated or non-automated, continuous or non-continuous electrochemical / electrical / optical detection or stimulation of cells, organoids, or tissue samples. The device can also allow for high-throughput assessment of the electronic state of cells or organoids or tissue samples, potentially including any given chemical to the cells or organoids or tissue samples.

[0036] Optionally, the microelectrode array 100 may further comprise one or more reference electrodes 60. The signal from the reference electrode 60 may be used to remove noise from the electrode pad signal. Alternatively, the reference electrode 60 may be provided within the glass capillary as an external Pt, Ag or Ag / AgCl wire or an Ag / AgCl reference electrode. Reference electrodes are used in electrochemistry (e.g., in a three-electrode cell with wire, counter and reference) to sense the potential of the working electrode in order to accurately apply the desired potential. The reference electrode provides a stable, well-known potential.

[0037] FIG. 2 shows a schematic cross-sectional view of a portion of a microelectrode array 100. Although the features are not shown to scale, it is clear that an optically transparent substrate 10 supports electrode pads 20 that are connected to external contact pads 50 via conductive traces 40. Between the electrode pads 20, an insulating material 70 may be provided, potentially also covering the conductive traces 40. This may be advantageous to ensure that electrical signals do not dissipate through the conductive traces 40 or travel along undesired paths between the electrode pads 20. It may also help to obtain spatial resolution of the microelectrode array so that cells / organoids or populations of cells can be separated at the site of interest observed under a microscope. The insulating material helps to electrically isolate the conductive traces 40 from the culture medium using a dialysis coating. This insulating material 70 may comprise a screen printable polymer, including siloxane or phenoxyphenylsilane, a UV-curable epoxy-based polymer such as SU-8, or a material based on liquid glass or spin-on glass. These materials may also be applied by alternative coating techniques such as inkjet printing, 3D printing, spin coating, dip coating, or conventional UV or e-beam, or micro- or nanoimprint lithography techniques.

[0038] Alternative designs of the microelectrode array 100 are envisioned since the traces can be placed slightly above or below the measurement pad area. Optionally, external contact pads 50 can be placed slightly above or below the traces. The contact pads 50 are optionally made on the edges of the microelectrode array 100 and / or substrate 10. These contact pads 50 can be made of any of the transparent materials mentioned above, or alternatively, if the contact pads 50 are not within the imaging field of view, an opaque conductive material such as metal can be used.

[0039] The conductive traces 40 may be insulated with an insulator material that is transparent in the wavelength range of optical and fluorescent microscopy (250-900 nm), but is not necessarily limited to this wavelength range. The insulator material may also include an absorption peak in the wavelength range of 250-900 nm if it does not interfere with fluorescent microscopy. The MEA device may be insulated with a screen printable material, including but not limited to those based on siloxane or phenoxyphenylsilane, UV curable epoxy-based polymers such as SU-8, liquid glass, or spin-on glass. The insulator material may also be applied by alternative coating techniques such as inkjet printing, 3D printing, spin coating, dip coating, physical vapor deposition, chemical vapor deposition, atomic layer deposition, or conventional UV or e-beam or micro / nanoimprint lithography techniques. If alternative coating techniques are used, commonly used insulator materials may be used, including but not limited to metal oxides such as silicon dioxide, silicon nitride, aluminum oxide or titanium oxide, or transparent polymers that do not autofluoresce in the wavelength range of 250-800 nm, such as Parylene C.

[0040] FIG. 3 is an illustration of one potential use of the present invention. The microelectrode array device includes a substrate 10 on which a microelectrode array 100 is located. The device comprises a well 200 adapted to contain a liquid medium above the substrate 10 and the microelectrode array 100. The device may be placed on an optical microscope and / or a fluorescent microscope. At the same time, the contact pads 50 of the array are connected to a voltage source 300, a potentiostat and / or an oscilloscope, which can provide a current through the electrode pads 20. Due to the optical transparency of the electrode pads 20, any cells or tissues present in the well 200 can be imaged at the same time as the cells / tissues are electrically stimulated via the electrode pads 20. Alternatively or additionally, the electrode pads 20 may measure electrical activity generated by the cells or tissues via the connection of the contact pads 50 to a potentiostat and / or an oscilloscope.

[0041] The contact pads 50 can be connected to an external voltage source 300 and a computer and / or an external control system. The control system can be implemented to provide the appropriate potential, current, frequency, pulse width, etc. to the electrode pads 20. At the same time, the electrode pads 20 can be monitored for changes in impedance and potential resulting from cellular activity.

[0042] One potential embodiment of the microelectrode array device employs an optically transparent microelectrode array 100 for measuring action potentials in combination with high / low time resolution combined with electrochemical analysis. In this implementation, the action potential is typically around 1 kHz and the required impedance is low. The recommended electrode diameter for single cells is in the range of 10 μm to 1000 μm, preferably 100 μm to 1000 μm. In this embodiment, carbon nanotube networks can be used as both the conductive film 22 and the conductive traces 40 of the electrode pad. Optionally, the electrode pad may also include additional metal and / or conductive polymer coatings.

[0043] In another embodiment, a large diameter electrode pad 20 (1 mm) with low impedance can be manufactured to monitor network cellular or tissue phenomena in combination with single cell electrical activity. 2 From 100mm 2 Between 1 mm and 2 mm, preferably 2 From 10mm 2 For example, to study voltage-gated sodium channels in cancer cells, the ability to measure lower amplitude orders of magnitude is required. The small electrodes of commercially available microelectrode arrays 100 have impedances that are too high for such measurements. However, a microelectrode array device according to the present invention can effectively measure signals from voltage-gated sodium channels.

[0044] In other embodiments, the microelectrode array device may be shaped as a neural probe, with the electrode pad locations being arranged along the probe. Furthermore, optogenetic modulation of cells, organoids, or tissue samples can potentially be performed on the microelectrode array device. Optogenetic modulation of ion channels or proteins can be performed and the effect can be measured directly on the measurement pads. This can be used, for example, in studying how neurotransmitter vesicles fuse with the plasma membrane, in addition to many other applications. Furthermore, due to the electrochemical activity of the electrode pads and their transparency, electrochemiluminescent cell imaging is also possible. In a further embodiment, the microelectrode array device of the present invention can be combined with a microfluidic device.

[0045] In one embodiment, single-walled carbon nanotube (SWCNT) networks were produced in a laminar flow reactor by chemical vapor deposition and collected using a membrane filter. SWCNT network samples were pressed onto transparent polymer substrates, and then the networks were densified by adding 99.9% ethanol (obtained from Anora, Finland) and evaporating in air. Electrical contact was made by adding conductive silver paste (obtained from Electrolube, UK) and drying overnight in air, and then the silver paste was contacted to a copper slide using conductive copper tape (obtained from Ted Pella, USA). The electrodes were insulated from other areas using inert PTFE tape (obtained from Irpola, Finland, or CHR 2255 modified high modulus PTFE membrane coated with high temperature silicone adhesive from Saint-Gobain, France). Before using the electrode pads, their surfaces were treated by electrochemical oxidation in Dulbecco's phosphate buffered saline (PBS obtained from Gibco, ThermoFisher Scientific, USA) at a potential of 1.5 V (vs. Ag / AgCI) for 30 s, followed by a second treatment at 0.4 V (vs. Ag / AgCI) for 60 s to stabilize the surface.

[0046] Figure 4 provides a practical example of the microelectrode device of the present invention in use. There, an immunofluorescence image of primary cortical astrocytes adhered on top of the microelectrode array 100 is provided. Imaging is performed via the electrodes. In this example, the electrode pads 20 are formed by CNT network electrode material. The electrode surface has not been treated in any way to improve cell adhesion or viability, highlighting the excellent biocompatibility of the material itself. Cells were stained for nuclei (DAPI, blue), focal adhesions (vinculin, green) and actin cytoskeleton (phalloidin, red). Commonly used imaging filters (DAPI, FITC, TXRED) were selected that represent the entire wavelength range of the fluorescence microscope. The filters used are labeled above each image.

[0047] Figure 5 provides experimental evidence of the excellent chronoamperometric performance of the electrode pads. In this example, the electrode pads are formed from a transparent CNT network sheet sample. Current was measured during a series of dopamine injections (0 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM dopamine) in cell culture medium. Each line represents an individual electrode pad. As can be seen, the electrode pads respond highly linearly to stimulation within this physiologically relevant dopamine concentration range (R 2 =0.9905±0.0074).

[0048] Figure 6 provides further experimental evidence for the chronoamperometric performance of the electrode pads. This example utilizes another example of a transparent CNT network sheet sample. Oxidation currents were measured during a dopamine injection series (0 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1000 nM dopamine) in cell culture medium. The average individual electrodes perform highly linearly in this physiologically relevant dopamine concentration range (R 2 =0.9833±0.0189).

[0049] FIG. 7 is a graph of experimental results of light transmission through a glass reference slide and a glass reference side (lower line) on which the carbon nanotube network (CNT) used in the electrode pad according to the invention was deposited using press transfer. UV-VIS-NIR spectroscopy measurements were performed of the carbon nanotube network on the glass slide compared to the glass slide reference. Measurements were taken through the entire stack and the absorbance includes all materials in the stack. As can be seen, the electrode pad according to the invention maintains very high transmission over the wavelength spectrum from 250 nm to 3000 nm. The light transmission within this range is consistently above 70% and generally around 80%. Thus, the electrode pad described herein is highly advantageous for optical imaging purposes.

[0050] The absorbance of the SWCNT materials was characterized using a UV-vis spectrometer (Agilent Cary 5000 UV-vis-NIR spectrometer, Agilent Technologies, Inc., USA). The transmittance was calculated according to the relationship between absorbance and transmittance based on the Beer-Lambert law as follows:

number

[0051] Figure 8 shows two examples of chronoamperometric data from one electrode pad with a high-iron (AC) SWCNT conductive film and a second electrode pad with a low-iron (DF) SWCNT conductive film. Each spike in the graph corresponds to the addition of dopamine to the medium as described in connection with Figure 6. Both types of electrode pads show robust and time-resolved signal transduction. By changing the amount of ferrocene available in the gas phase, high-iron and low-iron samples were produced at different collection rates, 100% and 500%. The amount of iron can be controlled by varying the temperature of the ferrocene cartridge, as this changes the vapor pressure of ferrocene. SWCNT networks synthesized with a slower recovery of 100% are referred to as low-iron SWCNTs, and samples with a recovery of 500% are referred to as high-iron SWCNTs. With prior art microelectrode arrays, it is often very difficult to obtain these types of sensitivities in the presence of culture medium due to contamination of the electrodes. Most electrodes that function at this level in buffered media are very rapidly inactivated in the buffered media. However, with the electrode pad of the present invention, such a problem does not occur.

Claims

1. 1. A microelectrode array device for biological imaging, comprising: a well for containing a liquid medium; an optically transparent substrate; 1. A microelectrode array comprising: a plurality of electrode pads, each comprising a conductive film having a thickness between 5 nm and 1000 nm, the conductive film comprising a carbon nanotube network and / or graphene; At least one counter electrode; and a microelectrode array comprising: a microelectrode array connecting the electrode pads to a voltage source; and a conductive trace connecting the at least one counter electrode to the voltage source such that a potential difference can be provided between the electrode pads and the counter electrode.

2. 10. The microelectrode array device of claim 1, wherein the substrate is made of at least one of glass, quartz, a transparent polymer, a transparent metal oxide, and a cellulose film.

3. Each electrode pad is 1 μm ２ to 1000 μm ２ Between 1 μm and 2 μm, preferably 1 μm ２ From 500 μm ２ More preferably between 10 μm ２ to 100 μm ２ 3. The microelectrode array device of claim 1 or 2, comprising a surface area between

4. Each electrode pad is 1 mm ２ From 100 mm ２ Between 1 mm and 2 mm, preferably ２ From 10 mm ２ 3. The microelectrode array device of claim 1 or 2, comprising a surface area between

5. 5. A microelectrode array device according to any one of claims 1 to 4, wherein each electrode pad has a light transmittance within the wavelength spectrum of 250 nm to 900 nm of at least 70%, preferably at least 80%, more preferably at least 90%, or each electrode pad has a light transmittance within the wavelength spectrum of 250 nm to 3000 nm of at least 70%, preferably at least 80%, more preferably at least 90%.

6. 6. The microelectrode array device of claim 1, wherein the conductive film is deposited using press transfer, spin coating, dip coating, liquid phase adsorption, spray coating, inkjet printing, screen printing, electrochemical deposition, arc discharge, evaporation, sputtering, physical vapor deposition, chemical vapor deposition (CVD) or plasma enhanced CVD, preferably the film is deposited using aerosol chemical vapor deposition (CVD) dry deposited by press transfer.

7. 6. The microelectrode array device of claim 5, wherein the carbon nanotube network film has a thickness between 10 nm and 400 nm, preferably between 30 nm and 200 nm, more preferably between 30 nm and 100 nm.

8. 8. The microelectrode array device of claim 1, wherein the conductive traces are formed of the same material as the electrode pads.

9. The microelectrode array device of claim 1 , wherein the plurality of electrode pads are arranged in a grid or repeating pattern.

10. 9. The microelectrode array device of claim 8, further comprising contact pads disposed on the optically transparent substrate adjacent the grid of electrode pads, one contact pad for each electrode pad, and wherein the conductive traces connect each pair of electrode pads and contact pads.

11. The microelectrode array device of any one of claims 1 to 10, wherein the plurality of electrode pads are further coated with a cell growth promoting agent, preferably the growth promoting agent comprises collagen, laminin, poly-L-ornithine and / or extracellular matrix factors.

12. A method for cell imaging using a device according to any one of claims 1 to 11, comprising the steps of: i. depositing cells or tissue onto the microelectrode array in a well; ii. Measuring the voltage and / or current from each electrode pad; iii. imaging said cells or tissues using a light microscope and / or a fluorescent microscope.

13. The cellular imaging method of claim 12, wherein steps ii and iii are performed simultaneously, and / or step iii comprises fluorescent imaging of fluorescently labeled cells or tissue, and / or step ii comprises performing chronoamperometric, voltammetric and / or impedance measurements of the electrode pads.

14. 14. The cellular imaging method according to claim 12 or 13, comprising the further step of introducing a biochemical reagent into the well during step ii, preferably wherein the biochemical reagent comprises a neurotransmitter, a pharmacological agent and / or a cell signaling agent.

15. 15. The method of cell imaging according to any one of claims 12 to 14, comprising the further step of stimulating the cells or tissue by establishing a voltage difference between the electrode pad and at least one counter electrode.

Images