Electrochemical microelectrode array for analyzing neurotransmitter release and electrophysiological activity of neurons

Abstract

A chip for electrochemical and electrophysiological measurements of neuronal medium, comprising a base substrate and at least one array of microelectrodes fabricated on the base substrate, wherein the array comprises : an electrochemical sensing subarray which includes a counter microelectrode positioned in the center of the subarray and multiple working microelectrodes placed essentially equidistantly from the counter microelectrode, and optionally one or more reference microelectrodes; an electrophysiology recording subarray which includes at least one microelectrode for in vitro extracellular recording, positioned in close proximity to the electrochemical sensing subarray, and a reference electrode for the electrophysiological electrical circuit; leads extending from the microelectrodes to connect the microelectrodes to contacts placed at the distal end of each respective lead.

Description

[0001] Electrochemical microelectrode array for analyzing neurotransmitter release and electrophysiological activity of neurons

[0002] Background of the invention

[0003] Brain-Machine Interfaces (BMI s ) are designed to establish direct communication pathways between the brain ' s electrical activity and external electronic devices , thereby facilitating the diagnosis and treatment of neurological disorders . The electrical activity of the brain is predominantly generated by neurons that communicate through various types of electrical and chemical signals . At the heart of neuronal communication are action potentials — rapid electrical impulses generated by neurons in response to various stimuli . These impulses propagate along the axons , playing a critical role in synaptic transmission and enabling neurons to interact with one another at synapses .

[0004] Current BMI approaches ef fectively transduce electrical neuronal activity by employing various neural recording modalities . One of the foremost methods is electrophysiology, which is recogni zed as the gold standard for assessing electrical neuronal activity and facilitating the study of both normal and pathological neuron functions .

[0005] Electrophysiology offers several key advantages when studying the nervous system, including high temporal resolution for capturing rapid neural events , real-time monitoring of neuronal activity, and direct measurement of synaptic events , such as excitatory and inhibitory postsynaptic potentials . However, electrophysiology lacks the ability to record biochemical molecules ( such as neurotransmitters ; NTs ) and to obtain fundamental information to better understand the neurophysiological mechanisms of functions . Neurotransmitters are the body's chemical messengers. These chemical substances are used in the nervous system to transmit messages either between or from neurons to muscles. Neurotransmitters are released at the distal end of the axon of a neuron in response to the arrival of a nerve impulse that, by diffusing a cross a synapse, is capable of transmitting the impulse to e.g., another neuron or a muscle cell. Neurotransmitters include amino acids, small peptides, or derived chemicals, such as acetylcholine, dopamine, gamma- aminobutyric acid, glutamine, serotonin, and norepinephrine.

[0006] Many neurotransmitters are electro-active and are therefore electrochemically detectable, i.e., they can be oxidized or reduced over an electrode surface that generates a quantifiable and dose-dependent electrochemical signal. For example, the neurotransmitter dopamine can undergo 2e~ / 2H+electrochemical oxidation-reduction at an applied potential. The current produced by the transfer of two electrons and release of two protons is proportional to the concentration of dopamine :

[0007] [see Banerjee, S., McCracken, S., Hossain, M. F., & Slaughter, G. 2020. Electrochemical detection of neurotransmitters.

[0008] Biosensors, 10 (8) , 101. https ; / / doi . org / 10.3390 / biosl0080101. ]

[0009] Selective detection of specific neurotransmitters was reported, e.g., by Tavakolian-Ardakani et al. [Tavakolian- Ardakani, Z . , Hosu, 0., Cristea, C., Mazloum-Ardakani , M., & Marrazza, G. 2019. Latest trends in electrochemical sensors for neurotransmitters: A review. Sensors, 19(9) , 2037. https: / / doi.org / 10.3390 / sl9092037.] For example, electrodes were modified with a selective layer of a composite of graphene quantum dots / multiwalled carbon nanotubes (GQDs- MWCNTs) to detect dopamine in human serum and live PC12 cells, with a limit of detection of 0.87 nM [ (Facure, M. H. M., Schneider, R., Lima, J. B. S., Mercante, L. A., & Correa, D. S. 2021. Graphene quantum dots-based nanocomposites applied in electrochemical sensors: A recent survey. Electrochem, 2 (3) , 490-519. https: / / doi.org / 10.3390 / electrochem2030032] . Another study reported an ultrasensitive nonenzymatic electrochemical glutamate sensor fabricated by synthesizing copper oxide nanostructures and combining them with MWCNTs on a screen- printed carbon electrode, demonstrating a 20 pM to 200 pM linear detection response and a limit of detection (LOD) of 17.5 pM [Ali, M. Y., Knight, D., & Howlader, M. M. R. 2023. Nonenzymatic electrochemical glutamate sensor using copper oxide nanomaterials and multiwall carbon nanotubes. Biosensors, 13 (2) , 237. https: / / doi.org / 10.3390 / biosl3020237] .

[0010] A different type of sensor, incorporating both electrophysiological and electrochemical sensing into a single microelectrode array chip was reported in GN 103031246. The chip is shown in Figures 1A and IB appended herein. The chip (1) includes an array of microelectrodes (2) , arranged in a chessboard configuration (8x8) ) , counter electrodes (3) , reference electrodes (4) , electrode leads and contacts. It is stated in GN 103031246 that nerve cells can be cultivated on microelectrodes and that the chip can be used for sensing electrophysiological signals of nerve cells and electrochemical signals of neurotransmitters . However, no experimental work attesting to the survival of neurons on the microchip and detection of their electrochemical activity through sensing of neurotransmitters by e.g. , voltammetry techniques, was reported in GN 103031246. The invention

[0011] An artistic scheme illustrating the invention is shown in Figure 2. A well accommodating neuronal culture medium is placed on a chip. The well is located atop of, and in contact with, an array of microelectrodes (useful designs are shown in Figures 3 and 4) . The array of microelectrodes is used for extracellular recordation of electrophysiological and electrochemical signals from the neuron cells simultaneously. The electrophysiological signals consist of action potentials (a membrane potential versus time plot is shown in Figure 2) . The electrochemical signals (e.g., current versus potential plot, acquirable by voltammetry, as shown in Figure 2) are assigned to neurotransmitters released from the cell. This dual detection allows direct assessment of the coupling between action potentials and neurotransmitter secretion at the synapse. The information gained may serve various purposes, e.g., differentiation between normal coupling observed in healthy neurons, as opposed to states of neurological disorders where the coupling can be disrupted.

[0012] The invention is based on a novel holistic approach that combines 1) in vitro extracellular recording concurrently with 2) electrochemical analysis of multiple redox-active neurotransmitters released by neurons. As shown below, the electrochemical method of choice is preferably based on a high temporal resolution technique (fast-scan cyclic voltammetry; e.g., 8.5 ms transient readings) . This technique was utilized to record signals generated by the neurotransmitters' profile using a suitably designed microfabricated array of microelectrodes. Specifically, electrochemical signals from motor neurons derived from induced pluripotent stem cells that were cultured on the microelectrode array were recorded by voltammetry. The experimental results reported below further show a concentration-dependent increase in the electrochemical signals generated by the neurons upon chemical stimulation with potassium chloride (KC1; a chemical known to induce depolarization and enhance neuronal firing) . That is, distinct differences in electrochemical responses between stimulant- added neurons and stimulant-free samples were observed, attesting to the efficiency of the microfabricated array of microelectrodes .

[0013] Figures 3A-3B show a preferred design for a microfabricated chip suitable for use according to the invention. On chip (1) , an electrochemical sensor (2) consisting of an array of discshaped microelectrodes (3, 4) is deployed. A counter electrode (3) is encircled by microelectrodes (4) such that the electrochemical sensor possesses rotational, e.g., circular symmetry. Microeletrodes (4) are intended to function as working electrodes, but one of them may be suitably coated to serve as a reference electrode (5) for the electrochemical cell. An electrophysiological electrode (6) is placed adjacent to the electrochemical sensor (2) . The reference electrode needed for the electrophysiological recordings is marked by numeral (7) . The two arcs (8) symbolize the well where a neuronal culture medium is held, in contact with the set of electrodes deployed beneath the medium. Each electrode is connected by a lead (9) to a contact (10) placed at the distal end of a respective lead, at the edge of chip (1) .

[0014] A modified design is shown in Figures 4A, 4B and 4C. It is seen that the major structural motif, namely, an electrochemical sensor (2) possessing rotational symmetry, comprising a central counter microelectrode (3) surrounded by working (4 ) / ref erence (5) microelectrodes that are placed equidistantly from the counter electrode on a perimeter of a circle, is found also in the modified design. But in addition, multiple electrophysiological electrodes (6) are positioned so as to create a second circle around the counter electrode , such that the two circles are arranged concentrically, having the same center, that is occupied by the counter electrode ( 3 ) . The electrodes for electrochemical sensing constitute the inner circle , whereas the recording electrodes for electrophysiology are placed on the perimeter of the outer circle .

[0015] A design possessing rotational symmetry, with all working electrodes located equidistantly from the counter electrodes , allows ef ficient electrochemical sensing . Such design also enables locali zation of the di f ferent types of electrodes in a manner that facilitates the dual detection of electrophysiological and electrochemical signals from the same tiny subarea on the surface of the chip . That is , in the designs shown in Figures 3A-B and 4A-C the electrophysiological and electrochemical electrodes are positioned in close proximity, sharing the same or contiguous recording sites . Because the same neuron ( s ) are in the vicinity of both types of electrodes , it is possible to pick up action potentials from neuron ( s ) and simultaneously detect the neurotransmitters released from the axon of the same neuron ( s ) .

[0016] Accordingly, the invention is primarily directed to a chip for electrochemical and electrophysiological measurements on neuronal medium, comprising : a base substrate and at least one array of microelectrodes fabricated on the base substrate , wherein the array comprises : an electrochemical sensing subarray which includes a counter microelectrode positioned in the center of the subarray and multiple working microelectrodes placed essentially equidistantly from the counter microelectrode , lying, for example , on a polygonal line or on a circumference of an arc or a circle , wherein one or more of the working microelectrodes is ( are ) optionally surface-modi fied, with one or more reference microelectrodes optionally lying on said polygonal line , or on a circumference of an arc or a circle ; an electrophysiology recording subarray which includes at least one microelectrode for in vitro extracellular recording, positioned in close proximity to the electrochemical sensing subarray, and a reference electrode for the electrophysiological electrical circuit ; leads extending from the electrodes to connect the electrodes to contacts placed at the distal end of each respective lead .

[0017] In a preferred design shown in Figure 4C, the electrochemical sensing subarray possesses rotational symmetry, i . e . , the working microelectrodes ( 4 ) lie on a circumference of a regular polygon, e . g . , hexagon, with circumradius Ri, or a circle with radius ri . It is also seen that the electrophysiology recording subarray includes multiple recording microelectrodes that are located essentially equidistantly from the counter microelectrode of the electrochemical subarray, lying on the circumference of a regular polygon with circumradius R2 ( R2>RI ) , or a circle with radius ( r2>ri ) . That is , an individual array can combine the electrochemical sensing subarray and the electrophysiology recording subarray by placing the corresponding microelectrodes on the perimeters of two concentric regular polygons or circles . The common center is occupied by the counter electrode of the electrochemical sensing subarray . Owing to this configuration, the array of microelectrodes possesses rotational / circular symmetry ( the axis of rotation is defined by the counter electrode 3 ) . Slight deviations from rotational symmetry (embodied in the term "essentially equidistantly") is possible, as the radial distances measured from the center of the counter electrode to the individual electrodes may show variation of up to 10%., or arrangement of the microelectrodes on the perimeter of an oval or an ellipse.

[0018] In the electrochemical sensing subarray, the working microelectrodes are preferably equal circular discs with diameter in the range from 5 to 15pm, e.g., 7 to 13 pm, e.g., ~10pm. The reference microelectrode corresponds in size and shape to the working microelectrodes. Alternatively, a reference electrode externally to the subarray, immersed in the medium, can be used. The counter microelectrode also has the shape of a circular disc, but its diameter is at least twice as large, e.g., >20pm, e.g., from 20 to 40pm, e.g., from 25 to 35pm, ~30 pm. The total number of working microelectrodes and reference microelectrode patterned in an individual electrochemical sensing subarray (this number is denoted ni) is preferably at least 5, e.g., 5<ni<10, 5<ni<7. That is, ni circular discs are placed essentially equidistantly from the counter microelectrode so that their centers lie on the vertices of ni-polygon with circumradius Ri, or the circumference of a circle with radius ri, wherein Ri and ri, which designate the distance between the center of the counter electrode and the centers of the working / ref erence microelectrodes is from 40 to 60 pm, e.g., ~ 50 pm. The ni circular discs are preferably regularly spaced on the ni- polygonal line or the circle with radius ri.

[0019] In the electrophysiology recording subarray, the recording microelectrodes are preferably equal circular discs, with diameter comparable to that of the counter electrode, namely, >20pm, e.g., from 20 to 40pm, e.g., from 25 to 35pm, ~30 pm. The total number of electrophysiological recording microelectrodes patterned in an individual subarray (this number is denoted by the letter 112) is preferably at least 3, e.g., 5<n2^10, e.g., 5<n2^7. That is, 112 circular discs are placed essentially equidistantly from the counter microelectrode so that their centers lie on the vertices of n- polygon of circumradius R2, or the circumference of a circle with radius r2, wherein R2 and r2, which designate the distance between the center of the counter electrode and the centers of the electrophysiological recording microelectrodes, is from 100 to 140 pm, e.g., ~ 120 pm. The 112 circular discs are preferably regularly spaced on the n2-polygonal line or the circle with radius r2. ni and n2 are preferably equal, for example, ni=n2=5, 6 or 7 (in the specific design shown in Figure 3, ni=n2=6) . It should be noted that the working microelectrodes and the electrophysiology recording microelectrodes are angularly separated, namely, neighboring microelectrodes of the two subarrays do not lie in the same radial direction. The diameter of an individual array of microelectrodes (consisting of both subarrays in a concentric design) is usually from 200 to 300 pm e.g., ~250 pm.

[0020] Figure 4C shows the design of an individual array of microelectrodes with rotational symmetry as described in detail above. Deployment of multiple arrays (e.g., three) on the surface of the chip is shown in Figure 4B. The arrays are essentially tangentially positioned, i.e., imaginary lines drawn around each individual array form three mutually tangent circles. As shown in Figure 4A, the proposed design enables the positioning of the respective contacts (10) on three different sides of the rectangular chip (each contact is spaced 2-3 mm, e.g., ~2.54 mm from the adjacent contact) , so connections to the chip are made on its edges, with a bundle of leads (9) extending from each of the three mutually tangential arrays to three sides of the chip. The leads (9) and contacts (10) are usually made of the same metal like the microelectrodes. The width of a lead is from 10-200 pm (increases as the lead approaches the contact) .

[0021] The microfabrication of the electrodes array on the chip involves techniques such as photolithography and etching. The chip is usually made of transparent material, e.g., glass such as borosilicate or quartz glass, polycarbonate and polydimethylsiloxane. The electrodes are usually placed atop of an adhesion layer which is applied onto the chip after its surface was thoroughly cleaned. The chip (1) is usually rectangular or square with side length in the range from 1 to 10 cm and thickness of ~ 0.5 to 1 mm.

[0022] The counter and working microelectrodes (3, 4) are usually made of noble metals, e.g., gold, platinum, rhodium and iridium, but other types of electrodes, such as carbon electrodes (chiefly glassy carbon electrodes) , can be used. The working microelectrodes can be surface modified, i.e., they can be coated with thin conductive films as described in detail below. The reference electrode, when incorporated alongside the working microelectrodes, is made of the same metal / carbon, but is coated with Ag / AgCl, i.e., an electroplated Ag / AgCl reference microelectrode is used as (5) .

[0023] The electrophysiological recording microelectrodes (6) are made of the same noble metals mentioned above, steel or tungsten but are used in a bare (uncoated) form. The reference electrode (7) is placed on the "free" area of the chip as shown in Figure 4A and has a large surface area to prevent artifacts in the recorded signal due to increased impedance. The microfabrication of the chip of the invention can be accomplished by photolithography techniques, described, for example, in a review paper by Huang et al. [" Microelectrode Arrays for Electrochemistry: Approaches for Fabrication" Small 2009, 5, No. 7, 776-788] . A photolithography process can include the following steps. The base substrate is cleaned, a first photoresist is applied (either negative or positive) , e.g., by spin coating, spray coating or dip coating, to produce a thin uniform layer on the substrate, followed by soft baking. A first mask is aligned, to transfer the pattern corresponding to electrodes' sites onto the surface of the substrate. The photoresist is exposed through the pattern on the mask with UV light, followed by a development step. Next, bare microelectrodes are deposited in the intended sites, e.g., first an adhesion layer (e.g., made of titanium) and then electrode material (e.g., gold) followed by lift-off procedure that results in a gold microelectrode array on glass substrate. In order to define the electrode effective surface area, another lithography step is done using, e.g., SU-3005 photoresist (to create an insulting coating that is patterned with another suitable mask to expose only the active area of the electrodes) . See the experimental section below for complete protocols of microfabrication of different array configurations .

[0024] The system described herein can measure the overall neuronal electrochemical activity, i.e. , using bare electrodes . But the array of macroelectrodes can be turned into an electrochemical tongue, by suitably modifying the surface of the working microelectrodes, to enable detection of individual neurotransmitters with the aid of chemometric models. That is, having patterned the microelectrodes on the substrate, coatings can be applied on the working microelectrodes in the electrochemical subarray, for example, by electrodeposition.

[0025] In general, formation of a coating onto the surface of a microelectrode can be accomplished from a deposition solution by the following electrodeposition techniques:

[0026] (i) galvanostatic electrodeposition (chronopotentiometry) , in which a constant current is passed through the microelectrode ( s ) to be coated;

[0027] (ii) potentiostatic electrodeposition (chronoamperometry) , in which a constant potential is applied on the working microelectrode ( s ) to be coated; or

[0028] (iii) cyclic voltammetry electrodeposition.

[0029] When on-chip reference Ag / AgCl microelectrode ( s ) are desired, then it is better to start the surface modification of the multielectrode array with the production of these reference microelectrodes, i.e., by creation of Ag / AgCl coatings onto one or more microelectrodes. This is achieved via a two-step process; 1) Ag electroplating, and 2) Ag anodization in a chloride solution.

[0030] In the first step, a suitable Ag electrodeposition solution is prepared by dissolving in water a silver source (e.g., AgNCh or Ag2SO4, at a concentration from 3 to 10% by weight) . Stabilizers such as piperazine which prevent the silver ions from forming nanoparticles can also be added. The solution is made somewhat alkaline (e.g., 8<pH<10) by addition of a weak base such as ammonium hydroxide. The electrodeposition is driven effectively in a continuously stirred Ag+deposition solution (agitation rate is from 50 to 400 RPM) , using a two- electrode cell configuration, with the application of a constant current from a DC source or a galvanostat. A cathodic current , fixed in the range from 0 . 1 to 100 A im2, is passed between the electrodes . In case that more than one reference microelectrode is sought to be included in the array, then these microelectrodes are connected simultaneously to the counter electrode to become electroplated with silver (Ag° ) layer by the reduction of Ag+from the solution . In general , the electroplating lasts a few minutes , usually not more than fi fteen minutes , depending on the concentration of the deposition solution, agitation rate , etc .

[0031] In the second step, about quarter to third of the amount electrodeposited metallic silver on the microelectrode is converted into AgCl ( s ) through electrochemical anodi zation of the silver-coated microelectrodes in a continuously stirred aqueous chloride solution such as hydrochloric acid or sodium chloride , at a constant voltage . A three-electrode configuration can be used, which includes the silver-coated microelectrodes as working electrode ( s ) , Ag / AgCl as reference electrode , and a ring or wire Pt counter electrode . A fixed voltage in the range from 0 to 0 . 5 V, for example , about 0 . 2V (vs Ag / AgCl ) is applied to the Ag-coated microelectrodes over five to fi fteen minutes . Through this two-step process , two or more Ag / AgCl reference microelectrodes are incorporated into the multielectrode array .

[0032] Next , the working microelectrodes are modi fied to create the desired coatings onto their surface ( each subgroup of working microelectrodes possesses the same coating) . The order of generation of the coatings is according to the pH of the deposition solution, from the lowest pH coating solution to highest pH coating solution . A reverse order of coatings ' formation is undesired because strongly acidic coating solutions may af fect previously prepared coatings . Platinum black film can be generated via galvanostatic electrodeposition onto one or more microelectrodes, by passing a constant current (a cathodic current, with current density fixed in the range of 4 to 6 mA / cm2, for 3 to 7 minutes; for example, a current density of 5 mA / cm2is supplied over five minutes) , through a deposition solution in which a suitable platinum source is dissolved, e.g., by electrochemical reduction of chloroplatinic acid dissolved in DI water at a concentration in the range of l% (v / v in water) to 3% (v / v in water) , in the presence of about 0.05% (v / v in water) of lead acetate. Lead acetate enhances the electrode reaction (i.e. the reduction of Pt) in presence of platinum black solution and it also strengthens the adhesion of the coating to the electrode. The pH of the deposition solution is shifted to the strongly acidic by addition of hydrochloric acid. A two-electrode configuration can be used, which includes the microelectrode to be coated as working electrode and a ring or wire Pt counter electrode.

[0033] Another type of film-forming material that is applied to create film-coated microelectrode ( s ) in the chip of the invention is reduced graphene oxide. The deposition solution is prepared by known methods, e.g., the Hummers' method, where oxidation of graphite flakes or powder takes place upon adding the graphite to a cold solution of sulfuric acid (e.g., 0°C) followed by gradual addition of sodium nitrate and potassium permanganate under continuous stirring. For example, on a laboratory scale, the addition time of each of the successively added NaNOs and KMNO4 reagents is not less than ten to fifteen minutes. On completion of reagent's addition, the reaction mixture is heated to about 35-45°C and kept under stirring for a couple of hours, e.g., not less than two hours. The reaction is terminated by addition of water and hydrogen peroxide which removes excess permanganate. The graphene oxide is recovered by centrifugation and freeze dried and used to prepare deposition solution with concentrations in the range from 0.1 to 0.9 mg / ml GO. A deposition solution can also be prepared by a modified Hammers procedure, which consists of adding the graphite powder (or flakes) to a mixed sulfuric acid / phosphoric acid solution (e.g., proportioned about 9:1 by volume) , followed by the slow addition of KMnO4. The mixture is kept under stirring for couple of hours at a slightly elevated temperature (at 30-35 °C) until the mixture acquires a dark green color. Termination of the reaction is achieved by slow addition of H2O2 aqueous solution (e.g., the commercial 30% w / w solution) . Graphene oxide is recovered through acidification of the mixture by hydrochloric acid (e.g., addition of commercial 32% HC1 solution and DI) , centrifugation of the resulting solution, washing of the supernatant with HCl / water, drying of the washed solution (e.g., at 90 °C in an oven) and collecting the GO powder. The dried GO powder is dissolved in DI, usually up to concentration of 0.5 g / L GO concentration. Addition of an electrolyte to the GO solution affords the GO electrodeposition solution. Next, r-GO is obtained electrochemically from the GO solution onto the microelectrode (Au) surface, using cyclic voltammetry electrodeposition, in a three-electrode cell configuration consisting of the microelectrode ( s ) as working electrode ( s ) ; an externally applied Pt wire as counter electrode and Ag / AgCl as reference electrode. A potential window, for example from -1.4. to 1.4V (versus Ag / AgCl) is scanned at rate of in the range of to 50 to 500 mV / s, with number of cycles varying from 1 to 5.

[0034] Electrodeposition of M0S2 and WS2 films on the surface of one or more microelectrode ( s ) can be carried out by cyclic voltammetry, using a three-electrode configuration, which includes the microelectrode ( s ) to be coated as working electrode ( s ) , Ag / AgCl as reference electrode and a ring or wire Pt counter electrode, by scanning the potential window of 0 to 1.4 V (versus Ag / AgCl) at rate of 50 to 100 mV / s, with number of cycles varying from 10 to 20. Suitable deposition solutions include 1-2 mg / mL M0S2 or WS2 dissolved in 0. IM sulfuric acid solution. See also WO 2022 / 137236 for M0S2 and WS2 electrodeposition.

[0035] Electrodeposited chitosan film-coated microelectrode can be prepared with the aid of a deposition solution with chitosan concentration in the range from 0.5 to 2 wt%, preferably from 0.8 to 1.2 wt%, prepared by dissolving chitosan in a strongly acidic environment, whereby the amino groups undergo protonation to reach a slightly acidic pH (5-6) . As pointed out above, conductive additives can be included in the deposition solution; these additives will co-deposit and affect the film properties. The concentration of the additives in the deposition solution (e.g., carbon nanotubes (abbreviated herein CNT) , gold nanoparticles and platinum nanoparticles) is in the range from 0.1 to 2 %, preferably from 0.8 to 1.8 wt . % . For example, chitosan-CNT electrodeposition solution can be prepared by mixing a chitosan solution as previously described with CNTs, followed by ultra-sonication. The arrayed chip is immersed in the chitosan deposition solution (or chitosan / CNT solution) and electrodeposition is achieved by the chronopotentiometry technique, i.e., selected microelectrodes to be coated are biased to the negative potential against a counter electrode with constant (cathodic) current being applied between the electrodes for a period of time of 0.5 to 5 min, supplied by a DC current source; typically the current is set in the range from 3 to 6 pA / cm2. A two-electrode configuration can be used, i.e., the counter electrode is shorted to reference terminal. Weakly bound chitosan is removed from the microelectrode surface, by immersing the device in a buffer solution.

[0036] Typical thicknesses of the films produced (can be measured by atomic force microscopy or prof ilometry ) are: polysaccharide (e.g., chitosan) film: from 5 to 20 pm; polysaccharide (e.g., chitosan) film with conductive additives: from 5 to 60 pm; platinum black film: from 6 to 10 pm; reduced graphene oxide film: 350 to 550 nm;

[0037] M0S2 film: 10 to 20 nm (very thin nanoflakes) ; and WS2 film: 10 to 20 nm (very thin nanoflakes) .

[0038] Upon completion of the surface modification of the working microelectrodes in the electrochemical sensing subarray by the various electrodeposition techniques set out above, the chip device is rinsed to remove non-deposited material and is ready for use. It should be noted that one or more bare electrodes may remain in the electrochemical subarray, i.e., not all the working microelectrodes are surface modified. The electrophysiological recording microelectrodes that are patterned on the chip are also used as is, i.e., uncoated (e.g., the electrophysiological recording microelectrodes are bare metal (e.g., gold) electrodes or coated with suitable materials to enhance the attachment of the cells as described below .

[0039] Experimental results reported below show the good electrochemical performance of the microelectrodes patterned on a chip by photolithography. The electrochemical performance was measured by cyclic voltammetry in ferrocyanide / ferricyanide redox couple [Fe(CN)63~ Fe (CN) 64~] solution - a benchmark frequently used to assess the acceptability of microfabricated electrodes. The results indicate that the working microelectrodes and the counter microelectrode ( i . e . , the components of the electrochemical sensing subarray) , and the electrophysiological recording electrode ( in the electrophys iological subarray) exhibit acceptable vo It ammo grams . For example , voltammograms recorded for the working microelectrodes show well-defined anodic and cathodic peaks , and anodic-to-cathodic peak current ratio close to 1 . 0 , attesting to the reversibility of the redox process over the microfabricated electrodes .

[0040] In addition, experimental work reported below shows that not only does the microchip sensor of the invention able to support the viability of neuronal cells thereon, but also their complex electrochemical action . Speci fically, detectability of electro-active neurotransmitters generated by acetyl cholinergic neurons derived from Human Induced Pluripotent Stem Cells (hiPSCs ) was investigated . To this end, cholinergic neurons were di f ferentiated from hiPSCs through a three-stage protocol comprising neuroepithelial induction, motor neuron precursor generation on laminin-coated plates , and maturation into induced motor neurons ( iMNs ) . A microfabricated electrochemical sensing subarray with rotational symmetry as previously described ( albeit with electrodes of larger dimension) was used to record electrochemical signals near the iMNs .

[0041] To record the signals , the fast- scan cyclic voltammetry ( FSCV) technique was employed with a scan rate of 400 V s-1, corresponding to a frequency of approximately 235 Hz ( 8 . 5 ms ) , a technique chosen for its high temporal resolution and sensitivity in detecting rapid NT dynamics . To test the performance of the sensor, the chemical stimulant potassium chloride was added to the cells ( expected to increase the secretion rate of NTs ) , and the generated electrochemical signals were recorded . The results reported below show an enhancement in the electrochemical activity of the neurons with increasing concentration of the KC1 stimulant . For example , in a set of experiments reported below, voltammograms were recorded from neurons cultured on the microelectrode array . That is , from neurons alone , and from neurons to which the stimulant KC1 was added at concentrations of 30 mM and 90 mM . The voltammograms indicate that the higher the concentration of the stimulant in the medium, the higher the electrochemical currents . Charge trans fer associated with the voltammograms was measured and results are shown in a tabular form below :

[0042] *p-value < 0 . 05 compared with the neurons without the stimulant

[0043] * *p-value < 0 . 01 compared with the neurons without the stimulant

[0044] The charge versus time analysis revealed that neuronal electrochemical activity increases with increased KC1 concentrations . With 30 mM, the charge increases by 18 . 6% , and with 90 mM KC1 , the charge increases by 35 . 8 % compared with the cell without stimulant condition; the latter produced the most signi ficant electrochemical response . The overall duration of the reduction and oxidation reactions is 8 . 5 ms , corresponding to the typical time for neuronal firing and neurotransmitter release.

[0045] Accordingly, another aspect of the invention is a method of simultaneous electrophysiological and electrochemical analysis of neuronal activity, comprising the steps of : culturing neuron cells on a microelectrode array; and extracellularly recording action potentials while concurrently measuring redox reaction (s) of neurotransmitter ( s ) released by said neuron cells with the aid of fast-scan cyclic voltammetry .

[0046] The array of microelectrodes for use in the method of the invention comprises a subarray of electrophysiological microelectrodes and a subarray of electrochemical microelectrodes that are arranged in a concentric fashion, with a counter electrode occupying the common center, such that the same neuron (s) are in the vicinity of both types of microelectrodes. Preferably, the microelectrode array is fabricated on a base substrate in the form of a chip, as described in detail above.

[0047] Voltammetry, namely, FSCV, is recorded over bare working microelectrodes, to determine the overall neuronal electrochemical activity. Alternatively, voltammetry, e.g., FSCV, is recorded over surface-modified working microelectrodes to detect multiple redox-active neurotransmitters in a discriminant manner and identify individual neurotransmitters.

[0048] To cultivate neuron cells on the chip, protocols such as those described in Vatine et al., 2017 PMID: 28526555 (Cell Stem Cell 20, 831-843. el-e5, June 1, 2017) and Ben-Zvi et al., 2022 PMID: 36555735 (Int. J. Mol. Sci. 2022, 23, 16092. https: / / doi.org / 10.3390 / ijms232416092) can be used. To facilitate attachment of the cells onto the surface of the chip, adhesion layers such as Polyethylenimine (PEI) followed by laminin, can be applied to the surface. Neuron cells that can be analyzed by the method of the invention include Primary rodent neural cells, or stem cell-derived human neural cells. For example, the neurons are derived from induced pluripotent stem cell-derived neurons, specifically spinal motor neurons or cortical neurons. Figure 8C shows the chip, with a well placed on the chip to allow sufficient medium to feed the cells .

[0049] After the cells are attached and cultured on the chip, the electrochemical activity of the neurons in the cell medium is recorded using the FSCV technique. In operation, the microelectrodes in the electrochemical subarray are electrically connected to potentiostat to vary the potential of the working microelectrodes and create a data set of electrochemical signals when the microelectrodes are in contact with the neuronal medium.

[0050] For example, beginning at an initial potential higher than -0.7V, versus RE (Ag / AgCl) , for example, Einitiai = -0.1 V versus RE (Ag / AgCl) , the potential is swept at a constant scan rate of not less than 100 V s-1, e.g., >200 V s-1, >300 V s-1, preferably from 350 to 450 V s-1, e.g., more preferably around 400 V s-1, over the potential range from Ei=-0.4V versus RE, to £2= 1.3V versus RE (the switching potentials) , conducting two or more cycles. The current passing at each of the working microelectrodes is measured across the potential range, to create the voltammograms .

[0051] Multiple bare working microelectrodes can be used for repeatability. In case of surface-modified microelectrodes, the data set of electrochemical signals, i.e., the voltammograms recorded by FSCV, is analyzed by a processor applying one or more chemometric techniques as described, for example, in Polymers 2022, 14, 717 ( https: / / doi.org / 10.3390 / polyml4040717 or https : / / www .mdpi . com / 2073-4360 / 14 / 4 / 717, WO 2018 / 225058 and

[0052] US 2023 / 0104086. To record action potentials , an electrophysiological system is evaluated and activated within an in vitro multichannel setup (MEA2100 ) . Electrophysiological signals are acquired and can be analyzed using software tools provided by the multichannel system, including the Multichannel Data Manager, Multichannel Analyzer, and Multichannel Experimenter .

[0053] In summary, the micro-system of the invention enables realtime , in-situ, high-resolution monitoring of ( 1 ) multiple neurotransmitters and ( 2 ) action potentials simultaneously . The results reported below demonstrate the potential for studying neurochemical communication and thereby advancing personalized therapies for neurological disorders . By enabling in si tu neurotransmitter profiling from patient-derived cells , of fering valuable insights into patient-speci fic diagnostics and treatment strategies . Speci fically, the proposed sensor enables a personali zed detection of neurological disorders , including Parkinson ' s disease and the rare neurological disorder Syntaxin Binding Protein 1 ( STXBP1 ) encephalopathy .

[0054] The non-invasive nature of the sensor makes it a versatile tool for diagnosing neurodegenerative diseases , evaluating drug ef ficacy, and advancing the understanding of neuronal function . Notably, the proposed sensor of the invention holds promise for personali zed diagnostics , enabling tailored treatments for neurological disorders by assessing neurotransmitter release dynamics in patient-speci fic models . Utili zing the proposed sensor for neuron behavior studies enables the exploration of electrochemical responses in healthy and pathological neuronal states , as well as across di f ferent neuron subtypes , to further validate neurotransmitter-related fundamental metabolic pathways . Integrating this technology into advanced diagnostic and therapeutic platforms could open new frontiers in neuroscience and neuropharmacology . In the drawings

[0055] Figures 1A-1B show a microelectrode array chip for multiparameter detection of nerve cells (prior art, CN 103031246) .

[0056] Figure 2 is an artistic scheme illustrating the invention.

[0057] Figures 3A-3B show a preferred microelectrode array design possessing rotational symmetry (multiple electrochemical electrodes and a single electrophysiological recording electrode) .

[0058] Figures 4A-4C show a preferred microelectrode array design possessing rotational symmetry (multiple electrochemical electrodes and multiple electrophysiological recording electrodes) .

[0059] Figures 5A-5C: (A) Microfabrication of the array of gold disk microelectrodes using photolithography and thin film deposition techniques. (I) Borosilicate glass substrate cleaning, (II) AZ-5214 negative photoresist coating, (III) 1stmask exposure, (IV) Titanium and gold deposition onto the patterned wafer, (V) Photoresist removal leaving the deposited metals in the desired pattern ( 'lift-off process' ) , (VI) SU-8 3005 negative photoresist coating, (VII) 2ndmask exposure, and (VIII) Photoresist development to create a pattern that reveals only the active surface area of the gold electrodes, followed by a hard bake to remove any residues or impurities on the substrate. (B) Drawings of the designed photomask with multiple microelectrode arrays on a single wafer, a closeup drawing of a single array of microelectrodes and an inside view of the electrochemically active area of the array containing 11 microelectrodes and a single counter electrode. (C) A photo of a microchip with gold microelectrodes patterned thereon, one of which is electroplated Ag / AgCl reference electrode . Figures 6A-6C: Electrochemical validation of the fabricated microelectrode array microchip of Example Al. (A) Recorded cyclic voltammograms at a scan rate of 0.1 V s-1from an 11- gold microelectrode array chip in the presence of a 5 mM ferrocyanide / ferricyanide redox couple solution. (B) An average cyclic voltammogram derived from the 11-microelectrode array. (C) Electrochemical validation of the fabricated microelectrode array microchip of Example A2 : cyclic voltammogram recorded from Ag / AgCl microelectrode patterned.

[0060] Figures 7A-7B: Electrochemical characterization of the exposed surface area of the gold microelectrode. (A) Recorded and the average cyclic voltammograms at increasing scan rates (0.1 [solid circle blue] , 0.15 [dashed cross orange] , 0.2 [dash dot square yellow] , 0.25 [dotted diamond purple] , and 0.3 [solid upward triangle green] V / s) in the presence of a 5 mM ferricyanide / ferrocyanide solution. (B) The dependence of the anodic (black circles) and the cathodic (red squares) peak currents on the square root of the scan rate (R2= 0.999) .

[0061] Figures 8A-8C: Motor neurons growing on the microelectrode array microchip. (A) Micrograph of neurons cultured on a plastic dish on day 35. (B) Micrograph of neurons cultured on the microelectrodes ' array chip on day 37 (C) Micrograph of a chip with the well placed thereon.

[0062] Figures 9A-9B: Electrochemical signals recorded in the presence and the absence of neuronal cells. (A) Average voltammograms recorded from medium in the presence (full red) and the absence (dashed black) of neurons. (B) Average total electrochemical charge calculated from the voltammograms of medium in the presence and the absence of neurons. Figures 10A-10C: Average total electrochemical charge recorded for 8.5 ms from the medium in the presence (dashed black) and the absence (full red) of neurons.

[0063] Figures 11A-11B: The effect of chemical stimulation on the electrochemical signals recorded from neuronal cells. (A) Average voltammograms recorded from neurons in the absence of a stimulant (solid red) and in the presence of either 30 mM (dashed black) or 90 mM (dotted blue) KC1 stimulant. (B)

[0064] Average total electrochemical charge calculated from the voltammograms of neurons in the absence of a stimulant and in the presence of either 30 mM or 90 mM KC1 stimulant (p < 0.05 and p < 0.01) .

[0065] Figures 12A-12C: Average (A) anodic, (B) cathodic and (C) total electrochemical charge recorded over 8.5 ms from neurons in the absence of a stimulant (solid red) and in the presence of either 30 mM (dashed black) or 90 mM (dotted blue) KC1 stimulant .

[0066] Figures 13A-13B: (A) cyclic voltammograms recorded at a scan rate of 0.1 V / s for the counter and electrophysiological electrodes in the presence of 5 mM ferrocyanide / ferricyanide solution (B) Cyclic voltammograms recorded at a scan rate of 0.1 V / s from the 10 gold working electrodes in the presence of 5 mM ferrocyanide / ferricyanide solution.

[0067] Examples

[0068] Data recording and analysis: all electrochemical measurements were performed using a PalmSens4 and MUX16 multiplexer plug-in (PalmSens, Ltd.) . The data were analyzed using MATLAB MathWorks R2018a. Chemicals: Methanol (001368052100, Bio-Lab, Ltd.) , acetone (376, Bio-Lab, Ltd.) , potassium chloride (11595, Alfa Aesar) , and 2-propanol (1301221, BioLab, Ltd.) , sulfuric acid (258105, Bio-Lab, Ltd.) , hydrogen peroxide (1.07210.1000, Merck) , potassium hexacyanof errate ( 11 ) trihydrate ( 'Ferrocyanide' , 1.04984.0100, Merck) , and potassium hexacyanoferrate (HI) ( 'Ferricyanide' , 1.04973.0100, Merck) . Deionized water ( 'DI' ; resistivity > 18 MQ) was obtained from a Super Q water system (Millipore) . Phosphate buffered saline (PBS) tablets (BP2944- 100, ThermoFisher) were used to prepare a 0.01M PBS solution. Biological materials for neuronal differentiation: IMDM (12440061, LifeTech) , F12 (11765062, LifeTech) , B27 (+vitamin A) (17504044, LifeTech) N2 (1780240, LifeTech) , NEAA (1114050, Gibco) , GlutaMax (35050061, LifeTech) , Antibiotic antimycotic (15240062, LifeTech) , CHIR99021 (13122, Cayman Chemicals) , LDN193189 (S2618, Selleck Direct) , SB431542 (1614, Tocris) , SAG (Sonic Hedgehog Agonist) (11914, Cayman Chemicals) , AllTrans Retinoic Acid (040021, Stemgent) , BDNF (45002, Peprotech) , GDNF (45010, Peprotech) Ascorbic acid (A4403, Sigma) , Compund E (565790, Calbiochem) , DAPT (13917, Cayman Chemicals) , db-cAMP (28745, Millipore) , Accutase (SCR005, Millipore) , Laminin (Mouse) (L2020, Sigma) , Matrigel (Growth factor reduced) (354230, Corning) , PolyHEMA (P3932, SIGMA) , NutriStem hESC XF medium (05-200-1A , Sartorius) 6 well plates (0877233, Fisher) , and pre-separation filters 30uM (130041407, Miltenyi Biotec (Macs) ) . The stem cells utilized in this experiment were healthy iPSCs obtained from the Regenerative Medicine and Stem Cells (RMSC) Research Center, Ben-Gurion University of the Negev (BGU) . Example 1

[0069] Fabrication of microelectrode array microchip and validation by cyclic voltammetry

[0070] Al: microelectrode array (working and counter electrodes)

[0071] A photomask was created using a Clewin4 software (version 4.3.6.0, WieWeb) design utilizing a chip that included an array of 11 working microelectrodes, each with a diameter of 100 pm, along with a counter electrode with a diameter of 1.5 mm. The fabrication of the chip utilized a photolithography lift-off process shown in Figure 5A. It began with wafer preparation, where the glass substrate (prime grade, wafer diameter 100 mm, wafer thickness 500 pm, and double side polished, University Wafer, Ltd.) was thoroughly cleaned to ensure a contaminant-free surface, needed for proper photoresist adhesion. The glass substrate was cleaned with a 'piranha' solution (a 1:3 ratio mixture of hydrogen peroxide and sulfuric acid, HMxSquare SUSS MicroTec system) , followed by dehydration on a contact hot plate at 120°C for 10 min. The cleaned substrate was then left at room temperature for 10 min to cool down. A negative photoresist AZ-5214 (product no. 97, Micro chemicals) was spin-coated (80RCDelta, Universal Spin- Coating system, SUSS MicroTec) onto the glass wafer (2,200 RPM for 12 s at an acceleration rate of 800 RPM s-1) using a vacuum chuck, which spun the wafer to form a uniform layer of photoresist. The coated substrate was left at room temperature in the spinner chamber for 5 min to settle down, followed by a 'soft bake' step on the contact hot plate (110°C for 2.5 min) . Next, the substrate was cooled down to room temperature for 10 min, followed by exposure to a transparency mask (a light flux of 7.6 mW cm-2for 65 s; Karl Suss Mask Aligner MA6 system, SUSS MicroTec) . A 'post-exposure bake' was performed on the contact hot plate (120°C for 2.5 min) , and the substrate was left to cool down to room temperature for 10 min. A flood exposure step was performed (with a light flux of 7.6 mW cm-2) , and the exposed substrate was developed (AZ 726 MIF developer, Micro Chemicals) for 6 min, then rinsed with distilled water for 5 min, and dried with nitrogen gas, followed by oxygen plasma cleaning for 30 s. Next, 20 nm-thick titanium and 200 nm-thick gold layers were evaporated onto the developed substrate using an E-gun deposition system (VST Service, Ltd.) . The substrate was then dipped in an acetone solution for 60 min, followed by rinsing with distilled water and drying with nitrogen gas.

[0072] SU8-3005 (STS-Science Technology and Services Ltd) was used to determine the microelectrode array chamber; this allows the microelectrode array to be cleaned with an AMI (acetone, methanol, and isopropanol rinsing) cleaning protocol without destroying the chamber before its use. First, SU8-3005 was spun-coated at 3000 RPM for 30 s, followed by a soft bake on a hot plate at 95°C for 15 min. Next, the photoresist was exposed to light using a hard contact mode of 7.6 mW cur2for 50 s at a Mask Aligner (MA6, SUSS MicroTec) . Then, a postexposer bake was conducted for 5 min at 95°C. The exposed wafer was then developed in PGMA ERB developer solution for 8 min and washed in isopropanol for 10 s. The hard bake on a contact hot plate at 150°C for 5 min was carried out to remove any hydration on the substrate, and oxygen plasma cleaning (30W, 500 mTorr, 2 min, and 3 seem) was used after the hard bake to remove any residues or impurities on the substrate. Finally, the fabricated substrate (Figure 5B) was diced into single microchamber chips (Dicer ADT-7100, ADT) .

[0073] Prior to electrochemical testing, the microfabricated chips were sequentially cleaned by AMI solutions for 5 min in each solution. The chips were then rinsed with double-distilled water (DDW) and dried using nitrogen gas. Next, the chips were immersed in a mild piranha solution (7:1 mixture of concentrated sulfuric acid and hydrogen peroxide and left to react for 30 min) for 1 min, followed by a thorough rinse with DDW followed by drying with nitrogen gas.

[0074] A2 : microelectrode array (working, counter and reference electrodes)

[0075] The procedure of Part Al was repeated, but this time a total of 12 microelectrodes, each with a diameter of 100 pm, along with a counter electrode with a diameter of 1.5 mm, were patterned on the microchip. One of the microelectrodes was converted into a reference electrode by electroplating with Ag / AgCl as previously described (see Example 6 of WO 2022 / 137236) . The microchip is shown in Figure 50, where the Ag / AgCl reference electrode is marked by a red circle.

[0076] Bl : electrochemical validation of the microchip Al

[0077] The electrochemical activity of the microelectrode array was determined by recording cyclic voltammograms (CV) . The electrochemical cell used for the CV measurements consisted of a three-electrode configuration: the fabricated microelectrode array of Part Al (which includes the working electrodes [WE] and the counter electrode [CE] ) , as well as an externally applied commercial Ag / AgCl reference electrode [RE] (CHI111P, CH Instruments) .

[0078] CV was performed by scanning the initial potential E±=-0.2V versus RE, vertex #1 potential Ei = -0.2V versus RE, vertex #2 potential E2 = 0.65 V versus RE, scan rate = 0.1 V s-1, the number of cycles = 2 using a 5mM solution of the reversible redox pair ferrocyanide / ferricyanide in PBS. This way the Nernstian reversibility characteristics of the electrochemical reaction, indicated by the typical "duck-shaped" voltammogram, can be determined . The voltammograms recorded for each of the eleven working electrodes are shown in Figure 6A. All voltammograms exhibit well-defined anodic and cathodic peaks, indicative of Nernstian behavior. The individual voltammograms were averaged, yielding the representative signal displayed in Figure 6B . Electrochemical performance was calculated by calculating the ratio of the anodic peak current to the cathodic peak current to obtain the average signal. The anodic-to-cathodic peak current ratio was 0.98210.023, attesting to the reversibility of the redox process.

[0079] CV was conducted at scan rates of 0.1, 0.15, 0.2, 0.25, and 0.3 V s-1to determine the effective surface area of the microelectrode array using the Randles-Sevcik equation. This value was then compared to the theoretical diameter using the dimensions from the mask design created in CleWin, where the theoretical diameter of the working electrodes was 100 pm.

[0080] The Randles-Sevcik equation has the following form: where iP(A) is the peak current, (cm2) is the effective electrode surface area, v (V s-1) is the scan rate, F is Faraday's constant (96485.339 C mol-1) , R is the universal gas constant (8.31447 J Kmol-1) , T (K) is the absolute temperature (298.15 K) , C (mol cm-3) is the concentration of the redoxactive species, D (cm2s-1) is the diffusion coefficient of the electroactive species (0.72 x 10-5cm2s-1for ferricyanide and 0.67 x 10-5cm2s-1for ferrocyanide (see Shukla RP, Ben-Yoav H. A. 2019. Adv Healthc Mater. 8 (15) , 31240866. https ; / / dor , org / 10 ■ 1002 / adhm. ) , and n is the number of electrons transferred in the redox reaction. The Randles-

[0081] Sevcik equation shows a linear relationship between the peak current and the square root of the scan rate, and the surface area may be calculated from the slope of the linear relationship .

[0082] The voltammograms recorded for each scan rate are shown in Figure 7A. The anodic and cathodic peak currents are plotted against v0-5in Figure 7B (black (upper ) and red (lower) curves respectively, showing the linear relationship. For the anodic peak current, the slope was found to be 0.327 ± 0.003 pAs°-5V~°-5, whereas the intercept was calculated as 0.026 ± 0.002 pA. In contrast, for the cathodic peak current, the slope was found to be -0.27 ± 0.01 pAs0-5V~°-5, with the intercept at -0.035 ± 0.001 pA. The mean slope obtained from both the anodic and cathodic fits was 0.296 ± 0.005 pAs0-5V~°-5. When normalized by the electrode surface area, this corresponds to 3.89*10~3pAs°-5cm2V~°-5. Based on these findings, the average effective surface area of the working electrode was calculated to be (77.1 ± 1.2) * 10~6cm2with a corresponding diameter of 98.9 ± 0.8 pm (1.1% difference between the theoretical (100 pm) and experimental values) .

[0083] B2 : electrochemical validation of the microchip A2

[0084] The cyclic voltammogram recorded in Figure 6C shows the electrochemical behavior of the patterned Ag / AgCl reference electrode when subjected to a scan rate of 0.1 V / sec within a potential range of -0.2 to 0.65 V. The electrolyte used for the measurements was a 5 mM ferro / ferricyanide solution.

[0085] Example 2

[0086] Electrochemical detection of signals generated by neurons

[0087] Part A: generation of iPSC-derived cholinergic motor neurons The differentiation process began with the culture of hiPSCs, which were expanded on Matrigel for at least two weeks using NutriStem to support optimal growth and expansion. Prior to initiating differentiation, the hiPSCs were plated at a low density on Matrigel-coated plates. Cells were maintained daily and monitored to ensure they reached an ideal confluence of 30-40% at the start of the differentiation process. The first stage in the process involved neuroepithelial induction. Briefly, hiPSCs were cultured in SIM medium for six days to facilitate their differentiation into neuroepithelial cells. The composition of the medium is listed in Table SI:

[0088] Table SI: SIM medium (Stage 1 Medium) - Neuroepithelium Induction Medium (S1M / NIM) day 0-day 6.

[0089] In the second stage, the neuroepithelial cells were further differentiated into motor neuron precursors (MNPCs) , a mixed population of neural stem cells (NSCs) , and neural progenitor cells (NPCs) . The neuroepithelial cells were rinsed once with 1 mL of lx PBS, which was added slowly to avoid cell detachment. Next, 1 mL of Accutase was added per well (6 wells per plate) , and the cells were incubated at 37°C for 5 min. After incubation, PBS was added to each well, and the cells were gently scraped from the surface using a 10 mL serological pipette. The cell suspension was transferred to a 15 mL conical tube and centrifuged at 200 g for 5 min at room temperature. To determine cell density, two samples (10 pL each) were removed for counting. The cell suspension was diluted with 0.04% Trypan Blue in lx PBS to an appropriate dilution factor (i.e., 1:20) . Viable and dead cells were counted using a hemocytometer and the LUNA-FL™ dual fluorescence cell counter (L20001, logobiosystems) . Then the cells were seeded onto plates coated with 1 mg / mL laminin and cultured in S2M medium for six days to facilitate their further differentiation. The composition of the medium is listed in Table S2 :

[0090] Table S2 : S2M medium (Stage 2 Medium) - MN Precursor

[0091] Generation Medium (S2M / MNPM) day 6-day 12. The final stage focused on the maturation of the induced motor neurons (iMNs) . Cells were cultured in S3M medium for an additional six days to promote terminal differentiation and maturation. The composition of the medium is listed in Table S3:

[0092] Table S3: S3M medium (Stage 3 Medium) - Terminal iMN Maturation

[0093] Medium (S3M / TMNMM) day 12-day 18

[0094] On day 35 of the differentiation, the motor neurons were transferred to a microchip substrate coated with polyethyleneimine (PEI) and laminin for neural cell adhesion and growth. Figure 8A shows neurons that differentiated from hiPSCs at day 35 post-differentiation on a plastic surface prior to their transfer onto the microchip. Figure 8B illustrates neurons cultured on the microchip sensor. The successful survival of neurons on the microchip sensor attests to biocompatibility of the electrode environment with neuronal maintenance and health.

[0095] Part B: Recording the electrochemical signals generated from neurons

[0096] After the cells were cultured on the chip, the electrochemical activity of the neurons in the cell medium was recorded using the FSCV technique with a scanning initial potential Ei=-0.1V versus RE, vertex #1 potential Ei = -0.4V versus RE, vertex #2 potential £2= 1.3V versus RE, scan rate= 400 V s-1, and the number of cycles=2. A three-electrode cell configuration consisting of the microfabricated electrodes (i.e., the array of working electrodes and a counter electrode, labeled Al above) , as well as an externally applied commercial Ag / AgCl electrode (CHI111P, CH Instruments, reference electrode) . Figure 8C shows the chip, with a well placed on and fixed to the chip.

[0097] Figure 9A shows the average electrochemical signals obtained from neurons cultured on the chip sensor (red voltammogram) , along with the average control signal obtained from measurements conducted in the absence of cells (control: growth medium without cells, black voltammogram) .

[0098] Figure 9B is a bar diagram showing the calculated total charge transferred in the anodic and cathodic electrochemical reactions (the sum of the positive charge and the absolute value of the negative charge) , for both sets of signals (left bar: control; right bar: neurons culture) . To calculate the total charge, the current obtained from the FSCV measurements was used, using the relationship between charge (Q) and current ( I) that is given by Equation 2: where t is the time (s) , which can be expressed in terms of the scan rate ( v; units of V s-1) and the potential range (AE; units of V) as follows (Eq. 3) :

[0099] (3) v = AE / t t = AE / v

[0100] Figure 9B shows that the charge obtained from the neuronal measurements was 2320 ± 30 pC and was statistically different from the charge recorded from the control measurements (168 ± 14 pC; p < 0.001) . This difference suggests that neuronal cells were not only viable — they also functioned effectively on the microchip. The higher charge observed in the neuronal measurements indicates that the neurons were actively generating and releasing electroactive substances, whereas the minimal charge observed under control conditions highlights the absence of active biological processes in the medium without the cells.

[0101] The observed difference in charge between the neuronal and control conditions further confirms the viability of the neuronal cells and demonstrates the microchip's ability to support the complex electrochemical behavior associated with neuronal function.

[0102] Figure 10A, 10B and 10C show average electrochemical charge plots versus time over time interval of 8.5 ms (Figure 10A for the anodic reaction, Figure 10B for the cathodic reaction, and Figure IOC for the total electrochemical charge) . Two distinct step increases are observed in the charge profile: the earlier one corresponds to the reduction reaction (Figure 10B) and the later to the oxidation reaction (Figure 10A) . The earlier reduction step increase indicates a disparity in the rates of the oxidation and reduction processes. This imbalance could be due to differences in the availability of the reduced and oxidized species or to the inherent kinetics of the reduction reaction. The overall duration of the process is 8.5 ms, which can be calculated using Equation 4:

[0103] 2-AE 2’1.7

[0104] (4) - = - = 8.5 ( s' scs rate 480

[0105] The calculated time corresponds well with the typical duration of neuronal firing, during which action potentials are generated and neurotransmitters are released.

[0106] Part C: electrochemical response of neurons on the microelectrodes ' array to chemical stimulation

[0107] To stimulate the neurons, a stock solution of KC1 was prepared in PBS and added to the neuronal culture medium to achieve final concentrations of 30 and 90 mM (i.e., to the well shown in Figure 8C) . Measurements were initiated immediately after adding the KC1 solution.

[0108] Figure 11A presents the average voltammograms recorded from neurons cultured on the microelectrode array, comparing the baseline activity of neurons alone (solid red) to the measurements taken after adding chemical stimulants KC1 at concentrations of 30 mM (dashed black) and 90 mM (dotted blue) . Average was calculated based on the eleven individual working microelectrodes patterned on the chip) . The voltammograms shown in Figure 11A indicate that the higher the concentration of the stimulant in the medium, the higher the electrochemical currents .

[0109] Figure 11B is a bar diagram showing the calculated charge trans fer associated with the voltammograms of Figure 11A; the calculation is as described above . The results are shown in a tabular form below :

[0110] The data shows an enhancement in the electrochemical activity of the neurons with increas ing concentration of the KC1 stimulant . KC1 enhances neuronal excitability, with higher concentrations o f KC1 leading to greater depolari zation of the neuronal membrane and consequently, higher electrochemical activity .

[0111] Figures 12A, 12B and 12C show average electrochemical charge plots versus time over time interval of 8 . 5 ms ( Figure 12A for the anodic reaction, Figure 12B for the cathodic reaction, and Figure 12C for the total electrochemical charge ) . In each figure , three curves are shown, based on the data recorded from a medium containing the neurons alone ( solid red) with 30 mM KC1 ( dashed black) and 90 mM KC1 ( dash-dotted blue ) KC1 . It is seen that the di f ferences in the reduction reaction are less pronounced compared with the changes in charge for the oxidation reaction across the three di f ferent conditions : neurons , neurons with 30 mM KC1 , or 90 mM KC1 . Example 3 Fabrication of microelectrode array microchip and validation by cyclic voltammetry

[0112] A microelectrode array having the design shown in Figures 3A- 3B, consisting of a counter microelectrode encircled by ten working microelectrodes and an adj acent ( single ) electrophysiological recording microelectrode was microfabricated by photolithography akin to the protocol described above , and each of the electrodes was validated by cyclic voltammograms ( CV) . The electrochemical cell used for the CV measurements consisted of a three-electrode configuration : the fabricated microelectrode array (which includes the working electrodes [WE ] , as well as an externally applied commercial Ag / AgCl reference electrode [RE ] ( CHI 111P, CH Instruments ) . A separate platinum electrode was used as the counter electrode .

[0113] CV was performed by scanning the initial potential E± = -0 . 2 V versus RE , vertex #1 potential Ei = -0 .2 V versus RE , vertex #2 potential E2 = 0 . 65 V versus RE , scan rate = 0 . 1 V s-1, the number of cycles = 2 using a 5mM solution of the reversible redox pair ferrocyanide / ferricyanide in PBS .

[0114] The cyclic voltammograms recorded at a scan rate of 0 . 1 V / s for the counter and electrophysiological electrodes in the presence of 5 mM ferrocyanide / ferricyanide solution are shown in Figure 13A. The cyclic voltammograms recorded at a scan rate of 0 . 1 V / s from ten gold working microelectrodes in the presence of 5 mM ferrocyanide / ferricyanide solution are shown in Figure 13B .

Claims

Claims1 ) A chip for electrochemical and electrophysiological measurements of neuronal medium, comprising a base substrate and at least one array of microelectrodes fabricated on the base substrate , wherein the array comprises : an electrochemical sensing subarray which includes a counter microelectrode positioned in the center of the subarray and multiple working microelectrodes placed essentially equidistantly from the counter microelectrode , and optionally one or more reference microelectrodes ; an electrophysiology recording subarray which includes at least one microelectrode for in vitro extracellular recording, positioned in close proximity to the electrochemical sensing subarray, and a reference electrode for the electrophysiological electrical circuit ; leads extending from the microelectrodes to connect the microelectrodes to contacts placed at the distal end of each respective lead .2 ) A chip according to claim 1 , wherein in the electrochemical sensing subarray, the multiple working microelectrodes and the optional reference electrode lie on a polygonal line , an arc, or a circumference of a circle .3 ) A chip according to claim 1 or 2 , wherein the electrochemical sensing subarray includes at least one surface-modi fied microelectrode .4 ) A chip according to any one of claims 1 to 3 , comprising : an electrochemical sensing subarray possess ing rotational symmetry, which includes a counter microelectrode positioned in the center of the subarray and multiple working microelectrodes placed equidistantly from the counter microelectrode , wherein the working microelectrodes and theoptional reference electrode lie on a circumference of a regular polygon with circumradius Ri, or on a circumference of a circle with radius ri ; an electrophysiology recording subarray which includes multiple recording microelectrodes that are located equidistantly from the counter microelectrode of the electrochemical subarray, lying on the circumference of a regular polygon with circumradius R2 ( R2>RI ) , or a circumference of a circle with radius r2 ( r2>ri ) ; such that the two subarrays are arranged on the circumferences of two concentric regular polygons or circles , and the common center is occupied by the counter electrode of the electrochemical sensing subarray .5 ) A chip according to claim 4 , wherein the working microelectrodes are circular discs with diameter in the range from 5 to 15pm, the counter microelectrode is a circular disc with diameter in the range from 20 to 40pm, and the total number (ni) of working and reference microelectrodes patterned in an individual electrochemical sensing subarray is at least 5 , wherein the ni circular discs are placed equidistantly from the counter microelectrode so that their centers lie on the vertices of ni-polygon with circumradius Ri, or the circumference of a circle with radius ri, wherein Ri or ri is from 40 to 60 pm .6 ) A chip according to claim 5 , wherein the electrophysiology recording microelectrodes are circular discs , with diameter in the range from 20 to 40pm, and the total number (112 ) of electrophysiological recording microelectrodes patterned in an individual subarray is at least 3 , wherein the 112 circular discs are placed equidistantly from the counter microelectrode so that their centers lie on the vertices of n2-polygon ofcircumradius R2 , or the circumference of a circle with radius r2 , wherein R2 or r2 is from 100 to 140 pm .7 ) A chip according to claim 6 , wherein ni and 112 are equal .8 ) A chip according to claim 6 or 7 , wherein the working microelectrodes and the electrophysiology recording microelectrodes are angularly separated .9 ) A chip according to any one of claims 4 to 8 , wherein the number of arrays of microelectrodes fabricated on the base substrate is three , each possessing rotational symmetry, and the arrays are essentially mutually tangential .10 ) A method of simultaneous electrophysiological and electrochemical analysis of neuronal activity, comprising the steps of : culturing neuron cells on a microelectrode array; and extracellularly recording action potentials while concurrently measuring redox reaction ( s ) of neurotransmitter ( s ) released by said neuron cells with the aid of fast-scan cyclic voltammetry .11 ) A method according to claim 10 , wherein the microelectrode array comprises a subarray of electrophysiological microelectrodes and a subarray of electrochemical microelectrodes that are arranged in a concentric fashion, with a counter electrode occupying the common center, such that the same neuron ( s ) are in the vicinity of both types of microelectrodes .12 ) A method according to claim 10 or 11 , wherein the microelectrode array is fabricated on a base substrate in the form of a chip, as defined in any one of claims 1 to 9 .13 ) A method according to any one of claims 10 to 12 , wherein the voltammetry is recorded over bare working microelectrodes to determine the overall neuronal electrochemical activity .14 ) A method according to any one of claims 10 to 12 , wherein the voltammetry is recorded over surface-modi fied working microelectrodes to detect multiple redox-active neurotransmitters in a discriminant manner and identi fy individual neurotransmitters .15 ) A method according to any one of claims 10 to 14 , wherein the neurons are induced pluripotent stem cell-derived neurons .

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