WO2018031675A1

WO2018031675A1 - Development of a multichannel carbon fiber microelectrode array for electrochemical measurements

Info

Publication number: WO2018031675A1
Application number: PCT/US2017/046126
Authority: WO
Inventors: Michael L. HEIEN; James R. SIEGENTHALER
Original assignee: Arizona Board Of Regents On Behalf Of The University Of Arizona
Priority date: 2016-08-09
Filing date: 2017-08-09
Publication date: 2018-02-15

Abstract

Tonic levels of dopamine can be measured with single carbon fiber microelectrodes using fast-scan cyclic voltammetry ("FSCV") and fast-scan controlled-absorption voltammetry ("FSCAV"). A major limitation to these measurement techniques is the regional localization based on the microelectrode placement. In an effort to overcome this challenge, a multi-channel carbon fiber microelectrode array has been developed for both FSCV and FSCAV measurements. The array features a 2x8 microelectrode placement with 300 micron spacing between fibers, providing the ability to span regions of the brain. Characterization of the array includes single channel analysis by slow can voltammetry and FSCAV.

Description

DEVELOPMENT OF A MULTICHANNEL CARBON FIBER MICROELECTRODE ARRAY FOR ELECTROCHEMICAL MEASUREMENTS

FIELD OF THE INVENTION

[0001] The present invention relates to detection and measurement of an electroacfive chemical located in the brain of an animal using Fast Scan Cyclic Voltammetry or Fast- scan Controlled Absorption Voltammetry.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No, R21 DA035425 awarded by N!H, The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Tonic levels of dopamine can be measured with single carbon fiber microelectrodes (or alternately, CF Es) using fast-scan cyclic voltammetry ("FSCV") and fast-scan controiled-absorption voltammetry ("FSCAV"). A major limitation to these measurement techniques is the regional localization as each electrode only samples from 10 microns around its environment based on the microeiectrode placement. In an effort to overcome this challenge, a multi-channel carbon fiber microeiectrode array has been developed for both FSCV and FSCAV measurements. In an exemplary embodiment, the array features a 2x8 microeiectrode placement with 300 micron spacing between fibers, providing the ability to span regions of the brain. Characterization of the array includes single channel analysis by slow scan voltammetry and FSCV.

[0004] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

SUMMARY OF THE INVENTION

[0005] The present invention features a multi-channel CFME array effective for optimizing an acquisition of electrochemical measurements via FSCV or FSCAV. The array may comprise an array structure having a plurality of parallel, equally-sized, and equally-spaced gold traces. In some embodiments, the plurality of gold traces and a plurality of insulating barriers are arranged in an alternating pattern in the array structure such that each insulating barrier and each gold trace alternate with each other.

[0006] In other embodiments, a plurality of CFMEs, each having an upper portion and a lower portion, also comprises the array. In an embodiment, the upper portion of each CFME contacts one of the gold traces, while the lower portion of each CFME extends beyond a bottom of the array structure. Additional embodiments may feature an adhesive affixing each CFME to a gold trace. In another embodiment, the upper portion of each CFME and each gold trace has a conductive coating disposed thereon.

[0007] In further embodiments, the lower portion of each CFME has a conductive tip. The lower portion of each CFME, excluding the conductive tip, may be insulated with an insulation material.

[0008] In supplementary embodiments, an insulating seal overlays the entire array structure. This insulating seal, along with the plurality of insulating barriers, provides a double layer of insulation to the plurality of CFMEs. In this way, the overall array is effectively insulated while maintaining an isolated eiectroactive area at the end of each carbon fiber microeiecfrode.

[0009] The lower portion of each CFME may serve as a channel for acquiring electrochemical measurements. In exemplary embodiments, the electrochemical measurements are acquired by applying a voltage to each CFME and measuring the resultant current. In preferred embodiments, the array is coupled to a custom amplifier for amplifying the resultant current before measurements are taken. Further, as a result of having multiple channels, the array optimizes the acquisition of the electrochemical measurements, as compared to single CFMEs, by obtaining simultaneous measurements from a plurality of regions of, for example, a test solution or an animal brain.

[0010] One of the unique and inventive technical features of the present invention is the use of multiple channels in the microelectrode array, which allow for the acquisition of multiple electrochemical measurements that span several regions of, for example, an animal brain. Presently known prior references and work limit the acquisition of electrochemical measurements to single carbon-fiber microeiectrodes due to the problems associated with fabricating and insulating multi-channel carbon-fiber microelectrode arrays.

[0011] Multi-channel arrays using CFMEs have been developed for acquiring measurements in other applications, but are not compatible with FSCV and FSCAV measurements because of the resistance and capacitance of the arrays. The resistance and capacitance are directly related to the effective insulation of the carbon fibers; thus, the multi-channel array must effectively insulate the carbon fibers while providing a defined eiectroactive area at the end of each carbon fiber. To overcome this difficulty, the present invention selectively insulated the carbon fibers, leaving an exposed eiectroactive area at each end. A removable masking material was placed on the end of each carbon fiber prior to insulating the present multi-channel array. After the array was insulated, using electrodeposition, the mask was removed with a compatible solvent (i.e., that would not dissolve any of the other dried epoxies or paint already on the array).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0013] FIGs. 1A-1 E are a diagrammatic detailing of the steps to making the multichannel CFME array.

[0014] F!G. 1 A shows an empty array structure with a plurality of gold traces.

[0015] FIG. 1 B shows the array structure with torr Seal Epoxy insulating barriers added between the gold traces.

[0016] FIG. 1 C shows CFMEs tacked into the gold traces using 2-ton epoxy.

[0017] FIG. 1 D shows a conductive silver paint applied to the CFMEs for increased conductivity of cells.

[0018] FIG. 1 E shows the array structure overlaid with an insulating 2-ton epoxy.

[0019] FIG. 2 shows a diagrammatic representation of the multi-channel CFME array, [0020] FIG. 3 shows an alternate diagrammatic representation detailing of the steps to making the multichannel CFME array.

[0021] FIG. 4A shows an FSCV calibration curve constructed for dopamine (top left, bottom left).

[0022] FIG. 4B shows the flow profile for measured dopamine (top right, bottom right).

[0023] FIG. 5 shows that the selective insulation of a carbon fiber was achieved by masking the carbon fiber with polypropylene followed by an electrodeposited insulating layer. The mask was then removed exposing a defined electroactive area on the carbon fiber.

[0024] FIG. 6A shows the insulated carbon fiber was cut and imaged using SEM to reveal the insulating film of ClearClad HSR® measured to be approximately 1 -1 .2 m in thickness.

[0025] FIG. 6B shows the SEM imaging of a carbon fiber that had been selectively insulated as the insulating material.

[0026] FIG. 7A shows a calibration plot for dopamine (alternately, "DA").

[0027] FIG. 7B shows selected flow profiles of 1000nM dopamine for each carbon fiber.

[0028] FIG. 7C shows flow profile of dopamine for two carbon fibers.

[0029] FIG. 8A shows a calibration curve for each microeiectrode of the dual electrode system.

[0030] FIG. 8B shows the flow profiles for each microeiectrode of the dual electrode system.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0031] Referring now to FIGs. 1 A-8B, the present invention features a multi-channel carbon fiber microeiectrode array (100) effective for optimizing an acquisition of electrochemical measurements via fast-scan cyclic voitammetry or fast-scan absorption voitammetry. The array (100) may comprise an array structure (101 ) having a plurality of parallel, equally-sized, and equally-spaced gold traces (102a, ... , 102n). In some embodiments, the center-to-center spacing between gold traces is 300 microns. In other embodiments, the plurality of gold traces (102a, ... , 102n) and a plurality of insulating barriers (103a, ... , 103m) are arranged in an alternating pattern in the array structure (101 ) such that each insulating barrier and each gold trace alternate with each other as shown in FIG. 3. In an alternate embodiment, each gold trace is disposed between two insulating barriers as can be seen in FIGs. 1 B-1 E and F!G. 2. In an exemplary embodiment, the number of gold traces can be greater than, or alternatively, less than the number of insulating barriers. In an embodiment, the number of gold traces varies from about 2 to about 18, In another embodiment, the number of gold traces is at least about 2. In a further embodiment, the number of insulating barriers range from about 2 to about 14. The number of insulating barriers may be at least 2. However, since an insulating barrier is disposed between each electrode, the number of insulating barriers depend on the number of carbon fiber electrodes in the array. Moreover, it can be appreciated that any number of carbon fiber electrodes can be included in the array.

|0032] In a preferred embodiment, the plurality of insulating barriers (103a, ..., 103m) are composed of torr seal epoxy. In further embodiments, a plurality of carbon fiber microeiectrodes (104a, ...,104n), each having an upper portion and a lower portion, also comprises the array (100). The number of carbon fiber microeiectrodes may be, at most, equal to the number of gold traces. A variety of ratios of the length of the upper portion to the length of the lower portion of the carbon fiber microeiecfrode may be employed, where the ratio is restricted only by the requirement that the upper portion is in electrical contact with a gold trace. Non-limiting examples of the ratio between the length of the upper portion to the length of the lower portion include 50:50, or 75:25, or 25:75, etc.

[0033] In other embodiments, the upper portion of each carbon fiber microelectrode contacts one of the gold traces, while the lower portion of each carbon fiber microelectrode extends beyond a bottom of the array structure (101 ). Additional embodiments may feature an adhesive (105a, ...,105n) (e.g., 2-ton epoxy) affixing each carbon fiber microelectrode to a gold trace. In another embodiment, the upper portion of each carbon fiber microelectrode and each gold trace has a conductive coating (106a, ...,106n) disposed thereon. In an embodiment, the conductive coating is either conductive colloidal silver paint or conductive carbon paint.

[0034] In supplementary embodiments, the lower portion of each carbon fiber microelectrode has a conductive tip. The lower portion of each carbon fiber microelectrode, excluding the conductive tip, may be insulated with an insulation material. In some embodiments, the insulation material is an insulative polymer or a silica capillary. In other embodiments, a predetermined length of the lower portion of each carbon fiber microelectrode may be (i.e., cut). |0035] In preferred embodiments, an insulating seal (107) overlays the entire array structure (101 ). This insulating sea! (107), along with the plurality of insulating barriers (103a,... ,103m), provides a double layer of insulation to the plurality of carbon fiber microelectrodes (104a, ... , 104n).

[0036] In this way, the overall array is effectively insulated while defining an isolated electroactive area for each carbon fiber microelectrode for acquisition of said electrochemical measurements. The lower portion of each carbon fiber microelectrode is the isolated electroactive area serving as a channel for acquiring eiectrochemical measurements. As a result of having multiple channels, the array (100) optimizes the acquisition of electrochemical measurements, as compared to single carbon fiber microelectrodes, by obtaining simultaneous measurements from a plurality of regions of a brain.

[0037] Consistent with previous embodiments, the array (100) may be employed for fast- scan cyclic voltammetry or fast-scan absorption voltammetry with an average scan rate of 400 volts per second and a total current of less than 2000 microamps. This configuration may yield an overall array resistance between 50 Ohms and 100 Ohms and a maximum collective capacitance of 5 nano Farads.

Microelectrode Array Fabrication

[0038] Fabrication of the multi-channel carbon fiber microelectrode array of the present invention was done by hand under a microscope. Initial designs of the array included eight gold traces spaced 300pm apart, center-to-center. The total distance across the array was 2.61 mm.

[0039] Tools necessary for the construction of the array include a surgical scalple, pulled glass capiilarys, and microforcepts. To construct the array, a barrier of Hysoi 2-part epoxy was applied between each gold trace, forming a barrier between the traces. The epoxy was allowed to dry overnight. A carbon fiber with a length between 1500μητι and 2500μηι was paced onto each gold trace and tacked in place by placing a small drop of epoxy at the end of the PCB using clear 2-Ton epoxy applied wih a pulled glass capillary. The epoxy was allowed to harden fully by placing the array in an oven at 1 15°C for 2hrs. Electrical contact between each gold trace and carbon fiber was made by painting the carbon fibers and gold traces with either conductive collodiai silver paint or conductive carbon paint by immersing the paint in acetone or isopronyl, aspirating a pulled capillary with the paint, and dispensing the paint using capillary action. The electrically connected carbon fibers were then insulated by painting the connection with 2-ton epoxy, which was allowed to dry either overnight, or by placement in an oven at 1 15°C for 2hrs. The carbon fibers that exteneded from the epoxy edge were then trimmed to a length of 1000μΜ from the epoxy edge.

[0040] An alternative method to placing raw carbon fibers and electricially insulating them to the desired length is to place preinsulated carbon fibers in place. This can be done by modifying the method of Phillips et. ai. Breifley, carbon fibers are aspirated while submerged in isoprolyl alcohol into a silica capillary that is 1500μηι long, and having an outer diameter of 90μηι. The aspirated capillaries are removed from the solution and allowed to dry. A small bead of epoxy is placed on the carbon fiber, and the carbon fiber is gently pulled through the capillary allowing the epoxy to wick inside the capiiary. A second bead is placed and the carbon fiber is further pulled. The bead is pulled until! it just touches the capillary and not allowed to wick inside. The capillary is allowed to dry. The carbon fiber extending from the dired epoxy is then trimmed to length, typically 50-100 μιτι. The aspirated capillary is placed on the PCB and tacked in place. The electrical connection process and insulation procedures remain unaltered.

[0041] The array was tested to determine if an electrical connection was made between the gold traces and carbon fibers by placing the array in a solution of artificial cerebral spinal fluid and cycling the array from -0.4 to 1.3V at 400V/s repeated at 60Hz. Upon successful determination, a single carbon fiber on the PCB was trimmed to be 100μηι in length from the epoxy edge and was calibrated to determine the linearity of the response (see FIGs. 3A-3B. The flow-cell color plot (FIG. 5B) and calibration curve (FIG. 4A) demonstrates that the array is capable of quantitativly measuring dopamine with a linear response from 50nM to 1000nM.

Microe!ectmde Array Insulation

[0042] To utilize the multi-channel carbon fiber microelectrode array of the present invention with FSCV without using pre-insuiated carbon fibers, the exposed Ι ΟΟΟμηΊ carbon fibers must be reduced to have an eiectroactive area of a tunable size between 50-100μηι. Insulation of the carbon fibers was achieved by using a commercially available electro-depositable paint, CiearClad HSR© using modified deposition parameters from Sripirom, et. al. CiearClad HSR© is a polyurethane suspension that is applied by cathodic electrodeposition, creating a solvent and electrically resistive insulating film. The thickness can be tuned based on applied deposition voltage and time. To selectively apply the CiearClad HSR© to the array, the tip of each carbon fiber were masked with melted polypropylene ("PP"). Electric deposition of the film was conducted by using chronoamperometry in a 2-electrode setup with a silver wire as the counter electrode. The coating was applied in two coats, the first was applied at a -4V potential for 120 seconds. The carbon fiber was then briefly rinsed in deionized water and heat cured in an oven at 1 15°C for 20 minutes. A second coating was completed by applying -8V for 120 seconds followed by a brief rinse and heat cure at 1 15°C. The array was then suspended in toluene and sonicated for 10 minutes to remove the PP mask (FSG. 4). The above parameters deposit an insulating layer of ~1 μηη thickness when a cross section of each coated carbon fiber is cut and imaged using scanning electron microscope ("SEM") imaging (FIGs. 6A-8B). SEM also shows that each carbon fiber is selectively insulated from where the mask was placed and removed indicating a defined electroactive area that is reduced from the original size. To determine the compatibility of the insulated array with FSCV, the array was placed in a modified flow- cell setup. Artificial cerebral spinal fluid ("ACSF") was flowed across the array, and dopamine was injected as a bolus into the flow path. While the ACSF was flowed, a dopamine triangle waveform from -0.4 to 1.3V at 400V/s was applied to two individual carbon fibers on the array and measured sequentially. Dopamine was measured using the CiearClad HSR© insulated carbon fibers to investigate the linearity of the calibration curve as well as the flow dynamics in solution.

[0043] Investigation of the array was done using carbon fibers that were SOO m long from the epoxy edge and were insulated to be within l OOpm of the edge of the carbon fiber, making a 1 Q0pm cylinder electrode. Flow cell analysis demonstrated that linearity for a calibration curve of dopamine was realized. Data collected from the flow-ceil also demonstrated a new tool that could probe flow dynamics of solution based on the time that it takes for dopamine to reach each carbon fiber within the flow-ceil once injected. The determination of the selective coating of dopamine was measured using two microelectrodes on the array individually as shown in FIGs. 7A-7C, The carbon fiber was 500 m long from the edge of the epoxy, and insulated using CiearClad HSR© exposing 100 m at the end.

[0044] A homebui!t duai-e!ectrode potentiostat was utilized to measure two carbon fibers simultaneously for the detection of dopamine. A linear calibration curve was generated and solution flow dynamics were probed utilizing the array. Simultaneous detection of dopamine on multiple fibers was determined to be possible.

[0045] To determine the capabilities of measuring dopamine with the arrays simultaneously, the array was setup with a dual microelectrode system to measure two of the four electrodes together. Electrodes E1 and E3 were measured simultaneously, while electrodes E2 and E4 were measured simultaneously. This was done with cutting the carbon fibers to be 500 m from the epoxy seal edge. FIG. 8A shows a calibration curve for each microelectrode and FIG. 8B shows the flow profiles for each microelectrode.

[0046] As used herein, the term "about" refers to plus or minus 10% of the referenced number.

[0047] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fail within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0048] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. Reference numbers recited in the claims are exemplary and for ease of review by the patent office only, and are not limiting in any way. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase "comprising" includes embodiments that could be described as "consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase "consisting of is met.

[0049] The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.

[0050] REFERENCES:

[0051] Patei, P. R.; Na, K.; Zhang, H.; Kozai, T. D. Y.; Kotov, N. A.; Yoon, E.; Chestek, C. A. Insertion of Linear 8.4 Mm Diameter 16 Channel Carbon Fiber Electrode Arrays for Single Unit Recordings. J. Neural Eng. 2015, 12 (4), 46009.

[0052] Clark, J. J.; Sandberg, S. G.; Wanat, M. J.; Gan, J. O.; Home, E. A.; Hart, A. S.; Akers, C. A.; Parker, J. G.; Willuhn, I.; Martinez, V.; et al. Chronic Microsensors for Longitudinal, Subsecond Dopamine Detection in Behaving Animals. Nat. Methods 2010, 7 (2), 126-129.

[0053] Sripirom, J.; Kuhn, S.; Jung, U.; Magnussen, O.; Schulte, A. Pointed Carbon Fiber Ultramicroelectrodes: A New Probe Option for Electrochemical Scanning Tunneling Microscopy. Anal. Chem. 2013, 85 (2), 837-842.

Claims

WHAT IS CLAIMED IS:

1. A mulii-channei carbon fiber microelectrode array (100) effective for optimizing an acquisition of electrochemical measurements via fast-scan cyclic voitammetry or fast- scan absorption voitammetry, wherein the array (100) comprises:

(a) an array structure (101 ) comprising:

i. a plurality of gold traces (102a, ...,102n) that are parallel, equally-sized, and equally-spaced;

ii. a plurality of insulating barriers (103a, ...,103m), the plurality of insulating barriers and the plurality of gold traces are disposed in an alternating pattern on the array structure (101 ) such that each insulating barrier and each gold trace alternate with each other;

iii. a plurality of carbon fiber microelectrodes (104a, ...,104n) each having an upper portion and a lower portion, wherein for each gold trace, the upper portion of one of the carbon fiber microelectrode contacts said gold trace, wherein the lower portion of each carbon fiber microelectrode extends beyond a bottom of the array structure (101 ); and

iv. a conductive coating (106a, ...,106n) disposed on the upper portion of each carbon fiber microelectrode and on each gold trace; and

(b) an insulating seal (107) overlaying the array structure (101 ),

wherein the insulating seal (107) and the plurality of insulating barriers (103a, ...,103m) provide a double layer of insulation to the plurality of carbon fiber microelectrodes (104a,... ,104n), thus the overall array is effectively insulated while defining an isolated electroactive area for each carbon fiber microelectrode for acquisition of said electrochemical measurements, wherein the lower portion of each carbon fiber microelectrode is the isolated electroactive area serving as a channel for acquiring electrochemical measurements, wherein as a result of having multiple channels, the array (100) optimizes the acquisition of electrochemical measurements, as compared to single carbon fiber microelectrodes, by obtaining simultaneous measurements from a plurality of regions.

2. The array (100) of claim 1 , wherein a center-to-center spacing between each pair of gold traces is about 300 microns.

3. The array (100) of claim 1 , wherein the plurality of insulating barriers (103a, ... , 103m) are made of a torr seal epoxy.

4. The array (100) of claim 1 , wherein an adhesive (105a, ...,105n) affixes each carbon fiber microe!ectrode to its corresponding gold trace.

5. The array (100) of claim 4, wherein the adhesive (105a, ..., 105n) is an epoxy.

6. The array (100) of claim 1 , wherein the lower portion of each carbon fiber microelectrode has a conductive tip, wherein the lower portion of each carbon fiber microelectrode, excluding the conductive tip, is insulated with an insulation material.

7. The array (100) of claim 8, wherein the insulation material is an insuiative polymer or a silica capillary.

8. The array (100) of claim 1 , wherein the conductive coating (106a, ...,106n) is a conductive colloidal silver paint or conductive carbon paint.

9. The array (100) of claim 1 , the array (100) being employed for fast-scan cyclic voitammetry or fast-scan absorption voitammetry with an average scan rate of about 400 volts per second and a total current of less than about 2000 microamps, wherein the overall array resistance is about 50 Ohms and about 100 Ohms.

10. The array of claim 9, wherein a collective capacitance of the array (100) has a maximum value of about 5 nano Farads.