Patent ID: 12195718

DETAILED DESCRIPTION

Different embodiments will now be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown. Many different forms can be set forth and described embodiments should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope to those skilled in the art.

In accordance with a non-limiting example, a microfabrication process may employ 3D printing technology as described herein to produce a high-throughput (HT), self-insulated, 3D microelectrode array (MEA) that may be used for in vitro electrophysiological applications, such as electrophysiological measurements. The 3D microelectrode array has in vitro electrophysiological applications, including a lab-on-a-chip and neuropharmacological testing devices. The 3D microelectrode array may be used for cardiotoxicity assessment, disease modeling, and pre-clinical drug discovery. Because it is designed as a high-throughput device, it may be used for high-throughput phenotypic screening of drug candidates. New electrophysiological models may be developed on top of the 3D microelectrode array and be used for 3D micro-physiological system integration and evaluation and 3D organoid and spheroid integration. The 3D microelectrode array may be used in the development of new modes of sensing and integrating electrical sensing to other modes, including energy and biological sensing applications.

The 3D printing, fabrication process may incorporate micro-stereolithography (μSLA) based 3D printing technology, which not only allows for the production of 3D microelectrode geometries, but also enables the monolithic integration of all components on a “bio plate” as a 3D printed well plate that includes 3D printed standard culture wells in order to realize the high-throughput, American National Standards Institute (ANSI)/Society for Lab Automation and Screening (SLAS)-compatible geometry in 1 to 768 (or more) culture well configurations. The microelectrode array includes a bifacial configuration where the 3D printed well plate includes a top face and bottom face. The 3D microelectrodes are formed on the top face via conductive paste or ink filled microchannels in an example, that are 3D printed in each of the 3D printed culture wells. The conductive traces for electrical connections may be 3D printed on the bottom face to form the self-insulated 3D microelectrode array after metal ink casting and printing and ink filling of the 3D printed vertical microchannels. These high-throughput 3D microelectrode arrays include optical, SEM (scanning electron microscopy), Impedance Spectroscopy and well-well impedance variations, allowing a rapid, accurate, cost-effective, two step scaling-up technique to microfabricate the high-throughput 3D printed microelectrode arrays in several multi-well configurations that are compatible with standard, high-throughput assay equipment such as plate readers, robotic handlers and electrophysiological systems.

Referring now toFIGS.1A-1I,2A-2F, and3A-3F, in an example, a three-dimensional (3D) microelectrode array is illustrated generally at20for in vitro electrophysiological applications and may be formed from a 3D printing resin, including a photopolymer clear resin. The microelectrode array20includes a 3D printed well plate24having a top face28and bottom face32(FIGS.2A-2D and3A-3F), and a plurality of culture wells36formed by 3D printing on the top face of the 3D printed well plate, and in this example, 3D printed to form what are cylindrical configured culture wells. Each culture well36contains an array of 3D printed hollow vertical microchannels40formed as cylindrical microtower structures and may be arranged in an ordered or random array within each culture well (FIGS.1B,1F,3A, and3C).

The well plate24forming the microelectrode array20may have anywhere from 1 to 768 culture wells36also arranged in an ordered or random array, such as the illustrated example of two rows of three culture wells (6-well microelectrode array) or three rows of four culture wells (12-well microelectrode array) (FIGS.1A,1B,1E,2A-2D). A 24-well microelectrode array20is described in detail below with reference toFIGS.6A-6D. The well plate24may include an outer peripheral wall42as best shown inFIGS.1A,10, and1E. Each culture well36includes the plurality of 3D printed hollow, vertical microchannels formed as hollow microtowers40on the top face28(FIG.3A).

Each of the plurality of vertical microchannels40are also cylindrically configured and form an ordered or random array as best shown inFIG.3A, in this example showing two rows of five vertical microchannels. Any array and number may be used, however, depending on specific requirements of the microelectrode array20. A plurality of microtroughs44or small indented channel lines are formed on the bottom face32(FIGS.1G,3B, and3D) and communicate with the vertical microchannels40via an orifice45(FIG.3D) that communicates with the microchannel. A conductive paste50(FIG.3F), which in an example, is formed as a silver ink, fills the microtroughs44on the bottom face32and the vertical microchannels as the hollow microtowers40on the top face28, forming on the top face in an example an array of self-isolated microelectrodes54(FIGS.1E,1H,2F, and3E) in the culture wells36, and on the bottom face, conductive traces58(FIGS.1I,2A-2D,2E,2F, and3F) that communicate with the array of self-isolated microelectrodes54, such that in this example, one conductive trace connects to one microelectrode.

In an example, each self-isolated microelectrode54may include an enlarged top contact section62(FIGS.1H,1I, and3E) at the upper portion of the microchannel40formed from the conductive paste50as silver ink in an example and extends from the top face28in a microbullet configuration, but could be a mushroom type configuration. The conductive traces58may terminate into contact pads66(FIGS.2E and2F) that are configured to interface with a electrophysiological circuit component to which the 3D microelectrode array connects. Each culture well36may include about 5 to about 15 microelectrodes configured in an array or a non-array in a non-limiting example, yet in another example, may preferably include between about 2 and 64 microelectrodes, and in another example, up to about 1,000 microelectrodes. The 3D printed well plate24may be formed as a photopolymer clear resin to allow a user to see the conductive traces58, contact pads66, and microelectrodes54, including the enlarged top contact sections62of mushroom and/or microbullet configuration, which allows integration of the 3D microelectrode array20with an optical microscope. Top to bottom microscopy is appropriate for the microelectrodes. Bottom-up microscopy is more difficult due to translucency of the resin and 3D structures.

A method of forming the three-dimensional microelectrode array20for in vitro electrophysiological applications may include 3D printing the well plate24having the top face28and bottom face32. The method further includes 3D printing on the top face28the plurality of culture wells36formed on the top face of the 3D printed well plate24. Each culture well includes the plurality of 3D printed, hollow, vertical microchannels40formed as microtowers on the top face, and further 3D printing microtroughs44on the bottom face32that communicate with the microchannels40. The method includes filling the microtroughs44on the bottom face32and the microchannels40on the top face28with the conductive paste50to form on the top face the self-isolated microelectrodes54in each of the culture wells, and forming on the bottom face the conductive traces58that communicate with the self-isolated microelectrodes.

The structure as described forms a high-throughput, 3D printed microelectrode array20. In those examples ofFIGS.1A-1I,2A-2F, and3A-3F, the 3D microelectrode array20is shown as configured and shown as either a 6-well or a 12-well microelectrode array, or as shown and described further below, the microelectrode array is shown as a 24-well bio-plate configuration that is formed using the 3D printing fabrication techniques. Example 3D printing technologies that may be used to form the 3D microelectrode array20include those plate and microchip systems manufactured by Axion BioSystems, MultiChannel Systems, Maxwell Biosystems, and Acea Biosciences/Agilent. Other commercially available technologies may be used, including those 3D printing systems that operate to produce planar configurations, e.g., 2D microelectrodes in some examples.

The 3D printing and similar fabrication technology that may be used to form the 3D microelectrode array20as illustrated and described may require only two steps in the example of 3D printing and silver ink casting/printing as shown and described to complete substantially the 3D microelectrode array. This two-step 3D printing fabrication process for the microelectrode array20is possible since the microelectrode array includes the unique bifacial configuration having the top face28and bottom face32that allows for two distinct advantages: 1) The array of 3D microelectrodes54are self-insulated and self-isolated, and 2) there is no requirement for any insulation strategy on the top face28.

The conductive traces58may terminate into contact pads66and may extend upward into the microchannels40are formed on the bottom face32, as opposed to the top face28, which makes the integration with commercial biological/electrical amplification systems hassle-free because no through-vias are required to transition the conductive traces58to the bottom face32. The fabrication components of the microelectrode array20, such as the 3D printing resin and silver epoxy, are usually inexpensive, and the material costs to produce a 6-well, a 12-well and up to 768-well bio-plate (corresponding to the 3D printed well plate24) for the 3D microelectrode array20are affordable. It is possible to configure different, arbitrary designs and conductive well changes. Rapid development of prototypes is possible for the microelectrode array20.

The advent of additive manufacturing and microelectromechanical systems (MEMS) that are tailored to this technology are ideal to tackle the microfabrication and packaging challenges when manufacturing the microelectrode array20. Different 3D printers and related devices may be based on different technologies, such as stereolithography (SLA), digital light processing (DLP), fused deposition modeling (FDM), selective laser sintering (SLS), selective laser melting (SLM), electronic beam melting (EBM), laminated object manufacturing (LOM) and 2-photon polymerization (2PP). These 3D printing processes may be pushed to their limits with other additive/subtractive technologies to realize high-throughput 3D microelectrode arrays20as described.

It is possible to use micro selective laser melting (μSLM). This process has been found effective for the fabrication of high density, low throughput 3D printed stainless steel microelectrode arrays. However, this fabrication technique still requires an insulation step during fabrication when manufacturing devices in 3D. In accordance with a non-limiting example, the microelectrode array20may be fabricated by a combination of inexpensive polymer 3D printing, which is followed by techniques that add conductive functionalities, thus resulting in self-insulated and high-throughput 3D microelectrode arrays in two steps.

The current microelectrode array20as described provides commercial and academic scientists with tools to improve their ability to monitor, control and evaluate electro-active tissue in a high-throughput process. The fabrication process as described is more accurate, productive and cost-effective in scaling up a tool traditionally presented in a single sample analysis format to a high-throughput, ANSI/SLAS-compatible format. The bifacial design that includes the 3D printed well plate24having the top face28and bottom face32includes the 3D self-isolated microelectrodes54on the top face and configured in an array (or non-array) within each culture well36. The conductive traces50for electrical connections on the bottom face32are added in a second step such as by silver ink casting/printing into the microtroughs44, and metal casting/printing into the 3D printed, 3D hollow vertical microchannels40. This eliminates the need for insulation any for microelectrodes54and reduces multiple assembly processing steps, thereby saving time and costs.

In a non-limiting example, the microelectrode array20may be designed using the Solidworks software platform, e.g., the 2020×64 bit edition, which permits rapid design concepts to be generated. Three design configurations of this high-throughput, multi-well microelectrode array20were developed: 1) a 6-well configuration (FIGS.1A and1B); 2) a 12-well configuration (FIGS.1C and1D); and 3) a 24-well configuration, shown inFIGS.6A-6D. Each of the culture wells36in the 6/12 well 3D printed well plate24includes ten (10) 3D printed and self-isolated microelectrodes54that had been 3D printed as an array of 3D hollow vertical microchannels40and had been subsequently filled with the conductive paste50, e.g., the silver ink. The bifacial design includes the top face28having the 3D self-isolated microelectrodes54printed as 3D hollow, vertical microtowers formed as microchannels40, which in a non-limiting example, have a height of about 600 μm in one embodiment, an outer diameter of about 900 μm, and an inner diameter that is filled with the conductive paste50of about 700 μm.

Ink casting may be performed from the bottom face32, enabling self-insulation. The microtroughs44on the bottom face32are configured in this example with a width of about 540 μm, and a depth of about 300 μm that permit the ink casting process to form the conductive traces58. The microtroughs44that are filled with the conductive paste50may terminate into hollow microreservoirs, which in an example, may have a depth of about 600 μm (not illustrated in detail), which may subsequently serve as contact pads66. These contact pads66may be configured to interface with commercial electronics amplifier systems, e.g., an Axion BioSystems Maestro and AxIS software.

Depending on end use requirements, the 6-well, 12-well and later described 24-well well plate24, in a non-limiting example, may have an overall length of about 127.8 mm, a width of about 85.5 mm, and a height of about 20.13 mm with a ±3 mm variation to cover most well plate designs. These dimensions may vary, of course, depending on the end use and types of culture wells that are required. Individual culture wells36may have an inner diameter and wall thickness and be configured in a cylindrical configuration as illustrated of about 34.6 mm and 1.4 mm (6-well) and about 22.4 mm and 1.4 mm (12-well) respectively. As explained below for the 24-well microelectrode array20, dimensions may vary and may include a height of about 18.4 mm, an ID of about 16.5 mm, and an OD of about 19.5 mm. Each culture well36may include its array (or non-array) of isolated microelectrodes54.

It should be understood that the microelectrodes54may be arranged in any configuration, but an array such as two rows of five microelectrodes as shown inFIG.3Ahas been found feasible. The stereolithography file for 3D printing in the examples as described may be printed in FormLabs Form3, and may be operable with a μSLA 3D printer having a laser wavelength of about 405 nm, using a photopolymer clear resin, such as FLGPCL04 from FormLabs. The X- and Y-printing resolution may be determined by the spot size of the laser, which is about 85 μm in a non-limiting example. The axial resolution in the Z direction may be kept at about 100 μm.

To obtain an optimized print quality for the various features that constitute the microelectrode array20, it was printed in one example at 20° with respect to an anchor/substrate holder used with the 3D printing system, but other angles as described below were later used. The 3D printing bifacial configuration having the top face28and bottom face32allows for ink casting (FIG.1E) using, for example, Epo-Tek® EJ2189 from Epoxy Technologies Inc. on the bottom face of the 3D printed well plate24so that the hollow microtowers as the microchannels40(FIG.1F) are filled with the conductive paste along with the microtroughs44to form the conductive traces58for electrical connections (FIGS.1F-1I), and forming the completed self-insulated 3D microelectrode array20having self-isolated microelectrodes54on the top face28(FIG.1H). Impedance spectroscopy was performed using a Bode 100 from Omicron Labs with Dulbecco's Phosphate Buffer Solution such as from Thermo Fisher Scientific, Waltham, MA, US, as the electrolyte. The collected data cluster was imported into a MATLAB® (MathWorks) and Origin Software software program for processing and analysis.

Referring now toFIGS.2A and2B, there are shown images of the monolithically 3D printed 6-well high-throughput 3D microelectrode array20showing the top and bottom faces28,32respectively. Referring now toFIGS.2C and2D, there are shown images of the monolithically 3D printed 12-well high-throughput 3D microelectrode array configuration showing the top and bottom faces28,32respectively. The silver ink as the conductive paste50in this example selectively fills the 3D printed microtroughs44on the bottom face32to form the conductive traces58and the microchannels as the microtowers40on the top face28to form the microelectrodes54.

Referring now toFIGS.2E and2F, there are shown photomicrographs of a single culture well36in the 6- and 12-well configuration respectively. The images show the patterned and self-isolated 3D isolated microelectrodes54on the top face28and the conductive traces58on the bottom face32of the 3D printed microelectrode array20. The images additionally depict the monolithic construction with features such as the culture well36, and contact pads66, and the structure may include alignment features as explained in greater detail below.

FIGS.3A and3Bare SEM images of a single culture well36looking towards the respective top and bottom faces28,32respectively, and showing the 3D printed microchannels40and microtroughs44. The close-up micrographs ofFIGS.3C and3Dshow a 3D hollow, vertical microchannel40(FIG.3C) that has not yet been filled with the conductive paste50and a single microtrough44(FIG.3D) showing its end having the via or orifice45. This excellent design-to-device transition to form the microelectrode array20may be achieved using the selective ink casting process. The actual inside diameter of the 3D hollow, vertical printed microchannels40in an example are smaller than the design parameters with actual dimensions of about 600 μm±30 μm (N=10), and the design diameter of about 700 μm. The height of the 3D, hollow vertical microchannels40in an example are about 600 μm±5 μm (N=10), with the design parameters being about 600 μm.

Referring toFIG.3E, there is shown a SEM image illustrating the hollow 3D printed microchannel40post-ink casting looking from the top face28, which when filled with the ink or other conductive paste50, forms the self-isolated microelectrode54once the ink, i.e., conductive paste50has filled the structure.

Referring toFIG.3F, there is shown a SEM image of the bottom face32of the completed 3D microelectrode array20in which the microtrough44has been filled or ink cast with silver conductive ink or paste50to form the conductive trace58and contact pad66. After ink casting from the bottom face32in an example, a desired, high top surface area as a finished “mushroom shaped” and/or microbullet, enlarged top contact section62of the microelectrode54is formed on the top section of the microchannel40as best shown inFIGS.1H and3E. The radius of curvature of this “mushroom” and/or microbullet as the top contact section62as shown in the enlarged image ofFIG.1Hand the SEM image atFIG.3Eis about 208 μm in a non-limiting example. Additionally, the “mushroom” and/or microbullet shaped top contact section62of the microelectrode54makes an obtuse angle of about 135° with the top of the microchannel40.

Referring now toFIG.4, there is a graph showing the impedance in ohms on the left vertical axis, the phase in degrees on the right vertical axis, and the frequency on the horizontal axis. The average full spectrum impedance as the real part and the phase as the imaginary part for N=10 microelectrodes40for both the 6-well and 12-well microelectrode arrays20across several culture wells36depict ideal microelectrode behavior. Further analysis on the consistency of impedance is shown in the graphs ofFIGS.5A and5B, which show the impedance in ohms at 500 Hz, 1 KHz, and 3 KHz for the well-to-well variations in the 6-well and 12-well 3D printed well plate configurations respectively. Those listed electrophysiologically relevant frequencies may pertain to cardiac/neural applications and correspond to the 500 Hz, 1 kHz, and 3 kHz frequencies. These values remain in close tolerance for M=3 electrodes for C=6 wells for M×C=18 total, and for M=3 electrodes for C=12 wells for M×C=36 total, for the 6-well and 12-well 3D printed well plate24.

From the graphs, it is seen that that the mean of the real part of the impedance varies by only 4.9%, 4.8% and 4.9% at the 500 Hz, 1 kHz, and 3 kHz frequencies among the 6-well and 12-well configuration. This evidence shows the excellent reproducibility of the fabrication process. The diameter of the culture wells36may vary as noted before, and in an example, includes about 5 to about 15 microelectrodes per culture well, but may preferably include between about 2 and 64 microelectrodes, and in another example, up to 1,000 microelectrodes.

As the culture well36count has increased, it has been found that the printing angle and device orientation has an effect on the 3D printing fabrication of these high-throughput (HT), self-isolated, 3D printed 3D microelectrode arrays20for in vitro biotechnology applications. Makerspace based development using micro-Stereolithography (μSLA) 3D printing and ink casting techniques has increased efficiency and in an example developed a 24-well microelectrode array20(FIGS.6A-6D) with self-isolated 3D microelectrodes54having 100% yield, and in an example, seven microtowers per well36, acting as supports for the microelectrodes54. This 24-well microelectrode array20configuration as shown inFIGS.6A-6Dwas analyzed by Optical Microscopy, Electrical Impedance Spectroscopy (EIS), Laser Scanning Confocal Microscopy, and Scanning Electron Microscopy, and the process optimization results were interpreted using established μSLA theory, which allows for greater understanding of the process relationships for these high-throughput, 3D printed 3D microelectrode arrays. These studies permitted further scalability, denser electrodes, and higher culture well36counts for high-throughput screening in biotechnology.

As the number of culture well36counts increased, fill factors increased for the resulting microelectrode arrays20, and the micro-Stereolithography (μSLA) 3D printing process was optimized as a combination of multiple variables: lateral (X, Y) and vertical (Z) print resolutions, support structure placement, device orientation with respect to a substrate holder, printing angle, laser power (P), maximum exposure energy (Emax) and critical energy (Ec), feature distances, and other variables. Some of these parameters were predefined in the μSLA 3D printer used to make the microelectrode arrays20, but other parameters were manipulated in order to optimize the final printing and microelectrode array characteristics. Some techniques optimized the printing process using a simulation of sensitive parameters, such as temperature and photo initiator properties, and increased the degrees of freedom in the ultraviolet source and substrate.

The manufacture of the 24-well microelectrode array20as a bioplate having seven microelectrodes54per culture well36with self-isolated microelectrodes54was optimized. Testing and characterization of the 24-well microelectrode arrays20as bioplates after variation in the printing angle and device orientation with respect to the base plane significantly improved the isolation of the conductive traces58and was supported by the correlation between numerical and experimental findings. It was determined that the need for any mechanical or chemical post-processing steps for finishing the surfaces of the microelectrode array20was reduced. With these results, the 3D printing process may be scaled to higher density, high-throughput 3D microelectrode arrays20by optimizing the different processing and manufacturing variables as discussed further below.

Optimizing 3D printing is considered by some skilled in the art as a process of maximization. For example, having a maximum number of parallel or perpendicular faces with respect to a base plane, and referenced as a horizontal plane created by the resin surface, during a μSLA 3D printing process, for example, may increase the surface quality, and thus, minimize the stepping phenomena created by devices as the microelectrode array20that are oriented using various tilt angles. Using this criteria, four different printing angles with respect to the base plane (0°, 12°, 20° and 24°) were evaluated.

A Stepping Line-Width Artifact (SLWA) phenomenon was tracked and defined during these observations in both the vertical microchannel or microtower40vias or openings45and microtrough44definition. Some increase in the SLWA was found in tilted versions of the printed structure forming the microelectrode array20.

During the ink casting step, the conductive silver ink was defined in the microtroughs44and unintended connected conductive traces58, and hence, shorting may result. From initial theory, the 0° tilt printed version was expected to not have SLWA due to the maximization on the number of parallel and perpendicular surfaces due to its orientation. A flat and smooth surface on the conductive traces58side and no short circuits were expected in the 3D printing along with full 3D definition of the microtowers as the vertical microchannels40.

In order to overcome the trace-shorting expected at various tilt angles, 3D printed surfaces due to SLWA, an additional step could be performed as was known to those skilled in the art to minimize the short circuit, i.e., employing fine mechanical sanding over the surface, or acetone polishing. This additional step was eliminated with the optimization process as developed and described below aiding in manufacturing efficiency.

The 24-well high-throughput, 3D microelectrode array20was designed in this example using Solidworks 2020 CAD software (Dassault Systěmes) as shown in the example ofFIGS.6A-6Dusing ANSI/SLAS standards with overall dimensions (L×W×H): about 127.8 mm×85.5 mm×20 mm. The device as the microelectrode array20was 3D printed at 0°, 12° and 24° tilt angle using a Form3printer (FormLabs) and rapid prototyping resin Clear v4 FLGPCL04, having the support structures placed on the culture wells, top side looking towards the top face28. One additional version was printed at a 20° tilt angle with the support structures on the conductive traces/back side as the wells-side orientation.

In this example, a total of 168 isolated 3D microelectrodes54, i.e., seven per well, were designed. The 3D microelectrodes54were formed from the microtowers as the microchannels40with open ports or vias45and having the following dimensions: about 800 μm height, 1000 μm outer diameter (OD) and 800 μm inner diameter (ID). These microtowers40were connected to individual microtroughs44that formed the conductive traces58on the backside of the microelectrode array20and in an example dimensioned (L×D): about 400 μm×400 μm, and forming 3D microelectrodes54upon metallization/ink casting.

The μSLA process used a laser wavelength of about 405 nm at 250 mW power, and a 85 μm laser spot size in a non-limiting example. Print resolutions for the various printed configurations were maintained at Z (100 μm) and X/Y resolution (25 μm). A bottom-up printing process was used, although other similar 3D printing processes may be used. The devices as the microelectrode arrays20were immersed twice in Isopropyl Alcohol (IPA) (Sigma-Aldrich) for 15 minutes and air dried for 30 minutes. A final UV curing step (at about 405 nm) for 15 minutes at about 60° C. was performed to enhance the mechanical strength and robustness of the structure forming the microelectrode array20. Optical characterization of the resultant high-throughput, 3D microelectrode array20structure was performed using a Stereoscope (SMZ800, Nikon), to obtain more precise information about the definition of the microtowers40that formed the microelectrode54and conductive traces and microtroughs.

Trace metallization for the conductive traces58and microelectrodes54was performed using an ink casting technique with two silver conductive pastes50: 1) Silver Paste EP3HTSMED (Masterbond), and 2) Prima-Solder EG8050 (AI Technology). Both conductive pastes50were cured at about 60° C. for about 20 hours to obtain full strength. Further end-to-end, e.g., from the backside at the bottom face32to the top face28of the 3D microelectrode array20on the front, resistance measurements were performed using a Source Meter Mod. 2400 (Keithley, Tektronix). Gold electroplating on top of the defined silver 3D microelectrodes54as the top contact section62in an example was made using Sulfite Gold Plating Solution TSG-250 (Transene Company) and a Platinum (Pt) counter electrode with a 50% AC cycle square pulse of amplitude 1 A/cm2 for 30 s.

Electrical Impedance Spectroscopy (EIS) was performed using a Vector Network Analyzer (Bode 100, Omicron Lab) with Phosphate Buffer Solution (PBS) as an electrolyte and Pt/Ti wire as a counter electrode. The collected data was post processed using Origin software (OriginLab) for statistical and impedance/phase analysis during frequency sweeping in the range of 1-10 MHz. High precision imaging was performed using Scanning Electron Microscopy (SEM, TM3000, Hitachi) and Laser Scanning Confocal Microscope (VK-X1000, Keyence) in order to characterize the stepping effect, shape, dimensions and profile.

Table I presents a summary of the different 3D printing conditions used for the high-throughput, microelectrode arrays20and basic observations on the percentage of open microchannels as microtowers40and microtroughs44that formed the conductive traces58after preliminary optical observations under a stereoscope.

TABLE IProcess Conditions and General Characterization forAll the 3D Printed Microelectrode ArraysSupportOpenPrintedPrint-3D printingOrientationStructuresMicro-OpenResinPrintedingpost-(TiltOrientationtowersTracesVolumeLayersTimeprocessingAngle °)(Attachment)(%)(%)(mL)(n)(min)(conditions)0°Topside/Wells10010062.26270465IPA Bath × 212°Topside/Wells98.210076.4643958515 min each20°Bottomside/10050.664.48543570Air DryingTraces30 min24°Topside/Wells27.410058.34591540UV Curing @60° C. 15 min

The best results were obtained at a tilt angle of 0° for all the metrics measured. The microtowers40that later form the microelectrodes54were fully open for a 20° tilt angle but the orientation, support structures and SLWA reduced the conductive traces58definition efficiency down to about 50% for 85 out of 168, necessitating additional post-processing. The 12° tilt angle orientation resulted in comparable performance to the 0° tilt angle 3D printing, however, that 3D printing at the 12° tilt angle suffered in the print volume, number of layers, and other measures. The 24° orientation resulted in the worst definition of the 3D microtower40structures with only 27.4% of the microtowers that would later form the 3D microelectrodes54being fully open.

Referring again toFIGS.6A-6D, an example of a 24-well microelectrode array20. The outer peripheral well36in the configuration shown inFIGS.6A and6Bshow a stepped configuration with a step37or ledge, and inFIG.6Bshowing schematically by an example of the three holes inFIG.6Eon the middle left side, one or more alignment features37a, which allow alignment. The alignment features37acan be different configurations as grooves, slots, holes, or other features to aid in alignment for end-use microelectrode array uses, such as vertical grooves shown at43ainFIG.6B.FIGS.60and6Dare optical images. The images ofFIGS.6E and6Fare enlarged views and show a number of culture wells36from the bottom face32view and the microtroughs44and vias45, which are shown in greater detail of the enlarged view of a single culture well inFIG.6F. Another view looking from the top face28down into a single culture well36and showing the microelectrodes54and conductive traces58.

FIGS.7A and7Bdepict optical microimages of the 0° tile angle and the ink casting of the defined 3D microelectrodes54and conductive traces58at two orientations, i.e., wells-side looking down towards the top face28(FIG.7A) and with an example microelectrode54shown by the dotted circle, and the traces-side looking down towards the bottom face32(FIG.7B).FIGS.7C to7Fshow laser scanning confocal microscopic 3D images (FIGS.7C and7E) and corresponding optical images (FIGS.7D and7F) of respective filled (FIGS.7C,7D) and unfilled (FIGS.7E,7F) vias45forming the microelectrodes54as shown IFIGS.7C and7Din the high-throughput microelectrode array20that was printed at 0° tilt angle. There did not appear to be any trace of SLWA on the conductive traces58as may have been predicted by theory, and the completely isolated conductive traces did not require further processing. The 3D microelectrodes54are well defined and may have the mushroom shaped top contact section62that may benefit 3D organoid stimulation applications.

Laser confocal microscope images are shown in the 3D isometric view looking towards the bottom face32ofFIG.8A, and plan view looking down towards the bottom face inFIG.8B, and depicting the profile images using laser confocal microscope for the 24° tilt 3D printing of the microelectrode array20and having lateral profiling. The tilt angle may range, however from 0° to 90°. The tilt angle may be based upon the orientation of a support surface against the top or bottom faces during 3D printing. The printing angle and orientation may be changed to optimize printing and isolation of the well plate, conductive traces, and microchannels. A scaled profile of the SLWA showing a triangular shaped curve is illustrated in the graph ofFIG.8Cwith the Y-axis at 20 μm scale and the X-axis at the 100 μm scale. Derivation of this SLWA can be performed by applying geometric calculations using the 3D printing process for inclination and orientation as shown in the geometric configurations depicted in the drawing ofFIG.9, indicating the stepping effect and line width artifact with the SLWA denoted as “h” inherent to SLA 3D printing, and showing the SLWA in the top row, the base plane in the second row with the different tilt angles.

This calculation depicts the wider SLWA for increased inclinations of 3D printing, thus creating uneven surfaces and unintended short-circuits among microtroughs44and resulting conductive traces58in the 24-well high-throughput 3D microelectrode arrays20. These calculations reveal the relationships between the angles and SLWA distances may be provided by the equations below:
α+β=90°
β=90°−α  (1), and
h=z/Cos(β)  (2),

where “α” is the angle of tilt in 3D printing and “z” is the vertical resolution of the 3D printer. The variable of interest is “h,” which is the mathematical representation of SLWA. Theoretically, h increases when lowering the inclination angle, and for 0° becomes infinite giving a smooth printed surface defined by the parallel face optimization.

Theoretical “h” can be calculated as 246 μm (tilt angle: about 24°; z resolution: about 100 μm). These values correlate with the experimental dimensions presented in the graphs ofFIGS.10A-10D, with SEM images inFIGS.10A and10B, and calculated SLWAs represented by profiles in the graphs shown inFIGS.100and10D(220 μm and 235 μm). Differences from theory can be attributed to changes in the actual 3D printed resolution in the “z” resolution due to energy-dependent curing. These experimental results for SLWA in the 24° tilt angle 3D printed version are shown in these images ofFIGS.10A and10Bwith the selection of one and two SLWA on the traces-side looking down at the bottom face32and their profiling depicted in the graphs forFIGS.100and10Drespectively, with scales forFIG.10C: X-axis: 10 μm, Y-axis: 10 μm, and the scales forFIG.10D: X-axis: 20 μm, Y-axis: 10 μm.

SEM images and measurements of the microelectrode array20are presented in the images ofFIGS.11A and11Bthat demonstrate the surface flatness observed in the high-throughput, 3D microelectrode array20printed at 0° tilt angle showing inFIG.11Aunfilled microtroughs44and the filled conductive traces58inFIG.11B. The relatively smooth surface observed due to a SLWA value of infinity as calculated theoretically correlated to printing an optimized surface. On the other hand, for tilted versions as presented in the images ofFIGS.12A and12B, appreciable SLWA is observed and this becomes an undesirable feature and showing the microtroughs44.

The SEM images of the microelectrode array20on the traces-side at different inclination angles show the Stepping Line-Width Artifact where the 24° tilt device and orientation are shown with support structures on wells-side looking down at the top face28(FIG.12A), and the 20° tilt device and orientation with support structures on traces-side looking down at the bottom face32(FIG.12B) are shown.

The graph ofFIG.13shows the impedance and phase signature for gold 3D microelectrodes with an average of N=6, from a single well on the 24-well high-throughput microelectrode array printed at 0° tilt angle.

This averaged (N=6) end-to-end microelectrodes54resistance for 0° tilt angle printed version was 0.41Ω (Ohms). As noted above, the graph ofFIG.13shows the full spectrum impedance averaged for N=6 gold plated electrodes in the optimized version printed at a tilt angle of 0°. The curves depict the desired microelectrode54behavior for biological applications with mean values of 317Ω (Ohms) and −11.2° (Degrees) at the electrophysiological relevant frequency of 1 kHz. The histogram ofFIG.14Aillustrates the phase versus frequency, and the histogram ofFIG.14Billustrates the impedance versus frequency.

It is evident that the tilt angle and device orientation have an effect on the μSLA 3D printing of complex structures for the high-throughput microelectrode arrays20as described. Minimization of the remnant line width artifact inherent to the μSLA process was best shown at the 0° tilt, wells-side (looking down at the top face28) oriented design.

This application is related to copending patent application entitled, “METHOD OF FORMING HIGH-THROUGHPUT 3D PRINTED MICROELECTRODE ARRAY,” which is filed on the same date and by the same assignee and inventors, the disclosure which is hereby incorporated by reference.

Many modifications and other embodiments of the invention will come to the mind of one skilled in the art having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is understood that the invention is not to be limited to the specific embodiments disclosed, and that modifications and embodiments are intended to be included within the scope of the appended claims.