Implanted neural probes such as microwires, tetrodes, and silicon-based microelectrodes are among the most important techniques in both fundamental and clinical neuroscience. Scientifically, they remain the only option to temporally resolve the fastest electrophysiological activities of individual neurons, thus providing critical information to dissect neural circuitry. Clinically, neural electrodes have been successfully used in the treatment of neurological disorders such as Parkinson's disease, epilepsy, and obsessive compulsive disorder. Moreover, neural electrodes allow for direct communication between brain and man-made devices, which can enable applications such as human brain-machine interfaces and neuroprosthetics.
However, these conventional neural electrical probes typically have dimensions substantially larger than neurons and capillaries, which fundamentally precludes the possibility of interrogating the whole neuronal population in a functional brain region. For example, microwire electrodes, tetrodes, and Utah arrays host only one recording site at the tip of each wire, and therefore cannot simultaneously record neural activity at multiple depths. Micro-Electro-Mechanical System (MEMS)-based silicon probes have significantly increased the number of recording sites on a single probe. However, these silicon probes typically have cross-sectional areas around or greater than 10 μm2, which yields a volume per electrode similar to that of microwires and tetrodes, at least two orders of magnitude larger than the average size of a neuronal soma. In addition, these probes are strongly invasive to living brain tissue. Therefore, their highest implantation density is typically limited to maintain tissue vitality, by allowing at most 1-2% of the enclosed volume to be occupied by the electrode array. Therefore, the smallest inter-probe spacing is limited to at least several hundred microns for both microwire array and silicon probes.
In an effort to further reduce electrode size and increase packing density, advanced lithography techniques such as electron-beam lithography (EBL) have been used to fabricate silicon-based microelectrodes that enable closely-packed recording sites along the length of a probe. However, these nanofabricated probes have similar dimensional limits on implantation density. Ultra-flexible nanoelectronic neural probes, using a substrate-less, multi-layer layout, markedly reduce the cross-section to the subcellular range, but demonstrate limited electrode density along the probe due to the fabrication resolution of planar photolithography techniques.
Understanding brain functions at the circuit level requires time-resolved simultaneous measurement of a large number of densely distributed neurons, which remains a great challenge for current neural technologies. Currently available probes preclude the high implant density that is necessary for mapping large neuronal populations with full coverage.
Thus, there is a need in the art for an improved intracortical electrode array. The present invention satisfies that need.
In one aspect, a probe comprises a probe body having a distal end and a proximal end, at least one flexible nanoelectronic thread extending from the distal end of the probe body, the nanoelectronic thread comprising at least one electrode positioned on a top surface of the nanoelectronic thread, and at least one nanoscale wire having a first portion electrically connected to the at least one electrode and a second portion encapsulated by a dielectric insulator, at least one external trace electrically connected to the at least one nanoscale wire at a proximal end of the at least one nanoelectronic thread, and an interface connector near the proximal end of the probe body, electrically connected to the at least one electrode via the at least one nanoscale wire and the at least one external trace, wherein the at least one nanoscale wire has a width less than 1 μm, and wherein the nanoelectronic thread has a cross-sectional area that is less than 100 μm2. In one embodiment, the at least one electrode comprises a plurality of electrodes positioned on the top surface of the flexible nanoelectronic thread, the plurality of electrodes being evenly spaced along a length of the nanoelectronic thread, and the at least one nanoscale wire comprises a plurality of nanoscale wires, the nanoscale wires having a pitch of less than 500 nm. In one embodiment, the plurality of electrodes are arranged in a linear array, and wherein the cross-sectional area of the flexible nanoelectronic thread is less than 10 μm2.
In one embodiment, the at least one electrode comprises at least one array of electrodes, the electrodes in the array being separated by a distance of less than 10 μm. In one embodiment, each of the electrodes has a sensing surface having an area less than 100 μm2. In one embodiment, the at least one nanoelectronic thread comprises at least 8 nanoscale wires. In one embodiment, the at least one nanoelectronic thread comprises at least 16 nanoscale wires. In one embodiment, the at least one nanoelectronic thread further comprises a hole having a diameter of less than 10 μm. In one embodiment, the at least one external trace has a width greater than the width of the at least one nanoscale wire.
In another aspect, a method of fabricating a flexible probe comprises the steps of patterning at least one external trace on a substrate using photolithography, fabricating a flexible nanoelectronic thread using a process comprising the steps of depositing a sacrificial layer on a substrate, depositing a first insulating layer on top of the sacrificial layer, patterning at least one nanoscale wire on top of the insulating layer using electron beam lithography, depositing a second insulating layer on top of the at least one nanoscale wire, thereby encapsulating at least a first portion of the at least one nanoscale wire and leaving at least a second portion of the at least one nanoscale wire exposed, patterning at least one electrode on top of the second insulating layer, wherein the at least one electrode is in electrical contact with the second portion of the at least one nanoscale wire, and removing the sacrificial layer and releasing the finished probe from the substrate, wherein the electron beam lithography and photolithography are overlapped by at least 4 μm in all directions in all layers.
In one embodiment, the sacrificial layer comprises nickel and is removed with a nickel etchant. In one embodiment, the first and second insulating layers comprise SU8. In one embodiment, the electron beam lithography is controlled at a resolution of 0.5 μC/cm2.
In another aspect, a method of implanting a flexible probe in a tissue of a subject comprises the steps of attaching a flexible probe to a rigid implantation assist device at an attachment point, inserting the flexible probe into a tissue of a subject by piercing the tissue with the rigid implantation device and inserting the rigid implantation assist device such that the attachment point is within the tissue, detaching the flexible probe from the rigid implantation assist device, and withdrawing the rigid implantation device from the tissue.
In one embodiment, the step of attaching further comprises the step of inserting a micro post on the rigid implantation device into a hole near a distal end of the flexible probe. In one embodiment, the rigid implantation device and the flexible probe are inserted at a speed of 5 μm/second. In one embodiment, the flexible probe is attached to the rigid implantation device via a chemical bond. In one embodiment, the chemical bond comprises polyethylene glycol. In one embodiment, the detaching step further comprises dissolving the polyethylene glycol. In one embodiment, the rigid implantation assist device comprises a linear actuator and a carrier block slidably attached to a linear guide and fixedly attached to the flexible probe, and wherein the method further comprises actuating the linear actuator thereby implanting the flexible probe into the tissue. In one embodiment, the linear actuator comprises a solenoid. In another aspect, a probe is made according to one or more methods described above.
In another aspect, a system for implanting a flexible probe in a tissue of a subject comprises a flexible probe and a rigid implantation assist device, wherein the flexible probe is configured to engage the rigid implantation device. In one embodiment, the rigid implantation assist device comprises a post configured to be inserted into a hole positioned at the distal end of the flexible probe. In one embodiment, the rigid implantation device comprises a linear actuator and a carrier block slidably attached to a linear guide and fixedly attached to the flexible probe.
The foregoing purposes and features, as well as other purposes and features, will become apparent with reference to the description and accompanying figures below, which are included to provide an understanding of the invention and constitute a part of the specification, in which like numerals represent like elements, and in which:
It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.
Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any whole and partial increments therebetween. This applies regardless of the breadth of the range.
Embodiments of the present invention relate to an ultra-flexible electrode array platform that can reliably detect and stimulate the electrical activity of individual or groups of cells (e.g., neurons, peripheral nerves, etc.). In certain instances, the platform allows for detection and/or stimulation with minimal immune responses over chronic time scales of months to years. Further, the present invention provides a strategy to chronically implant these electrodes in living tissue at high-packing density in three dimensions. The present invention is based, in part, upon the demonstrated ability to drastically reduce the dimensions of the probe by multiple orders of magnitude from currently-used microelectrodes, and the ability to integrate these electrodes with a minimally-invasive implantation mechanism for high-density, large-scale, parallel delivery into living tissue. The separation of electrodes in all three dimensions can be reduced to sub-100 μm, which will allow for complete coverage of all enclosed cells in a given volume. Because the ultra-flexible electrodes also have great long-term biocompatibility, the platform of the present invention also enables stable long-term recording and stimulation without the artifact of scar-encapsulation, continuous blood leakage and other tissue response to conventional electrodes. Therefore, this platform will open new opportunities for neuroscience research, clinical therapeutic applications for neuronal diseases, and development of brain machine interfaces. Probes of the present invention may be used for example to detect and monitor signals in a subject having a disability or condition, including but not limited to paralysis, amputation, blindness, or hearing loss. Other relevant conditions include neurological disorders, including but not limited to epilepsy, depression, addiction disorder, or neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and cerebrovascular diseases such as stroke. Probes of the present invention may also be used for fundamental and applied neuroscience research in experimental animal models in laboratories.
The present invention generally relates to nanoscale wires, nanoelectronic threads comprising multiple nanoscale wires and multiple electrodes, and, to probes comprising one or more nanoelectronic threads for use in detecting and/or stimulating electrical activity of biological tissue, including but not limited to the brain, spinal cord, heart, or peripheral nerves. Probes according to the present invention may be formed from relatively flexible materials, and in some cases, the probes may comprise nanoscale wires or other electronic components. Other embodiments are generally directed to systems and methods of making, using, or implanting such probes, kits involving such probes, and the like.
The various components described above may best be understood with reference to the figures. With reference to
One aspect of the present invention is generally directed to a probe for insertion into a tissue, or other material. The probe can be fully or partially inserted into the tissue or other material. The probe may be used to determine a property of the tissue or other material, and/or provide an electrical signal to the tissue or other material. This may be achieved using one or more nanoscale wires on the probe. For example, in certain embodiments, a probe comprising one or more electrodes may be inserted into an electrically-active tissue, such as the heart or the brain, and the one or more electrodes may be used to determine electrical properties of the tissue, e.g., action potentials or other electrical activity. In some cases, the one or more nanoelectronic threads of the probe are relatively porous to allow cells, axons, blood vessels, or other biological structures to infiltrate the threads. This may be useful for long-term applications, for example, where the probe is to be inserted and used for days, weeks, months, or years within the tissue. In some embodiments, neurons or cardiac cells may be able to grow around and/or into the one or more threads while they are inserted into the brain or the heart over extended periods of time.
In one embodiment, a system of the present invention comprises a probe comprising one or more nanoelectronic threads. In certain instances, elements of the probe are manufactured with a substrate-less device architecture and using electron-beam lithography to define the smaller features. Nanoelectronic thread of the present invention may comprise a densely-packed electrode array and versatile design patterns, with each thread comprising one or more electrodes. In some embodiments, each thread comprises at least eight electrodes. Each probe may comprise one or more nanoelectronic threads, wherein some embodiments of individual threads have a cross-sectional area of less than about 10 μm2. In other embodiments, individual threads have a cross-sectional area of less than about 5 μm2, less than about 2 μm2, or less than about 1 μm2.
Referring now to
While in vivo components of probes of the present invention are ideally minimized, ex vivo components may be larger as convenient to promote reliability and ease of use. In one embodiment, the ex vivo component of a probe of the present invention is about 5 mm×30 mm, but in other embodiments it might be 10 mm×30 mm, 30 mm×30 mm, or 2 mm×10 mm, or any other suitable size. By contrast, the in vivo components, i.e. the one or more nanoelectronic threads, ideally have minimal cross-sectional area, but may be long if the tissue sought to be instrumented is deep within the subject. In one embodiment, the nanoelectronic threads are differently sized in order to maximize geometric granularity. In various embodiments, the one or more nanoelectronic threads have a length in the range of about 0.1 mm to about 10 cm. For example, in certain embodiments, the one or more nanoelectronic threads are 0.5 mm long, 1 mm long, 2 mm long, 5 mm long, or multiple centimeters long. The individual nanoelectronic threads on a given probe need not be the same length as one another, and each thread may be a different length from any other thread on the same probe as needed.
Referring now to
Referring now to
The number and spacing of electrodes per thread and number of threads per probe may vary depending on the specific application. Each thread may have one electrode, or may have as many as 20, 50, 100, 300, or 500 electrodes. The maximum number of electrodes per thread is typically governed by the number of nanoscale wires necessary to connect each electrode to the probe. More wires means a larger thread cross-sectional area, which, in certain instances, increases the likelihood of negative impacts on the subject tissue due to implantation. In the embodiments shown in the figures, one layer of nanoscale wires is used, but multiple layers of nanoscale wires could be used in order to minimize the cross-sectional area per electrode. In one embodiment, the cross sectional area tapers down from the proximal end of the thread to the distal end of the thread, but in other embodiments the cross sectional area is approximately the same along the length of the thread. Each probe may also comprise a single thread or multiple threads. The number of threads per probe is limited only by the implantation footprint, which varies widely among subjects and implantation locations. Multi-thread embodiments described in the present disclosure are therefore not intended to limit the number of threads per probe in any way. Where multiple threads are used, the threads may be spaced apart from each other at a distance of about 10 μm apart to about 1 mm apart. For example, in certain instances, the threads may be about 15 μm apart, about 20 μm apart, about 25 μm apart, about 30 μm apart, about 35 μm apart, about 40 μm apart, about 45 μm apart, about 50 μm apart, about 75 μm apart, about 100 μm apart, about 150 μm apart, about 200 μm apart, about 250 μm apart, about 300 μm apart, about 400 μm apart, about 500 μm apart, about 750 μm apart, or about 1 mm apart. Threads may be spaced evenly or irregularly as desired for electrode positioning.
Individual electrodes may be evenly spaced apart as shown in
Referring now to
In some embodiments, the electrodes are finished with gold film, but the electrodes may alternatively be fabricated with lower-impedance materials, including but not limited to platina (Pt), Iridium Oxide, (IrOx) or a conductive polymer such as poly(3,4-ethylenedioxythiophene) (PEDOT) or poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). Electrodes should ideally be capable of detecting single unit action potentials with a high signal-to-noise ratio (SNR). In some embodiments, electrodes may include noise-suppression measures, for example a local reference electrode. In some embodiments, a system of the present invention comprises a computing device configured to perform offline analysis using a common mode reference to remove reference noise from the measured signal.
Exemplary electrode sizes are 5×8 μm and 12×15 μm, but electrodes may be larger or smaller as needed to measure the subject tissue. Typical electrodes could have a width (as measured horizontally in
Systems of the present invention may further include one or more rigid implantation assist devices, including for example a micro-shuttle device as shown in
Further aspects of the present invention relate to a system and method for implanting intracortical probes comprising one or more nanoelectronic threads in the tissue of a subject. In certain instances, by themselves, ultra-flexible nanoelectronic threads are not rigid or strong enough to be inserted into brain tissue unassisted. In one embodiment, a micro-shuttle device is used, with a “needle-and-thread” strategy to deliver the nanoelectronic threads to the desired location and depth in tissue. Referring now to
One alternative system and method of implantation is shown in
In some embodiments, the system of the present invention further comprises one or more controllers, connected to supply power and signals to, and to measure signals received from, probes of the present invention. In one embodiment, a controller is connected to a wired communication port of a probe, but in another embodiment the connection may be implemented via a wireless link. Power may be supplied to the controller via wires or wirelessly, and the controller may be positioned in vivo or ex vivo.
Systems of the present invention may further comprise one or more signal processing modules including but not limited to filtering, amplification, storage, and analysis modules, connected via wires or wirelessly to one or more probes. In some embodiments, the various signal processing modules are implemented as dedicated hardware circuitry, but the signal processing functions may also be implemented as software on a computing device. The purpose of the signal processing modules is to generate data and draw inferences from the measurements gathered from the various probes of the present invention. Filtering modules may include, but are not limited to high-pass, low-pass, or band-pass filters, Kalman filters, or any other filtering module used in the art. Amplification modules of the present invention may comprise one or more operational amplifiers or transistors, or may alternatively accomplish amplification through software means such as multiplication of analog values to add gain to some or all of the signals received. Storage modules may include any suitable means of data storage, including but not limited to hard disk drives, solid state storage, or flash memory modules.
In some embodiments, one or more of the threads of the present invention may be used for electrochemical sensing applications. For example, one or more electrodes on a thread may be coated with a capture agent capable of binding with a biochemical species of interest. Binding of the biochemical species to the capture agent results in a measurable change in conductance in the electrode. In this way, signal measurements from the electrodes on a nanoelectronic thread may be used to detect the presence, absence, or concentration of a chemical compound.
The present invention further includes a method of making an intracortical probe comprising one or more nanoelectronic threads. In one embodiment, a hybrid method involving both EBL and optical lithography is used to fabricate nanoelectronic thread devices with high throughput. EBL is used to define the implanted section (the “thread”) where dimensional constraints are more stringent, and photolithography with a more relaxed resolution requirement is used for larger structures that are not intended to be implanted in tissue. An example is shown in
Different components may be constructed with different EBL techniques, wherein each production step is individually optimized for high yield, throughput, and fabrication resolution. In one embodiment, the nanoscale wires and electrodes are produced using standard EBL with poly(methyl methacrylate) (PMMA) as the positive resist followed by metallization and lift-off. Nanoscale wire width and pitch are typically minimized, but may be optimized to accommodate for stitching errors, and in one embodiment nanoscale wires are fabricated with a minimum line width of 200 nm and a pitch of 400 nm, in order to accommodate stitching errors of 40-50 nm and to improve fabrication yield.
One or more insulation layers may be fabricated using non-standard EBL, using a negative resist, including but not limited to PMMA or SU-8. SU-8 requires a very low exposure dose of less than 4 microcoulombs per square centimeter (μC/cm2) and therefore a controlled dose of EBL is necessary to achieve the desired resolution and thickness. In one embodiment, EBL with 0.5 μC/cm2 was used. In another embodiment, EBL with 2.0 μC/cm2 was used.
Referring now to
When manufactured with sub-micron thickness, certain embodiments of the invention exhibit ultra-flexibility that precludes free standing in air.
Another aspect of the present invention is a method of implanting intracortical probes comprising one or more nanoelectronic threads in the tissue of a subject. A method of implantation using a device such as the one depicted in
In some embodiments, a solenoid may drive the one or more nanoelectronic threads into the tissue at a speed in a range of 1-10 m/s. The speed and position of the carrier block 940 may be monitored, for example using a digital encoder, a capacitive sensor, or other linear position monitoring means. Once the array of nanoelectronic threads 938 pierces the tissue 937, in some embodiments the micromanipulator may be employed to further change the implantation depth, at a much slower speed than the implantation speed of the linear actuator. In embodiments where a dissolvable adhesive is used, the method may include the step of waiting for the adhesive between the nanoelectronic threads and micro shuttles to dissolve, at which point the micro shuttles may be retracted from the tissue, leaving only the nanoelectronic threads behind, embedded in tissue 937 (as shown in 904). Although the linear actuator in the embodiment of
An alternative method of implantation involves parallel implantation of multiple nanoelectronic threads with the aid of an implantation assistance device as described above. Such methods allow for high throughput, parallel insertion of nanoelectronic threads with cellular-sized surgical footprints. As shown in
In some embodiments, fine control over the implantation process is achieved by adjusting the molecular weight of the PEG coating, so as to precisely control the duration of adhesion between the shuttle-device and the nanoelectronic threads. In this way, the implantation method may target deep brain structures. The microtrenches and microconduits may be made from any suitable material, including but not limited to tungsten microwires or carbon nanotubes. Many of the delivery methods of the present invention use low-cost components that can be readily adapted to the implantation of other flexible neural implants.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the system and method of fault-tolerant and scalable parallel computing of the present invention. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
Different components of the probes of the present invention require different EBL techniques to construct, which were individually optimized for high yield, throughput and fabrication resolution. Standard EBL using PMMA as the positive resist followed by metallization and lift-off was performed to define the interconnects and the electrodes. The highest resolution was required for the nanoscale wire interconnect traces, which is determined by the EBL sample stage stitching error, because the length of the thread (about 1 mm) was much larger than the size of the writing field in the EBL tool used in this work (80 μm). A minimum linewidth of 200 nm (pitch of 400 nm) was used for the nanoscale wires to accommodate stitching errors at 40-50 nm and to improve fabrication yield. Non-standard EBL was performed to construct the insulation layers, for which both PMMA and SU-8 were evaluated as negative EBL resists. These two resists were chosen for their good insulation, excellent tensile strength after hard-baking, and tunable thickness depending on the exposure doses. Because PMMA requires a high dose of about 40,000 μC/cm2 to crosslink, the required EBL exposure times are prohibitively long, even at high currents. Thus, SU-8 was chosen for higher fabrication throughput. However, SU-8 requires a very low exposure dose at less than 4 μC/cm2, which makes the fabrication outcome highly sensitive to small changes in EBL conditions and to the design patterns. Therefore, the EBL dose was tested and controlled with 0.5 μC/cm2 accuracy to achieve the necessary resolution and targeted thickness for every pattern.
A detailed step-by-step diagram is shown in
The resulting fabrication yield was about 70%, with little variation among different design patterns that required fabrication resolution ranging between 200 nm to 400 nm. The fabrication process has been thoroughly examined, and yield loss is believed to be mainly due to fabrication defects. All fabrication procedures, including the four EBL steps, seven photolithography steps, and five metal deposition steps, were performed manually, which makes it difficult to completely avoid micro-particles and scratches especially on the long, narrow nanoscale wire interconnect traces. The EBL exposure time was relatively short. For example, an entire 4″ wafer that contains 8 nanoelectronic threads and a total of 256 electrodes typically requires exposure times of approximately 5 minutes for the insulating layers composed of SU-8, 2 hours for the nanoscale wire interconnects and 15 minutes for the electrode layers. However, the fabrication throughput was mostly limited by the two-step iterative alignment process during EBL that was necessary for sub-100 nm registration accuracy across the entire wafer. Despite the relatively low throughput compared to photolithography, the application of EBL greatly improved the fabrication resolution and reduced the probe's overall dimensions. It is believed that the fabrication yield, throughput and resolution can be further improved using more advanced EBL equipment.
The electrode impedance of the EBL defined probes was typically above 1 MΩ at 1 kHz, which was consistent with the small dimensions of the probes. Although single unit action potentials were detected with a sufficiently high SNR, the high impedance leads to a relatively high noise level and a narrow detection range. The signaling characteristics may be improved by using low-impedance materials for constructing the electrodes, such as Pt, IrOX and PEDOT coating. The noise levels during awake recording were elevated due to motion-induced coupling. This noise can be suppressed by using a local reference electrode or by offline analysis using a common mode reference.
Several technical challenges were overcome in the fabrication of probes, including precise inter-layer alignment across the entire wafer and the application of SU-8 as a sensitive negative e-beam resist to construct the insulation layers. A minimum linewidth of 200 nm was achieved (pitch of 400 nm) for the nanoscale wire interconnect traces and sub-100 nm inter-layer alignment. The total thickness of the nanoelectronic threads was determined mostly by the thickness of the two SU-8 layers, and was precisely controlled to be 0.8-1 μm by fine tuning the e-beam exposure dose of SU-8 with 0.5 μc/cm2 accuracy.
As shown in
A straight segment of carbon fiber was attached to a stainless steel micro needle (prod #13561-10, Ted Pella, Inc.) for convenient handling. It was then cut to the designed length (2-3 mm) using a focused ion beam (FIB). An anchor post was micro-milled at the tip of the shuttle device using FIB to shape a well-defined micro-post (approx. 3 μm in diameter, 4 μm in height).
Wild type male mice (C57BJ/6, 8 weeks old, Taconic) were used in the experiments. Animals were housed at 22° C. (12 hour light/dark cycle, food and water ad libitum).
Mice were anesthetized using isoflurane (3% for induction and maintained at 1-2%) in medical grade oxygen. The skull was exposed and prepared by scalping the crown and removing the fascia, and then was scored with the tip of a scalpel blade. A 3 mm×3 mm square craniotomy was performed with a surgical drill over the somatosensory cortex. Dura mater was carefully removed to facilitate the delivery. After probe implantation, the remaining flexible segment of the probe, which connected the bonding pad on the substrate with the electrodes inside the brain, was routed to the edge of the cranial opening. The exposed brain was then protected by artificial cerebrospinal fluid (ACSF) and a coverslip #1 (Fisher Scientific) fit into the cranial opening. The space between the coverslip and the remaining skull was filled with Kwik-sil adhesive (World Precision Instruments). After the skull was cleaned and dried, a layer of low viscosity cyanoacrylate was applied over the skull. An initial layer of C&B-Metabond (Parkell Inc.) was applied over the cyanoacrylate and the Kwik-sil. A second layer of Metabond was used to cement the coverslip and the probe carrier chip to the skull.
In typical procedures, a flexible section of a nanoelectronic thread was placed on the brain surface where dura mater was removed. The shuttle device was mounted on a micromanipulator (MP-285, Shutter instrument) vertically, and positioned atop the engaging hole at the end of the probe. Referring to
Mice were allowed a 1-2 week recovery period after surgery. For recording, mice were head-constrained on a custom-made, air-supported spherical treadmill to allow walking and running, similarly to setups previously used for optical imaging. The treadmill was made of an 8-inch diameter Styrofoam ball (Floracraft) levitated by a thin cushion of air between the ball and a casting containing air jets. Voltage signals from the probes were amplified and digitized using a 32-channel RHD 2132 evaluation system (Intan Technologies) with a bare Ag wire inserted into the contralateral hemisphere of the brain as the grounding reference. The sampling rate was 20 kHz, and a 300 Hz high-pass and a 60 Hz notch filter were applied for single-unit recording. Mice and the amplifier were placed in a noise-attenuated, electrically shielded chamber. Impedance of the recording electrodes was measured using the same setup at 1 kHz prior to recording.
Two-photon (2P) imaging was performed in a laser scanning microscope (Praire Technology) equipped with a 20× water immersion objective (NA 1.0; Zeiss) and a Ti: sapphire excitation laser (MaiTai DS, Spectra-Physics) at a few weeks to months post-implantation. The laser was tuned to 810-910 nm for 2P excitation (power 3.0-50 mW, dwell time 4.0-6.0 μs). Fluorescence emissions were detected simultaneously by two standard photomultiplier tubes with a 595/50 nm filter (Semrock, US) for “red” fluorescence emission and a 525/70 nm filter (Semrock, US) for “green” fluorescence emission. Mice were anesthetized using isoflurane (3% for induction and 1.5% during experiment) in medical grade oxygen to maintain full immobility during imaging and placed in a frame that stabilized the head on the microscope stage. Anesthetized animals were given FITC-dextran (0.1 mL, 5% w-v, Sigma) retro-orbitally to label blood vessels prior to imaging. To facilitate imaging the probe-tissue interface beyond superficial cortical layers, the threads were doped with sulferhodamine 6G (Sigma) in the insulating layers and delivered at an angle of about 45 degrees with respect to the skull.
To prepare the histological samples, mice were given lethal intraperitoneal injections of 0.15 mL ketamine mixed with xylazine (10 mg/ml xylazine in 90 mg/mL ketamine) and then perfused intracardially with oxygenated, cold (˜4° C.) modified ACSF (2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2, 7 mM dextrose, 205.5 mM sucrose, 1.3 mM ascorbic acid, and 3.7 mM pyruvate) followed by 4% paraformaldehyde in 0.02 M phosphate-buffered saline (PBS). Brains were cryoprotected in a 30% sucrose/4% paraformaldehyde solution overnight. Tissue was sectioned into 20-50 μm slices perpendicular to the probe using a Leica CM1950 cryostat (Leica Microsystems). The slices were washed (3×5 min) and incubated in hot sodium citrate solution (85° C.-95° C., 0.01 M in H2O) for 30 mins for antigen retrieval. Then, the slices were washed (3×5 min), incubated in blocking solution and permeabilized (0.5% Triton X and 10% normal goat serum (Sigma) in PBS) for 3 hours at room temperature, washed (4×5 min), and incubated in fluorophore conjugated antibodies for 24 hours at 4° C. Reagents used for neurons are (Millipore): Alexa Fluor 488 conjugated anti-NeuN antibody, clone A60.
The spatial-temporal correlation of the four electrodes improved the detection and sorting fidelity of single-unit action potentials (
Straight Tungsten microwires of a variety of diameters from 25-50 μm (W5607, Advent Research Materials) were manually cut to the desired length of 4-6 mm. Microtrenches of desired width and pitch were microfabricated on silicon substrates (4″ wafer, 900 nm SiO2, n-type 0.005 V*cm, University Wafer) using standard planar photolithography. SU-8 photoresist (SU-8 2075, MicroChem Corp.) was spun on the silicon substrate at 4000 rpm for 45 seconds, followed by the standard SU-8 photolithography process which defined microtrench structures with a height of 60 μm. After hard baking (180° C., 3 hours), the 4″ wafer was cut to small rectangular pieces using a dicing saw (7100 Dicing system, ADT), which provided the carrier chip for shuttle devices. The width of the shuttle device carrier chip matched the width of the multi-shank thread substrate at 2-4 mm, and the length was about 3 mm, which provided sufficient margin for microwire alignment and convenience of handling. Tungsten microwires were placed into the trenches manually, resulting in a linear array of microwires at the pitch predefined on the carrier chip. The microwires protruded from the edge of the carrier chip by a few millimeters, where the ultra-flexible segment of nanoelectronic thread is attached. A thin layer of super glue (Loctite) was used to fix microwires in the trenches on the carrier chip without overflowing. Small drops of epoxy (Loctite) were placed on all four corners of the carrier chip to act as spacers between the chip and the probes when assembled together face-to-face. The shuttle device was then baked at 180° C. for 30 minutes for the epoxy to cure. Electrochemical etching (0.8 M KOH, graphite as anode, at 2.5 V for 30 seconds) was performed after the assembly procedure to sharpen the tungsten microwires.
Nanoelectronic threads were microfabricated using planar photolithography and multi-layer architecture, and diced to individual devices. A connector to external I/O was mounted on the contact pads and the flexible segment was released from the substrate by etching off the sacrificial layer. After the nanoelectronic thread was rinsed in phosphate-buffered saline (PBS) and allowed to dry in air, a shuttle device was placed on the nanoelectronic thread. The shuttle device was manually aligned under a stereomicroscope (A605, Leica microsystems), so that the lateral position of individual microwires matched the positions of nanoelectronic threads, and the microwires protruded from the end of the nanoelectronic threads by 10-20 μm. A customized clip was placed to gently press together the shuttle device carrier chip and the substrate, while a small gap between them was created by the epoxy drops at the corners of the shuttle device to protect the threads. After being rinsed in distilled water, the thread-shuttle device pair was dipped into a PEG solution (4% w/v, molecular weight of 4-5000 kDa, Alfa Aesar) and slowly pulled out so that the surface tension at the solution-air interface aligned the threads with the microwires. A 34-gauge syringe needle was used to manually assist attaching the threads onto the microwires at the air-solution interface. After the microwires and threads were aligned and pulled out of the solution, additional PEG was applied between the shuttle device carrier chip and the thread substrate to attach them together. The clip was then released and the assembled device was allowed to dry in air before implantation.
Methods of fabricating the integrated microtrench array (see
Methods of fabricating microwire arrays guided by microconduit arrays differ further from the methods described above. PTFE tubes (sub-lite-wall tubing, Zeus) of outer diameter 200 μm, inner diameter 100 μm and length of 6 mm were manually stacked to form a variety of structures, including linear, rectangular and triangular arrays. Epoxy (Loctite) was applied to permanently fix the structures. Pre-cut straight tungsten microwires as used in previous methods were inserted into a selection of the conduits. The microwires protruded from the tube edge by 3-5 mm on both ends while PEG solution was applied at one end of the conduits to temporarily fix the microwires. The microconduit array was then mounted on the nanoelectronic thread substrate using Epoxy (Loctite), and aligned under a stereomicroscope so that the lateral position of individual microwires best matched the positions of threads and the microwires protruded from the ends of the threads by 50-100 μm. The assembled device was then partially soaked in Ni etchant (TFB, Transene Co. Inc.) for 2 to 4 hours at 25° C. to release the flexible segment of threads. After being rinsed in distilled water, the threads were attached to the microwires in PEG solution as described above. For some structures, manual manipulation of the threads using a 34-gauge syringe needle was necessary to attach threads to the designated microwires. The assembled device was allowed to dry in air for implantation.
Parallel implantation of nanoelectronic thread linear arrays was demonstrated with the assistance of microwire arrays. In order to conveniently and precisely align microwires with threads, a microtrench array was fabricated on a shuttle device base chip (
Parallel implantation of nanoelectronic thread arrays with integrated microtrenches was demonstrated, and is described with reference to
Parallel implantation of nanoelectronic thread arrays with microconduit arrays was demonstrated, and is described with reference to
In the various parallel implantation solutions of the present invention, PEG solution was used to attach nanoelectronic threads to microwires, the PEG dissolving shortly after contact with brain tissue. The ability to control the release time of the threads is crucial for precisely controlling the insertion speed, which often affects the degree of surgical tissue damage. Typical insertion speeds for neural probes range from slow insertion at about 5 μm/s to fast insertion at about 1 mm/s, which requires attachment durations from 2 seconds to 400 seconds for targeting subcortical brain structures such as the hippocampus. The dissolution time of PEG in living tissue may be controlled by the molecular weight of PEG. For quantitative comparison, a constant concentration of PEG solution (4% w/v) was used, and the molecular weight was systematically varied to control the thread attachment duration. In-vitro tests were performed in PBS, and the dissolution time was found to be monotonically dependent on the molecular weight (
Two months after implantation, the tissue-thread interface was examined, with the results shown in
The above experiments demonstrated the possibility of integrating neural electrode arrays within a subcellular form factor and their implantation in a high-density, scalable manner. By applying nanofabrication techniques on unconventional substrate-less design of neural probes, the physical dimensions of neural probes were drastically reduced. Combining these nanofabricated ultra-flexible probes with minimally-invasive implantation methods at subcellular surgical footprints, a practical approach was developed to overcome current physical limits in the design and implantation of intracortical neural electrodes, which paves the way for full-coverage neural recording and complete circuit-level mapping of neural activity. More importantly, because little tissue response was observed for single or densely packed nanoelectronic threads after long-term implantation, the technique has the unique potential to chronically track the evolution of neuronal circuits for longer-term evaluation of brain development, for example learning and memory.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application is a '371 national phase entry of PCT application no. PCT/US18/49853, filed on Sep. 7, 2018, which claims priority to U.S. provisional patent application No. 62/555,798, filed on Sep. 8, 2017, both of which are incorporated herein by reference in their entireties.
This invention was made with government support under Grant no. W81XWH-16-1-0580 awarded by the Army Medical Research and Material Command, ARMY/MRMC. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US18/49853 | 9/7/2018 | WO | 00 |
Number | Date | Country | |
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62555798 | Sep 2017 | US |