Robust minimally-invasive microfabricated stainless steel neural interfaces for high resolution recording

Domain: chemistry
Published: 2026-01-09
Editorial outcome: minor_revision

Abstract
Understanding brain function and developing effective neurotherapeutics require high-resolution electrophysiologyical recording across large primate brains. To achieve this goal, minimally invasive, compact, long, and high-density implantable neural probes are needed. Conventional silicon-based probes, designed for rodents, cannot be directly scaled to larger brains because silicon’s brittleness restricts long, high–aspect-ratio designs. Stainless steel is a biocompatible, resilient, and less brittle alternative for making neural probes, though its microfabrication is less explored. Here, we introduce “steeltrodes”, customizable microfabricated stainless steel neural probes enabling high-density multi-layer electrode integration. We demonstrate 8 cm long, ~300 µm wide probes, featuring rigid shanks with optional flexible cables for in vivo high-resolution laminar recording from macaque auditory cortex. In rats, these probes can be safely implanted through intact dura with minimal cortical damage. High-fidelity recordings of single units and local field potentials in macaques highlight the potential of steeltrodes for translation to human applications, enabling both inter- and intraoperative neural recordings.

Introduction
Over the last decades, novel manufacturing and microfabrication processes have revolutionized neural probe technology for rodents1–5. Based in large parts on the adaptation of silicon as the material platform of choice, it has been possible to massively increase recording channel density using high-resolution lithography and microfabrication processes and to add new functionalities such as optical stimulation and imaging and chemical sensing5–8. However, silicon has a low fracture toughness and therefore, it is very brittle. As a result, it has been challenging to adapt these new tools to the larger brains of primates. Probes for primates need to be an order of magnitude longer (4–15 cm long as opposed to just a few mm-long rodent probes), while at the same time, maintaining a very small cross-section to minimize tissue damage9. As a result of its high-aspect ratio, a typical NHP (non-human primate) probe requires substantial rigidity and robustness to endure the stresses of implantation and post-implantation when inserted without rigid inserters.

Nevertheless, silicon-based neural probes have been used in NHPs and humans. However, the implantable length of those probes is usually shorter than 3 cm which limits the recording to the superficial cortical regions of large animals7,10,11 and humans12. There are also reports of silicon probes shattering during insertions into human brains12. Michon et al. reported thin silicon probes with 4-cm-long shanks integrated with a custom-designed microdrive array for recording from large ensemble of neurons in rats13. These thin silicon probes are somewhat flexible. However, the implantable shank length was limited to 7 mm due to physical constraints of the custom microdrive array. Recently, a silicon-based high-density NHP probe with 45 mm shank has been reported for recording from deep brain structures in macaques14. However, due to silicon’s inherent fragility, the use of specialized equipment and implantation hardware are essential for ensuring proper alignment of such probes to reduce the likelihood of the probe breaking during insertion into the deeper areas of the brain9,15.

In addition to silicon, polymer materials have also been used to implement neural probes for rodents and large animals. Compared to silicon, polymers are much less vulnerable to fracturing and much more flexible. Polymer-based neural probes have been shown to minimize tissue damage upon implantation due to their significantly smaller Young’s modulus16–19. However, lower rigidity of polymeric probes makes them more difficult to implant, which necessitated designing new schemes for implantation, including temporarily stiffening neural probes by coating bioresorbable materials to overcome the implantation force without buckling20,21. Pothof et al. reported microfabricated polymer-based Stereo-electroencephalography (SEEG) probes with high-density electrodes on a long implantable shank of 3 cm22. In this work, first, planar flexible polymer neural probes are microfabricated and then manually assembled to form a cylindrical shank, infilled with an epoxy. These neural probes have many electrodes (32–128) and the cylindrical cross section is rather large (~0.8 mm diameter). This work exhibits an interesting combination of microfabrication process and manual assembly towards realizing high-density neural probes for large animals.

In summary, the material platform developed for rodent probes cannot be directly translated to fulfill the challenging design requirements of high density, compact, yet robust neural probes for primates and humans. This gap calls for new material platforms and novel microfabrication processes. Given its highly desirable mechanical and biomedical properties, austenitic stainless steel is an excellent candidate for implementing large aspect ratio neural implants. Stainless steel 316 has a fracture toughness of 112–278 MPa m23, nearly two orders of magnitude higher than that of silicon, which is only 0.7–0.9 MPa m24. Fracture toughness, K Ic is a material property that characterizes the material’s robustness to fracturing. Fracture toughness is a function of stress (\sigma c) required to fracture the bulk material for a given crack length (a), as described in this equation: K IC=\sigma c \pi a. Therefore, for the same crack length (a), compared to silicon, which has a much lower fracture toughness24, it would take almost two orders of magnitude higher stress to fracture a stainless steel device. As a result, a neural probe with extreme aspect ratio fabricated on stainless steel will be significantly less likely to fracture compared to a silicon neural probe. Therefore, stainless steel neural probes would be more robust and resistant to fracturing. In addition, stainless steel 316 has a high Young’s modulus of 190–203 GPa23, which is comparable to that of silicon (140–180 GPa24). Since the overall stiffness is determined by the device geometry and the material elastic modulus, a stainless steel probe offers a comparable level of stiffness to a silicon probe with similar dimensions, but with much less vulnerability to fracture. Therefore, unlike polymer-based probes, stainless steel probes can be implanted reliably without the need for additional stiffener layers.

Stainless steel also has high corrosion resistance and is biocompatible. It has been used to manufacture biomedical implants such as prosthetics and coronary stents25–29. Most devices fabricated using this material platform are made using macro-scale manufacturing and machining techniques. Commercially available stainless steel neural probes for NHP are manually assembled (Plexon Inc.). Such neural probes are used for NHP experiments because of their robustness and reliability. The manual manufacturing of such neural probes puts constraints on the density of microelectrode channels (e.g., currently, up to 64 channels with a minimum pitch of 50 μm) and limit the yield. In addition, the probes are very expensive30 and while these probes are typically of very high quality, they are nevertheless subject to human error due to the non-scalable hand-assembly processes.

Optimized scalable microfabrication and micromachining processes are necessary to leverage the excellent material and mechanical properties of stainless steel to design miniaturized biomedical devices such as high-channel density neural probes with micron-scale features on stainless steel. In the case of silicon probes, fabrication and micromachining process had benefitted from decades of research and development in the MEMS/NEMS (Micro-/Nano-Electromechanical Systems) and CMOS (Complementary metal–oxide–semiconductor) electronic industries1,31–33. However, the same processes cannot be readily translated to stainless steel. Moreover, the micro- and nanofabrication processing for stainless steel is quite challenging and comparatively underdeveloped and underexplored. In this paper, we show that it is possible to overcome these challenges and enable micro- and nanofabrication for stainless steel. Robustness of stainless steel material combined with the unmatched scalability of planar microfabrication paves the way for realization of different biomedical devices with extreme aspect ratios, not currently feasible using traditionally used material platforms. These devices will be robust and reusable, customizable and can be mass produced.

In this paper, we show case the material platform and a scalable microfabrication process by realizing high-density, ultra-compact stainless steel neural probes (aka steeltrodes), that are long enough to record from anywhere in the NHP brain. Recording neural activity across different areas of brain with high resolution and minimal damage to the brain tissue, especially in larger animal models and humans has remained elusive mainly due to the lack of a robust and minimally invasive implantable neural interface platform that can be mass produced and reliably used. The steeltrodes discussed in this paper provide a path towards high-resolution distributed neural recording from various brain areas, as shown in Fig. 1a, where multiple steeltrodes are implanted at different depths within the brain. Each of the penetrating neural probes is a hybrid polymer-stainless steel neural probe with a rigid shank that can be designed to be fully stiff (Fig. 1b) or monolithically connected to a flexible cable (Fig. 1c, d) to route the recorded signals to the backend circuitry, while minimizing the tethering force and damage to the brain tissue due to brain micromotions34. Steeltrode features high-density microelectrodes with highly customizable size and arrangement (Fig. 1e–g). This high degree of flexibility in the design, combined with a repeatable high-throughput microfabrication process, enables a plethora of applications, ranging from basic science research on rodent and NHP models to acute and chronic neural interfacing with human neural tissue for clinical and therapeutic applications. As a demonstration of the capabilities of the platform, steeltrodes with long implantation length (8 cm) with a very thin cross section of 140 µm × 280 µm with high-density microelectrodes (16–100 channels, with pitches ranging from 25 µm to 2.5 mm) were fabricated and tested in macaque auditory cortex which is located deep in the lateral fissure and is notoriously difficult to reach. With an implanted steeltrode, we were able to reliably record single-unit and multi-unit activity as well as local field potentials (Fig. 1i). Given their compactness, high-density design of electrodes and their exquisite robustness, these steeltrodes can be translated to clinical applications such as intraoperative neural recording from human subjects, in the future.

While our steeltrode design was optimized for fabrication of NHP probes, it can be readily scaled down to make mm-long probes for rodents (inset of Fig. 1b), where we can still benefit from the robustness of stainless steel over conventional silicon-based neural probes to minimize the risk of failures during implantation and experiment and introduce a robust and re-usable neural recording platform. Despite having smaller aspect ratios, rodent silicon neural probes are still susceptible to fracturing owing to the material properties which makes the manipulation of such probes challenging. Moreover, implantation of these conventional silicon neural probes usually requires removing the protective dural layer, that can cause trauma and exposing a region of the brain larger than the probe cross section, that can compromise the cranial pressure. In contrast, since steeltrodes are resilient against fracture, they can be reliably inserted into the neural tissue multiple times and their handling and manipulation is safe and repeatable. Moreover, these robust neural probes can be implanted through rodent dura, obviating the need for removing dura, thus reducing damage to the superficial layers of the neural tissue. Therefore, in addition to enabling high-fidelity recording from deep brain regions of large animals, the microfabrication process on stainless steel discussed in this work can also enable manufacturing of mechanically robust stainless steel probes with smaller implantable shanks for recording from small animals without the need for surgical removal of dura.

Results
High resolution neuronal recording from large brains using steeltrodes
Our large-animal steeltrodes feature up to 8-cm-long stainless steel shanks with a compact small cross-section of 140 µm × 280 µm. Stainless steel has high resistance to fracture, making our implant mechanically robust even at such an extreme form factor. We have demonstrated probes with 16–100 electrical recording electrodes on a single shank, with much larger than the channel density of commercially available stainless steel neural probes for large animals30. Such high-density electrodes can be implemented on both fully rigid shanks or on hybrid rigid-flex shanks, where the implantable rigid stainless steel shank is attached to the backend circuitry via highly flexible tether cables. Figure 1h shows SEM image of the tip of a steeltrode showing its smooth sidewalls, and 15-µm-diameter microelectrodes with a pitch of 50 micron. Images of fully rigid and hybrid rigid-flex steeltrodes are shown in Fig. 1b, c, respectively. This flexible cable in the rigid-flex design allows the implantable portion of the probe to move independently of the backend part that accommodates electrical connectors and circuitry. As a result, once inserted into the brain, the implanted shank of the stainless steel probe can move independently of the backend, reducing tissue damage and preserving signal quality by mitigating tethering forces from skull attachment during brain micromotions and sudden bodily movements57,58. This is particularly helpful for long-term neural recordings. Steeltrodes are packaged with flexible PCBs and zero-insertion-force (ZIF) connectors to ensure a compact footprint. Due to its small form-factor packaging and narrow cross section, multiple steeltrodes can potentially be implanted at different depths of the non-human primate brain for simultaneous high-density neural recording from different brain regions (Fig. 1a). A long and robust implantable stainless steel shank enables access to deep parts of macaque brain. With 8-cm-long steeltrodes, we have demonstrated high-resolution electrophysiology recording from the auditory cortex of macaque brain (Fig. 1i). In summary, our novel neural interface utilizes the excellent mechanical properties of stainless steel and the unmatched customizability of lithographically defined microscale features to enable distributed simultaneous recording of neural activity from deep regions of the large animal brain.

High-resolution lithography on commercially available rough stainless steel using a planarization process
We have developed a fabrication process that allows high-density electrodes and electrical traces lithographically defined on flexible polymer layers monolithically integrated on the stainless steel substrate. This manufacturing method utilizes planar micro- and nano-fabrication processes on stainless steel substrates and mostly incorporates parallel fabrication processes and can be scaled for mass production of such devices. Commercially available thin (thickness of 125 µm and lower) stainless steel sheets have high surface roughness. Topography and surface roughness of the stainless steel complicate high-resolution lithography on these substrates. It is possible to polish and smoothen the surface of thin stainless steel substrates. Such wafers are even commercially available (Valley Design Corp., Shirley, MA, USA). However, the polishing process can induce excessive stress in the thin film, which can lead to severely bowed wafers which are not suitable for planar fabrication processes. Therefore, a surface planarization process is essential to achieve high-resolution lithography on commercially available low-stress stainless steel substrates (SS 316, McMaster Carr), which have an inherent surface roughness with a coarse texture. Conformal deposition of a thin-film polymer insulation material is not sufficient to planarize the surface roughness of stainless steel. For example, 7 µm Parylene C, deposited via a chemical vapor deposition process, only marginally reduced the surface roughness. Due to this roughness, lithographic resolution on Parylene C on stainless steel was therefore limited to ~2.5 µm. Whereas spin-coating SU-8 2010 of the same thickness (7 µm) proved to be much more effective in planarization; features with dimensions down to ~1 µm can be patterned on stainless steel (Fig. 2a). SU-8-based planarization process was used for realizing steeltrodes with fully rigid shanks (details discussed in Methods section). In addition to planarization, SU-8 functions as the top and bottom insulation layer for the neural probe. Moreover, since SU-8 is photodefinable, both the device outlines and the electrode openings can be patterned directly with photolithography in SU-8, without the need for plasma-based dry etching processes. Dry etching processes used for Parylene C, PDMS, and Polyimide (non-photodefinable) can lead to oxidization of the Platinum electrode surface that necessitates an additional electrochemical cleaning processor a physical etching process to restore the electrode impedance to its typical values59,60. Therefore, using SU-8 insulation not only contributes to expedite the fabrication process for rapid prototyping of different variants of rigid steeltrodes, but also mitigates the need for post-fabrication treatments of the electrode surfaces. Figure 2b shows a simplified fabrication process flow for realizing steeltrodes with SU-8 insulation.

To ensure the flexibility of tether cable for rigid-flex steeltrodes, the insulation material need to have low flexural modulus. Compared to SU-8, Parylene C has almost 1000 times lower flexural modulus and as a result, its mechanical properties are better suited for steeltrodes with flexible tether cables. However, as shown in Fig. 2a, Parylene C itself is inadequate to planarize the stainless steel substrate to enable high-resolution lithography. To address this issue, we have developed a process for planarizing stainless steel with a combination of PDMS and Parylene C. PDMS has even lower Young’s modulus compared to Parylene C and is suitable for realizing integrated flexible tether cables52. However, PDMS is porous and has a high level of permeability, which makes it unsuitable as an insulation layer5. Moreover, its low surface energy leads to poor adhesion to photoresist, making photolithography directly on PDMS layer problematic61. To overcome these issues, a 7-µm-thick conformal layer of Parylene C is deposited on the PDMS layer at room temperature. Parlyene C layer combined with the PDMS layer forms the bottom insulation layer for the probe and functionalizes the PDMS surface for contact photolithography. As shown in Fig. 2a, a bilayer stack of PDMS–Parylene C provides sufficient planarization to enable high-resolution lithography (~1 µm features) to manufacture high-channel density steeltrodes with flexible tether cables for potential long-term recording in different animal models. With a 1 µm space and trace, the crosstalk between adjacent traces over the entire length of the probe is minimal, as shown in the previously reported crosstalk analyses44. Additional fabrication details are provided in the Methods section, with process steps shown in Supplementary Fig. S2.

Multilayer lithography on stainless steel substrate facilitates large number of channels while retaining a small cross section of the probe
We can leverage multilayer metal trace fabrication on stainless steel to drastically increase the channel count of the neural probe without increasing the shank width. Such multilayer processes have been demonstrated for flexible probes with Parylene C62,63 and SU-848 insulation. On Stainless steel substrate, we have demonstrated the feasibility of multilayer process with SU-8 insulation. Figure 2c illustrates a simplified microfabrication process flow diagram using which we have implemented a multilayer steeltrode on SU-8 with two vertically stacked metal layers (Pt/Au/Pt or Cr/Au/Pt stacks). Figure 2e, f shows micrographs of vertically stacked metal layers and the microelectrodes realized on stainless steel with SU-8 insulation and intermediate layers. We have demonstrated a 32-channel stainless steel probe implemented by vertically stacking two layers of metal traces separated by 2.5-µm-thick SU-8. In both layers, metal traces are 4 µm wide with 4 µm pitch, similar to the 16-channel variant of the probe with a single metal layer. Using this multilayer stacking of metal traces, we can scale up the number of channels significantly with minimal increase of the shank thickness. Moreover, this approach obviates the need for even narrower interconnect features using higher resolution lithographic techniques (i.e., deep-UV lithography, or electron-beam lithography) to retain the cost-effectiveness and scalability of the manufacturing process. When using narrower interconnects, especially in multi-layer designs, the fabrication process has to be optimized to achieve a high yield.

Highly customizable, high-density electrode arrangements on steeltrode enabled by high resolution single and multi-layer lithography
Taking advantage of photolithography-based microfabrication process that we have developed on stainless steel, we could realize steeltrodes with highly customizable recording electrodes with variable channel densities and electrode arrangements. We have fabricated devices with 16 and 32 channels with inter-electrode separation as small as 25 µm. Different versions of these probes with different electrode arrangements have also been realized. Figure 1e–g shows microscope images of steeltrodes with linear, edge and honeycomb arrangements. Such devices can enable systematic study of the effect of recording electrode sizes, densities, and arrangements in terms of single-unit isolation yield as well as temporal and spatial frequencies in neural activities in the primate brain.

Apart from high degree of customizability and channel density, our proposed microfabrication platform also facilitates realization of high-channel density robust neural probes on stainless steel. The 16 and 32 channel devices that we have demonstrated utilize conservative metal traces with 2.5 µm width. However, as shown in Fig. 2a, we can pattern metallic features with much smaller dimensions on stainless steel. Using a conventional contact photolithography process (at the wavelength of 320 nm, Karl Suss MA6), we have demonstrated line-widths of 1 µm with 1 µm inter-trace space on stainless steel substrate. This critical dimension enables fabrication of probes with ~100 channels on a narrow shank spanning 250 µm, using only a single layer of metallic traces. Moreover, by leveraging the multilayer fabrication process discussed in the last section, we can massively scale up the number of channels. For example, using a 4-layer metallic trace lithography, we can accommodate ~400 channels on the same 250-µm-wide shank with an active recording span of 20 mm (assuming linearly arranged microelectrodes with 50 µm pitch). Therefore, we can realize steeltrodes with substantially larger channel counts compared to commercially available stainless steel neural probes using the conventional lithography process. It is worth noting that there is potential to further increase the channel count and density by employing advanced lithography techniques such as deep-UV lithography or electron-beam lithography to pattern sub-micrometer wide metallic traces on polymer layers43,55. However, it’s important to acknowledge that these advanced lithographic processes are sequential in nature compared to conventional contact lithography. Consequently, the increase in channel count and density comes at the expense of manufacturing scalability. In contrast, our utilization of conventional photolithography processes ensures the fabrication of steeltrodes with substantially higher channel counts and density while retaining the crucial aspects of manufacturing scalability for high-throughput production.

High-throughput laser micromachining process for precise singulation of individual steeltrodes
Following wafer-level microfabrication, we employ a customized laser micromachining process (Micron Laser Technology, Inc., Hillsboro, OR, USA) to release the steeltrode devices with long implantable shanks from the stainless steel substrate. A 200W fiber laser (kerf: 20 µm, 300 PSI air assist) was utilized to ablate the stainless steel around the probe outlines to release each of the probes from the wafer. Using a combination of computer-aided-design (CAD) file and lithographically defined alignment marks on the stainless steel wafer, the laser’s position and movements were precisely controlled within ±10 µm. This process allows customization of the tip profile (shape, opening angle, etc.) through CAD specification. Supplementary Fig. S3a shows the laser etching trajectory used to singulate a steeltrode on a processed stainless steel wafer, along with representative images (Supplementary Fig. S3b) of the steeltrodes after their release via laser micromachining.

Adapting commonly used planar microfabrication processes for stainless steel, followed by precision laser micromachining makes our neural interface platform promising for highly scalable wafer-scale fabrication.

Monolithic flexible tethering cable is realized by selective electrochemical etching of stainless steel
Brain micromotions is reported to be one of the factors causing tissue response during chronic recording. Rigid probes tethered to the skull can impart force on the brain tissue due to the relative motion of brain with respect to skull. Flexible tether cables on probes allow rigid neural probe to move with brain micromotion and minimize tethering forces. In our proposed hybrid stainless steel-polymer platform, we have demonstrated post-fabrication processing to manufacture probes with highly flexible polymer tether cables that separate the rigid implantable portion of the probe from the backend ZIF connector that connects to the recording interfaces. This process is based on electrochemical etching (details available in Methods section) of some portion of stainless steel shank of the microfabricated rigid stainless steel probe insulated with PDMS–Parylene C. Steeltrode variant with PDMS–Parylene C is preferred over SU-8 for implementing tether cables since SU-8 has few orders of magnitude higher flexural modulus compared to Parylene C. Therefore, compared to SU-8, tether cables made from Parylene/PDMS are significantly more flexible. Figure 1c, d show micrographs of steeltrode with PDMS/Parylene C tether cable.

Rigid stainless steel backend allows fast, efficient, and reusable ZIF connectors for interfacing with the recording electronics
Packaging of high-channel-count neural implants with the recording amplifier circuitry is challenging. Wire bonding, one of the most widely used packaging processes, is time-consuming and requires professional expertise to achieve high yield when bonding to high-density contacts to minimize the form factor of the probe backend. It also requires epoxy encapsulation to protect the fragile connections. Our stainless steel probes are interfaced with a custom-designed flexible printed circuit board (fPCB) via a compact, low-pitch zero insertion force (ZIF) connector (Hirose FH26 Series). The backend bond pads of our probes are designed to complement the contacts of the ZIF connector. Stainless steel support layer underneath the bond pads of the backend of the probes makes it convenient for plugging the probes directly into the ZIF connector sockets without any additional stiffener layers. Moreover, the probes can be easily unplugged from the connectors and the same ZIF connector and PCB assembly can be reused multiple times with different probes. The simplified packaging process enabled by the stainless steel backend provides effortless, hassle-free, and swappable interface to recording circuits with high yield while maintaining a compact profile of the probe backend.

Electrodeposition of conductive polymers on electrode surface to enable high-fidelity neural recording
Electrochemical impedance of recording electrodes is an important criterion that determines the extracellular recording quality and the background noise. We have characterized the electrochemical impedance of 15-µm-diameter Pt electrodes on steeltrodes in 1X phosphate-buffered saline (PBS). At 1kHz frequency, the electrodes yield an impedance of 1.07 ± 0.093 MΩ (N=14 channels) in PBS solution (Supplementary Fig. S4a). Electrochemical impedance of < 2 M-ohms has been reported to be sufficient for single unit isolation even with commercial neural amplifiers with relatively low input impedances64. However, lower impedances are desired to reduce thermal and biological noises during recording. Different materials such as Pt grass, Pt black, Au, PEDOT:PSS, and PEDOT:CNT are often deposited post-fabrication to reduce electrode impedances, while keeping the electrode size unchanged64–68. For our stainless steel probe, we electrodeposited conductive polymer PEDOT:PSS on the bare Pt electrodes (details in Methods). As a result, PEDOT:PSS coated electrodes (N=14) on steeltrodes exhibited impedances of 51.2 ± 2.26 kΩ at 1kHz (Supplementary Fig. S4a), representing a ~40 fold reduction compared to bare Pt electrodes.

Bending experiments validate the mechanical durability and electrical performance of steeltrodes
A key requirement for long-aspect ratio intracortical neural probes, particularly those intended for repeated use or for applications involving challenging implantation scenarios in non-human primates, is the ability to withstand significant mechanical stress without fracturing. While conventional silicon-based probes are prone to brittle failure, steeltrode’s stainless steel substrate provides a much higher fracture toughness, thereby mitigating concerns of breakage during or after insertion. To experimentally quantify this mechanical robustness, we performed bending tests on fully fabricated steeltrode devices and monitored their electrical functionality before and after each deformation.

In our experiments, a 3D-printed mount was used to secure the backend of the steeltrode, while a motorized indenter (driven by a linear actuator, controlled via a microcontroller) applied a bending force to the tip of the probe (Fig. 3a). The tip was displaced from its neutral (undeflected) position by 10, 20, 30, and 40 mm, respectively, and was then returned to the neutral position. Figure 3b illustrates three representative displacement levels of the probe tip under this controlled bending load. The steeltrode used for this experiment was 78 mm long. After each displacement step, the steeltrode was immersed in 1X phosphate-buffered saline (PBS), and the electrochemical impedance of all microelectrode channels was measured using the same three-electrode configuration described previously. As shown in Fig. 3c, d, the electrochemical impedance magnitude (1.55 ± 0.13 MΩ at 1kHz) and phase (−70 ± 1 deg at 1kHz) of 25 recording electrodes remained virtually unchanged relative to baseline even after the maximum applied deflection of 40 mm.

These observations confirm that the monolithically micro-fabricated stainless steel probe and patterned electrode layers are highly resilient to large bending forces. Consequently, the electrical functionality of the steeltrode remained stable despite being repeatedly subjected to substantial mechanical stress that would likely fracture silicon-based probes with similar dimensions. This robust mechanical performance demonstrates steeltrode’s suitability for neural interfacing in scenarios where significant bending or incidental loading may occur during implantation, explantation, or handling.

Single-unit and multi-unit auditory-evoked potential recorded in macaque brain using steeltrodes
The functionalities of the probes were tested in adult Macaca mulatta by recording both spontaneous and stimulus-locked evoked response from auditory cortex. Our goals were to show (i) that the steeltrodes are compatible with standard recording setups used for acute recordings in NHPs, (ii) that the probes were long enough to reach deep brain structures such as auditory cortex, and (iii) that the quality of the neural signals was good enough for detecting single and multi-unit activity, as well as laminar local field potentials, current-source densities and frequency following responses.

The non-human primate experiments were conducted on an awake head-fixed macaque in a sound-attenuated recording booth. Steeltrode was implanted in the brain through an implantation grid (targeting auditory cortex) in a recording chamber placed over frontal cortex. Prior to steeltrode insertion, the dura was punctured using a stainless steel guide tube. The steeltrode was connected to a neural amplifier for neural data acquisition. Details of the implantation process are available in the Method section.

Spontaneous activity recording from auditory cortex
We recorded spontaneous neural activity at multiple depths corresponding to different layers of auditory cortex using a 16 channel steeltrode with high-density microelectrodes with 25-µm inter-electrode separation. To confirm accurate positioning of the steeltrode in the auditory cortex, we relied on identifying responses evoked by sound, a method detailed in the following section. We used an in-house neural data analysis pipeline based on Mountainsort69 to isolate high-fidelity single and multi-unit activities from the raw neural recording data. The raw data were first band-pass filtered between 500 and 7500 Hz. Spikes were then identified by setting a threshold of 4 times the standard deviation of noise for each channel. Figure 4c shows the high-pass filtered signal from 15 functional recording channels, with threshold crossing events identified. Putative units were clustered using principal component analysis (PCA). Figure 4d shows average waveforms across laminar recording channels for 2 putative units identified from spike sorting. Inter-spike interval (ISI) histograms are also shown for these 2 units. For Unit 1, we note that almost 2% of the spiking events occur within the typical neuronal refractory period of 2.5 ms. This suggests that Unit 1 corresponds to a multi-unit response, as multiple neurons may be contributing to the spiking activity. For Unit 2, none of the spiking events violate the refractory period threshold, suggesting that it is a single unit.

Identifying recording locations in auditory cortex
To ensure precise placement of the steeltrode in the auditory cortex, a passive white-noise burst paradigm was employed. This protocol used 25-ms-long white noise bursts with 5-ms-long rise and fall time at 5 different intensities (46, 56, 66, 76, 86 dB SPL (sound pressure level)). These sounds were presented every 400 ms, with an additional ± 50 ms of jitter to introduce variability (as illustrated in Fig. 5a). Each session of the auditory stimulation protocol comprised 250 trials. The flat frequency spectrum and the fast rise time ensured strong responses across the entire tonotopic map of auditory cortex.

We employed current source density (CSD) analysis on the local field potential (LFP) signals to identify the specific neural generators responsive to auditory stimulation. This analysis played a crucial role in determining the accurate placement of the steeltrode within the auditory cortex. Additionally, we analyzed sound-evoked multi-unit activity. Although an evoked response in the multi-unit band may suggest activity in fibers of passage of stimulus-responsive neurons, the presence of neural generator features identified through CSD analysis provides a more robust and reliable indication of local synaptic response within the auditory cortex70.

To analyze the local field potentials (LFP), we first applied a low-pass filter with a cutoff frequency of 300 Hz to the raw neural signal. Next, using synchronization triggers presented at the sound onset, we segmented the filtered signals into 300 ms long epochs, ranging from −50 ms to +250 ms around the tone onset. Each electrode’s epochs were then averaged over multiple trials after subtracting the common average response from all recording channels. The averaged evoked LFP responses across the laminar electrodes were visualized in Fig. 5b.

To compute the CSD, we used trial-averaged LFP signals from five adjacent electrodes to calculate the hamming window smoothened second-order spatial derivative71,72. Figure 5c displays CSD features, revealing post-synaptic response of an ensemble of auditory responsive pyramidal cells near the tip of the probe as indicated by dipole-like neural features located near the bottom part of steeltrode.

To extract multi-unit activity (MUA), we first band-pass filtered the raw neural data in the range of 300–7500 Hz. Afterward, we applied full wave-rectification and a low-pass filter with a cutoff of 100 Hz to obtain the envelope of power in the frequency band of spiking activity. We computed the trial-averaged MUA for each channel by averaging the responses from epochs around the onset of auditory stimuli. Figure 5d depicts the auditory-evoked average MUA profiles across the cortical depth.

These analyses were performed online as the probe was advanced in small steps of 500–2000 µm. We considered the probe to have reached auditory cortex if (i) we identified a polarity inversion of the first LFP deflection that peaks at around 25 ms after sound onset and (ii) we identified robust sound-evoked multi-unit activity with a peak latency on the order of 20 ms around and below the depth of polarity inversion. We also analyzed single and multi-unit sound-evoked activity. Figure 5e, f display the average multichannel waveforms, peri-stimulus time histogram, and raster plot for a putative unit whose firing rate exhibits a strong correlation with the auditory stimulation. Notably, we observed a significant increase in firing rate ~20 ms after the tone onset, which also aligns with peak latency of trial-averaged multi-unit activity.

Tonal response field (TRF) for the most responsive channel in a specific recording session. Figure 6d displays this TRF map, illustrating how the response varies with tone frequency and stimulus intensity, and further highlighting the strong tuning at 7.1 kHz. The best frequency, identified as the frequency for which the evoked neural response is the strongest, varies at different regions of auditory cortex. TRF maps from two additional recording sessions—where the steeltrode was implanted at different locations in auditory cortex—are shown in Fig. 6e, f. These sites were located ~4.5 mm posterior, and 6.0 mm posterior with an additional 1.5 mm lateral offset, respectively, relative to the location corresponding to the TRF map in Fig. 6d. These spatial offsets were calculated based on the known 1.5 mm pitch of the cortical positioning grid. To enable direct comparison across sessions, each electrode’s MUA response was normalized by the maximum response observed across all frequencies, intensities, and electrodes within that session. This yielded a normalized scale from 0 to 1, allowing standardized comparisons across recordings. The resulting TRF maps reveal distinct best frequencies of 17.4 kHz and 20.5 kHz at the two more posterior sites (Fig. 6e, f, respectively), reflecting regional variability in frequency tuning along the auditory cortex. Recordings were made at depths estimated to correspond to cortical layer 3, based on microdrive depth tracking, known cortical thickness, and characteristic tone-evoked MUA profiles.

Given the tonotopic organization of auditory cortex and the orthogonal insertion angle, adjacent contacts on the same shaft during each recording session showed very similar best frequencies; larger frequency gradients would emerge if steeltrode was inserted parallel to the superior temporal plane from posterior to anterior. Although we observed uniform best‑frequency tuning across contacts at a single implantation site, future studies can leverage the steeltrode’s high‑density array to resolve millisecond‑scale response‑latency differences among neurons that may share similar best frequencies. Additionally, this fine spacing will facilitate probing of local synaptic interactions, which are strongest among closely spaced cells. The successful acquisition of TRF maps using steeltrode demonstrates its effectiveness and strong potential for probing functional organization in deep brain structures, offering valuable insights for neuroscientific research.

Frequency following response (FFR) in auditory cortex
Recording yield/quality of steeltrode in the low frequency band were characterized by acquiring frequency following response from macaque auditory cortex. FFR is a neural response in auditory cortex that is synchronized with the phase of the auditory stimulus waveform. There are some inherent challenges associated with FFR detection. These challenges stem from FFR’s characteristically low signal-to-noise ratio and also elusive nature of the specific locations of FFR neural generators which has been the source of continued scientific debates. We aim to demonstrate high-density steeltrode's capability in detecting such challenging signals and its utility in potentially providing new insights into triangulating FFR neural generators. FFR is a useful tool for understanding the auditory system and provides information about how sound and speech are encoded and processed in the brain. FFR is also useful in diagnosis of neurodevelopmental, speech and learning disorders74,75.

FFR is characterized by neural response in the frequency band ranging from 70 to 500 Hz, which follows the temporal periodicity envelope of the auditory stimuli. The click train stimuli used in this study were specially designed to correspond with the range of fundamental frequencies (F0) found in a Mandarin tone, corresponding to the vowel /yi/76. It is typical for Mandarin words to be used in FFR paradigm, as it is a tonal language, which means that the pitch and tone of the word can change its meaning. The chosen mandarin tone had a time varying F0 contour that changed from 89 to 111 Hz. The stimulus click train (Fig. 7a) was synthesized from this tone to match the timing and amplitude characteristics of the fundamental frequency. Specifically, the F0 cycle timing was determined by the moment of peak pressure, and the intensity of the clicks was set at double the absolute peak amplitude76. The presentation of the stimuli was randomized, with varying inter-stimulus intervals ranging from 300 to 500 ms. During each recording session, which lasted 40 min, a total of 500 repetitions of each tone and polarity were presented, amounting to 4000 sweeps in total.

FFR from the recorded data was extracted by first band pass filtering the raw signal at each of the microelectrodes of steeltrode in the range of 70–500 Hz and then by averaging the time-locked epochs around the onset of each type of tone. Epochs spanning 350 ms around the tone were considered. Figure 7b shows the trial averaged FFR at a single channel of steeltrode corresponding to the stimulus click train. Robust high-density electrodes on steeltrode can be a valuable tool to study the sources of neural generators for FFRs. Laminar distribution of FFR (Fig. 7c) can be calculated from the electrode array of steeltrode. Moreover, neural generators for FFR can be identified and isolated by calculating CSD across the cortical depth. In particular, high-density electrode on steeltrode, we can facilitate extraction of highly localized information regarding FFR sources that can be used to improve our understanding of speech encoding and auditory processing in primate and non-human primate brain.

To assess the impact of electrode spacing on the spatial accuracy in identifying neural generators of FFRs, we compared the CSD results from recording with three different electrode densities, i.e., 50 µm, 100 µm and 150 µm pitch. This involved synthesizing two datasets from the original raw data (recorded with 50 µm pitch): one using every other channel to achieve an effective pitch of 100 µm, and the other using every second channel for an effective pitch of 150 µm. From these laminar FFR data, CSD can be calculated by taking the second-order spatial derivative. Figure 7d shows the laminar CSD for FFR measurement using steeltrode with 50 µm pitch. As it can be seen, in the top half of the probe, we can observe two distinct dipole-like field arrangements corresponding to sinks and sources that can be attributed to local neural generators of FFR. However, with increased electrode spacing, there is a noticeable decrease in spatial resolution for identifying these neural generators, as dipole-like features spread out and lose definition (Fig. 7e, f). Particularly at a 150 µm pitch, crucial spatial information about upper neural generators is significantly diminished or lost. This underscores the utility of high-density steeltrodes for precise detection of local field potentials, current sources densities and shows their significance in advancing our understanding of auditory neuroscience and speech processing.

Steeltrode with smaller shank is rigid enough to puncture rat dura and remains fully functional and reusable after repeated insertions
Using the highly customizable photolithographic patterning and laser micromachining process of our hybrid stainless steel polymer probe platform, we can also implement rodent steeltrodes with smaller shanks. We have fabricated 16- and 32-channel rodent probes with 1–2-cm-long stainless steel shanks. Much like the NHP probes with ultra-long shanks, these shorter rodent probes also benefit from mechanical robustness of stainless-steel substrate. In addition to being highly resistant to fracturing, these short steeltrodes are mechanically resilient enough to puncture rodent dura. Conventionally, for inserting silicon-based rodent probes, dura is surgically removed before the probe is implanted. However, this surgical process may lead to accidental tissue and vascular damage and tissue swelling. The mechanically superior material properties of stainless steel make the steeltrodes rigid and robust enough to withstand the force needed for penetration through rodent dura. Therefore, the probe itself can be used to puncture the dura and that precludes the need for a surgical procedure for dura removal. As a result, the risks associated with surgical removal of dura can be avoided.

We successfully demonstrated the efficacy of steeltrodes in puncturing the intact dura in a rat model. To achieve this, the probe was inserted into the rat brain at a controlled speed of 100 µm/s through the intact dura to a depth of 3.5 mm, targeting the somatosensory cortex and hippocampus. Following acute insertion and recovery, spontaneous neural activity was recorded, with the probe remaining in place for 2 h. Figure 8a shows different stages of steeltrode implantation through rat dura. To evaluate the benefits of dura piercing using the probe, a histological comparison (Fig. 8b) was conducted between probe-assisted dura penetration, probe insertion following surgical dura removal and no insertion. After electrophysiological recording, the animals were sacrificed, and their brains were fixed, sectioned and then processed using Nissl staining (NeuroTrace) to evaluate neural density in the superficial layers of the target site. For analysis, a region of interest (ROI) measuring 900 µm × 500 µm was chosen to consistently sample the superficial cortical layers and the expected local effect produced by dura removal or piercing, while limiting heterogeneity introduced by deeper layers or adjacent fields. This ROI size balance spatial coverage and measurement precision and supports robust biological replication. To account for inter-experimental variability, cellular densities from each biological replicate were divided by the highest control value and expressed on a normalized scale. This ensure that all reported values range from 0 to 100% relative to the maximal control response and allows direct comparison of relative reductions or increases across experimental conditions. Normalization to the maximum cell density of the control is appropriate here because the ROI area and sampling method were consistent across all groups, and the intent was to represent relative cellular preservation or loss rather than absolute density differences. Detailed procedures for in vivo rat experiments and histological analysis are provided in the Methods section. Histological examination revealed that dura removal resulted in a significant cell density reduction compared to the control case (~44.3% decrease versus control), whereas dura piercing caused only a modest reduction (~16.8% decrease compared to the control case). Cellulardensity in the dura removal case was also significantly lower compared to dura piercing (~33% decrease) as shown in Fig. 8c. These results suggest that direct piercing of dura with steeltrode preserves more cells compared to surgical removal of dura and then inserting the steeltrode.

We also characterized the mechanical robustness and reusability of these rodent steeltrodes, highlighting their suitability for repeated use. We took a representative steeltrode (15 mm long) and carried out 10 successive dura penetrations in four rats. After every few insertions, we measured the electrochemical impedance of the electrodes on the probe (15 channels). Figure 8d, e compare the magnitude and phase of the electrochemical impedances at the baseline (before any implantation) and after 4, 8, and 10 insertions. Notably, minimal changes in electrode impedances were observed, indicating negligible degradation of the steeltrode integrity. Since the electrochemical impedance stability is closely related to the noise floor for in vivo recordings64, this result implies that the steeltrode remains viable for high-fidelity neuronal recording after multiple penetrations. Furthermore, on the 10th implantation, we used the same steeltrode to record high-fidelity spiking activity in rat hippocampus across multiple channels. Representative average waveforms and the corresponding ISI histogram of putative units with amplitudes on the order of 100 µV are shown in Fig. 8h, i.

In summary, our rodent steeltrodes provides sufficient mechanical robustness for directly piercing through dura, which can minimize damage to superficial cortical layers by obviating the surgical removal of dura. Our experiments also demonstrate electrochemical stability of steeltrode’s electrodes after multiple implantations through rodent dura, indicating its potential for reusability for recording high-fidelity neural signals.

Methods
Fabrication process of steeltrode
PDMS–parylene C-based fabrication process. Commercially available stainless steel substrate is first spin coated with ~7-µm-thick PDMS (Supplementary Fig. S2). To achieve this thickness, PDMS is diluted with hexane at a ratio of 1:3 and then spin coated on stainless steel at a spin speed of 4000 rpm for 180 s. Then the wafer is put in vacuum chamber at 120 °C temperature to cure and degas for 3 h. A thin layer (7 µm) layer of Parylene C is deposited on the wafer. Parylene C is deposited by using SCS Labcoter-2 (dimer mass 8.3 g, furnace setpoint 690 °C, vaporizer setpoint 135 °C, vacuum setpoint 35 mTorr). Then, metal traces are patterned lithographically on Parylene C (AZ5214E photoresist). Metal stack (5 nm Pt/120 nm Au/5 nm Pt) is deposited by electron-beam evaporator. Then, lift-off of metal traces is done at room temperature by soaking the sample in Acetone for 2 h. Afterwards, another thin-film (7 µm) of insulating Parylene C is deposited with the same process parameters as the first layer. Then, we deposit 100 nm of Chromium (Cr) using electron-beam evaporator and then photolithographically define (AZ 4110 photoresist and AZ 400K developer) the probe outlines and the electrode openings. Then Chromium is etched using Cr1020 Etchant. Cr was used as a hard mask for subsequent parylene C etch step, as photoresist has poor selectivity to Parylene C. We etch Parylene C to expose electrodes, bondpads, and the probe outlines by O 2 reactive ion etching (RIE) (14.0 sccm O 2, 50 mTorr, 50 W RF). After etching, the Cr hard mask is stripped using Cr1020 Etchant. For etching the underlying PDMS layer to define the device outlines, another Cr hard mask is deposited and patterned using the same parameters as mentioned previously. PDMS is etched via reactive ion etching (RF=120 W, Pressure=35 mTorr) in SF 6 (22.5 sccm) and O 2 (9 sccm) chemistry. The final step of our fabrication process is to release each of the probes from the stainless steel wafer with laser micromachining process. With a high-powered fiber laser, stainless steel is etched away along the outlines of the probe. Lithographically defined alignment marks on wafer and CAD designs are used to accurately align and trace the borders of the probe with laser.

SU-8-based steeltrode fabrication with single layer metal. SU-8 2010 is spin coated on stainless steel wafer at 7000 rpm to produce 7-µm-thick bottom insulation layer. Next, the device outlines are defined in this layer via photolithography. Then we pattern the metal layer using AZ5214E photoresist. Then, we use electron beam evaporator to deposit 5 nm Cr/120 nm Au/5 nm Pt and subsequent lift-off in acetone to realize the metal traces and electrodes. Next, another 7-µm-thick SU-8 layer is spin coated and subsequently patterned to expose the electrode openings and device outline. SU-8 layers are baked at 180 °C for 10 min after photolithography. Finally, individual stainless steel probes are released from the wafer using a laser micromachining process.

SU-8-based probe with multi-layer metal. Before each metal layer, we have spin coated diluted SU-8 2010 (SU-8 2010 to SU-8 Thinner ratio of 1.8 by weight) at 4000 rpm to form a 2.5-µm-thick film. Probe outlines and electrode openings for the previous metal layers were photolithographically defined. Each SU-8 layer is baked at 180 °C for 10 min. Next, we pattern the metal layer using AZ5214E photoresist. Then, we use electron beam evaporator to deposit 5 nm Cr/120 nm Au/5 nm Pt and subsequent lift-off in acetone to realize the metal traces and electrodes. These steps are repeated for each metal layer. A 2.5-µm-thick SU-8 layer is deposited and patterned to form the top insulation layer exposing the recording electrodes and contacts. Finally, individual stainless steel probes are released from the wafer using a laser micromachining process.

Electrochemical etching process to implement flexible tether cable on steeltrode. We have utilized an electrochemical etching process with oxalic acid to selectively etch stainless steel underneath the polymer insulation layers. In this process, we mask the implantable rigid portion of the shank with Crystalbond and immerse the part of the probe that we want to etch in 10% oxalic acid solution at 35 °C which is being stirred at 300 rpm. Then we apply positive voltage to the stainless steel substrate of the probe with respect to a stainless steel counter electrode and impose 0.3 A current to the circuit with a Kiethley sourcemeter. This electrochemical etching process is continued until the stainless steel shank exposed to Oxalic acid is etched away completely. Then the Crystalbond covering the implantable shank of the probe is removed in Acetone. In this process, we can get probes where the implanted portion of shanks can freely move with brain micromotions with little or no tethering force and can retain electrical connection to the recording amplifier through a highly flexible PDMS–Parylene C cable.

Electropolishing of steeltrode sidewalls. To achieve a smooth surface finish on the stainless-steel sidewalls while maintaining polymer insulation integrity, we utilized an eco-friendly electropolishing process in a NaCl solution, following a procedure adapted from Han et al.84. The electrolyte consisted of 500 mL pure ethylene glycol solution with 1 M NaCl. The setup included a titanium mesh counter electrode, an Ag/AgCl reference electrode, and a custom 3D-printed jig to precisely hold the steeltrode, ensuring a uniform distance between the steeltrode and the mesh counter electrode. The electropolishing process was conducted at 50 °C, with an agitation speed of 300 rpm, while voltage was applied using an Autolab PGSTAT302N system. An electropolishing voltage in the range of 5.3-5.6 V was used to achieve polishing uniformity and prevent excessive material removal. This method produced a smooth finish while maintaining the structural integrity and electrical functionality of the steeltrode.

Electrochemical impedance characterization and electrode surface modifications. Electrical performance of the probes was characterized by Electrochemical Impedance Spectroscopy (EIS) measurements in 1X PBS (Phosphate-Buffered Saline) solution. The characterization was conducted with a PGSTAT302N potentiostat (Metrohm, Netherlands) with a 3-electrode configuration in potentiostatic mode. The counter/auxiliary electrode was a Platinum wire electrode (MW-1032, BASI Inc) and the reference electrode was Ag/AgCl electrode (MF-2052, BASI Inc.). Electrochemical impedances of different channels of a typical probe were measured over the frequency range of 0.1 Hz–100 kHz. We used 50 mV (rms) sinusoidal signal at open circuit potential (OCP) to do the electrochemical measurements over this frequency range.

PEDOT:PSS electrodeposition. Conductive polymer PEDOT:PSS was electrochemically deposited on the surface of bare Pt electrode from an aqueous solution of 0.5 M 3,4-ethylenedioxythiophene (EDOT, Sigma-Aldrich, USA) and 0.6 wt% of poly(sodium 4-styrenesulfonate) (PSS, Sigma-Aldrich, USA). A 3-electrode configuration with Pt counter electrode and Ag/AgCl reference electrodes was used under galvanostatic condition. For each of the microelectrodes, conductive polymer is electrodeposited by applying constant current of 1 nA, until a charge density of 1 µC/cm2 is reached. Each electrode was inspected optically and through EIS measurement to ensure successful electrodeposition.

Probe bending experiment. Housing for the backend of the probe was 3D printed using a polylactic acid (PLA) based filament. Probe was attached to the housing using UV curable resin (Piccasio UV Resin Clear) and placed inside a holder. Indenter was also 3D printed using PLA filament and taped to a motorized linear stage (YunDuan model T06). Stage was driven by an open loop stepper motor (NEMA 11 size) and controlled by a stepper motor controller (TB6600 based microstepping driver) and microcontroller development board (Arduino Nano). The linear stage was moved until the desired displacement of the probe tip was achieved. Later, the indenter was returned to initial position while the probe returned to its original shape.

Animal experiments
In-vivo measurements in macaque
Animal model and surgical preparation. All experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh. A single adult Macaca mulatta was used for acute intracranial electrophysiology, which remained awake throughout all the experiments discussed in the paper. To enable head fixation during recording sessions, a stainless-steel head post (Christ Instruments) was surgically implanted, along with a cranial recording chamber over the frontal cortex. The recording chamber had been in place for over a year prior to the steeltrode experiments to ensure long-term stability and accessibility to the cortical surface.

Recording chamber grid and microdrive assembly. A custom 3D-printed grid was designed to fit tightly into the recording chamber and provide precise alignment for guide tubes and electrodes. The grid was secured using set screws, ensuring stability throughout the recording session. A horizontal ledge was integrated into the grid to mount a microdrive, which was affixed with an additional set screw. The microdrive arm featured a micro-clamp to hold the steeltrode securely in place.

To maintain sterility, the microdrive assembly was first tested in a mock recording chamber before being sterilized using ethylene oxide. On the day of the experiment, the sterilized microdrive–grid assembly was transferred to the implanted recording chamber on the animal’s head, ensuring aseptic conditions. A 23-gauge extra-thin-walled stainless-steel cannula (guide tube) was inserted through a selected guide hole to puncture the dura, after which the steeltrode was fed into the guide tube and aligned with the grid.

Electrode insertion and recording procedure. Once properly positioned, the steeltrode was secured to the microdrive, and its position was adjusted to align the electrode shaft with the grid hole. The steeltrode was then advanced in 0.5–2.0 mm increments using the microdrive until it reached the target depth within the auditory cortex. The entire recording procedure took place inside a sound-attenuating booth (Eckel) to minimize external noise interference. Neural signals were acquired using an Intan RHD 256-channel recording system (Intan Technologies, Los Angeles, CA, USA) at a sampling rate of 30 kS/s. The signals were referenced to the stainless-steel substrate of the probe. Initial recordings were performed to assess spontaneous neural activity, after which auditory stimuli were presented via free-field speakers to confirm probe placement within the auditory cortex. Local field potential (LFP) and multi-unit activity (MUA) data were analyzed using custom-developed MATLAB 2024a scripts. Single-unit data were processed and analyzed using custom Python scripts developed based on the Mountainsort69.

In-vivo measurements in rat. All rodent experiments conducted in this study received ethical approval from the Institutional Animal Care & Use Committee (IACUC) of Carnegie Mellon University. All procedures were conducted as acute experiments, where probes were implanted and recordings performed during a single terminal session under anesthesia. For probe implantation, 3-month-old Sprague Dawley (SD) rats of both sex within a weight range of 200–300 g were used. To begin the experiments, rats were anesthetized with Ketamine/Xylazine cocktail (80 mg/kg and 8 mg/kg body weight) and placed on a feedback-controlled heating blanket maintained at 36 °C (Kent Scientific, Torrington, CT) mounted on a stereotaxic frame (Neurostar, Tubingen-Germany). An incision was made in the scalp, and craniotomy was performed by drilling a 1 mm diameter hole above one hemisphere with the coordinates AP −2.5 mm and ML +3 mm targeting the somatosensory cortex and hippocampus. A screw hole was implanted on other hemisphere to provide a reference ground to the recording probe. Following craniotomy, a steeltrode probe was inserted at the target site at a depth of DV −3.5 mm to target CA1 regions of the hippocampus using a motorized stereotaxic arm at a speed of 100 µm/s. Following insertion, 10 min recovery was allowed for the acclimatization of tissue to the external material before starting spontaneous recordings. Spontaneous response was recorded for twenty minutes at 30 kHz sampling rate using a neural recording amplifier (Intan Technologies, Los Angeles, CA, USA). Spike band (single and multi-unit activity) data were processed and analyzed using Offline Sorter from Plexon Inc.

For histological evaluation, rats were transcardially perfused with fixative after 2 h of implantation. We transcardially perfused rats with PBS followed by 4% paraformaldehyde (PFA) to fix the tissues. Brain was isolated and kept in 4% PFA overnight followed by sequentially incubating in gradient sucrose solution (10%, 20% and 30%) for cryo-sectioning. Then 30 µm sectioned slices were prepared using cryostat and stored in PBS. Sections were further stained using NeuroTrace™ 500/525 Green Fluorescent Nissl Stain (Cat No. N21480, Thermofisher scientific), colabelled with DAPI (Nuclear Stain) and mounted on slides. To assess the effect of insertion on dura, images were captured at insertion site with the region of interest (ROI) measuring 900 µm × 500 µm covering dura and superficial cortical layers. Images were analyzed to measure cellular density using ImageJ and data was represented as normalized cellular density in each condition. Histology data was averaged for multiple insertions and normalized to max of control group.

Probe cleaning. After explanting the steeltrode at the end of in-vivo experiment, it is first rinsed off with Deionized (DI) water. Then the probe tip is immersed in 1% Tergazym solution for 2 h and then rinsed off again with DI water.

Statistical power, replication, and exclusion criteria (rodent). As no prior data were available to estimate sample sizes for this exploratory study, a formal a priori power analysis was not feasible. A total of four rats were used based on feasibility, ethical considerations, and alignment with similar proof-of-concept studies aimed at evaluating experimental parameters and generating preliminary data to inform future, adequately powered investigations.

Different hemispheres from each rat served as an independent biological replicate, with hemispheres allocated across three experimental conditions: three hemispheres for control (no insertion), two for dura removal, and three for dura piercing. This within-animal design maximized the amount of experimental data obtained while minimizing overall animal use. Only one insertion was performed per hemisphere to prevent localized tissue effects from multiple penetrations.

Two additional animals were excluded prior to histological analysis due to physiological instability during the procedure, including irregular respiration and inconsistent anesthesia depth despite adjustments. Data from the remaining four rats were included in all reported analyses.

Anesthesia depth and physiological parameters were carefully monitored to ensure procedural consistency, and all efforts were made to minimize both technical and biological variability. Quantitative histological comparisons were conducted at the level of individual tissue sections (6–9 per hemisphere), treated as technical replicates. Follow-up studies with larger sample sizes are planned to refine the quantification of these findings and explore condition-specific effects more comprehensively.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Title: Steeltrode: Robust Minimally-invasive Microfabricated Stainless Steel Neural Interfaces for High Resolution\nRecording.\nThis innovative work presents a new compact and high-density neural probes which can be implanted in different areas of\nthe brain of rodents and non-human primates (NHP). While silicon has been the material of choice for rigid neural probes for\nrodents, its brittleness, and fragility are problematic for long aspect ratio silicon probes for large animals. Stainless steel is\npresented in this work as a biocompatible and less brittle material able to create robust neural probes, especially for large\nanimal and human recordings. Moreover, stainless steel is more resilient and has a high modulus of elasticity. However, its\nmicrofabrication and micromachining technology has been less developed and is more challenging.\nThe authors discuss in this manuscript a novel microfabricated customizable stainless steel neural probe design for high-\nresolution recording in large animals. A rigid implantable stainless-steel shank and a flexible cable to relieve tethering force\nfrom the skull fixture have been created for this new type of probe. The design of high-density stainless steel and their\nimplementation using a novel scalable microfabrication process can be repeated in multiple layers increasing the density\nand the number of recording electrodes.\nThese new probes have been tested using high-resolution in vivo neural recordings of single and multiple units across\ncortical laminae from the auditory cortex in rhesus macaques. Safe electrode implantation through intact dura in rats has\nbeen proven to record high action potentials, proving the robustness of electrodes for recording high-fidelity neural signals\non rodent and NHP models to long-term chronic electrophysiology recording.\nThe work demonstrates the potential of stainless steel to be translated to humans and the possible\napplicability in epilepsy source localization and deep-brain-stimulation (DBS) electrode\nimplantation.\nThe article is easy to read and well-explained. This new type of probe is innovative and it can be a great advance in animal\nresearch, especially with non-human primates. Researchers urgently need robust electrodes that simplify and make less\ntime-consuming the experimental part and data acquisition. Thus, I find this work useful and interesting.\nI have some comments and questions for the authors:\n- Abstract: I think you should avoid citation sources in the abstract. But if you want to keep it, I think it should be cited in\nthe Introduction part too.\n- Page 4: “long enough to record from anywhere in the non-human primate brain”. Later in the article has been written\nthat the 8 mm electrode can raise the auditory cortex, as the experiment showed. It is not clear to me if this type of electrode\nallows recordings into the brainstem.\n- Page 5: “The steeltrodes were able to reliably record single unit and multi-unit activity as well as local field potentials\n((Figure 1I)”. There are two parenthesis before the word Figure.\n“In contrast, since steeltrodes are resilient against fracture, they can be safely inserted into the neural tissue multiple times\nand their handling and manipulation are reliable and safe. Moreover, these robust neural probes can be implanted through\nthe dura, obviating the need to remove dura, thus reducing damage to the superficial layers of the neural tissue”. It is a good\nthing to have the possibility to insert the electrodes multiple times. However, if the penetrations are too close it can damage\nthe tissue and create undesirable effects or symptoms in the animal. Reading all the article I understood that dura removing\ncan be avoided only in rats, but not in NHP. However, this sentence is not specific.\n- Page 8: “As a result, steeltrode itself can be used to puncture the dura of small animals during implantation, without the\nneed for surgical removal of the dura. The ability to puncture through the intact dura layer without surgical removal is critical\nboth for retaining the cranial pressure and for reducing the volume of removed tissue for probe implantation”. I agree about\nthe intracranial pressure undesirable effects when a dura removal is done. However, in my opinion, penetrating directly with\nthe electrode can also damage the tissue with the pieces of dura that can go into the brain while the electrode is going in.\nThis will contaminate the results and perhaps damage the electrode.\n- Page 11: “For passive electrical neural probes, scaling up he the number of channels means increasing the number of\ntraces connected to each recording electrode”. Is the “he” an error in this sentence?\nThe citation 42 is cited two times in the same paragraph.\n- Page 15: Things to keep in mind about the “brain micromotions”. Sometimes the monkeys make pressure from the\ninside of the brain, making force with their bodies. Thus, they increase their intracranial pressure. When this happens the\nbrain goes up and cells are killed and the brain tissue is detached from the probe influencing the data.\n- Page 22: How many monkeys have been used to test the probes? Sometimes the animals have different behaviors that\ncan influence the data or the probe's brittleness. For example, if it is a big male with aggressive behavior it will be more\nchallenging to keep the electrode safe than if it is a female, even if both are head fixated.\nHow much time the researcher spent to insert the electrode into the brain?\nWhat was the total duration of the experiment? (This has an influence on the behavior of the monkey as I explained above)?\nHow many times the probes can be used while keeping their data quality threshold?\nHow the electrode should be cleaned?\nDid you check it into subcortical areas?\n- Page 31: “Histological examination demonstrated that surgical removal of dura reduces the neural density along the\ntract…”. When did this histological examination take place? The same day of the procedure or days later?\n- Page 33: “Such devices can be implanted into deep cortical regions while being free to move with brain micromotions\nensuring minimized chronic tissue damage due to significantly reduced tethering force”. At the beginning of the article is\nwritten that electrodes can arise anywhere in the brain. It would be interesting to be precise if subcortical regions such as the\nbrainstem can be recorded.\n- Page 34: “We can also readily adapt our fabrication process to realize probes with multiple shanks”. In this case, you\nshould keep in mind the number of probes that the researcher uses at the same time. The arrangement of the instrument\naround the recorded zone has to have enough place and to be separated enough to allow several probes at the same time.\nMoreover, the position degree will influence if the shanks will touch each other.", "reviewer_2": "Key results:\nThis paper demonstrates the novel fabrication process to integrate PDMS / Parylene C / SU8 and platinum electrodes (that\ncan be vertically stacked to increase channel count) on commercially available stainless steel. This process allows\nfabrication of high aspect ratio neural implants that are less fragile for implantation in vivo. The authors showcase the\nrecording ability of such an implant in the macaque brain for auditory cortex recordings.\nMain comment:\nThe claims that high-aspect ratio, high-density, versatile, stainless-steel probes, fabricated using\nmicrofabrication/micromachining processes, that can record with high-resolution from deep regions of the brain is supported\nby the data presented in the paper. However, the robustness/resilience against fracture of the stainless-steel probes is not\nthoroughly demonstrated in the present version of the manuscript, in my opinion. A figure does show the force required to\nfracture/plastically deform silicon vs. stainless steel, but no/little demonstration of what happens to the integrity of the\nelectrodes integrated on the stainless-steel under stress, before/after bending, after multiple insertions, etc., is shown.\n“Reusability” of such devices, or “safety for multiple insertions” is not clearly demonstrated here.\nData and methodology questions/comments:\nMain, P.5: “In contrast, since steeltrodes are resilient against fracture, they can be safely inserted into the neural tissue\nmultiple times and their handling and manipulation is reliable and safe”. It would be important to provide data to demonstrate\nthis: the paper as it is does not really show “safe multiple insertion”, nor that the probe can be safely manipulated. This would\nbe important, not only regarding the stainless-steel part, but also with the integrated electrodes, as part they are the critical\npart of the whole recording probe.\nMain, P.7: “…whereas the steeltrode only deforms and bends", "reviewer_tissue”": "same here, what about the impact on the electrodes contact integrity of bending/deformation ?\nMain, P.9: “We have fabricated probes with electrodes as small as 10-μm diameter and as large as 40-μm diameters.”: I\nbelieve no data shows this directly in the manuscript. It could be interesting to see some sort of quantification", "reviewer_25": "ave you done any cross-talk measurements on this configuration?\nMain, P. 22: “As a result, we have obtained ~40-fold reduction in impedance”. Could you mention the actual impendence\nvalues in the result? Also, in Figure S3A, could it be possible to see the range from multiple contacts ? Is this just n = 1 ?\nFigure 4B: Would be interesting to see the impedance profiles for these recordings electrodes to see consistency of the\nspectrum over multiple contacts.\nResults, P.31: “For assessing cellular damage, brain sections were stained using fluorescent Nissl stain (NeuroTrace) to\nevaluate neural density along the insertion tract. Histological examination demonstrated that surgical removal of dura\nreduces the neural density along the tract by 32% when compared to the group with no insertion, whereas in the case of\nprobe insertion by piercing through dura, the reduction was only 18% when compared to control group (Figure 7C), thereby\nshowing a significant improvement.” I believe more details are needed here to evaluate the strength of these results. In\nparticular: how many animals does this represent? And/or how many insertions? Error bar in Figure 7C suggests there was\nmore than 1. When was the brain collected for analysis following insertion: right after, or after some time (tissue settling)?\nHow long after implantation where the probes removed: right away or after some time? Was the whole tract considered for\ncalculation of neural density, or just the top around dura as shown in the images on Figure 7? If only the top, how did you\nexactly define the region of the “superficial cortical layers” ? Was the intensity (Figure 7C) normalized? If yes, how? Here\nalso, it could be useful to have electrode integrity before and after insertion (or even multiple insertions through the dura), or\nmimicking insertion forces, buckling, bending, to show that indeed that they are robust to manipulation and insertion, as the\nclaim is that “Even for rodents, it is highly desired to design robust neural probes that can be safely used for multiple\ninsertions.”\nClarity and context questions/comments:\nStructure suggestion: Nature Communications probably allows for quite flexible paper structures, but I would suggest some\ntext restructuring to make the manuscript clearer for the reader to navigate and find useful information when needed:\n- keep the “Main” free of specific results (the different design constraints are interesting to mention), but actual results of what\nwas achieved can be kept for the result sections\n- keep the result section free of methods (except the main elements for fabricating the steeltrode, which are part of the main\nresults of this paper, but for the electrophysiology and in vivo, I would suggest focusing on the actual results of the\nrecordings and keep the methodology for the method section). This would alleviate the text and help have a more direct look\nat what was achieved, and the specifics of how it was done in the methods.\nAbstract, P.1, last line: “from auditory cortex in rhesus macaques.” : was this done in multiple animals? If not, keep\n“macaque” singular, to be precise.\nAbstract, P.2, “to long-term chronic electrophysiology recording”: no demonstration of long-term implantation of these probes\nis demonstrated in this paper, from what I could understand, so I would refrain from saying that these experiments “showcase\nthe robustness of our steeltrodes […] in a gamut of applications ranging from […] to long-term chronic”. Or mention that this is\nthe first step towards future experiments for long-term chronic electrophysiology for next generation BMI.\nMain, P.2: “NHP probes have extremely high aspect ratios and thus need to be very rigid”: they have to be rigid in the case\nwhere no guide/shuttle is used, but they don’t need to be per se. I would maybe rephrase or add the mention that this applied\nfor probes implanted without inserter/shuttles, to be more specific.\nMain, P.4: “The steeltrodes discussed in this paper address this issue by enabling high resolution distributed neural\nrecording from different areas of the brain as envisioned in Figure 1A, where multiple steeltrodes are implanted at different\ndepths within the brain. Each of the penetrating neural probes is a hybrid polymer-stainless steel neural probe with a rigid\nshank that can be designed to be fully stiff (Figure 1B) or monolithically connected to a flexible cable (Figure 1C and 1D) to\nroute the recorded signals to the backend circuitry, while minimizing the tethering force and damage to the brain tissue due\nto brain micromotions”: This is the vision here, to have multiple steeltrodes implanted, but from what I understand, this hasn’t\nbeen done directly in this paper. So I would not say that the steeltrodes “enable high resolution distributed neural recording\nfrom different areas”, but they do definitely lead the path towards this vision. Make clearer that this is the vision, i.e. goal,\nbut not demonstrated specifically in this paper. This paper (and if not, perhaps make it clearer) shows single insertion of a single\nprobe with a fully rigid shank (Fig. 3A).\nMain, P.5 : ((Figure 1E – 1G)) and ((Figure 1I) (extra parentheses).\nMain, P.5: “As a demonstration of the capabilities of the platform", "reviewer_microelectrodes”": "Could you add the actual number of electrodes +\npitch also here?\nMain, P.6: “With these dimensions", "reviewer_570": "In order to better compare, can you give the equivalent aspect ratio of other relatable probes\n?\nNo reference to figure 1H in the main text ? Also for Figure 1H, would be nice to have important dimensions directly\nhighlighted, such as thickness, width, main materials, pitch, for a more comprehensive figure.\nIn general, I would try to be more specific when using the term “reliable”: with respect to what ?\nFabrication yield", "reviewer_4": "The manual manufacturing of such neural probes puts constraints on the density of microelectrode channels\n(e.g., currently, up to 64 channels with a minimum of 50 μm) “: Can you maybe comment again in the discussion/conclusion,\non how your device compares in terms of max channel count and pitch for these stainless steel probes?\nMain, P.15: “Due to its small form-factor packaging and narrow cross section, multiple steeltrodes can potentially be\nimplanted at different depths of the non-human primate brain for simultaneous high-density neural recording from different\nbrain regions (Figure 1A): (if) this has not been actually tested, I would suggest to move this to the discussion as possible\nfuture next steps, not in the result section.\nFigure 2A: scale bars on the images\nFigure 2: “Metal", "reviewer_22": "Some details are missing, which are important to mention. One vs. multiple animal ? Single vs. multiple\ninsertions ? Acute vs. chronic ? Awake vs. Sedated ? If sedated, give information on anesthetics. All of these should be at\nleast accessible in the methods.\nFigure 3 and corresponding methods: Can you provide more info on this microdrive and implantation grid ? How was it\nimplanted ? When in time, compared to the insertion of the probe ? What is placed acutely, or kept chronically to allow\ninsertions at multiple times ? Can you comment on how this microdrive + guide tube + implantation chamber compares with\n“the use of specialized equipment and hardware […] for ensuring proper alignment of such probes” (ex. Neuropixel NHP)\nas mentioned in the introduction ?\nResults, P.24: “We recorded spontaneous neural activity at multiple depths corresponding to different layers of auditory\ncortex using a 16 channel steeltrode with high density microelectrodes with 25-μm inter- electrode separation. Why only 16\nchannels ? Are these just the channels at the tip ?\nFigure 3B: scale bar for dimensions.\nFigure 4C: Colorbar scale missing.\nFigure 4B", "reviewer_d": "Y-scale missing.\nResults, P.26: Tone duration and inter-stimulus duration for the TRF experiments ? This should be in the method section,\nactually.\nFigure 5: Can the chosen channels used to show mapping (Frequency vs. Intensity) be somehow highlighted on the\nschematic of the probe? Also, did you get different best frequency for two electrode contacts when the probe was in the\nsame position ? Showing this (or better highlighting in the current figure) could help understand how useful the high\nresolution (small electrode pitch) is in differentiating best frequencies between close-by contacts.\nFigure 5 legend: Labels in legend C,D,E,F don’t match figure.\nFigure 5 E-F: Can the depth at which these were recorded be given ? Or at least the distance in the tissue between the two\nmeasurements ?\nFigure 5: “normalized MUA response”: be more specific in the methods how this was computed and especially normalized.\nFigure 5D: Y-label “Response” ? Is the also the normalized MUA response?\nDiscussion, P.33: “as well as long-term chronic brain interfacing”: there is no demonstration of chronic use of these probes\nhere, unless I missed it. I would discuss this as a future next step, but not as something readily demonstrated here.\nDiscussion, P.33: “Using selective electrochemical etching of stainless steel, we have created completely flexible tether\ncables attached to rigid shuttles and demonstrated hybrid rigid-flexible probe architectures for non-human primates. Such\ndevices can be implanted into deep cortical regions, while being free to move with brain micromotions ensuring minimized\nchronic tissue damage due to significantly reduced tethering force.” This was fabricated, but not implanted and tested (or at\nleast not shown in this manuscript). I would more clearly mention that this would be an interesting improvement to the\narchitecture used in the present NHP recordings, especially for chronic implants, and that is technically feasible, and\ncould/should readily be tested.\nDiscussion, P.33: “different depths of auditory cortex in macaque monkeys”. If n = 1 monkey, keep “monkey” singular.\nFigure S2: “metal”: can you be more specific as to which ones ?\nFigure S3: (c) legend should be labeled as (b).\nWhat about stimulation using the steeltrode ? Could this be achieved ? Has it been tested ? It could be interesting to\nmention/discuss this in the discussion. To the authors’ discretion.\nReferences:\nMain, P.2: missing some references to support the claim that very small cross-section is important to minimize tissue\ndamage.\nMain, P.2: Ref. 13 and 14 are actually the same. Also, more recent literature could be added and interesting to refer to:\nhttps://doi.org/10.1016/j.jneumeth.2023.110016\nFigure 4: Could be interesting to put this into perspective/discuss with more recent literature using Plexon stainless steel\nelectrode: https://doi.org/10.1016/j.neuroimage.2023.120364\nMy expertise:\nSections regarding the microfabrication and micromachining of the steeltrode themselves and how novel the approach is,\nare out of the scope of my expertise. I leave it to the other reviewers to judge the feasibility, soundness and novelty of this\nprocess."}, "author_response": "Response to the reviewer’s comments:\nWe thank the reviewers for the very helpful comments and constructive feedback. We\nperformed new experiments, revised the manuscript and carefully addressed all the comments.\nThese revisions have enhanced the manuscript and further substantiate the messages of this\npaper. Below, we summarize our revisions and responses to the specific points raised by the\nreviewers.\nReviewer 1:\nTitle: Steeltrode: Robust Minimally-invasive Microfabricated Stainless Steel Neural Interfaces for\nHigh Resolution Recording.\nThis innovative work presents a new compact and high-density neural probes which can be\nimplanted in different areas of the brain of rodents and non-human primates (NHP). While silicon\nhas been the material of choice for rigid neural probes for rodents, its brittleness, and fragility are\nproblematic for long aspect ratio silicon probes for large animals. Stainless steel is presented in\nthis work as a biocompatible and less brittle material able to create robust neural probes,\nespecially for large animal and human recordings. Moreover, stainless steel is more resilient and\nhas a high modulus of elasticity. However, its microfabrication and micromachining technology\nhas been less developed and is more challenging.\nThe authors discuss in this manuscript a novel microfabricated customizable stainless steel neural\nprobe design for high-resolution recording in large animals. A rigid implantable stainless-steel\nshank and a flexible cable to relieve tethering force from the skull fixture have been created for\nthis new type of probe. The design of high-density stainless steel and their implementation using\na novel scalable microfabrication process can be repeated in multiple layers increasing the\ndensity and the number of recording electrodes.\nThese new probes have been tested using high-resolution in vivo neural recordings of single and\nmultiple units across cortical laminae from the auditory cortex in rhesus macaques. Safe electrode\nimplantation through intact dura in rats has been proven to record high action potentials, proving\nthe robustness of electrodes for recording high-fidelity neural signals on rodent and NHP models\nto long-term chronic electrophysiology recording.\nThe work demonstrates the potential of stainless steel to be translated to humans and the possible\napplicability in epilepsy source localization and deep-brain-stimulation (DBS) electrode\nimplantation.\nThe article is easy to read and well-explained. This new type of probe is innovative and it can be\na great advance in animal research, especially with non-human primates. Researchers urgently\nneed robust electrodes that simplify and make less time-consuming the experimental part and\ndata acquisition. Thus, I find this work useful and interesting.\nI have some comments and questions for the authors:\n- Abstract: I think you should avoid citation sources in the abstract. But if you want to keep\nit, I think it should be cited in the Introduction part too.\nResponse: Citations are removed from the abstract and we have added those citations to the\nintroduction\n- Page 4: “long enough to record from anywhere in the non-human primate brain”. Later in\nthe article has been written that the 8 mm electrode can raise the auditory cortex, as the\nexperiment showed. It is not clear to me if this type of electrode allows recordings into the\nbrainstem.\nResponse: Depending on the approach angle, reaching the brainstem requires traversing on the\norder of 5 cm of brain tissue. Hence, it is in principle possible to reach the brain stem. There are\ntwo issues to note here. (1) Most NHP recording setups impose a rather large ‘dead’ space built\nin, meaning that a sizable portion of the shank can not be inserted into the brain and has to stay\nout due to the arrangement of the microdrive and the attachments. (2) While the current\nsteeltrodes were 8cm long, the proposed fabrication platform can be used to manufacture much\nlonger probes. For example, rather than using a 4-inch wafer, a 6-inch substrate can be employed\nto fabricate probes longer than 12 cm. Therefore, by optimizing the approach angle, reducing\ndead space, and using the longer probes, it is indeed possible to reach the entire brain, including\nbrain stem.\nThe scalability of our proposed neural probe fabrication platform to make steeltrodes with >12 cm\nlong shanks are noted in the Discussion section.\nRevision: (In Discussion Section)\n“The 8-cm steeltrode easily enables reaching subcortical regions in macaques. In our\nexperiments, the approach angle, chamber design and microdrive arrangement dictated\nthat between 2 and 4 cm of the shank length remained out of the brain tissue, thus leading\nto an effective electrode length of 4 to 6 cm. Given that the dorso-ventral extent of the\nmonkey brain is below 5 cm, the current setup would provide access to most of the brain,\nincluding most subcortical targets. A redesign of the setup can further increase the\neffective length to around 7 cm, thus providing access to the entire monkey brain. For use\nin larger species, it will also be possible to use longer probes. The length of the neural\nprobe was limited by the size of the substrate (i.e., a 4-inch-diameter wafer) that we made\nfor photolithography. Since we made this wafer out of large stainless steel sheets, the size\nof the wafer can be easily made larger (i.e., 6 inches or even 8 inches) to microfabricate\nneural probes, with implantable shanks of up to 18 cm, if needed.”\n- Page 5: “The steeltrodes were able to reliably record single unit and multi-unit activity as\nwell as local field potentials ((Figure 1I)”. There are two parenthesis before the word Figure.\nResponse: Thank you for pointing it out. This has been corrected in the revised manuscript\n“In contrast, since steeltrodes are resilient against fracture, they can be safely inserted into the\nneural tissue multiple times and their handling and manipulation are reliable and safe. Moreover,\nthese robust neural probes can be implanted through the dura, obviating the need to remove dura,\nthus reducing damage to the superficial layers of the neural tissue”. It is a good thing to have the\npossibility to insert the electrodes multiple times. However, if the penetrations are too close it can\ndamage the tissue and create undesirable effects or symptoms in the animal. Reading all the\narticle I understood that dura removing can be avoided only in rats, but not in NHP. However, this\nsentence is not specific.\nResponse: We have updated the text to make this claim specific for rodent dura.\nRevision: Here is the updated text:\n(Page 5): “Moreover, these robust neural probes can be implanted through rodent dura,\nobviating the need to remove dura, thus reducing damage to the superficial layers of the\nneural tissue. ”\n- Page 8: “As a result, steeltrode itself can be used to puncture the dura of small animals\nduring implantation, without the need for surgical removal of the dura. The ability to puncture\nthrough the intact dura layer without surgical removal is critical both for retaining the cranial\npressure and for reducing the volume of removed tissue for probe implantation”. I agree about\nthe intracranial pressure undesirable effects when a dura removal is done. However, in my\nopinion, penetrating directly with the electrode can also damage the tissue with the pieces of dura\nthat can go into the brain while the electrode is going in. This will contaminate the results and\nperhaps damage the electrode.\nResponse: Thank you for raising this important concern. Steeltrode design features a\ncustomizable probe tip angle/sharpness so that it can slice through the dura cleanly, thereby\nminimizing the accumulation and transport of dural fragments.\nTo address the concern about compromising electrode integrity in the dura piercing process, we\nconducted additional experiments to evaluate the probe’s robustness during repeated insertions\nthrough the rat dura. In these tests, the same probe was inserted 10 consecutive times in rats.\nWe monitored the electrode impedance after several insertions and observed no significant\nvariation, which confirms that the probe's structural and functional integrity remains intact.\nMoreover, even after the 10th insertion through dura, the probe successfully recorded putative\nsingle and multi-unit activities, demonstrating high-fidelity neural recordings. These results\nindicate that any potential contamination from small dural fragments does not compromise\nelectrode performance, addressing the concern about tissue damage and electrode degradation.\nRevision: We have included these findings in the revised manuscript in the Results\nsection in the subsection titled “Steeltrode with smaller shank is rigid enough to\npuncture rat dura and remains fully functional and reusable after repeated\ninsertions”\n- Page 11: “For passive electrical neural probes, scaling up he the number of channels\nmeans increasing the number of traces connected to each recording electrode”. Is the “he” an\nerror in this sentence? The citation 42 is cited two times in the same paragraph.\nResponse: Corrections were made to the revised manuscript.\n- Page 15: Things to keep in mind about the “brain micromotions”. Sometimes the monkeys\nmake pressure from the inside of the brain, making force with their bodies. Thus, they increase\ntheir intracranial pressure. When this happens the brain goes up and cells are killed and the brain\ntissue is detached from the probe influencing the data.\nResponse: We acknowledge this is an important consideration, and we plan to further investigate\nthese effects in future studies to ensure that the interface between the steeltrode and brain tissue\nremains reliable under all physiological conditions. The text has been revised to reflect that both\nhead movements and brain micromotions in monkeys contribute to tethering force induced tissue\ndamage and data quality degradation for neural probes.\nRevision: On Page 15:\n“As a result, once inserted into the brain, the implanted shank of the stainless steel probe\ncan move independently of the backend, reducing tissue damage and preserving signal\nquality by mitigating tethering forces from skull attachment during brain micromotions and\nsudden bodily movements (Polanco et. al., Biosensors (2016) and Harris et. al., Nat.\nNeuroscience (2016 ). ”\n- Page 22: How many monkeys have been used to test the probes? Sometimes the animals\nhave different behaviors that can influence the data or the probe's brittleness. For example, if it is\na big male with aggressive behavior it will be more challenging to keep the electrode safe than if it is\na female, even if both are head fixated.\nResponse: For the experiments reported in this paper, steeltrode was tested on a large male\nmacaque, weighing above 15 kgs. With properly restrained and head-fixated monkey, we have\nnot encountered any difficulty with the implantation of steeltrode, regardless of the individual\nmonkey's behavior on the day of the experiment. It is our understanding that animal movement\nmay play a role with extremely brittle silicon probes. For the steeltrodes, this is no concern at all.\nThere is a small amount of risk when the electrodes get mounted to the microdrive (this happens\nwithout the animal present). At that stage, it is possible that the experimenter drops the assembly\nand probes bend. But once the electrodes have been attached to the microdrive, the process of\nmounting them to the recording chamber and inserting them into the brain is without risk to the\nsteeltrode.\nHow much time the researcher spent to insert the electrode into the brain?\nResponse: The insertion of the steeltrode into the macaque brain took approximately 15 minutes.\nThis duration also includes the time required to connect the neural amplifier, ground wires, and\nother necessary components.\nWhat was the total duration of the experiment? (This has an influence on the behavior of the\nmonkey as I explained above)?\nResponse: The typical macaque experiments had an approximate duration of 7 hours.\nHow the electrode should be cleaned?\nResponse: After explanting the probe, it is first rinsed off with Deionized (DI) water. Then the\nprobe tip is immersed in Tergazyme solution for 2 hours, and then rinsed off again with DI water.\nThe cleaning procedure is added to the methods section related to the animal experiments\nRevision: We have added the following information to the Methods section:\nProbe Cleaning\nAfter explanting the steeltrode at the end of invivo experiment, it is first rinsed off\nwith Deionized (DI) water. Then the probe tip is immersed in Tergazym solution for\n2 hours and then rinsed off again with DI water.\nDid you check it into subcortical areas?\nResponse: Thank you for your question. As noted in our previous response regarding the\nfeasibility of reaching the brain stem with steeltrodes, by optimizing our approach—specifically by\nreducing dead space in implantation hardware and employing extended-length steeltrodes—it is\nindeed very feasible to reliably target subcortical areas, including the brain stem. However, in this\npaper, we focus on demonstrating the feasibility and robustness of steeltrodes for recording from\nthe entire cortical laminae.\nHow many times the probes can be used while keeping their data quality threshold?\nResponse: This is a very important point. We have observed that even after insertion of and re-\nuse of the same probe multiple times in NHP brain, the electrode impedances and the recorded\nsignal quality (i.e., SNR) did not significantly change. We note that after each insertion the\nelectrodes were cleaned using DI water and Tergazyme and sterilized using ethylene oxide before\neach re-use. Despite these aggressive cleaning and sterilization processes, the performance and\nsignal quality did not degrade.\nWe have also conducted additional experiments to evaluate the probe's robustness and\nperformance under repeated use. These experiments involved 10 consecutive insertions through\nrat dura using the same probe. We observed no significant changes in electrode impedance after\nmultiple insertions, validating both the probe's structural integrity and the microelectrodes'\nfunctional integrity during repeated use.\nGiven the stable electrode impedance, it is reasonable to infer that the neural recording noise\nfloor and subsequent data quality would exhibit similar stability, considering the inherent\ncorrelation between noise floor and electrochemical impedance. Furthermore, the successful\nacquisition of putative single- and multi-unit activities following the 10th insertion indicates that\nthe electrodes retained their capacity for high-fidelity neural recordings even after substantial\nreuse.\nRevision: The results of these experiments are added to the manuscript under the section\ntitled “Steeltrode with smaller shank is rigid enough to puncture rat dura and\nremains fully functional and reusable after repeated insertions”\nApart from the aforementioned section, we have added the following text to the Discussion\nsection:\n“In this paper, we report that even after extensive reuse and multiple insertions,\nthe steeltrodes preserved electrode integrity and continued to record high-quality\nneural signals, attesting to their robustness. However, a systematic evaluation to\ndetermine the threshold at which signal fidelity deteriorates under chronic use or\nrepeated acute implantations lies beyond the scope of this study.”\nPage 31: “Histological examination demonstrated that surgical removal of dura reduces the neural\ndensity along the tract…”. When did this histological examination take place? The same day of\nthe procedure or days later?\nResponse: Brain sample for histological assessment was collected 2 hrs following implantation\nof probe to study the acute tissue damage/response following insertions.\nRevision: The following is added to the Methods section to clarify this point:\n(Page 46) “For histological evaluation, rats were transcardially perfused with\nfixative after 2 hours of implantation. Briefly, rats were transcardially perfused with\nPBS followed by 4% paraformaldehyde (PFA) to fix the tissues. Brain was isolated\nfollowing fixation and kept in 4% PFA overnight followed by gradient sucrose\nsolution (10%, 20% and 30%) as preprocessing for cryosectioning. Further 30m\nsectioned slice was prepared using cryostat and stored in PBS. Sections were\nfurther stained using NeuroTrace™ 500/525 Green Fluorescent Nissl Stain (Cat\nNo. N21480, Thermofisher scientific), co labelled with DAPI (Nuclear Stain) and\nmounted on slides.”\nPage 33: “Such devices can be implanted into deep cortical regions while being free to move with\nbrain micromotions ensuring minimized chronic tissue damage due to significantly reduced\ntethering force”. At the beginning of the article is written that electrodes can arise anywhere in the\nbrain. It would be interesting to be precise if subcortical regions such as the brainstem can be\nrecorded.\nResponse: The 8 cm steeltrode we have demonstrated in this work could in principle reach the\nbrainstem, since reaching the brainstem only requires traversing about 5 cm of brain tissue,\ndepending on the approach angle. However, as mentioned in a previous response, there are two\nthings to consider: (1) Most NHP recording setups impose a ‘dead’ length, meaning a large part\nof the shank can't be inserted into the brain, due to hardware constraints of the implantation rig.\n(2) The current 8 cm steeltrodes can be made much longer with the proposed fabrication platform.\nBy optimizing the approach angle, reducing dead space, and using longer probes, it's possible to\nreach the entire brain, including the brain stem.\nRevision: To clarify this issue in the manuscript, we have added the following to the\nDiscussion section of the manuscript:\n“The 8-cm steeltrode easily enables reaching subcortical regions in macaques. In\nour experiments, the approach angle, chamber design and microdrive\narrangement dictated that between 2 and 4 cm of the shank length remained out\nof the brain tissue, thus leading to an effective electrode length of 4 to 6 cm. Given\nthat the dorso-ventral extent of the monkey brain is below 5 cm, the current setup\nwould provide access to most of the brain, including most subcortical targets. A\nredesign of the setup can further increase the effective length to around 7 cm, thus\nproviding access to the entire monkey brain. For use in larger species, it will also\nbe possible to use longer probes. The length of the neural probe was limited by\nthe size of the substrate (i.e., a 4-inch-diameter wafer) that we made for\nphotolithography. Since we made this wafer out of large stainless steel sheets, the\nsize of the wafer can be easily made larger (i.e., 6 inches or even 8 inches) to\nmicrofabricate neural probes, with implantable shanks of up to 18 cm, if needed.”\nPage 34: “We can also readily adapt our fabrication process to realize probes with multiple\nshanks”. In this case, you should keep in mind the number of probes that the researcher uses at\nthe same time. The arrangement of the instrument around the recorded zone has to have enough\nplace and to be separated enough to allow several probes at the same time. Moreover, the\nposition degree will influence if the shanks will touch each other.\nResponse: We acknowledge the importance of probe spacing and the backend headstage\narrangement when implanting multiple probe/shanks simultaneously. We will carefully consider\nthese factors in our future investigations to ensure optimal multi-shank probe configurations.\nRevision: We have revised the manuscript to mention this important point:\nPage 39: “We can also readily adapt our fabrication process to realize probes with\nmultiple shanks. We should however note that the while implanting multiple multi-\nshank probes next to each other for high-density neural recording, the backend\nheadstage arrangement should be optimized”.\nReviewer 2:\nKey results:\nThis paper demonstrates the novel fabrication process to integrate PDMS / Parylene C / SU8 and\nplatinum electrodes (that can be vertically stacked to increase channel count) on commercially\navailable stainless steel. This process allows fabrication of high aspect ratio neural implants that\nare less fragile for implantation in vivo. The authors showcase the recording ability of such an\nimplant in the macaque brain for auditory cortex recordings.\nMain comment:\nThe claims that high-aspect ratio, high-density, versatile, stainless-steel probes, fabricated using\nmicrofabrication/micromachining processes, that can record with high-resolution from deep\nregions of the brain is supported by the data presented in the paper. However, the\nrobustness/resilience against fracture of the stainless-steel probes is not thoroughly\ndemonstrated in the present version of the manuscript, in my opinion. A figure does show the\nforce required to fracture/plastically deform silicon vs. stainless steel, but no/little demonstration\nof what happens to the integrity of the electrodes integrated on the stainless-steel under stress,\nbefore/after bending, after multiple insertions, etc., is shown. “Reusability” of such devices, or\n“safety for multiple insertions” is not clearly demonstrated here.\nResponse: We appreciate your detailed feedback regarding the need to further demonstrate the\nrobustness and resilience of our stainless-steel steeltrodes. In the revised manuscript, we have\nconducted additional experiments aimed specifically at addressing your concerns:\nMechanical Stress Testing:\nTo demonstrate functional robustness, we performed bending experiments and measured\nelectrode impedances before and after aggressive bending. The impedance values remained\nunchanged, indicating that the probe and microelectrodes did not degrade under stress. The\ndetails of these experiments are included in the manuscript in the section titled “Bending\nExperiments Validate the Mechanical Durability and Electrical Performance of Steeltrode”.\nReusability and in vivo Multiple-Insertion Trials:\nTo further demonstrate and validate the reusability of the probes, we conducted in vivo\nexperiments in which the same steeltrode was used for ten consecutive dura insertions in rodents.\nThe electrochemical impedance of the electrodes remained stable across insertions. The probe\ncontinued to record putative single unit activities, confirming no significant changes in impedance\nor electrode failure and that both the substrate and the microelectrode array remain fully\noperational during multiple uses. The methodology and results of these experiments are now\ndiscussed in Section “Steeltrode with smaller shank is rigid enough to puncture rat dura\nand remains fully functional and reusable after repeated insertions” in the revised\nmanuscript.\nData and methodology questions/comments:\nMain, P.5: “In contrast, since steeltrodes are resilient against fracture, they can be safely inserted\ninto the neural tissue multiple times and their handling and manipulation is reliable and safe”. It\nwould be important to provide data to demonstrate this: the paper as it is does not really show\n“safe multiple insertion”, nor that the probe can be safely manipulated. This would be important,\nnot only regarding the stainless-steel part, but also with the integrated electrodes, as part they\nare the critical part of the whole recording probe.\nResponse: We have added a section on our new in vivo experiments (Section titled “Steeltrode\nwith smaller shank is rigid enough to puncture rat dura and remains fully functional and\nreusable after repeated insertions”), specifically demonstrating the ability to insert and reuse\nour steeltrodes multiple times without compromising probe integrity even after rather aggressive\ncleaning and handling steps. We performed 10 successive dura penetrations in four animals using\nthe same 15 mm steeltrode and measured electrode impedances before implantation and after\n4, 8, and 10 insertions. We observed negligible impedance changes, indicating that integrated\nmicroelectrodes were functionally and mechanically intact and robust. Crucially, the probe\nmaintained high-fidelity spiking recordings from the rat hippocampus, confirming not only\nstructural stability but also sustained electrophysiological performance. These results directly\nsupport our claim that the steeltrode can be repeatedly inserted, safely handled and manipulated,\nand remain highly reliable for neural recordings.\nMain, P.7: “…whereas the steeltrode only deforms and bends, thus causing much less and often\nreversible damage to the neural tissue”: same here, what about the impact on the electrodes\ncontact integrity of bending/deformation ?\nResponse: In the revised manuscript (see “Bending Experiments Validate the Mechanical\nDurability and Electrical Performance of Steeltrode”), we have included a new set of\nexperiments specifically designed to address this issue.\nThe following figure shows the experimental setup and the measurements.\nIn this experiment, a 78 mm-long steeltrode was secured at its backend using a custom 3D-printed\nmount, while a linear actuator, controlled by a microcontroller, applied bending forces to the free\ntip of the probe. The tip was incrementally displaced by 10, 20, 30, and 40 mm from its neutral\n(undeflected) position, and the probe was then returned to neutral after each step. Following every\nbending cycle, the electrochemical impedances of all channels of the steeltrode were measured.\nOur data (Figures 3(C) and 3(D) in the revised manuscript) shows that neither the impedance\nmagnitude (1.55 ± 0.13 MΩ at 1 kHz) nor the phase (−70 ± 1° at 1 kHz) deviated significantly from\nthe baseline, even after the maximum deflection of 40 mm. This high level of stability indicates\nthat the microelectrodes—along with their insulation layers—remained structurally and electrically\nintact, with no detectable microcracking, delamination, or adhesion failures.\nMain, P.9: “We have fabricated probes with electrodes as small as 10-μm diameter and as large\nas 40-μm diameters.”: I believe no data shows this directly in the manuscript. It could be\ninteresting to see some sort of quantification"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate and thanks to authors about the responses to my comments. All of my concerns have been adequately addressed, and I have no further suggestions. I support the publication of the revised manuscript.", "reviewer_2": "Thank you for addressing my concerns. I believe the manuscript has gained in clarity and additional data that was previously missing to support some of the main claims.\nI have only a few additional small comments/remarks:\nP.6 last paragraph end: “while minimizing the tissue damage. Damage.” : Typo, twice “damage”.\nP.7 l.3: “the buckling force for the will be 40mN”: word missing after “the”.\nP.9 l.21: “For steeltrodes, the devised microfabrication …”: “devised”?\nP.22: “Error reference not found” x2\nLegend Fig. 8 C) / P.34 l. new paragraph / “Statistical Power", "reviewer_section": "Can you make more clear in all these sections what N you are mentioning (and make sure you mention the appropriate N): is it N = number of animals or N = number of insertions or N = number of cross sections, and how many insertions/animals/sections per group/condition (control, dura removal, dura piercing) for comparison analysis? You mention in your response “The histology data were collected from 5 different insertions in 3 animals. Two insertions were performed in each animal.”: was the second insertion performed on another hemisphere for example to avoid tissue effect due to the first insertion when looking at histology ? What happened to the 6th insertion (if 2 insertions/animals) ?\nReference 30 and 56 are the same ?\nFig. 8 A: can a scale bar be added for the images ?", "reviewer_3": "The authors have provided a detailed and systematic procedure on fabrication of steeltrode used as a brain sensor. The design aspects of the probe seem fine in my view, though foll0wing minor issues may be considered.\n- Though fabrication steps have been provided in details, still it is not very clear that how did the authors made the long shank which is inserted in the brain. some clear figure may be provided.\n- Also, in the figure 2S the first and second figure are same. Please look into this."}, "author_response": "Response: We thank the reviewer for the thoughtful feedback and kind words. We are pleased\nto know that all concerns have been adequately addressed and that the revised manuscript meets\nyour expectations.\nResponse: Thank you for pointing these out. These errors are now corrected in the revised\nmanuscript.\nResponse: Thank you. We have corrected the figure citation error.\nResponse : We thank the reviewer for this important comment and apologize for the earlier\nambiguity regarding sample sizes and experimental design. We have now revised the manuscript\nto provide a clear and detailed account of the number of animals, hemispheres, insertions, and\nhistological sections used in each condition. Specifically, we clarify that all insertions were\nperformed in 5 hemispheres and no insertion was performed in 3 hemispheres from a total of 4\nanimals. The hemispheres were allocated as follows: 3 for control (no insertion), 2 for dura\nremoval, and 3 for dura piercing. From each hemisphere, 6-9 coronal sections were analyzed,\nand statistical comparisons were conducted at the section level. We have also corrected and\nadded justification for the use of a total 4 animals (including control) in this exploratory study.\nThese revisions are reflected in the updated Methods section under “Statistical Power,\nReplication, and Exclusion Criteria (Rodent).”\nResponse: Thank you for pointing this out. This error is now corrected in the manuscript.\nResponse: The scale bars have been added to the images.\nResponse: Thank you for your comment. We have included an additional figure (Figure S3) to\nthe supplementary material to clarify the steeltrode release process.\nTo fabricate steeltrode in a high throughput process, we use planar microfabrication processes\non stainless steel wafers to deposit insulation layers, deposit and pattern high-density metal\ntraces, and define selective openings to expose the recording electrodes, backend contact pads,\nand probe outlines. The details of these planar microfabrication processes is discussed in the\nMethods section.\nAfter wafer-level processing is completed, we employ a laser micromachining process to release\nthe steeltrodes with long implantable shanks from the stainless-steel wafer. In this step, the outline\nof each steeltrode device is precisely etched using a 200 W fiber laser through laser ablation. The\nlaser is precisely guided along the device (steeltrode) boundaries based on a CAD layout, using\nalignment fiducials patterned on the wafer during the planar microfabrication process. An example\nof the laser etching path for a steeltrode on a processed stainless steel wafer is shown in Figure\nS3. By repeating this automated process for each steeltrodes on a wafer, the individual\nsteeltrodes are cleanly released from the substrate. Representative images of the released\nsteeltrodes following laser micromachining are also shown in Figure S3.\nResponse: Thank you for pointing this out. This figure is now corrected in the manuscript."}]

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