Neural probes that contain microelectrode arrays are employed as tools to understand better the intricate networks in the nervous system by recording neural activity in the form of action potentials and local field potentials, as well as therapeutic approaches to treat and restore lost sensorimotor functions via neuromodulation, for example, electrical stimulation. Nonetheless, the implementation of devices that match the three-dimensional (3D) topology of nervous tissues is still challenging. Aiming to capture and interact with neural activity from single to groups of neurons in the 3D space of nervous tissues, this work presents the development and implementation of fully flexible multisite 3D penetrating neural probes (NPs) for in vitro and in vivo neural applications.

Combining thin film technology and surface micromachining processes with two-photon lithography (2PL), we developed 3D-printed NPs based on self-aligned template-assisted electrodeposition processes [1]; Kiri-NPs based on two-dimensional (2D) designs followed by a kirigami cut-and-fold approach; and 3D NP-stacks, based on the stacking of 2D NPs with key-lock systems. Furthermore, we optimized the cross-sectional dimensions to reduce the bending stiffness of the penetrating probes, thereby allowing the implementation of an atraumatic insertion by reducing the effective length of the penetrating shanks of the NPs.

Exploring the scope of our 3D neurotechnology, we tested the NPs in different neuronal models, spanning from explanted rodent retinas and human brain slices in vitro to the cortex of rodents in vivo. While all three devices enabled 3D laminar recordings across neural models capturing both single units and neuronal population activity, 3D-printed NPs, and Kiri-NPs simultaneously allowed the additional recording of neuronal potentials from the tissue surface. On the other hand, 3D NP-stacks promise access to deeper neural structures, by facilitating the implementation of additional insertion aids, such as dissolvable polymer braces.

Achieving an aspect ratio of 3D microelectrodes of up to 33:1, multiple multisite shanks up to 128 shanks in a standalone device, and a density of 44 shanks/mm², 3D-printed NPs, Kiri-NPs, and 3D NP-stacks, respectively, comprise a pool of 3D neurotechnologies that is highly customizable and adaptable to the anatomy of different neural structures. Hence, these technologies hold the potential of easily increasing the topological dimensionality of neuroelectronic interfaces to address single or groups of neurons, to networks across neural layers and regions. The deployment of such technology is then versatile, from the investigation of micro-seizures in epileptic models in vitro to the further development of visual prostheses to enable a bidirectional communication for the simultaneous recording and stimulation of the visual pathway [2], [3].