Nano Research 1 https://doi.org/10.1007/s12274Nano Res -018-2127-4
Nano functional neural interfaces
1,§ 2,§ 3,4,§ 5 2 2 Yongchen Wang , Hanlin Zhu , Huiran Yang , Aaron D. Argall , Lan Luan , Chong Xie (), and Liang Guo3,6 ()
Nano Res., Just Accepted Manuscript • https://doi.org/10.1007/s12274-018-2127-4 http://www.thenanoresearch.com on Jun. 12, 2018
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TABLE OF CONTENTS (TOC)
Nano functional neural interfaces
Yongchen Wang1†, Hanlin Zhu2†, Huiran Yang1,3†, Aaron D. Argall1, Lan Luan2, Chong Xie2*, and Liang Guo1*
1 The Ohio State University, U.S.A. 2 The University of Texas at Austin, U.S.A. 3 Nanjing Tech University, China
† Equal contribution Engineered functional neural interfaces serve as essential abiotic-biotic transducers between an engineered system and the nervous system. This review covers the exciting developments and applications of functional neural interfaces that rely on nanoelectrodes, nanotransducers, or bionanotransducers to establish an interface with the nervous system.
Guo Lab: http://guolab.engineering.osu.edu Xie Lab: http://faculty.engr.utexas.edu/xie
Nano Research Nano Res. Nano Res. 1 DOI Review Article
Nano functional neural interfaces
1† 2† 3,4† 5 2 2 Yongchen Wang , Hanlin Zhu , Huiran Yang , Aaron D. Argall , Lan Luan , Chong Xie (), and Liang 3,6 Guo ()
1 Department of Biomedical Engineering, The Ohio State University, Columbus 43210, USA 2 Department of Biomedical Engineering, The University of Texas at Austin, Austin 78712, USA 3 Department of Electrical and Computer Engineering, The Ohio State University, Columbus 43210, USA 4 Key Laboratory of Flexible Electronics and Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing 211816, China 5 Biomedical Sciences Graduate Program, The Ohio State University, Columbus 43210, USA 6 Department of Neuroscience, The Ohio State University, Columbus 43210, USA † Equal contribution
Received: day month year ABSTRACT Revised: day month year Engineered functional neural interfaces (fNIs) serve as essential abiotic-biotic Accepted: day month year transducers between an engineered system and the nervous system. They (automatically inserted by convert external physical stimuli to cellular signals in stimulation mode, or read the publisher) out biological processes in recording mode. Information can be exchanged using electricity, light, magnetic fields, mechanical forces, heat, or chemical © Tsinghua University Press signals. fNIs have found applications for studying processes in neural circuits and Springer-Verlag Berlin from cell cultures to organs to whole organisms. fNI-facilitated signal Heidelberg 2014 transduction schemes, coupled with easily manipulable and observable external physical signals, have attracted considerable attentions in recent years. This KEYWORDS enticing field is rapidly evolving toward miniaturization and biomimicry to achieve long-term interface stability with great signal transduction efficiency. Neural interface, Not only a new generation of neuroelectrodes has been invented, but advanced neurotechnology, fNIs that explore other physical modalities of neuromodulation and recording nanoelectrode, have started to bloom. This review covers these exciting developments and nanomaterial, applications of fNIs that rely on nanoelectrodes, nanotransducers, or neural recording, bionanotransducers to establish an interface with the nervous system. These neural stimulation nano fNIs are promising in offering a high spatial resolution, high target specificity, and high communication bandwidth by allowing for a high density and count of signal channels with minimum material volume and area to dramatically improve the chronic integration of the fNI to the target neural tissue. Such demanding advances in nano fNIs will greatly facilitate new opportunities not only for studying basic neuroscience, but also for diagnosing and treating various neurological diseases.
Address correspondence to Prof. Liang Guo, [email protected]; and Prof. Chong Xie, [email protected]
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fNIs are of a major focus. Such demanding advances in fNIs will greatly facilitate new opportunities not 1. Introduction only for studying basic neuroscience, but also for diagnosing and treating various neurological 1.1 What are functional neural interfaces? diseases. Engineered functional neural interfaces (fNIs) serve as essential abiotic-biotic transducers between 1.3 Why nano? an engineered system and the nervous system. They Conventional electrode-based neurotechnologies convert external physical stimuli to cellular signals in are facing two major hurdles: (1) susceptibility of the stimulation mode, or read out biological processes in abiotic-biotic interface to immune responses and (2) recording mode. Information can be exchanged using communication inefficiency through the electricity, light, magnetic fields, mechanical forces, abiotic-biotic interface [2]. Nano fNIs are compelling heat, or chemical signals. fNIs have found in offering more effective solutions to both of these applications for studying processes in neural circuits two aspects. from cell cultures to organs to whole organisms. fNI-facilitated signal transduction schemes, coupled 1.3.1 Chronic stability with easily manipulable and observable external Even though many electrode-based physical signals, have attracted considerable neurotechnologies have made great strides during attentions in recent years. This enticing field is the preceding few decades in proving their feasibility rapidly evolving toward miniaturization and in treating and restoring impaired neural functions, biomimicry to achieve long-term interface stability their clinical potential is severely restricted by issues with great signal transduction efficiency. This review in integration of the neural interface within the covers the developments and applications of fNIs complex tissue environment. Not only do these that rely on nanoelectrodes, nanotransducers, or mechanically and chemically distinct neural bionanotransducers to establish an interface with the interfaces cause significant infection, but the nervous system. communication at the electrode-tissue interface is significantly diminished as the implant is isolated by 1.2 Why are fNIs important? fibrosis over time as a consequence of the In the past decade, the field of fNIs has foreign-body reactions [3]. This discovery of fibrotic experienced a dramatic revolution. The once encapsulation developing around the implanted electrical-engineering concentrated field has evolved neuroelectrodes over a short time window of a few to a new stage that has absorbed an ever-large months [4-7], which physically screens the electrical research population and ever-diverse sensors from accessing to the target neurons, has multidisciplinary approaches. Not only a new largely shaped the thinking and practice in the field generation of neuroelectrodes has been invented, but in the past decade. The resulting new concepts in advanced fNIs that explore other physical modalities neural interfacing have motived both the of neuromodulation and recording have started to development of a new generation of miniaturized bloom, partially stimulated by the great success of neuroelectrodes [2, 8-10] and the exploration of optogenetics [1]. This new stage is facilitated by alternative approaches that feature minimum or even advocations and funding supports on brain-related none invasiveness. Reduction of the footprint of the research across the globe. Specifically, in the USA, the neural implant down to the nanoscale to make it Brain Research through Advancing Innovative less “sensible” to the host tissue environment has Neurotechnologies (BRAIN) and Stimulating proven to dramatically improve the chronic stability Peripheral Activity to Relieve Conditions (SPARC) of the neural interface [8-10]. Alternatively, to Initiatives aim to significantly promote brain and mitigate the problems associated with the bioelectric medicine research by accelerating the conventional electrode-based approaches, such as the development and application of novel and requirement for implantation of the bulky interface paradigm-shifting neurotechnologies, among which into the immediate target neural tissue [11],
Nano Res. 3 nonspecific and variable activation, bio-fouling, potentials (spikes) provides insufficient information motion artifacts, and tissue damage [3], remotely to establish definite correlation with individual controlled approaches that leverage nanotransducers neurons, and often bias towards frequently firing or bionanotransducers in the pursuit of minimum neurons. Unavoidable invasiveness of implanted invasiveness, high spatial resolution, and cell-type electrodes greatly restricts the number and density of specificity have drawn unprecedented attentions. electrodes that can be implanted into the brain, These alternative nano fNIs hold great potentials for which leads to sparse sampling of the neural circuitry. better integration to the target neural tissue at the Furthermore, conventional neuroelectrodes typically tissue and cellular levels. fail to provide consistently stable, high-quality neural recordings over both the short- and long-terms 1.3.2 Functional efficiency [20-22]. In time scales as short as hours, substantial Realizing neural-realistic communication while changes to the recording conditions often occur due minimizing side-effects to neural circuits is of great to micro-movements of the implanted electrodes interest in the field. It requires developing fNIs that with respect to the brain tissue [23-25]. Over weeks to comply with the biological mechanism of neural months, deterioration in recording efficacy and signaling. Unfortunately, signal transduction at the fidelity is caused by biotic and abiotic failures [26-29], macro electrode-tissue interface is inefficient and can including sustained foreign body reactions at the lead to tissue injuries due to mismatched tissue-probe interface such as neural degeneration, communication mechanisms between the electronics reoccurring blood leakage in capillaries and glial scar and target tissue [12-14]. Neuronal activity is formation [23, 30-34]. Recently, there have been modulated by ion channel-mediated action potential increasing interests in taking advantage of nano and signaling, and thus ion channels naturally become micro technologies to address the aforementioned the direct target of physical stimuli. Working at a challenges in neuroelectrodes. scale close to the dimensions of single ion channels, 2.1.1 Nanostructure-enabled intracellular access nano fNIs are promising in offering a high spatial resolution, high target specificity, and high Two major types of electrophysiological recording communication bandwidth by allowing for a high methods, intracellular and extracellular, have been density and count of signal channels with minimum developed to measure action potentials with material volume and area to minimize tissue complementary capabilities. Traditional volumetric displacement and foreign-body reactions [15, 16]. intracellular recording methods such as the whole-cell patch clamp requires rupturing a portion of the plasma membrane to access the cell interior 2. Nano fNIs for neural recording directly [35]. Whole-cell patch clamp (recording tip 2.1 Nanoelectrodes diameter commonly ranging from close to 1 micron [36] to several micrometers [37]) is the most Detecting the complex and dynamic activities of sensitive method to record electrophysiological the nervous system requires precise measurements of the basic functional units—neurons. Neuroelectrodes events of neurons, but is highly invasive and provide one of the most useful neurotechnologies by technically difficult to implement, which precludes allowing for time-resolved electrical detection of long-term or large-scale recording. On the other neural activities and direct stimulation of neural hand, extracellular recording methods such as tissues. Therefore, pushing the limits of multi-electrode arrays utilize micropatterned electrophysiological recording and stimulation is of electrodes to afford long-term and multiplexed in great scientific and clinical interests [17-19]. Despite important and unique capabilities, conventional vitro measurements [38-40]. However, extracellular neuroelectrodes have significant limitations. recording suffers significantly in signal strength Intrinsically, electrophysiological recording of action and quality. This has therefore resulted in the need
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 4 Nano Res. for electrophysiological methods that combine the silicon nanowire arrays that enabled intracellular advantages of both intracellular and extracellular access to both record from and stimulate neurons (Figure 1c and 1d) [45]. Abbott et al. further extended recording methods. In the past several years, there this strategy by fabricating vertical nanoelectrodes on have been developments that aim at achieving a CMOS multiplexer, so that an array of 32 X 32 intracellular recording with extracellular nanoelectrodes can be simultaneously addressed [46]. nanoelectrodes or transistors while possibly A similar strategy was also reported by Liu et al. [47]. allowing for advantages of minimal invasiveness Field effect transistor (FET) sensors, due to their and easy scalability. Although some structures sensing mechanism, do not suffer from thermal noise reviewed in this section fall into microscales instead, as the sensor size decreases, which is the major limitation of passive electrodes. Tian et al. their working mechanisms rely on nanoscale developed FET sensors based on kinked silicon structures or interactions. nanowires [48]. Using cultured cardiomyocytes as an It has been shown in multiple works that micro- in vitro model, they showed a clear transition from and nanostructures can promote tighter contacts at extracellular to intracellular recording, as the tip of the cell-electrode interface, which enhance recording the device slowly penetrated the cellular membrane outcomes. Hai et al. pioneered in creating (Figure 1e-1j). A free standing version of the kinked mushroom-shaped micrometer-sized gold electrode silicon nanowire FET sensor was created by Qing et arrays that could detect attenuated intracellular al. [49], in which individual branches of the kinked action potentials [41]. Xie et al. fabricated vertical nanowire could be adjusted to target specific cell platinum nanopillar electrodes, with which they under a standard microscope. Duan et al. reported demonstrated that these electrodes formed tight the recording of a full amplitude intracellular action junctions with cultured cardiomyocytes (Figure 1a potential in cardiomyocytes by silicon FET sensors and 1b) [42, 43]. By local electroporation, the coupled with silicon dioxide nanotubes [50]. Fu et al. nanopillar electrodes could enhance the action further pushed the electrical detection limit by potential recording with more than ten times greater producing a silicon nanotube and nanowire hybrid amplitudes and intracellular-like waveforms. Lin et FET electrode with the recording tip size less than 10 al. discovered that nanotubes were advantageous nm [51]. To probe fine structures in the neuron, than nanopillars in making and maintaining tight Jayant et al. took advantage of quantum dot (QD) junctions with the cell [44]. It was demonstrated that coated nanopipette electrodes to recover full back iridium oxide nanotubes enabled a more effective propagation details of action potentials along and stable intracellular access to cultured targeted dendritic spines [52]. cardiomyocytes. Robinson et al. fabricated vertical
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Figure 1 Representative nanoelectrodes. (a) Scanning electron microscopy (SEM) image showing that the nanopillar electrodes were strongly engulfed by a cell [42]. (b) Demonstration of the change in cellular membrane and corresponding recorded signal before and after electroporation. Nanopores were formed in the plasma membrane, enabling intracellular recording [42]. (c) SEM image of a 3-by-3 array of vertical nanowire electrodes [45]. Scale bar: 1 µm. (d) SEM image of nanowires interacting with a rat cortical neuron [45]. (e)-(j) Nanowire FET [48]. (e)-(g) Schematic diagrams showing the recording configurations. (h)-(j) Transition of the recorded signal from extracellular, transition period, to steady-state intracellular, as the probe slowly entered a cell. (a) and (b) adapted with permission from Springer Nature Ref. [42]; (c) and (d) adapted with permission from Springer Nature Ref. [45]; (e)-(j) adapted with permission from The American Association for the Advancement of Science Ref. [48].
2.1.2 Nanomaterials to reduce electrode impedance capacity comparing to titanium nitride and similar to carbon nanotube (Figure 2a-2c) [55]. Park et al. As the electrode size decreases to increase identified an optimal surface roughness factor of 233 recording density or to minimize invasiveness, the to maximize the performance for electrical impedance of the electrode increases dramatically. stimulation and recording by nanoporous platinum The thermal noise associated with the electrode and yielded an impedance of 0.039 Ω·cm2 and a impedance becomes a major limitation. Nano charge injection capacity of 3 mC/cm2 (400 μs) [56]. structures and materials have great potential in Lee et al. deposited highly roughened nonporous significantly lowering the electrode impedance and platinum film on gold tips of traditional silicon extending the electrode size limitation. electrode and greatly decreased the interface Owning to its highly porous structure that impedance to 0.029 Ω·cm2 [57]. Chung et al. used CF4 increases the electrochemical surface area and plasma treatment to increase the surface roughness subsequently decreases the impedance, platinum of gold from 1.7 nm to 22 nm, drastically decreasing black has long been used in electrical recording. Kim the impedance by 98% [58]. Plasma treated electrodes et al. improved the mechanical stability and electrical recorded signal with lower background noises and property of platinum black by depositing bioinspired evoked local field potentials (LFPs) with higher adhesive polydopamine film in a layer-by-layer amplitudes from anterior cingulate cortex in rats. manner [53]. Nanostructured porous platinum was Chen et al. fabricated a carbon nanotube-based also demonstrated to increase the surface area of electrode which had a lower impedance and 6 times electrodes by more than 30 times, thus reducing the higher charge transfer capacity than gold impedance by 77% [54]. Weremfo et al. fabricated microelectrodes [59]. (More about CNT electrodes are surface-roughened platinum electrodes that had covered in section 3.1 and Figure 2d and 2e.) Kim et nano features and a superior charge injection
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 6 Nano Res. al. developed Au-nanotube composite electrodes and a cathodic charge storage capacity (CSCc) 6, 2.8 which reduced the impedance of gold electrodes by and 2.7 times higher than those of electrodes with 99.3% [60]. They demonstrated in vitro extracellular bare Pt, Pt gray and IrOx, respectively. recordings from mouse cortical neurons, which had Deng et al. [68] invented a one-step an average signal-to-noise ratio of 92. Ju-Hyun et al. electrochemical process to deposit graphene modified electrodes with gold nanoflakes which led oxide/polypyrrole composite sheet on platinum to a reduction of impedance from 1.15 MΩ to 26.7 kΩ electrodes in order to increase electrode surface at 1 kHz [61]. Kim et al. enhanced the charge storage roughness (Figure 2h). The coated probe reduced the capacity as well as decreasing the impedance of impedance of bare platinum electrode by 90% and electrodes by depositing gold nanopourous increased the charge capacity density for more than structures by dealloying Ag-Au alloy [62]. two orders of magnitude. Bruggemann et al. demonstrated the fabrication of Besides reducing impedance, neurons seeded on vertical gold nanopillars on electrodes in an effort to Graphene oxide doped poly(3,4-ethylene achieve a higher electrode surface area, which led to dioxythiophene) (PEDOT) was found to grow longer a decreased electrode impedance [63]. Zhou et al. neurites than on PEDOT/PSS (polystyrene sulfonate). incorporated gold nanorods onto polyimide Functional biomolecules such as laminin peptides substrate to create a flexible thin-film microelectrode could be easily bonded to the graphene surface to array [64]. The nanostructure was able to bring down increase neurite outgrowth [69]. Such an electrode the impedance by 25 folds. Zhao et al. reported a demonstrated an improved sensitivity of 151 nA/μM relatively simple method to reduce electrode to dopamine comparing to a glass carbon electrode impedance [65]. Electrodes were first and minimized interference from ascorbic acid, a electro-co-deposited Au-Pt-Cu alloy nanoparticles competing analyte [70]. followed by etching away Cu. The remaining Au-Pt In addition to being used as a coating material, composite exhibited a rough surface with pores of graphene oxide could serve as the electrode material different sizes. The electrode impedance was reduced directly. Ng et al. [71] fabricated reduced graphene to 4.7% of that of the bare gold. oxide into disk microelectrodes of 10 μm diameter Kim et al. took a hybrid approach by depositing a and 60 μm pitch using nanoimprint lithography. The layer of iridium oxide on nanoporous gold, which electrodes featured a high sensitivity of 91 nA/μM to further reduced the impedance and increased the dopamine with a detection limit of 0.26 μM without charge storage capacity of the electrode (Figure 2f) using any functionalization process. The detection [66]. By combining the merits of platinum gray and capability was robust to highly resistive media, iridium oxide, Zeng et al. [67] deposited Pt gray and continuous flow and mechanical stress. IrOx on bare Pt in a layer by layer setup (Figure 2g). A decent adhesion of IrOx was enabled by the large surface area of nanocone-shaped Pt. They achieved impedance value of 2.45 kΩcm2 at 1 kHz
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Figure 2 Nanomaterials used to reduce electrode impedance. (a)-(c) Atomic force microscopy (AFM) images showing the morphologies of smooth, 3-min roughtened, 5-min roughened platinum surfaces [55]. (d) SEM images illustrating the nanofibrous network of multi-walled nanotube (MWNT) bundle coated on an electrode surface. Scale bar: 500 nm [72] . (e) Carbon nanotube outperformed traditional electrode materials by having a lower impedance [72]. (f) Scanning transmission electron microscope energy-dispersive X-ray spectroscopy (STEM-EDS) element mapping image of iridium oxide coating on a nanoporous gold electrode [66]. (g) Morphology of IrOx/Pt gray coatings [67] . (h) Field emission scanning electron microscope (FESEM) image showing PPy grafted at the surface of graphene oxide sheets (circled) [68]. (a)-(c) adapted with permission from American Chemical Society Ref. [55]; (d)-(e) adapted with permission from American Chemical Society Ref. [72]; (f) adapted with permission from American Chemical Society Ref. [66]; (g) adapted with permission from Elsevier Ref. [67]; (h) adapted with permission from Elsevier Ref. [68].
2.1.3 Minimizing tissue invasiveness of neuroelectrodes to the host tissues or apply stresses. Such micro movements not only prevent the tracking of the same It is commonly agreed that a long-term stable neurons as recording waveforms change accordingly, neural interface that can provide reliable neural but also induce neuron degeneration and bleeding recording and stimulation with minimal tissue [75], which further cause chronic inflammatory invasiveness is of paramount importance in responses and accumulation of reactive oxygen advancing our knowledge of brain functions and species, and eventually result in glial scarring and dysfunctions, as well as developing the next accelerated electrode corrosion [76]. generation neurotherapies [73]. A number of failure Seymour et al. discovered that thin electrodes (5 modes have been identified and many approaches μm in thickness) placed as close as 25 μm from the have been hypothesized and tested for improving the main silicon electrode shank (50 μm in thickness) chronic stability of neural electrodes, including but caused noticeably fewer neuronal cell death while not limited to physical (dimension and mechanical attracting less immune cells [77]. Schouenborg et al. stiffness), chemical (delamination, corrosion of compared the neuro-inflammatory responses of two electrodes) and biological (neuro-inflammatory, and probes having identical surgical damage profile but foreign body response) [74]. Among those highly different total surface areas [78]. Despite of the same interplayed failure factors, geometry and mechanical initial damage, the silicon lattice electrode with the mismatches between neural tissue and electrodes smaller surface area activated and attracted less plays a critical role in determining the long-term macrophages than their solid silicon counterpart. performance of the electrode-tissue interface. As a Karumbaiah et al. investigated the probe result of such mismatches, traditional neural geometry-dependent inflammation by comparing the electrodes may have micro movements with respect foreign body response in both acute and chronic time
8 Nano Res. scale of silicon electrode arrays that had disparate neuro-inflammatory responses was found for the soft thicknesses [79]. A 15 μm version outperformed a 50 probe comparing to its rigid counterpart. Sohal et al. μm version in terms of histological analysis and investigated long-term (26–96 weeks) foreign body activated immune cell density around the probe on responses in rabbit cortex induced by flexible both 3- and 12-week checkpoints. These studies all parylene-C based electrodes with microwire underscored the importance of electrode geometrical electrodes as the control [87]. Less gliosis and greater dimensions in mitigating tissue reactions. neuronal density were observed for the flexible probe. Carbon fibers have recently been a popular choice More importantly, such effects were more prominent to fabricate thin neuroelectrodes. Kozai et al. towards the chronic time scale. demonstrated ultra-small 7 μm diameter carbon fiber It is clear that the switch from conventional rigid electrodes for high quality extracellular recording materials, such as silicon and metals, to softer [80]. These electrodes were shown to have polymers, such as polyimide and parylene, helps significantly reduced foreign body responses and mitigate tissue reactions and promote the recording record single unit activities for over 4 weeks in vivo. stability. However, these polymers are still orders of Guitchounts et al. employed bundles of 5 μm magnitude stiffer than the brain tissue [88], and diameter carbon nanofibers to fabricate penetrating therefore the mechanical mismatch is still prominent. electrodes, and demonstrated more stable single-unit On the other hand, it is impractical, at present, to recordings in vivo [81]. Patel et al. fabricated fabricate functional devices with materials that are as 16-channel arrays of 8.4 μm diameter carbon fiber soft as tissues. This dilemma led researchers to try electrodes and demonstrated high-quality unit different approaches besides alternative materials. recordings for up to 3 months post implantation [82]. Because most neuroelectrodes are constructed in No neuronal density change and minimal to none high-aspect ratio probe geometries, bending is the astrocyte activation were observed in post-mortem major deformation happening at the interface. histology. Vitale et al. fabricated carbon nanotube Therefore, the primary goal of mitigating the fiber electrodes, which were softer than carbon fibers electrode-tissue mismatch is to minimize the bending and offered smaller impedance and greater charge stiffness of the implanted probe, which is given by Ks injection capacity, making them more suitable for = Eswh3/12 for a probe with a rectangular stimulation [83]. In addition, these softer fiber cross-section [10], where Es is the elastic modulus of electrodes also elicited less chronic tissue responses. the material, w is the probe width, and h is the probe Many efforts have also been put to make thickness. It is clear that the geometry of the probe neuroelectrodes flexible. Mercanzini et al. created (thickness and width) plays an important role in polyimide-based flexible probe to record LFPs, single modulating the bending stiffness. Therefore, and multi-unit activities in mouse cortex [84]. nanoelectronic devices could have unique and Histology results demonstrated a reduction in important impact in promoting tissue integration of inflammatory response comparing to rigid electrodes. neuroelectrodes. Wu et al. developed a flexible intracortical neural A series of recent work has demonstrated neural electrode with an 8 μm thick flexible construction probes made by nanoelectronic devices based on [85]. The electrodes were shown to record neural ultrathin polymer structures (Figure 3) [9, 10, 89-93]. activities for over six weeks. Du et al. compared the The key feature of 1 μm thickness yielded chronic tissue reactions of a novel ultraflexibility that allowed for a great reduction in poly(3,4-ethylenedioxythiophene)-polyethylene the mechanical mismatches at the tissue-electrode glycol (PEDOT-PEG) based ultrasoft microwire and interface. This enabled chronically stable recording of conventional tungsten wire electrodes [86]. At both 1 the same neurons over periods of multiple months. and 8 weeks post-surgery, a significant reduction in
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Figure 3 Increased flexibility improved tissue-electrode interface. (a) Photoacoustic images of two flexible carbon nanotube (CNT) electrodes inserted in the brain [93]. (b) Micrograph of a 3D mesh nanoelectronic brain probe suspended in buffer with a cylindrical shape [9]. (c) Chronic tracking of two detected neurons over a course of 4 months post-surgery [10]. Scale bar: vertical, 200 µV; horizontal, 1 ms. (d) Nanoelectronic probe as fabricated on a substrate [10]. Scale bar: 50 µm . (e) Two-photon imaging demonstrating little dislocation between neurons and an ultraflexible electrode during one month [10]. Scale bar: 50 µm. (f) Confocal micrograph of immunochemically labeled brain slice 5 months post-surgery, showing neurons surrounded the probe with minimum microglia activation [10]. Neurons were labeled in orange; electrode labeled as a green rectangle; and microglia indicated by white arrows. Scale bar: 50 µm. (a) adapted with permission from American Chemical Society Ref. [93]; (b) adapted with permission from Springer Nature Ref. [9]; (c)-(f) adapted with permission from American Association for the Advancement of Science Ref. [10].
2.1.4 High-density neural recording
One of the primary challenges of al. demonstrated the use of organic transistors to neurotechnologies is to simultaneously record from a record in vivo neural signals [100]. The transistors large number of neurons. The nature of extracellular were made of PEDOT on a highly flexible parylene recording determines that the electrode must be substrate with a total thickness of 4 μm. These within the close vicinity (~100 μm) of a neuron to devices are capable of amplifying and multiplexing precisely detect its activity [94]. To record from many signals locally, and potentially support high-density neurons inevitably requires a large array of surface recordings. Viventi et al. fabricated a electrodes placed on the surface of the brain or high-density electrode array multiplexed by silicon inserted into non-superficial structures. Besides, nanomembrane transistors on a flexible recording throughout a region requires that all polyimide substrate [101]. LFP recording on the brain neurons be within the range of a recording site and surface was demonstrated in an animal seizure that recording sites be densely packed to allow model. accurate spike sorting. In particular, the detection There are also significant recent efforts on making range of each recording site is typically 50–100 μm penetrating neuroelectrodes with nanofabrication. [95, 96], and spike sorting requires an electrode Du et al. nanofabricated a 64-channel probe with spacing of 20–50 μm center-to-center [97, 98] to interconnect width and spacing of 290 nm and resolve more single neurons. electrode pitch from 28 μm to 40 μm [95]. They found In the efforts to push limits of recording density, that only 50% of all the identified putative thalamic channel count and single unit yield, significant neurons were detectable with electrodes separated challenges arise, such as addressing individual more than 40 μm, which justifies the importance of channels and tissue displacement/injury. electrode array density. Similarly, Rios et al. Guo and DeWeerth developed an effective lift-off nanofabricated electrodes with features as small as method to pattern gold wires at high density on 300 nm to enable high-density 3D recording [102]. stretchable substrates [99]. Based on this technique, They achieved a minimum inter-electrode distance of high-density compliant and stretchable electrode 16 μm, totaling 1024 channels (64 channels per shank) arrays were fabricated and demonstrated by and covering a tissue volume of 0.6 mm3. Single units interfacing with muscular tissue [88]. Khodagholy et were found to show up on more than 6 channels,
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 10 Nano Res. allowing for neuron trilateration. Unique width of 100 μm, among which 52 could record layer-specific field potential signatures across the simultaneously, using 180 nm CMOS technology whole hippocampus facilitated the determination of [106]. Lopez et al. further developed this technology layer boundaries, which was subsequently validated and achieved 384 configurable channels out of 966 by histology result. An even denser array was created electrodes on a 70 μm wide shank with 130 nm by Scholvin et al. with 200 nm wiring width and 400 CMOS technology [107]. Recording with two of such nm spacing [103]. 1000 electrodes (200 channels per active CMOS probes were demonstrated in freely shank) were patterned on silicon probes with 11 μm moving rats (Figure 4f) [108]. A total of 700 single electrode separation (Figure 4a and 4b). A typical units were recorded from 5 brain structures, recorded unit could manifest on more than 10 demonstrating the capability of acquiring large-scale channels. Obien et al. fabricated a 128-channel activities of hundreds of neurons. electrode array with on-chip μLED for High density nanosensors also enabled mapping high-resolution electrophysiology coupled with of electrochemical activities of neurons at superior optogenetics [104]. Wei et al. nanofabricated spatial resolution. With more than 20,000 sensors per high-density electrode arrays on an ultraflexible cell. Kruss et al. [109] fabricated single wall nanotube structure (Figure 4c-4e) [105]. The interconnect traces based sensors that detected dopamine release at 100 had a width of 200 nm and a pitch of 400 nm, ms resolution. They were able to investigate how addressing electrodes spaced as small as 20 μm. The chemical communication of neurons were affected by overall probe cross-section area was as small as 10 cell morphology and developed models to optimize μm², which minimized chronic tissue reactions. their probe design based on the spatially preserved In addition to the passive approach, CMOS-based data [110]. active recording devices with buffer amplifiers For a detailed review of high-density neural placed closely to individual electrodes were also electrode array architectures and their respective developed for high-density penetrating electrodes. merits and challenges, readers are referred to Refs. Lopez et al. patterned 455 electrodes on a shank [111] and [112]. .
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Figure 4 Nanofabricated high-density neural electrodes. (a) Individual high-density probe with 200 electrode channels [103]. The electrodes were spaced by 11 µm in a 1D arrangement. (b) Recorded spikes using the probe in (a). (c)-(e) High-density ultraflexible probes with 20 µm electrode pitch and 800 nm thickness [105]. (f) Schematic diagram and microscope image of an active CMOS high-density probe [108]. (a) and (b) adapted with permission from IEEE Ref. [103]; (c)-(e) adapted with permission from Wiley-VCH Ref. [105]; (f) adapted with permission from Springer Nature Ref. [108].
neural activities can be recorded [114]. Thus, directly 2.2 Nanotransducer-assisted functional neural imaging the transmembrane potential of neurons imaging with high spatiotemporal (micrometer and sub-millisecond) resolutions, sensitivity up to Imaging the electrical activity of neurons and subthreshold activity, and versatility for both neural networks is of fundamental importance in excitation and inhibition, is greatly desired. Although understanding their physiological functions and the voltage-sensitive dyes (VSDs) for voltage imaging treating their pathological dysfunctions. Their (discussed in details below) have a high electrical activity can be recorded through the spatiotemporal resolution and capability of imaging electrodes in a patch clamp or microelectrode array. a large region of interest, they are constrained by These electrical recording techniques are invasive sensitivity, photobleaching, and phototoxicity [115]. and incapable of recording neural activity from a Thus, semiconducting nanotransducers, especially large region of interest, which has promoted the QDs, with higher photoluminescence intensity and optical imaging approaches. As calcium ion (Ca2+) is a photostability have attracted attention for potential critical indicator of neuronal activity, Ca2+ imaging is application to voltage sensing in neurons. a powerful tool to study the neural activity [113]. Due to quantum confinement, QDs can be excited However, the Ca2+ dynamics is slower than that of the by light, creating excitons, pairs of separated actual action potential, and only suprathreshold negatively charged electrons and positively charged
12 Nano Res. holes [116]. An electric field applied onto the excited 2.3 Bionanotransducer-enabled functional neural QDs leads to a quantum-confined Stark effect, imaging decreasing the energy of electrons and holes [117]. As a result, their photoluminescence intensity is Comparing to electrode-based neural recording quenched, and the emission peak is red-shifted and approaches, optical techniques offer complementary, broadened. This property well aligns with the needs yet compelling, advantages for imaging the for optical voltage imaging. A theoretical study transmembrane potentials, including less showed that the membrane potential of an action invasiveness and superior spatial resolution [120]. potential could result in such photoluminescence Optical recording or imaging can capture both the quenching and red-shift in type-II QDs, and that QDs electrical activities of each neuron in the circuit and would have a higher sensitivity comparing to VSDs map with micrometer resolution and sub-millisecond [118]. In a follow-up work, the use of type-I and precision. quasi-type-II QDs to image a voltage resembling an Nanobiotechnologies for optical neural imaging action potential with millisecond temporal resolution are mostly based on genetically encoded proteins, for was experimentally confirmed [115]. In this work, it their remarkable biocompatibility and optical was also claimed that the photoluminescence properties, fast response dynamics, high effective quenching by QDs was attributed to an increase of sensitivity, and easy functional modification. ionized QDs and that quasi-type-II QDs had a higher Protein-based optical sensors can be used to measure sensitivity than type-I QDs. In a more recent transmembrane potentials in single cell, tissue, and theoretical study, modeling showed that type-I and living animals through collection of the exhibited type-II semiconducting nanorods could have even fluorescent signals. A number of optical imaging higher sensitivity than QDs, while the sensitivity of methods based a variety of mechanisms have been type-II nanorods was higher than that of type-I explored for the benefits of neuroscience research. nanorods [119]. However, the nanorods needed to 2.3.1 Voltage-sensitive dye imaging vertically and symmetrically penetrate the plasma membrane, which was technically challenging. Traditional voltage-sensitive dye imaging (VSDI) Up to now, the QD- or semiconducting is based on a fluorescent change of small molecular nanorod-assisted voltage imaging has not been dyes when subjected to a change of a local electric biologically validated. The physiological scenario is field. For its good spatial (up to 20 m) and temporal far more complicated than theoretical simulation. (up to tens of microseconds) resolutions, this Once tested in neurons, there are still major practical approach offers a great potential in visualization of challenges associated with their voltage sensitivity, the information processing in the nervous system placement and biocompatibility. Voltage imaging [121, 122]. After decades of development and requires the nano sensors to be placed in the vicinity optimization, VSDI exhibits facile delivery into living of or within the plasma membrane. The QDs preparations for long-term observation, from delivered to the plasma membrane are, however, dissociated neurons to frogs, rats, and nonhuman rapidly internalized, and the placement of QDs is primates [123-125]. dynamically changing. Considering the fast voltage Typical VSDs possess an amphiphilic structure, in dynamics comparing to the endocytosis dynamics, which the hydrophobic components work as anchors this should not affect a single measurement within a into the cellular membrane, and the hydrophilic short period of time but will require repetitive dosing components, mostly chromophores, array for separate measurements, which will worsen the perpendicularly to the cellular membrane. The cytotoxicity of QDs. Thus, exploring nanotransducers intensity changes of the fluorescent signal correlate to that can convert voltage to an observable and the transmembrane voltage changes, and the neural readable signal and meanwhile can serve as stable activity can be measured accordingly. The most and biocompatible interfaces is essential for the common imaging mechanism is redistribution development of nanotransducer-assisted voltage (Figure 5a). A change in the transmembrane potential imaging in neurons. causes the charged chromophores to move in or out
| www.editorialmanager.com/nare/default.asp Nano Res. 13 of the cell, leading to a regional concentration change such as intracellular Ca2+ concentration, of the chromophores with a corresponding change in transmembrane potentials, and small metabolites the fluorescence intensity. Another mechanism is and other ions. Two major classes of genetically called reorientation (Figure 5a), which is determined encoded optical indicators have been developed: one by the interaction of the intermolecular electric fields. is genetically encoded calcium indicators (GECIs), Additionally, electrical modulation of the electronic which have a Ca2+-binding domain and a Ca2+ structure and fluorescent resonance energy transfer concentration response conformation; another is (FRET) can cause fluorescence changes in response to genetically encoded voltage indicators (GEVIs), changes of the transmembrane potential (Figure 5b). which have a domain responsive to the In addition to the commonly used VSDs (ANEP transmembrane potential changes. and RH families) [126], QD-based approaches, as Genetically encoded calcium indicators: Developed since mentioned above, provide an alternative opportunity. 1997 [128], GECIs monitor changes to the Functional modification using biomolecules can both intracellular Ca2+ concentration caused by action reduce the cytotoxicity of QDs and provide potentials. As a ubiquitous second messenger, Ca2+ is functional targeting sites to the neuronal membrane of great significance in all aspects of cellular for voltage imaging [118]. Recently, Nag et al. physiology. Triggered by an action potential, an reported a QD-peptide-fullerene nanobioconjugate increase in its intracellular concentration, from 20-100 for imaging membrane potentials in living cells [127]. nM (initial concentration) to 5-10 M (peak This alanine/leucine-rich peptide was designed to be concentration), causes a change to the fluorescence helix-forming to promote membrane insertion. It also [129]. Briefly, the design of GECIs involves a fusion of could append the fullerene component at discrete two parts: a naturally evolved Ca2+-binding protein fixed distances for the signal detection and facilitate component that undergoes a conformational state energy transfer via tunneling. The imaging of PC-12 transition in response to Ca2+ binding (calmodulin cells showed a 20- to 40-fold improvement in F/F (CaM) [128] or troponin-C (TnC) [130]) and a with no sacrifice in responsivity. Thus, reporter component based on a protein-modified nanotransducers also play conformation-sensitive fluorescent protein (FP). important roles in specific targeting to the cellular Two types of signals are recorded according to the membrane, enhancement of biocompatibility, and type of reporter component (Figure 5c). Simple connection between two FRET components, with fluorescence signal is collected through the minimum effect on the fluorescent signal. conformational transition of single FP component 2.3.2 Genetically encoded fluorescent imaging [131]. A pair of FPs exhibits FRET, which could provide a measure of Ca2+ concentration by the ratio Neural activities are encoded in dynamic fluorescence signal between the two FPs [128]. FRET fluctuation of the transmembrane potential, signal is a better alternative due to its independence including both subthreshold and action potentials. To on probe concentration, low excitation light intensity, visualize the dynamic changes of the transmembrane and low absorption in the optical path. For example, voltage, which range from ~1 mV (e.g., postsynaptic as a typical GECI, yellow cameleon (YC) 3.60 has a potential caused by a single vesicle release) to over pair of enhanced cyan FP (ECFP) as donor and Venus 100 mV (i.e., action potential) and span from 100 ms protein FP with circularly permuted conformation (axonal conduction) to 10 s (plateau potential), (cpYFP) as acceptor. In the presence of Ca2+, its genetically encoded optical indicators offer a great intramolecular conformation changes lead to a promise. Nanobiotechnological engineering reduced spatial distance and illuminate the Venus approaches have been used to fuse the fluorescent protein, and the ratio change of YFP/CFP is up to 6 chromophores with other domains that undergo folds with a high Ca2+ dynamics up to 0.25 M [132]. conformational changes in response to cellular events
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Figure 5 Bionanotransducer-enabled functional neural imaging. (a) and (b) Mechanisms of VSD imaging: redistribution (a) left, reorientation (a) right, and FRET (b). (c) GECIs based on single fluorophore and FRET mechanisms. After binding of Ca2+ to GCaMP, conformational changes induce an increase in emission (left) or a change to the ratio of emission intensities (YFP/CFP) [131, 132]. (d) Mechanisms of GEVIs. Left, single FP-based VSFPs exhibit fluorescent quenching upon membrane depolarization; right, FRET-based VSFP-Butterfly family has a fused four-transmembrane-segment voltage-sensitive domain between two FPs [133]. (e) VSFP2.3 reported transmembrane voltage transients through two different FPs [134]. (f) Extracellular electrical stimulation induced an increase in intracellular Ca2+ concentration in myotubes with the expression of GCaMP [131]. Figure credits: (e) adapted with permission from Ref. [134]; (f) adapted with permission from Ref. [131].
The mostly optimized GECIs are single-wavelength A number of improved variants of the original blue, green indicators based on the original GCaMP cyan, yellow and red FPs [133] have been sensor. The reporter domain of GCaMPs is a developed to improve the brightness, photostability, circularly permuted enhanced green FP (EGFP), tissue penetration and signal-to-noise ratio. which is flanked between Ca2+ binding protein To avoid interferential interactions of CaM with CaM and CaM-binding peptide M13. In the endogenous binding factors and improve the presence of Ca2+, CaM-M13 interactions lead to detection precision, two effective approaches are conformational change and an increase in pursued: one is the replacement of CaM with fluorescent emission [131, 135]. A number of troponin C variants, which is the Ca2+ binding engineered variants of GCaMP have been reported, protein in muscle cells and does not have an and these indicators are composed of constituent endogenous binding site in neurons [139] [130]; the molecules to achieve specific manipulation of other is the modification of binding interference. sensor applications [136-138]. Palmer et al. reengineered the binding interface Since the first GECI was reported with the between CaM and a target peptide to generate chromophore green FP (GFP), the modulation of selective and specific binding pairs that could be no indicator color has been well explored. Directed longer perturbed by large excesses of native CaM, mutation of the side-chains comprising the and the Ca2+ detection sensitivity turned over a chromophore can tune the excitation and emission. 100-fold range at 0.6-160 M [140].
Nano Res. 15
In addition, optimization of GECIs focuses on signals based on FRET between a pair of the modulation for specific applications and better fluorescent moieties [134, 149], which are both optical properties, such as optimization on the Ca2+ involved in the molecular interactions and motions, binding constant [135, 141], subcellular targeting leading to ratio changes in fluorescence intensity [142], brightness, and red-shifting of the fluorescent (Figure 5d). Signals from a single FP have limited indicators for large penetration and good sensitivity, while the ratiometric FRET signals can resolution [143]. significantly reduce the effects of motion and blood GECIs can be used in combination with flow in complex environments. two-photon imaging to achieve improved Several GEVIs based on different fluorescent measurements in highly scattering medium with a chromophore families are developed. For example, greater signal-to-noise ratio without the need of the fluorescent Shaker (FlaSh) and sodium channel averaging. Mank et al. generated a GECI as protein-based activity reporting construct (SPARC) TN-XXL for two-photon ratiometric imaging in are based on simple fluorescent signal detection, visual cortex. They rearranged the Ca2+ sensing while the voltage sensitive FP (VSFP) uses an FP moiety TnC within the indicator and mutagenesis pairs based on FRET [150-152]. of selected amino acid to increase overall signal Siegel et al. reported an early attempt on FlaSh strength and sensitivity in the low Ca2+ regime [150]. They constructed a GFP-Shaker fusion [144]. protein, using a nonconducting mutant of a GECI fluorescence imaging is a good proxy to voltage-gated K+ channel as the voltage-responsive record average action potential changes, but it has site and an FP inserted into the K+ channel protein an inherent inadequacy. Because Ca2+ transients are as a reporter. This GEVI had a maximal fractional 100-1000 folds slower than the underlying electrical fluorescent change of 5.1%. Guerrero et al. waveforms, most subthreshold changes of expanded the FlaSh GEVIs by modifying the transmembrane potentials cannot elicit a change to kinetics, dynamic range, and color. The improved the Ca2+ concentration, and the Ca2+ dynamics is folding enhanced the detection sensitivity, and the confounded by the complicated interactions availability of different FP colors promoted wide between different Ca2+ sources and intrinsic or adoptions [153]. The SPARC was developed with a extrinsic Ca2+ buffers. Hence, weak and brief strategy to insert a GFP molecule into a rat muscle changes of the transmembrane potentials may not Na+ ion channel subunit, which exhibited rapid be recorded by the Ca2+ indicators [114, 145]. response kinetics without significant inactivation [151]. Genetically encoded voltage indicators (GEVIs): GEVIs As the key component in the FP-based GEVIs, may be preferred over GECIs, for their capability of VSFP underwent a lot of research. The first directly reporting both synaptic and action generation of VSFPs was derived from the potentials and capturing the entire voltage voltage-sensing domain of a K+ channel subunit dynamics. Several different approaches to build [152]. This VSFP consisted of a voltage sensing GEVIs have been developed since 1997. Most domain of a K+ channel and a pair of cyan and GEVIs consist of two components: a yellow emission mutants of FPs. Although this voltage-sensitive domain from an ion channel as generation of VSPFs could optically report changes the voltage sensor and a component that binds in in the transmembrane potential, their application to the plasma membrane and experiences the voltage functional imaging of mammalian neurons was changes. limited by their low targeting capability to the Same as GECIs, two types of fluorescent signals membrane [154]. The second generation of VSPF based on different response mechanisms can be (VSPF2) was based on Ci-VSP (Ciona intestinalis collected: one is the single fluorescent signal from a voltage sensor-containing phosphatase), exhibited single fluorescent moiety [146-148], which better membrane targeting and formed a big undergoes a significant conformational change that Ci-VSP GEVI family [120]. Akemann et al. alters its spectra; and the other is ratiometric developed FRET-based VSFP2s by changing the
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 16 Nano Res. enzyme domain of Ci-VSP with two tandem sensitivity (< 5% F/F per 100 mV) and slow fluorescent domains, which exhibited more kinetics (10-200 ms). The second generation of efficient targeting to the cellular membrane and Ci-VSP-based GEVIs had a novel GEVI (Arclight) higher responsiveness to the transmembrane consisting of a Ci-VSP and a super ecliptic potentials [134]. The variant VSFP2.3 was pHluorin that carried the point mutation A227D developed as a FRET-based indicator possessing [146]. Arclight A242, derived from Arclight, the capacity of imaging both spontaneous action exhibited a fluorescent intensity increase by 35% and synaptic potentials in neurons [155]. The F/F per 100 mV, but the response time (10 ms) was indicator VSPF3.1 was the fusion of Ci-VSP and a much slower, hindering action potential detection. single red-shift protein, which offered the In another GEVI design, called ASAP1, a circularly advantages of a red-shifted spectrum and relative permuted FP was inserted in an extracellular loop fast overall kinetics [156]. The VSPF Butterfly series, of a voltage-sensing domain [148]. ASAP1 with a sandwiched structure between two FPs and exhibited high sensitivity (18-29% F/F per 100mV) a middle voltage sensitive domain, showed a and high kinetic speed (2 ms) and could be used for subthreshold detection range and fast kinetics for action potential detection at waveforms up to 200 single-cell synaptic responses in applications from Hz. single neurons to the brain [157]. GECIs and GEVIs have exhibited the capability Besides of FP-based GEVIs, microbial rhodopsin of recording neuron potentials in multiple could also be used as the fluorescent voltage mammals and neuron types, but the use of them in reporter [158, 159]. This retinal chromophore humans seems unlikely at present, because its shows very weak near-infrared fluorescence, while delivery involves viral vectors. The development upon light-driven transport of protons, a change in and optimization of genetically encoded indicators the chromophore emission was induced. still continue. The ideal GECIs and GEVIs should Comparing to FP-based GEVIs, GEVIs based on the process properties such as: large voltage/Ca2+ microbial rhodopsin have a sub-millisecond induced fluorescent changes over a physiological response time and better voltage sensitivity range, quick response kinetic time [158-160]. GEVIs based on non-pumping mutants (sub-millisecond), targeting capability to different of Archaerhodopsin 3 (Arch) have voltage neuron types, good photostability, large brightness sensitivities between 30-90% per 100 mV and and signal-to-noise ratio, and red/near infrared half-maximal response times between 50 s and 1.1 emission for in vivo application. ms at room temperature [160]. However, Arch-derived GEVIs exhibited weak fluorescence, more than 30-fold weaker than that of FPs. 3. Nano fNIs for neural stimulation Meanwhile, to illuminate microbial 3.1 Nanoelectrodes rhodopsin-based GEVIs, the excitation laser power needs to be much higher, typically 300-1000 W/cm2, 3.1.1 Microstimulation with nanomaterial coatings much more than that of FP-based GEVIs (10 W/cm2) Nanostructured surfaces and nanomaterial [160]. The low brightness of rhodopsin-based coatings have been shown to improve the charge GEVIs presents a challenge for widespread use. storage capacity and charge injection limit of Current efforts on the development of GEVIs microelectrodes, which results in more effective primarily focus on optimization of existing sensor stimulation including smaller voltage, greater constructs, including the voltage range, color, power efficiency, and less tissue damage. brightness, response kinetics, and cellular targeting Carbon nanotube-based electrodes were found properties. As key indicators to evaluate sensing to offer lower impedance and improved charge performance, response sensitivity (F/F), injection comparing to platinum, making it a half-maximal response times, signal-to-noise ratio suitable candidate for electrical recording and and excitation power all need to be considered. The stimulation. Wang et al. first reported the use of first generation of GEVIs exhibited modest voltage vertically aligned multiwall carbon nanotube to
| www.editorialmanager.com/nare/default.asp Nano Res. 17 stimulate excitable cells (rat hippocampus neurons) modulation techniques featuring minimal or even [161]. Tsang et al. reported the use of Au-carbon none invasiveness with greatly improved nanotube composite electrode fabricated on flexible spatiotemporal precision and cellular targeting polyimide substrate [162]. As a result of the lower specificity. stimulation voltage and less power consumption, Like wireless charging, in which the the stimulation could be done wirelessly. The electromagnetic energy is transmitted through the resulting device forms an insect machine interface intermedium and converted to electrical currents in which the flight path of moths could be biased by an antenna in the target battery, a primary by selectively stimulating one side of the moth wireless signal (e.g., optical, magnetic or acoustic) body. Yi et al. developed vertically aligned carbon can be transmitted through tissues and converted nanotube electrodes on flexible substrates and used to a secondary local signal (e.g., electric, thermal, them to stimulate rat sciatic nerve and record from optical or mechanical signal) by nano rat spinal nerve with a signal-to-noise ratio as high antenna-transducers to modulate the target neuron. as 12.5 [163]. Jan et al. quantified the impedance These nanotransducers are essential for delivering and charge storage capacity of iridium oxide, the highly localized secondary signal with spatial PEDOT, and layer-by-layer synthesized multiwall precision, target specificity, and improved carbon nanotube [72]. Carbon nanotube efficiency. Due to their small size and novel outperformed traditional electrode materials by physicochemical properties, these having a lower impedance and higher cathodic nanomaterial-based nanotransducers perfectly charge storage capacity. In addition, no sign of align with these demands. Firstly, they can be failure was observed after 300 cycles of cyclic delivered to the target region via voltammetry scan. minimally-invasive local or intravenous injection. Secondly, their diverse energy transduction 3.2 Nanotransducer-assisted neural modulation schemes allow the conversion of a medically safe, tissue-penetrating primary signal to a localized cell The emerging concept of going wireless has modulatory secondary signal at the been revolutionizing the contemporary nanotransducer-neuron interface [167]. technologies and reshaping the modern lifestyle. It Additionally, their ease of surface modification and is also driving the advancement of medical devices bio-conjugation can facilitate specific targeting to [164]. Conventional neural modulation techniques the targeted neuron population at cellular and even deliver electrical signals to the target neural subcellular levels, dramatically improving the population. Due to fast dissipation and attenuation target specificity, selectivity and spatiotemporal of the electrical signals through tissues, these resolution. traditional techniques often demand invasive Over the past few decades, particularly last placement of the electrodes in the immediate decade, a great deal of efforts has been made to vicinity of the target neural regions and develop such nanotransducer-assisted wireless implantation of a pulse generator wiring to the neural modulation techniques. In the literature, electrodes to deliver the electrical signals. Surgical these techniques are commonly categorized by the procedures cause tissue damage and surgical primary wireless signals. In contrast, in the present complications [165], and chronic inflammation review these techniques are categorized by the around the electrodes causes scar tissue formation secondary local signals to elucidate the concepts and early device failure [166]. Alternative strategies and design rationales, as the secondary signal is the to neural modulation, which rely on direct wireless ultimate modulatory signal to the target neuron. delivery of magnetic field, ultrasound, and infrared For example, manipulation of ion channels plays light to the target neurons, unnecessitate the an important role in neural modulation, and there electrodes but are limited in spatial resolution are various types of ion channels, including and/or power efficiency. These deficiencies have voltage-gated, temperature-gated, motivated the development of a new generation of mechanosensitive, and light-gated. Thus, the local nanotransducer-assisted wireless neural
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 18 Nano Res. secondary signal needs to be generated by the secondary signal should be controlled to exceed the nanotransducers to gate the corresponding ion threshold for cellular modulation but not to exceed channels and eventually modulate the neuronal the safety limit that can cause cell damages. activities. Here, these diverse types of Theoretical simulation can be used to help assess nanotransducers can be classified into the feasibility of a signal transduction scheme and electrotransducers, thermotransducers, guide the experimental design and optimization. optotransducers, and mechanotransducers based Additionally, the temporal resolution of the on the type of secondary signal they generate. modulation significantly depends on how fast the To develop such nanotransducer-assisted neurons respond to the secondary signal. wireless neural modulation techniques, there are Nanotransducer biosafety and biostability: The several key factors to consider, including the nanotransducers need to serve as stable neural primary signal, secondary signal, nanotransducer’s interfaces within the modulation period, and they biosafety, biostability, placement, and modification, must be safe in the physiological environment. as well as cell modification. They should cause no toxicity and minimal Primary signal: The primary signal needs to be safe inflammation. Moreover, they should remain intact and wirelessly transmittable through tissues to to maintain the functional interfaces and not to reach the target region. Although light, magnetic release or leak toxic substances. Their biostability field, and ultrasound can all meet these two criteria can be challenging. First, certain nanotransducers to possibly serve as a primary signal, their are prone to degradation due to the physiological interactions with tissues affect their penetration acidic pH and enzymatic activities. Second, the depth and spatial resolution (i.e., how well the exogenous nanotransducers are also prone to primary signal can be focused on the target region). clearance, so their placement is dynamic instead of Light has superior spatial resolution of 10-7 m, static. while it can barely pass the cranium (10-6 m travel Nanotransducer placement and distribution: Overall, depth) and only penetrate dermal tissues as deep nanotransducers can either be in a as 4 millimeters [168, 169]. Thus, for deep neural dispersed/injectable form or immobilized onto a tissue modulation, implantation of a light source is substrate or microelectrode (Figure 6a). The usually required. Transcranial focused ultrasound dispersed nanotransducers can be placed travelled through skull and modulated the cortical extracellularly, targeted to the plasma membrane activity 30 mm deep in human brains with spatial (e.g., onto the membrane, receptors, anchoring resolution of 10-3-10-2 m [170]. The penetration proteins or ion channels), or internalized. depth and spatial resolution of transcranial Immobilization of nanotransducers onto a magnetic stimulation are highly dependent on the substrate or microelectrode facilitates the build-up coil design. For example, 50 coil design has of the secondary signal to reach the threshold and penetration depth and spatial resolution of 10-2 m control of the nanotransducer distribution, but the [171]. For these primary signals, there is a tradeoff implantation increases invasiveness. On the between penetration depth and spatial resolution contrary, injectable and monodispersed [170, 171]. Additionally, the interactions of primary nanotransducers minimize invasiveness, but their signals with tissues may also generate noxious heat, distribution needs to be carefully controlled, as limiting the maximum intensity of the primary their placement and distribution affect the signal. cytotoxicity, modulation efficacy, efficiency, and Secondary signal: The secondary signal needs to be consistency. For example, unbound and able to modulate neurons by gating the respective extracellularly distributed nanotransducers ion channels, activating cell signaling pathway, prolong the stability of the interface but tend to be changing the plasma membrane capacitance, etc. removed by the fluid circulation, such as the Possible secondary signals include electric field, cerebrospinal fluid in the central nerve system. On heat, light, and mechanical force. Generation of the the other hand, binding of nanotransducers to the
| www.editorialmanager.com/nare/default.asp Nano Res. 19 plasma membrane, receptors or ion channels enables and optimizes the cells’ response to the improves the specificity and washout resistance, corresponding secondary local signal. Neurons can but they can be rapidly internalized. The also be genetically modified to express binding internalized nanoparticles can cause toxicity and sites for nanotransducer docking and create modulation inconsistency [172, 173]. Additionally, a hallmarks for evaluating the modulatory effect. complete retrieval or clearance of the These cell modification strategies make the nanotransducers after the modulation can be modulation technique versatile and adaptable for challenging for dispersed nanoparticles. different types of cells. They can also improve the modulation specificity and selectivity through Nanotransducer modification: Nanotransducers are selective engineering of the target neuron usually surface-modified so that their dispersity population. However, the viral vectors used for the and biocompatibility can be improved. They can be cell modification can cause immune responses, and immobilized on a substrate, specifically targeted to the transfection is usually irreversible. Thus, cell a neural population or even to the receptors and modification by genetic engineering brings safety ion channels. However, surface modification can and moral concerns and thwarts their clinical attenuate the secondary local signals and increase applications. the distance between the nanotransducers and the Considering these critical factors, we herein modulation target. Moreover, the surface chemistry review four main types of nanotransducers for needs to be well controlled, because certain surface wireless neural stimulation: electrotransducers, chemistry, even without inducing clear cytotoxicity, thermotransducers, optotransducers, and may cause false modulatory effects due to plasma mechanotransducers. Each type is further membrane perturbation [174]. subdivided by the types of nanomaterial used. Cell modification: Genetically engineering certain These nanotransducers are summarized regarding types of neurons to express the target ion channels these factors in Table S1.
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Figure 6 Nanotransducer-assisted neural modulation. (a) Placement of nanotransducers: nanotransducers can be dispersed extracellularly (1), coated on a substrate (2), bound to the plasma membrane (3), targeted to receptors or anchoring molecules in the plasma membrane (3’), targeted to ion channels in the plasma membrane (3”), and internalized (4). (b) and (c) Semiconducting nanorods-based electrotransducers, adapted with permission from Ref. [175]. (b) Semiconducting nanorods convert light to electric fields. (c) A train of extracellular spikes triggered by a light pulse. (d) and (e) Magnetoelectric nanomaterials-based electrotransducers, adapted with permission from Ref. [176]. (d) Magnetoelectric nanomaterials convert a magnetic field to electric fields. (e) Modulated EEG waveforms. (f) and (g) Piezoelectric nanomaterials-based electrotransducers, adapted with permission from Ref. [177]. (f) Piezoelectric nanomaterials convert ultrasound to electric fields; (g) Ca2+ influxes following an ultrasound pulse. (h) and (i) Gold nanomaterial-based thermotransducers, adapted with permission from Ref. [178]. (h) Gold nanomaterials convert light to heat. (i) A train of action potentials, of which each was evoked by a light pulse. (j) and (k) Superparamagnetic nanoparticle-based thermotransducers, adapted with permission from Ref. [168]. (j) Superparamagnetic nanoparticles convert a magnetic field to heat. (k) P