Nano Research 2018, 11(10): 5065–5106

https://doi.org/10.1007/s12274-018-2127-4

Nano functional neural interfaces

Yongchen Wang1,§, Hanlin Zhu2,§, Huiran Yang3,4,§, Aaron D. Argall5, Lan Luan2, Chong Xie2 (), and
Liang Guo3,6 ()

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
§
Yongchen Wang, Hanlin Zhu and Huiran Yang contributed equally to this work.

Received: 15 March 2018 ABSTRACT

Revised: 5 June 2018 Engineered functional neural interfaces (fNIs) serve as essential abiotic–biotic
Accepted: 6 June 2018 transducers between an engineered system and the nervous system. They convert
external physical stimuli to cellular signals in stimulation mode or read out
© Tsinghua University Press biological processes in recording mode. Information can be exchanged using
and Springer-Verlag GmbH electricity, light, magnetic fields, mechanical forces, heat, or chemical signals.
Germany, part of Springer fNIs have found applications for studying processes in neural circuits from cell
Nature 2018 cultures to organs to whole organisms. fNI-facilitated signal transduction schemes,
coupled with easily manipulable and observable external physical signals, have
KEYWORDS attracted considerable attention in recent years. This enticing field is rapidly
neural interface, evolving toward miniaturization and biomimicry to achieve long-term interface
neurotechnology, stability with great signal transduction efficiency. Not only has a new generation
nanoelectrode, of neuroelectrodes been invented, but the use of advanced fNIs that explore other
nanomaterial, physical modalities of neuromodulation and recording has begun to increase.
neural recording, This review covers these exciting developments and applications of fNIs that
neural stimulation rely on nanoelectrodes, nanotransducers, or bionanotransducers to establish an
interface with the nervous system. These 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 Liang Guo, guo.725@osu.edu; Chong Xie, chongxie@utexas.edu

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1 Introduction
1.1 What are functional neural interfaces?
Engineered functional neural interfaces (fNIs) serve
as essential abiotic-biotic transducers between an
engineered system and the nervous system. They
convert external physical stimuli to cellular signals in
stimulation mode or read out biological processes in
recording mode. Information can be exchanged using
electricity, light, magnetic fields, mechanical forces,
heat, or chemical signals. fNIs have found applications
for studying processes in neural circuits from cell
cultures to organs to whole organisms. fNI-facilitated
signal transduction schemes, coupled with easily
manipulable and observable external physical signals,
have attracted considerable attention in recent years.
This enticing field is rapidly evolving toward
miniaturization and biomimicry to achieve long-term
interface stability with great signal transduction
efficiency. This review covers the developments and
applications of fNIs that rely on nanoelectrodes,
nanotransducers, or bionanotransducers to establish
an interface with the nervous system.
1.2 Why are fNIs important?
In the past decade, the field of fNIs has experienced a
dramatic revolution. The once electrical-engineering
concentrated field has evolved to a new stage that
has absorbed an ever-large research population and
ever-diverse multidisciplinary approaches. Not only
a new generation of neuroelectrodes has been invented,
but the use of advanced fNIs that explore other physical
modalities of neuromodulation and recording has
begun to increase, partially stimulated by the great
success of optogenetics [1]. This new stage is facilitated
by advocations and funding support of brain-related
research across the globe. Specifically, in the USA,
the Brain Research through Advancing Innovative
Neurotechnologies (BRAIN) and Stimulating Peripheral
Activity to Relieve Conditions (SPARC) initiatives
aim to significantly promote brain and bioelectric
medicine research by accelerating the development
and application of novel and paradigm-shifting
neurotechnologies, among which fNIs are a major
focus. Such demanding advances in fNIs will greatly
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Nano Res. 2018, 11(10): 5065–5106
facilitate new opportunities not only for studying basic
neuroscience but also for diagnosing and treating
various neurological diseases.
1.3 Why nano?
Conventional electrode-based neurotechnologies
are facing two major hurdles: (1) susceptibility of
the abiotic–biotic interface to immune responses and
(2) communication inefficiency at the abiotic–biotic
interface [2]. Nano fNIs are compelling in offering
more effective solutions to both of these two aspects.
1.3.1 Chronic stability
Even though many electrode-based neurotechnologies
have made great strides during the preceding few
decades in proving their feasibility in treating and
restoring impaired neural functions, their clinical
potential is severely restricted by issues in the
integration of the neural interface within the complex
tissue environment. Not only do these mechanically
and chemically distinct neural interfaces cause
significant infection, but the communication at the
electrode-tissue interface is significantly diminished
as the implant is isolated by fibrosis over time as a
consequence of the foreign body reactions [3]. This
discovery of a fibrotic encapsulation developing around
the implanted neuroelectrodes over a short time
window of a few months [4–7], which physically
screens the electrical sensors from accessing to the
target neurons, has largely shaped the thinking and
practice in the field in the past decade. The resulting
new concepts in neural interfacing have motivated
both the development of a new generation of minia-
turized neuroelectrodes [2, 8–10] and the exploration
of alternative approaches that feature minimum or
even no invasiveness. The reduction of the footprint
of neural implants down to the nanoscale to make
them less “sensible” to the host tissue environment
has proven to dramatically improve the chronic
stability of the neural interfaces [8–10]. Alternatively, to
mitigate the problems associated with the conventional
electrode-based approaches, such as the requirement
for implantation of the bulky interface into the
immediate target neural tissue [11], nonspecific and
variable activation, bio-fouling, motion artifacts, and
tissue damage [3], remotely controlled approaches
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that leverage nanotransducers or bionanotransducers
in the pursuit of minimum invasiveness, high spatial
resolution, and cell-type specificity have drawn
unprecedented attention. These alternative nano fNIs
hold great potentials for a better integration into the
target neural tissue at the tissue and cell levels.
1.3.2 Functional efficiency
Realizing neural-realistic communication while
minimizing side-effects to neural circuits is of great
interest in the field. It requires developing fNIs that
comply with the biological mechanism of neural
signaling. Unfortunately, signal transduction at the
macro electrode–tissue interface is inefficient and can
lead to tissue injuries due to mismatched communi-
cation mechanisms between the electronics and the
target tissue [12–14]. Neuronal activity is modulated
by ion channel-mediated action potential signaling,
and, thus, ion channels naturally become the direct
target of physical stimuli. Working at a scale close to
the dimensions of single ion channels, 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
minimize tissue volumetric displacement and foreign
body reactions [15, 16].
2 Nano fNIs for neural recording
2.1 Nanoelectrodes
Detecting the complex and dynamic activities of the
nervous system requires precise measurements of
its basic functional units—neurons. Neuroelectrodes
provide one of the most useful neurotechnologies by
allowing for time-resolved electrical detection of neural
activities and direct stimulation of neural tissues.
Therefore, pushing the limits of electrophysiological
recording and stimulation is of great scientific
and clinical interest [17–19]. Despite important and
unique capabilities, conventional neuroelectrodes have
significant limitations. Intrinsically, electrophysiological
recordings of action potentials (spikes) provide
insufficient information to establish a definite correla-
tion with individual neurons and often bias towards
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frequently firing neurons. Unavoidable invasiveness
of implanted electrodes greatly restricts the number
and density of electrodes that can be implanted into
the brain, which leads to sparse sampling of the neural
circuitry. Furthermore, conventional neuroelectrodes
typically fail to provide consistently stable, high-quality
neural recordings over both the short and long term
[4, 20, 21]. In timescales as short as a few hours,
substantial changes to the recording conditions often
occur due to micro-movements of the implanted
electrodes with respect to the brain tissue [22–24].
Over weeks to months, deterioration in recording
efficacy and fidelity is caused by biotic and abiotic
failures [25–28], including sustained foreign body
reactions at the tissue–probe interface such as neural
degeneration, reoccurring blood leakage in capillaries,
and glial scar formation [5, 22, 29–32]. Recently, there
has been an increasing interest in taking advantage
of nano and micro technologies to address the
aforementioned challenges in neuroelectrodes.
2.1.1 Nanostructure-enabled intracellular access
Two major types of electrophysiological recording
methods, intracellular and extracellular, have been
developed to measure action potentials with com-
plementary capabilities. Traditional intracellular
recording methods such as the whole-cell patch clamp
technique requires rupturing a portion of the plasma
membrane to access the cell interior directly [33].
Whole-cell patch clamping (recording tip diameter
commonly ranging from close to one micron [34] to
several micrometers [35]) is the most sensitive method
to record electrophysiological events in neurons, but is
highly invasive and technically difficult to implement,
which precludes long-term or large-scale recordings.
On the other hand, extracellular recording methods
such as multi-electrode arrays utilize micropatterned
electrodes to afford long-term and multiplexed in
vitro measurements [36–38]. However, extracellular
recording suffers significantly in signal strength
and quality. This has, therefore, resulted in the need
for electrophysiological methods that combine the
advantages of both intracellular and extracellular
recording methods. In the past several years, there
have been developments that aim at achieving
intracellular recordings with extracellular nano-
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electrodes or transistors while possibly allowing for
advantages of minimal invasiveness and easy scalability.
Although some structures reviewed in this section fall
into microscales instead, their working mechanisms
rely on nanoscale structures or interactions.
It has been shown in multiple works that micro-
and nanostructures can promote tighter contacts at
the cell–electrode interface, which enhance recording
outcomes. Hai et al. pioneered in creating mushroom-
shaped micrometer-sized gold electrode arrays that
could detect attenuated intracellular action potentials
[39]. Xie et al. fabricated vertical platinum nanopillar
electrodes, with which they demonstrated that these
electrodes formed tight junctions with cultured
cardiomyocytes (Figs. 1(a) and 1(b)) [40, 41]. By local
electroporation, the nanopillar electrodes could
enhance the action potential recordings with more
than ten times greater amplitudes and intracellular-
like waveforms. Lin et al. discovered that nanotubes
were advantageous than nanopillars in making and
maintaining tight junctions with the cell [42]. It was
demonstrated that iridium oxide nanotubes enabled
a more effective and stable intracellular access to
cultured cardiomyocytes. Robinson et al. fabricated
vertical silicon nanowire arrays that enabled intra-
cellular access to both record from and stimulate
neurons (Figs. 1(c) and 1(d)) [43]. Abbott et al. further
extended this strategy by fabricating vertical nano-
electrodes on a complementary metal–oxide–
semiconductor (CMOS) multiplexer, so that an array
of 32 × 32 nanoelectrodes can be simultaneously
addressed [44]. A similar strategy was also reported
by Liu et al. [45].
Field effect transistor (FET) sensors, due to their
sensing mechanism, do not suffer from thermal noise
as the sensor size decreases, which is the major
limitation of passive electrodes. Tian et al. developed
FET sensors based on kinked silicon nanowires [46].
Using cultured cardiomyocytes as an in vitro model,
they showed a clear transition from extracellular to
intracellular recording, as the tip of the device slowly
penetrated the cellular membrane (Figs. 1(e)–1(j)). A
free-standing version of the kinked silicon nanowire
FET sensor was created by Qing et al. [47], in which

Figure 1 Representative nanoelectrodes. (a) Scanning electron microscopy (SEM) image showing that the nanopillar electrodes were
strongly engulfed by a cell [40]. (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 recordings [40]. (c) SEM image of a
3-by-3 array of vertical nanowire electrodes [43]. Scale bar: 1 µm. (d) SEM image of nanowires interacting with a rat cortical neuron
[43]. (e)–(j) Nanowire FET [46]. (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 Ref. [40], © Springer Nature 2012; (c) and (d) adapted with permission from Ref. [43], © Springer Nature 2012; (e)–(j)
adapted with permission from Ref. [46], © The American Association for the Advancement of Science 2010.

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Nano Res. 2018, 11(10): 5065–5106
individual branches of the kinked nanowire could be
adjusted to the target-specific cell under a standard
microscope. Duan et al. reported the recording of
a full-amplitude intracellular action potential in
cardiomyocytes by silicon FET sensors coupled with
silicon dioxide nanotubes [48]. Fu et al. pushed the
electrical detection limit further by producing a
silicon nanotube and nanowire hybrid FET electrode
with a recording tip size of less than 10 nm [49]. To
probe fine structures in the neuron, Jayant et al. took
advantage of quantum dot (QD)-coated nanopipette
electrodes to recover full backpropagation details of
action potentials along targeted dendritic spines [50].
2.1.2 Nanomaterials to reduce electrode impedance
As the electrode size decreases to increase recording
density or to minimize invasiveness, the impedance
of the electrode increases dramatically. The thermal
noise associated with the electrode impedance becomes
a major limitation. Nano structures and materials have
great potential in significantly lowering the electrode
impedance and extending the electrode size limitation.
Owing to its highly porous structure that increases
the electrochemical surface area and subsequently
decreases the impedance, platinum black has long
been used in electrical recordings. Kim et al. improved
the mechanical stability and electrical property of
platinum black by depositing bioinspired adhesive
polydopamine film in a layer-by-layer manner [51].
Nanostructured porous platinum was also demon-
strated to increase the surface area of electrodes by
more than 30 times, thus reducing the impedance by
77% [52]. Weremfo et al. fabricated surface-roughened
platinum electrodes that had nano features and a
superior charge injection capacity comparing to
titanium nitride and similar to carbon nanotube
(Figs. 2(a)–2(c)) [53]. Park et al. identified an optimal
surface roughness factor of 233 to maximize the
performance for electrical stimulation and recording
by nanoporous platinum and yielded an impedance
of 0.039 Ω·cm2 and a charge injection capacity of
3 mC/cm2 (400 μs) [54]. Lee et al. deposited highly
roughened nonporous platinum film on gold tips of
traditional silicon electrodes and greatly decreased
the interface impedance to 0.029 Ω·cm2 [55]. Chung
et al. used CF4 plasma treatment to increase the surface
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roughness of gold from 1.7 to 22 nm, drastically
decreasing the impedance by 98% [56]. Plasma-treated
electrodes recorded signals with lower background
noises and evoked local field potentials (LFPs) with
higher amplitudes from the anterior cingulate cortex
in rats.
Chen et al. fabricated a carbon nanotube-based
electrode which had a lower impedance and a six
times higher charge transfer capacity than gold
microelectrodes [57]. (More about carbon nanotube
(CNT) electrodes are covered in section 3.1 and Figs. 2(d)
and 2(e).) Kim et al. developed Au-nanotube composite
electrodes which reduced the impedance of gold
electrodes by 99.3% [58]. They demonstrated in vitro
extracellular recordings from mouse cortical neurons,
which had an average signal-to-noise ratio of 92. Kim
et al. modified electrodes with gold nanoflakes which
led to an impedance reduction from 1.15 MΩ to
26.7 kΩ at 1 kHz [59]. Kim et al. enhanced the charge
storage capacity while decreasing the impedance of
electrodes by depositing gold nanoporous structures
by dealloying Ag-Au alloy [60]. Bruggemann et al.
demonstrated the fabrication of vertical gold nano-
pillars on electrodes in an effort to achieve a higher
electrode surface area, which led to a decreased
electrode impedance [61]. Zhou et al. incorporated
gold nanorods into a polyimide substrate to create
a flexible thin-film microelectrode array [62]. The
nanostructure was able to decrease the impedance by
25 fold. Zhao et al. reported a relatively simple method
to reduce the electrode impedance [63]. Electrodes
were first electro-co-deposited Au-Pt-Cu alloy nano-
particles followed by etching away the Cu. The
remaining Au-Pt composite exhibited a rough surface
with pores of different sizes. The electrode impedance
was reduced to 4.7% of that of a bare gold electrode.
Kim et al. took a hybrid approach by depositing
a layer of iridium oxide on nanoporous gold, which
further reduced the impedance and increased the
charge storage capacity of the electrode (Fig. 2(f)) [64].
By combining the merits of platinum gray and iridium
oxide, Zeng et al. [65] deposited Pt gray and IrOx on
bare Pt in a layer-by-layer setup (Fig. 2(g)). A decent
adhesion of IrOx was enabled by the large surface area
of nanocone-shaped Pt. They achieved an impedance
value of 2.45 kΩcm2 at 1 kHz and a cathodic charge
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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 roughened, and 5-min roughened platinum surfaces [53]. (d) SEM image illustrating the nanofibrous
network of multiwalled nanotube (MWNT) bundle coated on an electrode surface. Scale bar: 500 nm [66]. (e) Carbon nanotube
outperformed traditional electrode materials by having a lower impedance [66]. (f) Scanning transmission electron microscope
energy-dispersive X-ray spectroscopy (STEM-EDS) element mapping image of iridium oxide coating on a nanoporous gold electrode
[64]. (g) Morphology of IrOx/Pt gray coatings [65]. (h) Field emission scanning electron microscope (FESEM) image showing
polypyrrole (PPy) grafted at the surface of graphene oxide sheets (circled) [67]. (a)–(c) adapted with permission from Ref. [53], © the
American Chemical Society 2015; (d)–(e) adapted with permission from Ref. [66], © the American Chemical Society 2009; (f) adapted
with permission from Ref. [64], © the American Chemical Society 2016; (g) adapted with permission from Ref. [65], © Elsevier 2017;
(h) adapted with permission from Ref. [67], © Elsevier 2011.

storage capacity (CSCc) 6, 2.8, and 2.7 times higher
than those of electrodes with bare Pt, Pt gray, and
IrOx, respectively.
Deng et al. [67] invented a one-step electrochemical
process to deposit graphene oxide/polypyrrole com-
posite sheet on platinum electrodes in order to increase
the electrode surface roughness (Fig. 2(h)). The coated
probe reduced the impedance of bare platinum
electrode by 90% and increased the charge capacity
density by more than two orders of magnitude.
Besides reducing the impedance, neurons seeded
on graphene oxide doped poly(3,4-ethylene dioxy-
thiophene) (PEDOT) were found to grow longer
neurites than those on PEDOT/polystyrene sulfonate
(PSS). Functional biomolecules such as laminin
peptides could be easily bonded to the graphene
surface to increase neurite outgrowth [68]. Such an
electrode demonstrated an improved sensitivity of
151 nA/μM to dopamine compared to a glass carbon
electrode and minimized interference from ascorbic
acid, a competing analyte [69].
In addition to being used as a coating material,
graphene oxide can directly serve as the electrode
material. Ng et al. [70] fabricated reduced graphene
oxide into disk microelectrodes of 10 μm diameter
and 60 μm pitch using nanoimprint lithography. The
electrodes featured a high sensitivity of 91 nA/μM to
dopamine with a detection limit of 0.26 μM without
using any functionalization process. The detection
capability was robust to highly resistive media,
continuous flow, and mechanical stress.
2.1.3 Minimizing tissue invasiveness of neuroelectrodes
It is commonly agreed that a long-term stable neural
interface that can provide reliable neural recording
and stimulation with minimal tissue invasiveness is
of paramount importance in advancing our knowledge
of brain functions and dysfunctions, as well as
developing next-generation neurotherapies [71]. A
number of failure modes have been identified, and
many approaches have been hypothesized and tested
for improving the chronic stability of neural electrodes,

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Nano Res. 2018, 11(10): 5065–5106
including but not limited to physical (dimension,
mechanical stiffness), chemical (delamination, corrosion
of electrodes), and biological (neuroinflammatory,
foreign body response) [72]. Among those highly
interplayed failure factors, geometry and mechanical
mismatches between neural tissue and electrodes
play a critical role in determining the long-term per-
formance of the electrode–tissue interface. As a result
of such mismatches, traditional neural electrodes may
exert micro-movements with respect to their host
tissue or apply stress. Such micro-movements not only
prevent the tracking of the same neurons as recorded
waveforms change accordingly, but also induce neuron
degeneration and bleeding [73], which further cause
chronic inflammatory responses, accumulation of
reactive oxygen species, and eventually result in glial
scarring and accelerated electrode corrosion [74].
Seymour et al. discovered that thin electrodes (5 μm
in thickness) placed as close as 25 μm from the main
silicon electrode shank (50 μm in thickness) caused
noticeably fewer neuronal cell death while attracting
less immune cells [75]. Skousen et al. compared the
neuroinflammatory responses of two probes having an
identical surgical damage profile but different total
surface areas [76]. Despite the same initial damage,
the silicon lattice electrode with the smaller surface area
activated and attracted fewer macrophages than their
solid silicon counterpart. Karumbaiah et al. investigated
at both an acute and a chronic timescale the probe
geometry-dependent inflammation by comparing the
foreign body response to silicon electrode arrays
that had disparate thicknesses [77]. A 15 μm version
outperformed a 50 μm version in terms of histological
analysis and activated immune cell density around
the probe at both 3- and 12-week checkpoints. These
studies all underscore the importance of electrode
geometrical dimensions for mitigating tissue reactions.
Carbon fibers have recently been a popular
choice to fabricate thin neuroelectrodes. Kozai et al.
demonstrated ultra-small 7-μm diameter carbon fiber
electrodes for high-quality extracellular recording [8].
These electrodes were shown to have significantly
reduced foreign body responses and to record
single-unit activities for over 4 weeks in vivo.
Guitchounts et al. employed bundles of 5-μm diameter
carbon nanofibers to fabricate penetrating electrodes
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and demonstrated more stable single-unit recordings
in vivo [78]. Patel et al. fabricated 16-channel arrays
of 8.4-μm diameter carbon fiber electrodes and
demonstrated high-quality unit recordings for up to
3 months post implantation [28]. No neuronal density
change and minimal to none astrocyte activation
were observed in post-mortem histology. Vitale et al.
fabricated carbon nanotube fiber electrodes, which
were softer than carbon fibers and offered smaller
impedance and greater charge injection capacity,
making them more suitable for stimulation [79]. In
addition, these softer fiber electrodes also elicited less
chronic tissue responses.
Many efforts have also been put into making
neuroelectrodes flexible. Mercanzini et al. created a
polyimide-based flexible probe to record LFPs, single-
and multi-unit activities in the mouse cortex [80].
Histology results demonstrated a reduction in the
inflammatory response compared to rigid electrodes.
Wu et al. developed a flexible intracortical neural
electrode with an 8-μm thick flexible construction [81].
The electrodes were shown to record neural activities
for over six weeks. Du et al. compared the chronic
tissue reactions to a novel PEDOT-polyethylene glycol
(PEG)-based ultrasoft microwire with conventional
tungsten wire electrodes [82]. At both 1 and 8 weeks
post-surgery, a significant reduction in neuroin-
flammatory responses was found for the soft probe
compared to its rigid counterpart. Sohal et al.
investigated long-term (26–96 weeks) foreign body
responses in the rabbit cortex induced by flexible
parylene-C-based electrodes with microwire electrodes
as the control [83]. Less gliosis and greater neuronal
density were observed for the flexible probe. More
importantly, those effects were more prominent
towards the long-term timescale.
It is clear that the switch from conventional rigid
materials, such as silicon and metals, to softer polymers,
such as polyimide and parylene, helps mitigating tissue
reactions and promotes recording stability. However,
these polymers are still orders of magnitude stiffer than
the brain tissue [84], and therefore the mechanical
mismatch is still prominent. On the other hand, it is
impractical, at present, to fabricate functional devices
with materials that are as soft as tissues. This
dilemma led researchers to try different approaches
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besides using alternative materials. Because most
neuroelectrodes are constructed in high-aspect-ratio
probe geometries, bending is the major deformation
happening at the interface. Therefore, the primary
goal of mitigating the electrode-tissue mismatch is to
minimize the bending stiffness Ks of the implanted
probe, which is given by Ks = Eswh3/12 for a probe
with a rectangular cross-section [10], where Es is the
elastic modulus of the material, w is the probe width,
and h is the probe thickness. It is clear that the probe
geometry (thickness and width) plays an important
role in modulating the bending stiffness. Therefore,
nanoelectronic devices could have a unique and
important impact in promoting tissue integration of
neuroelectrodes.
A series of recent work has demonstrated neural
probes made by nanoelectronic devices based on
ultrathin polymer structures (Fig. 3) [9, 10, 85–89]. The
key feature of 1-μm thickness yielded ultra-flexibility
that allowed for a great reduction in mechanical
mismatches at the tissue–electrode interface. This
enabled chronically stable recordings from the same
neurons over multiple months.
2.1.4 High-density neural recording
One of the primary challenges of neurotechnologies
is to simultaneously record from a large number
of neurons. The nature of extracellular recordings
determines that the electrode must be within the
close vicinity (~ 100 μm) of a neuron to precisely
detect its activity [90]. To record from many neurons
inevitably requires a large array of electrodes placed
on the surface of the brain or inserted into non-
superficial structures. Besides, recording throughout
a region requires that all neurons are within the
range of a recording site and that recording sites are
densely packed to allow accurate spike sorting. In
particular, the detection range of each recording site is
typically 50–100 μm [91, 92], and spike sorting requires
an electrode spacing of 20–50 μm center-to-center
[93, 94] to resolve more single neurons.
In the efforts to push limits of recording density,
channel count, and single-unit yield, significant
challenges arise, such as addressing individual channels
and tissue displacement/injury.
Guo and DeWeerth developed an effective lift-off
method to pattern gold wires at high density on
stretchable substrates [95]. Based on this technique,
high-density compliant and stretchable electrode arrays
were fabricated and demonstrated by interfacing with
muscular tissue [84]. Khodagholy et al. demonstrated
the use of organic transistors to record neural signals

Figure 3 Increased flexibility improves the tissue-electrode interface. (a) Photoacoustic images of two flexible CNT electrodes inserted
in the brain [89]. (b) Micrograph of a three-dimensional (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 ultra-flexible electrode for one month [10]. Scale bar: 50 µm. (f) Confocal micrograph of an
immunochemically-labeled brain slice 5 months post-surgery, showing neurons surrounding the probe with minimal microglia activation
[10]. Neurons labeled in orange, electrode labeled as a green rectangle, and microglia indicated by white arrows. Scale bar: 50 µm.
(a) adapted with permission from Ref. [89], © the American Chemical Society 2013; (b) adapted with permission from Ref. [9], © Springer
Nature 2015; (c)–(f) adapted with permission from Ref. [10], © the American Association for the Advancement of Science 2017.

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Nano Res. 2018, 11(10): 5065–5106
in vivo [96]. The transistors were made of PEDOT on
a highly flexible parylene substrate with a total
thickness of 4 μm. These devices are capable of
amplifying and multiplexing signals locally and
potentially support high-density surface recordings.
Viventi et al. fabricated a high-density electrode array
multiplexed by silicon nanomembrane transistors on
a flexible polyimide substrate [97]. LFP recording
on the brain surface was demonstrated in an animal
seizure model.
There are also significant recent efforts on making
penetrating neuroelectrodes with nanofabrication.
Du et al. nanofabricated a 64-channel probe with
interconnect width and spacing of 290 nm and electrode
pitch from 28 to 40 μm [91]. They found that only
50% of all identified putative thalamic neurons were
detectable with electrodes separated more than 40 μm,
which justifies the importance of the electrode array
density. Similarly, Rios et al. nanofabricated electrodes
with features as small as 300 nm to enable high-
density 3D recordings [98]. They achieved a minimum
inter-electrode distance of 16 μm, totaling 1,024
channels (64 channels per shank) and covering a tissue
volume of 0.6 mm3. Single units were found to show
up on more than six channels, allowing for neuron
trilateration. Unique layer-specific field potential
signatures across the whole hippocampus facilitated
the determination of layer boundaries, which was
subsequently validated by histology results. An even
denser array was created by Scholvin et al. with
200 nm wiring width and 400 nm spacing [99]. 1,000
electrodes (200 channels per shank) were patterned
on silicon probes with 11 μm electrode separation
(Figs. 4(a) and 4(b)). Typically, a recorded unit could
manifest on more than 10 channels. Obien et al.
fabricated a 128-channel electrode array with on-chip
micro light emitting diode (μLED) for high-resolution
electrophysiology coupled with optogenetics [100].
Wei et al. nanofabricated high- density electrode arrays
on an ultra-flexible structure (Figs. 4(c)–4(e)) [101].
The interconnect traces had a width of 200 nm and
a pitch of 400 nm, addressing electrodes spaced as
small as 20 μm. The overall cross-section area of the
probe was as small as 10 μm2, which minimized chronic
tissue reactions.
In addition to the passive approach, CMOS-based
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active recording devices with buffer amplifiers placed
closely to individual electrodes were also developed
for high-density penetrating electrodes. Lopez et al.
patterned 455 electrodes on a shank width of 100 μm,
among which 52 could simultaneously record, using
180 nm CMOS technology [102]. Lopez et al. further
developed this technology and achieved 384 con-
figurable channels out of 966 electrodes on a 70 μm
wide shank with 130 nm CMOS technology [103].
Recordings with two of such active CMOS probes
were demonstrated in freely moving rats (Fig. 4(f))
[104]. A total of 700 single units were recorded from
five brain structures, demonstrating the capability of
acquiring large-scale activities of hundreds of neurons.
High-density nanosensors also enabled mapping
of electrochemical activities of neurons at superior
spatial resolution. With more than 20,000 sensors per
cell, Kruss et al. [105] fabricated single-wall nanotube-
based sensors that detected dopamine release at 100 ms
resolution. They were able to investigate how the
chemical communication of neurons was affected by
cell morphology and developed models to optimize
their probe design based on the spatially preserved
data [106].
For a detailed review of high-density neural
electrode array architectures and their respective merits
and challenges, readers are referred to Refs. [107, 108].
2.2 Nanotransducer-assisted functional neural
imaging
Imaging the electrical activity of neurons and neural
networks is of fundamental importance in under-
standing their physiological functions and treating
their pathological dysfunctions. Their electrical activity
can be recorded through the electrodes in a patch clamp
or microelectrode array. These electrical recording
techniques are invasive and incapable of recording
neural activity from a large region of interest, which
has promoted the optical imaging approaches. As
calcium ions (Ca2+) are a critical indicator of neuronal
activity, Ca2+ imaging is a powerful tool to study
neural activity [109]. However, the Ca2+ dynamics is
slower than that of the actual action potential, and
only suprathreshold neural activities can be recorded
[110]. Thus, directly imaging the transmembrane
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5074 Nano Res. 2018, 11(10): 5065–5106

Figure 4 Nanofabricated high-density neural electrodes. (a) Individual high-density probe with 200 electrode channels [99]. The
electrodes were spaced by 11 µm in a one-dimensional (1D) arrangement. (b) Recorded spikes using the probe in (a). (c)–(e)
High-density ultra-flexible probes with 20 µm electrode pitch and 800 nm thickness [101]. (f) Schematic diagram and microscope image
of an active CMOS high-density probe [104]. (a) and (b) adapted with permission from Ref. [99], © IEEE 2016; (c)–(e) adapted with
permission from Ref. [101], © Wiley-VCH 2018; (f) adapted with permission from Ref. [104], © Springer Nature 2017.

potential of neurons with high spatiotemporal (micro-
meter and sub-millisecond) resolutions, sensitivity
up to subthreshold activity, and versatility for both
excitation and inhibition is greatly desired. Although
the voltage-sensitive dyes (VSDs) for voltage imaging
(discussed in detail below) have a high spatiotemporal
resolution and capability of imaging a large region
of interest, they are constrained by sensitivity,
photobleaching, and phototoxicity [111]. Thus, semi-
conducting nanotransducers, especially QDs, with
higher photoluminescence intensity and photostability
have attracted attention for potential application to
voltage sensing in neurons.
Due to quantum confinement, QDs can be excited
by light, creating excitons, pairs of separated negatively
charged electrons and positively charged holes [112].
An electric field applied to the excited QDs leads to a
quantum-confined Stark effect, decreasing the energy
of electrons and holes [113]. As a result, their
photoluminescence intensity is quenched, and the
emission peak is red-shifted and broadened. This
property well aligns with the needs for optical voltage
imaging. A theoretical study showed that the membrane
potential of an action potential could result in such
photoluminescence quenching and red-shift in type-II
QDs and that QDs would have a higher sensitivity
compared to VSDs [114]. In a follow-up work, the use
of type-I and quasi-type-II QDs to image a voltage
resembling an action potential with millisecond
temporal resolution was experimentally confirmed
[111]. In this work, it was also claimed that the
photoluminescence quenching by QDs was attributed
to an increase of ionized QDs and that quasi-type-II
QDs had a higher sensitivity than type-I QDs. In a
more recent theoretical study, modeling showed that
type-I and type-II semiconducting nanorods could
have an even higher sensitivity than QDs, while the
sensitivity of type-II nanorods was higher than that
of type-I nanorods [115]. However, the nanorods
needed to penetrate the plasma membrane vertically
and symmetrically, which was technically challenging.
Up to now, the QD- or semiconducting nanorod-
assisted voltage imaging has not been biologically
validated. The physiological scenario is far more
complicated than the theoretical simulation. Once
tested in neurons, there are still major practical

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Nano Res. 2018, 11(10): 5065–5106
challenges associated with their voltage sensitivity,
placement, and biocompatibility. Voltage imaging
requires the nanosensors to be placed in the vicinity
of or within the plasma membrane. The QDs delivered
to the plasma membrane are, however, rapidly
internalized, and the placement of QDs is dynamically
changing. Considering the fast voltage dynamics
compared to the endocytosis dynamics, this should
not affect a single measurement within a short period
of time but will require repetitive dosing for separate
measurements, which will worsen the cytotoxicity
of QDs. Thus, exploring nanotransducers that can
convert voltage to an observable and readable signal
and meanwhile can serve as stable and biocompatible
interfaces is essential for the development of
nanotransducer-assisted voltage imaging in neurons.
2.3 Bionanotransducer-enabled functional neural
imaging
Compared to electrode-based neural recording
approaches, optical techniques offer complementary,
yet compelling, advantages for imaging the tran-
smembrane potentials, including less invasiveness
and superior spatial resolution [116]. Optical recording
or imaging can capture both the electrical activities of
each neuron in the circuit and map with micrometer
resolution and sub-millisecond precision.
Nanobiotechnologies for optical neural imaging
are mostly based on genetically encoded proteins, for
their remarkable biocompatibility and optical properties,
fast response dynamics, high effective sensitivity, and
easy functional modification. Protein-based optical
sensors can be used to measure transmembrane
potentials in single cells, tissues, and living animals
through detection of the exhibited fluorescent signals.
A number of optical imaging methods based on a
variety of mechanisms have been explored for the
benefits of neuroscience research.
2.3.1 Voltage-sensitive dye imaging
Traditional voltage-sensitive dye imaging (VSDI) is
based on a fluorescent change of small molecular dyes
when subjected to a change in a local electric field.
For its good spatial (up to 20 m) and temporal (up
to tens of microseconds) resolutions, this approach
offers a great potential in the visualization of the
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information processing in the nervous system [117,
118]. After decades of development and optimization,
VSDI exhibits facile delivery into living preparations
for long-term observation, from dissociated neurons
to frogs, rats, and nonhuman primates [119–121].
Typical VSDs possess an amphiphilic structure, in
which the hydrophobic components work as anchors
into the cellular membrane, and the hydrophilic
components, mostly chromophores, which array per-
pendicularly to the cellular membrane. The intensity
changes of the fluorescent signal correlate to the
transmembrane voltage changes, and the neural activity
can be measured accordingly. The most common
imaging mechanism is redistribution (Fig. 5(a)). A
change in the transmembrane potential causes the
charged chromophores to move in or out of the cell,
leading to a regional concentration change of the
chromophores with a corresponding change in the
fluorescence intensity. Another mechanism is called
reorientation (Fig. 5(a)), which is determined by
the interaction of the intermolecular electric fields.
Additionally, electrical modulation of the electronic
structure and fluorescent resonance energy transfer
(FRET) can cause fluorescence changes in response to
changes of the transmembrane potential (Fig. 5(b)).
In addition to the commonly used VSDs
(aminonaphthylethenylpyridinium (ANEP) and Rina
Hildesheim (RH) families) [122], QD-based approaches,
as mentioned above, provide an alternative opportunity.
Functional modifications using biomolecules can
both reduce the cytotoxicity of QDs and provide
functional targeting sites to the neuronal membrane
for voltage imaging [114]. Recently, Nag et al. reported
a QD-peptide-fullerene nanobioconjugate for imaging
membrane potentials in living cells [123]. This
alanine/leucine-rich peptide was designed to form a
helix to promote its membrane insertion. It also could
append the fullerene component at discrete fixed
distances for the signal detection and facilitate energy
transfer via tunneling. The imaging of PC-12 cells
showed a 20- to 40-fold improvement in F/F with
no sacrifice in responsivity. Thus, protein-modified
nanotransducers also play important roles in specific
targeting to the cellular membrane, enhancement of
biocompatibility, and connection between two FRET
components, with minimum effect on the fluorescent
signal.
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5076 Nano Res. 2018, 11(10): 5065–5106

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 in the ratio of emission intensities (YFP/CFP) [124, 125].
(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 [126].
(e) VSFP2.3 reported transmembrane voltage transients through two different FPs [127]. (f) Extracellular electrical stimulation induced
an increase in intracellular Ca2+ concentration in myotubes with the expression of GCaMP [124]. (c) adapted with permission from
Refs. [124, 125], © Springer Nature 2001 and National Academy of Sciences 2004, respectively; (d) adapted with permission from Ref.
[126], © Springer Nature 2015; (e) adapted with permission from Ref. [127], © Springer Nature 2010; (f) adapted with permission from
Ref. [124], © Springer Nature 2001.

2.3.2 Genetically encoded fluorescent imaging
Neural activities are encoded by dynamic fluctuations
of the transmembrane potential, including both
subthreshold and action potentials. To visualize the
dynamic changes of the transmembrane voltage, which
range from ~ 1 mV (e.g., a postsynaptic potential
caused by a single vesicle release) to over 100 mV (i.e.,
an action potential) and span from 100 ms (axonal
conduction) to 10 s (plateau potential), genetically
encoded optical indicators offer a great promise.
Nanobiotechnological engineering approaches have
been used to fuse the fluorescent chromophores with
other domains that in response to cellular events
undergo conformational changes such as intracellular
Ca2+ concentrations, transmembrane potentials, small
metabolites, and other ions. Two major classes of
genetically encoded optical indicators have been
developed: One contains genetically encoded calcium
indicators (GECIs), which have a Ca2+-binding domain
and a Ca2+ concentration response conformation;
another comprises genetically encoded voltage in-
dicators (GEVIs), which have a domain responsive to
transmembrane potential changes.
Genetically encoded calcium indicators: Developed
since 1997 [128], GECIs monitor changes to the

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Nano Res. 2018, 11(10): 5065–5106
intracellular Ca2+ concentration caused by action
potentials. As a ubiquitous second messenger, Ca2+ is
of great significance in all aspects of cellular physiology.
Triggered by an action potential, an increase in its
intracellular concentration, from 20–100 nM (initial
concentration) to 5–10 M (peak concentration), causes
a change to the fluorescence [129]. Briefly, the design
of GECIs involves the fusion of two parts: a naturally
evolved Ca2+-binding protein component that undergoes
a conformational state transition in response to
Ca2+ binding (calmodulin (CaM) [128] or troponin-C
(TnC) [130]) and a reporter component based on a
conformation-sensitive fluorescent protein (FP).
Two types of signals are recorded according to
the type of reporter component (Fig. 5(c)). A simple
fluorescence signal is collected through the confor-
mational transition of a single FP component [124]. A
pair of FPs exhibits FRET, which could provide a
measure of the Ca2+ concentration by the ratiometric
fluorescence signal of the two FPs [128]. The FRET
signal is a better alternative due to its independence
from probe concentration, low excitation light intensity,
and low absorption in the optical path. For example,
as a typical GECI, yellow Cameleon (YC) 3.60 has a
pair of enhanced cyan FP (ECFP) as the donor and
Venus protein FP with circularly permuted confor-
mation (cpYFP) as the acceptor. In the presence of
Ca2+, its intramolecular conformation changes lead to
a reduced spatial distance and illuminate the Venus
protein, and the ratio change of YFP/CFP is up to six
folds with a high Ca2+ dynamics up to 0.25 M [125].
The mostly optimized GECIs are single-wavelength
green indicators based on the original GCaMP sensor.
The reporter domain of GCaMPs is a circularly
permuted enhanced green FP (EGFP), which is flanked
by the Ca2+ binding protein CaM and the CaM-binding
peptide M13. In the presence of Ca2+, CaM-M13
interactions lead to conformational changes and an
increase in fluorescent emission [124, 131]. A number
of engineered variants of GCaMP have been reported,
and these indicators are composed of constituent
molecules to achieve specific manipulation of sensor
applications [132–134].
Since the first GECI was reported with the chromo-
phore green FP (GFP), the modulation of indicator
color has been well explored. Directed mutation of
the side-chains comprising the chromophore can tune
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the excitation and emission. A number of improved
variants of the original blue, cyan, yellow, and
red FPs [126] have been developed to improve the
brightness, photostability, tissue penetration, and
signal-to-noise ratio.
To avoid interferential interactions of CaM with
endogenous binding factors and improve the detection
precision, two effective approaches are pursued: One
is the replacement of CaM with troponin C variants,
which is the Ca2+ binding protein in muscle cells and
does not have an endogenous binding site in neurons
[130, 135]; the other is the modification of binding
interference. Palmer et al. reengineered the binding
interface between CaM and a target peptide to generate
selective and specific binding pairs that could be no
longer perturbed by large excesses of native CaM, and
the Ca2+ detection sensitivity turned over a 100-fold
range at 0.6–160 M [136].
In addition, the optimization of GECIs focuses on
their modulation for specific applications and better
optical properties, such as optimization on the Ca2+
binding constant [131, 137], subcellular targeting [138],
brightness, and red-shifting of the fluorescent indicators
for large penetration and good resolution [139].
GECIs can be used in combination with two-photon
imaging to achieve improved measurements in a highly
scattering medium with an increased signal-to-noise
ratio without the need for averaging. Mank et al.
generated a GECI as TN-XXL for two-photon ratio-
metric imaging in the visual cortex. They rearranged
the Ca2+ sensing moiety TnC within the indicator and
mutagenesis of selected amino acid to increase
overall signal strength and sensitivity in the low Ca2+
regime [140].
GECI fluorescence imaging is a good proxy to
record average action potential changes, but it has an
inherent inadequacy. Because Ca2+ transients are
100–1,000 fold slower than the underlying electrical
waveforms, most subthreshold changes of tran-
smembrane potentials cannot elicit a change in the
Ca2+ concentration, and the Ca2+ dynamics is confounded
by the complicated interactions between different
Ca2+ sources and intrinsic or extrinsic Ca2+ buffers.
Hence, weak and brief changes of the transmembrane
potentials may not be recorded by the Ca2+ indicators
[110, 141].
GEVIs: GEVIs may be preferred over GECIs, for
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their capability of directly reporting both synaptic
and action potentials as well as capturing the entire
voltage dynamics. Several different approaches to
build GEVIs have been developed since 1997. Most
GEVIs consist of two components: a voltage-sensitive
domain from an ion channel as the voltage sensor
and a component that binds in the plasma membrane
and experiences the voltage changes.
Same as for GECIs, two types of fluorescent signals
based on different response mechanisms can be
collected: One is the single fluorescent signal from a
single fluorescent moiety [142–144], which undergoes
a significant conformational change that alters its
spectrum; and the other is a ratiometric signal based
on FRET between a pair of fluorescent moieties [127,
145], which are both involved in the molecular
interactions and motions, leading to ratio changes
in fluorescence intensities (Fig. 5(d)). Signals from a
single FP have limited sensitivity, while the ratiometric
FRET signals can significantly reduce the motion and
blood flow effects in complex environments.
Several GEVIs based on different fluorescent
chromophore families have been developed. For
example, the fluorescent Shaker (FlaSh) and the sodium
channel protein-based activity reporting construct
(SPARC) are based on simple fluorescent signal
detection, while the voltage-sensitive FP (VSFP) uses
an FP pair based on FRET [146–148].
Siegel et al. reported an early attempt on FlaSh
[146]. They constructed a GFP-Shaker fusion protein,
using a nonconducting mutant of a voltage-gated K+
channel as the voltage-responsive site and an FP
inserted into the K+ channel protein as a reporter. This
GEVI had a maximal fractional fluorescent change of
5.1%. Guerrero et al. expanded the FlaSh GEVIs by
modifying kinetics, dynamic range, and color. The
improved folding enhanced the detection sensitivity,
and the availability of different FP colors promoted
wide adoptions [149]. The SPARC was developed
with a strategy to insert a GFP molecule into a rat
muscle Na+ ion channel subunit, which exhibited rapid
response kinetics without significant inactivation [147].
As the key component in the FP-based GEVIs, VSFP
underwent a lot of research. The first generation of
VSFPs was derived from the voltage-sensing domain
of a K+ channel subunit [148]. This VSFP consisted of
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Nano Res. 2018, 11(10): 5065–5106
a voltage sensing domain of a K+ channel and a pair
of cyan and yellow emission mutants of FPs. Although
this generation of VSPFs could optically report changes
in the transmembrane potential, their application to
functional imaging of mammalian neurons was limited
by their low targeting capability to the membrane
[150]. The second generation of VSPF (VSPF2) was
based on Ciona intestinalis voltage sensor-containing
phosphatase (Ci-VSP), exhibited better membrane
targeting, and formed a big Ci-VSP GEVI family
[116]. Akemann et al. developed FRET-based VSFP2s
by changing the enzyme domain of Ci-VSP with two
tandem fluorescent domains, which exhibited more
efficient targeting to the cellular membrane and higher
responsiveness to the transmembrane potentials [127].
The variant VSFP2.3 was developed as a FRET-based
indicator possessing the capacity of imaging both
spontaneous action and synaptic potentials in neurons
[151]. The indicator VSPF3.1 was the fusion of Ci-VSP
and a single red-shift protein, which offered the
advantages of a red-shifted spectrum and relatively
fast overall kinetics [152]. The VSPF-Butterfly series,
with a sandwiched structure between two FPs and
a middle voltage-sensitive domain, showed a
subthreshold detection range and fast kinetics for
single-cell synaptic responses in applications from
single neurons to the brain [153].
Besides of FP-based GEVIs, microbial rhodopsin
could also be used as the fluorescent voltage reporter
[154, 155]. This retinal chromophore shows very weak
near-infrared fluorescence, while upon light-driven
transport of protons, a change in the chromophore
emission was induced. Comparing to FP-based
GEVIs, GEVIs based on the microbial rhodopsin have
a sub-millisecond response time and better voltage
sensitivity [154–156]. GEVIs based on non-pumping
mutants of Archaerhodopsin 3 (Arch) have voltage
sensitivities between 30%–90% per 100 mV and
half-maximal response times between 50 s and 1.1 ms
at room temperature [156]. However, Arch-derived
GEVIs exhibit weak fluorescence, more than 30-fold
weaker than that of FPs. Meanwhile, to illuminate
microbial rhodopsin-based GEVIs, the excitation
laser power needs to be much higher, typically 300–
1,000 W/cm2, much more than that of FP-based GEVIs
(10 W/cm2) [156]. The low brightness of rhodopsin-
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based GEVIs presents a challenge for its widespread
use.
Current efforts on the development of GEVIs
primarily focus on the optimization of existing sensor
constructs, including the voltage range, color, brightness,
response kinetics, and cellular targeting properties.
As key indicators to evaluate sensing performance,
response sensitivity (F/F), half-maximal response
times, signal-to-noise ratio, and excitation power all
need to be considered. The first generation of GEVIs
exhibited modest voltage sensitivity (< 5% F/F per
100 mV) and slow kinetics (10–200 ms). The second
generation of Ci-VSP-based GEVIs had a novel GEVI
(Arclight) consisting of a Ci-VSP and a super ecliptic
pHluorin that carried the point mutation A227D [142].
Arclight A242, derived from Arclight, exhibited a
fluorescent intensity increase by 35% F/F per 100 mV,
but the response time (10 ms) was much slower,
hindering action potential detection. In another GEVI
design, called ASAP1, a circularly permuted FP was
inserted in an extracellular loop of a voltage-sensing
domain [144]. ASAP1 exhibited high sensitivity
(18%–29% F/F per 100 mV) and high kinetic speed
(2 ms) and could be used for action potential detection
at waveforms up to 200 Hz.
GECIs and GEVIs have exhibited the capability of
recording neuron potentials in multiple mammals
and neuron types, but the use of them in humans
seems unlikely at present because its delivery involves
viral vectors. The development and optimization of
genetically encoded indicators still continue. The ideal
GECIs and GEVIs should process properties such as
large voltage/Ca2+ induced fluorescent changes over
a physiological range, quick response kinetic time
(sub-millisecond), targeting capability to different
neuron types, good photostability, large brightness and
signal-to-noise ratio, and red/near-infrared emission
for in vivo applications.
3 Nano fNIs for neural stimulation
3.1 Nanoelectrodes
3.1.1 Microstimulation with nanomaterial coatings
Nanostructured surfaces and nanomaterial coatings
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have been shown to improve the charge storage
capacity and charge injection limit of microelectrodes,
which results in more effective stimulation including
smaller voltage, greater power efficiency, and less
tissue damage.
Carbon nanotube-based electrodes were found to
offer lower impedance and improved charge injection
compared to platinum, making it a suitable candidate
for electrical recording and stimulation. Wang et al.
first reported the use of vertically aligned multiwall
carbon nanotubes to stimulate excitable cells (rat
hippocampus neurons) [157]. Tsang et al. reported
the use of Au-carbon nanotube composite electrodes
fabricated on flexible polyimide substrate [158]. As a
result of the lower stimulation voltage and less power
consumption, the stimulation could be done wirelessly.
The resulting device forms an insect–machine interface
in which the flight path of a moth could be biased by
selectively stimulating one side of the moth’s body. Yi
et al. developed vertically aligned carbon nanotube
electrodes on flexible substrates and used them to
stimulate rat sciatic nerves and record from rat spinal
nerves with a signal-to-noise ratio as high as 12.5 [159].
Jan et al. quantified the impedance and charge storage
capacity of iridium oxide, PEDOT, and layer-by-layer
synthesized multiwall carbon nanotube [66]. Carbon
nanotubes outperformed traditional electrode materials
by having a lower impedance and higher cathodic
charge storage capacity. In addition, no sign of failure
was observed after 300 cycles of cyclic voltammetry
scans.
3.2 Nanotransducer-assisted neural modulation
The emerging concept of going wireless has been
revolutionizing the contemporary technologies and
reshaping the modern lifestyle. It is also driving the
advancement of medical devices [160]. Conventional
neural modulation techniques deliver electrical signals
to the target neural population. Due to fast dissipation
and attenuation of the electrical signals through tissues,
these traditional techniques often demand invasive
placement of the electrodes in the immediate vicinity
of the target neural regions and implantation of a
pulse generator wired to the electrodes to deliver the
electrical signals. Surgical procedures cause tissue
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5080
damage and surgical complications [161], and chronic
inflammation around the electrodes causes scar
tissue formation and early device failure [14].
Alternative strategies to neural modulation, which
rely on direct wireless delivery of magnetic field,
ultrasound, and infrared light to the target neurons,
unnecessitate the electrodes but are limited in spatial
resolution and/or power efficiency. These deficiencies
have motivated the development of a new generation
of nanotransducer-assisted wireless neural modulation
techniques featuring minimal or even no invasiveness
with greatly improved spatiotemporal precision and
cellular targeting specificity.
Like wireless charging, in which the electromagnetic
energy is transmitted through the intermedium and
converted to electrical currents by an antenna in the
target battery, a primary wireless signal (e.g., optical,
magnetic, or acoustic) can be transmitted through
tissues and converted to a secondary local signal (e.g.,
electric, thermal, optical, or mechanical) by nano
antenna-transducers to modulate the target neuron.
These nanotransducers are essential for delivering the
highly localized secondary signal with spatial precision,
target specificity, and improved efficiency. Due to
their small size and novel physicochemical properties,
these nanomaterial-based nanotransducers perfectly
align with these demands. Firstly, they can be delivered
to the target region via minimally-invasive local or
intravenous injection. Secondly, their diverse energy
transduction schemes allow the conversion of a
medically safe, tissue-penetrating primary signal to
a localized cell modulatory secondary signal at the
nanotransducer–neuron interface [15]. Additionally,
their ease of surface modification and bio-conjugation
can facilitate specific targeting to the targeted neuron
population at cellular and even subcellular levels,
dramatically improving the target specificity, selectivity,
and spatiotemporal resolution.
Over the past few decades, particularly the last
decade, a great deal of efforts has been made to
develop such nanotransducer-assisted wireless neural
modulation techniques. In the literature, these
techniques are commonly categorized by the primary
wireless signals. In contrast, in the present review,
these techniques are categorized by the secondary
local signals to elucidate the concepts and design
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Nano Res. 2018, 11(10): 5065–5106
rationales, as the secondary signal is the ultimate
modulatory signal to the target neuron. For example,
manipulation of ion channels plays an important role
in neural modulation, and there are various types of
ion channels, including voltage-gated, temperature-
gated, mechanosensitive, and light-gated. Thus, the
local secondary signal needs to be generated by the
nanotransducers to gate the corresponding ion channels
and eventually modulate the neuronal activities.
Here, these diverse types of nanotransducers can be
classified into electrotransducers, thermotransducers,
optotransducers, and mechanotransducers based on
the type of secondary signal they generate.
To develop such nanotransducer-assisted wireless
neural modulation techniques, there are several
key factors to consider, including the primary and
secondary signals, the nanotransducer’s biosafety,
biostability, placement, and modification, and cell
modification.
Primary signal: The primary signal needs to be safe
and wirelessly transmittable through tissues to reach
the target region. Although light, magnetic field, and
ultrasound can all meet these two criteria to possibly
serve as a primary signal, their interactions with tissues
affect their penetration depth and spatial resolution
(i.e., how well the primary signal can be focused on
the target region). Light has superior spatial resolution
of 10–7 m, while it can barely pass the cranium (10–6 m
travel depth) and only penetrate dermal tissues as
deep as 4 mm [163, 164]. Thus, for deep neural tissue
modulation, implantation of a light source is usually
required. Transcranial focused ultrasound traveled
through the skull and modulated the cortical activity
30 mm deep in human brains with a spatial resolution
of 10–3–10–2 m [165]. The penetration depth and spatial
resolution of transcranial magnetic stimulation are
highly dependent on the coil design. For example,
the 50 coil design has a penetration depth and spatial
resolution of 10–2 m [166]. For these primary signals,
there is a tradeoff between penetration depth and
spatial resolution [165, 166]. Additionally, the interac-
tions of primary signals with tissues may also generate
noxious heat, limiting the maximum intensity of the
primary signal.
Secondary signal: The secondary signal needs to be
able to modulate neurons by gating the respective ion
Nano Res. 2018, 11(10): 5065–5106
channels, activating cell signaling pathways, changing
the plasma membrane capacitance, etc. Possible
secondary signals include electric fields, heat, light,
and mechanical forces. Generation of the secondary
signal should be controlled to exceed the threshold for
cellular modulation but not to exceed the safety limit
that can cause cell damages. Theoretical simulation
can be used to help assess the feasibility of a signal
transduction scheme and guide the experimental
design and optimization. Additionally, the temporal
resolution of the modulation significantly depends
on how fast the neurons respond to the secondary
signal.
Nanotransducer biosafety and biostability: The nano-
transducers need to serve as stable neural interfaces
within the modulation period, and they must be safe
in the physiological environment. They should cause
no toxicity and minimal inflammation. Moreover,
they should remain intact to maintain the functional
interfaces and not release or leak toxic substances.
Their biostability can be challenging. First, certain
nanotransducers are prone to degradation due to the
physiological acidic pH and enzymatic activities.
Second, the exogenous nanotransducers are also prone
to clearance, so their placement is dynamic instead of
static.
Nanotransducer placement and distribution: Overall,
nanotransducers can either be in a dispersed/
injectable form or immobilized onto a substrate or
microelectrode (Fig. 6(a)). The dispersed nanotran-
sducers can be placed extracellularly, targeted to the
plasma membrane (e.g., onto the membrane, receptors,
anchoring proteins, or ion channels), or internalized.
Immobilization of nanotransducers onto a substrate
or microelectrode facilitates the build-up of the
secondary signal to reach the threshold and control of
the nanotransducer distribution, but the implantation
increases invasiveness. On the contrary, injectable
and monodispersed nanotransducers minimize inva-
siveness, but their distribution needs to be carefully
controlled, as their placement and distribution affect
the cytotoxicity, modulation efficacy, efficiency, and
consistency. For example, unbound and extracellularly
distributed nanotransducers prolong the stability of
the interface but tend to be removed by the fluid
circulation, such as the cerebrospinal fluid in the central
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nervous system. On the other hand, binding of
nanotransducers to the plasma membrane, receptors,
or ion channels improves the specificity and washout
resistance, but they can be rapidly internalized. The
internalized nanoparticles can cause toxicity and
modulation inconsistency [167, 168]. Additionally, a
complete retrieval or clearance of the nanotransducers
after the modulation can be challenging for dispersed
nanoparticles.
Nanotransducer modification: Nanotransducers are
usually surface-modified so that their dispersity
and biocompatibility can be improved. They can be
immobilized on a substrate, specifically targeted to a
neural population or even to the receptors and ion
channels. However, surface modification can attenuate
the secondary local signals and increase the distance
between the nanotransducers and the modulation
target. Moreover, the surface chemistry needs to be
well controlled, because certain surface chemistry,
even without inducing clear cytotoxicity, may cause
false modulatory effects due to plasma membrane
perturbation [169].
Cell modification: Genetically engineering certain
types of neurons to express the target ion channels
enables and optimizes the cells’ response to the
corresponding secondary local signal. Neurons can
also be genetically modified to express binding sites
for nanotransducer docking and create hallmarks
for evaluating the modulatory effect. These cell
modification strategies make the modulation technique
versatile and adaptable for different types of cells.
They can also improve the modulation specificity
and selectivity through selective engineering of the
target neuron population. However, the viral vectors
used for the cell modification can cause immune
responses, and the transfection is usually irreversible.
Thus, cell modification by genetic engineering causes
safety and moral concerns and thwarts their clinical
applications.
Considering these critical factors, we herein review
four main types of nanotransducers for wireless neural
stimulation: electrotransducers, thermotransducers,
optotransducers, and mechanotransducers. Each type
is further subdivided by the types of nanomaterial
used. These nanotransducers are summarized regar-
ding these factors in Table S1 in the Electronic
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5082 Nano Res. 2018, 11(10): 5065–5106

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 nanorod-based
electrotransducers [170]. (b) Semiconducting nanorods convert light to electric fields. (c) A train of extracellular spikes triggered by a
light pulse. (d) and (e) Magnetoelectric nanomaterial-based electrotransducers [171]. (d) Magnetoelectric nanomaterials convert a
magnetic field to electric fields. (e) Modulated EEG waveforms. (f) and (g) Piezoelectric nanomaterial-based electrotransducers [172].
(f) Piezoelectric nanomaterials convert ultrasound to electric fields; (g) Ca2+ influxes following an ultrasound pulse. (h) and (i) Gold
nanomaterial-based thermotransducers [173]. (h) Gold nanomaterials convert light to heat. (i) A train of action potentials, each evoked by a
light pulse. (j) and (k) Superparamagnetic nanoparticle-based thermotransducers [163]. (j) Superparamagnetic nanoparticles convert a
magnetic field to heat. (k) Peristimulus time histogram. (l) and (m) Upconverting luminescent nanomaterial-based optotransducers
[174]. (l) Upconverting luminescent nanomaterials convert NIR light to visible light. (m) A train of action potentials, each evoked by an
NIR light pulse. (n) and (o) Superparamagnetic nanoparticle-based mechanotransducers [175]. (n) Superparamagnetic nanoparticles
convert a magnetic field to mechanical forces. (o) Peristimulus time histogram. (b) and (c) adapted with permission from Ref. [170], ©
the American Chemical Society 2014; (d) and (e) adapted with permission from Ref. [171], © Elsevier 2015; (f) and (g) adapted with
permission from Ref. [172], © the American Chemical Society 2015; (h) and (i) adapted with permission from Ref. [173], © Elsevier
2015; (j) and (k) adapted with permission from Ref. [163], © The American Association for the Advancement of Science 2015; (l) and
(m) adapted with permission from Ref. [174], © Royal Society of Chemistry 2015; (n) and (o) adapted with permission from Ref. [175],
© the American Chemical Society 2016.

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Nano Res. 2018, 11(10): 5065–5106
Supplementary Material (ESM).
3.2.1 Nano electrotransducers
Electrical signals are used to modulate neurons in the
conventional modulation techniques. Instead of being
transmitted from an electrical stimulator to electrodes
through wires, electrical signals can also be converted
locally by nanotransducers from other wirelessly
transmitted physical signals to target voltage-gated
ion channels for neural modulation [176, 177]. For
example, light, magnetic field, and ultrasound can be
converted by QDs, magnetoelectric nanomaterials, or
piezoelectric nanomaterials, respectively, into local
electric fields to modulate neurons.
QDs—From light to electric field: Upon excitation,
QDs convert light to an electric dipole (electron–hole
pair) via quantum confinement. The electric field
created by the electric dipole can serve as a secondary
signal to modulate neurons [178]. The excitation
wavelengths of QDs range from ultraviolet via visible
to near-infrared (NIR), and the tissue penetration
depth and spatial resolution of light depend on its
wavelength. The shorter the wavelength, the lower
the penetration and the higher resolution. NIR light
within the first (700–950 nm) and second (1,000–
1,350 nm) NIR windows has minimal interactions
(scattering and absorption) with tissues [179, 180], so
this NIR light has maximal penetration through dermal
tissues, as deep as 4 mm, but the lowest resolution
[164]. Thus, selection of QDs with their excitation
wavelengths depends on the target application. For
example, light of short wavelengths is only suitable
for superficial neural modulation, such as in retinal
prostheses, while light of longer wavelengths can be
used for deeper intervention. As to the electric field,
mathematical modeling shows that an excited QD
could evoke an action potential if the distance between
the QD and a voltage-gated ion channel is within
2 nm [181, 182]. Thus, intimate contacts of QDs to the
neuron’s membrane are realized by targeting the QDs
to the plasma membrane or by coating them onto a
neuronal culturing substrate.
However, due to their tiny size (2–6 nm), QDs are
easily internalized through endocytosis. Attempts to
create an fNI by targeting bio-conjugated QDs directly
to the plasma membrane rarely succeeded [182–184].
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Thus, efforts were mainly focused on improving
the interface stability by immobilizing QDs onto a
substrate.
The initial contrivance of fNIs by immobilizing QDs
onto salinized and poly-D-lysine-coated substrates
achieved slightly improved stability for 1 and 3 days,
respectively [182]. By culturing NG108-15 cells on a
layer-by-layer film containing repeating layers of HgTe
QDs and poly(dimethyldiallylammonium chloride),
Pappas et al. demonstrated that cells were depolarized
and even stimulated to fire action potentials upon
irradiation with 532 nm laser pulses (duration: 500 ms;
irradiance: 800 mW/cm2) [185]. This was the first study
revealing an optical-to-electrical cellular modulation
assisted by QDs. Following the same strategy,
CHO cells, RBL cells, NG108-15 cells, and primary
hippocampal neurons were cultured on a stacked film
containing repeating layers of QDs and adhesives,
and irradiation with 380 nm laser pulses depolarized
all types of cells and induced Ca2+ influxes and action
potentials in NG108-15 cells and primary hippocampal
neurons [186]. However, LnCap cells cultured on a
stacked film of CdTe QDs were hyperpolarized upon
illumination with 430 nm laser pulses [181]. In this
latter work, a simple coating strategy was used to
drop-cast a film of CdSe QDs with van der Waals
force as the anchoring force. Cortical neurons cultured
on top were randomly hyperpolarized or depolarized
upon irradiation with 550 nm laser pulses, and
cytotoxicity was found in these neurons. A CdSe
QD-coated micropipette fell short to achieve consistent
stimulation on cortical neurons, either.
It became clear that direct coating of QDs onto a
substrate could not achieve a long-term stable fNI.
Although layer-by-layer QD films achieved some
modulatory effects, their modulation efficiency and
consistency were fairly low. In order to improve the
stability of the fNI, instead of physically stacking layers
of QDs, they were covalently bound to the substrate.
Core–shell structured CdSe/CdS semiconducting
nanorods were bound to highly dense carbon nanotube
films via carbodiimide crosslinking. Embryonic chick
retinas, unresponsive to light, were cultured on the
composite film and stimulated by 405 nm light pulses
(duration: 100 ms; irradiance: 3–12 mW/cm2; Figs. 6(b)
and 6(c)) [170]. The interface was stable for up to
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5084
21 days, with a significant improvement. However,
due to the small photocurrents, the spatial resolution
of this platform was low, with long latency and limited
synchronization.
Up to now, these QD-assisted wireless fNIs have
only been studied in vitro, and the focus is still on
establishing a stable interface to achieve effective and
consistent modulation. The inherent tiny size of QDs
makes it extremely challenging to use dispersed QDs.
One solution is to anchor QDs onto a substrate, and,
for this strategy, chemical bonding creates a more
stable interface than physical stacking or direct coating.
Another solution is to explore semiconducting
nanorods as an alternative. Even when the fNI is
optimized with both chemical bonding and semicon-
ducting nanorods, its spatial resolution still needs
improvement. Additionally, the majority of QDs
are cytotoxic. Although surface modification and
encapsulation have been used to alleviate this issue,
degradation of the QDs by the physiological acidic
pH and enzymes is inevitable.
Magnetoelectric nanomaterials—From magnetic field to
electric field: Magnetoelectric nanomaterials can convert
a magnetic field to an electric field via magnetoelectric
transduction. A computational study showed that
such a transduction could theoretically recover the
deteriorated electric pulses in Parkinson’s disease [187].
However, the model used a simplified assessment
of nanoparticles in an aqueous solution without
considering many practical factors, such as the
physiological environment, placement of the nano-
particles, etc. A follow-up in vivo study demonstrated
that 30 nm CoFe2O4-BaTiO3 magnetoelectric core–shell
nanoparticles administrated into a mouse brain under
a low-energy magnetic field (100 Oersted; 0–20 Hz)
could modulate the electroencephalography signals
(Figs. 6(d) and 6(e)) [171]. However, further studies
are needed to assess the basics underlying this finding,
such as the cellular distribution of the nanoparticles,
biostability of the interface, spatial selectivity, temporal
precision, biochemistry, and, most importantly, the
electrophysiological evidence of the cellular modulation
both in vitro and in vivo.
Piezoelectric nanomaterials—From ultrasound to electric
field: Piezoelectric nanomaterials convert mechanical
stress to an electric field via mechanoelectrical
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Nano Res. 2018, 11(10): 5065–5106
transduction. Ultrasound, a longitudinal wave with a
frequency higher than 20 Hz, can cause a nanoparticle
to vibrate along the transmission path and exert
mechanical stress on it. Ultrasound can penetrate
deeply into soft tissues, and low-intensity, low-
frequency ultrasound can travel through the skull
and be focused onto a specific deep brain region [188].
Thus, ultrasound can serve as a primary wireless signal
to activate piezoelectric nanotransducers to create a
secondary local electric field.
Zinc oxide nanowires, one of the most studied
piezoelectric nanomaterials, however, induce cyto-
toxicity in excitable cells and fall short in maintaining
a stable interface in a physiological environment [189].
Other piezoelectric nanomaterials, including boron
nitride nanotubes [190, 191] and barium titanate
nanoparticles [172, 192], have been used to develop
ultrasound-driven wireless cellular modulation
techniques.
Similar to the development of QD-based fNIs, in
the early efforts, dispersed piezoelectric boron nitride
nanotubes were studied. Dispersed piezoelectric
boron nitride nanotubes were readily internalized
by pheochromocytoma PC-12 cells, neuroblastoma
SH-SY5Y cells, and myoblast C2C12 cells. When
coupled with ultrasound, the piezoelectric boron nitride
nanotubes promoted neurite outgrowth of PC-12 cells
and SH-SY5Y cells as well as myogenesis of C2C12
cells [190, 191]. Dispersed gum Arabic-coated barium
titanate nanoparticles, electrostatically attached to
the plasma membrane of cell bodies and neurites of
SH-SY5Y cells, induced high-amplitude Ca2+ influxes
when coupled with ultrasonic treatment (Figs. 6(f)
and 6(g)) [172]. Following a similar placement strategy,
dispersed gum Arabic-coated barium titanate nano-
particles were electrostatically attached to the plasma
membrane of rat hippocampal neurons and cortical
neurons within neuronal networks cultured on a
microelectrode array [193]. Ultrasound treatment
increased the spiking activity of these neuronal networks
in a temporal pattern that matched the ultrasound
pulses.
Besides being applied in a dispersed form,
piezoelectric nanomaterials were also immobilized onto
substrates by blending piezoelectric nanoparticles
into polymers. A micropatterned photoresist blended
Nano Res. 2018, 11(10): 5065–5106
with barium titanate nanoparticles synergistically
promoted the osteogenesis of osteosarcoma Saos-2
cells under ultrasound [194]. With the same immo-
bilization strategy, piezoelectric polyvinylidene fluoride
trifluoroethylene (p(VDF-TrFE)) blended with barium
titanate piezoelectric nanoparticles promoted neurite
outgrowth and induced Ca2+ transients in SH-SY5Y
cells cultured on both the p(VDF-TrFE) film and the
blended film under ultrasound [192]. However, both
films decreased the cellular proliferation compared
to a control Ibidi film.
It is worth noting that also piezoelectric polymers
coupled with ultrasound were explored for neural
modulation. A piezoelectric p(VDF-TrFE) film coupled
with ultrasound promoted the neurogenesis of PC-12
cells [195]. Moreover, electrospun piezoelectric p(VDF-
TrFE) microfibers alone promoted neurite extension
of dorsal root ganglion neurons [196]. However, no
synergetic treatment of piezoelectric polymer nanofibers
and ultrasound has been reported yet.
Although theoretically piezoelectric nanomaterials
can convert mechanical stimuli into an electric field,
no direct evidence has validated the generation of an
electric field from dispersed piezoelectric nanomaterials
under ultrasound. Many questions still remain to be
answered: How does ultrasound interact with the
dispersed nanoparticles? Can adequate electric fields
be generated? How do the generated electric fields
distribute? Additionally, the long-term interface stability
and biocompatibility of dispersed piezoelectric nano-
materials must also be addressed.
3.2.2 Nano thermotransducers
Gold nanomaterials—From light to heat: Rod- and
sphere-shaped gold nanoparticles can convert NIR
and visible light to localized heat through surface
plasmon resonance, respectively [197]. Like QDs, the
excitation (resonant) wavelength of gold nanomaterials
affects the penetration depth and spatial resolution,
and its selection depends on the target application.
The resonant wavelength can be tuned by controlling
the aspect ratio [197]. Incident irradiation with the
corresponding resonant wavelength excites confined
electron oscillation and collision at the nanoparticle
surface, resulting in heat generation and dissipation.
The generated local heat can change the capacitance
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5085
of the plasma membrane [198] and/or modulate
temperature-sensitive ion channels [199], therefore
modulating neural activities. Cell differentiation, such
as neurite outgrowth, has been used as an initial
hallmark to show the feasibility of this stimulation
technique. More affirmative validations have also
been provided involving in vitro and in vivo
electrophysiological tests.
Internalized gold nanorods coupled with NIR light
treatment were initially shown to promote neuronal
differentiation. Gold nanorods (resonant wavelength:
780 nm) were internalized in NG108-15 neuronal cells.
Neurite outgrowth was promoted upon irradiation
with a 780 nm continuous-wave laser (duration: 1 min;
irradiance: 0–13.75 W/cm2) [200], and intracellular
Ca2+ transients were induced upon irradiation with
780 nm laser pulses (duration: 20–100 ms; radiant
exposure: 0.07–407 J/cm2) [167].
Untargeted dispersed gold nanorods were found to
stimulate neurons with NIR illumination proportionally
to the laser duration or irradiance. In vitro rat auditory
neurons were treated with silica-coated gold nanorods
(resonant wavelength: 780 nm), and the nanorods
were distributed extracellularly, contacted the plasma
membrane, or were internalized [201]. A laser pulse
of 780 nm (duration: 0.025–50 ms; power: 90 mW;
irradiance: unknown) induced inward transmembrane
currents and membrane depolarization proportional
to the pulse duration. However, the neural responses
were not consistent among the tested neurons, and a
train of action potentials matching the pulse pattern
was not achieved. Gold nanorods (resonant wavelength:
977 nm) were injected into a rat sciatic nerve in vivo
and extracellularly distributed [202]. Irradiation
with a 980-nm laser pulse (duration: 1 ms; irradiance:
159–1,046 W/cm2) enhanced the amplitude of compound
nerve action potentials proportionally to the radiant
exposure. Comparing to a null control without gold
nanorods, irradiation with gold nanorods increased
the responsivity by five times and substantially
lowered the threshold by two-thirds.
To improve the stimulation specificity and efficiency,
gold nanomaterials were targeted to the plasma
membrane and ion channels, and illumination induced
excitatory or inhibitory effects in stimulated cells.
Amine-terminated PEGylated gold nanorods (resonant
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5086
wavelength: 785 nm) were bound to the plasma
membrane of rat hippocampal, cortical, and olfactory
bulb neurons, and irradiation of 785 nm laser pulses
(duration: 10 s; irradiance: 1.5 W/cm2) inhibited the
spontaneous, electrically stimulated, and chemically
hyperexcited neural network activity, while the unbound
nanorods did not substantially affect the neural network
spike rate [203]. Cationized high-density lipoprotein-
coated gold nanorods (resonant wavelength: 785 nm)
were targeted to the plasma membrane of HEK293T
human epithelial cells, and irradiation with a 780 nm
continuous-wave laser (duration: 120 s; irradiance:
800 W/cm2) induced Ca2+ influxes [169]. Uncoated gold
nanoparticles (resonant wavelength: 565 nm) and
antibody-conjugated PEGylated gold nanoparticles
(resonant wavelength: 568 nm) were bound to the
plasma membrane of non-genetically engineered
and genetically engineered rat hippocampal neurons,
respectively, and irradiation with 800 nm laser pulses
(duration: 140 fs; irradiance: 0.27–1.02 mW/cm2)
induced Ca2+ transients at both the cellular and
subcellular level and evoked action potentials [204].
Streptavidin-coated gold nanorods (resonant wavelength:
977 nm) were targeted to the biotinylated antibodies
bound to the plasma membrane of rat hippocampal
neurons, and irradiation with a 980 nm laser pulse
(duration: 400 μs; irradiance: 75.25 W/cm2) improved
the stimulation efficiency, lowered the threshold, and
shortened the latency in comparison to the null
control without gold nanorods [205]. In this study,
antibody-conjugated gold nanorods were also injected
into the rat motor cortex, and irradiation with a 980-nm
laser pulse (duration: 1.5 ms; irradiance: 85.3 W/cm2)
induced whisker oscillations. Following the same
targeting strategy, streptavidin-coated gold nanorods
(resonant wavelength: 982 nm) were also targeted
to the biotinylated antibodies bound to the plasma
membrane of rat astrocytes, and irradiation with
a 980 nm laser pulse (duration: 950 μs; irradiance:
1,381 W/cm2) induced intracellular Ca2+ transients
[162]. Ligand-conjugated gold nanoparticles (resonant
wavelength: 523 nm) were bound to the ion channels
in the plasma membrane of rat dorsal root ganglion
neurons in vitro, and irradiation with 532 nm laser
pulses (duration: 1 ms; irradiance: 31 kW/cm2) evoked
a train of action potentials (Figs. 6(h) and 6(i)) [173]. In
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Nano Res. 2018, 11(10): 5065–5106
this work, the ligand-conjugated gold nanoparticles
were also injected into acute mouse hippocampal
slices ex vivo, and irradiation with 532 nm laser
pulses (duration: 10 ms; power: 140 mW) induced a
membrane depolarization indicated by a VSD.
To improve the interface stability and stimulation
efficiency, gold nanomaterials were immobilized
onto substrates. A monolayer of amine-terminated
PEGylated gold nanorods was coated onto a micro-
electrode array for rat hippocampal neuronal culturing,
and irradiation with 785 nm laser pulses (duration:
120 s; irradiance: 0.3–2.1 W/cm2) inhibited the neural
activities and blocked signal transmission between
neural clusters through axons [206]. The authors
claimed that the nature of the effects depended on the
radiant exposure, i.e., a higher radiant exposure excited
the neurons, whereas a lower radiant exposure inhibited
them. In another study, a gold nanoparticle (resonant
wavelength: 532 nm)-coated glass micropipette was
placed in the vicinity of a neuroblastoma SH-SY5Y
cell or a rat cardiomyocyte, and the cellular activity
could be stimulated (duration: 1–5 ms; power: 75–
120 mW) or inhibited (duration: 300 ms; power:
120 mW) upon irradiation with 532 nm laser pulses
[207]. The authors claimed that the excitatory or
inhibitory effect depended on the pulse duration, i.e., a
shorter pulse of a few milliseconds excited the neurons,
whereas a longer pulse of tens of milliseconds to
minutes inhibited the neurons. However, based on a
complete analysis of this topic (Table 1), neither
assumption could fully account for all the observations.
To discern the excitatory and inhibitory effects, the
consideration may not be solely on the primary
signal—the light, but should also be given to many
other factors, particularly the heat generated locally.
Heat is the secondary signal with which the neurons
directly interact. Both the amount and rate of heat
generation affect the neural responses, which are
determined by the light (duration and irradiance),
nanoparticle amount and placement, etc.
Moreover, gold nanomaterials also have a great
tendency to be rapidly internalized, making it difficult
to maintain a stable and effective fNI. The uptake of
gold nanomaterials can also cause inconsistency and
cytotoxicity [167, 201]. Furthermore, although NIR
light can penetrate deeper than visible light, their
Nano Res. 2018, 11(10): 5065–5106 5087

Table 1 Summary of gold nanomaterial-assisted thermotransduction

Reference Laser duration (ms) Laser irradiance (W/cm2) Irradiant exposure (mJ/cm2) Modulation effect
a
[201] 0.025–50 N/A N/A Excitatory
[202] 1 159–1,046 159–1,046 Excitatory
5 5
[203] 6 × 10 1.5 9 × 10 Inhibitory
6 9
[169] 7.2 × 10 800 5.76 × 10 Excitatory
–10 –4 –3 –14 –13
[204] 1.4 × 10 2.7×10 –1.02×10 3.78×10 –1.428×10 Excitatory
0.4 75.25 30.1 Excitatory
[205]
1.5 85.3 127.95 Excitatory
[162] 0.95 1,381 1,311.95 Excitatory
[173] 1 31,000 31,000 Excitatory
6 6 7
[206] 7.2 × 10 0.3–2.1 2.16×10 –1.512×10 Inhibitory
1–5 75–120 75–600 Excitatory
[207]
300 N/A N/A Inhibitory
a
N/A: no available information.

penetration depth is still limited to 4 mm in dermal
tissues [164]. Additionally, heat, as a localized secondary
signal, can be limited by its temporal responsivity
[167, 173, 201]. The heat generation must be carefully
controlled, as overheating can lead to protein
denaturation and cell damage [208].
Nevertheless, gold nanomaterials have a good
biocompatibility, and their surface plasmonic resonant
properties can be tailored based on the application
needs. Gold nanorods also have a higher heat
generation efficiency, thus requiring a nanoparticle
concentration almost 1,000 times less than the magnetic
nanoparticle-assisted thermotransduction discussed
below [169]. More importantly, they do not require
genetic engineering of the target cells. Thus, gold
nanomaterials have become one of the most developed
types of wireless nanotransducers.
Superparamagnetic nanomaterials—From magnetic field
to heat: Superparamagnetic nanoparticles generate
localized heat when exposed to an oscillating or
alternating magnetic field. Similar to the gold
nanomaterial-assisted thermotransduction, this localized
heating phenomenon via hysteresis triggered by
a magnetic field makes these nanoparticles good
candidates as wireless nanotransducers for cellular
modulation. This type of technique started with the
targeted placement of these nanoparticles.
Streptavidin-conjugated superparamagnetic nano-
particles were targeted to genetically engineered
protein markers in the plasma membrane of HEK293T
human epithelial cells and rat hippocampal neurons
expressing temperature-gated transient receptor
potential vanilloid 1 (TRPV1) ion channels. Application
of a radio-frequency (RF) magnetic field generated
highly localized heat, inducing Ca2+ influxes in
HEK293T cells and evoking action potentials in
hippocampal neurons within 45 s [11]. In this study,
the PEG-phospholipid-coated nanoparticles were also
injected into the amphids of C. elegans having TRPV1
ion channel-expressing sensory neurons, and appli-
cation of a magnetic field initiated the worm’s
thermal avoidance response within 5 s. Besides being
anchored onto the plasma membrane, antibody-
conjugated magnetic nanoparticles were also directly
targeted to the TRPV1 ion channels in the plasma
membrane of HEK293T cells and mouse embryonic
stem cells expressing a Ca2+-dependent promoter for
insulin secretion, and application of an RF magnetic
field resulted in cytosolic Ca2+ increase, enhanced
proinsulin release, and relevant gene expressions [209].
In this study, antibody-conjugated magnetic nano-
particles were also injected into xenografts of PC-12
pheochromocytoma cells expressing both TRPV1 ion
channels and Ca2+-dependent promoter for insulin
secretion in mouse, and application of a magnetic field
resulted in a decrease of blood glucose, an increase

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5088
of plasma insulin, and an increase of relevant gene
expressions. Additionally, genetically engineered cells
expressing ferritin fusion protein, TRPV1 ion channels,
and a Ca2+-dependent promoter for insulin secretion
produced magnetic ferritin nanoparticles intracellularly;
the application of a magnetic field also resulted in
increases of proinsulin release and relevant gene
expressions. In this publication, the Ca2+ influxes were
induced faster than in Ref. [11], and it was the first
one to use endogenous nanoparticles for cellular
modulation.
Stanley et al. extended the use of endogenous ferritin
nanoparticles for wireless cellular modulation in
another study [210]. In genetically engineered HEK293T
human epithelial cells and mouse mesenchymal
stem cells expressing ferritin proteins (cytoplasmic,
membrane-bound, or TRPV1-bound), TRPV1 ion
channels, and a Ca2+-dependent promoter for insulin
secretion, the intracellularly produced ferritin nano-
particles coupled with an RF magnetic field resulted
in increases in proinsulin release and relevant gene
expressions. In this publication, in vivo studies were
conducted by either implanting gelatin scaffolds con-
taining the genetically engineered mouse mesenchymal
stem cells in mouse or injecting adenoviruses to
transfect cells. The application of an RF magnetic field
resulted in a decrease of blood glucose, an increase of
plasma insulin, and an increase of relevant gene
expressions. The effectiveness was shown to last for at
least several weeks. Application of a static magnetic
field led to similar results in vitro and in vivo only for
TRPV1-bound ferritin proteins, indicating that both
heating and mechanical force could activate TRPV1
ion channels.
Untargeted placement of superparamagnetic nano-
particles was also implemented for cellular modulation
to suppress internalization and prolong the interface.
PEGylated poly(acrylic acid)-coated superparamagnetic
nanoparticles were distributed in the adjacent
surroundings of HEK-293FT cells and hippocampal
neurons, and application of an alternating magnetic
field induced Ca2+ influxes in HEK293-FT cells and
evoked trains of action potentials in hippocampal
neurons within 5 s (Figs. 6(j) and 6(k)) [163]. It is worth
mentioning that the cells were genetically engineered
to express higher levels of TRPV1 ion channels than
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Nano Res. 2018, 11(10): 5065–5106
naturally occurring. In this study, superparamagnetic
nanoparticles were also injected into the transfected
ventral tegmental area of the mouse brain, and
application of a magnetic field increased the c-fos
gene expression. These nanotransducers lasted more
than a month in vivo.
For magnetic nanoparticle-assisted thermotrans-
duction, heat was confirmed as the localized secondary
signal. However, mechanical force as another possible
secondary signal cannot be excluded [210]. Similar
to gold nanomaterial-based thermotransducers, the
temporal resolution of this type of techniques also
needs to be significantly improved. Interestingly, the
thermotransduction enabled by magnetic nanoparticles
generally requires genetic engineering to overexpress
TRPV1 ion channels and other binding proteins, while
gold nanomaterial-assisted thermotransduction usually
does not involve genetic engineering. Although some
of the differences can be attributed to the difference
in experimental designs, the exemption of genetic
engineering may have taken advantage of the higher
heating efficiency of gold nanomaterials. To generate
the same amount of heat, a much larger number of
magnetic nanoparticles is needed compared to gold
nanomaterials [169]. Nevertheless, superparamagnetic
nanoparticles have good biocompatibility and
biodegradability. And the primary signal, an RF
magnetic field, can penetrate more deeply than light
into tissues without causing tissue heating. Additionally,
magnetic nanoparticles can also convert a magnetic
field into mechanical forces, a secondary signal that
can also interact with cells, which will be discussed
in the mechanotransducers below.
3.2.3 Nano optotransducers
Upconverting luminescent nanomaterials—From NIR light
to visible light: Optogenetics (to be discussed in detail
below), artificially inserting light-sensitive ion channels
into the plasma membrane and modulating neural
activity with light, has become a powerful tool for
neural stimulation with its exceptional spatiotemporal
resolution and specificity [211]. The majority of light-
sensitive ion channels, such as the channelrhodopsins
(ChRs), respond to blue to green light with some
newly developed ChRs responding to orange to red
light, such as the red-activatable channelrhodopsin
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(ReaChR) [212], Chrimson [213], and Jaws [214]
through laborious engineering [174]. However, visible
light only penetrates soft tissues superficially. Thus,
optogenetics often demands the implantation of a light
source close to the target neuron population. On the
other hand, NIR light (650–1,450 nm) can penetrate
deeper into the soft tissues when the wavelength
lies within the biological window [215]. Additionally,
upconverting luminescent nanomaterials can be
excited by NIR light and emit ultraviolet/visible light
through an upconversion process. These inspire the
development of a new wireless neural modulation
technique by combining upconverting luminescent
nanomaterials with optogenetics. The upconverting
luminescent nanomaterial-assisted optogenetics, in
comparison to the original optogenetics, uses an
inexpensive light source and could possibly avoid
surgical implantation of the light source. The emission
wavelength of such optotransducers can be relatively
easily tailored instead of a laborious screening and
engineering of the ion channels [174].
Dispersed untargeted and targeted placement of
the upconverting luminescent nanomaterials could
be coupled with optogenetics to modulate cells.
Synergistic treatment by NaYF4:Sc/Yb/Er nanoparticles
(excitation: 980 nm; maxima emission: 543 nm) and a
976 nm NIR laser pulse induced photocurrents in
C1V1 (maxima absorbance: 539 nm) expressing mouse
neuroblastoma and rat neuron hybrid ND7/23 cells
[216]. However, the cellular distribution of the nano-
particles was not assessed in this study, and a single
light pulse induced a series of action potentials due
to the slow off-kinetics of C1V1 channels. Quasi-
continuous-wave NIR light (980 nm) was used to
excite extracellularly and intracellularly distributed
silica-coated NaYF4:Yb/Tm nanoparticles (excitation:
980 nm; maxima emission: 450 nm), and the emission
induced Ca2+ influxes in ChR2-expressing HEK293T
cells and elicited reversal responses in C. elegans
having ChR2-expressing mechanosensory neurons
[217]. Core–shell structured NaYF4:Yb/Tm@NaYF4
nanoparticles (excitation: 980 nm; maxima emission:
470 nm) were made to improve the upconversion
efficiency, and the nanoparticles were conjugated with
streptavidin and bound to the Ca2+ channels engineered
with a streptavidin-binding site [218]. Illumination
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with 980 nm continuous-wave NIR light induced Ca2+
influxes in HeLa cells, activated the Ca2+/NFAT pathway
in T lymphocytes, and promoted melanoma tumor
destruction by promoting dendritic cell maturation
and the sensitivity of T lymphocytes to antigen.
Immobilizing core–shell structured upconverting
luminescent nanomaterials onto substrates improved
the modulation efficiency and resolution. The ChR2
(maxima absorbance: 470–475 nm)-expressing mouse
hippocampal neurons were cultured on a poly(lactic-
co-glycolic acid) scaffold containing NaYF4:Yb/
Tm@NaYF4 core–shell structured nanoparticles (excitation:
980 nm; maxima emission: 475 nm), and irradiation
with a sequence of 980 nm NIR laser pulses induced
a train of action potentials (Figs. 6(l) and 6(m)) [174].
More importantly, in this study, a single pulse could
rapidly evoke a single action potential. Similarly,
ChR-expressing rat hippocampal neurons were cultured
on a cover glass over a poly(methyl methacrylate) film
containing IR-806-sensitized NaYF4:Yb/Er@NaYF4/Yb
core–shell structured nanoparticles (excitation: 800 nm;
maxima emission: ~ 540 nm), and irradiation with a
sequence of 800 nm continuous-wave NIR light pulses
evoked light intensity-dependent depolarization and
a train of action potentials, the temporal pattern
of which matched that of the light pulses [219]. The
improved efficiency and resolution could be due to
the enhanced upconversion efficiency of core–shell
structured nanoparticles and increased interface
stability.
It is worth noting that although the above excitation
wavelength (975–980 nm) is within the NIR range, its
absorption by water is still significant [220]. It is also
worth mentioning that NIR light can only penetrate
through dermal tissues up to 4 mm [164] and has
difficulties to penetrate through bones and the skull.
Additionally, for these upconverting luminescent
nanomaterial-assisted optogenetic techniques, both
the optotransducers and ion channels need to be well
designed. On the one hand, a high upconversion
efficiency is greatly demanded to use a low-power
NIR source to reach the light threshold for ion channel
activation while avoiding thermal hazards to the cells
[217]. The emission of ultraviolet light and its toxicity
also need to be assessed [217]. In addition, in vivo
biocompatibility of the upconverting luminescent
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nanomaterials needs to be further improved and
optimized for clinical stimulation [217]. On the other
hand, the ion channels should have rapid dynamics
and stable expression.
3.2.4 Nano mechanotransducers
Superparamagnetic nanomaterials—From magnetic field to
mechanical force: Comparing to other recently developed
nanotransducer-assisted cellular modulation techniques,
mechanical manipulation of cellular responses via
magnetic nanoparticles has been studied much earlier.
Magnetic nanoparticles were bound to the cell surface,
and application of a magnetic field generated either
piconewton forces to stretch the corresponding ion
channels and cytoskeleton via particle twisting or
pulling [221, 222] or femtonewton forces to induce
receptor clustering via particle aggregation. Inter-
nalized magnetic nanoparticles were also used to
manipulate protein distributions inside the cells [223,
224]. Interestingly, while the majority of magnetic
nanoparticle-assisted mechanical stimulations were
conducted on non-excitable cells, the concepts can be
transferred to neural modulation straightforwardly.
Magnetic nanoparticles can be bound to the cell
surface, and through twisting or pulling of these
nanoparticles, a magnetic field can stretch the ion
channels and the cytoskeleton. For example, endothelial
cells were bound to ferromagnetic microparticles via
a molecular linker, and shear stress was applied
to the integrin receptors via the molecular linker by
magnetic twisting, leading to cytoskeleton stiffening
[225–227]. Collagen-coated magnetic microbeads were
bound to human gingival fibroblasts, and the
application of magnetic force on the plasma membrane
promoted Ca2+ influxes via mechanosensitive channels
[228]. Antibody- or complex-conjugated magnetic
nanoparticles were targeted to a genetically added
loop region of the mechanosensitive TREK-1 ion
channels in the plasma membrane of COS-7 kidney
cells, and the application of a magnetic field induced
an increase of the outward current [229]. Similarly,
magnetic microparticles were bound to the integrin
receptors and chimeric integrin receptors of bovine
capillary endothelial cells, and the magnetic pulling on
these integrin receptors activated the mechanosensitive
transient receptor potential vanilloid 4 (TRPV4) ion
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Nano Res. 2018, 11(10): 5065–5106
channels and induced almost instantaneous Ca2+
fluxes [230].
Magnetic nanoparticles bound to the cell surface
receptors were magnetized and aggregated upon
exposure to a magnetic field. The receptor clustering
due to particle aggregation activated receptor
oligomerization-dependent cell signaling pathways.
Ligand-conjugated superparamagnetic nanoparticles
were targeted to the transmembrane FcεRI receptors
of RBL-2H3 rat mast cells, and application of a
magnetic field activated the Ca2+ signaling pathway,
increasing the cytosolic Ca2+ concentration [231].
Similarly, antibody-conjugated magnetic nanoparticles
were targeted to death receptors at the surface of
DLD-1 human colon cancer cells and injected into
zebrafish embryos, and magnetic field-induced receptor
clustering activated an apoptosis signaling pathway
[232]. Antibody-conjugated or streptavidin-functionalized
superparamagnetic nanoparticles were targeted to the
epidermal growth factor receptors (EGFRs) of A431
human epidermoid carcinoma cells and genetically-
engineered chimeric EGFRs of HeLa cells, respectively,
and magnetic field-induced receptor clustering activated
the EGFR signaling pathway [233].
Internalized magnetic nanoparticles could be con-
trolled by an external magnetic field to manipulate
the intracellular protein and organelle distribution.
The superparamagnetic nanoparticles internalized
into cortical neurons coupled with a magnetic field
exerted magnetic forces to modulate protein tau
distribution and cell polarization [223]. The super-
paramagnetic nanoparticles were microinjected into
HeLa cells, and a magnetic field was applied to
control the distribution of HaloTag proteins [224].
Similarly, anti-TrKB agonist antibody-functionalized
superparamagnetic nanoparticles were rapidly
internalized into TrKB signaling endosomes of primary
neurons and promoted neurite outgrowth [234]. A
focal magnetic force halted the neurite outgrowth via
modulation of the location of these TrKB signaling
endosomes.
Magnetic nanoparticles were recently employed to
modulate neurons. Starch-coated ferromagnetic nano-
particles were mainly bound to the plasma membrane
of cortical neurons, and chitosan-coated ferromagnetic
nanoparticles were mostly internalized (Figs. 6(n) and
Nano Res. 2018, 11(10): 5065–5106
6(o)) [175]. Application of a high gradient magnetic
field for both placements of nanoparticles induced
Ca2+ fluxes in the neural network via activation of
N-type mechanosensitive ion channels. This mechanism
was further validated in a follow-up study [208]. In
this work, starch-coated magnetic nanoparticles were
bound to the plasma membrane of the neurons in
which the N-type mechanosensitive ion channels were
over-expressed, and chronic treatment with a high
gradient magnetic field lowered the expression of these
N-type mechanosensitive ion channels by chronically
activating these channels. Further electrophysiological
validations are needed for these wireless mechano-
transducers to be used for neural modulation, and
their responsivity and temporal resolution need to be
improved.
Considering the widespread existence of mechano-
sensitive ion channels and cell signaling pathways
[222], these mechanical cellular modulations on non-
excitable cells can be adapted and tailored for neural
modulation. Magnetic microparticles are limited for
in vivo cellular modulation due to their relatively
large size [11]. Magnetic nanoparticles are usually
bio-friendly and can be rapidly cleared in the human
body, thus, these nanoparticles are used clinically in
magnetic resonance imaging. Magnetic nanoparticles
bound to receptors could also be rapidly internalized
[232, 233]. Their biodegradability and tendency for
internalization make it difficult to maintain a stable
fNI lasting for a long time [235]. Similar to the wireless
thermotransducers based on magnetic nanoparticles,
it is necessary to discern if the secondary signal is
heat or mechanical force so that the design can be
optimized.
3.3 Bionanotransducer-enabled functional neural
stimulation
3.3.1 Optogenetics
Nanobiotechnology utilizing light-sensitive proteins
can function as a switch to control neural activities like
intracellular signal transduction pathways, action
potential propagation, and even gene transcription
or protein–protein interactions. Optogenetics is the
combination of genetic and optical methods to allow
for targeted, fast control of precisely defined events
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in biological systems. Once stimulated by extrinsic
signals or stimuli, spatiotemporal changes within
neurons will lead to profound effects on behavior.
Optogenetics offers many advantages, such as cell-type
specific targeting, high spatiotemporal resolution
(subcellular and millisecond), and concurrent recording
with metal electrodes without the introduction of
aberrant artifacts. For a comprehensive overview, the
readers are referred to many excellent reviews,
including Refs. [1, 236–240].
In 2005, Boyden et al. reported that mammalian
neurons could be precisely modulated by light with
the introduction of a single microbial ChR gene [211].
Opsins are seven-transmembrane domain proteins
(structurally akin to G-protein coupled receptors)
possessing the chromophore retinal, which undergoes
a conformational change in response to photon
absorption and drives ion transport across the cell’s
membrane. Commonly, bacteriorhodopsin (BR), ChR,
and halorhodopsin (HR) are the most widely used
opsin families that can modulate neural activity in
response to different wavelengths of light (Fig. 7(a)).
Two different effects exhibited by these proteins
are excitation and inhibition of neural activity. BRs
are green-light-activated ion pumps that transport
hydrogen ions out the cytoplasm causing hyper-
polarization and preventing an action potential. ChRs
are blue-light-activated ion channels that conduct
various cations (Na+, H+, K+, and Ca2+) across the
cellular membrane and depolarize neurons, eliciting
an action potential. HRs are yellow-light-activated ion
pumps that transport Cl– ions intracellularly and
inhibit neuron firing [241].
Introduction of opsin genes is accomplished
via transgenic (Cre-LoxP) or viral delivery (adeno-
associated virus (AAV), Lentivirus) methods. Cre
transgenic mice can be crossed with another
mouse strain expressing the opsin of interest in a
Cre-dependent manner, an example of which was
accomplished with Ai39 mice with Cre-dependent
Halo3.0/eNpHR3.0 expression [242]. Viral gene delivery
using an AAV is another method where a plasmid
containing an opsin gene under the control of a
promoter (CMV) would be injected into a brain region
or transfected into cells in vitro and visualized with a
fluorescent reporter gene [243]. Owing to the specificity
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Figure 7 Optogenetic molecular applications. (a) Main opsin families that conduct ions upon light stimulation: BR (H+), HR (Cl–),
and ChR (Na+, K+, Ca2+, H+) activated via green, yellow, and blue lights, respectively [244]. (b) Schematic representation of ReaChR
consisting of the N-terminal domain from ChEF, the transmembrane domains from VChR1 except that domain F is from VChR2 and an
L171I mutation in domain C [212]. (c) and (d) Protein control via light stimulation [245]. (c) Transmembrane protein control over
intracellular signaling cascade upon light stimulation. (d) Caged ligands released upon light stimulation causing subsequent protein
conformational change and leading to activation. (e) Spectra of emission and activation wavelengths of luciferase and eNpHR3.0 with
peaks at ~ 550 and ~ 600 nm, respectively [246]. (f) and (g) Photoactivatable CRISPR-Cas9 system [247, 248]. (f) Cleaved Cas9 protein
whose photoinducible domains (pMag/nMag) dimerize upon blue-light stimulation, allowing for Cas9 fragments to reassociate and cut
DNA at a specific region. (g) Dead-Cas9 tagged with CIB1, targeted to a promoter region, serves as a transcription modulator upon
blue-light stimulation causing CRY2 tagged with a gene activator to bind to CIB1 and promote transcription. (a) adapted with
permission from Ref. [244], © Elsevier 2011; (b) adapted with permission from Ref. [212], © Springer Nature 2013; (c) and (d) adapted
with permission from Ref. [245], © Springer Nature 2014; (e) adapted with permission from Ref. [246], © Frontiers Media S. A. 2014;
(f) and (g) adapted with permission from Refs. [247, 248], © Springer Nature 2015 and Elsevier 2015, respectively.

of optogenetics, selection of similar promoter sequences
to target a cell subtype will allow a cell-specific
integration since cells that are expressing the same
promoters will have the required transcription factors
to initiate opsin gene synthesis. Transgenic mice
offer a robust and precise optogenetic activation or
suppression in vivo. While viral delivery methods have
a few limitations, including limited DNA packaging
space and transfection efficiency, it still remains a
viable option to genetically modify specific cells.
A distinct limitation of optical stimulation is its
tissue penetration with blue light having the shallowest
penetration depth (1–2 mm) and near-infrared light
having the deepest (4 mm). Aside from inserting an
optic fiber deep into the brain or using upconverting
luminescent optotransducers as discussed above,
several opsins have been discovered and engineered
to have shifted light sensitivities and faster ion
transport to compensate for this deficiency. An
inhibitory HR from Natronomonas pharaonis (Halo3.0/
eNpHR3.0) was developed, which possessed a
membrane localization tag that could hyperpolarize

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Nano Res. 2018, 11(10): 5065–5106
neurons upon yellow-light stimulation with increased
excitability and faster photokinetics [249–252]. VChR1,
a ChR from Volvox carteri, can be activated at 589 nm,
which is slightly red-shifted when compared to ChR2.
However, it expressed poorly in mammalian cells and
had minimal trafficking to the cellular membrane
[253]. VChR1 was further modified with components
of other ChR proteins and an L171I mutation
allowing for its optimal excitation to be red-shifted
further (630 nm); termed ReaChR, this opsin had
vastly improved membrane trafficking and kinetic
properties (Fig. 7(b)) [212]. Engineered for prolonged
periods of neural depolarization, bistable step-function
opsins (SFOs) allowed for orders of magnitude greater
light sensitivity and had lasting effects without the
need for continuous light stimulation [254]. Persistent
work on opsins for H+/Cl– conducting-based inhibition
is ongoing, for they could mediate the optical silencing
of significant neural activities over relevant timescales
[249, 255–258]. Opsins with faster kinetics that could
deliver more efficient photocurrents while also being
expressed at higher levels within the cell are of great
paramount [259, 260].
Aside from using light to stimulate an opsin
within a cell to drive a response, another way is to
use chemically modified or genetically encoded caged
ligands to control neural activities (Figs. 7(c) and 7(d))
[261, 262]. Light-sensitive proteins, engineered with
the attachment of photo-switchable tethered ligands,
could ultimately regulate the interaction between
ligand and receptor and allow for precise control over
intracellular signaling events. The correct wavelength of
light would effectively activate the ligand, transitioning
from a caged to an uncaged state, allowing for a
protein–protein interaction to occur. However, several
considerations have limited the application of caged
proteins, one being that cleaving the cage tends to be
irreversible and may produce toxic byproducts that
can alter cell signaling. Reversible caged ligands,
using a short connecting molecule called azobenzene,
controls the activity of ligands with the ability to
go through many photoactivation cycles without a
reduction in activity [245, 261]. An alternative approach,
typically used concurrently with other optogenetic
methods, uses constitutively expressed luciferin (a
light producing compound) within a specific cell type
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(Fig. 7(e)). Endogenous light production from those
cells would then stimulate nearby cells expressing an
opsin and further control cell signaling and intracellular
events [246].
Recent advances in gene editing and targeting
methods have supplemented the field of optogenetics
with the advent of photoactivatable CRISPR-Cas9.
One method split Cas9 into two domains, each tagged
with a photoinducible domain that, when exposed
to light, dimerized and brought the Cas9 domains
together allowing for it to target and edit the genome
based upon the guide RNA (gRNA) (Fig. 7(f)) [247].
The same group also demonstrated earlier the ability
to use dead-Cas9 (dCas9) as a photoactivatable
transcription system. dCas9 is a useful tool for gene
regulation since it can still target DNA with gRNA,
but lacks nuclease activity, and can be coupled to gene
activators and repressors. Tagged with CIB1, dCas9
induced gene transcription once CRY2, tagged with an
activator, heterodimerized with CIB1 upon blue-light
stimulation (Fig. 7(g)) [248]. Utilizing one or adopting
multiple methods concerning optogenetics can allow
for a high degree of specificity and spatiotemporal
control over neural stimulation.
3.3.2 Magneto(thermo)genetics
Due to its noninvasiveness and deep tissue penetration,
magnetic neural stimulation is of great interest. To
stimulate neurons with cell-type specificity, magnetic
nanoparticles combined with genetic engineering of
certain ion channels have been developed for nano-
transducer-assisted magnetic stimulation [263, 264],
as discussed earlier. The commonly used channels
include mechanosensitive channels (TREK) [175, 229]
and heat-sensitive capsaicin receptor channels (TRP)
[11, 163]. TREK-1 is a type of tandem-pore K +
channel (2PK+) and produces an outwardly rectifying
K+ leak current to regulate the resting membrane
potential and cellular excitability [265]. Hughes et al.
inserted a 6-histidine-repeat into the TREK-1 channel
and attached an anti-His antibody-grafted magnetic
nanoparticle to it to directly activate the mechano-
sensitive channel by exerting a mechanical force
through a permanent magnetic field (Fig. 8(a)) [229].
Wheeler et al. developed the Magneto, a new fusion
of the nonselective cation channel TRPV4 and the
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5094 Nano Res. 2018, 11(10): 5065–5106

paramagnetic protein ferritin (an iron-containing

storage protein), and applied it to mediate Ca2+ influx
upon magnetic stimulation to modulate the behavior
of freely moving zebrafish and mice [266]. TRP
channels form the most important temperature-
sensitive ion channel family, among which TRPM8
and TRPA1 are sensitive to cold temperatures, and
TRPV1, TRPV3, and TRPV4 respond to an elevation
in local temperature [267]. For example, TPRV1 opens
when the local temperature increases from normal
to 42 °C and causes an influx of Ca2+ to depolarize
the cell. These ion channels can be heated by an
RF magnetic field through nano thermotransducers
(e.g., superparamagnetic nanoparticles) or bionano
thermotransducers (e.g., ferritin) either attaching
to a ligand inserted into the membrane (Fig. 8(b))
or directly attaching to the ion channel through an
engineered ligand recognition (Fig. 8(c)) [11, 209, 210].
The latter approach of specific ion-channel binding
ensures a higher efficiency in signal transduction
while minimizing heat-induced side-effects. In their
study, Stanley et al. used genetically encoded ferritin
as the thermotransducers to control insulin expression
and blood glucose in mice [210]. The ferritin was
targeted to TPRV1 for gating under low RF waves, Figure 8 Mechanisms of magneto(thermo)genetics and sono-
which resulted in elevated intracellular Ca2+ concen- genetics. (a) Magnetomechanical stimulation with mechanotransducers
trations and induced the expression of proinsulin. to modulate the mechanosensitive channel (TREK1). His-tagged
magnetic nanoparticles are targeted to TREK1 with a 6-histidine
They also expanded this ferritin-TPRV1 magneto-
repeat inserted region [229]. (b) Magnetothermal stimulation with
thermogenetics system to target hypothalamic different nanoparticle placements. Top, streptavidin-conjugated
glucose-sensing neurons to control proinsulin release nanoparticles bound to the plasma membrane around TRPV1
in mice [268]. channels through a genetically engineered linker [11]. Bottom,
Additionally, Qin et al. reported a single magnetic through His tag binding, superparamagnetic nanoparticles (grey
circle) or ferritin proteins (purple circle) are attached to the
protein MagR for neural stimulation [268]. Long et al.
thermal-sensitive channel (TPR family) [209, 210]. (c) Potential
invented a noninvasive magnetogenetics that combined mechanism for sonogenetics, involving the mechanosensitive TRP-4
the genetic targeting of a magnetoreceptor with channel [271]. (a) adapted with permission from Ref. [229], © The
remote magnetic stimulation through the use of a Royal Society 2008; (b) top adapted with permission from
Ref. [11], © Springer Nature 2010; (b) bottom adapted with
genetically encoded magnetic protein [270], without
permission from Refs. [209, 210], © The American Association
the requirement for injection of exogenous super- for the Advancement of Science 2012 and Springer Nature 2015,
paramagnetic nanoparticles. Although these emerging respectively; (c) adapted with permission from Ref. [271],
techniques have aroused a great deal of enthusiasm in © Springer Nature 2015.
the field, there are still many technical obstacles, and
3.3.3 Sonogenetics
further efforts are required to decipher the specific
stimulation mechanism and develop new magnetic- Ultrasound can also be used to noninvasively stimulate
sensitive receptors. the nervous system. To enhance the power efficiency

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Nano Res. 2018, 11(10): 5065–5106
and target specificity, genetically encoded acoustic-
sensitive ion channels can be used as the bionano-
transducers. An early investigation by Ibsen et al. used
the misexpressed ultrasonic-sensitive channel TRP-4,
a pore-forming subunit of a mechanotransduction
channel, and ultrasound-amplified microbubbles to
manipulate the functions of interneurons and sensory
neurons in C. elegans for behavior control (Fig. 8(c))
[271]. They termed their technology “sonogenetics”.
Kubanek et al. measured the effects of ultrasound
on mechanosensitive ion channels embedded within
cellular membranes in the Xenopus oocyte system,
including potassium mechanosensitive channels
(TREK-1, TREK-2, TRAAK) and a sodium-sensitive
channel (Nav1.5) [272]. Ultrasound could modulate
the currents flowing through the ion channels by
up to 23%. Compared to other bionanotransducer-
enabled neural stimulation techniques, sonogenetics
has a much longer way to go. Much more work is still
needed to elucidate the stimulation mechanism, search
for more effective acoustic-sensitive ion channels that
can be transfected into other cells, and expend the
application to mammalian neuron stimulation.
4 Future perspectives
The three thematic nano fNIs covered in this review,
i.e., nanoelectrode-based, nanotransducer-assisted,
and bionanotransducer-enabled, represent the most
exciting new advances in the field of neural interface
engineering over the past 15 years, which address the
long-term physical integration issue of the neural
interface. These fascinating advances embody the
insensibility and indistinguishability principles that
we proposed earlier [2] through the pursuits of
miniaturization and biomimicry and the employment
of nature’s materials and designs.
Tackling the inefficiency problem of limited com-
munication bandwidth through the abiotic–biotic
interface has so far solely relied on increasing the
electrode’s spatial resolution, density, and number.
However, it has been largely ignored that, in the current
design of neuroelectrodes, the neuron’s “vocabulary”
(including action potentials, excitatory and inhibitory
synaptic potentials [273]) in forming the brain’s
language is “filtered” into an overly simplistic binary
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mode (“1” for action potential and “0” for no action
potential) by each electrode, with a great loss of the
information contents that can otherwise be conveyed
through the fNI. While some electrode types exist to
record the subthreshold neuronal activities [273],
they are, at present, limited to cell culture interfacing
only. The need to develop implantable versions of
such “analog” neural interfaces is urgent.
Furthermore, the plastic nature of neuron–neuron
communication has never been purposely emulated
at any fNI. We argue that the goal of neuroelectrode
design in the functional aspect should not be set to
achieve a static signal-transmission interface but rather
to achieve a dynamic interface that remodels based
on a memory of the signals transmitted by it. These
gaps in interface efficiency and plasticity, thus, call
for an urgent and serious rethinking of the design of
fNIs to capture the full vocabulary of a neuron and
implement plasticity into the interface. With such
new fNIs, it would be more effective and efficient to
wire a neuromorphic chip running biologically realistic
neuronal models to the brain to repair and even
augment the brain [274], as exemplified in the recent
attempts in building cognitive prostheses [275, 276].
Acknowledgements
L. G. is supported by The Defense Advanced Research
Projects Agency (No. D17AP00031) of the USA. The
views, opinions, and/or findings contained in this
article are those of the author and should not be
interpreted as representing the official views or policies,
either expressed or implied, of the Defense Advanced
Research Projects Agency or the Department of
Defense.
C. X. is supported by National Institute of
Neurological Disorders and Stroke through Grant #
R01NS102917, Welch Foundation through Grant #
F-1941-20170325, and Department of Defense through
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