Intaglio Contact Printing of Versatile Carbon Nanotube Composites and Its Applications for Miniaturizing High-Performance Devices.

Composites based on carbon nanotubes (CNTs) are promising patternable materials that can be engineered to incorporate the outstanding properties of CNTs into various applications via printing technologies. However, conventional printing methods for CNTs require further improvement to overcome the major drawbacks that limit the patterning resolution and target substrate. Herein, an intaglio contact printing method based on a CNT/paraffin composite is presented for realizing highly precise CNT network patterns without restrictions on the substrate. In this method, the CNT/paraffin composite can be patterned with a high resolution (<10 µm) and neatly transferred onto various substrates with a wide range of surface energies, including human skin. The patterned composite exhibits high durability against structural deformations, and structural damage caused by fatigue accumulation can be cured in a few seconds. In addition, miniaturized sensing and energy-harvesting applications are demonstrated with high performances. The present method facilitates the rapid fabrication of highly precise interdigitated electrodes via one-step printing, enabling high-performance operation and miniaturization of the devices. It is anticipated that these results will not only spur the further development of various applications of CNTs but also contribute to advances in soft lithography methods applicable to many fields of science and engineering.

An efficient cell type specific conjugating method for incorporating various nanostructures to genetically encoded AviTag expressed optogenetic opsins.

Here, we report genetically encoded AviTag conjugating system for Channelrhodopsin-2(ChR2) in order to attach various nanostructures to the membrane protein in a cell type specific manner. First, AviTag peptide sequence is cloned to N-terminal site of ChR2 construct and expressed at the membrane of primary-cultured hippocampal neurons via lentiviral transduction. Second, with the help of BirA enzyme and ATP, biotin coated quantum dots (Qdots) and streptavidin (SAv) coated Qdots are successfully bound to AviTag sites at the membrane where ChR2 is located and confirmed by fluorescence imaging. Moreover, we synthesize biotinylated Traptavidin-DNA conjugate probes containing a desthio-biotin that has weaker affinity than a regular biotin, and successfully exchange them with pre-conjugated Biotin-AviTag-ChR2 site at the membrane of neuronal cells which can potentially solve the crosslinking issue of Avidin linked probes. Therefore, we expect the AviTag-ChR2 fusion platform to become a great tool for incorporating various nanostructures at the specific sites of a cellular membrane in order to overcome the limits of optogenetic opsins.

Investigation of stereotactic surgery for avian brain stimulation by a fully implanted wireless system.

OBJECTIVE: The authors' goal was to study avian motor brain mapping via wireless stimulation to induce certain behaviors. In this paper, the authors propose an electrode design that is suitable for avian brain stimulation as well as a stereotactic implant procedure for the proposed electrode. METHODS: An appropriate breed for avian brain study was chosen. A fully implantable remote-controlled electrical stimulation system was inserted to minimize discomfort. A suitable electrode design and stereotactic surgery method based on the electrode design were investigated. RESULTS: Using a wireless stimulation system, flapping and rotation behaviors were induced by stimulating the ventral part of the nucleus intercollicularis and formatio reticularis medialis mesencephali both on the ground and during flight. CONCLUSIONS: The authors were able to implant the entire brain stimulation system inside the avian body without any surgical complications. Postoperative observations suggested that the bird did not find the implant uncomfortable.

Transparent intracortical microprobe array for simultaneous spatiotemporal optical stimulation and multichannel electrical recording.

Optogenetics, the selective excitation or inhibition of neural circuits by light, has become a transformative approach for dissecting functional brain microcircuits, particularly in in vivo rodent models, owing to the expanding libraries of opsins and promoters. Yet there is a lack of versatile devices that can deliver spatiotemporally patterned light while performing simultaneous sensing to map the dynamics of perturbed neural populations at the network level. We have created optoelectronic actuator and sensor microarrays that can be used as monolithic intracortical implants, fabricated from an optically transparent, electrically highly conducting semiconductor ZnO crystal. The devices can perform simultaneous light delivery and electrical readout in precise spatial registry across the microprobe array. We applied the device technology in transgenic mice to study light-perturbed cortical microcircuit dynamics and their effects on behavior. The functionality of this device can be further expanded to optical imaging and patterned electrical microstimulation.

Nucleolin-Targeting AS1411 Aptamer-Conjugated Nanospheres for Targeted Treatment of Glioblastoma.

Post-operative chemotherapy is still required for the treatment of glioblastoma (GBM), for which nanocarrier-based drug delivery has been identified as one of the most effective methods. However, the blood-brain barrier (BBB) and non-specific delivery to non-tumor tissues can significantly limit drug accumulation in tumor tissues and cause damage to nearby normal tissues. This study describes a targeted cancer therapy approach that uses AS1411 aptamer-conjugated nanospheres (100-300 nm in size) loaded with doxorubicin (Dox) to selectively identify tumor cells overexpressing nucleolin (NCL) proteins. The study demonstrates that the active target model, which employs aptamer-mediated drug delivery, is more effective than non-specific enhanced permeability and maintenance (EPR)-mediated delivery and passive drug delivery in improving drug penetration and maintenance in tumor cells. Additionally, the study reveals the potential for anti-cancer effects through 3D spheroidal and in vivo GBM xenograft models. The DNA-protein hybrid nanospheres utilized in this study offer numerous benefits, such as efficient synthesis, structural stability, high drug loading, dye labeling, biocompatibility, and biodegradability. When combined with nanospheres, the 1411 aptamer has been shown to be an effective drug delivery carrier allowing for the precise targeting of tumors. This combination has the potential to produce anti-tumor effects in the active targeted therapy of GBM.

A solid state nanopore device for investigating the magnetic properties of magnetic nanoparticles.

In this study, we explored magnetic nanoparticles translocating through a nanopore in the presence of an inhomogeneous magnetic field. By detecting the ionic current blockade signals with a silicon nitride nanopore, we found that the translocation velocity that is driven by magnetic and hydrodynamic forces on a single magnetic nanoparticle can be accurately determined and is linearly proportional to the magnetization of the magnetic nanoparticle. Thus, we obtained the magneto-susceptibility of an individual nanoparticle and the average susceptibility over one hundred particles within a few minutes.

An implantable neural sensing microsystem with fiber-optic data transmission and power delivery.

We have developed a prototype cortical neural sensing microsystem for brain implantable neuroengineering applications. Its key feature is that both the transmission of broadband, multichannel neural data and power required for the embedded microelectronics are provided by optical fiber access. The fiber-optic system is aimed at enabling neural recording from rodents and primates by converting cortical signals to a digital stream of infrared light pulses. In the full microsystem whose performance is summarized in this paper, an analog-to-digital converter and a low power digital controller IC have been integrated with a low threshold, semiconductor laser to extract the digitized neural signals optically from the implantable unit. The microsystem also acquires electrical power and synchronization clocks via optical fibers from an external laser by using a highly efficient photovoltaic cell on board. The implantable unit employs a flexible polymer substrate to integrate analog and digital microelectronics and on-chip optoelectronic components, while adapting to the anatomical and physiological constraints of the environment. A low power analog CMOS chip, which includes preamplifier and multiplexing circuitry, is directly flip-chip bonded to the microelectrode array to form the cortical neurosensor device.

Reduction of graphene damages during the fabrication of InGaN/GaN light emitting diodes with graphene electrodes.

Although graphene looks attractive to replace indium tin oxide (ITO) in optoelectronic devices, the luminous efficiency of light emitting diodes (LEDs) with graphene transparent conducting electrodes has been limited by degradation in graphene taking place during device fabrication. In this study, it was found that the quality of graphene after the device fabrication was a critical factor affecting the performance of GaN-based LEDs. In this paper, the qualities of graphene after two different device fabrication processes were evaluated by Raman spectroscopy and atomic force microscopy. It was found that graphene was severely damaged and split into submicrometer-scale islands bounded by less conducting boundaries when graphene was transferred onto LED structures prior to the GaN etching process for p-contact formation. On the other hand, when graphene was transferred after the GaN etch and p-contact metallization, graphene remained intact and the resulting InGaN/GaN LEDs showed electrical and optical properties that were very close to those of LEDs with 200 nm thick ITO films. The forward-voltages and light output powers of LEDs were 3.03 V and 9.36 mW at an injection current of 20 mA, respectively.

A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane.

Efficient plasma-membrane expression is critical for genetically encoded voltage indicators (GEVIs). To improve the plasma-membrane expression, we introduced multiple combinations of plasma-membrane trafficking motifs at different positions to members of the Bongwoori family of GEVIs. An improvement from 20% to 27% in the ΔF/F/100 mV depolarization of the plasma membrane was observed when a Golgi transport motif was inserted near the N-terminus in conjunction with an endoplasmic reticulum release motif near the C-terminus of the protein. Unfortunately, this variant was also slower. The weighted tau on of the variant (25 ms) was more than double the original construct (11 ms). The weighted tau off was >20 ms compared with 10 ms for the original GEVI. The voltage range of the GEVI was also shifted to more negative potentials. Insertion of spacer amino acids between the fluorescent-protein domain and the endoplasmic reticulum release motif at the C-terminus rescued the speed of both the tau on and tau off while restoring the voltage range and maintaining the improved voltage-dependent optical signal. These results suggest that while trafficking motifs do improve plasma-membrane expression, they may also mediate persistent associations that affect the functioning of the protein.

Eco-Friendly Keratin-Based Additives in the Polymer Matrix to Enhance the Output of Triboelectric Nanogenerators.

A triboelectric nanogenerator (TENG) is an energy generator that converts mechanical energy into electrical energy using triboelectricity at a nanoscale. Given their potential application as power sources in electronic devices, various attempts have been made to improve their output performance. Here, we present an eco-friendly, low-cost, and facile fabrication method to enhance TENG characteristics with keratin protein additives. Keratin sources, human and cat hair, are processed into powder and added to the friction layer, which increases their positive charge affinity, thereby boosting the output performance of the TENG. The output performances of the keratin-added TENG (K-TENG) are measured in the vertical contact-separation mode, with both additives having the highest output values at 5 wt % load. The K-TENG generates more output voltage and current values than the pristine TENG by 90 and 208%, respectively. Hence, we conclude that this method would potentially promote TENG as a strong candidate for a competitive "green" energy harvesting device in future electronics applications.

Self-Assembled DNA-Protein Hybrid Nanospheres: Biocompatible Nano-Drug-Carriers for Targeted Cancer Therapy.

We developed hybrid nanospheres comprised of two of the most important biomolecules in nature, DNA and proteins, which have excellent biocompatibility, high drug payload capacity, in vivo imaging ability, and in vitro/in vivo cancer targeting capability. The synthesis can be done in a facile one-pot assembly system that includes three steps: step-growth polymerization of two DNA oligomers, addition of streptavidin to assemble spherical hybrid nanostructures, and functionalization of hybrid nanospheres with biotinylated aptamers. To test the feasibility of cancer targeting and drug-loading capacity of the hybrid nanospheres, MUC1-specific aptamers (MA3) were conjugated to nanosphere surfaces (apt-nanospheres), and doxorubicin (Dox) was loaded into nanospheres by DNA intercalation. The successful construction of nanospheres and apt-nanospheres was confirmed by agarose gel electrophoresis and dynamic light scattering (DLS). Their uniform spherical morphology was confirmed by transmission electron microscopy (TEM). Fluorescence spectra of nanospheres demonstrated high Dox-loading capability and slow-release characteristics. In vitro MUC1-specific binding of the apt-nanospheres was confirmed by flow cytometry and confocal microscopy. Dox-loaded apt-nanospheres significantly increased cytotoxicity of the MUC1-positive cancer cells due to aptamer-mediated selective internalization, as shown via cell viability assays. Apt-nanospheres could also be imaged in vivo through the synthesis of hybrid nanospheres using fluorescent dye-conjugated DNA strands. Finally, in vivo specific targeting ability of apt-nanospheres was confirmed in a MUC1-positive 4T1 tumor-bearing mouse model, whereas apt-nanospheres did not cause any sign of systemic toxicity in normal mice. Taken together, our self-assembled DNA-streptavidin hybrid nanospheres show promise as a biocompatible cancer targeting material for contemporary nanomedical technology.

8-Channel Biphasic Current Stimulator Optimized for Retinal Prostheses.

Retinal prostheses substitute the functionality of damaged photoreceptors by electrically stimulating retinal ganglion cells (RGCs). RGCs, densely packed in a small region, needs a high spatial resolution of the microelectrode, which in turn raises its impedance. Therefore, the high output impedance circuit and the high compliance output voltage are the key characteristics of the current-source-based stimulator. Also, as the system is intended to implant in the retina, the stimulation parameter should be optimized for efficiency and safety. Here we designed 8-channel neural stimulator customized to the retinal ganglion cell. Designed IC is fabricated in the TSMC 0.18 µm 1P6M RF CMOS process with 3.3 V supply voltage, occupying the 1060 µm×950 µm area.

Wireless Addressable Cortical Microstimulators Powered by Near-Infrared Harvesting.

Ensembles of autonomous, spatially distributed wireless stimulators can offer a versatile approach to patterned microstimulation of biological circuits such as the cortex. Here, we demonstrate the concept of a distributed, untethered, and addressable microstimulator, integrating an ultraminiaturized ASIC with a custom-designed GaAs photovoltaic (PV) microscale energy harvester, dubbed as an "optical neurograin (ONG)". An on-board Manchester-encoded near-infrared downlink delivers incident IR power and provides a synchronous clock across an ensemble of microdevices, triggering stimulus events by remote command. Each ONG has a unique device address and, when an incoming downlink bit sequence matches with this device identification (ID), the implant delivers a charge-balanced current stimulus to the target cortex. Present devices use 7-bit metal fuses fabricated during the CMOS process for their device ID, laser-scribed in post-processing, allowing in principle for a stimulator network of up to 128 nodes. We have characterized small ensembles of ONGs and shown a proof of concept of the system both on benchtop and in vivo rat rodent model.

Current Stimulation of the Midbrain Nucleus in Pigeons for Avian Flight Control.

A number of research attempts to understand and modulate sensory and motor skills that are beyond the capability of humans have been underway. They have mainly been expounded in rodent models, where numerous reports of controlling movement to reach target locations by brain stimulation have been achieved. However, in the case of birds, although basic research on movement control has been conducted, the brain nuclei that are triggering these movements have yet to be established. In order to fully control flight navigation in birds, the basic central nervous system involved in flight behavior should be understood comprehensively, and functional maps of the birds' brains to study the possibility of flight control need to be clarified. Here, we established a stable stereotactic surgery to implant multi-wire electrode arrays and electrically stimulated several nuclei of the pigeon's brain. A multi-channel electrode array and a wireless stimulation system were implanted in thirteen pigeons. The pigeons' flight trajectories on electrical stimulation of the cerebral nuclei were monitored and analyzed by a 3D motion tracking program to evaluate the behavioral change, and the exact stimulation site in the brain was confirmed by the postmortem histological examination. Among them, five pigeons were able to induce right and left body turns by stimulating the nuclei of the tractus occipito-mesencephalicus (OM), nucleus taeniae (TN), or nucleus rotundus (RT); the nuclei of tractus septo-mesencephalicus (TSM) or archistriatum ventrale (AV) were stimulated to induce flight aviation for flapping and take-off with five pigeons.

Listening to Brain Microcircuits for Interfacing With External World-Progress in Wireless Implantable Microelectronic Neuroengineering Devices: Experimental systems are described for electrical recording in the brain using multiple microelectrodes and short range implantable or wearable broadcasting units.

Acquiring neural signals at high spatial and temporal resolution directly from brain microcircuits and decoding their activity to interpret commands and/or prior planning activity, such as motion of an arm or a leg, is a prime goal of modern neurotechnology. Its practical aims include assistive devices for subjects whose normal neural information pathways are not functioning due to physical damage or disease. On the fundamental side, researchers are striving to decipher the code of multiple neural microcircuits which collectively make up nature's amazing computing machine, the brain. By implanting biocompatible neural sensor probes directly into the brain, in the form of microelectrode arrays, it is now possible to extract information from interacting populations of neural cells with spatial and temporal resolution at the single cell level. With parallel advances in application of statistical and mathematical techniques tools for deciphering the neural code, extracted populations or correlated neurons, significant understanding has been achieved of those brain commands that control, e.g., the motion of an arm in a primate (monkey or a human subject). These developments are accelerating the work on neural prosthetics where brain derived signals may be employed to bypass, e.g., an injured spinal cord. One key element in achieving the goals for practical and versatile neural prostheses is the development of fully implantable wireless microelectronic "brain-interfaces" within the body, a point of special emphasis of this paper.

A Scalable and Low Stress Post-CMOS Processing Technique for Implantable Microsensors.

Implantable active electronic microchips are being developed as multinode in-body sensors and actuators. There is a need to develop high throughput microfabrication techniques applicable to complementary metal-oxide-semiconductor (CMOS)-based silicon electronics in order to process bare dies from a foundry to physiologically compatible implant ensembles. Post-processing of a miniature CMOS chip by usual methods is challenging as the typically sub-mm size small dies are hard to handle and not readily compatible with the standard microfabrication, e.g., photolithography. Here, we present a soft material-based, low chemical and mechanical stress, scalable microchip post-CMOS processing method that enables photolithography and electron-beam deposition on hundreds of micrometers scale dies. The technique builds on the use of a polydimethylsiloxane (PDMS) carrier substrate, in which the CMOS chips were embedded and precisely aligned, thereby enabling batch post-processing without complication from additional micromachining or chip treatments. We have demonstrated our technique with 650 µm × 650 µm and 280 µm × 280 µm chips, designed for electrophysiological neural recording and microstimulation implants by monolithic integration of patterned gold and PEDOT:PSS electrodes on the chips and assessed their electrical properties. The functionality of the post-processed chips was verified in saline, and ex vivo experiments using wireless power and data link, to demonstrate the recording and stimulation performance of the microscale electrode interfaces.

A handheld neural stimulation controller for avian navigation guided by remote control.

BACKGROUND: Animal learning based on brain stimulation is an application in a brain-computer interface. Especially for birds, such a stimulation system should be sufficiently light without interfering with movements of wings. OBJECTIVE: We proposed a fully-implantable system for wirelessly navigating a pigeon. In this paper, we report a handheld neural stimulation controller for this avian navigation guided by remote control. METHODS: The handheld controller employs ZigBee to control pigeon's behaviors through brain stimulation. ZigBee can manipulate brain stimulation remotely while powered by batteries. Additionally, simple switches enable users to customize parameters of stimuli like a gamepad. These handheld and user-friendly interfaces make it easy to use the controller while a pigeon flies in open areas. RESULTS: An electrode was inserted into a nucleus (formatio reticularis medialis mesencephalic) of a pigeon and connected to a stimulator fully-implanted in the pigeon's back. Receiving signals sent from the controller, the stimulator supplied biphasic pulses with a duration of 0.080 ms and an amplitude of 0.400 mA to the nucleus. When the nucleus was stimulated, a 180-degree turning-left behavior of the pigeon was consistently observed. CONCLUSIONS: The feasibility of remote avian navigation using the controller was successfully verified.

Origin of complex behaviour of spatially discordant alternans in a transgenic rabbit model of type 2 long QT syndrome.

Enhanced dispersion of repolarization has been proposed as an important mechanism in long QT related arrhythmias. Dispersion can be dynamic and can be augmented with the occurrence of spatially out-of-phase action potential duration (APD) alternans (discordant alternans; DA). We investigated the role of tissue heterogeneity in generating DA using a novel transgenic rabbit model of type 2 long QT syndrome (LQT2). Littermate control (LMC) and LQT2 rabbit hearts (n = 5 for each) were retrogradely perfused and action potentials were mapped from the epicardial surface using di-4-ANEPPS and a high speed CMOS camera. Spatial dispersion (Delta APD and Delta slope of APD restitution) were both increased in LQT2 compared to LMC (Delta APD: 34 +/- 7 ms vs. 23 +/- 6 ms; Delta slope: 1.14 +/- 0.23 vs. 0.59 +/- 0.19). Onset of DA under a ramp stimulation protocol was seen at longer pacing cycle length (CL) in LQT2 compared to LMC hearts (206 +/- 24 ms vs. 156 +/- 5 ms). Nodal lines between regions with APD alternans out of phase from each other were correlated with conduction velocity (CV) alternation in LMC but not in LQT2 hearts. In LQT2 hearts, larger APD dispersion was associated with onset of DA at longer pacing CL. At shorter CLs, closer to ventricular fibrillation induction (VF), nodal lines in LQT2 (n = 2 out of 5) showed persistent complex beat-to-beat changes in nodal line formation of DA associated with competing contribution from CV restitution and tissue spatial heterogeneity, increasing vulnerability to conduction block. In conclusion, tissue heterogeneity plays a significant role in providing substrate for ventricular arrhythmia in LQT2 rabbits by facilitating DA onset and contributing to unstable nodal lines prone to reentry formation.

Engineering Photoactivatability in Genetically Encoded Voltage and pH Indicators.

Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Förster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.

Multivalent Traptavidin-DNA Conjugates for the Programmable Assembly of Nanostructures.

Here, we explore the extended utility of two important functional biomolecules, DNA and protein, by hybridizing them through avidin-biotin conjugation. We report a simple yet scalable technique of successive magnetic separations to synthesize traptavidin-DNA conjugates with four distinct DNA binding sites that can be used as a supramolecular building block for programmable assembly of nanostructures. Using this nanoassembly platform, we fabricate several different plasmonic nanostructures with various metallic as well as semiconductor nanoparticles in predetermined ways. We also use the platform to construct dendrimer nanostructures using valency-controlled traptavidin-DNA conjugates in a programmable manner. These results suggest that our protein-DNA supramolecular building blocks would make a significant contribution to the assembly of multicomponent and complex nanostructures for numerous contemporary and future applications from molecular imaging to drug delivery.