Symposium SM03 : Engineered Functional Biointerfaces—From Electronics and Nanomaterials to Biocircuits and Bionanomaterials

To understand the mechanisms underlying the function and dynamics of the nervous system it is essential to develop tools capable of recording and modulating a diversity of signals employed by neurons and glia. In addition to matching the signaling complexity of the nervous system, these tools must match the mechanical and chemical properties of the tissue to avoid foreign body response and functional perturbation to local circuits. Our group relies on materials design to address these challenges. By leveraging fiber-drawing methods inspired by telecommunications and textile industries, we create flexible and stretchable multifunctional probes suitable for recording and stimulation of neural activity as well as delivery of drugs and genetic information into the brain and spinal cord. We use these tools to probe brain circuits involved in control of motor functions, anxiety, and fear and to promote recovery following spinal cord and peripheral nerve injury. In addition to polymer-based fibers, we develop a broad range of magnetic nanotransducers that can deliver thermal, chemical, and mechanical stimuli to neurons when exposed to externally applied magnetic fields. Magnetic nanoparticles can undergo hysteresis and dissipate heat in alternating magnetic fields. This local temperature increase can be used to directly stimulate activity of heat-sensitive neurons or to trigger release of pharmacological compounds from thermally responsive carriers. Since biological tissues exhibit negligible magnetic permeability and low conductivity, magnetic fields can penetrate deep into the body with no attenuation allowing us to apply the nanomagnetic transducers to remotely control deep brain circuits.

Nanomaterials offer promising platforms for establishing active interface with biological systems, owing to their unique electrical, optical, mechanical properties and large surface-to-volume ratio. Among various nanomaterials-based bioelectronic platforms, graphene has been shown to be an advanced building block over zero- and one-dimensional nanomaterials, owing to its compatibility with conventional top-down approaches (for scalable and low-cost process), high carrier mobility (i.e., sensitivity), and the complementary field-effect sensing capability (both at the p- and n-type regimes). Thus, a variety of graphene-based bioelectronic studies have been reported, such as the electrical recording of electrogenic cells (e.g., cardiac muscle cells). However, little work has been reported beyond other phenotypes, and no studies have demonstrated simultaneous optical stimulation and its electrical and optical recording. In this work, we report light-stimulation of optogenetically encoded mouse-derived skeletal muscle cell lines (C2C12) and their simultaneous imaging and electrical recording based on optically transparent graphene field-effect transistors (FETs). C2C12 transfected with channelrhodopsin-2 (ChR2) was used to express ChR2, and the transparent graphene FET array was prepared by the deposition of graphene on quartz followed by the standard photolithography processes. The light-stimulation with various frequencies (0.5 to 4.0 Hz) and pulse-widths (50 to 500 ms) generated reproducible contractions of the differentiated ChR2-expressing C2C12, which was recorded by the video as well as the biphasic current signals both at the p- and n-type regimes of graphene FET. Control experiments without cells showed no biphasic current peaks under the light-stimulation, demonstrating the current signals resulted from the myogenic action potentials instead of the photoresponse of graphene. Our work, for the first time, demonstrates electrical measurements of optically stimulated skeletal muscle cells based on optically transparent graphene FETs. The distinct and complementary properties of transparent graphene FET and its versatile sensing capabilities will open up unique opportunities in the field of nano-bioelectronics and electrophysiology in the future.

As our understanding of the brain’s physiology and pathology progresses, increasingly sophisticated technologies are required to advance discoveries in neuroscience and develop more effective approaches to treat brain diseases.
To meet this challenge, we propose a novel transistor architecture that provides an efficient interface with biological substrates, especially neural networks, through its channel’s intrinsic ion mobility. Because independent electronic gating can be applied to these transistors, they can be incorporated into integrated circuits, unlike their electrolyte-gated counterparts. The channel consists of a composite film based on highly conductive poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) enriched with D-sorbitol. At the gate electrode, an ion exchange membrane serves as ion conductor. To determine an optimal transistor configuration and material composition, we microfabricated transistor arrays of varying geometrical parameters. In doing so, we were able to extract conductivity, contact resistance, and electrochemical impedance values for all the critical interfaces of various composites. Furthermore, output characteristics and current temporal response of each configuration revealed the key driving physical parameters and provided insight into optimization of the device for various applications.
The resulting optimal transistors were tested as electroencephalography (EEG) interface and amplifier circuitry and compared with organic electrochemical transistors and surface electrodes showed promising signal-to-noise ratio and spatio-temporal resolution.

Neural recording/stimulation devices to chronically record / stimulate biosignal, i.e., neuropotentials, in our bodies have been of great interest to scientists due to their potential benefits to diagnosis and treatment. Existing recording/stimulating system all comprise active components such as amplifier and microcontroller. One of the main concerns of using active components is the heat generation from the electronics could lead to heat trauma. Our work overcome this disadvantage via fully-passive wireless biotelemetry. This unique wireless telemetry utilizes EM backscattering methods to record/stimulate biosignal. Their small size and ability to operate without any battery or energy harvester make them attractive and feasible for chronic recording/stimulation inside or on the body. We, for the first time, demonstrate wireless recorder having sensitivity of less than 60 micro-Vpp and stimulator having capability of more than 1 milli-A, all operating in a fully-passive manner.

Recent advances in materials, device designs and assembly techniques allow for electronic/optoelectronic systems capable of establishing intimate, chronically stable interfaces to the brain. This talk summarizes recent progress in two areas (1) cellular-scale optoelectronic devices that inject into targeted regions of the deep brain for optogenetic stimulation/inhibition and wireless recording of neural activity and (2) thin, conformal sheets of electronics that laminate onto the surfaces of the brain for large-area, high-speed mapping of electrophysiological behavior.

The present work reports effective methods that enabled neural interfaces to stabilize monitoring electrocorticogram (ECoG) on a rodent for 2 months. Integration of metal-nanowire-based tracks that showed high optical transparency and stretchability facilitated a simultaneous optogenetic stimulation and ECoG monitoring in vivo.
Neural interface monitoring and manipulating a target neural activity have realized versatile approaches in neurology [1, 2]. Specific example is a contribution on brain machine interfaces (BMIs) [3] with a feedback sensation. The bifunctional neural interface is also considered on genetic diagnoses and therapies of patients who suffers from intractable diseases. Such neural interfaces have been demanded to keep their function under long-term implantation, however, usually face on granulation tissue [4] that interrupts the access to the targeted neuron.
Here, we report highly stable neural interfaces integrated with transparent and stretchable metal-nanowire-based tracks. Au-plated Ag-nanowire-based (AgNW/Au) tracks were connected to 16-channel-microelectrode and encapsulated in thin polymer substrate. The microelectrode was fabricated with gel materials which endured over 2 months immersion in saline solution. Moreover, the AgNW/Au tracks that showed optical transparency over 70%, mechanical durability over 50% strain, and electrical reliability in the presence of water; Ag-nanowire plated with a noble metal increased track’s performance from pristine Ag-nanowire-based tracks without a significant loss of transparency. The transparent neural interface stabilized mechanically, electrically, and chemically realized in long-term implantation by the mean of an antithrombogenic polymer treatment. The sufficient optical transparency also achieved a simultaneous optogenetic stimulation and ECoG monitoring, indicating to open a way of multifaceted approaches for translational research.

[1] Dong-Wook Park et al., Nature Comm., 5, 5258, 2014.
[2] S. Royer et al., Eur. J. Neurosci., 31 (12), 2279–91, 2010.
[3] A. L. Miguel et. al., Nat. Rev. Neurosci., 10 (7), 530–40, 2009.
[4] J. M. Anderson, et al., Semin. Immunol., 20 (2), 86–100, 2008.

The performance of chronically implanted neural electrodes is dependent on the mechanical and electrical properties of the electrode as well as those of the electrode-tissue interface. We report on the electrochemical fabrication of PEDOT:heparin-coated gold nano-electrodes (hereafter called composite electrodes) and focus on optimizing the electrode-parameters of size, Young's modulus, electrical conductivity, and electrode-medium capacitance. The diameter and the Young's Modulus of neural electrodes determine the buckling threshold during penetration, and the size of the "kill zone" and the degree of immunological response (gliosis) once the electrode has been implanted. The composite electrodes reported here have diameters of ~500 nm. Their lengths, determined by the grower, can be hundreds of microns. The Young's modulus of the polymeric coating is 2.0 GPa. We demonstrate the insertion of ~100 micron long electrodes into single living cells as well as into dense aggregates of ~hundreds of cells (approximating brain tissue). This feat is possible because the Euler stress of the electrode exceeds the critical rupture stress of the cellular aggregate. Additionally, the "kill zone" is minimized by the electrodes' ~500 nm bore, which is much smaller than a cell diameter. The reduced size of the "kill zone" and the Young's modulus of the PEDOT:heparin coating (relative to conventional metals) are expected to reduce the degree of gliosis after implantation. The electrical conductivity and the capacitance of the electrode-medium interface strongly affect the bandwidth, noise, and signal attenuation of neural electrodes. By introducing a single crystalline gold core, these composite electrodes attain conductivities in excess of 10³ S cm^-1, significantly larger than the PEDOT nanofibers that we have previously grown.¹ The capacitive properties of these electrodes in PBS buffer solutions, as determined by measurements of their galvanostatic voltage-transients, will be discussed and related to their effective RC time constants and their ability to deliver high fidelity signals to an external amplifier. Taken together, these results show that these electrodes are reasonably successful at optimizing the contradictory set of requirements (as described above) for neural probes and, thereby, are interesting candidates for next-generation neural implants.

1. PS Thapa, DJ Yu, JP Wicksted, JA Hadwiger, JN Barisci, R Baughman & BN Flanders, "Directional growth of polypyrrole and polythiophene nanowires", Appl. Phys. Lett. 94, 033104, (2009).

*Supported by the NIH BRAIN Initiative (1 R21EY026392).

In vivo pharmacology and optogenetics hold great promise for selective examination and control of neural circuits and systems in conscious, freely moving animals. However, existing neural interface technologies, such as metal cannulas connected to external drug supplies for pharmacological infusions and tethered fiber optics for optogenetics, are not ideal for minimally-invasive, untethered studies on freely behaving animals. Here we present soft optofluidic probe systems that can provide wireless drug delivery and optical stimulation for spatiotemporal control of the targeted neural circuit in the brain. The optofluidic neural probes combine ultrathin, soft microfluidic drug delivery with cellular-scale inorganic light-emitting diode arrays. These probes are orders of magnitude smaller than cannulas, thus allowing minimally invasive integration with neural tissue. We have developed two different types of wireless optofluidic systems. One is a head-mounted system based on infrared wireless control and rechargeable batteries, which can be extended to incorporate replaceable/refillable drug reservoirs for long-term drug delivery and optogenetics. The other is a miniaturized, fully-implantable, battery-free system enabled by a tiny stretchable multichannel radiofrequency antenna, which eliminates the need for batteries for seamless implantation and operation in the body. Our proof-of-principle experiments and studies prove the feasibility of the wireless optofluidic systems for use in freely moving animals, demonstrating its potential for wireless in vivo pharmacology and optogenetics.

Biotechnology Center (BIOTEC), Center for Molecular and Cellular Bioengeneering (CMCB), Technische Universität Dresden

Neurological disorders affect up to a billion people worldwide. However, our ability to understand and to treat disorders is currently limited by the lack of tools capable of interfacing with the brain over extended periods of time. This is hypothesized to stem from the mismatch in mechanical and chemical properties between the neural probes and the neural tissues. To address the challenge, we developed a sensing and actuation platform mimicking the properties of the brain tissue while integrating thermally drawn polymer fibers and a tough alginate-based hydrogel.
To fabricate the hybrid probes, fibers including a waveguide, three electrode arrays, and three hollow channels were first thermally drawn and assembled by hydrogel dip-coating process. The optical waveguides consisted of a polycarbonate (PC) core and cyclic olefin copolymer (COC) cladding due to their high refractive index contrast, and the recording electrodes were composed of seven tin cores (10 µm) in poly(etherimide) (PEI) insulation. Hollow PEI microtubes served as the microfluidic channels. To establish strong bonding of resilient hydrogels onto polymer-based neural probes, their surfaces were treated with hexamethylenediamine (HMDA), which allowed the use of straightforward carbodiimide chemistry to covalently graft Ca-alginate/polyacrylamide hydrogels from the surfaces of the fibers.
To test the hypothesis that our probes reduce the mechanical damage of the brain tissue, we used finite element models to calculate the bending stiffness of the probes and the stress fields of brain tissue. The results indicated that our devices containing hydrogel bodies exhibited significantly lower bending stiffness than polymer fibers. This also resulted in lower stresses on the brain tissue during micromotion. Interestingly, dried hydrogel body rendered these hybrid structures an order of magnitude stiffer, which facilitated the fiber implantation into deep brain regions without buckling.
Lastly, the long-term performance of devices was assessed. The probes were implanted into the mouse basolateral amygdala (BLA) and ventral hippocampus (vHPC), as the neural projection between these regions is established in the context of anxiety-related behaviors. In this circuity, optogenetic experiments with viral injection, optical stimulation, and electrophysiological recording were conducted with simultaneous behavioral experiments. The foreign-body responses in the probe vicinity were also quantified by immunohistochemistry.
In summary, we designed hydrogel hybrid probes that will enable long-term studies of brain circuits in freely moving subjects. Our results provide an experimental evaluation of the hypothesis that matching the properties of neural tissue minimizes foreign-body response and extends the lifetime of neural interfaces. Our research offer a promising pathway for development of brain-machine interfaces needed to accelerate understanding and treatments of progressive neurological disorders.

The ability to reliably record from a large ensemble of neurons, map their functional connectivity, and track the activity over chronic time scale is of paramount importance to basic and clinical neuroscience, as a large number of brain functions are realized by coordinated activation of a neuronal population. Implanted electrodes provide one of the primary neurotechniques by allowing for time-resolved acquisition of individual neuron activity in the living brain. However, their recording stability and density pose major limitations on their scientific and clinical applications. We recently demonstrated that ultraflexible, cellular-dimensioned neural electrodes afford seamless integration with brain tissue and reliable recording of individual neurons for several months. Building upon this platform, we further demonstrate the capabilities of reliable detection and isolation of individual units, as well as functional mapping and chronic tracking of the local circuitry in a neuronal cluster over several months in behaving brain.

Recent advances in organic electronics have made available materials with a unique combination of attractive properties, including mechanical flexibility, mixed ionic/electronic conduction, and enhanced biocompatibility. These materials are currently being used in a variety of biological interfacing applications including neural prosthesis, tissue engineering, diagnostics, and drug delivery. Using these applications as examples, I will show how structure, electrical, and biological properties of organic electronic materials can be tuned to improve device performance.

We present a process which allows the patterning of fully stretchable organic sensors. The device consists of an active stretchable area connected with stretchable metallic interconnections. Such features allow the development of biosensors at the interface with the body; that can measure and address in real-time various physiological parameters at the interface with the body, but also with organs for in vivo experiments. This work is focused on sensors that will be fully stretchable, that is to say, all the materials are thought to be stretchable. The current research does not provide a completely simple and accurate process using the standard microelectronic techniques, allowing for the creation of such sensors.
In this work, we optimized the horseshoe shape of stretchable conductive interconnections encapsulated in polydimethylsiloxane (PDMS). An innovative patterning process based on the combination of laser ablation and thermal release tape ensures the fabrication of highly stretchable lines encapsulated in PDMS from conventional aluminum tape. State-of-the-art stretchability up to 70% combined with ultra-low mOhms resistance is demonstrated. We present a photolithographic process to pattern the organic active area on PDMS. Finally the formulation of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) is tuned to maximize its stretchability. The fabricated organic electrochemical transistor (OECT) shows state-of-the-art electrical performance with a high transconductance of 6.5 mS at no strain and a maximum stretchability of 38%, whilst maintaining a transconductance of up to 0.35 mS and channel current as high as 0.2 mA.

Advancements in electronics are driving the biomedical field and vice-versa. Convergence of biomaterials and electronics holds promise for many applications and drives the field of bioelectronics. Last decade has seen emergence of high-performance, multi-functional, flexible and printed electronic devices that have contributed to the development of the new age, conformal and non-invasive biomedical devices. Bioelectronics platforms are gaining widespread attention as they provide a template to study the interactions between biological species and electronics.
The talk will highlight our group’s work on printed electronics using aerosol jet technology using various materials on different substrates. 3D bioprinting of biomaterials particularly hydrogels with cells will be discussed for various applications. We will also highlight our work on interfacing printed electronics and biomaterials for bioelectronic platforms for added functionality. The work showcases engaging 3D printing techniques to build bioelectronic platforms, and how 3D printing is driving the bio-medical industry with specifications such as user customization, cost-effectiveness and short response time. We report printing, optimizing and characterizing electronic circuits on bio-scaffolds/ biomaterials for making complete devices, thus trying to understand 3D printing capabilities for such platforms. We fabricated a freestanding and flexible hydrogel based platform using 3D bioprinting. The fabrication process is simple, easy and provides a flexible route to print materials with preferred shapes, size and spatial orientation. Through the design of interdigitated electrodes and heating coil, the platform can be tailored to print various circuits for different functionalities. The biocompatibility of the printed platform is tested using C2C12 murine myoblasts cell line. Furthermore, normal human dermal fibroblasts (primary cells) are also seeded on the platform to ascertain the compatibility. Thus, The fabricated bioelectronics platform is compatible with cells and tissues, cost-effective and does not require any post-processing. Our research is focused on bringing electronics and biomaterials together on a single platform for futuristic applications like drug delivery, wound management etc.

Measuring electrochemical potentials in complex environments is crucial for better understanding of biological and chemical systems. Optical schemes for electrostatic and chemical potential detection are non-invasive, scalable and can be applied to many different systems, but usually have a slow response compared to electrical detection methods. Furthermore, methods using organic fluorophores often suffer from bleaching due to exposure to high-intensity light. These issues can be addressed by using non-organic optical sensors. Among such sensors, two-dimensional (2D) materials are attractive because they are thin, flexible, and compatible with photolithographic patterning.
We show that monolayer MoS₂ can be used as a 2D screen to optically detect real-time changes in potential in fluid environments. We characterize the photoluminescence response of MoS₂ to fluid potential by liquid-gate measurements in an aqueous electrolyte solution, showing the capability to detect sub-mV changes at timescales of a few milliseconds, only limited by our experimental setup sensitivity. By performing cyclic voltammetry near our MoS₂ devices in a solution of ferrocene, we demonstrate that the photoluminescence of electrically floating MoS₂ responds to the chemical potential of the solution in the presence of redox active molecules. Based on these findings, we use an array of MoS₂ “pixels” to image the ionic diffusion of ferrocene ions (ferrocenium) in real time. These results indicate that monolayer MoS₂ can be used as an optical detector of potentials of electrogenic cells and of redox-active biomolecules. Furthermore, the flexibility and ease of transfer of MoS₂ devices allows them to be placed on relevant structures such as flexible substrates and optical fibers in order to measure systems of interest.

Inspired by the cell membranes which can precisely control the ion flow, the artificial analogs are developed to reproduce biological energy conversion process. Using biomimetic smart nanopores to convert osmotic energy into electricity have been widely investigated. They are commonly composed of two different materials and require the complicated fabrication technique. The use of a single component macroporous conducting membrane to harvest the osmotic energy has not been reported.
Here, we developed a macroporous free-standing polypyrrole film with two types of pores via a straightforward chemical synthesis method. This flexible asymmetric macroporous membrane is conductive (~200 S/m). It could generate a voltage in the range of several to around 200 mV, reaching 207 mV when the gradient ratio was 10⁶. The power density harvested from a gradient using artificial seawater (0.5 M NaCl) and river water (0.01M NaCl) reached 0.087 W/m² with an external resistor of 5 KΩ. Impressively, its ionic conductance can be tuned by the applied potential. This PPy film has also been demonstrated to be a good substrate for Adipose-derived stem cell adhesion and proliferation. With its unique property of electrical and ionic conductivity, this biocompatible PPy membrane shows its promising application in the bioelectrogenesis and clean energy conversion field.

Graphene field-effect transistors (GFETs) have unique properties that make them ideal candidates for recording the activity of electrogenic cells. Graphene is mechanically strong yet flexible enough to conform to irregular shapes. Transistors made from graphene are capable of locally amplifying small voltage signals. Lastly, graphene is optically transparent and biocompatible. Here we present the use of ultra-flexible GFETs for recording action potentials from individual neurons. Measurements have been performed in two configurations. (1) A “standard” configuration with GFETs on a rigid substrate and neurons cultured on top of the GFETs. (2) A novel wearable graphene sensor configuration, in which the GFET is released from the substrate and placed over a neuron. The wearable graphene configuration makes use of graphene’s intrinsic strength and flexibility. Cells remain active while in contact with the wearable sensor and high signal-to-noise ratios are demonstrated. These proof-of-concept measurements demonstrate new possibilities for ultra-flexible brain-machine interfaces.

Electronic materials with percolating structures have been attracting tremendous interest in the fields of stretchable electrodes, transparent conducting electrodes, energy storage/conversion devices, wearable electronics and sensors, and bio-interfacing materials. Percolating structures can provide large effective surface area of electronic materials, enabling efficient interfacing with ionic systems such as biological, biochemical, and electrochemical systems as well as mechanical flexibility. Single-walled carbon nanotubes (SWNTs), rolled-up sheets of graphene, are attractive nanoscale electronic materials for fabricating percolating electronic materials owing to their extremely large aspect ratio and excellent electrical and mechanical properties. In this presentation, a biological material-based method to assemble electronic nanomesh of SWNTs in solution and produce patterns of SWNT-nanomesh on flexible substrates with excellent control of nanostructures will be introduced. In our approach, a genetically engineered filamentous M13 phage with strong binding affinity toward SWNTs controls and stabilizes the nanostructures of the SWNT-nanomesh during a hydrodynamic process. This unique biological material-based in-solution assembly process enables the realization of electronic nanomesh of SWNTs, independent of the substrate, as well as the delivery of intact nanostructures with excellent electrical and electrochemical properties onto large-scale flexible devices. The assembled SWNT-nanomesh can greatly reduce the in vivo contact impedance of a flexible 40-channel microarray and significantly increase the detection rate of high frequency brain signals (HFBS) on a mouse skull. In addition, flexible biosensors and bioelectronic devices are successfully realized by employing the SWNT-nanomesh as a key interface material.

Organic electronics is today explored and utilized in various electronic sensor and bioelectronics systems to transduce physical, chemical and biological signals into electronic ones, and vice versa. In organic electronic sensor-actuator systems, we explore this dual signal translation functionality to derive artificial neural networks, coordinated by a capacitively coupled intra-body network (IBN) technology. With organic electronics printed on medical packages, skin patches and in vivo electrodes, we aim at an IBN-based health care technology for advanced diagnostics, monitoring and therapy, targeting neurological disorders and diseases. In our IBN platform PEDOT:PSS serves as a key-component, being able to conduct and process both biochemical and electronic signals. Various signal processing, sensor and delivery devices, based on PEDOT:PSS, have been developed for various dedicated applications. These have then been applied to different in vitro and in vivo model systems, to combat for instance pathological pain and epileptic seizures.

In the last decade, significant progress has been made on the development of wearable and implantable flexible devices to improve clinical diagnosis and treatments of human health. As a new emerging field, organic bioelectronics has witnessed increasing interested due to recent technology advancements in the materials research field and electronics. Of particular interest is the use of plastic based electronic devices that can conform to the curvilinear shapes of organs, making them a more desirable alternative to rigid silicon microchips at the biotic/abiotic interfaces. (1) However, it is still in high demand the use of multifunctional flexible devices able to readily interact with the biotic interface and to undertake multiple tasks at once, e.g. on line recording and drug delivery.
In parallel to this, the development of reliable in vitro models for toxicological testing and pharmacological investigation of chemical substances is urgently required to move away from animal testing and animal models. The predictive ability of 2D in vitro based model is limited by its poor reproducibility of the microenvironments and the physiological behavior of tissue in organs. In this scenario, 3D cell models provide a valid alternative to their 2D counterparts. However, the transition into the third dimension poses severe limitations on the coupling of label free in-line monitoring systems, such as trans-epi/endothelial resistance (TEER), as previously shown by us. (2)
In this paper, I will present our parallel efforts on the novel use of parylene C (PaC) conformable neural probes for both in vivo and in vitro applications. For the former, the flexible parylene C neural probes have been integrated within a microchannel for the realization of a microfluidic ion pump (μFIP), capable of delivering a drug without the solvent through electrophoresis. (3) We demonstrate the low voltage operation, high drug delivery capacity of the μFIP and efficient cortical delivery in vivo while simultaneously recording cortical brain activity. In addition to this, I will show how by taking advantage of the flexibility and conformability of the PaC neural probes, it is possible to measure in a reliable manner the TEER of both epithelial and endothelial cells when grown on a collagen type I hydrogel.

(1) T. Someya, Z. Bao, G.G. Malliaras “The rise of plastic bioelectronics” Nature, 2016, 540, 379–385
(2) V.F. Curto, B. Marchiori, A. Hama, A. Pappa, M.P. Ferro, M. Braendlein, J. Rivnay, M. Fiocchi, G.G. Malliaras, R.M. Owens “An organic transistor platform with integrated microfluidics for in-line multi-parametric in vitro cell monitoring”, Microsystems and Nanoengineering, in press
(3) I. Uguz, C.M. Proctor, V.F. Curto, A. Pappa, M. J. Donahue, M. Ferro, R.M. Owens, D. Khodagholy, S. Inal, G.G. Malliaras “A Microfluidic Ion Pump for In Vivo Drug Delivery” Advanced Materials 10.1002/adma.201701217

With great progress in the field of implantable electronics, pushing towards an integration of technology with people, the biocompatibility of those electronics is a key issue. Collagen, one of the most abundant proteins in mammalian tissues, is a well-known biomaterial used in tissue engineering and bone scaffolds. Previous work has shown that collagen¹ can be used as a substrate for flexible electronics made using with E-Beam deposition by shadow mask. However, in order to make more advanced electronic devices, manufacturing strategies need to be developed in order to overcome limitations of collagen, namely temperature and mechanical stability in water. Transfer printing of electronics is one such strategy, using sacrificial layers of PMMA, however these also have their own temperature limitations. Germanium oxide is presented in this paper as novel water based sacrificial layer, which is amenable for high temperature processes such as the annealing and doping of Zinc Oxide (ZnO) via Pulse Laser Deposition. Some of the devices presented in this study include capacitors, transistors, and an integrated inverter transistor circuit. After etching in water overnight, devices made on wafers are lifted off and transferred to collagen films. By using cross linkers such as Riboflavin (Vitamin B2) and, a photochemical initiator, in the presence of blue light, devices built on collagen films can be programmatically enhanced to resist enzymatic digestion. Cross-linked collagen was shown to have enhanced mechanical and thermal properties while maintaining biocompatible aspects. Encapsulated integrated electrical devices transferred onto collagen were shown to have minimal effects on cell viability on assays on MC3T3 osteoblast and a549 epithelial cells. Together, this study demonstrates a manufacturing strategy of developing biocompatible integrated electrical devices on collagen.

The ability to spatially map intercellular communication hotspots as a function of different biochemical environments is important for disentangling the complex active processes in brain tissue. Organic electrochemical transistors (OECTs) have been proven to act as tissue-compatible biosensors for in vivo electrophysiological recording of neuronal circuits. However, arrays of OECTs are needed to glean useful spatial information about cell-cell communication in active tissues. Current approaches to building OECT arrays have two main limitations: the minimum achievable sensing channel length and maximum attainable device density on flexible substrates. Small channel lengths lead to accurate, pinpoint signal detection and high device density allows high throughput data acquisition, which can reveal important behavioral patterns.

Fabrics are theoretically ideal substrates for OECTs because many fabrics, such as medical gauze, are biocompatible, have the same mechanical features as biological tissue and readily encourage cell growth on their surfaces. However, fabrics are also highly-demanding substrates on which to construct electronic devices.

Here, we demonstrate that a combination of reactive vapor deposition and simple sewing can create a dense array of high-performing, small channel-length OECTs on textiles. Nylon fibers (100 μm diameter) vapor-coated with an electroactive conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), are used as the sensing channel for our OECT array. The resulting channel fiber and a stainless-steel gate electrode yarn are subsequently sewed onto a tight-woven silk textile in parallel. An array of 10 × 10 OECTs with identical channel lengths can be rapidly fabricated on a 2 × 2 inch² textile. The ruggedness of the vapor-deposited PEDOT coating on the nylon fiber allows this coated fiber to participate in mechanically-demanding sewing processes without leading to losses in electrical performance. The minimum achievable channel length of each OECT in the array is simply determined by the weave density of the underlying fabric substrate. A 200 μm channel length for each individual OECT was obtained, which is significantly smaller than previously-reported millimeter- to centimeter OECT channel lengths. Additionally, our sewn OECT arrays display maximum transconductance at zero gate voltage, which is useful for singly-powered transducer amplifiers.

Currently, implantable neuroprosthetic devices are made of materials with high elastic moduli. The mechanical mismatch between the device and soft neural tissue leads to a foreign body response, which results in encapsulation and can ultimately cause a loss of function. Recent approaches to make neuroprosthetic implants softer and more stretchable employ silicones, metallic thin-films or conductive composites as functional materials. Even though this approach allows for a drastic reduction in device stiffness, the overall mechanical behavior of devices resembles that of connective tissue. Hydrogels are a promising class of materials as they can have elastic moduli similar to that of neural tissues. However, at the same time they are typically brittle, do not support electronic conduction and are challenging to process with standard microfabrication technology.

In this work we design a hydrogel based material that is mechanically tough, electrically conductive and contains a network of interconnected cell-sized pores. To induce toughness, we employ a covalent polyacrylamide and a non-covalent agar-alginate network. Compared to the single polyacrylamide network the interpenetrating network shows superior toughness but only a minor increase in elastic modulus (about 40 time lower than the modulus of PDMS and only 2 times higher than the modulus of neural tissue). By cryogelation of the tough hydrogel with the addition of gelatin A at different temperatures between -10 to -20°C we induced pores with defined sizes ranging from about 100 µm down to 10 µm depending on the processing temperature. The porosity should allow for the growth of cells directly into the gels thus improving hydrogel-tissue integration. The pre-gel solution can be 3D printed using extrusion nozzles with a diameter down to 250 µm. We employed post-gelation polymerization of poly(3,4-ethylenedioxythiophene) around the hydrogel struts to electrically functionalize the material for potential neural interfacing applications. By the use of benzophenone or 3-(trimethoxysilyl)propyl methacrylate we polymerized gels directly to PDMS, glass and titanium surfaces.

Taken together our material shows promise for the fabrication of tailor made neuroprosthetic devices. The combination of toughness and softness may allow for minimally invasive implantation combined with reduced mechanical mismatch between device and host tissue.

Communication across neural interfaces currently relies on conventional metal electrodes. Typically, the electrical and biological performance of these electrodes is restricted by charge injection limits of metals and fibrosis. The hypothesis driving this research was that neural cell loaded coatings provide a more physiological interface able to integrate electrodes with the neural tissue without significantly reducing the charge transfer characteristics. The study aimed to develop and evaluate an in vitro and in vivo tissue-engineered, living electrode (LE) coating for brain-machine interfaces. LEs were fabricated by first coating the electrode with a conductive hydrogel (CH) and then overlaying the CH with primary neural progenitor cells encapsulated within a 3D biosynthetic hydrogel. Performance of the LE was compared with conventional platinum (Pt) electrodes. In vitro studies confirmed that charge storage capacity (CSC) and frequency dependent electrochemical impedance was not significantly impacted by addition of the neural cell layer on the CH. Mass loss and swelling of the CH and LE were also not significantly different. Implantation of LE coated and uncoated Pt electrodes on Neuronexus probes within rat brain confirmed that the LE did not cause any adverse events over 8 weeks implantation. Significantly higher signal to noise ratio was demonstrated in LE, and scar tissue was reduced at the implant interface when compared to Pt controls. These results demonstrate the potential for a tissue-engineered electrode to support development of more robust neural interfaces in vivo.

Acknowledgments
The research was supported by the Defense Advanced Research Projects Agency (DARPA) under contract number HR0011-16-C-0032.

Electroactive scaffolds have been intensively explored in cardiac tissue engineering, based on observations that the conductive components can enhance the ultrastructure and function of the tissue constructs. A prevalent hypothesis states that cardiac cells can be electrically bridged by conductive materials to achieve synchronization of cardiac action potentials. Thus, high conductivity is pursued as a property of priority in designing electroactive scaffolds. However, rational design of these scaffolds would be impossible without validating this hypothesis and revealing the underlying mechanism. In the current work, using equivalent circuit models, systems of cardiomyocytes-on-substrate and cardiomyocytes-via-nanowire are theoretically studied. Specifically, when one group of cells fires an action potential (AP) that is transmitted through the conductive material, the depolarizing effect on its adjacent cells is investigated. For the cardiomyocytes-on-substrate system, simulation results show that the seal resistance is the most sensitive factor to AP-induced depolarization of neighboring cells, while surface roughness and conductivity of the material have less impacts. For the cardiomyocytes-via-nanowire system, the required seal resistance for substantial depolarization is at least 10¹³ Ω/sqr because of the small interfacial area and large interfacial impedance. These analyses validate the electro-bridge hypothesis by confirming the positive role of conductive scaffolds and nanostructures in aiding seeded cardiac cells to electrically synchronize with each other, while revealing the cell-scaffold adhesion strength as a crucial factor to the performance. Although further experimental tests are needed to verify these analytical results, this work provides a theoretical basis to the rational design of electroactive scaffolds for enhanced cardiac tissue engineering.

The application of electrical stimulation to stem cells is currently being explored as a method to facilitate their differentiation into various cell lineages. The potential differentiation of adipose-derived stem cells (ADSCs) to cardiac or neural phenotypes is particularly interesting due to the ubiquitous nature of adipose cells throughout the body, their ease of extraction and rapid expansion. In this work, the electrical stimulation is delivered using penetrating nanoelectrodes Penetrating electrodes provide direct access to the cell’s interior in a minimally invasive fashion, and with enhanced cell-electrode coupling. Nanoelectrodes (pillars and fins) of controlled height, diameter, and density are fabricated on silicon using nanofabrication techniques. Extensive experiments confirmed the ability of the nanoelectrodes to penetrate cells. ADSC were then modulated by applying electrical stimulus of 500-500 mV/cm for 15 minutes per day for 5 days. The differentiated neural phenotypes were validated using various stem cell surface markers and electrophysiology measurements.

Piezoelectric materials can convert mechanical energy into electrical energy, and piezoelectric devices made of various inorganic materials and organic polymers have been demonstrated. However, synthesizing such materials often requires toxic materials, harsh conditions and/or complex procedures. Recently, it was shown that hierarchically organized natural materials, such as bones, collagen fibrils and peptide nanotubes, can display piezoelectric properties. In my presentation, I will show our innovative approach to produce virus-based piezoelectric energy generation. Recently, we establish that the piezoelectric and liquid crystalline properties of M13 bacteriophage (phage) can be used to generate electrical energy. Using piezoresponse force microscopy, we characterize the structure-dependent piezoelectric properties of phage at the molecular level. We then show that self-assembled thin films of phage can exhibit piezoelectric strengths of up to 7.8 pm/V. We also demonstrate that it is possible to modulate the dipole strength of phage, and hence tune their piezoelectric response by genetically engineering the phage’s major coat proteins. Finally, we develop a phage-based piezoelectric generator that produces up to 100 nA of current and 2 V of potential, and use it to operate a liquid crystal display and light emitting diodes. Because biotechnology techniques enable large-scale production of genetically modified phages, phage-based piezoelectric materials potentially offer a simple and environment-friendly approach to piezoelectricity generation.

Antibiotic resistant bacteria have increasingly become an issue in both the environment and in the human body. Photothermal lysis, using the plasmonic properties of metal nanoparticles (NPs), has been shown to be effective against bacteria, regardless of their drug resistance. Typically, these particles need to be functionalized with specific bacteria-targeting molecules and the particle geometries tailored to the desired spectral absorption range. Viruses provide an alternative approach to the synthesis of these bacteria targeting metal nanoparticles. Through genetic modification, a virus can be programmed to simultaneously serve as a scaffold for metal nanostructure assembly and have an affinity for a specific bacterial host. In this work, we used a gold-binding M13 bacteriophage to create a photothermal bactericide for e coli. The filamentous virus was modified to display an 8-mer peptide with gold affinity on the major coat protein, while the unmodified minor coat protein was used to specifically target e coli F pili. To create a nanoshell template, the virus underwent a simple chloroform treatment causing it to contract from its filamentous form to its spheroidal form. Two different types of gold nanoshells were formed: one by binding gold nanoparticles to the spheroid surface for visible light absorption and the other by synthesizing a gold shell on the spheroid surface for near infrared absorption. Transmission electron microscopy and spectrophotometry were used to evaluate shell morphology and optical absorption, respectively. Nanoshell photothermal activity was quantified under either green (532 nm) or near infrared (785 nm) laser illumination. Significant antibacterial activity was measured via colony titer count. The use of gold/M13 bacteriophage nanoshells as a photothermal bactericide was demonstrated in this study by utilizing the innate affinity of the unmodified M13 minor coat protein for e coli f pili. However, this potentially powerful approach can be generalized to target a variety of bacteria through the incorporation of affinity peptide fusions.

The leaves of certain plants, such as banana, pothos, and palm, are readily and cheaply available and possess amazing mechanical robustness, to the extent that they are used as eco-friendly, disposable food containers and other household wares in many countries. In theory, these plant leaves can also act as rugged, cheap, lightweight and flexible substrates for biocompatible and biodegradable organic electronic devices. However, the surfaces of these leaves are textured and nonplanar, and the leaves themselves are degraded by heating and/or various chemical treatments. Therefore, the typical fabrication methods currently used to create polymer devices cannot reliably produce leaf-based electronic technologies. Here, we describe our efforts to grow functional, high-performing electrode arrays and electrochemical transistors on various plant leaves using reactive vapor deposition (RVD). RVD is a nascent vapor coating technique in which a conjugated polymer film is formed in situ upon the mixing of separately-introduced oxidant and monomer vapors inside a specially-designed reaction chamber. RVD reliably and conformally creates conjugated polymer films on a diverse range of substrates, irrespective of surface chemistry or roughness/topography. Banana, pothos and palm leaves can be uniformly and conformally coated with highly-conductive conjugated polymer films using our custom reaction chamber while remaining unaltered and undamaged. Further, the electronic polymer coatings retain the unique, hierarchical native structure of each leaf, leading to electronically-active films that display intriguing mesoscale architectures. The performance of selected electrochemical transistors created on various plant leaves will be discussed.

We provide portable, on-demand micropower generation by developing paper-based biobatteries that can deliver on-chip energy to the next generation of point-of-care (POC) diagnostic platforms. This work creates a low-cost, disposable, long shelf life and eco-friendly micro-power source that can be easily integrated in paper-based POC devices and be readily activated by one drop of saliva, which is readily, available in any challenging area. We created a high-performance, paper based, bacteria-powered battery by building microbial fuel cells with inactive, lyophilized (or freeze-dried) exoelectrogenic cells, allowing for a long shelf life, and that generate power within minutes of adding saliva. An oxygen-tight interface and engineered conductive paper reservoir achieved significant performance boosts from maximized microbial electron transfer efficiency. Exoelectrogenic bacteria pre-inoculated in the paper battery was freeze-dried for long-term storage (in this work, the bacteria cells were stored for up to four months) and could be readily rehydrated for on-demand power generation. Sixteen MFCs were incorporated on a single sheet of paper while all were connected in series with two electrical switches mounted on a paper circuit board and produced more than enough electrical energy to power an on-chip, light-emitting diode (LED).

Across the globe, more than 400 million people are affected by an overactive bladder (OAB)^[1], and within Europe, it affects 17 % of people^, with that proportion increasing to 30-40 % for those over the age of 75^[2]. OAB can have a devastating effect on patients’ lives, since it produces a sudden and intense need to pass urine, with the patient having almost little or no control. It is known that stretch of the bladder wall is linked to the need to pass urine since it gives an indication of bladder fullness; however, until now, it has not been possible to fully determine bladder fullness without using complex and invasive surgical procedures. We present a fully biocompatible, soft, stretchable sensor device, capable of monitoring minuscule changes in the bladder wall, which can lead to the development of new treatment options for various urological conditions, including OAB.

We have developed sensors comprising thermally evaporated Cr/Au thin films (~ 150 nm) on compliant, stretchable polyurethane (PU) film. When stretched, sensor resistance changes from a few Ω’s, to tens or even hundreds of Ω’s depending on sensor dimensions. We demonstrate repeatable sensor response up to at least 70 % stretch, with sensitivity exceeding 20 Ω/mm. Furthermore, the sensors show almost no hysteresis when subjected to repeated cycling tests between 0 and 50 % stretch, making them suitable for application on the bladder. The sensors have also been tested in vitro on a pig’s bladder, subjected to repeated filling and emptying cycles to mimic natural bladder behaviour. Sensors were attached to the bladder wall using an optimised biocompatible adhesive hydrogel film, which allows sensor stretch to be retained, whilst providing strong adhesion as the bladder constantly cycles between full and empty. As the bladder fills, the sensor detects minute changes in the bladder wall (micromotions) using changes in resistance as a function of stretch; and this resistance is used to correlate stretch with fullness, and can provide an early warning system for OAB patients of the need to pass urine. Our soft, stretchable, biocompatible sensors pave the way for fully implantable health monitoring systems of the future.


[1] Irwin D.E. et al. BJU Int. 108 (7), 1132 (2013).
[2] Milsom I et al. BJU Int. 87 (9), 760 (2001).

It is well known that neural activity and function is inherently correlated to local availability of oxygen and oxygen consumption for energy in vivo. However, due to limitations in current technologies, tissue oxygenation levels cannot be easily measured at the tissue-implant interface. Current, minimally-invasive techniques to measure tissue oxygenation levels in vivo typically suffer from low spatial and temporal resolution around implant sites. In addition, oxygenation levels are either qualitative, indirect measurements (i.e BOLD fMRI and biophotonic techniques), or are quantitative only at the point of measurement (i.e highly invasive, polarographic methods). Therefore, there is a critical need for a minimally invasive, quantitative measurement of pO₂ with high spatiotemporal resolution that can serve as a biomarker for interfacial tissue health under chronic implantation conditions.

In this study, a technique to quantitatively map tissue oxygenation levels in the microenvironment surrounding implanted microprobes is performed using a novel tissue oximetry technique known as proton imaging of siloxanes to map tissue oxygenation levels (PISTOL). A polydimethyl siloxane (PDMSO, ~410 g/mol) contrast agent was embedded in a soft, PDMS based matrix that was coated around a silicone catheter (0.6-1.5 mm diameter) and various MR-compatible microwires such as platinum-iridium (75 µm diameter), tungsten (100 µm diameter), gold (50 µm diameter). Siloxane loaded implants were scanned in phantoms and ex vivo brains in a time-dependent manner using a Bruker 7T preclinical MRI system with either a volume or surface coil at room temperature. A PISTOL scan was conducted after suppression of the water/fat signal using a combination of pulse-burst saturation recovery with frequency-selective excitation of the PDMSO-siloxane resonance. After analysis with custom MATLAB algorithms, pO₂ levels were correlated to PDMSO-based siloxane T1 relaxation times where decreasing oxygen tension displays longer relaxation times. With the exception of thick, platinum based electrodes that displayed susceptibility related artifacts, the MR/PISTOL was sensitive enough to spatially map pO₂ levels around MR-compatible, microelectrode implants. Selective, spatial mapping of matrix embedded siloxane based sensors showed ambient air levels (~140-150 torr) close the ex vivo brain surface and decreasing pO₂ levels along the length of the shank going into the brain. The current spatial resolution of the mapping was ~150-300 µm around the implant based on the chosen field of view (FOV) during the imaging process. Preliminary results from this study demonstrate that PISTOL MR imaging technique can be useful for measuring tissue oxygenation around microscale brain implants. Quantitative tissue oximetry can potentially be used for deep brain imaging in conjunction with brain implants. Current studies are on-going to quantitate pO₂ levels in vivo under chronic conditions.

Overproduced reactive oxygen species (ROS) are closely related to various health problems including inflammation, infection, and cancer. The abnormally high ROS level can cause serious oxidative damage to biomolecules, cells, and, tissues. A series of nano- or micro-sized particles has been developed to reduce the oxidative stress level by delivering antioxidant drugs. However, most systems are often plagued by the slow molecular discharge driven by diffusion. In this work, we demonstrate an active antioxidizing polymeric particles that can increase the internal pressure in response to the abnormal ROS level and thus actively discharge antioxidants to protect cells and tissues from the oxidative damage. The on-demand pressurized particles particle was assembled by simultaneously loading water-dispersible manganese oxide (MnO₂) nanosheets and green tea-derived antioxidant into poly(lactic-co-glycolic acid) (PLGA) spherical shell. In the presence of H₂O₂, one of the ROS, MnO₂ nanosheets in the PLGA particle generated oxygen gas by decomposing H₂O₂ and increased the internal pressure. Accordingly, the active antioxidizing particle system could release a larger fraction of antioxidants and effectively protect endothelial cells and brain tissues from H₂O₂-induced oxidative damage. We believe that this H₂O₂-responsive, self-pressurizing particle system would be useful to deliver a wide array of molecular cargos in response to the oxidation level.

Biomprinting technology has been recently developed to capture proteins, viruses and entire living cells via their structural and chemical information. Bioimprinting techniques can permanently capture an impression of biological samples into polymer surfaces with promising approaches for early cancer diagnosis [1,2], developing selective antimicrobial therapies and formulations [3,4]. Here we report a novel in-vitro approach for the removal of myeloblasts from peripheral blood samples of acute myeloid leukaemia patients utilizing a cell shape recognition technology. Due to size and shape differences between myeloblasts and normal white blood cells, myeloblasts represent an ideal target for bioimprinting. In this work, we have developed the bioimprinting technology to replicate myeloblasts (AML cells) based on their cell shape and size. Myeloblasts were inactivated with fixatives to preserve the cells structural and morphological information. Monolayers of fixed myeloblast cells were prepared by immobilisation on a polyelectrolyte pre-treated glass slides and partially protected by a film of glucose solution. Curable polymer (PDMS) was used to the imprint the exposed part of the cell monolayer and was peeled off after curing. Positive replica of the PDMS bioimrint of AML cell monolayers was prepared and the surface pattern was replicated on a large scale by using roll-to-roll printing on PET foil. These bioimprints were surface modified to promote weak adhesion to the myeloblasts which allow them to be trapped selectively on the surface of the bioimprint based on cell shape recognition. We present the results of our myeloblast cell recognition experiments as a function of the cell concentration and surface coatings of the produced cell imprints. The results indicate that the cell imprinting technology can be used to capture the AML cells based on their shape and size. We demonstrate the selectivity of the cell imprints in retention of the cells of matching shape in a mixture with other cells. The removal of myeloblasts from the normal white blood cells based on interaction with a negative bioimprinted surface which selectively attracts and retains myeloblasts. This technology is expected to find application in AML cell separation devices capable of removing myeloblasts from peripheral blood of AML patients which can lead to new blood cancer therapies.

[1] J. Medlock, A.A.K. Das, L.A. Madden, D.J. Allsup, V.N. Paunov, Chem. Soc. Rev, 2017, 46, 5110.
[2] K. Ren, N. Banaei, R.N. Zare, ACS Nano, 2013, 7, 6031.
[3] J. Borovicka, W. J. Metheringham, L.A. Madden, C.D. Walton, S.D. Stoyanov, V.N. Paunov, V.N., J. Am. Chem. Soc., 2013, 135, 5282.
[4] J. Borovicka, S.D. Stoyanov, V.N. Paunov, Nanoscale, 2013, 5, 8560.

Organs-on-chips constitute an emerging class of microfluidic models of functional units of human organs. They combine the advantage to provide a more controllable environment than in vivo models while generating a more realistic physiological and pathological behavior of cells than conventional in vitro models made for drug discovery ^1,2. Impedance spectroscopy is a widely accepted technique for dynamic quantification of tight junction integrity in cell culture models. It is mostly performed using a two electrodes configuration. The transepithelial/transendothelial electrical resistance (TEER) extracted is a strong indicator of cell barrier integrity for high throughput toxicology screening³. However, for organ-on-chips the measurement of ionic cell barrier resistance require cells to be cultivated on a flat porous membrane otherwise the high resistance of the channel masks TEER value. This configuration is limiting parameters like cell curvature and cell-matrix interaction, which improve cell barrier characteristics particularly in the case of blood brain barrier (BBB) models ^4,5. Here we show the use of PEDOT.PSS based organic electrochemical transistors (OECTs) integrated with a 3D model of the BBB for monitoring of endothelial cell barrier resistance with high resolution. Our device combines microvasculature made of collagen and human brain capillary endothelial cells (hCMEC/D3) with OECT in an optically transparent platform. The application of physiologically relevant fluid shear stress on the endothelial cells has shown to increase ZO-1 tight junction expression compare to usual 2D cell culture and can be related to cell resistance measurements. Rat astrocytes could also be integrate in the collagen structure in order to biochemically interact with endothelial cells allowing a more relevant BBB model. This platform will enable high-content screening for in vitro drug discovery and toxicology testing and could also be easily adapted to other tissue models like gut or intestine.
References:
1. Bhatia, S. N. & Ingber, D. E. Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760–772 (2014).
2. Esch, E. W., Bahinski, A. & Huh, D. Organs-on-chips at the frontiers of drug discovery. Nat. Rev. Drug Discov. 14, 248–260 (2015).
3. Srinivasan, B. et al. TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20, 107–126 (2015).
4. Ye, M. et al. Brain microvascular endothelial cells resist elongation due to curvature and shear stress. Sci. Rep. 4, (2014).
5. Adriani, G., Ma, D., Pavesi, A., Kamm, R. D. & Goh, E. L. K. A 3D neurovascular microfluidic model consisting of neurons, astrocytes and cerebral endothelial cells as a blood–brain barrier. Lab Chip (2017).

Mechanically compliant conductive materials for neural interfaces are desired both for recording and stimulation applications, including electroencephalography (EEG) measurements and deep brain stimulation treatments. However, whereas brain tissue is extremely soft (elastic modulus < 1 kPa) and dynamic, most inorganic conductors and dry conducting polymers are stiff (elastic modulus > 1 GPa) and inflexible. By contrast, hydrogels made with conducting polymers are promising soft electrode materials due to their high water content. We have developed a novel method for fabricating highly conductive hydrogels comprising two interpenetrating networks: one is a connected network of the conducting polymer PEDOT:PSS, while the second affords orthogonal control over the gel’s mechanical properties. With this method, ultra-low elastic moduli down to 8 kPa can be achieved without compromising stretchability (>100%) or conductivity (>10 S/m). We investigate the feasibility of these materials as electrodes for mechanically compliant neural interfaces.

In the last two decades, nanowire (NW) materials featuring nanoscale geometrical effect have attracted numerous attention for potential applications of highly sensitive chemical sensors and bioFETs. In the bioFET system, the mobile ion in aqueous solutions can be regarded as an important role in controlling the fundamental properties of liquid-NW interface, accordingly implying the importance of the ionic strength. Here, we demonstrated the electrical characteristics of NW FETs upon various ionic strength conditions of KCl aqueous solutions for giving more clear understanding of the effect of ionic strength on the electrical characteristics of NW bioFETs. The size of the NW width of our silicon NW FETs were defined by using the electron-beam lithography technique. In our liquid-gated bioFET system, a gold microwire was immersed in the aqueous solution for serving as a liquid-gated electrode for achieving an ideal gate-to-channel capacitive coupling system, accordingly resulting in the subthreshold swing approaching the limit value of 60 mV/dec. We also found that, when the NWs were exposed to the KCl aqueous solutions with different ionic strengths ranging from 10 μM to 0.1 M, the low-field carrier mobility decreased with increasing the ionic strength. Smaller the size of the NW width, more pronounced the decreasing trend in the mobility. This report gives insight into the importance of the ionic strength on the modulation of the electrical characteristics of NW bioFETs for chemical sensors, bioFETs, and bioelectronic applications.

Electrodes have been the gold standard for investigating neuronal signaling due to their high sensitivity and temporal resolution. However, the bulkiness of the electrode limits the spatial resolution and robustness of the tool. In this era, optical methods have become a sensible approach to measure the electrical activities of neurons by using an optical probe that transduces the electrical signal into an optical signal. Electrochromic materials such as Prussian blue, iridium oxide and PEDOT were investigated to determine which had the best optical properties to serve as a sensor for the measurement of extracellular action potentials. These materials are known for their biocompatibility, insolubility in water, and stability. We hypothesized that the electrical potential of excitable membranes will modulate the spectral properties of the material which will be detected through a differential photodiode detector. These materials were electrodeposited onto ITO-coated glass slides and their optical properties were characterized. As a model for electrophysiology measurements, a clone line of modified HEK 293 cells that stably express Na_v1.3 and K_IR 2.1 and generate spontaneous electrical action potential were used. Herein, we demonstrate the ability to detect the extracellular action potentials of HEK 293 cells and evaluate its potential for imaging network of neuronal cells.

Here, we present a nanoparticle platform capable of physiological imaging, triggered by excitation in the NIR windows I and II. This will be achieved with a nanoemulsion that is activated with a continuous wave light source at low power densities (~mW/cm2) for optical imaging, phototherapy, fiducial markers and photoacoustic imaging. The visible light produced from photon upconversion in this hybrid platform will enable wide-field monitoring for longer periods of time than current methods (i.e. days compared to minutes) with the same level of resolution as confocal microscopy. It will decrease phototoxicity and background scattering, with the advantages of multiphoton absorption microscopy at a fraction of the cost by eliminating expensive femtosecond pulsed lasers and laser scanners. The proposed nanoemulsions are composed of clinically approved fluorous phases, perfluorinated molecular emitters, and quantum dots (QDs). These nanoemulsions can be tailored in terms of size, surface functionality, and composition, thus facilitating the development of unique solutions to any imaging problems.

Multifunctional devices for optogenetic stimulation and neural recording may offer benefits to the basic study of the nervous system. Since optogenetic experiments rely on viral delivery of opsin genes and require visible light, an invasive two-step surgery is inevitable. As the majority of optogenetic studies still rely on commercially available silica fibers outfitted with metallic or semiconducting electrodes, the modulus mismatch between these devices and the brain tissue lead to profound foreign-body response posing a barrier to long-term opto-electrophysiology. Consequently, there remains a need for flexible and multifunctional neural probes that seamlessly combine viral delivery with optogenetic stimulation and electrophysiological recording in freely moving rodents.
Here we introduce an all-polymer probe that integrates an optical waveguide, 6 electrodes, and 2 microfluidic channels. This device produced via a thermal drawing process has a cross sectional diameter of 200 μm (smaller than typical silica fibers used for optical control of rodent behavior), and connectorized with optical, electrical and microfluidic interfaces weighs <0.5g. The choice of materials enabled low-loss optical transmission, and the development of a custom conductive polyethylene (CPE) composite with graphite (5% by weight) yielded electrodes with linear dimensions of 20-30 µm and impedance comparable to that of metallic microwires (100s kΩ) enabling electrophysiological recordings of isolated action potentials with high signal to noise ratio (SNR) during recording process. The probes maintained the optical, electrical, and microfluidic properties even under mechanical deformation.
The utility of our devices was confirmed by recording the optically-induced neural activity 2 weeks following the delivery of the adeno-associated virus carrying a gene for channelrhodopsin 2 into medial prefrontal cortex (mPFC) of wild type mice. Optical stimulation in the premotor area resulted in increase of locomotor activity consistent with ChR2-facilitated excitation. Multiple implantations were also performed to allow optogenetic studies of projections from the basolateral amygdala to the mPFC or the ventral hippocampus (vHPC). These circuits exhibited distinctly different latencies of optically evoked signals, and furthermore the activity was correlated to the behavioral response. Consistent with prior studies, stimulation of the BLA inputs into the vHPC resulted in a decreased time spent in the center during a standard open field test.
Finally, the flexibility of our probes was manifested in their enhanced biocompatibility as corroborated by reduced glial response and blood-brain barrier breach following up to 90 days of implantation. As our device allowed for minimally-invasive optogenetics in freely moving mice with a one-step surgery, we anticipate its future applications in systems neuroscience studies.

Early detection of melanoma is important to improve survival, however, the technology for accurate early diagnosis is still challenging. C-reactive protein (CRP) is an acute phase reactanthas produced by hepatocytes when inflammation occurs in body, and it was found to be associated with melanoma and could serve as an independent prognostic biomarker in melanoma patients. Particularly, increased levels of CRP in plasma were correlated with disease stage in patients with melanoma. Here we report a novel polymeric biosensor that could provide superior sensitivity in the detection of potential early biomarkers (e.g. CRP) of melanoma. The biosensor is composed of a highly specific molecular recognition core and a highly responsive transducer made of conductive polypyrrole (PPy) nanofibers.

We prepared a polymeric matrix by polymerization of acrylamide (AM), methylenebisacrylamide (MBAA), N-Isopropylacrylamide (NIPAAm) and CRP/CRP-aptamer complex first. Next, taking advantage of the porous structure of this NIPAAm-AM-CRP-aptamer/CRP polymeric matrix, we synthesized a polypyrrole-based conductive nanofiber structure using copper phthalocyanine-3,4',4",4""-tetrasulfonic acid tetrasodium salt (CuPT) as a dopant counterion in situ to achieve uniformly dispersed nanofibers within the polymeric network of NIPAAm-AM-CRP-aptamer/CRP. After removing CRP recombinant protein, we can obtain a robust CuPT-PPy/NIPAAm-AM polymeric sensor for detection of CRP with high sensitivity and selectivity. A two-step signal amplification cascades are involved in this CRP-specific polymeric sensor: 1) CRP binding-induced polymeric network shrinkage; 2) shrinkage-triggered conductance change of the polymeric network. Therefore, serum CRP levels could be quantitatively analyzed through monitoring the conductance change caused by polymeric network shrinkage upon aptamer-CRP binding. The limit of detection (LOD) of the polymeric sensor for detection of human recombinant CRP reached 10⁻¹⁹ M. The Fourier transform infrared (FT-IR) spectra confirmed the chemical structure of the polymeric network, and the morphology was observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results of FT-IR reveal that CuPT-PPy nanofiber was successfully synthesized in situ. The nanostructure of the nanofibers was clearly observed using SEM and AFM, indicating that the diameter of nanofiber is about 20 nm. This biosensor and a commercial CRP ELISA kit were used to perform side-by-side measurement of serum CRP in melanoma patients.

Our results indicated that this CRP-specific conductive polymeric senor is highly sensitive and selective in accurately discriminating melanoma patients from healthy controls. The serum CRP levels detected using sensor are correlated well with that obtained using ELISA. Collectively, such a flexible conductive polymeric biosensor may hold great promise as a point-of-care device in the diagnostics of melanoma and other cancers.

Nanoparticles could induce micron sized gaps between endothelial cells which we coined as nanoparticles induced endothelial leakiness (nanoEL effect) even without any tumor cells in the vicinity of the blood vessels cells; thus NanoEL is a novel nanoparticle driven effect distinct from the EPR effect. This presents an opportunistic window to control access of nanomedicine and drugs to tumors and other tissues without relying on the uncontrollable EPR effect. Nanoparticle parameters that determine NanoEL are slowly emerging but under intense investigation. In this study, we showed that the negatively charged Au nanoparticles could induce the NanoEL effect several fold more effectively than positively charged Au nanoparticles of the same size. This effect occurred in the absence of any cellular uptake of the particles which suggested that the action occurs external to the cells. We showed with in silico modelling that negatively charged particles were repelled successively from the negatively charged glycocalyx surface of the endothelial cells towards the cell-cell junction. The high density of the Au particles disrupted the critical VE-cadherin interactions that hold neighbouring endothelial cells together and resulted in gaps that are at least 1000 fold larger in the hundreds of micron ranges.

Functional bio-electronics devices have attracted considerable attention for many years. One of the critical elements in device architecture is the interface material, known as bio-electrode, which bridges the signal communication between neural cells and inorganic semiconducting devices. In our laboratory, we have developed a variety of wet chemical baths for IrO₂ deposition on different substrates. In this work, we demonstrate the deposition of IrO₂ on active retina chips for cell culturing and we will present that the as-deposited IrO₂ enhances the growth of selective cells.

Cancer metastasis is one of the leading cause of death of breast cancer patients but currently there is lacking effective cure. Addressing this grand challenge, several promising chemotherapeutic reagents that target metastatic cancers have been successfully developed. Still, their long term clinical outcome has not been satisfactory and is significantly hurdled by major obstacles such as multidrug resistance (MDR), which is developed during repeated drug treatment. Additionally, due to the complex nature of tumor migration, and the strong side effects of anti-tumor drugs, MDR can be further compounded by the heterogeneous tumor microenvironment, which make it extremely challenging to prevent recurrence of metastasis.
To this end, we developed a hybrid multifunctional nanoparticle (Fe₃O₄@C@MnO₂) based drug delivery system (DDS) for effectively killing metastatic cancer in vitro and in vivo. Unlike conventional nanoparticle based anticancer strategies, our hybrid nanoparticle based DDS not only target-specifically delivers anti-cancer reagents, but also downregulates important drug resistant pathways to achieve sensitization of cancer cells. More specifically, the multimodal photothermal effect of nanoparticle induces high and long-term expression of heat shock factor-1 (HSF-1) trimers in a breast cancer metastaic model, downregulates NF-κB and further suppresses anticancer drug effluxing machinery through MDR-1/P-gp expression. Meanwhile, MnO₂ shell reacts with intracellular GSH in cancer cells, enabling target-specific drug release, generating Mn²⁺ for deep tissue tumor imaging, and directly sensitized metastasis cancer by modulating redox inside cancer cells in situ. Furthermore, iRGD conjugated on our hybrid nanoparticle significantly enhances the tumor homing and penetration, thereby increasing cancer-killing efficiency and reducing side effects simultaneously. By targeting heterogenous MDR pathways in metastatic cancers and delivering anti-cancer drugs in a single platform, our multifunctional hybrid nanoparticles represent a unique and promising solution for treating metastatic cancer in vivo.

The hybrid composites which consist of PEDOT:PSS/graphene oxides and PEDOT:PSS/reduced graphene oxide, were successfully constructed on Au microelectrodes. The graphene oxide was synthesized by using modified Hummer’s methods. The PEDOT:PSS/(r)GO composites were coated by electrochemical chronopotentiometry methods. The cyclic voltammetry test, biphasic electrical stimulation, electrochemical impedance spectroscopy, and durability test were performed to evaluate the stimulating efficiencies for the neural micro-electrodes. The electrochemical performances were also compared by varying the degree of reduction of GOs. Overall, PEDOT:PSS/(r)GOs composites were significantly better than the PEDOT:PSS in terms of electrochemical charge transports as well as charge injection durability.

This work presents the concept of silicone gasket as solid underfill for insulation of high-density interconnections of the polyimide based electrodes array and hermetic electronics package in an active implant. Two conventional underfilling techniques i.e. the capillary flow fluids and no-flow pastes are widely used as underfills in microsystems. The former one utilizes viscous polymers like silicone or fluidic expoxies and work on the principle of capillary flow for underfilling the narrow gap. The no-flow underfilling is a pre-bonding process and uses expoxies or adhesive pastes with good adhesion properties, especially in aqueous environment if targeted for physiological conditions. However, the existing technologies suffer from limitations regarding long-term reliability. The prominent challenges are achieving long-term adhesion with the polymer array and hermetic package, voids formation due to flow limitations or solvent evaporation and leakage between interconnects in long-run due to contamination of bonding residues. Most of the epoxies for no-flow either contain ions featuring hygroscopic properties or are not biocompatible when featured with good adhesion in aqueous environment.
Silicone rubber being biocompatible is widely used as capillary-flow underfill and encapsulation material for implants and has established adhesion properties for some other materials e.g. glass. Introducing the voids-free pre-structured silicone sheet as underfill gasket between the hermetic package and the polymer array allows proper cleaning of the surfaces with solvents as well as oxygen plasma at each assembly step. A 20μm thick silicone sheet is fabricated by spin coating polydimethylsiloxane (PDMS) precursor on a carrier tape. Openings (200μm in diameter with 400μm pitch) for interconnection pads are structured with picosecond laser. The resulting gasket is surface-activated with 80W oxygen plasma and aligned on the metallized ceramic substrate (electronics package) under microscope. In next step, the top surface of the gasket and the mating-side of polyimide array (with interconnect-pads at backend) are cleaned with plasma and properly aligned and left under weight for firm mechanical bond. The interconnect pads on the polyimide array and electronics package are electrically bonded with Microflex. The assembly is finally encapsulated with silicone in a custom mold. The gaskets in assemblies sustained high insulation resistance in phosphate buffered saline solution (PBS) at 60 °C. Insulation resistance between adjacent interconnects changed from 15.7 ± 0.62 MΩ to 11.8 ± 0.14 MΩ (1kHz electrochemical measurements) after 11 weeks of accelerated aging. In contrast, the reference samples with PDMS flow-underfill and epoxy encapsulation shown a large drop during initial few weeks. The gasket also retained high insulation of 5.8 MΩ while subjected to 1800 billion pulses (±1mA) in PBS and above 10 MΩ when stored in 20 mM H₂O₂ solution in PBS at 60 °C (4 weeks).

Controlling the interface between hard and soft moieties can produce functional materials and assemblies for imaging and sensing, regenerative medicine, and optoelectronics. Genetically engineered polymers enable design of specific and tunable materials properties at the DNA level with control over function and structure. The precise control afforded by these polymers, coupled with their biocompatibility, programmable assembly, and flexibility, transforms our ability to engineer materials for applications such as “smart skins,” and self-healing materials, and for the interfacial control with biological systems. We have created large (10^8) and diverse libraries of genetically encoded polymers and rapidly identified functional materials using a genetic technique akin to evolution. We will present our libraries, selection strategies and downselected polymers that induce mesenchymal stem cells to differentiate toward chondrocytes. Additionally we will present recent results tuning these genetically encoded polymers with electronic properties.

Oligonucleotides have emerged as extremely important molecules in nanoscience. With their unparalleled programming and self-assembly characteristics, these materials have allowed the development of many precision nanoscale systems. However, the primary importance of these molecules has remained in their biological function. In particular, RNA has recently attracted significant interest for a variety of applications: the sequences found in vivo provide direct information about gene expression, its role in cell regulation has become evident, and the fact that it is naturally amplified inside the cell makes it an ideal target for sensor applications. For all of these reasons it is important to understand the electronic properties of both DNA and RNA duplexes. While the electronic properties of DNA have been the subject of intense research over the last several decades, and incredible progress has been made using photochemical and electrochemical measurements to understand DNA’s charge transfer characteristics, studies into the charge transport properties of RNA have been more limited. Here we report on single-molecule conductance measurements performed on DNA duplexes and RNA:DNA hybrids using the Single-Molecule Break Junction (SMBJ) approach with the goal of creating a measurement platform capable of identifying specific pathogenic serotypes from an electrical conductance measurement of microbial RNA. In moving toward this goal, we will discuss the necessity of controlling the local environment of DNA and RNA in order to obtain reproducible conductance values and understand the inherent charge transport properties. We will explore the role of secondary structure on the charge transport characteristics by controlling this environment, and compare the results with alternative duplexes such as RNA:DNA hybrids. We will also discuss the effects of sequence changes and the roles of A:T, A:U, and G:C base pairs within the stack on the transport characteristics. Finally, we will discuss how understanding charge transport in these systems can be leveraged to develop functional sensor technologies, and demonstrate that single-molecule conductance measurements can be used to identify biologically relevant sequences for specific serotypes of pathogenic species such as E. coli. This work opens new possibilities for electrically-based sensor and diagnostic platforms for food safety, water and environmental protection, plant and animal pathology, clinical diagnosis and research, and bio-security.

Natural systems utilize multifunctional biocomposites by a bottom-up self-assembly of nanomaterials for creating multiscale, hierarchical, and multiphasic structures. While conventional man-made synthetic composites increase one functionality by sacrificing the others, the biocomposites often synchronistically maximize multi-funcitonality. Here, by molecularly organized layer-by-layer assembly as well as thermodynamically driven orientation, we will introduce multifunctional nanocomposites, recently developed from our lab, from natural biomaterials including high crystalline cellulose nanofibers, naturally extracted electrically conductive melanin nanoparticles, and photosynthetic protein complexes. High electrical conductivity, high mechanical strength, and photoelectric sensibility are demonstrated by combination with biocompatibility. These composites can be used as key functional materials in wide range of biomedical device applications such as implantable circuits, biosensors, drug delivery carriers, and neural interfaces.

Cephalopods possess remarkable camouflage capabilities, which are enabled by their complex skin structure and sophisticated nervous system. Such unique characteristics have in turn inspired the design of novel functional materials and devices. Within this context, recent studies have focused on investigating the self-assembly, optical, and electrical properties of reflectin, a protein that plays a key role in cephalopod structural coloration. Herein, we report the discovery that reflectin constitutes an effective material for the adhesion, proliferation, and differentiation of neural stem/progenitor cells, with an efficacy comparable to that of extracellular matrix proteins that possess cell binding domains. Our findings may hold relevance for developing novel neural stem/progenitor cell substrates and improved protein-based bioelectronic devices.

Cephalopods (squid, octopuses, and cuttlefish) have captivated the imagination of both the general public and scientists for more than a century due to their visually stunning camouflage displays, sophisticated nervous systems, and complex behavioral patterns. Given their unique capabilities and characteristics, it is not surprising that these marine invertebrates have recently emerged as exciting models for novel materials and systems. Within this context, our laboratory has developed various cephalopod-derived and cephalopod-inspired materials with unique functionalities. Our findings hold implications for next-generation adaptive camouflage devices, sensitive bioelectronic platforms, and advanced renewable energy technologies.

The photosynthetic apparatus is substantially located in the thylakoids. There is interest in stimulating this apparatus and extract photoelectrons through attached electrodes and use them as electric power sources. The thylakoid membrane is however not a well conducting electrolyte. We present an experimental study on spinach derived thylakoids deposited on gold electrodes and on TiO2 electrodes. The dark currents and light currents are determined under illumination with light ranging from 625nm to 470 nm under various DC bias. The expermentally obtained photocurrents scale with wavelength generally in analogy to the UV-vis absorption spectra. The photoelectrochemical response suggests that there is a chromatic change of the thylakoid layer towards longer wavelengths which can be attributed to the adsorption of the thylakoids on the electrode, in contrast to thylakoids in solution. The impedance spectra give account of the stark electric blocking properties of the lipid layer, underlining the necessity to further "widen" the gates for electrons in the thylakoid membrane. At first sight, there seems to be no influence of the nature of the electrode material (metal vs. metal oxide) to the effectivitiy of the charge transfer.

During photosynthesis plants and algae use Photosystem I (PSI), a supra-molecular protein complex, to harness solar energy with 100% quantum efficiency and drive stable charge separation. The unique photoactive electrochemical properties of PSI make it a promising candidate for applications in biohybrid materials and devices. Many works have achieved organized two-dimensional assemblies of PSI for photovoltaic electricty or solar fuel generation. However, three-dimensional architecture to house PSI, such as the native folded thylakoid membrane, has yet to be achieved in artificial systems. Metal organic frameworks (MOFs) are an increasingly explored category of hybrid material with the unique properties of high crystallinity and exceptionally high porosity, which allow for diverse functionality whose applications are rapidly expanding. These materials are formed from solutions of metal ion nodes connected by organic linkers to form infinite 3D porous networks. This work employs the zinc-based MOF, ZIF-8, as a scaffold to both coordinate 3D assembly of PSI and serve as a protective coating due to its robust chemical and thermal stability. Optimized reaction conditions for a mixture of the MOF constituents and PSI together in solution lead to the formation of ZIF-8 microparticles encapsulating PSI trimers. X-ray diffraction and BET isotherms confirm the quality and porosity of our hybrid material. The ensuing changes in absorption and fluorescence behavior of embedded PSI contribute to our understanding of chromophore network confinement. Pump-probe spectroscopy was utilized to measure the electron turnover rate of PSI, establishing the activity of ZIF-8/PSI compared to free PSI in both benign and protein denaturing environments.

Recent advances in mechanics and materials have allowed for a class of electronics that intimately couple to the soft, curvilinear surfaces of biological tissue. The low thermal mass, low contact resistances and ultrathin construction of these devices offer a compelling platform with which to thermally characterize living, biological systems. This talk summarizes a class of soft, bio-integrated electronics that combines low-power thermal actuation schemes with precise temperature sensing that has broad applications in dermatology, cardiovascular health, neurosurgery and neuroscience. Theoretical and experimental results uncover the physics of thermal transport in these systems and their versatility is illustrated through two target applications: (1) Skin mounted, ‘epidermal’ shunt failure monitors for hydrocephalus patients and (2) soft, implantable ‘cuffs’ for peripheral nerve interfaces that track nerve health.

Existing techniques for measuring oxygen concentration in blood heavily relies on non-invasive transmission-mode pulse oximetry - a ratiometric optical sensing method, where light absorption in oxygenated and deoxygenated blood is interpreted to a person’s oxygen saturation (SpO₂). Since transmitted light through tissues is used to generate the signal, transmission-mode pulse oximetry is restricted to only tissues that can be transilluminated, such as the ear and the fingers. Here, we present a reflection oximeter, which uses printed organic light-emitting diodes (OLEDs) and organic photodiodes (OPDs) to sense reflected light from tissues to determine the oxygen concentration. Using the reflection-mode, the sensor can be used beyond the conventional sensing locations. We used the reflection-mode sensor to measure SpO₂ on the forehead with 1.1% mean error. We also demonstrate a method to determine oxygen saturation in the absence of pulsatile blood. Additionally, printing techniques are utilized to fabricate the sensor on flexible plastic substrates, making the sensor both comfortable to wear and efficient at extracting high-quality biosignal.

The modulation of biological activities by non-invasive remote manipulation is object of intense research efforts that, in the latest years, led to the development of different stimulation approaches. For instance, magnetic fields, ultrasounds, heating, electric fields, and light irradiation can be used in combination with smart nanomaterials, that are thus exploited as real “nanotransducers” [1].
A new rationale in nanomedicine is emerging, consisting in the exploitation of the intrinsic properties of nanoparticles as active devices rather than as passive structural vectors for medications. According to this paradigm, the nanomaterial itself becomes the active "smart" device that responds to external stimuli by modifying its intrinsic chemical and/or physical characteristics, and that provides useful bio-stimulation and/or bio-signaling.
In this talk, piezoelectric nanomaterials and their applications in the nanomedicine field will be introduced, with particular attention to tissue engineering and regenerative medicine. Despite their impressive potentials, in fact, this kind of nanostructures has not yet received significant attention for bio-applications. Our results suggest that the exploitation of piezoelectric nanoparticles in nanomedicine is possible and realistic, and their impressive physical properties can be most useful for several applications, that range from sensors and transducers for the detection of biomolecules, to “sensible” substrates for tissue engineering or cell stimulation [2-3].
After a short introduction to the major classes of innovative piezoelectric nanoparticles that have gained interest in the recent years, attention will be focused on the research carried out in our laboratories, introducing barium titanate nanoparticles, boron nitride nanotubes, and polymeric composites based on these nanomaterials [2-6].

[1] Genchi G.G., […], Ciofani G. Advanced Healthcare Materials, 6(9): 1700002 (2017)
[2] Marino A., […], Ciofani G. ACS Applied Materials and Interfaces, 9(21): 17663-17680 (2017)
[3] Marino A., […], Ciofani G. Nano Today, 14: 9-12 (2017)
[4] Genchi G.G., […], Ciofani G. Advanced Healthcare Materials, 5(14): 1808-1820 (2016)
[5] Genchi G.G., […], Ciofani G. Nanomedicine: Nanotechnology, Biology and Medicine, 10.1016/j.nano.2017.05.006
[6] Marino A., […], Ciofani G. ACS Nano, 9(7): 7678-7689 (2015)

This research is partially supported by the Compagnia di San Paolo Starting Grant Number 55_AI16GC01.

In pursuing nanomaterial-based approaches for contactless neural stimulation, we develop ZnO nanowire-based materials and study their biological effects and energy transducing capability. Although ZnO has been generally regarded as biocompatible and used in numerous biomedical device designs, our study finds that it can selectively induce higher inhibitory effects on cell lines (rapidly dividing cells) than primary cells in the nanowire array form. We trace the primary mechanism to be ZnO's capability of generating reactive oxygen species (ROS) when in contact with cell culture medium as a result of its semiconducting property. This observation points out its potential use as a material agent for treating tumors. To further optimize the material's properties toward this application, we coat biocompatible conducting polymer polypyrrole around these nanowires to form a composite of ZnO-nanwires/polypyrrole and find an interesting photocatalytic property, which opens up a new material-based opportunity for using optical stimulation to selectively suppress tumor growth. This new photodynamic therapy has four prominent features: (1) Its inhibitory effects can be turned on and off using an external light source: without illumination, the composite material is biocompatible, as it is coated with polypyrrole, while it produces ROS upon illumination. (2) Its efficiency of ROS production can be finely controlled by the intensity of the optical stimulus and the coating thickness of polypyrrole. (3) Under illumination, this composite material has a much higher efficiency than a pure ZnO nanowire array in producing ROS. And (4) monitoring of the amount of ROS produced is conveniently achieved by recording the photovoltaic current, providing an integrate solution for closed-loop control.

Abstract

The limited light penetration depth has tremendously hindered the clinical translation of nano-photothermal therapy (NPTT) even with prominent characteristics of local treatment and high efficiency. On the other hand, failure of intraoperatively eliminating microscopic residual disease (MRD) during standard surgery causes lethal locoregional recurrence and postoperative metastasis for breast cancer. Herein, we have applied NPTT assisted with multimodality imaging and surgery navigation for intraoperatively eliminating MRD in the surgical bed without limitations of laser penetration depth and preoperatively cleaning tumor margins that facilitate the surgery. Remarkably, the synthesized “all-in-one” nanoparticles (NPTT, multimodality imaging and targeting) were found aggregated mainly within the tumor cells in vitro and in vivo, and exhibited facile and high accurate elimination of tumor cells in microscopic level. For resectable MRD, NPTT after fluorescence imaging-guided surgery prevented local recurrence and delivered 100% tumor-free survival. Furthermore, for unresectable MRD, NPTT could delay local tumor recurrence and improved animal survival by nearly twofold compared with standard surgery. Moreover, in tumors initially diagnosed as non-operable immediately, preoperative NPTT could shrink the cancerous lesion and cleaned tumor boundary that could enable surgery. Finally, we have self-made two sets of clinic equipment (open surgery and laparoscopic surgery) for translating adjuvant NPTT to the clinic, combining surgery navigation and NPTT, and obtained excellent imaging and therapeutic effects in vitro. Our work provides a new perspective for the clinical translation of NPTT in the preoperative and intraoperative treatment, and will certainly promote further applications of NPTT in various cancers.

Key References

1. Yan, H.; Kang, F. et al. General synthesis of high-performing magneto-conjugated polymer core-shell nanoparticles for multifunctional theranostics. Nano Res. 2017, 10 (2), 704.
2. Yan, H.; et al. Controlled Synthesis of Fe₃O₄ Single Crystalline Spheres in One Solvothermal System and Their Application in MRI. J. Nanosci. Nanotechnol. 2017, 17 (3), 1983.
3. Du, Y.; Tian, J. et al. DNA-Nanostructure-Gold-Nanorod Hybrids for Enhanced In Vivo Optoacoustic Imaging and Photothermal Therapy. Adv. Mater. 2016, 28 (45), 10000.
4. Shang, W.; Tian, J. et al. Core-Shell Gold Nanorod@Metal-Organic Framework Nanoprobes for Multimodality Diagnosis of Glioma. Adv. Mater. 2017, 29 (3), 1604381.
5. Yan, H.; Tian, J.; Kang, F. et al. “All-in-One” Nanoparticles for Tri-modality Imaging-Guided Intracellular Photo-magnetic Hyperthermia Therapy under Intravenous Administration. Adv. Funct. Mater. 2017, minor revision.

The goal of a biological modulation device is to alter the biochemical or biophysical signal flow in single cells or whole tissues in order to obtain a therapeutic benefit. Existing device examples include deep brain stimulators for the treatment of Parkinson’s disease and tremor, and spinal cord stimulators for chronic pain. While these devices have improved patients’ quality of life—and indeed have saved millions of lives—they are often limited by their bulkiness, mechanical invasiveness, and inability to target single cells. Our lab recently developed a few silicon-based free-standing materials and devices that form modulative interfaces with biological components. In this talk, I will discuss the utility of these interfaces by showing light-controlled non-genetic modulations of intracellular calcium dynamics, cytoskeleton-based transport and structures, cellular excitability, neural transmitter release from brain slices, and brain activities in vivo.

It is well established that the key to minimizing diabetes-associated complications, in both type 1 and type 2 diabetes, is tight regulation of blood glucose levels. Currently the major approach for regulating blood glucose levels in patients with diabetes relies on external blood glucose monitors. Conventional self-testing methods require a drop of blood for each glucose measurement. Poor patient compliance usually results in limited insights into the dynamic range of blood glucose levels and may lead to hyperglycemia or hypoglycemia. For point-of-care (POC) purposes, continuous glucose monitoring (CGM) devices are considered to be the best candidates for diabetes therapy. Consequently, there has been, and continues to be, considerable investment in the development of minimally-invasive continuous glucose monitoring technologies. The aim of this study is to test and develop a minimal-invasive CGM interstitial fluid (ISF) sensing device based on 600µm long, silicon micro needles dermal sensors. These silicon micro needles are equipped with nano-sensor field effect transistors at their tips, embedded in Glucose Oxidase containing hydrogels which catalyzes the oxidation of glucose to hydrogen peroxide affecting the electrical field around the nanowire and changes its conductivity. Once CGM development is establish, we believe our ISF sensing device will also allow the monitoring of a variety of metabolites in the blood quickly painlessly.

The large interest in graphene-based materials and other carbon-based polymorphs and nanomaterials are truly far-reaching. The industrial scale and possibility of applying these to advanced manufacturing and commercial application have resulted in high throughput synthesis and lower costs. There is primary interest on their electrical and thermal conductivity properties. However, other applications are based on their ability to form very stable colloidal complexes. This talk will focus on the class of graphene (G) and graphene oxide (GO) nanomaterials that have been prepared with high loading in polymer matrices and ultrathin films to result in: anti-corrosion coatings, semiconductor thin films, patterning, and anti-microbial properties. We describe the preparation of very stable dispersions that can be used for nanopatterning in films as well as anti-corrosion-superhydrophobic coatings. Interestingly, by preparation of colloidal particles that can be stabilized at liquid-liquid and air-liquid interfaces, functional Janus type nanoparticles can be prepared which have interesting uses in dispersants, rheological modifiers, and smart fluids.

Implantable medical devices, including pacemakers, cochlear implants, neural recording devices, and deep brain simulators, touch every part of the human function. These devices need to communicate with external acquisition equipment that supervise the status of the device and transmit patient data. An antenna is an essential component of this wireless communication system. The size of the antenna is determined by the frequency of the transmitted signal. Specifications for an appropriate half wave antenna in the MedRadio band (402-405MHz) are a length of 36 cm in air and 6 cm or more if it is implanted in the body. These antennae are significantly larger than the implanted device itself (in mm scale). Traditional antenna design restricts the antenna on the medical devices. Our recent novel work has developed a subdermal antenna remotely coupled with a small feed on the medical devices. This approach radically increases the options for antenna size, shape, and configuration (similar to wireless charging technology). However, current materials are mechanically or biologically unsuitable for subdermal applications. This study is focused on designing a flexible, conductive, and biostable nanocomposite for subdermal antenna.

The extracellular matrix (ECM) provides tissues and organs suitable mechanical properties and chemical signals to trigger cell adhesion and differentiation. In tissue engineering, biomaterials provide the cells with an appropriate environment. Fibrillar proteins from the ECM (collagen, fibronectin) and synthetic peptide-based biopolymers (e.g. Peptide Amphiphile (PA)) are widely used as structuring scaffold.^[1] PAs that self-assemble into microfibers are biocompatible, biodegradable and can incorporate signaling motifs from the ECM within their peptide sequence. This way the bioactive signals are displayed to cells to control their behavior.
Biological events are dictated by spatiotemporal heterogeneities of biomacromolecules, which means that the variation of the local concentration of biomolecules is a key signal to trigger a given cell behavior. It is then necessary to engineer biomaterials with well-defined clusters. This can be achieved by a composite approach where bio-inspired polymers are combined with nanoparticles. To this aim, silica particles (SiNP) are very interesting candidates due to their low cost, biocompatibility, easy synthesis and surface functionalization.^[2] Through the incorporation of bioconjugated SiNP into PA scaffolds, we aim at providing a unique way of tuning the scaffold bioactivity with improved modularity, by adjusting (i) the grafting of a bioactive peptide , (ii) the concentration of SiNP within the matrix, and (iii) by combining SiNP grafted with different peptide epitopes.
In this work we show how the 3D spatial positioning of the fibronectin derived epitope RGDS affects cell response. Fibroblasts cultured on RGDS-grafted SiNP@PA matrix where found to be more efficiently spread compared to RGDS-presenting PA nanofiber. Indeed, the required amount of RGDS signal was four times lower for RGDS-SiNPs@PA than for the RGDS-PA for a similar cell spreading. This is attributed to the clustering of the bioactive epitope at the SiNP surface. In addition, the hybrid SiNPs@PA material offers the possibility to incorporate several biological signals to act synergistically. This is particularly interesting when working with the ECM motifs RGDS and PHSRN that operate in a spacing-dependent manner to promote cell adhesion and spreading. We show that when the two peptides are on the PA matrix or grafted on two different populations of SiNPs, fibroblasts show no spreading. This can be improved when one is on SiNP and the second one on PA ensuring the close interaction between RGSD and PHSRN. The synergic effect is further improved when both peptides are grafted on the same particle.
This 3D biocompatible material displays all necessary signals in a controlled way without altering the self-assembly and mechanical properties of the biopolymer, being a unique model to mimic the natural ECM in a tissue engineering point of view.

[1] Hartgerink, J. D. et al. Science 2001, 294, 1684
[2] Aimé, C. et al. Nanoscale, 2012, 4, 7127

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina’s foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3⁺–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

Pro-oxidant therapy using chemical agents that can encourage oxidative stress in cancer cells has been attempted for cancer-specific therapy. Piperlongumine (PL) is known as an innovative pro-oxidant agent because it shows cancer-specific cytotoxicity via elevation of intracellular ROS in cancer cells through inhibition of PI3K/Akt/mTOR pathway. However, therapeutic utility of PL is hampered by its poor water-solubility, which requires appropriate drug carriers for PL delivery. In this study, poly(ethylene glycol)-poly(histidine) [PEG-poly(His)] copolymer was synthesized and formulated as micelles with PL. It was demonstrated that the PL-loaded PEG-poly(His) micelles [PL-PEG-poly(His)] induce pH-sensitive drug release due to protonation of the imidazole groups in the poly(His) under acidic conditions. Plain PEG-poly(His) micelles without PL were innocuous, indicating their high biocompatibility. Interestingly, PL-PEG-poly(His) micelles exhibited remarkable cytotoxicity in cancer cells over normal cells. PL-PEG-poly(His) micelles were further modified with folic acid (FA) to enhance their cancer-specificity. As compared to FA-free micelles, FA-PL-PEG-poly(His) micelles manifested more increased cellular uptake and anticancer efficacy against folate receptor-positive cancer cells. This study demonstrates that PL-PEG-poly(His) micelles have great potential as effective PL delivery and cancer-targeted pro-oxidant therapy.

For the interconnection between a synthetic material and living matter, hollow mesoporous silica particles have gained intense attention due to their high biocompatibility and stability in biological milieu. Related to their inner core they exhibit a high loading capacity suitable for drug delivery vehicles. In this work ellipsoidal hollow mesoporous silica (HMS) capsules were synthesized via hard template method, using ellipsoidal hematite particles as core material. The hematite core was synthesized through a solvothermal process, coated with a silica sol followed by acidic leaching, leading to HMS capsules. The porosity of as-prepared particles was analyzed using nitrogen adsorption-desorption method revealing a pore size of circa 4 nm and a high surface area of 308.8 m²/g. To determine cytotoxicity, cell viability test (MTT) towards human kidney cells (HEK293) was performed clearly demonstrating that no reduction of cell viability was observed even at high concentrations of 100 µg/ml. Uptake studies using confocal microscopy were carried out using human cervical cancer cells (HeLa) which could show the successful internalization over a period of 24 hours. For testing their capability as drug delivery vehicle, a hydrophilic antibiotic (ciprofloxacin) and a hydrophobic anticancer (curcumin) compound were loaded and a pH dependent release under physiological conditions at 37°C was monitored via UV-Vis spectroscopy. Ciprofloxacin-loaded HMS particles with a concentration of 10 µg/ml were also tested towards gram negative bacteria (E.coli) revealing a complete growth inhibition over 18 hours. This study demonstrates the suitability of as-prepared hollow silica capsules as drug delivery vehicles for a broad range of drugs.

The discovery of self-assembling peptides, has opened a realm of opportunity for designing short peptide sequences which have applications in regenerative medicine for tissue repair, scaffolding for tissue engineering, drug delivery and sutures. The properties of such peptides, namely; their complex and complementary structures, biocompatibility, chemical versatility and biological recognition abilities have lead them to their versatility. In this study, aromatic oligopeptide sequences were examined for their ability to form structures such as nanofibers, nanospheres as well as cationic, amphiphilic peptides which self-assemble into micelles and their applications as targeted drug carriers. Non-covalent interactions such as π-stacking, hydrogen bonding, and hydrophobic interactions promote peptide self-assembly and the resultant architectures can vary as nanofibers, nanospheres or nanotubes; based on the composition of the short peptide strands and the dominant non-covalent forces.
In this study, the influence of electrostatic forces on the assembly of biodegradable nanofibers and nanospheres was studied. Mass spectroscopy to understand the changes in the chemical composition, infrared spectroscopy (FT-IR) and circular dichroism (CD) spectroscopy to study the secondary structure along with differential scanning calorimetry (DSC) to investigate the thermal behavior of the obtained fibers and spheres were performed. Other amphiphilic peptide sequences were designed for the formation of micelles for applications as drug carriers. Polar flavonoids/polyphenols, including quercetin and pro-anthocyanidins, and non-polar triterpenoids including ursolic acid and derivatives are the two main categories of cranberry fruits natural compounds which were studied. Self-assembled micelles or other peptide-based carriers deposited via electrospraying techniques represent a possible route to target delivery of the flavonoids to tissues and organs of interest. Response to the external stimuli such as pH, temperature and presence of enzymes are some of the advantages that these nanostructures provide for drug delivery systems. We have begun to explore the fabrication of these structures, ability of drug loading and their mechanisms of drug release and their biodistribution both in vitro and in vivo. We will assess the suitability of these natural compounds for micellar delivery, the extent of delivery to cells, and whether tumor cell proliferation is reduced as a result.

Iridium oxide is an attractive material for bio-interface applications due to its desirable stability, electrochemical performance, and biocompatibility. Nanostructured iridium oxide possesses several advantageous properties including large surface to volume ratio, light weight, super hydrophilic surface, desirable electrochemical capability, and cell adhesion. Herein, we employ a reactive sputtering process to fabricate uniform nanostructured iridium oxide thin film. The iridium oxide nanotree thin film undergoes electrochemical analysis in charge storage capacity (CSC) and electrochemical impedance to evaluate its potential as stimulating electrodes for implantable devices. Image from scanning microscopes confirm the formation of uniform iridium oxide nanotree. In addition, the cycling lifetime of iridium oxide nanotree thin film is evaluated by performing CV scans for 1,000 cycles. The iridium oxide nanotree thin film reveals large CSC values and low electrochemical impedances which are attributed to its dendritic nanostructure with high surface area. To sum up, these excellent characteristics presented for the iridium oxide nanotree thin film make it a promising candidate for the implantable stimulating electrodes and other bio-interface applications. In addition, the finding in this work is expected to make impact on bioelectronics medicine applications given the significant electrochemical performance.

The ability to correlate single-cell genetic and protein expression information to cellular phenotypes will provide the kind of detailed insight into human physiology and disease pathways that is not possible to infer from bulk cell analysis. This capability is particularly pertinent to understanding cancer biology, given the highly heterogeneous nature of the disease. This presentation emphasizes the development of nanomaterials in my lab to enable a set of single-cell technologies for studying complex tissues and disease.

Bacterial cultures exposed to iron-doped apatite nanoparticles (IDANPs) prior to the introduction of antagonistic viruses experience up to 2.3 times the bacterial destruction as compared to control cultures. Maximum antibacterial activity of these bacteria-specific viruses, or phage, occurs after bacterial cultures have been exposed to IDANPs (0.77 mg/mL) for 1 hr prior to phage introduction, demonstrating that IDANP-assisted phage therapy would not be straight forward, but would instead require controlled time release of IDANPs and phage. These findings motivated the design of an electrospun (ES) nanofiber mesh treatment delivery system that allows burst release of IDANPs, followed by slow, consistent release of phage for treatment of topical bacterial infections. The Centers for Disease Control and prevention estimate that at least 2 million people in the United States become infected with antibiotic-resistant bacteria and at least 23,000 people die each year as a direct result of those infections. As an alternative to traditional antibiotics, phages capable of exponential bacterial destruction have been used. Phages kill bacteria through biological processes that differ from traditional antibiotics, and therefore can avoid bacterial resistance. IDANP adjuvants have increased phage killing of gram positive and gram negative strains of bacteria, and with RNA, DNA, contractile tailed, and non-contractile tailed phages. IDANPs resemble hydroxyapatite (HA), a biocompatible mineral analogous to the inorganic constituent of mammalian bone. Our cytotoxicity evaluations have shown minimal to no toxicity imposed by IDANPs in three mammalian cell lines after 24 hr of exposure. The composite nanofiber mesh we designed for IDANP-assisted phage therapy treatment of topical wounds consists of a superficial, rapid release layer of polyethylene oxide (PEO) fibers doped with IDANPs, followed by deeper, coaxial PEO/polycaprolactone (PCL) blend polymer fibers for slower phage delivery. Our recent investigations have established that IDANP-doped PEO fibers are effective vehicles for dissemination of IDANPs for bacterial exposure and resultant increased bacterial death by phage. While PEO dissolves immediately upon exposure to an aqueous environment, PCL dissolves over several weeks to months in these systems. At present, PEO/PCL blends are being coaxially ES into fibers containing a microfluidic channel meant to protect viable phage and to release phage approximately 1 hr after submersion in an aqueous system. Although phage exposure to solvents is minimal during electrospinning, it may be desirable to mitigate phage exposure to solvents which render the phage incapable of infecting bacteria. Melt electrospinning does not require solvent, but instead utilizes solid polymer which is melted by heat prior to fiber deposition. Bandages fabricated by solution or melt electrospinning will be evaluated for efficacy by plaque assay, and cytotoxicity by XTT and LDH assays.

Nature abounds with mysterious biological creatures and organisms that exhibit unique surface wettability, such as lotus leaves with the self-cleaning property, rice leaves and butterfly wings with directional adhesion property, the mosquito eyes with antifogging functionality, and the Namib desert beetle and spider silk with water collection ability. Here, a full-range droplet manipulation system with a extreme wetting substrate is developed for the multiplex bioassay of a single-droplet sample. To fabricate the extreme wetting substrate, adhesive and superomniphobic (SPO) nanoparticles are sequentially sprayed onto a stretchable polymer substrate and then subjected to oxygen plasma treatment to form superhydrophilic (SPI) patterned regions. The SPO-layer-coated substrate maintains its extreme liquid repellency even under high strain (100%) and repeated cyclic stretching and releasing (1000 cycles). Thus, various liquid droplets can be precisely manipulated and dispensed onto the pre-defined SPI patterns on the SPO PDMS by using the developed droplet manipulation system. This system enables a multiplex colorimetric bioassay capable of detecting multiple analytes, including glucose, uric acid, and lactate, from a single droplet of the sample. In addition, the detection of glucose concentration in a plasma droplet of diabetic mouse demonstrates the feasibility of the proposed system for efficient clinical diagnostic applications.

The development of nanotechnology has provided challenging innovations in the synthesis of multifunctional nanoparticles (NPs) for medicine. However, numerous drug delivery carriers reported so far suffer from serious limitations such as : (1) poor drug loading typically lower than 10 wt% inducing both poor efficacy of the active molecule and high toxicity or side-effects , (2) rapid (reported as « burst release ») or non-specific delivery of the encapsulated drug, (3) difficulty of by-passing physiological barriers. These drawbacks, together with an often insufficient Enhanced Permeability and Retention effect (EPR) efficacy, are at the origin of the current limited number of manufactured drug nanomedicines. This justifies the need of multifunctional and more efficient nano-objects for nanomedicine. Among the different concepts reported so far, the integration of multiple functionalities into a single nanomedicine is of tremendous importance, expanding the capabilities of nano-objects to perform multiple parallel tasks. In particular, the strategy of combining into a single nano-object the functions of diagnosis/imaging and therapy (called “theranostics”) has emerged as an essential breakthrough in nanomedicine.
NPs of Metal Organic Frameworks (MOFs) have emerged recently as promising drug delivery platforms or stimuli-responsive nano-carriers. They are a class of highly porous hybrid crystalline materials with a large chemical and structural diversity, most of them exhibiting a very large, tunable and regular porosity with pore sizes and volumes up to 60 Å and 4 cm³.g^-1. Their large surface area, amphiphilic internal microenvironment as well as the possibility to introduce easily in the pores and frameworks on a spatially controlled way functional groups (e.g. acidic, basic, redox, hydrophobic…) through the metal or the organic linker make these materials suitable to host a large variety of drugs with a high loading capacity.^,,,
The present communication deals with the synthesis of stable superparamagnetic nanovectors for the image-guided therapy by coupling the iron carboxylate nanoMOF MIL-100(Fe), with ultra-small superparamagnetic iron oxide (USPIO). As shown by combining ⁵⁷Fe Mössbauer spectrometry and vibrating sample magnetometry, these nano-objects present a well-defined stoichiometry and a high saturation magnetization, both requirements to tailor the relaxometric properties of MRI contrast agents. Interestingly, the low toxicity and high anti-tumoral activity of MIL-100(Fe)/USPIO once loaded with doxorubicin was also demonstrated while their MRI capability was shown in vivo.[1] These features are important advantages, and make this new composite nanomaterial of a great interest for future in vivo biomedical experiments.
[1] S. Sene, M. T. Marcos-Almaraz, N. Menguy, J. Scola, J. Volatron, R. Rouland, J.-M. Grenèche, S. Miraux, C. Menet, N. Guillou, F. Gazeau, C. Serre, P. Horcajada, N. Steunou. Chem, 3, (2017), 303-322.

The construction of artificial complex materials, incorporating biomolecules that respond to biological circumstances in a smart manner, is a big challenge in nano-biomedical research. As a component of complex materials, DNA has attracted researchers because of its advantages, such as programmability, predictability, and biocompatibility. Specific DNA sequences can alter their conformation depending on specific environments, therefore, DNA sequences have been utilized as a trigger for the dynamic structural changes of nanomachine. Moreover, various biomolecules and chemical drugs are easily incorporated via specific DNA hybridization, therefore, DNA-based nanomachines have also shown immense potential as a drug delivery vehicle. However, DNA alone is not sufficient for use as a biological nanomachine because of its lack of functionality; consequently, a hybrid system composed of DNA and other functional materials should be developed. Among various DNA-based hybrid systems, the gold nanoparticle-DNA (AuNP-DNA) hybrid system is widely used for biomedical applications because of the advantages including biocompatibility and plasmonic properties.
The development of a multi-functional nanomachine as a combinatorial therapeutic agent for the treatment of specific disease is a research goal in this study. Especially, the use of combinatorial therapeutic agents in anti-cancer therapy is considered a promising approach, as the complete treatment of cancer cannot be achieved using a single agent because of the intrinsic complexity of cancer. Among various therapeutic combinations, phototherapy with chemotherapy, is regarded as a potent combination for efficient anti-cancer therapy. Therefore, a combinatorial therapeutic agent which induces an appropriate photothermal and photodynamic effect, combined with chemotherapeutic effect is highly demanded to maximize the anti-cancer therapeutic effect.
Herein, we reveal the design, construction, and operation of a functional DNA-decorated dynamic Au nanomachine as a therapeutic agent for triple combinatorial anti-cancer therapy. Taking advantage of the intrinsic optical properties of Au nanoparticles, which depend on their size, a cytosine rich i-motif sequence was employed for intracellular pH-sensitive duplex dissociation and subsequent aggregation of the DNA-Au nanomachine, enabling anticancer drug release and photothermal ablation upon infrared light irradiation. Moreover, another functional DNA sequence, a G-quadruplex, was exploited for the stable loading and intracellular delivery of a photosensitizer to achieve effective photodynamic therapy under red light illumination. The therapeutic properties and dynamics of DNA-Au nanomachine were investigated. Furthermore, the combinatorial chemo, photodynamic, and photothermal therapeutic effects of the functional DNA-decorated Au nanomachines were evaluated in vitro and in vivo using a triple negative breast cancer model.

The upconversion nanoparticles (UCNPs) have applications in the biomedical industry, they can function as specific cell targets, either to identify them or to deliver drugs, especially in cancer cells thar can be differentiated from healthy cells. In this study, we synthesize with the sol-gel technique, UCNPs with excellent luminescent properties such as La₂O₃ codoped with Er^{3 +} and Yb^{3 +} and Y₂O₃ codoped with Yb^{3 +}, Ho^{3 +} and Er^{3 +} which present luminescence in the green (λ= 550 nm ) and red ( λ=660 nm) range. The upconversion process consists of the fact that the Yb^{3 +} ion absorbs a photon with a wavelength of 980 nm and transfers the energy to the activating ion that can be Er^{3 +} or Ho^{3 +}, the electron returning to its base state emits in the visible spectrum. The functionalization with aminosilanes consists of two stages: first is a coating of silica in the nanoparticles, to make them inert in biological components and the second part is to functionalize with amino ligands (NH₂). After this process, we did the folic acid funcionalization (FA-NH₂). The FA ligands of the UCNPs can bind to the FA receptors of the cancer cells, such as cervical and breast cancer.

Methods relating signals to specific cells deep inside the body hold great promise for basic sciences studying regulation of functional systems as well as potential as diagnosis and treatment tools. While electrical and optical methods have found wide adaption, the do require implantation of wires or optical fibers. Magneto-thermal cell simulation and silencing would be tetherless and remote. Its basis are thermal processes which can be trigger via local magnetic nanoparticle hyperthermia. We present progress towards magnetothermal activation and silencing of neuronal in-vivo to unraveling the connection between behavior and brain circuits. The application requires creating nanomaterials for local heating in alternating magnetic fields, ways to deliver these into the brain, tether them to specific neurons, measure the local heating and temperature sensitizing the neurons. These restrain practical particle size, dipole moment, water solubility and biocompatibility. Optimizing all conditions simultaneously requires combining various solutions. We characterize chemically or biologically synthesized as well as hybrid nanoparticle systems for local hyperthermia. Further, we compare various nanoparticle assemblies either formed by targeting to the cells, or preassembled on micron-size delivery vehicles. We show that it is possible to robustly activate motor cortex and striatum neurons magnetothermally in awake and moving animals, and that this lead to distinct motor behaviors. In addition, we demonstrate magnetothermal silencing of neurons in the Ventral Tegmental Area, leading to abolishing of a place preference.

Magnetic nanoparticles (MNPs) dissipate heat upon exposure to alternating magnetic fields (AMFs), which then can trigger thermally-sensitive ion channels in electro-active cells such as neurons. In mammals, peripheral nerve fibers express heat-sensitive cation channels from the transient receptor potential family. Consequently, local heating from MNPs can be converted into an electrochemical gradient across the neural membranes, leading to depolarization and firing of action potentials in response to the externally applied AMF. The use of MNPs eliminates the need for invasive and tissue-damaging electrodes. Moreover, AMF has higher penetration depth (>10cm) compared to other methods and therefore is more suitable for deep tissue stimulation. Previously, MNPs were primarily used to trigger heat-sensitive ion channels in neurons. However, it has been shown that heat sensitive ion channels, such as TRPV1, exist also in other tissues, for example in the adrenal gland. Abnormal regulations of hormones produced within the glands have been linked to altered stress responses in patients suffering from mental disease. The adrenal gland is therefore a high-value target for peripheral neuromodulation and control of hormones. We employed iron oxide MNPs 22 nm exposed to AMF to trigger hormone release from adrenal glands in vivo. Iron oxide MNPs were synthesized and surface functionalization was conducted by polymeric coating with polyethylene glycol. The size, concentration, saturation magnetization, and specific loss power of the MNPs were examined and tailored to the applied AMF. Adrenal cell culture was shown to respond robustly to magnetothermal stimulation. MNPs location of injection, concentration and volume was determined by a finite element model showing the heat distribution within the adrenal gland under the applied AMF. In order to investigate the response of adrenal gland to magnetothermal stimulation in vivo, a custom AMF apparatus including a resonant tank circuit was developed. The effects of magnetothermal adrenal stimulation were assessed in live rats by hormone levels in the rat blood comparing the levels before and after stimulation.

Most of our understanding of neural function has come from intracellular recordings from single neurons. Traditionally, glass micropipette electrodes integrated with cumbersome positioning systems have been used to obtain intracellular recordings in-vitro and in-vivo. These conventional interfaces have some limitations due to their large size and weight: (1) recordings are mostly obtained from anesthetized/head-fixed animals; (2) recordings are obtained from one neuron at a time; (3) recording durations in-vivo are short (45-60 min) due to mechanical instabilities at electrode-neuron interface. These limitations have precluded long-term intracellular recordings from a population of neurons in anesthetized and behaving animals. Here, we present novel polycrystalline silicon-based micro/nanoscale interfaces that will be integrated with MEMS technology and closed loop control algorithms for autonomous, multi-channel intracellular recording.

We fabricated and tested two distinct designs of surface micromachined polysilicon-based electrodes for intracellular recording. In the first approach, miniaturized glass micropipettes (<500 nm tip sizes) filled with electrolyte were integrated with polycrystalline silicon microelectrodes doped with phosphorous (10²¹/cm³). These electrodes reliably recorded good quality resting and action potentials from neurons in Aplysia (< -40 mV and > 70 mV) as well as rodent motor cortex (< -55 mV and > 60 mV). We also demonstrate the feasibility of autonomous intracellular recordings with these electrodes by interfacing them with our closed loop control algorithm and electrothermal microactuators. The electrode autonomously isolated, penetrated and recorded intracellular potentials from Aplysia neurons in n=10 trials using DC electrical impedance as feedback variable. In the second approach, we used focused ion beam (FIB) to mill down the tips of polycrystalline silicon microelectrodes to <300 nm with different taper geometries to optimally penetrate and maintain interface with single neurons in order to maximize the quality and duration of intracellular recordings. Polysilicon nanoelectrodes recorded 1-4 mV intracellular-like action potentials from Aplysia neurons, with minimal damage to neuronal membrane. We are currently modifying the design of these nanoelectrodes for in vivo intracellular recordings. The use of polycrystalline silicon for intracellular interfaces (1) allows immediate integration with other complex microscale mechanical structures fabricated using MEMS technology and (2) provides a scalable approach to realize intracellular recordings from ensemble of neurons from behaving animals.

To study neural circuits, it is imperative to measure and manipulate electrical activity of the network. Conventional planar-type microelectrode array platform has become an emering tool to investigate the electrophysiological function of in vitro neural tissues, e.g., cultured cells, slices, or organoids. To meet the recent trend of optical neurotechnology, it is desired to combine the microelectrode array with optical modulation modality. In this talk, I will introduce a novel electro-opto-thermall neural interface platform that allows electrical recording and optical modulation at the same time. This new platform technology is based on our recent finding of photothermal neural inhibition effect using near-infrared light and plasmonic metal nanoparticles. This new interface opens a chance for neuroscientists and engineers to modulate brain activity without genetic modification.

In FRET-based systems, a specific quencher is normally able to quench the fluorescence only from those fluorophore donors that have significant overlap of their emission spectra with the absorption spectrum of the quencher when the donor and quencher are brought into proximity. In our work, we construct a non-toxic FRET system choosing NIR-emitting CuInS₂/ZnS colloidal quantum dots (QDs) as donor while water-soluble IRDye QC-1 dark quencher (quenching range from ~500 to 800 nm) as acceptor. Highly luminescent CuInS₂/ZnS QDs (photoluminescence quantum yields ~55%) are synthesized by successive ionic layer adsorption and reaction, which are further transferred into water via cap exchange of the native ligands for amine-functionalized His-PIMA-PEG/NH₂ polymer. The water-soluble CuInS₂/ZnS QDs possess excellent pH resistibility over a broad range (3-13) and long-term colloidal stability more than one year (ambient condition at 4 °C) accompanied by slight decrease in photoluminescence quantum yields (from 22% to 15%). These amine-terminated QDs can be readily coupled with NHS ester group of the IRDye QC-1 dark dye. The polymer coated QD-dark quencher pair shows efficient FRET evidenced by significant decrease in both photoluminescence intensity and lifetime of the donor via tuning the number of dark quencher per QD.

The fabrication of complex topologies such as a braid, which consists of three or more interlaced strands of a soft material, represents a major challenge in nanotechnology. DNA is a promising material for braid fabrication because of its high flexibility and extraordinary programmability. Using a DNA origami approach, we demonstrate that double-stranded DNA braids can be created through self-assembly of three or more single strands of DNA and a number of staple strands that form the crossing points (nodes) of the braid. We show that successful braid formation requires the distance between nodes to be an odd number of half-turns (5 basepairs in B-DNA), which minimizes torsional stress. We also investigate the metallization of the DNA origami braid template to create "nano-Litz" wire, which has low electrical resistance at high frequencies. Such constructs could potentially be used as electrical interconnects at up to gigahertz frequencies.

Recently, DNA has been studied for electronics due to its intrinsic benefits such as its natural plenitude, bio-degradability, bio-functionality and low-cost. However, its applications only limit to passive components because of inherent insulating properties. In this report, the metal-insulator-metal tunnel diode with Au/DNA/NiOx junctions is presented. Through the self-aligning process of DNA molecules, a two dimensional DNA nanosheet is synthesized and used as a tunneling barrier, and semitransparent conducting oxide, NiOx is applied as a top electrode for resolving metal penetration issues. These molecular devices successfully operate as a non-resonant tunneling diode and temperature-variable current-voltage analysis proves that Fowler–Nordheim tunneling is a dominant conduction mechanism in the junctions. The DNA-based tunneling devices appear to be a promising prototype approach to Bio-Nano electronics using DNA nanosheets.

This work demonstrates fully stretchable (>140%) organic electrochemical transistors (OECTs) by the development of highly stretchable Au conductors. OECTs are the ideal platform to realize high-sensitivity wearable/implantable sensors for electrophysiology and chemicals because of the high transconductance.[1] However, stretchable OECTs have not been demonstrated due to the limited stretchability of Au films which are utilized for source/drain contacts and wirings in OECTs.[2] To solve this issue, we first developed highly stretchable Au which has sheet resistance of 11.8 Ohm/sq. at 0% strain and 28.5 Ohm/sq. at 100% strain. Remarkably, the high performance was maintained even after 10,000 cycles of 50% strain. This highly stretchable Au conductor was realized by controlling the Au thin film growth on elastomer substrates during the vacuum deposition. In contrast to conventional Au having limited numbers of short (< 1 μm) microcracks, newly developed stretchable Au film possesses many initial microcracks longer than 1 μm. Larger numbers of long initial microcracks resulted in reduced crack propagation by strain, leading to the small resistance change by strain. Furthermore, stretchable OECTs was fabricated using this highly stretchable Au conductor. Poly(3,4-ethyle nedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was used as an active layer and showed high stretchability in electrolyte solution. The transconductance is as high as 0.54 mS at 0% strain and 0.22 mS at 100% strain. The stretchability of OECTs would improve the conformability to human bodies, leading to reduction of the discomfort of wearing and improved signal integrity.

[1] D. Khodagholy, et al. Nature Communications 4, 1575 (2013).
[2] S. Lacour, et al. Proceedings of the IEEE 93, 1459-1467 (2005).

Electrochemical transistors with printed semiconductors have been attracting attentions in the biomolecule detection with very low concentration, quantitatively due to their simple processing and universal complexation of receptors. We recently, developed quasi two-dimensional (Quasi-2D) In₂O₃ semiconductor-based electrochemical field-effect transistors, which showed high sensitivity and large transconductance. pH, glucose, and neurotransmitters could be detectable with high sensitivity and selectivity. pH sensitivity was reached to over 60 mV/pH that was highly compatible with nanomaterial platforms. For the glucose detecting application, we confirmed that the detecting ranges were covered within human tear’s glucose level (0.1 to 1 µM). Furthermore, our conformal device structures enabled to contact on highly rough surface as well as on an artificial eye for the smart lens applications.
We also expended to the neurochemical sensing, which were consisted with dopamine sequenced aptamers to get a strong specific binding of the small molecule. The conformational change of negative charge backbone of aptamers with the dopamine binding could overcome Debye screening length in physiological environments.

To understand and diagnose neurological disorders, it is important not only to detect levels of a single chemical in the brain but to simultaneously examine the changing ratios of chemical and electrical signals in the brain. In pursuit of a device that can achieve this goal, we have developed a flexible MEA that allows for monitoring of long-term local tissue responses in an awake, freely moving animal. While the device itself can now survive for many months in the body, the enzymatic glutamate sensors integrated into these arrays are not yet robust enough to detect in vivo glutamate changes beyond a week. Here we demonstrate improved lifetimes for glutamate sensors thanks a combination of surface modifications that improve the sensitivity and robustness of these sensors. We’ve developed a unique procedure allows us to nondestructively roughen thin film platinum microelectrodes to improve adhesion of coatings like platinum iridium or iridium oxide to the electrode surface and increase sensitivity of chemical sensors 3-fold. This extreme roughening of a thin film without corrosion is possible via the use of a non-adsorbing electrolyte for the electrochemical roughening to prevents preferential grain boundary dissolution seen with adsorbing electrolytes. In addition, lifetime of these sensors has been improved thanks to modifications to enzyme immobilization and attachment to the array. The enzyme is now matrixed with special nanoScyl capsule that protects enzyme from breakdown or dissolution. Thanks to these modifications, our chemical sensors are moving towards a longer functional lifetime in vivo compatible with the longer lifetime of the arrays themselves.

We have successfully fabricated an ultraflexible, transparent electrode with low sheet resistance for in vivo or vitro experiments, and it was finely patterned on 1 µm thick parylene substrate. The multilayer of Indium-tin-oxide (ITO) and Au was patterned by photolithography and wet etching process so that small patterns such as 5 µm long gap for transistor channels is available.
By a sandwiching structure with an ultrathin (< 20 nm) Au film and two oxide films, lower sheet resistance (< 10 ohms/sq.) and better mechanical durability can be achieved without using high temperature process and chemically instable materials. Because reactive ion etching causes severe damage on flexible plastic substrate and photoresist, we employed step by step wet etching method for each ITO and Au layer.

Recently, flexible multi sensor arrays with transparent electrodes have attracted much interest because optical observation and stimulation to the tissues wrapped by the array are enabled [1]. Compared to the previous researches, ITO/Au/ITO multilayer has lower sheet resistance which is expected to enable multi sensor array with smaller pixel characteristics distribution. In our previous report, simple in vivo optogenetic experiment using this electrode has been already demonstrated [2], but the wiring was patterned in several hundred microns of width by shadow mask. The fine patterning process is crucial toward more integrative devices, especially for transistor based sensors and high density, large scale multi sensor arrays.

This work was supported by the Someya Bio-Harmonized ERATO grant.

[1] Kuzum, D. et al., Nat. Commun. 5, 5259 (2014).
[2] Jimbo, Y. et al., ACS Appl. Mater. Interfaces 9, 34744−34750 (2017).

Conducting polymers (CP), such as poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), are widely used to interface electronics with biology due to their mechanical, physical-chemical properties similar to biological tissue [1, 2]. In particular, CP electrodes present low impedance when interfaced to excitable tissues and it was demonstrated an improved adaptability to the brain tissue and an increased charge-transfer efficiency. In this work, we investigated the possibility to employ PEDOT: PSS modified with different amount of poly-ethylene glycol dyacrilate (PEG DA) to evaluate the effect on the conductivity of PEDOT PH1000 to realize efficient electrodes for organic photovoltaic solar cells for light-induced electrical field for cell inhibition/stimulation. PEG DA is an easily modifiable polymer that is widely used in hydrogel fabrication, including 2D and 3D scaffolds for tissue culture [3]. Here, we present a comparison between different PEDOT:PSS with and without PEG DA deposited by spin and spray - coating techniques in order to obtain different surface morphologies. The functionality and electrochemical behavior of electrodes and the morphology of the film was evaluated using electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). To investigate the biocompatibility of modified PEDOT: PSS in terms of cells vitality and to discriminate optimal cell adhesion conditions when different morphologies of polymeric film are considered, we performed a toxicity assay based on viability of U87-MG astrocyte like cells (human glioblastoma cell lines). Finally, we reported the adhesion and biocompatibility of the cells seeded on PEDOT [4], in order to produce 3D organic photovoltaic devices for light inhibition of U87 cells to reduce tumoral cells proliferation rate [5].

[1] Ulises A. Aregueta-Robles, Andrew J. Woolley, Laura A. Poole-Warren, Nigel H. Lovell, and Rylie A. Green Organic electrode coatings for next-generation neural interfaces, Front Neuroeng. 2014; 7: 15
[2]G. Cellot, P. Lagonegro, G. Tarabella, D. Scaini, F. Fabbri, S. Iannotta, M. Prato, G. Salviati, and L. Ballerini, PEDOT:PSS Interfaces Support the Development of Neuronal Synaptic Networks with Reduced Neuroglia Response In vitro, Front Neurosci.; 9: 521, (2015)
[3] Malar A. Azagarsamy, Navakanth R. Gandavarapu, Kristi S. Anseth, Versatile Cell Culture Scaffolds via Bio-orthogonal Click Reactions, Material Matters, v7, n3, (2012)
[4] F. Santoro, Wenting Zhao, L. Joubert, L. Duan, Jan Schnitker, Y. van de Burgt, Hsin- Ya Lou, B. Liu, A. Salleo, L. Cui, Y. Cui, and B. Cui Revealing The Cell-Material Interface With Nanometer Resolution By Focused Ion Beam/Scanning Electron Microscopy, in press, ACS Nano, (2017)
[5] Carr L, Bardet SM, Burke RC, Arnaud-Cormos D, Leveque P, O’Connor RP. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells. Scientific Reports, 7:41267(2017)

The interface between biological cells and non-biological materials has profound influences on cellular activities, chronic tissue responses, and ultimately the success of medical implants and bioelectronic devices. Materials in contact with cells can be metals, plastics, silicon, ceramics or other synthetic materials, and their surfaces vary widely in chemical compositions, stiffness and levels of roughness. In particular, the interaction of cells with diverse materials relies primarily on the specific adhesion of the plasma membrane on to the material surface. Many attempts have been carried out in the last decade for characterizing this interface, however major limitations have been presented in respect to resolution and artefacts introduced by typical substrate removal. Here, we present an advanced microscopy method (scanning electron microscopy/focused ion beam) based on ultra-thin resin plastificization which uniquely allows the visualization of the interface between cells and materials with 5-10 nm resolution(Santoro et al., 2017; Zhao et al., 2017). Furthermore, we prove that this method can be used for organic and inorganic 2D materials, planar substrates with 3D vertical features (Santoro et al., 2017b, Cui et al., 2018) of different shapes and ultimately 3D scaffold-like systems (Mollo et al., 2018).

References
Cui B., McGuire A., Santoro F., Interfacing Cells with Vertical Nanoscale Structures: Applications and Characterization, Annual Review of Analytical Chemistry, 2018-accepted.
Mollo V., Iandolo D., Pennacchio F., Rossi D., Dannhauser D., Langella A., Netti P., Cui B., Owens R., Santoro F., (in preparation). Nanoscale resolution of 3D scaffold- cell interaction by SEM/FIB, in preparation.
Santoro, F., Zhao, W., Joubert, L.-M., Duan, L., Schnitker, J., van de Burgt, Y., Lou, H.-Y., Liu, B., Salleo, A., Cui, L., Cui Y., Cui B., Revealing the Cell–Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy. ACS Nano, 2017.
Santoro, F., Van der Burgt Y., Keene S. T., Cui B., Salleo A., Enhanced cell–chip coupling by rapid femtosecond laser patterning of soft PEDOT: PSS biointerfaces.. ACS Applied Materials and Interfaces, 2017.
Zhao, W., Hanson, L., Lou, H.-Y., Akamatsu, M., Chowdary, P.D., Santoro, F., Marks, J.R., Grassart, A., Drubin, D.G., Cui, Y., Cui B., Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotechnology, 2017.

Bio-integrated electronics is of great interest in emerging fields of bio-injectable and bio-medical applications. One of recent technologies, transient electronics, has peculiar characteristics that suggest the different behavior: it completely dissolves/disappear over time in a prescribed and/or triggered manner. Electronic system that is ‘transient’ in this sense has unique advantages that cannot be addressed with conventional electronics, such as biomedical implants that can be operational for clinically useful time span, then physically degrades/resorbs into the body. One of key properties, the control of entire lifetime of the system still has limitation. Previous researches have been trying to overcome this drawback with regulating pH level, passivating electronic components with other materials, and varying thickness of transient materials. In spite of these efforts to control the lifetime of devices, those strategies are limited to solution-based control. Here, we suggest a new approach that transient devices are successfully compromised by external heat source using near infrared (NIR) light, and all experiments are confirmed by in vivo and in vitro tests, and simple analytical (theoretical and FEM) simulations.

In this work, we introduce a new generation of Glassy Carbon (GC) microelectrodes mounted on a highly mechanically compliant elastomer substrate (E = 1.2 MPa) using a recently introduced pattern transfer method. This new class of probes leverages our recently introduced pattern transfer technology for GC microelectrode array supported on polyimide (E = 2 GPa) substrate that has been demonstrated to offer a significant advance in neuroprosthetics technology. While various neural prosthetic interfaces have been engineered for sensing electrical as well as electrochemical neural signals since 1950, the issue of tissue reaction in the form of microglial scarring due to difference in material stiffness largely remains unsolved. The glial scarring may reduce the neural sensing ability of neural probe, especially for long-term probe. To address this, flexible and elastomeric polymer materials that offer a promising solution to this problem by lowering the mismatch in stiffness of dura mater and elastomeric device have recently been considered by several researchers. With Young’s Modulus in the low MPA range, such elastomers have stretchability that enables neural probes to follow the natural internal brain movement regulated by blood flow and respiration. For long-term implants, in fact, the neural probes experience rhythmic mechanical friction as the brain is a pulsatile organ. In this study, we, therefore, integrate GC microelectrodes supported by elastomer substrate to fabricate a new generation of neural sensing and stimulation probes with high mechanical compliance. This new class of probes called GC Elastomer probes are based on a recently introduced pattern transfer technique to microfabricate GC microelectrodes array (12 channels, thickness= 2 µm, diameter= 300 µm) and supported on insulating silicone elastomer layer. The interconnecting traces made of thin-film metal (Au with thickness = 100 nm) with high stretchable properties (horseshoe shape) and good electrical conductivity are deposited by metal sputtering on elastomer substrate and are mechanically connected to the GC microelectrodes designed for recording electrophysiological signals such as Electrocorticography (ECoG). After device fabrication, in vitro characterization is carried out to evaluate the quality of the stretchable electrodes. In particular, impedance magnitude and phase of the microelectrodes are measured after uniaxial deformation cycling up to 20% and ultimately up to the device failure. Furthermore the capacitive charging and injection as well as the stability under prolonged stimulation use are quantified through Cyclic Voltammetry and Power Pulse Techniques.