Concurrent functional ultrasound imaging with graphene-based DC-coupled electrophysiology as a platform to study slow brain signals and cerebral blood flow under control and pathophysiological brain states.

Current methodology used to investigate how shifts in brain states associated with regional cerebral blood volume (CBV) change in deep brain areas, are limited by either the spatiotemporal resolution of the CBV techniques, and/or compatibility with electrophysiological recordings; particularly in relation to spontaneous brain activity and the study of individual events. Additionally, infraslow brain signals (<0.1 Hz), including spreading depolarisations, DC-shifts and infraslow oscillations (ISO), are poorly captured by traditional AC-coupled electrographic recordings; yet these very slow brain signals can profoundly change CBV. To gain an improved understanding of how infraslow brain signals couple to CBV we present a new method for concurrent CBV with wide bandwidth electrophysiological mapping using simultaneous functional ultrasound imaging (fUS) and graphene-based field effect transistor (gFET) DC-coupled electrophysiological acquisitions. To validate the feasibility of this methodology visually-evoked neurovascular coupling (NVC) responses were examined. gFET recordings are not affected by concurrent fUS imaging, and epidural placement of gFET arrays within the imaging window did not deteriorate fUS signal quality. To examine directly the impact of infra-slow potential shifts on CBV, cortical spreading depolarisations (CSDs) were induced. A biphasic pattern of decreased, followed by increased CBV, propagating throughout the ipsilateral cortex, and a delayed decrease in deeper subcortical brain regions was observed. In a model of acute seizures, CBV oscillations were observed prior to seizure initiation. Individual seizures occurred on the rising phase of both infraslow brain signal and CBV oscillations. When seizures co-occurred with CSDs, CBV responses were larger in amplitude, with delayed CBV decreases in subcortical structures. Overall, our data demonstrate that gFETs are highly compatible with fUS and allow concurrent examination of wide bandwidth electrophysiology and CBV. This graphene-enabled technological advance has the potential to improve our understanding of how infraslow brain signals relate to CBV changes in control and pathological brain states.

Nanoporous graphene-based thin-film microelectrodes for in vivo high-resolution neural recording and stimulation.

One of the critical factors determining the performance of neural interfaces is the electrode material used to establish electrical communication with the neural tissue, which needs to meet strict electrical, electrochemical, mechanical, biological and microfabrication compatibility requirements. This work presents a nanoporous graphene-based thin-film technology and its engineering to form flexible neural interfaces. The developed technology allows the fabrication of small microelectrodes (25 µm diameter) while achieving low impedance (∼25 kΩ) and high charge injection (3-5 mC cm-2). In vivo brain recording performance assessed in rodents reveals high-fidelity recordings (signal-to-noise ratio >10 dB for local field potentials), while stimulation performance assessed with an intrafascicular implant demonstrates low current thresholds (<100 µA) and high selectivity (>0.8) for activating subsets of axons within the rat sciatic nerve innervating tibialis anterior and plantar interosseous muscles. Furthermore, the tissue biocompatibility of the devices was validated by chronic epicortical (12 week) and intraneural (8 week) implantation. This work describes a graphene-based thin-film microelectrode technology and demonstrates its potential for high-precision and high-resolution neural interfacing.

Single-Step Functionalization Strategy of Graphene Microtransistor Array with Chemically Modified Aptamers for Biosensing Applications.

Graphene solution-gated field-effect transistors (gSGFETs) offer high potential for chemical and biochemical sensing applications. Among the current trends to improve this technology, the functionalization processes are gaining relevance for its crucial impact on biosensing performance. Previous efforts are focused on simplifying the attachment procedure from standard multi-step to single-step strategies, but they still suffer from overreaction, and impurity issues and are limited to a particular ligand. Herein, a novel strategy for single-step immobilization of chemically modified aptamers with fluorenylmethyl and acridine moieties, based on a straightforward synthetic route to overcome the aforementioned limitations is presented. This approach is benchmarked versus a standard multi-step strategy using thrombin as detection model. In order to assess the reliability of the functionalization strategies 48-gSGFETs arrays are employed to acquire large datasets with multiple replicas. Graphene surface characterization demonstrates robust and higher efficiency in the chemical coupling of the aptamers with the single-step strategy, while the electrical response evaluation validates the sensing capability, allowing to implement different alternatives for data analysis and reduce the sensing variability. In this work, a new tool capable of overcome the functionalization challenges of graphene surfaces is provided, paving the way toward the standardization of gSGFETs for biosensing purposes.

Covalent functionalisation controlled by molecular design for the aptameric recognition of serotonin in graphene-based field-effect transistors.

In the last decade, solution-gated graphene field effect transistors (GFETs) showed their versatility in the development of a miniaturized multiplexed platform for electrophysiological recordings and sensing. Due to their working mechanism, the surface functionalisation and immobilisation of receptors are pivotal to ensure the proper functioning of devices. Herein, we present a controlled covalent functionalisation strategy based on molecular design and electrochemical triggering, which provide a monolayer-like functionalisation of micro-GFET arrays retaining the electronic properties of graphenes. The functionalisation layer as a receptor was then employed as the linker for serotonin aptamer conjugation. The micro-GFET arrays display sensitivity toward the target analyte in the micromolar range in a physiological buffer (PBS 10 mM). The sensor allows the in-flow real-time monitoring of serotonin transient concentrations with fast and reversible responses.

Full-bandwidth electrophysiology of seizures and epileptiform activity enabled by flexible graphene microtransistor depth neural probes.

Mapping the entire frequency bandwidth of brain electrophysiological signals is of paramount importance for understanding physiological and pathological states. The ability to record simultaneously DC-shifts, infraslow oscillations (<0.1 Hz), typical local field potentials (0.1-80 Hz) and higher frequencies (80-600 Hz) using the same recording site would particularly benefit preclinical epilepsy research and could provide clinical biomarkers for improved seizure onset zone delineation. However, commonly used metal microelectrode technology suffers from instabilities that hamper the high fidelity of DC-coupled recordings, which are needed to access signals of very low frequency. In this study we used flexible graphene depth neural probes (gDNPs), consisting of a linear array of graphene microtransistors, to concurrently record DC-shifts and high-frequency neuronal activity in awake rodents. We show here that gDNPs can reliably record and map with high spatial resolution seizures, pre-ictal DC-shifts and seizure-associated spreading depolarizations together with higher frequencies through the cortical laminae to the hippocampus in a mouse model of chemically induced seizures. Moreover, we demonstrate the functionality of chronically implanted devices over 10 weeks by recording with high fidelity spontaneous spike-wave discharges and associated infraslow oscillations in a rat model of absence epilepsy. Altogether, our work highlights the suitability of this technology for in vivo electrophysiology research, and in particular epilepsy research, by allowing stable and chronic DC-coupled recordings.

Characterization of optogenetically-induced cortical spreading depression in awake mice using graphene micro-transistor arrays.

Objective.The development of experimental methodology utilizing graphene micro-transistor arrays to facilitate and advance translational research into cortical spreading depression (CSD) in the awake brain.Approach.CSDs were reliably induced in awake nontransgenic mice using optogenetic methods. High-fidelity DC-coupled electrophysiological mapping of propagating CSDs was obtained using flexible arrays of graphene soultion-gated field-effect transistors (gSGFETs).Main results.Viral vectors targetted channelrhopsin expression in neurons of the motor cortex resulting in a transduction volume ⩾1 mm3. 5-10 s of continous blue light stimulation induced CSD that propagated across the cortex at a velocity of 3.0 ± 0.1 mm min-1. Graphene micro-transistor arrays enabled high-density mapping of infraslow activity correlated with neuronal activity suppression across multiple frequency bands during both CSD initiation and propagation. Localized differences in the CSD waveform could be detected and categorized into distinct clusters demonstrating the spatial resolution advantages of DC-coupled recordings. We exploited the reliable and repeatable induction of CSDs using this preparation to perform proof-of-principle pharmacological interrogation studies using NMDA antagonists. MK801 (3 mg kg-1) suppressed CSD induction and propagation, an effect mirrored, albeit transiently, by ketamine (15 mg kg-1), thus demonstrating this models' applicability as a preclinical drug screening platform. Finally, we report that CSDs could be detected through the skull using graphene micro-transistors, highlighting additional advantages and future applications of this technology.Significance.CSD is thought to contribute to the pathophysiology of several neurological diseases. CSD research will benefit from technological advances that permit high density electrophysiological mapping of the CSD waveform and propagation across the cortex. We report anin vivoassay that permits minimally invasive optogenetic induction, combined with multichannel DC-coupled recordings enabled by gSGFETs in the awake brain. Adoption of this technological approach could facilitate and transform preclinical investigations of CSD in disease relevant models.

Bidirectional Modulation of Neuronal Cells Electrical and Mechanical Properties Through Pristine and Functionalized Graphene Substrates.

In recent years, the quest for surface modifications to promote neuronal cell interfacing and modulation has risen. This course is justified by the requirements of emerging technological and medical approaches attempting to effectively interact with central nervous system cells, as in the case of brain-machine interfaces or neuroprosthetic. In that regard, the remarkable cytocompatibility and ease of chemical functionalization characterizing surface-immobilized graphene-based nanomaterials (GBNs) make them increasingly appealing for these purposes. Here, we compared the (morpho)mechanical and functional adaptation of rat primary hippocampal neurons when interfaced with surfaces covered with pristine single-layer graphene (pSLG) and phenylacetic acid-functionalized single-layer graphene (fSLG). Our results confirmed the intrinsic ability of glass-supported single-layer graphene to boost neuronal activity highlighting, conversely, the downturn inducible by the surface insertion of phenylacetic acid moieties. fSLG-interfaced neurons showed a significant reduction in spontaneous postsynaptic currents (PSCs), coupled to reduced cell stiffness and altered focal adhesion organization compared to control samples. Overall, we have here demonstrated that graphene substrates, both pristine and functionalized, could be alternatively used to intrinsically promote or depress neuronal activity in primary hippocampal cultures.

Versatile Graphene-Based Platform for Robust Nanobiohybrid Interfaces.

Technologically useful and robust graphene-based interfaces for devices require the introduction of highly selective, stable, and covalently bonded functionalities on the graphene surface, whilst essentially retaining the electronic properties of the pristine layer. This work demonstrates that highly controlled, ultrahigh vacuum covalent chemical functionalization of graphene sheets with a thiol-terminated molecule provides a robust and tunable platform for the development of hybrid nanostructures in different environments. We employ this facile strategy to covalently couple two representative systems of broad interest: metal nanoparticles, via S-metal bonds, and thiol-modified DNA aptamers, via disulfide bridges. Both systems, which have been characterized by a multitechnique approach, remain firmly anchored to the graphene surface even after several washing cycles. Atomic force microscopy images demonstrate that the conjugated aptamer retains the functionality required to recognize a target protein. This methodology opens a new route to the integration of high-quality graphene layers into diverse technological platforms, including plasmonics, optoelectronics, or biosensing. With respect to the latter, the viability of a thiol-functionalized chemical vapor deposition graphene-based solution-gated field-effect transistor array was assessed.

High-resolution mapping of infraslow cortical brain activity enabled by graphene microtransistors.

Recording infraslow brain signals (<0.1 Hz) with microelectrodes is severely hampered by current microelectrode materials, primarily due to limitations resulting from voltage drift and high electrode impedance. Hence, most recording systems include high-pass filters that solve saturation issues but come hand in hand with loss of physiological and pathological information. In this work, we use flexible epicortical and intracortical arrays of graphene solution-gated field-effect transistors (gSGFETs) to map cortical spreading depression in rats and demonstrate that gSGFETs are able to record, with high fidelity, infraslow signals together with signals in the typical local field potential bandwidth. The wide recording bandwidth results from the direct field-effect coupling of the active transistor, in contrast to standard passive electrodes, as well as from the electrochemical inertness of graphene. Taking advantage of such functionality, we envision broad applications of gSGFET technology for monitoring infraslow brain activity both in research and in the clinic.

Iridium oxide pH sensor for biomedical applications. Case urea-urease in real urine samples.

This work demonstrates the implementation of iridium oxide films (IROF) grown on silicon-based thin-film platinum microelectrodes, their utilization as a pH sensor, and their successful formatting into a urea pH sensor. In this context, Pt electrodes were fabricated on Silicon by using standard photolithography and lift-off procedures and IROF thin films were growth by a dynamic oxidation electrodeposition method (AEIROF). The AEIROF pH sensor reported showed a super-Nerstian (72.9±0.9mV/pH) response between pH 3 and 11, with residual standard deviation of both repeatability and reproducibility below 5%, and resolution of 0.03 pH units. For their application as urea pH sensors, AEIROF electrodes were reversibly modified with urease-coated magnetic microparticles (MP) using a magnet. The urea pH sensor provided fast detection of urea between 78µM and 20mM in saline solution, in sample volumes of just 50µL. The applicability to urea determination in real urine samples is discussed.

Cancer prognostics by direct detection of p53-antibodies on gold surfaces by impedance measurements.

The identification and measurement of biomarkers is critical to a broad range of methods that diagnose and monitor many diseases. Serum auto-antibodies are rapidly becoming interesting targets because of their biological and medical relevance. This paper describes a highly sensitive, label-free approach for the detection of p53-antibodies, a prognostic indicator in ovarian cancer as well as a biomarker in the early stages of other cancers. This approach uses impedance measurements on gold microelectrodes to measure antibody concentrations at the picomolar level in undiluted serum samples. The biosensor shows high selectivity as a result of the optimization of the epitopes responsible for the detection of p53-antibodies and was validated by several techniques including microcontact printing, self-assembled-monolayer desorption ionization (SAMDI) mass spectrometry, and adhesion pull-off force by atomic force microscopy (AFM). This transduction method will lead to fast and accurate diagnostic tools for the early detection of cancer and other diseases.

Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects.

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell-adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm(2)), with a threshold value of 4.0 pmol/cm(2) for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis. FROM THE CLINICAL EDITOR: The authors observed by AFM nonlinear dependence of cell adhesion on RGD gradient surfaces with different surface densities. The nonlinear characteristics may be explained by non-homogeneous RGD surface distribution at the nanometer scale, conditioned by the stochastic nature of the hydrolysis process.

NMe amide as a synthetic surrogate for the thioester moiety in thiocoraline.

Bridged N-methyl amides are used as isosteres for depsi and thiodepsi bonds in thiocoraline. The introduction of NMe-amides in bridges mimics the thioester bonds without imposing steric hindrance and allows conservation of the hydrogen bonding map of the natural product. NMe-azathiocoraline was constructed by solid-phase N-methylation of the side chain of diaminopropionic acid (Dap). The three consecutive N-methyl amino acids could be coupled in good yields by using HATU/HOAt/DIEA in DMF, and the final octapeptide was also obtained on solid phase following a 4 + 4 fragment coupling approach. NMe-azathiocoraline (NMA) displayed nanomolar activity in the same order as the natural product and the same mode of action. In fact, modeling of NMe-azathiocoraline bonded to a TCGA sequence showed how the methyl groups remained further away from the DNA strand without changing the recognition pattern of thiocoraline. Moreover, NMe-azathiocoraline displayed an increased stability in human serum as compared to the parent natural product. This approach could be used in other depsipeptides and side chain to side chain cyclic peptides.