Facile solid-phase synthesis of biotinylated alkyl thiols

Biotinylated alkyl thiols with the capacity to graft avidin proteins are in increasing demand for the development of self-assembled monolayers on gold. Here we propose 2-Chlorotrityl Chloride solid-phase resin as a new platform to produce these functionalized alkyl thiols. Biotinylated alkyl thiols of non-obvious solution synthesis were obtained rapidly using this method and without previous purification steps. © 2006 Elsevier Ltd. All rights reserved

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

[Abstract]: 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.This work has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 881603 (GrapheneCore3) and no. 785219 (GrapheneCore2). M. P. is AXA Professor and is supported by the European Research Council (ERC-AdG-2019, no. 885323), the Agencia Estatal de Investigación-AEI (“Proyectos I+D+i 2019-Modalidad Retos Investigación”, no. PID2019-108523RB-I00), by grant PRE2020-095099 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future.” Part of this work has made use of the Spanish ICTS Network MICRONANOFABS, partially supported by MICINN and the ICTS NANBIOSIS, more specifically by the Micro-NanoTechnology Unit U8 of the CIBER-BBN. This project was also funded by the Generalitat de Catalunya (2021SGR00495), by the Spanish Ministerio de Ciencia e Innovación (PID2021-126117NA-I00), by “ERDF A way of making Europe”, and by CIBER-BBN (CB06/01/0049). X. I., R. V., A. G. and E. P.-A. thank the financial support provided by CIBER-BBN and the Instituto de Salud Carlos III with assistance from the European Regional Development. A. C. acknowledges financial support by Grant ED431H 2020/17 funded by Xunta de Galicia, and by Grant RYC2020-030183-I funded by MCIN/AEI/10.13039/501100011033 and “ESF Investing in your future”.Generalitat de Catalunya; 2021SGR0049

Development of a novel microsensor for the study of oxygen profiles in biofilms

Understanding of the processes taking place inside biofilms is a key parameter to progress in the optimization of biofiltration technologies. This study was conducted with the aim of developing a novel dissolved oxygen (DO) microsensor specially designed for biofilms monitoring. The microsensor was fabricated through standard photolithography techniques, resulting in a microelectrodes array (MEA) of 11 gold circular working electrodes, with a diameter of 50 µm , and a gold reference electrode, which allows obtaining a snapshot oxygen profile of 1 mm of depth. The performance of the sensor was fully characterized under different conditions, inwhich the sensor presented high sensitivity and repeatability, and low detection and quantification limits. Monitoring of sensor performance showed a stable and reliable response. The developed sensor was used in obtaining micropofiles in an aerobic heterotrophic biofilm, showing similar response to Clark-type commercial microsensors. These studies concluded that the novel MEA sensor for DO monitoring allows obtaining oxygen profiles within biofilms, becoming a useful tool for the research of many biological applications.Postprint (author's final draft

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

[Abstract] 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.This work was funded by the European Union’s Horizon 2020 Research and Innovation Program under the Grant Agreements 785219 and 881603 of the Graphene Flagship. DS acknowledges the support of the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement no. 838902. MP as the recipient of the AXA Bionanotechnology Chair, is grateful to the AXA Research Fund for financial support. This work was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency-grant no. MDM-2017- 0720. AC thanks Xunta de Galicia for his research grant Atracción de Talento (no. ED431H 2020/17). GR acknowledges funding from RYC-2016-21412. HH acknowledges funding from Juan de la Cierva – Incorporación no. IJC-2018-037396-IXunta de Galicia; ED431H 2020/1

Biofilm oxygen profiling using an array of microelectrodes on a microfabricated needle

A novel microelectrode array (DO-MEA) sensor was designed and fabricated using microelectromechanical systems technology on a needle for real time measurement of dissolved oxygen (DO). The sensor consisted of eleven gold disk microelectrodes and a rectangular auxiliary electrode along them. The sensor can also be operated with an integrated reference system. Three different sensor designs were fabricated, and their responses were fully characterized and evaluated. The DO-MEA sensor presented a linear response in the 0-8 mg DO·L-1 concentration range in water, displaying high sensitivity and repeatability. Knowledge of bacterial activity inside biofilms is key to the optimization of applied biotechnologies. The developed sensor was validated against a commercial Clark-type microelectrode overcoming its drawbacks, by profiling a heterotrophic biofilm cultivated in a flat-plate bioreactor. The DO-MEA sensor provided a multipoint, simultaneous dissolved oxygen snapshot profile inside a biofilm with high spatial resolution due to its micrometric dimensions, thus becoming a powerful tool for the research of many similar biological-based processes and applications.Peer ReviewedPostprint (author’s final draft

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 multi-technique 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

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

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 (&lt;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.</p

Ethanol Solvation of Polymer Residues in Graphene Solution-Gated Field Effect Transistors

The persistence of photoresist residues from microfabrication procedures causes significant obstacles in the technological advancement of graphene-based electronic devices. These residues induce undesired chemical doping effects, diminish carrier mobility, and deteriorate the signal-to-noise ratio, making them critical in certain contexts, including sensing and electrical recording applications. In graphene solution-gated field-effect transistors (gSGFETs), the presence of polymer contaminants makes it difficult to perform precise electrical measurements, introducing response variability and calibration challenges. Given the absence of viable short to midterm alternatives to polymer-intensive microfabrication techniques, a postpatterning treatment involving THF and ethanol solvents was evaluated, with ethanol being the most effective, environmentally sustainable, and safe method for residue removal. Employing a comprehensive analysis with XPS, AFM, and Raman spectroscopy, together with electrical characterization, we investigated the influence of residual polymers on graphene surface properties and transistor functionality. Ethanol treatment exhibited a pronounced enhancement in gSGFET performance, as evidenced by a shift in the charge neutrality point and reduced dispersion. This systematic cleaning methodology holds the potential to improve the reproducibility and precision in the manufacturing of graphene devices. Particularly, by using ethanol for residue removal, we align our methodology with the principles of green chemistry, minimizing environmental impact while advancing diverse graphene technology applications

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.Peer reviewe