Diego Pallarola

Monolayer graphene is a suitable active material for several electronics and sensor applications, for which large-area individual sheets are necessary on insulating substrates. Chemical vapor deposition (CVD) enables the synthesis of large-area graphene of high quality on metallic supports. However, graphene grown in this manner has to be transferred to suitable target substrates, for which there are still several challenges involved to obtain intact and defect-free sheets on a large scale. Here, we present a deep-learning assisted workflow for quantifying the coverage of transferred graphene based on high resolution optical image data, offering an accessible and scalable method for quality assessment. The workflow is based on training a neural network-based classifier (U-Net classifier) using an initial set of images to automatically identify areas covered by graphene. The trained model is subsequently used to quantify graphene coverage on samples obtained by different transfer methods. Utilizing this methodology, we first show that appropriate substrate pre-treatment is essential to maximize the coverage of transferred graphene. Moreover, we demonstrate that a post-transfer annealing treatment significantly improves the stability of graphene sensors in aqueous environments, a critical factor for sensing applications.

Soft substrates are interesting for many applications, ranging from mimicking the cellular microenvironment to implants. Conductive electrodes on such substrates allow the realization of flexible, elastic, and transparent sensors. Single-layer graphene as a candidate for such electrodes brings the advantage that the active area of the sensor is transparent and conformal to the underlying substrate. Here, we overcome several challenges facing the routine realization of graphene cell sensors on a canonical soft substrate, namely, poly-(dimethylsiloxane) (PDMS). We have systematically studied the effect of surface energy before, during, and after the transfer of graphene. Thus, we have identified a suitable support polymer, optimal substrate (pre)-treatment, and an appropriate solvent for the removal of the support. Using this procedure, we can reproducibly obtain stable and intact graphene sensors on a millimeter scale on PDMS, which can withstand continuous measurements in cell culture media for several days. From local nanomechanical measurements, we infer that the softness of the substrate is slightly affected after the graphene transfer. However, we can modulate the stiffness using PDMS with differing compositions. Finally, we show that graphene sensors on PDMS can be successfully used as soft electrodes for real-time monitoring of the cell adhesion kinetics. The routine availability of single-layer graphene electrodes on a soft substrate with tunable stiffness will open a new avenue for studies, where the PDMS-liquid interface is made conducting with minimal alteration of the intrinsic material properties such as softness, flexibility, elasticity, and transparency.

Soft substrates are interesting for a range of applications from mimicking cellular micro-environment to implants. Conductive electrodes on soft substrates open a broad spectrum of possibilities such as electrical and electrochemical sensing coupled with the flexibility, elasticity and transparency of the underlying substrate. Single layer graphene on a soft substrate as a candidate for such flexible electrodes brings the additional advantage that the active area of the sensor is transparent and conformal to the underlying substrate. Here, we overcome several challenges facing the routine realization of graphene cell sensors on a canonical soft substrate namely poly(dimethylsiloxane) (PDMS). Specifically, we have systematically studied the effect of surface energy before, during and after the transfer of graphene. Based on this, we have identified a suitable support polymer, optimal substrate (pre-) treatment and an appropriate solvent for the removal of the support. Using this procedure, we can reproducibly obtain stable and intact graphene sensors in millimeter-scale on PDMS, which can withstand continuous measurements in cell culture media for several days. From local nanomechanical measurements with an AFM, we infer that the softness of the substrate is slightly affected after graphene transfer. However, we can modulate the stiffness using PDMS of differing composition. Finally, we show that graphene sensors on PDMS can be successfully used as electrodes for real-time monitoring of cell adhesion kinetics on a soft substrate. The routine availability of single layer graphene electrodes on a flexible soft substrate with tunable stiffness will open a new avenue for a range of studies, where the PDMS-liquid interface is made conducting with minimal alteration of the intrinsic material properties such as softness, flexibility, elasticity and transparency.

Graphene oxide has been widely deployed in electricalsensors formonitoring physical, chemical, and biological processes. The presenceof abundant oxygen functional groups makes it an ideal substrate forintegrating biological functional units to assemblies. However, theintroduction of this type of defects on the surface of graphene hasa deleterious effect on its electrical properties. Therefore, adjustingthe surface chemistry of graphene oxide is of utmost relevance foraddressing the immobilization of biomolecules, while preserving itselectrochemical integrity. Herein, we describe the direct immobilizationof glucose oxidase onto graphene oxide-based electrodes prepared byLangmuir-Blodgett assembly. Electrochemical reduction of grapheneoxide allowed to control its surface chemistry and, by this, regulatethe nature and density of binding sites for the enzyme and the overallresponsiveness of the Langmuir-Blodgett biofilm. X-ray photoelectronspectroscopy, surface plasmon resonance, and electrochemical measurementswere used to characterize the compositional and functional featuresof these biointerfaces. Covalent binding between amine groups on glucoseoxidase and epoxy and carbonyl groups on the surface of graphene oxidewas successfully used to build up stable and active enzymatic assemblies.This approach constitutes a simple, quick, and efficient route tolocally address functional proteins at interfaces without the needfor additives or complex modifiers to direct the adsorption process.

Cells interact with their microenvironment by constantly sensing mechanical and chemical cues converting them into biochemical signals. These processes allow cells to respond and adapt to changes in their environment, and are crucial for most cellular functions. Understanding the mechanism underlying this complex interplay at the cell-matrix interface is of fundamental value to decipher key biochemical and mechanical factors regulating cell fate. The combination of material science and surface chemistry aided in the creation of controllable environments to study cell mechanosensing and mechanotransduction. Biologically inspired materials tailored with specific bioactive molecules, desired physical properties and tunable topography have emerged as suitable tools to study cell behavior. Among these materials, synthetic cell interfaces with built-in sensing capabilities are highly advantageous to measure biophysical and biochemical interaction between cells and their environment. In this review, we discuss the design of micro and nanostructured biomaterials engineered not only to mimic the structure, properties, and function of the cellular microenvironment, but also to obtain quantitative information on how cells sense and probe specific adhesive cues from the extracellular domain. This type of responsive biointerfaces provides a readout of mechanics, biochemistry, and electrical activity in real time allowing observation of cellular processes with molecular specificity. Specifically designed sensors based on advanced optical and electrochemical readout are discussed. We further provide an insight into the emerging role of multifunctional micro and nanosensors to control and monitor cell functions by means of material design.

Several fields of applications require a reliable characterization of the photothermal response and heat dissipation of nanoscopic systems, which remains a challenging task both for modeling and experimental measurements. Here, we present an implementation of anti-Stokes thermometry that enables the in situ photothermal characterization of individual nanoparticles (NPs) from a single hyperspectral photoluminescence confocal image. The method is label-free, potentially applicable to any NP with detectable anti-Stokes emission, and does not require any prior information about the NP itself or the surrounding media. With it, we first studied the photothermal response of spherical gold NPs of different sizes on glass substrates, immersed in water, and found that heat dissipation is mainly dominated by the water for NPs larger than 50 nm. Then, the role of the substrate was studied by comparing the photothermal response of 80 nm gold NPs on glass with sapphire and graphene, two materials with high thermal conductivity. For a given irradiance level, the NPs reach temperatures 18% lower on sapphire and 24% higher on graphene than on bare glass. The fact that the presence of a highly conductive material such as graphene leads to a poorer thermal dissipation demonstrates that interfacial thermal resistances play a very significant role in nanoscopic systems, and emphasize the need for in situ experimental thermometry techniques. The developed method will allow addressing several open questions about the role of temperature in plasmon-assisted applications, especially ones where NPs of arbitrary shapes are present in complex matrixes and environments.

In this study we investigate the impact of ligand presentation by various molecular spacers on integrin-based focal adhesion formation. Gold nanoparticles (AuNPs) arranged in hexagonal patterns were biofunctionalized with the same ligand head group, cyclic Arg-Gly-Asp [c(-RGDfX-)], but with different molecular spacers, each of which couples the head group to the gold. Aminohexanoic acid, polyethylene glycol (PEG) and polyproline spacers were used to vary the distance between the binding motif and the substrate, and thus the presentation of integrin binding on anchoring points. Adherent cells plated on nanopatterned surfaces with polyproline spacers for peptide immobilization could tolerate larger ligand spacing (162 nm) for focal adhesion formation, in comparison to cells on surfaces with PEG (110 nm) or aminohexanoic acid (62 nm) spacers. Due to the rigidity of the polyproline spacer, enhanced access to the ligand-binding site upon integrin-cRGD complex formation increases the probability of rebinding and decreases unbinding, as measured by fluorescence recovery after photobleaching (FRAP) analysis, compared to the analogues with aminohexanoic acid or PEG-containing spacers. These findings indicate that focal adhesion formation may not only be stabilized upon tight integrin clustering, but also by tuning the efficiency of the exposure of the cRGD-based ligand to the integrin extracellular domains. Our studies clearly highlight the importance of ligand spatial presentation for regulating adhesion-dependent cell behavior, and provide a sound approach for studying cell signaling processes on nanometer-scale, engineered bioactive surfaces under chemical stimuli of varying intensities.