Real-Time Monitoring of Cell Adhesion onto a Soft Substrate by a Graphene Impedance Biosensor.

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.

Electrochemical Fine-Tuning of the Chemoresponsiveness of Langmuir-Blodgett Graphene Oxide Films.

Graphene oxide has been widely deployed in electrical sensors for monitoring physical, chemical, and biological processes. The presence of abundant oxygen functional groups makes it an ideal substrate for integrating biological functional units to assemblies. However, the introduction of this type of defects on the surface of graphene has a deleterious effect on its electrical properties. Therefore, adjusting the surface chemistry of graphene oxide is of utmost relevance for addressing the immobilization of biomolecules, while preserving its electrochemical integrity. Herein, we describe the direct immobilization of glucose oxidase onto graphene oxide-based electrodes prepared by Langmuir-Blodgett assembly. Electrochemical reduction of graphene oxide allowed to control its surface chemistry and, by this, regulate the nature and density of binding sites for the enzyme and the overall responsiveness of the Langmuir-Blodgett biofilm. X-ray photoelectron spectroscopy, surface plasmon resonance, and electrochemical measurements were used to characterize the compositional and functional features of these biointerfaces. Covalent binding between amine groups on glucose oxidase and epoxy and carbonyl groups on the surface of graphene oxide was successfully used to build up stable and active enzymatic assemblies. This approach constitutes a simple, quick, and efficient route to locally address functional proteins at interfaces without the need for additives or complex modifiers to direct the adsorption process.

A portable and affordable aligner for the assembly of microfluidic devices.

The incorporation of sophisticated capabilities within microfluidic devices often requires the assembly of different layers in a correct arrangement. For example, when it is desired to include electrodes inside microfluidic channels or to create 3D microfluidic structures. However, the alignment between different substrates at the microscale requires expensive equipment not available for all research groups. In this work, we present an affordable, compact and portable aligner for assembling multilayered composite microfluidic chips. The instrument is composed of aluminum machined pieces combined with precision stages and includes a digital microscope with a LED illumination system for monitoring the alignment process. An interchangeable holder was created for substrate fixing, allowing the bonding of PDMS with other materials. Microscopic visualization is achieved through any device with internet access, avoiding the need of a computer attached to the aligner. To test the performance of the aligner, the center of an indium tin oxide microelectrode on a glass substrate was aligned with the center of a microchannel in a PDMS chip. The accuracy and precision of the instrument are suited for many microfluidic applications. The small and inexpensive design of the aligner makes it a cost-effective option for small groups working in microfluidics.

In Situ Photothermal Response of Single Gold Nanoparticles through Hyperspectral Imaging Anti-Stokes Thermometry.

Several fields of applications require a reliable characterization of the photothermal response and heat dissipation of nanoscopic systems, which remains a challenging task for both 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.

Biosensors for Studies on Adhesion-Mediated Cellular Responses to Their Microenvironment.

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.

Highly Ordered Gold Nanopatterned Indium Tin Oxide Electrodes for Simultaneous Optical and Electrochemical Probing Cell Interactions.

The formation of new types of sensitive conductive surfaces for the detection and transduction of cell-extracellular matrix recognition events in a real time, label-free manner is of great interest in the field of biomedical research. To study molecularly defined cell functions, biologically inspired materials that mimic the nanoscale order of extracellular matrix protein fibers and yield suitable electrical charge transfer characteristics are highly desired. Our strategy to achieve this goal is based on the spatial self-organization of patches of cell-adhesive molecules onto a gold-nanoparticle-patterned indium tin oxide electrode. Fibroblast adhesion response to selective ligands for integrins α5ß1 and αvß3, which are both relevant in cancer progression, is investigated by simultaneous electrochemical impedance spectroscopy and optical microscopy. Adhesive cells on α5ß1-selective nanopatterns showed enhanced membrane dynamics and tighter binding, compared with cells on αvß3-selective nanopatterns. The surface of the electrode exhibits high sensitivity to small changes in surface properties, because of the constitution of specific cell-surface interactions. Moreover, such sensitivity enables differentiation between cell types. This is exemplified by analyzing distinct features in the electrochemical readout of MCF-7 breast cancer cells versus MCF-10A mammary epithelial cells, when subjected to individual adhesive nanopatches.

Recognition-driven assembly of self-limiting supramolecular protein nanoparticles displaying enzymatic activity.

We report the recognition-driven assembly of self-limiting protein nanoparticles displaying enzymatic activity. Solution self-assembly of concanavalin A lectin and glycoenzyme glucose oxidase leads to the spontaneous formation of biocolloids with well-defined dimensions, narrow size distribution and remarkable stability. These biocolloids successfully recognize a glycosylated modified electrode retaining the enzyme activity.

Effect of gold nanoparticles on the structure and electron-transfer characteristics of glucose oxidase redox polyelectrolyte-surfactant complexes.

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox-active polyelectrolyte-surfactant complex containing [Os(bpy)2Clpy](2+) (bpy=2,2'-bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron-transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing-incidence small-angle X-ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz-crystal microbalance with dissipation (QCM-D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron-transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five-fold increase in current response to glucose compared with analogous supramolecular AuNP-free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron-transfer process.

Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion.

The interaction of specific surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand's spacer chemistry and length reveal significant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization.

Real-time monitoring of electrochemical controlled protein adsorption by a plasmonic nanowire based sensor.

A plasmonic sensor composed of a vertically aligned gold nanowire array was fabricated and employed for the optical detection of protein adsorption induced by an electric field.

Biselectivity of isoDGR peptides for fibronectin binding integrin subtypes α5ß1 and αvß6: conformational control through flanking amino acids.

Integrins are the major class of cell adhesion proteins. Their interaction with different ligands of the extracellular matrix is diverse. To get more insight into these interactions, artificial ligands endowed with a well-defined activity/selectivity profile are necessary. Herein, we present a library of cyclic pentapeptides, based on our previously reported peptide motif c(-phg-isoDGR-X-), in which high activity toward fibronectin binding integrins α5ß1 and αvß6 and not on vitronectin binding integrins αvß3 and αvß5 has been achieved by changing the flanking amino acids. The structure of the most promising candidates has been determined using a combined approach of NMR, distance geometry, and molecular dynamics simulations, and docking studies have been further used to elucidate the peptide-integrin interactions at the molecular level. The peptides' binding affinity has been characterized by enzyme linked immunosorbent assay experiments, and the results have been verified by cell adhesion experiments on specifically functionalized surfaces.

Electron transfer properties of dual self-assembled architectures based on specific recognition and electrostatic driving forces: its application to control substrate inhibition in horseradish peroxidase-based sensors.

This work describes the synergistic combination of ionic self-assembly and recognition-directed assembly with the aim of creating highly functional bioelectrochemical interfaces compatible with the supramolecular design of a wide variety of biosensing platforms. A recently synthesized glycopolyelectrolyte constituted of polyallylamine bearing redox-active osmium complexes and glycosidic residues (lactose) is used to create a self-assembled structure with sodium dodecylsulfate. In turn, this supramolecular thin films bearing redox-active and biorecognizable carbohydrate units enable the facile assembly of functional lectins as well as the subsequent docking and "wiring" of glycoenzymes, like horseradish peroxidase (HRP) (an elusive enzyme to immobilize via noncovalent interactions). The assembly of this system was followed by quartz crystal microbalance and grazing-incidence small-angle X-ray scattering (GISAXS) studies confirming that spin-coated ionically self-assembled films exhibit mesostructured architectures according to the formation of self-organized lamellar structures. In-depth characterization of the electrocatalytic properties of the biosupramacromolecular assemblies confirmed the ability of this kind of interfacial architecture to efficiently mediate electron transfer processes between the glycoenzyme and the electrode surface. For instance, our experimental electrochemical evidence clearly shows that tailor-made interfacial configurations of the ionic self-assemblies can prevent the inhibition of the glycoenzyme (typically observed in HRP) leading to bioelectrocatalytic currents up to 0.1 mA cm(-2). The presence of carbohydrate moieties in the ionic domains promotes the biorecognition-driven assembly of lectins adding a new dimension to the capabilities of ionic self-assembly.

Surface Properties of Nanostructured Bio-Active Interfaces: Impacts of Surface Stiffness and Topography on Cell-Surface Interactions.

Due to their ability to confer key functions of the native extracellular matrix (ECM) poly(ethylene glycol) (PEG)-based and PEG-modified materials have been extensively used as biocompatible and biofunctionalized substrate systems to study the influence of environmental parameters on cell adhesion in vitro. Given wide-ranging recent evidence that ECM compliance influences a variety of cell functions, the detailed determination and characterization of the specific PEG surface characteristics including topography, stiffness and chemistry is required. Here, we studied two frequently used bio-active interfaces - PEG-based and PEG-modified surfaces - to elucidate the differences between the physical surface properties, which cells can sense and respond to. For this purpose, two sets of surfaces were synthesized: the first set consisted of nanopatterned glass surfaces containing cRGD-functionalized gold nanoparticles surrounded by a passivated PEG-silane layer and the second set consisted of PEG-diacrylate (PEG-DA) hydrogels decorated with cRGD-functionalized gold nanoparticlesAlthough the two sets of nanostructured materials compared here were highly similar in terms of density and geometrical distribution of the presented bio-ligands as well as in terms of mechanical bulk properties, the topography and mechanical properties of the surfaces were found to be substantially different and are described in detail. In comparison to very stiff and ultrasmooth surface properties of the PEG-passivated glasses, the mechanical properties of PEG-DA surfaces in the biologically relevant stiffness range, together with the increased surface roughness at micro- and nanoscale levels have the potential to affect cell behavior. This potential was verified by studying the adhesive behavior of hematopoietic KG-1a and rat embryonic fibroblast (REF52) cells on both surfaces.

Ionic self-assembly of electroactive biorecognizable units: electrical contacting of redox glycoenzymes made easy.

This work explores the synergistic combination of ionic self-assembly and recognition-directed assembly for the modification of electrode surfaces with redox glycoenzymes on the basis of electroactive glycopolyelectrolyte-surfactant complexes.

Recognition-driven layer-by-layer construction of multiprotein assemblies on surfaces: a biomolecular toolkit for building up chemoresponsive bioelectrochemical interfaces.

The development of soft bioelectronic interfaces with accurate compositional and topological control of the supramolecular architecture attracts intense interest in the fast-growing field of bioelectronics and biosensing. The present study explores the recognition-driven layer-by-layer assembly of glycoenzymes onto electrode surfaces. The design of the multi-protein interfacial architecture is based on the multivalent supramolecular carbohydrate-lectin interactions between redox glycoproteins and concanavalin A (Con A) derivatives. Specifically, [Os(bpy)(2)Clpy](2+)-tagged Con A (Os-Con A) and native Con A were used to direct the assembly of horseradish peroxidase (HRP) and glucose oxidase (GOx) in a stepwise topologically controlled procedure. In our designed configuration, GOx acts as the biorecognition element to glucose stimulus, while HRP acts as the transducing element. Surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation (QCM-D) results are combined to give a close representation of the protein surface coverage and the content of water in the protein assembly. The characterization is complemented with in situ atomic force microscopy (AFM) to give a topographical description of the layers assemblage. Electrochemical (EC) techniques were used to characterize the functional features of the spontaneously self-assembled biohybrid architecture, showing that the whole system presents efficient electron transfer and mass transport processes being able to transform micromolar glucose concentration into electrical information. In this way the combination of the electroactive and nonelectroactive Con A provides an efficient strategy to control the position and composition of the protein layers via recognition-driven processes, which defines its sensitivity toward glucose. Furthermore, the incorporation of dextran as a permeable interlayer able to bind Con A promotes the physical separation of the biochemical and transducing processes, thus enhancing the magnitude of the bioelectrochemical signal. We consider that these results are relevant for the nanoconstruction of functional biointerfaces provided that our experimental evidence reveals the possibility of locally addressing recognition, transduction and amplification elements in interfacial ensembles via LbL recognition-driven processes.

Two efficient methods for the conjugation of smooth-form lipopolysaccharides with probes bearing hydrazine or amino groups. I. LPS activation with cyanogen bromide.

This chapter presents a conjugation method for coupling probes bearing hydrazine or primary amino groups to a smooth(S)-form lipopolysaccharide (LPS). LPS is modified by the activation of the hydroxyl groups present in its O-antigen moiety with cyanogen bromide in aqueous acetone. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota with dansyl hydrazine and horseradish -peroxidase yields labeling ratios above 300 nmol dansyl per mg LPS, with nearly no loss of the original endotoxin activity. In the case of horseradish peroxidase, introducing a spacer, a ratio of 28 nmol HRP per mg LPS is obtained, preserving 65% of the original endotoxic activity.

Two efficient methods for the conjugation of smooth-form lipopolysaccharides with probes bearing hydrazine or amino groups. II. LPS activation with a cyanopyridinium agent.

This chapter presents a conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS). LPS is modified by the activation of the hydroxyl groups present in its O-antigen moiety with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota with dansyl hydrazine and horseradish peroxidase yields labeling ratios above 110 nmol dansyl/mg LPS, with nearly no loss of the original endotoxic activity. In the case of horseradish peroxidase, introducing a spacer, a ratio of 29 nmol HRP/mg LPS was obtained, preserving 65% of the original endotoxic activity and an enzymatic activity of 120 U/mg.

Redox-active concanavalin A: synthesis, characterization, and recognition-driven assembly of interfacial architectures for bioelectronic applications.

The convergence of chemistry, biology, and materials science has paved the way to the emergence of hybrid nanobuilding blocks that incorporate the highly selective recognition properties of biomolecules, with the tailorable functional capabilities of inorganic molecules. In this work, we describe for the first time the decoration of concanavalin A (Con A), a protein with the ability to recognize sugars and form glycoconjugates, with Os(II) redox-active complexes. This strategy enabled the construction of electroactive biosupramolecular materials whose redox potentials could be easily modulated through the facile molecular modification of the electroactive inorganic complexes. Small-angle X-ray scattering (SAXS), steady-state fluorescence, surface plasmon resonance (SPR) spectroscopy, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and differential-pulsed (DPV) and cyclic voltammetry (CV) were used to characterize the structural and functional features of the synthesized biohybrid building blocks as well as their respective supramolecular assemblies built up on gold electrodes. By harnessing the electroactive and carbohydrate-recognition properties of these tailor-made biohybrid building blocks, we were able to integrate glucose oxidase (GOx) onto gold electrodes via sugar-lectin interactions. The redox activity of the Os-modified Con A interlayer allowed the electronic connection between the multilayered GOx assemblies and the metal electrode as evidenced by the well-defined bioelectrocatalytic response exhibited by the biomolecular assemblies in the presence of the glucose in solution. We consider that this approach based on the spontaneous formation of redox-active biosupramolecular assemblies driven by recognition processes can be of practical relevance for the facile design of biosensors, as well as for the construction of new multifunctional bioelectrochemical systems.

Supramolecular assembly of glucose oxidase on concanavalin A--modified gold electrodes.

There is a growing quest for the construction of functional supramolecular architectures to efficiently translate (bio)chemical events into easily measurable signals. This interest originates from its inherent scientific relevance as well as from their potential applications in the ever-flourishing areas of bioelectronics and biosensing. Herein, we describe the immobilization of glycoproteins onto electrode surfaces based on recognition-mediated supramolecular processes. Quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) spectroscopy, and electrochemical (EC) measurements were used to characterize the structural and functional features of these bio-supramolecular systems. Carbohydrate-lectin interactions were successfully used to build up stable assemblies of glucose oxidase (GOx) layers mediated by the recognition properties of concanavalin A supramolecular architectures. The catalytic response of GOx indicates that the whole population of enzymes incorporated in the supramolecular architecture is fully active. Even though lectin-carbohydrate interactions are rather weak, the multivalency effects prevailing in the supramolecular assembly confer remarkable stability to the interfacial architecture, thus preventing the release of the enzyme from the surface even with high glucose (ligand) concentrations. This approach represents a simple and straightforward route to locally address functional glycoproteins at interfaces. In this context, we consider that the versatility of a supramolecular assembly using biological interactions could open up new ways of envisioning or to generate new ideas for the future development of highly efficient bioelectronic platforms.