2D materials in electrochemical sensors for in vitro or in vivo use.

Individual cells and cell populations are at the present time investigated with a myriad of analytical tools. While most of them are commercially available, some of these analytical tools are just emerging from research laboratories and are in the developmental phase. Electrochemical sensors which allow the monitoring of low molecular weight compounds released (and / or uptaken) by cells are among these emerging tools. Such sensors are increasingly built using 2D materials (e.g. graphene-based materials, transition metal dichalcogenides, etc.) with the aim of conferring better analytical performances to these devices. The present work critically reviews studies published during the last 10 years describing electrochemical sensors made with 2D materials and exploited to monitor small compounds (e.g. H2O2, ·NO, glucose, etc.) in living biological systems. It also discusses the very few 2D material-based electrochemical sensors which are wearable or usable in vivo. Finally, the present work includes a specific section about 2D material biocompatibility, a fundamental requirement for 2D material-based sensor applications in vitro and in vivo. As such, the review provides a critical view on the state of the art of electrochemical sensors made with 2D materials and used at cellular level and it evaluates the possibility that such sensors will be used on / in the human body on a wider scale.

Measurement of the Extracellular pH of Adherently Growing Mammalian Cells with High Spatial Resolution Using a Voltammetric pH Microsensor.

There are only a few tools suitable for measuring the extracellular pH of adherently growing mammalian cells with high spatial resolution, and none of them is widely used in laboratories around the world. Cell biologists very often limit themselves to measuring the intracellular pH with commercially available fluorescent probes. Therefore, we built a voltammetric pH microsensor and investigated its suitability for monitoring the extracellular pH of adherently growing mammalian cells. The voltammetric pH microsensor consisted of a 37 µm diameter carbon fiber microelectrode modified with reduced graphene oxide and syringaldazine. While graphene oxide was used to increase the electrochemically active surface area of our sensor, syringaldazine facilitated pH sensing through its pH-dependent electrochemical oxidation and reduction. The good sensitivity (60 ± 2.5 mV/pH unit), reproducibility (coefficient of variation ≤3% for the same pH measured with 5 different microsensors), and stability (pH drift around 0.05 units in 3 h) of the built voltammetric pH sensors were successfully used to investigate the acidification of the extracellular space of both cancer cells and normal cells. The results indicate that the developed pH microsensor and the perfected experimental protocol based on scanning electrochemical microscopy can reveal details of the pH regulation of cells not attainable with pH sensors lacking spatial resolution or which cannot be reproducibly positioned in the extracellular space.

Electrochemical push-pull probe: from scanning electrochemical microscopy to multimodal altering of cell microenvironment.

To understand biological processes at the cellular level, a general approach is to alter the cells' environment and to study their chemical responses. Herein, we present the implementation of an electrochemical push-pull probe, which combines a microfluidic system with a microelectrode, as a tool for locally altering the microenvironment of few adherent living cells by working in two different perturbation modes, namely electrochemical (i.e., electrochemical generation of a chemical effector compound) and microfluidic (i.e., infusion of a chemical effector compound from the pushing microchannel, while simultaneously aspirating it through the pulling channel, thereby focusing the flow between the channels). The effect of several parameters such as flow rate, working distance, and probe inclination angle on the affected area of adherently growing cells was investigated both theoretically and experimentally. As a proof of concept, localized fluorescent labeling and pH changes were purposely introduced to validate the probe as a tool for studying adherent cancer cells through the control over the chemical composition of the extracellular space with high spatiotemporal resolution. A very good agreement between experimental and simulated results showed that the electrochemical perturbation mode enables to affect precisely only a few living cells localized in a high-density cell culture.

Complementarity of EIS and SPR to reveal specific and nonspecific binding when interrogating a model bioaffinity sensor; perspective offered by plasmonic based EIS.

The present work compares the responses of a model bioaffinity sensor based on a dielectric functionalization layer, in terms of specific and nonspecific binding, when interrogated simultaneously by Surface Plasmon Resonance (SPR), non-Faradaic Electrochemical Impedance Spectroscopy (EIS), and Plasmonic based-EIS (P-EIS). While biorecognition events triggered a sensitive SPR signal, the related EIS response was rather negligible. Contrarily, even a limited nonspecific adsorption onto the surface of the metallic electrode, allowed by the intrinsic imperfect compactness of the functionalization layers, was signaled by EIS and not by SPR. The source of this finding has been addressed from both theoretical and experimental perspectives, demonstrating that EIS signals are mainly sensitive to adsorptions that alter the current pathway through defects of the functionalization layer exposing the electrode. These observations are of importance for those developing biosensors analyzed by SPR, EIS, or the novel combination of the two methods (P-EIS). A possible application of the observed complementarity of the two methods, namely assessment of sample purity in respect to a target analyte is highlighted. Moreover, the possibility of false-positive EIS responses (determined by nonspecific binding) when assessing samples containing complex matrices or consisting of small molecular weight analytes is emphasized.

Substrate-Controlled Bidirectional Pumping by a Bienzymatic Micropump.

The development of autonomous, miniaturized pumps remains a problem of much interest, particularly with a view on microfluidics-based devices with increased portability and simplicity of use by nonspecialists. Spatially localized patches of enzyme imprinted on walls have been shown to induce a hydrodynamic flow when supplied with the corresponding enzyme substrate. Thus, such enzymatic micropumps are seen as a possible way of providing the means for nonmechanical, structurally simple, autonomous pumping. Hereby, we extend the current knowledge of enzymatic micropumps in two ways. First, we introduce ß-glucosidase as an enzyme that facilitates building micropumps with robust inward flows in the presence of cellobiose (e.g., 2.51 ± 0.56 µm s-1 in the presence of 80 mM cellobiose). Second, we embed ß-glucosidase and urease within the same patch and thus obtain a bienzymatic micropump. The latter exhibits the so far missing capability of bidirectional pumping as it produces inward flows in the presence of cellobiose (e.g., 0.95 ± 0.37 µm s-1 in the presence of 20 mM cellobiose) and outward flows in the presence of urea (e.g., 1.46 ± 0.47 µm s-1 in the presence of 20 mM urea). This bienzymatic micropump is a significant step for the development of biocompatible micropumps with versatile, controlled, and on-demand hydrodynamic pumping capabilities.

Development of a label-free aptasensor for monitoring the self-association of lysozyme.

A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 µg mL(-1) and a linear range of 5-50 µg mL(-1). As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.

Analyte Sensing with Catalytic Micromotors.

Catalytic micromotors can be used to detect molecules of interest in several ways. The straightforward approach is to use such motors as sensors of their "fuel" (i.e., of the species consumed for self-propulsion). Another way is in the detection of species which are not fuel but still modulate the catalytic processes facilitating self-propulsion. Both of these require analysis of the motion of the micromotors because the speed (or the diffusion coefficient) of the micromotors is the analytical signal. Alternatively, catalytic micromotors can be used as the means to enhance mass transport, and thus increase the probability of specific recognition events in the sample. This latter approach is based on "classic" (e.g., electrochemical) analytical signals and does not require an analysis of the motion of the micromotors. Together with a discussion of the current limitations faced by sensing concepts based on the speed (or diffusion coefficient) of catalytic micromotors, we review the findings of the studies devoted to the analytical performances of catalytic micromotor sensors. We conclude that the qualitative (rather than quantitative) analysis of small samples, in resource poor environments, is the most promising niche for the catalytic micromotors in analytical chemistry.

Real Time SPR Assessment of the Structural Changes of Adaptive Dynamic Constitutional Frameworks as a New Route for Sensing.

Cross linked gold-dynamic constitutional frameworks (DCFs) are functional materials of potential relevance for biosensing applications, given their adaptivity and high responsivity against various external stimuli (such as pH, temperature) or specific interactions with biomolecules (enzymes or DNA) via internal constitutional dynamics. However, characterization and assessment of their dynamic conformational changes in response to external stimuli has never been reported. This study proves the capability of Surface Plasmon Resonance (SPR) assays to analyse the adaptive structural modulation of a functional matrix encompassing 3D gold-dynamic constitutional frameworks (Au-DCFs) when exposed to pH variations, as external stimuli. We analyse Au-DCFs formed from Au nanoparticles, (AuNP) connected through constitutionally dynamic polymers, dynamers, with multiple functionalities. For increased generality of this proof-of-concept assay, Au-DCFs, involving DCFs designed from 1,3,5-benzene-tricarbaldehyde (BTA) connecting centres and polyethylene glycol (PEG) connectors, are covalently attached to standard SPR sensing chips (Au nanolayers, carboxyl terminated or with carboxymethyl dextran, CMD top-layer) and analysed using state-of-the art SPR instrumentation. The SPR effects of the distance from the Au-DCFs matrix to the Au nanolayer of the sensing chip, as well as of Au-DCFs thickness were investigated. This study reveals the SPR response, augmented by the AuNP, to the conformational change, i.e., shrinkage, of the dynamer and AuNP matrix when decreasing the pH, and provides an unexplored insight into the sensing applicability of SPR real-time analysis of adaptive functional materials.

Electrochemically Synthesized Poly(3-hexylthiophene) Nanowires as Photosensitive Neuronal Interfaces.

Poly(3-hexylthiophene) (P3HT) is a hole-conducting polymer that has been intensively used to develop organic optoelectronic devices (e.g., organic solar cells). Recently, P3HT films and nanoparticles have also been used to restore the photosensitivity of retinal neurons. The template-assisted electrochemical synthesis of polymer nanowires advantageously combines polymerization and polymer nanostructuring into one, relatively simple, procedure. However, obtaining P3HT nanowires through this procedure was rarely investigated. Therefore, this study aimed to investigate the template-assisted electrochemical synthesis of P3HT nanowires doped with tetrabutylammonium hexafluorophosphate (TBAHFP) and their biocompatibility with primary neurons. We show that template-assisted electrochemical synthesis can relatively easily turn 3-hexylthiophene (3HT) into longer (e.g., 17 ± 3 µm) or shorter (e.g., 1.5 ± 0.4 µm) P3HT nanowires with an average diameter of 196 ± 55 nm (determined by the used template). The nanowires produce measurable photocurrents following illumination. Finally, we show that primary cortical neurons can be grown onto P3HT nanowires drop-casted on a glass substrate without relevant changes in their viability and electrophysiological properties, indicating that P3HT nanowires obtained by template-assisted electrochemical synthesis represent a promising neuronal interface for photostimulation.

High-resolution impedance mapping using electrically activated quantitative phase imaging.

Retrieving electrical impedance maps at the nanoscale rapidly via nondestructive inspection with a high signal-to-noise ratio is an unmet need, likely to impact various applications from biomedicine to energy conversion. In this study, we develop a multimodal functional imaging instrument that is characterized by the dual capability of impedance mapping and phase quantitation, high spatial resolution, and low temporal noise. To achieve this, we advance a quantitative phase imaging system, referred to as epi-magnified image spatial spectrum microscopy combined with electrical actuation, to provide complementary maps of the optical path and electrical impedance. We demonstrate our system with high-resolution maps of optical path differences and electrical impedance variations that can distinguish nanosized, semi-transparent, structured coatings involving two materials with relatively similar electrical properties. We map heterogeneous interfaces corresponding to an indium tin oxide layer exposed by holes with diameters as small as ~550 nm in a titanium (dioxide) over-layer deposited on a glass support. We show that electrical modulation during the phase imaging of a macro-electrode is decisive for retrieving electrical impedance distributions with submicron spatial resolution and beyond the limitations of electrode-based technologies (surface or scanning technologies). The findings, which are substantiated by a theoretical model that fits the experimental data very well enable achieving electro-optical maps with high spatial and temporal resolutions. The virtues and limitations of the novel optoelectrochemical method that provides grounds for a wider range of electrically modulated optical methods for measuring the electric field locally are critically discussed.

High spatial resolution electrochemical biosensing using reflected light microscopy.

If the analyte does not only change the electrochemical but also the optical properties of the electrode/solution interface, the spatial resolution of an electrochemical sensor can be substantially enhanced by combining the electrochemical sensor with optical microscopy. In order to demonstrate this, electrochemical biosensors for the detection of hydrogen peroxide and glucose were developed by drop casting enzyme and redox polymer mixtures onto planar, optically transparent electrodes. These biosensors generate current signals proportional to the analyte concentration via a reaction sequence which ultimately changes the oxidation state of the redox polymer. Images of the interface of these biosensors were acquired using bright field reflected light microscopy (BFRLM). Analysis showed that the intensity of these images is higher when the redox polymer is oxidized than when it is reduced. It also revealed that the time needed for the redox polymer to change oxidation state can be assayed optically and is dependent on the concentration of the analyte. By combining the biosensor for hydrogen peroxide detection with BFRLM, it was possible to determine hydrogen peroxide in concentrations as low as 12.5 µM with a spatial resolution of 12 µm × 12 µm, without the need for the fabrication of microelectrodes of these dimensions.

Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes.

A miniaturized on-column digestion system constructed for the sequential analysis of semi-purified protein analytes is presented. By utilizing fused silica capillary (diameter 150microm) packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18 material, a linear column set-up was constructed. The protein analytes (pmol amounts) were first digested in the 600nl trypsin reactor portion of the system. Next, the generated peptides were trapped in the C18 column shaped as an electrospray emitter. Finally, after washing the matrix free from salts and other hydrophilic impurities present in the sample, peptides were eluted. A stepwise increased concentration profile of organic solvent, created by a dual syringe pump system, promoted the release of bound peptides, which were identified by electrospray ionization MS/MS. This approach proved to be very efficient, achieving almost complete digestion of the proteins studied, with suitable operational stability maintained for more than 1 week. Further, a small nebulizer was designed and fitted to the electrospray emitter. A significant improvement of the spray stability was observed and droplet build-up on the capillary was avoided, even at flow rates well above 1500nl/min. The proteins chloroperoxidase, staphylococcal enterotoxin B and protein A (injection volume 0.3microl, salt concentration 0.2-1M) were sequentially digested, desalted, eluted, detected and conclusively identified by bioinformatics web tools with an analytical cycle time of 10min.

Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces.

Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples.

Versatile SPR aptasensor for detection of lysozyme dimer in oligomeric and aggregated mixtures.

A Surface Plasmon Resonance (SPR) sensor for the quantitation of lysozyme dimer in monomer-dimer mixtures, reaching a detection limit of 1.4nM dimer, has been developed. The sensor is based on an aptamer which, although developed for the monomeric form, binds also the dimeric form but with a strikingly different kinetics. The aptasensor was calibrated using a dimer obtained by cross-linking. Sensorgrams acquired with the aptasensor in monomer-dimer mixtures were analysed using Principal Components Analysis and Multiple Regression to establish correlations with the dimer content in the mixtures. The method allows the detection of 0.1-1% dimer in monomer solutions without any separation. As an application, the aptasensor was used to qualitatively observe the initial stages of aggregation of lysozyme solutions at 60°C and pH 2, through the variations in lysozyme dimer amounts. Several other methods were used to characterize the lysozyme dimer obtained by cross-linking and confirm the SPR results. This work highlights the versatility of the aptasensor, which can be used, by simply tuning the experimental conditions, for the sensitive detection of either the monomer or the dimer and for the observation of the aggregation process of lysozyme.

Sensing based on the motion of enzyme-modified nanorods.

Asymmetric modification with an enzyme confers nanorods an enhanced diffusive motion that is dependent on the concentration of the enzyme substrate. In turn, such a motion opens the possibility of determining the concentration of the enzyme substrate by measuring the diffusion coefficient of nanorods modified with the appropriate enzyme. Nanorods, with a Pt and a polypyrrole (PPy) segment, were fabricated. The PPy segment of such nanorods was then modified with glucose oxidase (GOx), glutamate oxidase (GluOx), or xanthine oxidase (XOD). Calibration curves, linking the diffusion coefficient of the oxidase-modified nanorods to the concentration of the oxidase substrate, were subsequently built. The oxidase-modified nanorods and their calibration curves were finally used to determine substrate concentrations both in simple aqueous solutions and in complex samples such as horse serum and cell culture media. Based on the obtained results we are confident that our motion-based approach to sensing can be developed to the point where different nanorods in a mixture simultaneously report on the concentration of different compounds with good temporal and spatial resolution.

Magneto-plasmonic biosensor with enhanced analytical response and stability.

We present novel solutions to surpass current analytic limitations of Magneto-Optical Surface Plasmon Resonance (MOSPR) assays, concerning both the chip structure and the method for data analysis. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogeneous metals, as currently used. This alloy presents improved plasmonic and magnetic properties, yet a structural stability similar to Au-SPR chips, allowing for bioaffinity assays in saline solutions. Analyzing the whole reflectivity curve at multiple angles of incidence instead of the reflectivity value at a single incidence angle provides a high signal-to-noise ratio suitable for detection of minute analyte concentrations. Based on assessment of solutions with known refractive indices as well as of a model biomolecular interaction (i.e. IgG-AntiIgG) we demonstrate that the proposed structure of the MOSPR sensing chip and the procedure of data analysis allows for long-time assessment in liquid media with increased sensitivity over standard SPR analyses.

Bienzyme biosensors for glucose, ethanol and putrescine built on oxidase and sweet potato peroxidase.

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.

Visualization of micropatterned complex biosensor sensing chemistries by means of scanning electrochemical microscopy.

Redox hydrogel-based micropatterned complex biosensor architectures, used as sensing chemistries in amperometric ethanol or glucose biosensors, were deposited on gold, graphite or glass. Well-localized immobilization of active hydrogels with variable compositions was achieved by dispensing 100 pl droplets of cocktails containing alcohol or glucose dehydrogenase, redox polymer (PVI(13)dmeOs) and crosslinker (PEGDGE) while moving the target surface relative to the position of the nozzle of a piezo-actuated microdispenser. The resulting structures were microscopic patterns of enzyme-containing lines of a redox hydrogel with a line width of about 100 microm. Scanning electrochemical microscopy (SECM) in the amperometric feedback mode was used to visualize the immobilized enzyme microstructures and their localized biochemical activity was observed with high lateral resolution by detecting the enzymatically consumed substrate using K(4)[Fe(CN)(6)] as a free-diffusing electron-transfer mediator.

Enzymatically induced motion at nano- and micro-scales.

In contrast to adenosine triphosphate (ATP)-dependent motor enzymes, other enzymes are little-known as "motors" or "pumps", that is, for their ability to induce motion. The enhanced diffusive movement of enzyme molecules, the self-propulsion of enzyme-based nanomotors, and liquid pumping with enzymatic micropumps were indeed only recently reported. Enzymatically induced motion can be achieved in mild conditions and without the use of external fields. It is thus better suited for use in living systems (from single-cell to whole-body) than most other ways to achieve motion at small scales. Enzymatically induced motion is thus not only new but also important. Therefore, the present work reviews the most significant discoveries in enzymatically induced motion. As we will learn, freely diffusing enzymes enhance their diffusive movement by nonreciprocal conformational changes which parallel their catalytic cycles. Meanwhile, enzyme-modified nano- and micro-objects turn chemical energy into kinetic energy through mechanisms such as bubble recoil propulsion, self-electrophoresis, and self-diffusiophoresis. Enzymatically induced motion of small objects ranges from enhanced diffusive movement to directed motion at speeds as high as 1 cm s(-1). In spite of the progress made in understanding how the energy of enzyme reactions is turned into motion, most enzymatically powered devices remain inefficient and need improvements before we will witness their application in real world environments.

Label free sensing platform for amyloid fibrils effect on living cells.

This study presents a multiparametric label-free analysis gathering surface plasmon resonance (SPR) and electrical impedance spectroscopy (EIS) for monitoring the progress of a model epithelial cell culture (Madin Darbey Canine Kidney - MDCK) exposed to a peptide with high bio-medical relevance, amyloid ß (Aß42). The approach surpasses the limitations in using the SPR angle for analyzing confluent cell monolayers and proposes a novel quantitative analysis of the SPR dip combined with advanced EIS as a tool for dynamic cell assessment. Long, up to 48h time series of EIS and SPR data reveal a biphasic cellular response upon Aß42 exposure corresponding to changes in cell-substrate adherence, cell-cell tightening or cytoskeletal remodeling. The equivalent circuit used for fitting the EIS spectra provided substantiation of SPR analysis on the progress of cell adhesion as well as insight on dynamics of cell-cell junction. Complementary endpoint assays: western blot analysis and atomic force microscopy experiments have been performed for validation. The proposed label free sensing of nonlethal effect of model amyloid protein at cellular level provides enhanced resolution on cell-surface and cell-cell interactions modulated by membrane related protein apparatus, applicable as well to other adherent cell types and amyloid compounds.