Direct, Rapid Detection of Pathogens from Urine Samples.

The problem of rapidly detecting pathogens directly from clinical samples poses significant analytical challenges. Addressing this issue in relation to urinary tract infections, we propose an effective protocol and related immunomagnetic test kits enabling versatile screening for the presence of pathogenic bacteria in unprocessed urine samples. To achieve this, the components of a typical immunomagnetic separation protocol were optimized towards the sensitive assessment of the aggregates formed out of immunomagnetically tagged target pathogens collected from clinical samples. Specifically, a dedicated immunomagnetic material was developed via the functionalization of standardized, micron-sized magnetic beads with generic antibodies against gram-specific bacterial constituents with mannan binding lectin. As such, we demonstrate efficient procedures for achieving the enhanced, specific, and pathogen-mediated cluster formation of these tailored affinity-coated magnetic beads in complex samples. We further show how cluster analysis, in conjunction with the use of nonspecific, inexpensive fluorescent dye, allows for a straightforward optical assessment of the bacterial load directly from urine samples. The optimized sensing protocol and related kits provide, in less than 60 min, qualitative (positive/negative) information on the bacterial load with 85% specificity and 96% sensitivity, which is appropriate to empower clinical microscopy with a new analytic dimension. The procedure is prone to automation, can be conveniently used in clinical microbiology laboratories and, since it preserves the viability of the captured bacteria, can be interfaced with downstream analyses and antimicrobial susceptibility testing. Moreover, the study emphasizes a suite of practical validation assays that are useful for bringing the tool-box of immunomagnetic materials outside the academic laboratory and into real-life applications.

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.

Advanced Optogenetic-Based Biosensing and Related Biomaterials.

The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.

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.

Cellular sensing platform with enhanced sensitivity based on optogenetic modulation of cell homeostasis.

We demonstrate a new biosensing concept with impact on the development of rapid, point of need cell based sensing with boosted sensitivity and wide relevance for bioanalysis. It involves optogenetic stimulation of cells stably transfected to express light sensitive protein channels for optical control of membrane potential and of ion homeostasis. Time-lapse impedance measurements are used to reveal cell dynamics changes encompassing cellular responses to bioactive stimuli and optically induced homeostasis disturbances. We prove that light driven perturbations of cell membrane potential induce homeostatic reactions and modulate transduction mechanisms that amplify cellular response to bioactive compounds. This allows cell based biosensors to respond more rapidly and sensitively to low concentrations of bioactive/toxic analytes: statistically relevant impedance changes are recorded in less than 30 min, in comparison with >8 h in the best alternative reported tests for the same low concentration (e.g. a concentration of 25 µM CdCl2, lower than the threshold concentration in classical cellular sensors). Comparative analysis of model bioactive/toxic compounds (ouabain and CdCl2) demonstrates that cellular reactivity can be boosted by light driven perturbations of cellular homeostasis and that this biosensing concept is able to discriminate analytes with different modes of action (i.e. CdCl2 toxicity versus ion pump inhibition by ouabain), a significant advance against state of the art cell based sensors.

A short review on cell-based biosensing: challenges and breakthroughs in biomedical analysis.

Current cell-based biosensors have progressed substantially from mere alternatives to molecular bioreceptors into enabling tools for interfacing molecular machineries and gene circuits with microelectronics and for developing groundbreaking sensing and theragnostic platforms. The recent literature concerning whole-cell biosensors is reviewed with an emphasis on mammalian cells, and the challenges and breakthroughs brought along in biomedical analyses through novel biosensing concepts and the synthetic biology toolbox. These recent innovations allow development of cell-based biosensing platforms having tailored performances and capable to reach the levels of sensitivity, dynamic range, and stability suitable for high analytic/medical relevance. They also pave the way for the construction of flexible biosensing platforms with utility across biological research and clinical applications. The work is intended to stimulate interest in generation of cell-based biosensors and improve their acceptance and exploitation.

Modulation of Cellular Reactivity for Enhanced Cell-Based Biosensing.

Cell-based sensing platforms provide functional information on cellular effects of bioactive or toxic compounds in a sample. Current challenges concern the rather extended length of the assays as well as their limited reproducibility and sensitivity. We present a biosensing method capable of appraising, on a short time scale and with exquisite sensitivity, the occurrence and the magnitude of cellular alterations induced by low levels of a bioactive/toxic compound. Our method is based on integrating optogenetic control of non-electrogenic human cells, modified to express light sensitive protein channels, into a non-invasive electro-optical analytical platform enabling quantitative assessment of the stimulus dependent, dynamical cellular response. Our system exploits the interplay between optogenetic stimulation and time lapse fast impedance assays in boosting the platform sensitivity when exposing cells to a model exogenous stimulus, under both static and flow conditions. The proposed optogenetically modulated cell-based sensing platform is suitable for in field applications and provides a new paradigm for impedance-based sensing.

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.

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.

Functional Micrococcus lysodeikticus layers deposited by laser technique for the optical sensing of lysozyme.

Whole cell optical biosensors, made by immobilizing whole algal, bacterial or mammalian cells on various supports have found applications in several fields, from ecology and ecotoxicity testing to biopharmaceutical production or medical diagnostics. We hereby report the deposition of functional bacterial layers of Micrococcus lysodeikticus (ML) via Matrix-Assisted Pulsed Laser Evaporation (MAPLE) on poly(diallyldimethylamonium) (PDDA)-coated-glass slides and their application as an optical biosensor for the detection of lysozyme in serum. Lysozyme is an enzyme upregulated in inflammatory diseases and ML is an enzymatic substrate for this enzyme. The MAPLE-deposited bacterial interfaces were characterised by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Fourier-Transformed Infrared Spectroscopy (FTIR), Raman and optical microscopy and were compared with control interfaces deposited via layer-by-layer on the same substrate. After MAPLE deposition and coating with graphene oxide (GO), ML-modified interfaces retained their functionality and sensitivity to lysozyme's lytic action. The optical biosensor detected lysozyme in undiluted serum in the clinically relevant range up to 10µgmL-1, in a fast and simple manner.

Quantitative assessment of specific carbonic anhydrase inhibitors effect on hypoxic cells using electrical impedance assays.

Carbonic anhydrase IX (CA IX) is an important orchestrator of hypoxic tumour environment, associated with tumour progression, high incidence of metastasis and poor response to therapy. Due to its tumour specificity and involvement in associated pathological processes: tumourigenesis, angiogenesis, inhibiting CA IX enzymatic activity has become a valid therapeutic option. Dynamic cell-based biosensing platforms can complement cell-free and end-point analyses and supports the process of design and selection of potent and selective inhibitors. In this context, we assess the effectiveness of recently emerged CA IX inhibitors (sulphonamides and sulphocoumarins) and their antitumour potential using an electrical impedance spectroscopy biosensing platform. The analysis allows discriminating between the inhibitory capacities of the compounds and their inhibition mechanisms. Microscopy and biochemical assays complemented the analysis and validated impedance findings establishing a powerful biosensing tool for the evaluation of carbonic anhydrase inhibitors potency, effective for the screening and design of anticancer pharmacological agents.

Biosensing Based on Magneto-Optical Surface Plasmon Resonance.

In spite of the high analytic potential of Magneto Optical Surface Plasmon Resonance (MOSPR) assays, their applicability to biosensing has been limited due to significant chip stability issues. We present novel solutions to surpass current limitations of MOSPR sensing assays, based on innovative chip structure, tailored measurements and improved data analysis methods. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogenous metals with magnetic and plasmonic properties, as currently used. This new approach presents improved plasmonic and magnetic properties, yet a structural stability similar to standard Au-SPR chips, allowing for bioaffinity assays in saline solutions. Moreover, using a custom-designed measurement configuration that allows the acquisition of the SPR curve, i.e., the reflectivity measured at multiple angles of incidence, instead of the reflectivity value at a single-incidence angle, a high signal-to-noise ratio is achieved, suitable for detection of minute analyte concentrations. The proposed structure of the MOSPR sensing chip and the procedure of data analysis allow for long time assessment in liquid media, a significant advancement over existing MOSPR chips, and confirm the MOSPR increased sensitivity over standard SPR analyses.

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.

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.

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.

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.

Functional and molecular characterization of the effect of amyloid-ß42 on an in vitro epithelial barrier model.

Recently, the blood-brain barrier (BBB) has been pointed to as an active player in neurodegenerative disorders, albeit the actual succession of pathogenic events remains to be elucidated. Amyloid-ß (Aß) is an important pathogenic player in Alzheimer's disease, and it is cleared from the brain partly by transportation across the BBB. In this work we asked the question whether Aß-induced alteration of tight junction (TJ) protein expression is a result of the complex in situ microenvironment of the BBB or if it can be replicated in an externalized environment, such as an in vitro epithelial barrier, where barrier property changes can be investigated without confounding factors. Therefore, we treated barrier forming MDCKI and II epithelial cells with Aß42 and investigated TJ occludin and claudin-2 protein levels and cellular distribution through western blot and immunofluorescence. To assess barrier function, we measured transepithelial resistance (TEER) and studied cell polarity through atomic force microscopy (AFM). We found that Aß42 cell treatment increased occludin expression and decreased claudin-2 expression. With TEER, an increase in paracellular resistance was noted, which started at 10 hours and peaked at 20 hours of Aß42 treatment. AFM analysis demonstrated an associated morphological alteration of the cell monolayer. In conclusion, we demonstrated that Aß42 is able to modify TJ protein expression and to functionally alter barrier properties in vitro and that this effect is not conditioned by other pathogenic Alzheimer's disease events taking place in the complex brain microenvironment.

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.

Assessment of pathogenic bacteria using periodic actuation.

A new analytical platform for the assessment of pathogenic bacteria is presented. It is based on a robust technology which is able to amplify the signal to noise ratio providing fast and sensitive detection of target pathogenic bacteria. The system uses a custom made AC electrical impedance analyser to measure, using a lab on a chip platform, the oscillations of magnetically labelled analytes when applying a periodic magnetic field. The concentration of pathogenic Escherichia coli O157:H7 chosen as bacterial model was determined based on the amplitude of the electrical impedance oscillations at a selected AC frequency. The analytical platform provides a limit of detection of 10(2) cells ml(-1), has a fast analysis time, and is amenable for the detection of other target cells. The system has simple design suitable for portability and automated operation.