ABSTRACT
Biomarkers play key roles in the diagnosis, risk assessment, treatment and supervision of cardiovascular diseases (CVD). Optical biosensors and assays are valuable analytical tools answering the need for fast and reliable measurements of biomarker levels. This review presents a survey of recent literature with a focus on the past 5 years. The data indicate continuing trends towards multiplexed, simpler, cheaper, faster and innovative sensing while newer tendencies concern minimizing the sample volume or using alternative sampling matrices such as saliva for less invasive assays. Utilizing the enzyme-mimicking activity of nanomaterials gained ground in comparison to their more traditional roles as signaling probes, immobilization supports for biomolecules and for signal amplification. The growing use of aptamers as replacements for antibodies prompted emerging applications of DNA amplification and editing techniques. Optical biosensors and assays were tested with larger sets of clinical samples and compared with the current standard methods. The ambitious goals on the horizon for CVD testing include the discovery and determination of relevant biomarkers with the help of artificial intelligence, more stable specific recognition elements for biomarkers and fast, cheap readers and disposable tests to facilitate rapid testing at home. As the field is progressing at an impressive pace, the opportunities for biosensors in the optical sensing of CVD biomarkers remain significant.
Subject(s)
Biosensing Techniques , Cardiovascular Diseases , Nanostructures , Humans , Artificial Intelligence , Biomarkers , Biosensing Techniques/methods , Cardiovascular Diseases/diagnosisABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Biosensing Techniques/instrumentation , Cytotoxins/toxicity , Dielectric Spectroscopy/instrumentation , Electric Impedance , Equipment Design , HEK293 Cells , Humans , Light , Optogenetics/instrumentation , Toxicity Tests/instrumentationABSTRACT
Electro-optical measurements, i.e., optical waveguides and plasmonic based electrochemical impedance spectroscopy (P-EIS), are based on the sensitive dependence of refractive index of electro-optical sensors on surface charge density, modulated by an AC electrical field applied to the sensor surface. Recently, P-EIS has emerged as a new analytical tool that can resolve local impedance with high, optical spatial resolution, without using microelectrodes. This study describes a high speed image acquisition and processing system for electro-optical measurements, based on a high speed complementary metal-oxide semiconductor (CMOS) sensor and a field-programmable gate array (FPGA) board. The FPGA is used to configure CMOS parameters, as well as to receive and locally process the acquired images by performing Fourier analysis for each pixel, deriving the real and imaginary parts of the Fourier coefficients for the AC field frequencies. An AC field generator, for single or multi-sine signals, is synchronized with the high speed acquisition system for phase measurements. The system was successfully used for real-time angle-resolved electro-plasmonic measurements from 30 Hz up to 10 kHz, providing results consistent to ones obtained by a conventional electrical impedance approach. The system was able to detect amplitude variations with a relative variation of ±1%, even for rather low sampling rates per period (i.e., 8 samples per period). The PC (personal computer) acquisition and control software allows synchronized acquisition for multiple FPGA boards, making it also suitable for simultaneous angle-resolved P-EIS imaging.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Carbonic Anhydrase IX/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/pharmacology , Coumarins/pharmacology , Electric Impedance , Sulfonamides/pharmacology , Tumor Hypoxia/drug effects , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Coumarins/chemical synthesis , Coumarins/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HT29 Cells , Humans , Molecular Structure , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Biosensing Techniques/methods , Magnetics , Surface Plasmon Resonance/methods , Alloys , Animals , Biosensing Techniques/instrumentation , Equipment Design , Humans , Nanotechnology/instrumentation , Nanotechnology/methods , Statistics as Topic/methods , Surface Plasmon Resonance/instrumentationABSTRACT
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.
Subject(s)
Graphite/chemistry , Micrococcus luteus/metabolism , Muramidase/blood , Muramidase/metabolism , Surface Plasmon Resonance/methods , Adsorption , Animals , Cattle , Cells, Immobilized/metabolism , Gold/chemistry , Oxides/chemistryABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , Muramidase/analysis , Protein Multimerization , Surface Plasmon Resonance/methods , Animals , Chickens , Limit of Detection , Protein AggregatesABSTRACT
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.
Subject(s)
Biosensing Techniques , Glucose Oxidase/chemistry , Glucose/isolation & purification , Nanotubes/chemistry , Diffusion , Enzymes, Immobilized/chemistry , Glucose/chemistry , Polymers/chemistry , Pyrroles/chemistryABSTRACT
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.
Subject(s)
Biosensing Techniques/instrumentation , Immunoassay/instrumentation , Magnetometry/instrumentation , Surface Plasmon Resonance/instrumentation , Equipment Design , Equipment Failure Analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
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.
Subject(s)
Biosensing Techniques/methods , Dielectric Spectroscopy , Surface Plasmon Resonance , Animals , Cattle , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Protein Binding , Serum Albumin/chemistry , Serum Albumin/metabolismABSTRACT
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.
Subject(s)
Amyloid/isolation & purification , Biosensing Techniques/methods , Dielectric Spectroscopy/methods , Surface Plasmon Resonance , Animals , Dogs , Madin Darby Canine Kidney CellsABSTRACT
Nanorods were decorated with different hemeproteins that are able to convert hydrogen peroxide. When dispersed into hydrogen peroxide solutions, most of these nanorods are characterized by diffusion coefficients which increase with the concentration of hydrogen peroxide. Such a behaviour does not characterize unmodified nanorods.
Subject(s)
Hemeproteins/chemistry , Hydrogen Peroxide/chemistry , Nanotubes/chemistry , Diffusion , Models, Molecular , SolutionsABSTRACT
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.
Subject(s)
Escherichia coli O157/isolation & purification , Lab-On-A-Chip Devices , Electrodes , Magnetic Fields , Magnets/chemistry , Microspheres , Time FactorsABSTRACT
Surface quality of the Surface Plasmon Resonance (SPR) chips is a major limiting issue in most SPR analyses, even more for supported lipid membranes experiments, where both the organization of the lipid matrix and the subsequent incorporation of the target molecule depend on the surface quality. A novel quantitative method to characterize the quality of SPR sensors chips is described for L1 chips subject to formation of lipid films, injection of membrane disrupting compounds, followed by appropriate regeneration procedures. The method consists in analysis of the SPR reflectivity curves for several standard solutions (e.g. PBS, HEPES or deionized water). This analysis reveals the decline of sensor surface as a function of the number of experimental cycles (consisting in biosensing assay and regeneration step) and enables active control of surface regeneration for enhanced reproducibility. We demonstrate that quantitative evaluation of the changes in reflectivity curves (shape of the SPR dip) and of the slope of the calibration curve provides a rapid and effective procedure for surface quality assessment. Whereas the method was tested on L1 SPR sensors chips, we stress on its amenability to assess the quality of other types of SPR chips, as well.
Subject(s)
Biosensing Techniques/methods , Membrane Lipids/isolation & purification , Surface Plasmon Resonance , Membrane Lipids/chemistry , Membranes/chemistry , Microchip Analytical Procedures , Surface PropertiesABSTRACT
The complexity of the cellular response, induced even by the simplest experimental stimulus, requires an increased number of cellular parameters to be simultaneously monitored. An all electrochemical system allowing the simultaneous and real-time monitoring of both cell adherence and superoxide release into the extracellular space was developed to address this challenge. Cell adherence (to neighboring cells and to substrate) was monitored using non-faradaic impedance spectroscopy while the superoxide release was monitored using a cytochrome c-based amperometric biosensor. The system was used to observe for the first time how these two cellular parameters are changing in real-time for renal cells exposed to calcium oxalate, a calculus-forming salt. It was discovered that calcium oxalate crystals decrease cell adherence and in the same time induce oxidative stress by an overproduction of superoxide. Subconfluent cells, without fully developed tight junctions, appear to be more vulnerable than confluent cells with tight junctions indicating the important protective role of these junctions.
Subject(s)
Calcium Oxalate/pharmacology , Dielectric Spectroscopy , Electrochemical Techniques , Epithelial Cells/drug effects , Animals , Biosensing Techniques , Cell Adhesion , Cells, Cultured , Cytochromes c/metabolism , Kidney/cytology , Superoxides/metabolism , Xenopus laevisABSTRACT
A novel low-cost platform to assess biomolecular interactions was investigated using surface plasmon resonance and an aptamer-based assay for thrombin detection. Gold SPR surface functionalized with a carboxylated cross-linked BSA film (cBSA) and commercially available carboxymethylated dextran chip (CM5) were used as immobilization platforms for the thrombin binding aptamer. The high end commercial instrument Biacore 3000 and a custom made FIA set-up involving TI Spreeta sensor (TSPR2K23) were used to assess different concentrations of thrombin within the range 0.1-150 nM both in buffer and in a complex matrix (plasma) using the obtained aptasensors. Based on data derived from both CM5 and cBSA platforms, the cBSA aptasensor exhibited good selectivity, stability and regeneration ability, both in buffer and in complex matrices (plasma), comparable with CM5.