Extremozyme-Based Biosensors for Environmental Pollution Monitoring: Recent Developments.

Extremozymes combine high specificity and sensitivity with the ability to withstand extreme operational conditions. This work presents an overview of extremozymes that show potential for environmental monitoring devices and outlines the latest advances in biosensors utilizing these unique molecules. The characteristics of various extremozymes described so far are presented, underlining their stability and operational conditions that make them attractive for biosensing. The biosensor design is discussed based on the detection of photosynthesis-inhibiting herbicides as a case study. Several biosensors for the detection of pesticides, heavy metals, and phenols are presented in more detail to highlight interesting substrate specificity, applications or immobilization methods. Compared to mesophilic enzymes, the integration of extremozymes in biosensors faces additional challenges related to lower availability and high production costs. The use of extremozymes in biosensing does not parallel their success in industrial applications. In recent years, the "collection" of recognition elements was enriched by extremozymes with interesting selectivity and by thermostable chimeras. The perspectives for biosensor development are exciting, considering also the progress in genetic editing for the oriented immobilization of enzymes, efficient folding, and better electron transport. Stability, production costs and immobilization at sensing interfaces must be improved to encourage wider applications of extremozymes in biosensors.

Progress in the Optical Sensing of Cardiac Biomarkers.

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.

Two-Dimensional Nanostructures for Electrochemical Biosensor.

Current advancements in the development of functional nanomaterials and precisely designed nanostructures have created new opportunities for the fabrication of practical biosensors for field analysis. Two-dimensional (2D) and three-dimensional (3D) nanomaterials provide unique hierarchical structures, high surface area, and layered configurations with multiple length scales and porosity, and the possibility to create functionalities for targeted recognition at their surface. Such hierarchical structures offer prospects to tune the characteristics of materials-e.g., the electronic properties, performance, and mechanical flexibility-and they provide additional functions such as structural color, organized morphological features, and the ability to recognize and respond to external stimuli. Combining these unique features of the different types of nanostructures and using them as support for bimolecular assemblies can provide biosensing platforms with targeted recognition and transduction properties, and increased robustness, sensitivity, and selectivity for detection of a variety of analytes that can positively impact many fields. Herein, we first provide an overview of the recently developed 2D nanostructures focusing on the characteristics that are most relevant for the design of practical biosensors. Then, we discuss the integration of these materials with bio-elements such as bacteriophages, antibodies, nucleic acids, enzymes, and proteins, and we provide examples of applications in the environmental, food, and clinical fields. We conclude with a discussion of the manufacturing challenges of these devices and opportunities for the future development and exploration of these nanomaterials to design field-deployable biosensors.

Exhaled breath biomarker sensing.

This goal of this minireview is to introduce the reader to the area of research concerned with exhaled breath analysis for the purpose of detecting abnormal levels of physiologically-relevant chemical markers reflective of respiratory diseases. Two main two groups of sensing methods are reviewed: mass spectrometry and (bio)sensors. The discussion focuses on biosensor applications for EB and EBC analyses, which are presented in detail. The review finishes with conclusions and future perspectives, including recommendations for future near-term and long-term development of EBC biomarker sensing.

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives.

Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors' selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods.

Collision-Based Electrochemical Detection of Lysozyme Aggregation.

Proteins are utilized across many biomedical and pharmaceutical industries; therefore, methods for rapid and accurate monitoring of protein aggregation are needed to ensure proper product quality. Although these processes have been previously studied, it is difficult to comprehensively evaluate protein folding and aggregation by traditional characterization techniques such as atomic force microscopy (AFM), electron microscopy, or X-ray diffraction, which require sample pre-treatment and do not represent native state proteins in solution. Herein, we report early tracking of lysozyme (Lyz) aggregation states by using single-particle collision electrochemistry (SPCE) of silver nanoparticle (AgNP) redox probes. The method relies on monitoring the rapid interaction of Lyz with AgNPs, which decreases the number of single AgNPs available for collisions and ultimately the frequency of oxidative impacts in the chronoamperometric profile. When Lyz is in a non-aggregated monomeric form, the protein forms a homogeneous coverage onto the surface of AgNPs, stabilizing the particles. When Lyz is aggregated, part of the AgNP surface remains uncoated, promoting the agglomeration of Lyz-AgNP conjugates. The frequency of AgNP impacts decreases with increasing aggregation time, providing a metric to track protein aggregation. Visualizations of integrated oxidation charge-transfer data displayed significant differences between the charge transfer per impact for AgNP samples alone and in the presence of non-aggregated and aggregated Lyz with 99% confidence using parametric ANOVA tests. Electrochemical results revealed meaningful associations with UV-vis, circular dichroism, and AFM, demonstrating that SPCE can be used as an alternative method for studying protein aggregation. This electrochemical technique could serve as a powerful tool to indirectly evaluate protein stability and screen protein samples for formation of aggregates.

Flow injection enzymatic biosensor for aldehydes based on a Meldola Blue-Ni complex electrochemical mediator.

Carbon nanofibers (CNF) are efficient electrode modifiers in electrochemical biosensors that enhance the electrochemical active area, induce electrocatalytic effect toward the oxidation of the enzymatic cofactor nicotinamide adenine dinucleotide (reduced form, NADH), and enable the quantitative immobilization of enzymes. Combining CNF with efficient and stable mediators radically augments the speed of electron transfer between NADH and solid electrodes and leads to electrochemical sensors characterized by high sensitivity and stability. The main aim of this work was to investigate the performance of a novel mediator for NADH with advantageously low solubility in an electrochemical detector based on a screen-printed CNF electrode as well as its potential in biosensing. Using a mediator, prepared from Meldola Blue and Ni hexamine chloride, a stable and sensitive electrochemical NADH sensor is provided with a detection limit of 0.5 µmol L-1. Further on, covalent immobilization of a recently described aldehyde dehydrogenase from the Antarctic Flavobacterium PL002 strain on the surface of the mediator-modified electrode produced a stable biosensor for the detection of aldehydes. When integrated in a flow injection analysis (FIA) setup with amperometric detection at 0.1 V vs. Ag/AgCl, the measurement of benzaldehyde with a detection limit of 10 µmol L-1 over a linear range of 30-300 µmol L-1 is possible. Determination of trace benzaldehyde impurities in a pharmaceutical excipient was also demonstrated and results compared with a chromatographic method. Graphical abstract.

Electrochemical biosensing with odorant binding proteins.

The development of sensors that mimic the natural smell sensing mechanism and selectively recognizes the odorants remains highly challenging. Electrochemical based sensing approaches aiming at monitoring molecular recognition events between surface receptors and analytes in solution or in the gas phase, are one possible transduction platforms among others for the construction of an artificial nose. The principle of electrochemical detection lies on the shift of the potential/current during the recognition event, which is proportional to the concentration of the analyte, in our case the odorant. A tremendous amount of efforts has been put into making electrochemical sensors sensitive and selective to the analyte of interest through the use of nanomaterials, development of different detection schemes and application of innovative receptor ligands for selective detection of the analyte. There have been significant advances in electrochemical based odorant sensing by using odorant binding proteins (OBP) as surface receptors, small soluble proteins present in nasal mucus at millimolar concentrations where the hydrophobic binding pocket gives the ability to reversibly bind odorant molecules. As OBPs are robust and easy to produce receptors, they are good candidates for the design of biosensors. In this chapter, we focus on the progress made on the detection of odorant molecules using OBPs as a bioreceptor and electrochemistry as a transduction method.

Advances in the Detection of Dithiocarbamate Fungicides: Opportunities for Biosensors.

Dithiocarbamate fungicides (DTFs) are widely used to control various fungal diseases in crops and ornamental plants. Maximum residual limits in the order of ppb-ppm are currently imposed by legislation to prevent toxicity problems associated with excessive use of DTFs. The specific analytical determination of DTFs is complicated by their low solubility in water and organic solvents. This review summarizes the current analytical procedures used for the analysis of DTF, including chromatography, spectroscopy, and sensor-based methods and discusses the challenges related to selectivity, sensitivity, and sample preparation. Biosensors based on enzymatic inhibition demonstrated potential as analytical tools for DTFs and warrant further research, considering novel enzymes from extremophilic sources. Meanwhile, Raman spectroscopy and various sensors appear very promising, provided the selectivity issues are solved.

Detection of Biomedically Relevant Stilbenes from Wines by Mass Spectrometry.

Stilbenes represent a class of compounds with a common 1,2-diphenylethylene backbone that have shown extraordinary potential in the biomedical field. As the most well-known example, resveratrol proved to have anti-aging effects and significant potential in the fight against cardiovascular diseases and some types of cancer. Mass spectrometry is an analytical method of critical importance in all studies related to stilbenes that are important in the biomedical field. From the discovery of new natural compounds and mapping the grape metabolome up to advanced investigations of stilbenes' potential for the protection of human health in clinical studies, mass spectrometry has provided critical analytical information. In this review we focus on various approaches related to mass spectrometry for the detection of stilbenes-such as coupling with chromatographic separation methods and direct infusion-with presentation of some illustrative applications. Clearly, the potential of mass spectrometry for assisting in the discovery of new stilbenes of biomedical importance, elucidating their mechanisms of action and quantifying minute quantities in complex matrices is far from being exhausted.

Electrochemical Aptamer-Based Biosensors for the Detection of Cardiac Biomarkers.

Rapid and accurate diagnostic technologies for early-state identification of cardiovascular abnormalities have become of high importance to prevent and attenuate their progression. The capability of biosensors to determine an increase in the concentration of cardiovascular protein biomarkers in circulating blood immediately after a myocardial infarction makes them ideal point-of-care platforms and alternative approaches to electrocardiograms, chest X-rays, and different laboratory-based immunoassays. We report here a generic approach toward multianalyte sensing platforms for cardiac biomarkers by developing aptamer-based electrochemical sensors for brain natriuretic peptide (BNP-32) and cardiac troponin I (cTnI). For this, commercial gold-based screen-printed electrodes were modified electrophoretically with polyethyleneimine/reduced graphene oxide films. Covalent grafting of propargylacetic acid integrates propargyl groups onto the electrode to which azide-terminated aptamers can be immobilized using Cu(I)-based "click" chemistry. To ensure low biofouling and high specificity, cardiac sensors were modified with pyrene anchors carrying poly(ethylene glycol) units. In the case of BNP-32, the sensor developed has a linear response from 1 pg mL-1 to 1 µg mL-1 in serum; for cTnI, linearity is observed from 1 pg mL-1 to 10 ng mL-1 as demanded for early-stage diagnosis of heart failure. These electrochemical aptasensors represent a step further toward multianalyte sensing of cardiac biomarkers.

Detection of Antibiotics and Evaluation of Antibacterial Activity with Screen-Printed Electrodes.

This review provides a brief overview of the fabrication and properties of screen-printed electrodes and details the different opportunities to apply them for the detection of antibiotics, detection of bacteria and antibiotic susceptibility. Among the alternative approaches to costly chromatographic or ELISA methods for antibiotics detection and to lengthy culture methods for bacteria detection, electrochemical biosensors based on screen-printed electrodes present some distinctive advantages. Chemical and (bio)sensors for the detection of antibiotics and assays coupling detection with screen-printed electrodes with immunomagnetic separation are described. With regards to detection of bacteria, the emphasis is placed on applications targeting viable bacterial cells. While the electrochemical sensors and biosensors face many challenges before replacing standard analysis methods, the potential of screen-printed electrodes is increasingly exploited and more applications are anticipated to advance towards commercial analytical tools.

Advances in Enzyme-Based Biosensors for Pesticide Detection.

The intensive use of toxic and remanent pesticides in agriculture has prompted research into novel performant, yet cost-effective and fast analytical tools to control the pesticide residue levels in the environment and food. In this context, biosensors based on enzyme inhibition have been proposed as adequate analytical devices with the added advantage of using the toxicity of pesticides for detection purposes, being more "biologically relevant" than standard chromatographic methods. This review proposes an overview of recent advances in the development of biosensors exploiting the inhibition of cholinesterases, photosynthetic system II, alkaline phosphatase, cytochrome P450A1, peroxidase, tyrosinase, laccase, urease, and aldehyde dehydrogenase. While various strategies have been employed to detect pesticides from different classes (organophosphates, carbamates, dithiocarbamates, triazines, phenylureas, diazines, or phenols), the number of practical applications and the variety of environmental and food samples tested remains limited. Recent advances focus on enhancing the sensitivity and selectivity by using nanomaterials in the sensor assembly and novel mutant enzymes in array-type sensor formats in combination with chemometric methods for data analysis. The progress in the development of solar cells enriched the possibilities for efficient wiring of photosynthetic enzymes on different surfaces, opening new avenues for development of biosensors for photosynthesis-inhibiting herbicides.

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.

Electrophoretic Approach for the Simultaneous Deposition and Functionalization of Reduced Graphene Oxide Nanosheets with Diazonium Compounds: Application for Lysozyme Sensing in Serum.

Electrophoretic deposition (EPD) of reduced graphene oxide nanosheets (rGO) offers several advantages over other surface coating approaches, including process simplicity, uniformity of the deposited films, and good control of the film thickness. The EPD conditions might also be of interest for the reduction of diazonium salts, which upon the release of N2 molecules and generation of radicals, can form covalent bonds with the sp2 hybridized carbon lattice atoms of rGO films. In this work, we report on the coating of gold electrodes in one step with rGO/polyethylenimine (PEI) thin films and their simultaneous modification using different phenyl (Ph) diazonium salt precursors bearing various functionalities such as -B(OH)2, -COOH, and -C≡CH. We show further the interest of such interfaces for designing highly sensitive sensing platforms. Azide-terminated lysozyme aptamers were clicked onto the rGO/PEI/Ph-alkynyl matrix and used for the sensing of lysozyme levels in patients suffering from inflammatory bowel disease (IBD), where lysozyme levels are up-regulated. The approach attained the required demand for the determination of lysozyme level in patients suffering from IBD with a 200 fM detection limit and a linear range up to 20 pM without signal amplification.

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.

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens.

Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface.

A single use electrochemical sensor based on biomimetic nanoceria for the detection of wine antioxidants.

We report the development and characterization of a disposable single use electrochemical sensor based on the oxidase-like activity of nanoceria particles for the detection of phenolic antioxidants. The use of nanoceria in the sensor design enables oxidation of phenolic compounds, particularly those with ortho-dihydroxybenzene functionality, to their corresponding quinones at the surface of a screen printed carbon electrode. Detection is carried out by electrochemical reduction of the resulting quinone at a low applied potential of -0.1V vs the Ag/AgCl electrode. The sensor was optimized and characterized with respect to particle loading, applied potential, response time, detection limit, linear concentration range and sensitivity. The method enabled rapid detection of common phenolic antioxidants including caffeic acid, gallic acid and quercetin in the µM concentration range, and demonstrated good functionality for the analysis of antioxidant content in several wine samples. The intrinsic oxidase-like activity of nanoceria shows promise as a robust tool for sensitive and cost effective analysis of antioxidants using electrochemical detection.

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.

Vertically Aligned Nitrogen-Doped Carbon Nanotube Carpet Electrodes: Highly Sensitive Interfaces for the Analysis of Serum from Patients with Inflammatory Bowel Disease.

The number of patients suffering from inflammatory bowel disease (IBD) is increasing worldwide. The development of noninvasive tests that are rapid, sensitive, specific, and simple would allow preventing patient discomfort, delay in diagnosis, and the follow-up of the status of the disease. Herein, we show the interest of vertically aligned nitrogen-doped carbon nanotube (VA-NCNT) electrodes for the required sensitive electrochemical detection of lysozyme in serum, a protein that is up-regulated in IBD. To achieve selective lysozyme detection, biotinylated lysozyme aptamers were covalently immobilized onto the VA-NCNTs. Detection of lysozyme in serum was achieved by measuring the decrease in the peak current of the Fe(CN)6(3-/4-) redox couple by differential pulse voltammetry upon addition of the analyte. We achieved a detection limit as low as 100 fM with a linear range up to 7 pM, in line with the required demands for the determination of lysozyme level in patients suffering from IBD. We attained the sensitive detection of biomarkers in clinical samples of healthy patients and individuals suffering from IBD and compared the results to a classical turbidimetric assay. The results clearly indicate that the newly developed sensor allows for a reliable and efficient analysis of lysozyme in serum.