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Lysozyme, also called “muramidase”, is an antimicrobial enzyme with antibacterial activity with importance in medicine, food industry, protein studies and enzymology. Lysozyme is also a model analyte in the biosensing field. Optical biosensors have been proposed as alternative analytical tools to the standard detection of lysozyme by ELISA, chromatographic methods or capillary electrophoresis. These biosensors combine specific recognition via antibodies, aptamers, peptides, Micrococcus lysodeikticus bacteria and molecularly imprinted polymers (MIPs) with sensitive detection based on luminescence, surface plasmon resonance (SPR), surface enhanced Raman spectroscopy (SERS), colorimetry etc. To deliver shorter response time, a better sensitivity, but also a lower cost, many of the sensing schemes included nanomaterial and enzyme-enabled amplification. Several biological and food matrices were explored for the potential applications of optical biosensors. The review presents the bioreceptors used and provides illustrative details on the sensing schemes, discussed in relation to the method of detection. Challenges for real samples applications and perspectives of optical biosensors for lysozyme are also presented.

In part 1 (A. Vasilescu et al., Porous reduced graphene oxide modified electrodes for the analysis of protein aggregation. Part 1: Lysozyme aggregation at pH 2 and 7.4 Electrochem. Acta, 254 (2017) 375–383) we proposed porous reduced graphene oxide coated glassy carbon electrode (GC/prGO) in combination with differential pulse voltammetry as a new analytical tool for aggregation studies of proteins. Lysozyme was used as a model to follow its aggregation by electrochemical means at pH 2 and pH 7.4, leading to the formation of amyloid and amorphous aggregates, respectively. Part 2 of this work widens the scope of this approach by investigating a biopharmaceutical product, as the aggregation of peptide based drugs affects their therapeutic activity and can induce allergic reactions in patients. The salmon polypeptide calcitonin (sCT) was chosen as an example of a bioactive peptide with limited pharmaceutical potential due to a tendency to form cytotoxic aggregates and amyloid fibrils. For practical applications, screen printed electrodes (SPE) and flexible electrodes (FE) modified with polydiallyldimethylammonium (PDDA) and prGO by using the layer-by-layer deposition technique have been developed for the detection of sCT. The results indicate that these electrodes can differentiate between formation of amyloid aggregates of calcitonin (2 mg mL−1) in citrate buffer to no aggregation in acetate buffer. It was further demonstrated that these electrodes are able to analyze a pharmaceutical drug product of low potency, Miacalcic (8.3 μg mL−1), where no aggregation was observed.

Electrochemistry provides an array of methods to investigate protein aggregation and determine biomarkers of neurodenenerative diseases. Biosensors detecting monomeric or oligomeric biomarkers of Alzheimer's disease and Parkinson's disease evolved toward femtomolar, multiplexed detection in blood and biological fluids for less invasive diagnosis. The biosensors also serve as complementary tools in studies investigating putative biomarkers for the assessment of patient's cognitive decline. The study of protein aggregation via the direct electrochemical oxidation focused recently on enhanced sensitivity and on establishing correlations between protein structure and aggregation propensity. Departing from classic approaches, nanopore resistive pulse sensing and single-particle collision electrochemistry enable studying aggregates in solution. Growing applications converge toward accurate evaluation of aggregate populations and method adoption beyond proof of principle.

The combination between the specific recognition abilities of aptamers and the catalytic power of enzymes represents a powerful tool in bioanalysis to achieve the highly selective and sensitive detection of various molecules. Enzymes attached to biomolecules and nanomaterials facilitate the highly amplified detection of electroactive compounds. Meanwhile DNAzymes, small DNA strands showing enzyme-like activity are increasingly used as replacements of natural enzymes in electrochemical aptasensors. Moreover, nucleic acid catalysts have been integrated together with aptamer domains in aptazymes, where the aptamer-ligand interaction acts as a switch for the enzyme-like activity. The review discusses enzyme and DNAzyme based amplification, as the preferred approaches in electrochemical aptasensors. DNA and nanomaterial based signal enhancement is briefly mentioned, while a larger discussion is dedicated to aptasensors for the measurement of enzymatic activity and for the development of “universal” detection tools based on the aptamer-enzyme combination. This includes assays combining aptamers with glucometers for the detection of various molecules and promising “aptameric enzyme subunits”-yet to be integrated with electrochemical detection. The outlooks of the electrochemical biosensors coupling aptamers and enzymes are presented.

Protein instability due to misfolding and aggregation is of big concern for protein based therapeutics because it impacts the bioavailability and immunogenicity of such drugs. The development of simple and cost-effective methods for the analysis of pharmaceutical formulations, indicating the presence or absence of protein aggregates, is consequently of high importance. This work proposes a novel electrochemical interface based on porous reduced graphene oxide coated glassy carbon electrode (GC/prGO) allowing for the early and sensitive identification of protein aggregation by following the change in the oxidative current of the proteins. The novelty of this work lies in the exploration of the ability of GC/prGO interfaces to capture different aggregation behaviors. Lysozyme is used as a model to follow by electrochemistry its aggregation at two pH values, pH 2 and pH 7.4, leading to the formation of amyloid and amorphous aggregates, respectively. Comparing the oxidation peak of lysozyme by differential pulse voltammetry (DPV) for different electrode architectures allowed validating the higher sensitivity of the GC/prGO interface versus bare glassy electrodes or electrodes coated with non-porous reduced graphene oxide. Parallel experiments were performed by fluorescence with thioflavin T, size exclusion chromatography and Atomic Force Microscopy (AFM) imaging. These tests further highlighted the usefulness of GC/prGO electrode to visualize in a fast and reliable manner the changes in the protein structure and the differences between the processes occurring at pH 2 and pH 7.4. In particular, the ability to emphasize changes related to the first steps in aggregation that could be indicative of the aggregation course, recommend the GC/prGO electrode in combination with DPV as a new analytical tool for aggregation studies of biopharmaceuticals. Part 2 of this work will demonstrate later the utility of this approach for the analysis of a fast acting injectable human insulin formulation, Humulin R, used for diabetes treatment as well as for calcitonin.

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.

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.

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.

Development of highly sensitive analytical procedures for food contaminants is one of the critical points in addressing new challenges related to food safety worldwide. Electrochemical aptamer-based sensors have been intensively investigated as potential analytical tools providing the desired portability, fast response, sensitivity and specificity in addition to lower cost and simplicity versus classical methods. The paper summarizes the aptasensors reported in the literature in the last 3 years, that have been used in applications related to food safety. New trends pertaining to increasing the sensitivity of detection by using nanomaterials and engineering of new aptamers are briefly discussed. With the recent development of new aptamers and following the lead of aptasensors devoted to biomedical field, the next years will witness an avalanche of new exciting electrochemical aptasensors for food safety.

The optimization of an aptasensor with SPR detection allowed the determination of an allergen protein, lysozyme, with high accuracy, good sensitivity and a detection limit of 2.4 nM for spiked red and white wines. The interaction of lysozyme with specific phenolic compounds from wine was evidenced by circular dichroism and SPR. Quantitative recovery factors higher than 88% for wine lysozyme were achieved based on a sample pre-treatment procedure with high concentration of sodium chloride and Tween-20. This aptasensor is useful to monitor the lysozyme levels during winemaking. Also reported is a new chromatographic method based on size exclusion that provided a good separation of lysozyme from wine components, with a detection limit of 35 nM or 0.5 μg/mL.

Electropolymerization of Meldola Blue was carried out by cyclic voltammetry in the range from -0.6 to +1.4 V vs. Ag/AgCl, thus defining a new immobilization procedure of the phenoxazine mediator on screen-printed graphite electrodes. Evidence of polymer formation was provided by electrochemical and Fourier transform infrared spectroscopy (FTIR) data. Following polymerization, Meldola Blue preserved the ability to catalyze NADH oxidation allowing to achieve a detection limit of 2.5 x 10(-6) mol l(-1) and a sensitivity of 3713 microA l mol(-1) in amperometric determinations at 0 V vs. Ag/AgCl. In addition, the polymeric mediator was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase. Typical calibration at -0.1 V vs. Ag/AgCl shows a detection limit of 8.5 x 10(-5) mol l(-1), a sensitivity of 494 microA l mol(-1) and a linear range from 2.5 x 10(-4) to 5 x 10(-3) mol l(-1) hydrogen peroxide.