Alina Vasilescu

Lysozyme detection on aptamer functionalized graphene-coated SPR interfaces.

The paper reports on a surface plasmon resonance (SPR)-based approach for the sensitive and selective detection of lysozyme. The SPR sensor consists of a 50 nm gold film coated with a thin film of reduced graphene oxide (rGO) functionalized with anti-lysozyme DNA aptamer. The SPR chip coating with rGO matrix was achieved through electrophoretic deposition of graphene oxide (GO) at 150 V. Electrophoretic deposition resulted in partial reduction of GO to rGO with a thickness depending on the deposition time. For very short time pulses of 20 s, the resulting rGO film had a thickness of several nanometers and was appropriate for SPR sensing. The utility of the graphene-based SPR sensor for the selective and sensitive detection of proteins was demonstrated using lysozyme as model protein. Functionalization of rGO matrix with anti-lysozyme DNA aptamer through π-stacking interactions allowed selective SPR detection of lysozyme. The graphene-based SPR biosensor provides a means for the label-free, concentration-dependent and selective detection of lysozymes with a detection limit of 0.5 nM.

Screen-printed electrodes with electropolymerized Meldola Blue as versatile detectors in biosensors

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.

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

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

Cobalt phthalocyanine tetracarboxylic acid modified reduced graphene oxide: a sensitive matrix for the electrocatalytic detection of peroxynitrite and hydrogen peroxide

The quantification of peroxynitrite (ONOO−, PON) and hydrogen peroxide (H2O2) is intrinsically difficult as both species show similar oxidative features located within a narrow potential. The sub-second lifetime of ONOO− at neutral pH further complicates the analysis. In this paper, we examine the electrocatalytic activity of cobalt phthalocyanine tetracarboxylic acid (CoPc–COOH) loaded reduced graphene oxide (rGO) films towards peroxynitrite and hydrogen peroxide detection. The rGO/CoPc–COOH matrix is synthesized by the reaction of graphene oxide (GO) and CoPc–COOH at 90 °C for 5 h under ultrasonication. The integration of CoPc–COOH and the reduction of GO to rGO was confirmed by X-ray photoelectron spectroscopy, FTIR, Raman, UV-vis spectroscopy and electrochemistry. The rGO/CoPc–COOH film showed high electrocatalytic activity and specificity for ONOO− at anodic potential with a sensitivity of ≈11.5 ± 1 nA nM−1 and a peroxynitrite detection limit of ≈1.7 nM. The rGO/CoPc–COOH films further exhibited electrocatalytic reduction of H2O2 with a sensitivity of 14.5 μA mM−1 and a detection limit of ≈60 μM for H2O2.

Label free aptasensor for Lysozyme detection: A comparison of the analytical performance of two aptamers

This work presents a comparison of two different aptamers (Apts) (COX and TRAN) for the detection of a ubiquitous protein Lysozyme (Lys) using Apt-based biosensors. The detection is based on the specific recognition by the Apt immobilized on screen printed carbon electrodes (SPCEs) via diazonium coupling reaction. The quantitative detection of Lys protein was achieved by electrochemical impedance spectroscopy (EIS). A very good linearity and detection limits for the quantitation of Lys were obtained from 0.1 to 0.8 μM and 100 nM using Apt COX and from 0.025 to 0.8 μM and 25 nM using Apt TRAN respectively. The obtained results showed that the developed aptasensors exhibit good specificity, stability and reproducibility for Lys detection. For real application, the aptasensors were tested in wine samples and good recovery rates were recorded in the range from 94.2 to 102% for Lys detection. The obtained recovery rates confirm the reliability and suitability of the developed method in wine matrix. The developed method could be a useful and promising platform for detection of Lys in different applications.

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.

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.

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.

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.