Serban F. Peteu

A Clark-type oxidase enzyme-based amperometric microbiosensor for sensing glucose, galactose, or choline

Abstract Microbiosensors for glucose, galactose, or choline were constructed by attaching the respective oxidase enzyme to the tip of a Clark-type oxygen microelectrode. The enzyme is immobilized on the electrode tip in a polyacrylamide matrix and then coated with a polyurethane membrane. The analyte concentration in the sample controls the amount of oxygen consumed by the electrode and, hence, the biosensors output. These microbiosensors had tip diameters of 15–40 μm, response times of 0.5–5s, and could detect as little as 2 μM of analyte. The linear range of response was dependent on the thickness of the polyurethane coating, and extended up to 10 mM for glucose and galactose biosensors. The glucose sensors were the most stable and could remain operational for up to 6 months. Galactose sensors remained operational for at least 1 month. Choline sensors remained operational for about 2 weeks and were generally less sensitive. The specific activity of the enzyme was a key determinant in the longevity and linearity of the biosensor response. In continuous operation tests, the glucose sensors were relatively drift-free and showed little deterioration of response over 72 h. These sensors exhibited little stirring dependence. As a result a glucose sensor accurately measured the glucose gradient in an unmixed, semisolid gel. These microsensors should prove to be versatile tools for measuring specific analytes in unstirred environments with a spatial resolution of 100 μm or less, and with extremely rapid response times.

Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor

Abstract An olfactory biosensor based on a reduced graphene oxide (rGO) field‐effect transistor (FET), functionalized by the odorant‐binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real‐time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand–receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K d=4 μm to K d=3.3 mm. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

Peroxynitrite activity of hemin-functionalized reduced graphene oxide

Conducting interfaces modified with reduced graphene oxide (rGO) have shown improved electrochemical response for different analytes. The efficient formation of functionalized rGO based materials is thus of current interest for the development of sensitive and selective biosensors. Herein, we report a simple and environmentally friendly method for the formation of a hemin-functionalized rGO hybrid nanomaterial that exhibits remarkable sensitivity to peroxynitrite (ONOO(-)) in solution. The hemin-functionalized rGO hybrid nanomaterial was formed by mixing an aqueous solution of graphene oxide (GO) with hemin and sonicating the suspension for 5 h at room temperature. In addition to playing a key role in biochemical and electrocatalytic reactions, hemin has been proven to be a good reducing agent for GO. The sensitivity of the peroxynitrite sensor is ≈7.5 ± 1.5 nA mM(-1) with a detection limit of 5 ± 1.5 nM.

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.

Nitro-oxidative species in vivo biosensing: challenges and advances with focus on peroxynitrite quantification.

The importance of the so-called reactive nitrogen and oxygen species (RNOS) in biology and food technology has been widely recognized. However when these species are in excess, the steady-state maintained by physiological processes is disturbed. At this point, the nitro oxidative metabolic stress develops and its action in vivo over time leads to nitro-oxidative reactions in food and in living organisms, but also results in chronic degenerative diseases. Analytical methods enabling the assessment of the total antioxidant activity of a biological sample or a plant extract is therefore largely sought after. The ability of biosensors for rapid and real-time analysis that decreases the assay time and the possibility of automated and multi-analyte analysis at low cost has also allowed the quantitative and qualitative detection of RNOS. Among these RNOS, peroxynitrite (ONOO(-)) is a well-known inflammatory mediator during a number of physiological and pathological processes. Consequently, many efforts are underway to detect peroxynitrite in the biomedical field. This urgent demand makes the development of ONOO(-) specific probes of great interest. Not only they can be useful for the detection of disease states, but they will also allow for a screening-type analysis of potential signal transduction pathways in the cells. This invited review will critically discuss for the first time the very latest advancements and the challenges in the field of peroxynitrite biosensors and probes for in vivo and in vitro studies. Also, the main trends will be extracted, in order to chart the future directions and hence create an instrumental outlook.

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.

In situ mapping of community-level cellular response with catalytic microbiosensors.

Chemotaxis, the migration of cells in the direction of a spatial chemical gradient, is important in disease progression, microbial ecology, and bioremediation. The ability to map chemoattractant gradients and the corresponding cellular growth and motility patterns is essential to the study of chemotaxis. Microelectrodes and microbiosensors have the potential to measure chemoattractant gradients with high spatial resolution. In this study, Clark-type amperometric microelectrodes and microbiosensors were used to measure solute concentrations gradients generated by a chemotactic band of Escherichia coli in a semi-solid gel. A computerized image analysis system was used to simultaneously measure the cellular concentration profile across the chemotactic band. The experimental results compared favorably with a mathematical model of solute and cell transport in the gel. Scanning electron micrographs (SEM) of micro(bio)sensor tips taken after 6 months of use showed evidence of degradation, including adhesion of foreign particles to the glass body, the adhesion of a small gel capsule to the sensor tip, and separation of the bio-interface from the tip. A needle-type microbiosensor was constructed to better protect the tip and hence increase the ruggedness of the microbiosensors.

Electrochemical detection of peroxynitrite using hemin–PEDOT functionalized boron-doped diamond microelectrode

Peroxynitrite is a potent nitroxidation agent and highly reactive metabolite, clinically correlated with a rich pathophysiology. Its sensitive and selective detection is challenging due to its high reactivity and short sub-second lifetime. Boron-doped diamond (BDD) microelectrodes have attracted interest because of their outstanding electroanalytical properties that include a wide working potential window and enhanced signal-to-noise ratio. Herein, we report on the modification of a BDD microelectrode with an electro-polymerized film of hemin and polyethylenedioxythiophene (PEDOT) for the purpose of selectively quantifying peroxynitrite. The nanostructured modified polymer layer was characterized by Raman spectroscopy and scanning electron microscopy (SEM). The electrochemical response to peroxynitrite was studied by voltammetry and time-based amperometry. The measured detection limit was 10 ± 0.5 nM (S/N = 3), the sensitivity was 4.5 ± 0.5 nA nM(-1) and the response time was 3.5 ± 1 s. The hemin-PEDOT BDD sensors exhibited a response variability of 5% or less (RSD). The stability of the sensors after a 20-day storage in 0.1 M PB (pH 7.4) at 4 °C was excellent as at least 93% of the initial response to 50 nM PON was maintained. The presence of PEDOT was correlated with a sensitivity increase.

A catalase microbiosensor for detecting hydrogen peroxide

A microbiosensor for hydrogen peroxide (H2O2) was constructed by immobilizing catalase in a polyacrylamide gel on the tip of a Clark-type oxygen microelectrode. The outer tip diameter was 15–40 μm. The sensors had response times of 0.7–1.2 s, and could detect as little as 2–4 μM H2O2. They could measure with a spatial resolution of about 100μm and remained operational for up to three weeks.

Comparative study of optical fluorescent nanosensors (PEBBLEs) and fiber optic microsensors for oxygen sensing

In this paper we report the use of phase sensitive fluorometry to obtain preliminary results from opto-chemical fluorescent oxygen nanosensors. PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensors were fabricated by immobilizing tris(4,7-diphenyl-1,10-phenanthroline)Ru(II) chloride and tris(1,10-phenanthroline)Ru(II) chloride within a polyacrylamide matrix. PEBBLEs have diameters of 20 - 200 nm and exhibit excellent performance for dissolved oxygen detection. Their performance is compared with micrometer-sized (10 - 20 micrometer) optical fiber sensors and free dye in solution. Oxygen sensing ability of PEBBLEs was tested in the presence of other quenchers and compared with free dyes in solution. While PEBBLEs have been developed for minimally invasive intracellular chemical analysis they show additional advantages, such as increased dynamic range, compared to microsensors, and an absence of interference (quenching) by heavy ions in contrast to free dye solutions.