Mediatorless Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized on 4‐Carboxyphenyl Film Electrografted on Gold Electrode

A novel method to fabricate a third‐generation hydrogen peroxide biosensor was reported. The electrode was first derivatized by electrochemical reduction of in situ generated 4‐carboxyphenyl diazonium salt (4‐CPDS) in acidic aqueous solution yielded stable 4‐carboxyphenyl (4‐CP) layer. The horseradish peroxidase (HRP) enzyme was then covalently immobilized by amidation between NH₂ terminus of enzyme and COOH terminus of 4‐CP film making use of the carbodiimide chemistry. Electrodeposition conditions used to control electrode functionalization density and film electron transfer kinetics were assessed by chronoamperometry and electrochemical impedance spectroscopy. The immobilized HRP displayed excellent electrocatalytic activity towards the reduction of hydrogen peroxide (H₂O₂) without any mediators. The effect of various operational parameters was explored for optimum analytical performance. The reported biosensor exhibited fast amperometric response (within 5 s) to H₂O₂. The detection limit of the biosensor was 5 μM, and linear range was from 20 μM to 20 mM. Furthermore, the biosensor exhibited high sensitivity, good reproducibility, and long‐term stability.     

Characterization of self-assembled monolayers (SAMs) on silicon substrate comparative with polymer substrate for Escherichia coli O157:H7 detection

This article presents the characterization of two substrates, silicon and polymer coated with gold, that are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the anti-Escherichia coli O157:H7 polyclonal purified antibody.A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed. Immobilization of goat anti-E. coli O157:H7 antibody was performed by covalently bonding of thiolate mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon coated with gold. The F(ab′)₂ fragments of the antibodies have been used for eliminating nonspecific bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best results.These experimental results revealed the necessity to investigate each stage of the immobilization process taking into account in the same time the factors that influence the chemistry of the surface and the further interactions as well and also provide a solid basis for further studies aiming at elaborating sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.     

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS²–MS³) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by β-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Δ-[IdoA-GalNAc]. By optimized CID MS²–MS³, fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Δ-[GlcA-GalNAc]. The site of oversulfation was determined by MS²–MS³, which provided sequence patterns consistent with a rare GlcA-3-sulfate–GalNAc-6-sulfate structural motif.      

Top–down glycolipidomics: fragmentation analysis of ganglioside oligosaccharide core and ceramide moiety by chip‐nanoelectrospray collision‐induced dissociation MS2–MS6

We developed a straightforward approach for high‐throughput top–down glycolipidomics based on fully automated chip‐nanoelectrospray (nanoESI) high‐capacity ion trap (HCT) multistage mass spectrometry (MSⁿ) by collision‐induced dissociation (CID) in the negative ion mode. The method was optimized and tested on a polysialylated ganglioside fraction (GT1b), which was profiled by MS¹ and sequenced in tandem MS up to MS⁶ in the same experiment. Screening of the fraction in the MS¹ mode indicated the occurrence of six [M ? 2H]^2? ions which, according to calculation, support 13 GT1 variants differing in their relative molecular mass due to dissimilar ceramide (Cer) constitutions. By stepwise CID MS²–MS⁵ on the doubly charged ion at m/z 1077.20 corresponding to a ubiquitous GT1b structure, the complete characterization of its oligosaccharide core including the identification of sialylation sites was achieved. Structure of the lipid moiety was further elucidated by CID MS⁶ analysis carried out using the Y₀ fragment ion, detected in MS⁵, as a precursor. MS⁶ fragmentation resulted in a pattern supporting a single ceramide form having the less common (d20 : 1/18 : 0) configuration. The entire top–down experiment was performed in a high‐throughput regime in less than 3 min of measurement, with an analysis sensitivity situated in the subpicomolar range. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry

We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS² mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.      

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O₂^.−, an unstable Reactive Oxygen Species (ROS), and its relatives, H₂O₂ and O₂, was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a well-resolved reversible redox signature for O₂^.− detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H₂O₂ at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H₂O₂ (2 electrons/2 H⁺) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.     

Determination of Antioxidant Capacity by Using Xanthine Oxidase Bioreactor Coupled with Flow‐through H2O2 Amperometric Biosensor

A new flow system for antioxidant capacity (AOC) estimation, consisting of a bioreactor, containing immobilized xanthine oxidase (XOD), coupled with a H₂O₂ amperometric biosensor, based on Os‐wired horseradish peroxidase, was developed. The H₂O₂, resulting from the enzymatic reaction between xanthine (XA) and XOD, was amperometrically monitored at ?0.1 V vs. Ag/AgCl/KCl_sat, in order to avoid the electrochemical interferences. Two protocols were used to perform the AOC evaluation: “steady‐state”, when the antioxidant (AOX) was injected in the XA flow, and “transient state”, when XA and AOX were simultaneously injected in the carrier flow. The AOC of some commercial beverages were evaluated and compared with those obtained with 2,2‐diphenyl‐1‐picrylhydrazyl radical and Folin–Ciocalteu methods.     

Discrimination of GalNAc (4S/6S) sulfation sites in chondroitin sulfate disaccharides by chip-based nanoelectrospray multistage mass spectrometry

Sulfation pattern within chondroitin sulfate (CS) glycosaminoglycan (GAG) chains is an important post-translational modification that regulates their interaction with proteins. In this context, development of highly efficient and reproducible analytical methods for the investigation of CS sulfation patterns is of high necessity. In this study we report a novel method for straightforward determination of N-acetylgalactosamine (GalNAc) sulfation sites in chondroitin sulfate disaccharides. Our protocol involves combining fully automated chip-based nanoelectrospray (nanoESI) for analyte infusion and ionization in negative ion mode with multistage (MSⁿ) collision-induced dissociation (CID) high capacity ion trap (HCT) mass spectrometry for generation of sequence ions diagnostic for identification of sulfate ester group position within GalNAc residues. The feasibility of this approach is here demonstrated on chondroitin 6-O-sulfate and chondroitin 4-O-sulfate disaccharides. Fragmentation patterns obtained by MS² and MS³ sequencing stages provided first mass spectrometric data from which sulfation site(s) within GalNAc monosaccharide ring could be unequivocally deciphered. Hence, the method allowed discriminating 4S/6S sulfation sites solely on the basis of MS and multistage MS evidence.      

Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics

The conventional protocol for protein identification by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a significant progress to the field, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifications in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for efficient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identification. This advanced analytical platform, integrating robotics, microfluidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modified/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of significantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics.