Synthesis of the 3'-O-sulfated TF antigen with a TEG-N3 linker for glycodendrimersomes preparation to study lectin binding.

The synthesis of gram quantities of the TF antigen (ß-ᴅ-Gal-(1→3)-α-ᴅ-GalNAc) and its 3'-sulfated analogue with a TEG-N3 spacer attached is described. The synthesis of the TF antigen comprises seven steps, from a known N-Troc-protected galactosamine donor, with an overall yield of 31%. Both the spacer (85%) and the galactose moiety (79%) were introduced using thioglycoside donors in NIS/AgOTf-promoted glycosylation reactions. The 3'-sulfate was finally introduced through tin activation in benzene/DMF followed by treatment with a sulfur trioxide-trimethylamine complex in a 66% yield.

A Multifunctional Electrochemical Sensor for the Simultaneous Detection of Ascorbic Acid, Dopamine, Uric Acid, and Nitrite.

BACKGROUND: Ascorbic acid (AA), dopamine (DA), uric acid (UA), and nitrite (NO2-) are essential biomarkers for human metabolism and can be used to indicate some chronic diseases and metabolic disorders, including scurvy, Parkinson's disease, hyperuricemia, and kidney disease. OBJECTIVE: A multifunctional electrochemical sensor that can integrate the detection of these species was constructed using nanoporous gold (NPG) as a recognition element to modify glassy carbon electrode (GCE). METHODS: The electrochemical performance of the multifunctional electrochemical sensor was investigated toward AA, DA, UA, and NO2- in citrate buffer solution (CBS, 100 mM, pH 4.0) and human serum using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. RESULTS: In the quaternary mixture detection, the resulting NPG/GCE electrode displayed four independent oxidation peaks with wide peak separations. Further, the NPG/GCE electrode showed good linear responses with the sensitivities of 32, 1103, 71, and 147 µA/mM/cm2 and the detection limits of 1.58, 0.17, 0.37, and 0.36 µM for AA, DA UA, and NO2-, respectively. Additionally, the NPG/GCE electrode exhibited great anti-interference and was successfully applied in human serum samples. CONCLUSIONS: These results indicate that the NPG/GCE electrode can simultaneously and selectively detect AA, DA, UA, and NO2-, which has the potential for application and diagnosis in the screening and diagnosis of chronic diseases and metabolic disorders. HIGHLIGHTS: A multianalyte electrochemical sensor was fabricated for human metabolites detection. The sensor displayed good performance in the simultaneous detection of AA, DA, UA, and NO2- and applied to human serum samples.

Recombinant mucin-type proteins carrying LacdiNAc on different O-glycan core chains fail to support H. pylori binding.

The ß4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a ß1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of 125I-LDN-BSA to various clinical H. pylori isolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a ß1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a 125I-labelled LDN-BSA neoglycoconjugate to clinical H. pylori isolates was identified.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Highly sensitive microbial biosensor based on recombinant Escherichia coli overexpressing catechol 2,3-dioxygenase for reliable detection of catechol.

A highly sensitive whole cell based electrochemical biosensor was developed for catechol detection in this study. The carE gene of Sphingobium yanoikuyae XLDN2-5 encoding catechol 2,3-dioxygenase (C23O), a key enzyme in the biodegradation of aromatic compound, was cloned and over-expressed in Escherichia coli BL21 (E. coli BL21). Compared to Sphingobium yanoikuyae XLDN2-5, the recombinant E. coli BL21 over-expressed C23O exhibited higher catalytic activity towards catechol. Moreover, the whole cells provided a better environment for C23O to maintain its catalytic activity and stability compared with crude enzyme. The distinctive features of the recombinant E. coli BL21 over-expressed C23O made it an ideal bio-recognition element for the fabrication of a microbial biosensor. Additionally, nanoporous gold (NPG) with unique properties of structure and function was selected as a support to immobilized the recombinant E. coli BL21 over-expressed C23O. Based on the synergistic effect of C23O and NPG, the E. coli BL21-C23O/NPG/GCE bioelectrode showed a good linear response for catechol detection ranging from 1 µM to 500 µM with a high sensitivity of 332.24 µA mM-1 cm-2 and a low detection limit of 0.24 µM. Besides, the E. coli BL21-C23O/NPG/GCE bioelectrode exhibited strong anti-interference and good stability. For the detection of catechol in wastewater samples, the concentrations detected by the E. coli BL21-C23O/NPG/GCE bioelectrode were in good agreement with the standard concentrations that added in the wastewater samples, which make the E. coli BL21-C23O/NPG/GCE bioelectrode an ideal tool for reliable catechol detection.

Bacterial electroactivity and viability depends on the carbon nanotube-coated sponge anode used in a microbial fuel cell.

The anode material is vital to improve the power generation of a microbial fuel cell (MFC). In this study, a carbon nanotube (CNT)-coated sponge with macro-porous structure, high surface area, and high conductivity was constructed as an anode to encapsulate Escherichia coli K12 (E. coli K12) cells. To achieve high power generation of the MFC, the optimal concentration of the CNT coating the sponge was found to be 30mgmL-1. At this concentration, a maximum power density of 787Wm-3 and a chemical oxygen demand (COD) removal of 80.9% were obtained with a long stable electricity generation process in batch mode. This indicates that the biofilm on the CNT (30mgmL-1)-coated sponge possessed excellent electroactivity and stability. Scanning electron microscope (SEM) images confirmed that the CNT-coated sponge provided a suitable microenvironment for E. coli K12 cells to maintain their attachment and colonization. Additionally, a CNT-dependent viability phenomenon of the E. coli K12 cells was discovered after electricity generation. This CNT-dependent viability of the E. coli K12 cells was stable and sustainable after storage at -20°C in a milk tube for one year.

Nanoporous gold-based microbial biosensor for direct determination of sulfide.

Environmental pollution caused by sulfide compounds has become a major problem for public health. Hence, there is an urgent need to explore a sensitive, selective, and simple sulfide detection method for environmental monitoring and protection. Here, a novel microbial biosensor was developed using recombinant Escherichia coli BL21 (E. coli BL21) expressing sulfide:quinone oxidoreductase (SQR) for sulfide detection. As an important enzyme involved in the initial step of sulfide metabolism, SQR oxidizes sulfides to polysulfides and transfers electrons to the electron transport chain. Nanoporous gold (NPG) with its unique properties was selected for recombinant E. coli BL21 cells immobilization, and then glassy carbon electrode (GCE) was modified by the resulting E. coli/NPG biocomposites to construct an E. coli/NPG/GCE bioelectrode. Due to the catalytic oxidation properties of NPG for sulfide, the electrochemical reaction of the E. coli/NPG/GCE bioelectrode is attributed to the co-catalysis of SQR and NPG. For sulfide detection, the E. coli/NPG/GCE bioelectrode showed a good linear response ranging from 50µM to 5mM, with a high sensitivity of 18.35µAmM-1cm-2 and a low detection limit of 2.55µM. The anti-interference ability of the E. coli/NPG/GCE bioelectrode is better than that of enzyme-based inhibitive biosensors. Further, the E. coli/NPG/GCE bioelectrode was successfully applied to the detection of sulfide in wastewater. These unique properties potentially make the E. coli/NPG/GCE bioelectrode an excellent choice for reliable sulfide detection.