Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification.

Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

Gold nanoparticles decorated reduced graphene oxide nanolabel for voltammetric immunosensing.

This study describes the development and testing of a simple and novel enzyme-free nanolabel for the detection and signal amplification in a sandwich immunoassay. Gold nanoparticles decorated reduced graphene oxide (rGOAu) was used as the nanolabel for the quantitative detection of human immunoglobulin G (HIgG). The rGOAu nanolabel was synthesised by one pot chemical reduction of graphene oxide and chloroauric acid using sodium borohydride. The pseudo-peroxidase behaviour of rGOAu makes the nanolabel unique from other existing labels. The immunosensing platform was fabricated using self-assembled monolayers of 11-mercaptoundecanoic acid (11-MUDA) on a gold disc electrode. The covalent immobilisation of antibody was achieved through the bonding of the carboxyl group of 11-MUDA and the amino group of the antibody using chemical linkers [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and N-hydroxysuccinimide. The fabricated immunosensor exhibited a linear range that included HIgG concentrations of 62.5-500 ng ml-1. The sensor was also used for the testing of HIgG in the blood sample.