Glycosylation in extracellular vesicles: Isolation, characterization, composition, analysis and clinical applications.

This review provides a comprehensive overview of our understanding of the role that glycans play in the formation, loading and release of extracellular vesicles (EVs). The capture of EVs (typically with a size of 100-200 nm) is described, including approaches based on glycan recognition with glycan-based analysis offering highly sensitive detection of EVs. Furthermore, detailed information is provided about the use of EV glycans and glycan processing enzymes as potential biomarkers, therapeutic targets or tools applied for regenerative medicine. The review also provides a short introduction into advanced methods for the characterization of EVs, new insights into the biomolecular corona covering EVs and bioanalytical tools available for glycan analysis.

How to choose proper magnetic particles for bioaffinity interactions? The case for immobilised glyconanoconjugate.

In this study, an assay for detection of the cancer biomarker Thomsen-nouvelle (Tn) antigen on the ELISA plates format was designed and developed. The effects of size and the interfacial density of the negative charge of magnetic beads (MBs) on the specific sensitivity of the bioaffinity interaction were studied. In particular, glyconanoconjugate, i.e. glycan Tn antigen conjugated to bovine serum albumin (BSA) was covalently immobilised on MBs for the bioaffinity detection of anti-Tn antibodies as cancer biomarkers. Six different MBs were used in the study, i.e. carboxy-modified MBs of 250 nm, 500 nm, 1000 nm and 2800 nm and epoxy-modified MBs of 2800 nm and 4500 nm. In order to evaluate which MBs are the best suited for detection of the analyte anti-Tn antibodies, sensitivities of detection (slopes from calibration curves) were calculated. Next, specific sensitivities were calculated for each type of MBs as a ratio of sensitivity of detection to the mass of MBs. From zeta potential ζ for each type of MBs, the interfacial charge density on MBs was calculated, expressed as the density of zeta potential ζd (ratio of zeta potential to surface area of MBs, i.e. ζd = Î¶/A). Then, we evaluated the effect of size and ζd on the specific sensitivity of detection of anti-Tn antibodies in order to understand the immobilisation process on nanoscale. We also identified an optimal value of ζd on MBs; this was essential to achieve highly sensitive detection of the analyte, which made it possible to attain limit of detection (LOD) of (0.31 ± 0.01) ng mL-1 or (2.10 ± 0.04) pM for analyte detection. In addition, the optimal assay configuration was highly selective and enabled reliable detection of the analyte in human serum with a recovery index in the range of 102-104%.

Amplified suspension magnetic bead-based assay for sensitive detection of anti-glycan antibodies as potential cancer biomarkers.

The development of a novel SUspension Magnetic-Bead-based Assay (SUMBA) for the detection of antibodies against aberrant glycans (AGA) as potential cancer biomarkers is presented here. The SUMBA method was extensively optimised by choosing proper commercially available AGA able to specifically, and with high affinity, recognise aberrant glycans, which were attached to the protein backbone working as a molecular scaffold (a glycoconjugate). The whole SUMBA was optimised using several analytical techniques such as Surface Plasmon Resonance and Energy Dispersive X-ray Spectroscopy. Additionally, the SUMBA method was extensively optimised for signal enhancement. With all steps optimised, we were able to detect AGA ultrasensitively with a limit of detection of 0.45 pM. Moreover, AGA could be detected in serum samples with a recovery index in the range of 98%-104%.

Glycan Nanobiosensors.

This review paper comprehensively summarizes advances made in the design of glycan nanobiosensors using diverse forms of nanomaterials. In particular, the paper covers the application of gold nanoparticles, quantum dots, magnetic nanoparticles, carbon nanoparticles, hybrid types of nanoparticles, proteins as nanoscaffolds and various nanoscale-based approaches to designing such nanoscale probes. The article covers innovative immobilization strategies for the conjugation of glycans on nanoparticles. Summaries of the detection schemes applied, the analytes detected and the key operational characteristics of such nanobiosensors are provided in the form of tables for each particular type of nanomaterial.

A Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody.

The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc-O-Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V vs. Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L-1. Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.

Antibodies against aberrant glycans as cancer biomarkers.

Introduction: The review provides a comprehensive overview about applicability of serological detection of autoantibodies against aberrant glycans as cancer biomarkers.Areas covered: Clinical usefulness of autoantibodies as cancer biomarkers is discussed for seven types of cancers with sensitivity and specificity of such biomarkers provided. Moreover, an option of using serological antibodies against a non-natural form of sialic acid - N-glycolylneuraminic acid (Neu5Gc), which is taken into our bodies together with red meat, as a potential cancer biomarker is discussed shortly as well.Expert opinion: In the final part of the review, we discuss what measures need to be applied for selective implementation of autoantibody assays into a clinical practice. Moreover, we discuss key challenges ahead for reliable and robust detection of autoantibodies against aberrant glycans as biomarkers for disease diagnostics and for stratification of cancer patients.

Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody: Application to the Construction of an Ultrasensitive Glycan Biosensor.

The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t50% = 137 s, t50% = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t50% = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.