Glycan-Based Flow-Through Device for the Detection of SARS-COV-2.

The COVID-19 pandemic, and future pandemics, require diagnostic tools to track disease spread and guide the isolation of (a)symptomatic individuals. Lateral-flow diagnostics (LFDs) are rapid and of lower cost than molecular (genetic) tests, with current LFDs using antibodies as their recognition units. Herein, we develop a prototype flow-through device (related, but distinct to LFDs), utilizing N-acetyl neuraminic acid-functionalized, polymer-coated, gold nanoparticles as the detection/capture unit for SARS-COV-2, by targeting the sialic acid-binding site of the spike protein. The prototype device can give rapid results, with higher viral loads being faster than lower viral loads. The prototype's effectiveness is demonstrated using spike protein, lentiviral models, and a panel of heat-inactivated primary patient nasal swabs. The device was also shown to retain detection capability toward recombinant spike proteins from several variants (mutants) of concern. This study provides the proof of principle that glyco-lateral-flow devices could be developed to be used in the tracking monitoring of infectious agents, to complement, or as alternatives to antibody-based systems.

Physicochemical Approach to Understanding the Structure, Conformation, and Activity of Mannan Polysaccharides.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium Psychrobacter arcticus strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from Saccharomyces cerevisiae. Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from P. arcticus bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.

Mimicking the Ice Recrystallization Activity of Biological Antifreezes. When is a New Polymer "Active"?

Antifreeze proteins and ice-binding proteins have been discovered in a diverse range of extremophiles and have the ability to modulate the growth and formation of ice crystals. Considering the importance of cryoscience across transport, biomedicine, and climate science, there is significant interest in developing synthetic macromolecular mimics of antifreeze proteins, in particular to reproduce their property of ice recrystallization inhibition (IRI). This activity is a continuum rather than an "on/off" property and there may be multiple molecular mechanisms which give rise to differences in this observable property; the limiting concentrations for ice growth vary by more than a thousand between an antifreeze glycoprotein and poly(vinyl alcohol), for example. The aim of this article is to provide a concise comparison of a range of natural and synthetic materials that are known to have IRI, thus providing a guide to see if a new synthetic mimic is active or not, including emerging materials which are comparatively weak compared to antifreeze proteins, but may have technological importance. The link between activity and the mechanisms involving either ice binding or amphiphilicity is discussed and known materials assigned into classes based on this.

Comparison of RAFT derived Poly(vinylpyrolidone) verses Poly(oligoethyleneglycol methacrylate) for the Stabilization of Glycosylated Gold Nanoparticles.

Carbohydrates dictate many biological processes including infection by pathogens. Glycosylated polymers and nanomaterials which have increased affinity due to the cluster glycoside effect, are therefore useful tools to probe function, but also as prophylactic therapies or diagnostic tools. Here, the effect of polymer structure on the coating of gold nanoparticles is studied in the context of grafting density, buffer stability and in a lectin binding assay. RAFT polymerization is used to generate poly(oligoethyleneglycol methacrylates) and poly(N-vinyl pyrolidones) with a thiol end-group for subsequent immobilization onto the gold. It is observed that poly(oligoethylene glycol methacrylates), despite being widely used particle coatings, lead to low grafting densities which in turn resulted in lower stability in biological buffers. A depression of the cloud point upon nanoparticle immobilization is also seen, which might compromise performance. In comparison poly(vinyl pyrolidones) resulted in stable particles with higher grafting densities due to the compact size of each monomer unit. The higher grafting density also enabled an increase in the number of carbohydrates which can be installed per nanoparticle at the chain ends, and gave increased binding in a lectin recognition assay. These results will guide the development of new nanoparticle biosensors with enhanced specificity, affinity and stability.

Ultra-Low Dispersity Poly(vinyl alcohol) Reveals Significant Dispersity Effects on Ice Recrystallization Inhibition Activity.

Polymer mimics of antifreeze proteins are emerging as an exciting class of macromolecular cryoprotectants for the storage of donor cells and tissue. Poly(vinyl alcohol), PVA, is the most potent polymeric ice growth inhibitor known, but its mode of action and the impact of valency (DP) are not fully understood. Herein, tandem RAFT polymerization and column chromatography are used to isolate oligomers with dispersities <1.01 to enable the effect of molecular weight distribution, as well as length, to be probed. It is found that polymers with equal number average molecular weight, but lower dispersity, have significantly less activity, which can lead to false positives when identifying structure-property relationships. The minimum chain length for PVA's unique activity, compared to other non-active poly-ols was identified. These results will guide the design of more active inhibitors, better cryopreservatives and a deeper understanding of synthetic and biological antifreeze macromolecules.

Polymer mimics of biomacromolecular antifreezes.

Antifreeze proteins from polar fish species are remarkable biomacromolecules which prevent the growth of ice crystals. Ice crystal growth is a major problem in cell/tissue cryopreservation for transplantation, transfusion and basic biomedical research, as well as technological applications such as icing of aircraft wings. This review will introduce the rapidly emerging field of synthetic macromolecular (polymer) mimics of antifreeze proteins. Particular focus is placed on designing polymers which have no structural similarities to antifreeze proteins but reproduce the same macroscopic properties, potentially by different molecular-level mechanisms. The application of these polymers to the cryopreservation of donor cells is also introduced.

Impact of sequential surface-modification of graphene oxide on ice nucleation.

Base-washed graphene-oxide which has been sequentially-modified by thiol-epoxy chemistry, results in materials with ice-nucleation activity. The role of hydro-philic/phobic grafts and polymers was evaluated with the most potent functioning at just 0.25 wt%. These 2-D hybrid materials may find use in cryopreservation and fundamental studies on ice formation.

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

"Grafting to" of RAFTed Responsive Polymers to Glass Substrates by Thiol-Ene and Critical Comparison to Thiol-Gold Coupling.

Surface-grafted polymers have been widely applied to modulate biological interfaces and introduce additional functionality. Polymers derived from reversible addition-fragmentation transfer (RAFT) polymerization have a masked thiol at the ω-chain end providing an anchor point for conjugation and in particular displays high affinity for gold surfaces (both flat and particulate). In this work, we report the direct grafting of RAFTed polymers by a "thiol-ene click" (Michael addition) onto glass substrates rather than gold, which provides a more versatile surface for subsequent array-based applications but retains the simplicity. The immobilization of two thermoresponsive polymers are studied here, poly[oligo(ethylene glycol) methyl ether methacrylate] (pOEGMA) and poly(N-isopropylacrylamide) (pNIPAM). Using a range of surface analysis techniques the grafting efficiency was compared to thiol-gold and was quantitatively compared to the gold alternative using quartz crystal microbalance. It is shown that this method gives easy access to grafted polymer surfaces with pNIPAM resulting in significantly increased surface coverage compared to pOEGMA. The nonfouling (protein resistance) character of these surfaces is also demonstrated.

Multivalent Glycopolymer-Coated Gold Nanoparticles.

Glycosylated noble metal nanoparticles are a useful tool for probing biological binding events due to their aggregation-induced color changes, particularly for lectins that have multiple binding sites. To overcome the challenges of colloidal instability, which leads to false-positive results, it is essential to add polymeric coatings to these particles. Here we describe a versatile, and reliable, approach to enable coating of gold nanoparticles using well-defined polymers, with carbohydrate end groups. This produces multivalent nanoparticles that are both colloidally stable, but still retain their rapid colorimetric responses to lectin binding.

Probing the Biomimetic Ice Nucleation Inhibition Activity of Poly(vinyl alcohol) and Comparison to Synthetic and Biological Polymers.

Nature has evolved many elegant solutions to enable life to flourish at low temperatures by either allowing (tolerance) or preventing (avoidance) ice formation. These processes are typically controlled by ice nucleating proteins or antifreeze proteins, which act to either promote nucleation, prevent nucleation or inhibit ice growth depending on the specific need, respectively. These proteins can be expensive and their mechanisms of action are not understood, limiting their translation, especially into biomedical cryopreservation applications. Here well-defined poly(vinyl alcohol), synthesized by RAFT/MADIX polymerization, is investigated for its ice nucleation inhibition (INI) activity, in contrast to its established ice growth inhibitory properties and compared to other synthetic polymers. It is shown that ice nucleation inhibition activity of PVA has a strong molecular weight dependence; polymers with a degree of polymerization below 200 being an effective inhibitor at just 1 mg.mL(-1). Other synthetic and natural polymers, both with and without hydroxyl-functional side chains, showed negligible activity, highlighting the unique ice/water interacting properties of PVA. These findings both aid our understanding of ice nucleation but demonstrate the potential of engineering synthetic polymers as new biomimetics to control ice formation/growth processes.

Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding.

Carbohydrate arrays are a vital tool in studying infection, probing the mechanisms of bacterial, viral and toxin adhesion and the development of new treatments, by mimicking the structure of the glycocalyx. Current methods rely on the formation of monolayers of carbohydrates that have been chemically modified with a linker to enable interaction with a functionalised surface. This includes amines, biotin, lipids or thiols. Thiol-addition to gold to form self-assembled monolayers is perhaps the simplest method for immobilisation as thiolated glycans are readily accessible from reducing carbohydrates in a single step, but are limited to gold surfaces. Here we have developed a quick and versatile methodology which enables the use of thiolated carbohydrates to be immobilised as monolayers directly onto acrylate-functional glass slides via a 'thiol-ene'/Michael-type reaction. By combining the ease of thiol chemistry with glass slides, which are compatible with microarray scanners this offers a cost effective, but also useful method to assemble arrays.

Oxidation of tertiary amine-derivatized surfaces to control protein adhesion.

Selective oxidation of ω-tertiary amine self-assembled thiol monolayers to tertiary amine N-oxides is shown to transform the adhesion of model proteins lysozyme and fibrinogen upon them. Efficient preparation of both secondary and tertiary linker amides as judged by X-ray photoelectron spectroscopy (XPS) and water droplet contact angle was achieved with an improved amide bond formation on gold quartz crystal microbalance (QCM) sensors using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate methanaminium uronium (HATU). Oxidation with hydrogen peroxide was similarly assessed, and adhesion of lysozyme and fibrinogen from phosphate buffered saline was then assayed by QCM and imaged by AFM. Tertiary amine-functionalized sensors adsorbed multilayers of aggregated lysozyme, whereas tertiary amine N-oxides and triethylene glycol-terminated monolayers are consistent with small protein aggregates. The surface containing a dimethylamine N-oxide headgroup and ethyl secondary amide linker showed the largest difference in adsorption of both proteins. Oxidation of tertiary amine decorated surfaces therefore holds the potential for selective deposition of proteins and cells through masking and other patterning techniques.