Polymer-tethered glycosylated gold nanoparticles as versatile tools for bio-sensing

Abstract

Glycans direct many biological recognition and signalling processes, spanning basic cell biology to diagnostics and therapy. Multivalent presentation of glycans is well established to increase avidity (but not always selectivity) and hence glycosylated (nano)materials have emerged as versatile scaffolds to probe glycobiological processes and for application in biomedicine. In this Feature article we review our laboratory's contributions to the development of polymer-tethered glycosylated gold nanoparticles. This approach exploits the steric stability of the polymer coating, ease of glycan capture and the plasmonic signal-generation of the gold core. The article covers the synthetic rationale for the polymeric tether, glycan capture modalities and application to screen (un)natural glycans binding patterns and translation into point-of-care lateral flow glyco-diagnostics and solution-phase colourimetric biosensors. We also suggest areas for future development and comparison to related systems.

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Sarah-Jane Richards

Dr Sarah-Jane Richards is a Research Fellow at the University of Manchester. She obtained her PhD from the University of Warwick, where she focused on the design and synthesis of glycosylated nanomaterials. Her research combines polymers and nanoparticles to interrogate protein–carbohydrate interactions, providing insights into biological recognition processes. By integrating synthetic chemistry with nanotechnology, she aims to design innovative platforms for studying complex biomolecular systems and translating these findings into practical applications. A key focus of her work is the development of rapid point-of-care diagnostic technologies, leveraging molecular recognition to enable early detection and improved healthcare solutions.

Matthew I. Gibson

Professor Matthew Gibson holds a Chair in Sustainable Biomaterials at the University of Manchester, UK. After a PhD at the University of Durham, and a post-doc at EPFL (Switzerland), he established his group at the University of Warwick (2009–2023). His multidisciplinary research group develops new materials to address challenges in Biotechnology, Healthcare and Sustainability. He has held ERC Starter and Consolidator Grants and undertaken technology transfer including as a Royal Society Industry Fellow and cofounding spin out(s). Matt has been awarded prizes including the Corday Morgan, McBain, Dextra and MacroGroup Young Researcher's medals and an RSC Horizon Prize.

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1. Introduction

Carbohydrates (glycans) are one of the four major classes of biological macromolecules, in addition to nucleic acids, proteins and lipids.¹ These complex macromolecules fulfil a variety of tasks ranging from structural and metabolic function to regulating development, cell signalling, cell adhesion, and host–pathogen interactions. It has been reported that more than 50% of the human proteome is glycosylated and that 2% of the human genome is dedicated to glycosylation processes.² Lectins are the primary carbohydrate-binding proteins (other than enzymes and antibodies) that act as the ‘readers’ of glycans. The binding affinity of a carbohydrate to its lectin target is typically weak (K_a = 10³–10⁶ M⁻¹), compared to antibody-antigen interactions which are typically greater than 10⁹ M⁻¹.³ Nature circumvents this limitation through the “cluster-glycoside effect”, where multiple copies of the same glycan are presented on a cell surface, dramatically enhancing overall binding strength via multivalent interactions.^3–5 This avidity gain arises from cooperative binding, reduced dissociation rates, and increased local concentration of ligands at the interface.⁶ Multivalency is a fundamental principle in biological recognition, underpinning processes such as viral adhesion, immune signalling, and cell–cell communication. It enables selectivity and robustness in systems where individual interactions are inherently weak, inspiring synthetic strategies for inhibitors, vaccines, and diagnostics.^7,8 Glycosidases and glycosyltransferases act as the ‘writers/erasers’⁹ of glycans and their complex interplay based on their spatiotemporal distribution dictates the formation and presentation of glycans.¹⁰ It is also important to highlight there are anti-glycan antibodies.^11,12

The wide range of functions carried out by carbohydrates is reflected in their structural diversity. Glycan structural and chemical diversity is determined by the specific combination of selected elements from a set of monosaccharide building blocks: the different glycosidic linkages used to link these monosaccharides, the stereochemical configuration of the glycosidic bonds, branching and site-specific modifications (such as sulphation and phosphorylation) lead to the complexity of the glycome,¹³ making them the most structurally diverse biomacromolecule. This heterogeneity makes isolation of pure samples, and in sufficient amounts, from biological sources challenging in many cases.¹⁴ Several techniques are typically required to be used in parallel such as high-performance liquid chromatography (HPLC), mass spectroscopy (MS), nuclear magnetic resonance (NMR) and array technologies (glycan and lectin arrays).^2,15–22 In contrast, a single technique can be used to interrogate RNA, DNA or proteins including gene mutation, over-expression, and silencing which is challenging in glycobiology as glycans are not template-encoded. Fluorescent labelling can be genetically included to tag proteins for visualisation to study localisation.¹⁷ Protease enzymes are available to digest proteins and assignment of the protein sequence can be carried out by MS. A difficulty of MS for glycan characterisation is the discrimination of several structural isomers, such as enantiomers (e.g. D/L-glucose), diastereoisomers (e.g. glucose/mannose), anomers (α/β), and linkages and branching (e.g. 1–4/1–6).⁴ Progress in glycoscience has been hindered by the lack of analytical methods that can accurately map glycan structures, particularly distinguishing α/β anomeric linkages in oligosaccharides. Flitsch and co-workers have demonstrated that the anomeric configuration is preserved after gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS²). By integrating MS² with ion mobility spectrometry (IM-MS²), differentiation of α- and β-linkages in natural, underivatised carbohydrates can be achieved. This was validated on oligosaccharide standards and applied to sequencing of plant glycoconjugates, revealing that anomeric signatures persist in fragments from larger glycans.^23–25 For nucleic acids sequencing and microarray technologies can be used alongside amplification which are not possible for carbohydrates²⁶ adding to the challenge.

Carbohydrate arrays (glycoarrays) have emerged as the primary high-throughput tool for studying carbohydrate/lectin binding (Fig. 1).¹¹ Glycans can be covalently or non-covalently linked to a solid support surface (e.g. alkyne-azide ‘click’, thiol-ene ‘click’, activated esters) which can require non-trivial multi-step, protection and deprotection sequences. The non-covalent attachment of glycans has been performed by Feizi et al.,²⁷ using neoglycolipids (NGL) that adsorb onto hydrophobic surfaces, but retain some mobility to form clusters for optimal protein binding, as occurs in cell membranes. As an example, this technology was used to interrogate human and swine influenza strains against a panel of 86 glycans (80 sialylated and 6 neutral (control)). A clear difference in receptor-binding was observed between pandemic H1N1 viruses (Cal/09, Ham/09) and the seasonal virus Mem/96. Cal/09 and Ham/09 bound broadly to α2-6-linked sialyl sequences regardless of backbone type or length, and to many α2-3-linked sequences. In contrast, Mem/96 bound exclusively to α2-6-linked sequences.²⁸ More recently solution phase arrays based on chemically glycosylated phages have emerged, enabling genetic encoding and high-throughput screening.²⁹

Fig. 1 Overview of construction and analysis of glycoarrays, Reproduced from Heimburg-Molinaro et al. ref. 40 with permission from Elsevier, copyright 2024.

Another powerful tool to probe intracellular glycan function is metabolic oligosaccharide engineering (MOE) which uses unnatural glycans to hijack a cell's biosynthetic machinery, to allow unnatural (and bio-orthogonal^30,31) glycans to be integrated into glycoconjugates spanning the cell surface,^32–34 glycoproteins³⁵ and glycoRNA.^36–38 The majority of these are secreted or cell surface glycoproteins, but some are found inside the cell.³⁹ Metabolic oligosaccharide engineering has been applied to myriad problems in glycobiology; to disrupt glycan biosynthesis, chemically modify cell surfaces,³² probe metabolic flux inside cells, and for proteomics analysis of glycosylation.

The above summary showed examples of advances in glycomics which are generating new opportunities to exploit glycan binding processes in biosensing and diagnostics. For example, many respiratory pathogens (including SARS-CoV, SARS-CoV-2, influenza) and bacterial pathogens (such as uropathogenic E.coli) target and anchor glycans as key stages in their infection cycle and many cancers are associated with glycosylation changes.^41,42 New tools, including those suitable for point of care diagnostics, are needed to intercept, detect and diagnose these crucial pathways and processes which are discussed in the following section.^43–45

1.1. Gold nanoparticles as biosensors

Gold nanoparticles (AuNPs) exhibit characteristic optical properties that are dependent on surface plasmon resonance (SPR), which arises due to the collective oscillation of the conduction-band electrons. The maximum wavelength and shape of the local surface plasmon resonance (LSPR) are determined by the particle size and shape. AuNPs have extremely high extinction coefficients, which are three to five orders of magnitude greater than those of traditional molecular dyes and is stronger than other metal plasmonic nanoparticles. The SPR band intensity and wavelength depends on the factors affecting the electron charge density on the particle surface such as the metal type, particle size, shape, structure, composition and the dielectric constant of the surrounding medium, as theoretically described by Mie theory.⁴⁶ As a result of the above, spherical AuNPs between 10 nm and 80 nm appear red in colour (due to absorption of blue-green portion of the spectrum ∼450 nm). As the interparticle distance decreases to less than that of the particle diameter, coupling and dipole–dipole interactions between the plasmons of neighbouring particles result in a broadening and a shift to longer wavelengths of the surface plasmon absorption band, resulting in the AuNPs appearing blue. It is this colourimetric aggregation that is exploited for the use in bioassays which can be detected spectrophotometrically, or visually by eye or with cameras.⁴⁷

Colourimetric assays for investigating biomolecular interactions using AuNPs functionalised with biomolecules such as proteins,^48,49 peptides,^50,51 antibodies,^52–54 and DNA,^55,56 have been developed based on inducing particle aggregation (Fig. 2A/B). The detection of bacterial DNA using AuNPs originated in the pioneering work of Mirkin et al.⁵⁵ who demonstrated that as little as 10 fmol of an oligonucleotide analyte could be detected by exploiting the aggregation phenomenon of AuNPs.

Fig. 2 Gold nanoparticles in biosensing. (A) General procedure for functionalising AuNPs with biomolecules; (B) principle of aggregation-induced colour change of AuNPs in response to a stimulus; (C) AuNP in lateral flow diagnostics.

Lateral-flow diagnostics (LFD) are another sensing modality which deploy gold nanoparticles, most famously the home-pregnancy test,⁵⁷ which make use of the high extinction coefficient (strong colour) of the gold nanoparticles. Traditional LFDs use antibodies (lateral flow immuno diagnostics) as the detection units immobilised to both the stationary phase (e.g., nitrocellulose paper) and the mobile phase (AuNPs), forming a “sandwich” with the analyte in the middle, giving a red line due to concentration of the AuNPs (Fig. 2C). LFDs are typically low cost and require little or no clinical infrastructure or training to use. Immuno-LFDs tend to have lower sensitivity (some false negatives) but high selectivity (few false positives). The cost-effectiveness and clinical usefulness of these inexpensive devices has been demonstrated by various studies of malaria rapid diagnostic tests^58,59 in the diagnosis of cutaneous leishmaniasis⁶⁰ and were found to compare well to the more expensive RT-PCR for Ebola diagnosis.⁶¹ In addition to antibodies, other recognition unit such as nucleic acids,⁶² glycans, and lectins⁶³ could be used, provided they have suitable characteristics (affinity, selectivity, specificity). During the COVID-19 pandemic LFDs were extensively explored due to their low cost and rapid turnaround time which could enable regular mass testing of large populations.⁶⁴ This could find asymptomatic individuals spreading the virus, who would not be identified by a symptomatic RT-PCR testing pathway.^65–67 The first LFDs for the COVID-19 pandemic were designed to detect antibodies in patient blood samples produced in response to SARS-CoV-2 infections to repot prior infections.^68–70 Antigen LFDs, in contrast, were designed to diagnose the presence of the virus i.e., an active infection. Several antigen lateral-flow tests, by late 2020, had passed Phase 3 testing in the United Kingdom,⁷¹ gained WHO “Emergency Use Listing” approval,⁷² or had emergency approval granted by The United States Food & Drug Administration. These devices all utilise antibodies as detection/capture units. A university-based validation testing between LFDs and PCR confirmed that LFDs cannot detect lower viral loads but were estimated to be capable of identifying up to 85% of infections in the cohort trialed⁶⁷ showing their potential for frequent, low-cost testing when deployed appropriately.

Considering the above applications of gold nanoparticles in biosensing/diagnostics and the critical role played by glycans in cellular recognition processes, our team (and others) have been developing the tools and applications for integrating glycans onto gold nanoparticle surfaces to re-purpose these for glycobiology. This Feature Article focusses on reviewing our laboratory's development of polymer-tethered glycans, using the polymers as adapters to allow capture of the glycans and immobilisation onto the gold and to provide steric (colloidal) stabilisation. We review the synthetic principles behind this platform, the integration into tools and then the deployment as new arrays, diagnostics and biosensors, using native and unnatural glycans.

2. Polymer-tethered glycosylated gold nanoparticles

Glycosylated gold nanoparticles that change colour due to lectin-mediated aggregation are well known for their use as biosensors, but we and others identified a key challenge associated with direct immobilisation of glycans onto AuNPs: that the glycans provided insufficient colloidal stability in buffer or complex media, meaning aggregation (and hence false positives) was not always due to ‘binding’, but due to colloidal instability. To exemplify this problem we immobilised 1-thio-1-deoxy-glucose onto AuNPs using the one-pot method of Watanabe et al.⁷³ Concanavalin A (Con A: a lectin with affinity to α-mannose/glucose) binding was studied by UV-Vis spectroscopy showing that larger AuNPs gave response at lower concentration compared to smaller, showing the detection principle. However, a very low salt buffer had to be used due to the instability of particles which readily aggregated to give false-positive ‘binding’ responses (Fig. 3B). To overcome this, a PEG (poly(ethylene glycol)) tether was introduced as a steric stabiliser to improve the saline stability. Due to the stabilisation, the signal generation was both slower and weaker, taking 2 hours to give equivalent response as 15 minutes with directly glycosylated AuNPs (Fig. 3B). These first experiments highlighted the crucial role of the steric stabiliser, which has to balance stabilisation with allowing aggregation (the output) to still occur.⁷⁴ This balance is shown in many studies using PEG as a tether, including work from Field and coworkers.^75–77 In related work, Miura and co-workers have made glycopolymers (with glycan side-chains, not end groups) for immobilisation onto gold nanoparticles for probing lectin-binding.^78,79 Parry et al. used a similar strategy to synthesise glycoparticles for immunisation.⁸⁰

Fig. 3 Delicate balance of nanoparticle colloidal stability to enable aggregation-induced outputs, shown by UV-vis analysis of nanoparticle aggregation. (A) AuNPs with a glucose directly immobilised to the surface readily aggregate in saline, but also responses rapidly to lectins; (B) PEG-linked glycosylated AuNPs are sterically stabilised against saline but show slow lectin binding response.

2.1. PEG alternatives from RAFT polymerisation

Whilst PEG is a useful linker, there are limits to what is commercially available in terms of hetero-bi-functionality and molecular weights, and in our hands some commercial PEGs can contain chain-end defects, limiting application. Therefore, we considered the use of reversible-addition fragmentation chain transfer (RAFT) polymerisation as it is a versatile controlled radical polymerisation (CRP) technique, applicable to a wide range of monomers.⁸¹ The benefits of RAFT have been widely explored, but our interest lay in:

(1) The ability to fine-tune polymer length to optimise stability of coated AuNPs⁸²

(2) Incorporation of (latent) functionality at the α-chain end (e.g. for glycan capture)

(3) The ω-thio-carbonyl-thio end-groups, which in the presence of a reducing agent, or nucleophiles, cleaves to reveal a thiol-end group for subsequent AuNP immobilisation.

We designed a telechelic polymer system, with a conjugatable pentafluorophenyl ester at the α terminus for facile immobilisation of amino-glycans (or other functions) and a masked thiol (RAFT agent) at the ω-terminus for gold surface immobilisation. Poly(N-hydroxyethylacrylamide) (PHEA) was chosen as the polymer of choice for several reasons (Fig. 4). Firstly, it is highly water soluble, but also in organic media, unlocking a variety of chemistries. PHEA is sterically smaller than oligo(ethyleneglycol)(meth)acrylates (OEG(M)As) which we hypothesised would impact grafting density, as previous shown when “grafting to” flat gold surfaces.⁸³ PHEA does not show lower critical solution temperature, unlike some other water-soluble monomers like OEG(M)As, which would complicate assay performance.⁸⁴ Finally, the amide (rather than ester) linkage is more stable, preventing thiol back-biting which has been reported for (meth)acrylates.⁸⁵ The use of PFP-terminated polymers, rather than using glycosylated RAFT agents, ensures that all the polymers in a series have the same initial chain length distribution and therefore reduce the variability between particle types (when varying particles size or glycan for example).

Fig. 4 Summary of the use of RAFT polymerisation to obtain heterotelechelic poly(hydroxyethyl acrylamide) tethers suitable for glycan capture and immobilisation onto gold nanostructures.

Using a panel of PHEAs of different molecular weights (degree of polymerisation, DP = 10–75) it was observed that polymers of DP <15 on AuNPs of 60 nm were unstable at raised [NaCl] (>0.2 M, which is close to physiological (0.137 M)), making them unsuitable for a biosensing scenario (Fig. 5A/B). While longer polymers (predictably) improved saline stability to an extent that signals were very low/slow, (Fig. 5C/D). This serves to highlight the delicate balance between stability and sensing for metal nanoparticles where the glycan alone is not the only consideration but the macromolecular tether structure, which justifies the use of precision polymerisation methods.

Fig. 5 Impact of PHEA degree of polymerisation on the performance of polymer-tethered glycosylated gold nanoparticles. (A) Aggregation (from UV-vis) as a function of PHEA DP on gold nanoparticles verses a NaCl gradient; (B) photographs showing the colour change in PBS; (C) Con A-induced aggregation kinetics of ManNH₂-termintated polymer coated 60 nm AuNPs as a function of PHEA DP in response to 1 mg mL⁻¹ Con A; (D) photographs showing Con A-induced aggregation of ManNH₂-terminated polymer coated 60 nm AuNPs. Reproduced from ref. 82 with permission from American Chemical Society, copyright 2014.

A further library-oriented screen of 27 nanoparticle formulations (AuNP 30, 50, 70 nm; DP 10–50 in steps of 5 units) was used to identify the optimum size and linker length.⁸⁶ Initial saline stability screening determined that a linker length above DP25 was required to stabilise all particle sizes, therefore lower DP polymers were omitted from follow on screening. To screen for binding, the GalNAc-binding lectin SBA (soybean agglutinin) was used to validate the methods and to establish which particle structural requirements gave the strongest outputs. For all polymer lengths, the largest changes were seen for the shortest polymer chain length (DP25) with a step-wise decrease as polymer length increases, and the biggest (70 nm) particles gave the largest outputs. It is important to note that individual glycans and lectins will give subtly different effects, but that the trends above are broadly true for lectin binding (Fig. 6A). The aggregation assay used above provides a convenient output of binding, but it cannot rule out the case where lectins bind but fail to agglutinate: for example, the longer polymer linkers can bind to SBA, but also provide a steric block against agglutination, highlighting the need for tuning of these parameters. Therefore, a second assay using biolayer interferometry was developed to probe for false negatives. In short, biotinylated SBA was immobilised onto streptavidin functional BLI sensors and the nanoparticles exposed to this. The total BLI signal (which is essentially proportional to mass bound) as a function of particle parameters is shown in Fig. 6B. The shorter polymers at all particle sizes gave the largest output, highlighting how glycan accessibility is a crucial parameter but also that larger non-aggregating polymer coatings do bind, but without downstream signal generation.

Fig. 6 Importance of polymer tether length and gold core size on the readout response by (A) aggregation of GalNH₂-functionalised AuNPs in response to 500 nM SBA and (B) biolayer interferometry using streptavidin coated biosensor with SBA immobilised in response to GalNH₂-functionalised AuNPs. Reproduced from ref. 86 with permission from American Chemical Society, copyright 2020.

Another important consideration is the optical density (standard method of reporting concentration) of the gold nanoparticle solution used. This is essential for reproducibility (as an easily accessible measurement), plus the probe concentration is expected to have a large impact on the signal outcomes.⁸⁷ At high concentrations of lectin, the signal begins to decrease due to saturation of the particle surface, so lectins only bind 1 particle (i.e. no cross-linking occurs) and hence no aggregation, therefore no signal. This is an important consideration to ensure lectins are tested in a concentration regime which allows true positive effects to be probed.

2.2. Impact of polymer structure

In addition to molecular weight, the architecture and chemical nature of multivalent systems will impact their outcomes.^88,89 Kiessling and coworkers showed the aggregation of mannose clusters (linear, dendritic) with Con A was architecture dependant, and the backbone-glycan linker can dramatically impact access into lectin binding sites.^90,91 Glycopolymer routing through dendritic cells is also shown to be antigen-size dependant.⁹² We have explored how the exact chemical structure of the polymeric tether impacts outcomes, demonstrating that minor structural changes can dramatically impact biosensing success. For example, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a widely used hydrophilic polymer and is accessible from RAFT polymerisation. We carried out a critical comparison of PHEA and PHPMA to ask if subtle differences in polymer structure would impact the signal detection outputs ?⁹³ Telechelic polymers were prepared by photoRAFT polymerisation to give a library of 30 particles with AuNP core sizes of 20, 30 and 40 nm and polymer lengths of ∼DP25–75 ((Fig. 7A/B)). The first observation from this study was that that PHEA led to higher grafting densities than PHPMA (as revealed by comparative X-ray photoelectron spectroscopy (XPS)), attributable to the increased steric bulk associated with methyl on the backbone. Initial aggregation tests using Gal-PHPMA@AuNPs particles did not give any significant increase in Abs₇₀₀, but the PHEA equivalents did show aggregation (Fig. 7C/D). To a first approximation, this would be interpreted as ‘no binding’ for the PHPMA which is a surprise considering the presence of the galactose residue, but we considered this might be a false-negative due to the differences in polymer structure. It is perfectly feasible that binding can occur without cross-linking depending on the architecture of the glycans. To ensure that the above observations (no aggregation of Gal-PHPMA) was not due to inaccessibility of the glycan we used a complementary technique to assess lectin binding, BLI as described above. By comparing Gal-PHPMA₃₆@AuNP₄₀ (no aggregation response) with Gal-PHEA₃₃@AuNP₄₀ (strong aggregation response) we could clearly see that both particles have affinity towards the immobilised SBA with similar dose–response curves being obtained (Fig. 7E/F). This proves that the two polymer coatings both present sufficient Gal for strong SBA binding but that the PHPMA coating prevents aggregation from occurring, whereas PHEA encourages it. Taken together this study demonstrates that the chemical nature, as well as the molecular weight, of the polymer linker has a dramatic impact on the outcomes of glyconanoparticle sensing platforms, enabling control over aggregative versus non-aggregative outputs and will help design robust nano-biosensors in the future. It also highlights the need for complementary assays to determine binding profiles.

Fig. 7 Binding of nanoparticles with different polymeric tethers (A, B) with SBA. Measured by UV-visible spectroscopy: (A) Gal-PHPMA₃₆@AuNP₄₀ and (B) Gal-PHEA₃₃@AuNP₄₀. Biolayer interferometry analysis of nanoparticles binding to immobilised SBA in response to (E) Gal-PHPMA₃₆@AuNP₄₀ and (F) Gal-PHEA₃₃@AuNP₄₀. Reproduced from ref. 85 with permission from Royal Society of Chemistry, copyright 2020.

2.3. Glycan capture methods

A key consideration for the rationale design of modular glycan capturing ligands, is to use chemistry that is versatile, orthogonal and universal, and allows both small and larger branched glycans to be captured: in essence to take the inspiration of micro-array systems which allow automated glycan functionalisation and capture. Early examples of glycosylated nanoparticles from Cameron and co-workers synthesised glycosylated methacrylates which were polymerised by RAFT, before immobilisation on the gold (Fig. 8A). Such methods require multiple synthetic steps and column purification before polymerisation and when comparing two glycans it is challenging to obtain the same molecular weight and dispersity.⁹⁴ This led to post-polymerisation modification emerging as a powerful technology for systematic functionalisation of glycomaterials.⁹⁵ For example, Haddleton and co-workers used glycosyl azides to modify alkyne-functionalised pre-polymers using azide–alkyne click cycloadditions.⁹⁶ Other examples of the use of ‘click’-type reactions include the following: thiol–ene,⁹⁷ thiol–yne,⁹⁸ thiol–chloro,⁹⁹ activated esters,^100,101 or thiolactones.¹⁰² While convenient, the bottleneck is often the installation of the reactive handle onto the glycan. To overcome this challenge, Tanaka¹⁰³ and Fairbanks¹⁰⁴ have used imidazolinium salts to introduce azides at the reducing terminus directly from reducing glycans, allowing protecting-group free one-pot two-step glycosylations. Another appealing strategy for glycan conjugation is to exploit the hemiacetal equilibrium at the terminus of reducing glycans which can react with hydrazides. Glyconanoparticle/polymer libraries have been prepared using hydrazide functional polymers,^105–107 but there are significant challenges. The hydrazone bond is intrinsically dynamic so (depending on pH) the glycans can detach and exchange in a dynamic equilibrium.¹⁰⁸ Furthermore, the hydrazone linkage is an equilibrium between a ring-opened and ring-closed form. Chemical reduction (using, e.g., NaBH₄) can increase stability but adds another step to the process. To overcome this, amino–oxy conjugates have emerged as alternatives due to their higher stability against hydrolysis^109,110 and at equilibrium exist in greater proportions as ring-closed structures (more like the native linkages) compared to hydrazides. We reported a method to generate glyconanoparticles from unprotected glycans by conjugation to polymer tethers bearing terminal amino–oxy groups, which are then immobilised onto gold nanoparticles. Using an ¹³C enriched glycan, the efficiency of this reaction was probed in detail to confirm conjugation, with 25% of end-groups being functionalised, predominantly in the ring-closed form.¹¹¹ Whilst useful, this method's low conversion and more challenging synthesis make it less suitable for large scale use or in diagnostics (where residual hydrazide/aminooxy end groups may cross-react). Photochemical crosslinking methods based on perfluorophenyl azide have also been employed by Yan and co-workers.^112–114

Fig. 8 Strategies for producing glycosylated AuNPs. (A) synthesis of polymers with pendant glycans, (B) glycan end-functionalised polymers, (C) incorporating more complex glycans on polymer end-terminus using bioorthogonal handles. All these approaches use a terminal thiol to immobilise into a gold particle.

During our investigations, our modular polymer with a pentafluorophenyl (PFP) end-group was identified as being the most versatile and convenient handle, as it has been widely used with amino-monosaccharides as a method for capturing glycans on the polymer terminus (Fig. 8B).^{82,86,87,93,115–121} The PFP is appealing compared to other active esters (such as NHS) as it is more reactive and selective towards amines compared to alcohols or carboxylic acids, ensuring chemoselective ligation. The PFP group also has a unique signature in ¹⁹F NMR, allowing reaction monitoring which is not always possible in ¹H NMR due to the large number of glycan peaks. This is particularly appealing with the rise of benchtop NMRs allowing for rapid and non-destructive reaction monitoring. Theato and co-workers pioneered the use of PFP in polymer science, incorporating these esters into acrylate and methacrylate monomers and polymerising using controlled radical methods such as RAFT polymerisation, producing well-defined polymers with reactive side groups. These polymers serve as versatile platforms for introducing diverse functionalities, efficiently and quantitatively under mild conditions (e.g., room temperature).^122,123 This method has been used to produce glycopolymers for a range of lectin binding studies.^124,125

A key consideration to note when using aminoglycans, is that they are typically obtained via the reduction of an azido-precursor. Therefore “click” chemistry strategies (to remove one glycan manipulation step) can accelerate nanoparticle preparation (Fig. 8C). The most versatile of these is copper-free strain-promoted alkyne-azide click (SPAAC), with dibenzocyclooctyne (DBCO) being the most used due to low cost and commercially availability.¹²⁶ By displacing the PFP with DBCO-NH₂ capture of rare glycans or sterically large is possible, which would be challenging with PFP, exemplified by the capture of mono- and bi-antennary azido sialyllactose and fluorinated Lewis^x glycans.^127–129 Budhadev et al. have used click methods to add multiple sugar ligands on a single short PEG tether, these clustered glycan arrangements accelerated nanoparticle aggregation in the presence of lectins compared to monovalent or sparsely distributed ligands, but can be more challenging to synthesise.¹³⁰

The polymer-tethered glycosylated AuNPs are typically characterised using a range of complementary techniques, necessitated by the challenge of detecting very small amounts of surface ligands. UV-Vis spectroscopy, dynamic light scattering (DLS) and zeta potential measurements provide insights overall particle stability. Transmission electron microscopy (TEM) visualises core size and morphology but cannot typical probe the organic polymer coating. X-ray photoelectron spectroscopy (XPS) is a powerful tool which we have widely used for surface composition analysis of AuNPs providing chemical identity of the presence of the ligands. Thermogravimetric analysis (TGA) quantifies organic content without distinguishing glycan species but requires a large amount of material.¹³¹ Nuclear magnetic resonance (NMR)¹³² and mass spectrometry¹³³ can provide structural and compositional details.

3. Glycosylated nanoparticles to dissect protein-glycan and protein unnatural-glycan interactions

The above sections summarise the synthetic development of polymer-tethered glycosylated gold nanoparticles, which was undertaken to unlock their biosensing capabilities. Understanding protein-carbohydrate interactions is crucial for drug design, vaccine development, and identifying disease biomarkers, as altered glycosylation patterns are linked to conditions like cancer and diabetes.^42,134 Additionally, insights into these interactions support advancements in biotechnology and industrial applications. Conventional approaches involve microarrays which require labelled proteins¹³⁵ or methods such as ITC which require relatively large amounts of sample and has limited throughput.¹³⁶ The polymer-tethered gold nanoparticle platform was identified as being a complementary technology, to explore the colorimetric responses of plasmonic gold cores with the modular nature of the polymer-steric stabilisers.^{75,76,78,79,112,114,137} In an early demonstration Otten et al. prepared a library of seven monosaccharide-terminated polymers; mannose (Man), galactose (Gal), glucose (Glc), N-acetylmannosamine (ManNAc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and fucose (Fuc) using the hydrazide conjugation method.¹⁰⁶ This library was used to interrogate a panel of plant lectins: concanavalin A (Con A), Ricinus communis agglutinin (RCA₁₂₀, which is a non-toxic variant of ricin), soybean agglutinin (SBA), peanut agglutinin (PNA), wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) in multiwell plates using aggregation (red-blue shift) as the output (Fig. 9A–D). Due to the volume of data generated with this tool, linear discriminant analysis (LDA) (Fig. 9E), a statistical and machine learning technique could be deployed to create ‘barcodes’ for multiplexed lectin detection, which has since been used with soluble polymers.¹³⁸

Fig. 9 High-throughput analysis using AuNPs and lectin discrimination. (A) Scanned image of a multiwell-plate with a serial dilution of a lectin in response to different glycosylated AuNPs, where purple/blue shows an interaction (aggregation) and pink/red is no interaction (no aggregation); (B) UV-vis traces of different glycosylated AuNPs in response to a serial dilution of a lectin; (C) binding isotherms from the absorbance measurement at 700 nm as a function of lectin concentration; (D) absorbance at 700 nm of Man-, Gal-, Glc-, ManNAc-, GalNAc- and GlcNAc-functionalised particles after 30 min incubation with 6.25 µg mL⁻¹ Con A, RCA₁₂₀, SBA, WGA, PNA, UEA. Each is the average of five measurements; (E) linear discriminant plot showing excellent segregation between the six lectins. Reproduced from ref. 74 with permission from Royal Society of Chemistry, copyright 2014.

An advantage of using polymeric tethers is that heterogeneous surfaces can be easily assembled through mixing, which to a first approximation mimics the complexity of the glycocalyx. Eleven combinations were prepared, from 100% galactosamine (GalNH₂) to 100% mannosamine (ManNH₂) in steps of 10%.¹¹⁵ The post-polymerisation route employed means that all the polymers have the same initial chain length distribution and therefore reduced the variability between particle types, but allows versatile end-group functionalisation to make libraries of a variety of particles with differing carbohydrate densities. This heterogeneous display lead to improved discrimination between lectins which had otherwise similar binding preferences (SBA and RCA₁₂₀) for homogeneous glyconanoparticles (Fig. 10).¹¹⁵

Fig. 10 (A) Dose-dependent binding isotherms of mixed Man/Gal@AuNPs in response to a serial dilution of SBA. (B) The LDA model generated to discriminate between Con A, RCA₁₂₀ and SBA from mixed Man/Gal@AuNPs. In the model each point represents a sample of that lectin and the ellipse represents one standard deviation from the average. Reproduced from ref. 115 with permission from Royal Society of Chemistry, copyright 2016.

The above examples were focussed on plant lectins and synthetically trivial monosaccharides, but the real potential of this approach is to explore more complex glycans and/or biomedically relevant glycan-binding partners. During the early stages of influenza infection, hemagglutinins on the viral surface engage sialic acids in their host. Simplistically, human influenza targets 2,6-linked sialic acids, but avian influenza targets 2,3-linked sialic acids, representing the abundance of each isomer in the host.^139,140 Zoonosis can occur when intermediate species, such as swine, presents both glycans allowing adaptation. We captured 2,6- and 2,3-sialyllactose onto PHEA tethers and assembled them onto the gold nanoparticles and used these to interrogate hemagglutinins from a range of influenza strains.⁸⁶ Using hemagglutinins bound to BLI (biolayer interferometry) sensors it was possible to rapidly assess binding preference, which was in line with the microarray data, showing the potential for these particles for rapid evaluation of viral threats. In 2020 when SARS-CoV-2 emerged, it was unknown if, or which, glycans it would bind to during initial stage of infection and represented a significant knowledge gap. Guided by the above influenza work, Baker et al. used polymer-tethered nanoparticles to discover that SARS-CoV-2 binds sialic acids (Fig. 11), which was validated by complimentary modelling and STD NMR¹¹⁷ and also by using anisotropic gold nanoparticle probes.¹²¹ These observations were later confirmed by others^141,142 and used to develop multivalent sialic acid decoys as potential anti-adhesion agents.¹⁴³

Fig. 11 (A) Sequence alignment between the S1 domains of the SARS-CoV-2 and MERS spike proteins. Regions important for sialic acid binding are highlighted by red boxes; (B) model showing the hypothesised sialic acid binding sites (yellow CPK colouring) for the SARS-CoV-2 spike protein trimer; (C) a comparison between the sialic acid binding sites from MERS (PDB entry 6Q04) and the SARS-COV-2 model (PDB entry 6VSB) in complex with a 2,3′-sialyllactose. Biolayer interferometry analysis of SARS-COV-2 spike protein with glyconanoparticles. (D) NeuNAc functionalised 16 nm AuNPs (NeuNAc-PHEA₅₀@AuNP₁₆) (E) biolayer interferometry analysis of SARS-CoV-2 spike protein with serial dilution of NeuNAc-PHEA₅₀@AuNP₁₆. Reproduced from ref. 117 with permission from American Chemical Society, copyright 2020.

The above examples of generating signal outputs upon glycan/protein binding showed potential of this platform but had been limited to glycans of low complexity and mostly with known and well-characterised binding partners which were all available at unchallenging (mg) scale. An advantage of the modular nanoparticle platform compared to e.g. microarrays, is that it is also suitable for smaller numbers of glycans (e.g. 1–10), whereas microarrays’ power scales with the number of targets. Using chemoenzymatic synthesis nine sequentially modified fluoro-lacto-N-biose glycans were prepared and captured onto the particles, achieved using µg quantitates, with conjugation demonstrated using X-ray photoelectron spectroscopy (XPS) due to the limited amounts where e.g. NMR would not be appropriate. Using the colourimetric aggregation assay it was shown how the precise location of the fluorine atom impacted binding towards two galectins (galectin-3 and galectin-7, galectin-1 showed no binding to any of the probes), and led to increased selectivity, with a tetra-fluorinated lacto-N-biose showing a complete switch in affinity between galectin-3 and -7.¹²⁸ This work was further expanded for the study of fluorinated Lewis^x glycans, including a side-by-side comparison of the nanoparticle approach with convention lipid-linked microarrays studies, for the first time (Fig. 12).¹²⁹ This side-by-side analysis showed close correlation between IC₅₀ (nanoparticles) and total signal (arrays) in binding to the cholera toxin, showing how fluorination could drive a 1000-fold difference in IC₅₀. These particles were also shown, in a proof-of-concept, to be suitable for lateral flow glyco detection of the cholera toxin.

Fig. 12 (A) Lewis^x-functionalised AuNPs nanoparticles; (B) principle of detection due to gold nanoparticle red-blue shift upon aggregation with CTB; (C) UV-visible spectra for gold nanoparticles with native Lewis^x (LeX1) and fluorinated LeX22; (D) dose-dependent response of library of Lewis^x glyconanoparticles to CTB. Data is presented as mean normalised Abs₇₀₀ from UV-visible spectroscopy ± standard error of 3 replicates. (E) Correlation of glycan array and glyconanoparticle binding data. Reproduced from ref. 129 with permission from Springer Nature, copyright 2024.

The above examples were limited to trisaccharides as the largest glycan cargo, but to be truly competitive and complimentary to array-based technologies large and branched glycans are also required. Chemoenzymatic synthesis was used to make unnatural biantennary glycans (2 trisaccharide arms) and these were successfully captured and immobilised using both amine-PFP and ‘click’ coupling routes.¹²⁷ The particles were optimised to generate signal in the presence of whole virus (in this case, vaccine-strain influenzas) meaning glycan-binding preferences could be extracted by simple colourimetric changes in a microplate (Fig. 13). Compared to arrays, this technology can be deployed in situ in a biosafety hood (for example) without needing to move to a central array platform and can be considered a new bench-side tool. The sterically large glycans did not impact the overall stability of the particles, showing the polymer tethers performed this steric stabilisation role even with large end-groups. These interactions were studied in the presence of neuraminidase inhibitors (oseltamivir carboxylate and zanamivir), ensuring results were not biased by neuraminidase cleavage of sialic acids. Due to glycosidic O-linkages being prone to cleavage by glycosidases in biological fluids, risking premature loss of glycan ligands before they engage their targets, alternative, enzyme-resistant linkages such as C-glycosides which retain their conformation have been explored.^144,145

Fig. 13 Sialyllactose-functional nanoparticles binding to the live attenuated influenza vaccine (LAIV) virus. (A) Schematic representation of the aggregation assay, where GlycoAuNP particles cross-linking virus; (B) dose-response of LAIV viruses versus 2,3 sialyllactose AuNPs; (C) dose-response of LAIV viruses versus 2,6 sialyllactose AuNPs; Error bars are the standard error of the mean from n = 3. (D) Schematic representation of the BLI assay with immobilisation of the LAIV virus to the sensor; (E) BLI response of LAIV virus to 2,3 SL AuNPs; (F) BLI response of LAIV virus versus 2,6 SL AuNPs. BLI was undertaken at 0.375× dilution of LAIV virus with AuNPs at OD₅₄₀ (optical density@540 nm) = 1. In all assays, neuraminidase inhibitors, 25 µM oseltamivir carboxylate and 100 µM zanamivir, were used. Reproduced from ref. 127 with permission from Wiley-VCH GmbH, copyright 2025.

4. Lateral flow diagnostics

A lateral flow device (LFD) is a rapid diagnostic tool commonly used for point-of-care testing. It is a self-contained paper-based device designed to detect the presence (or absence) of a target analyte in a liquid sample. LFDs are often used for medical diagnostics, food safety, and environmental testing. LFDs typically rely on antibodies as recognition units, owing to their high specificity. A typical LFD has antibody immobilised on both the mobile phase (gold particle) and stationary phase (nitrocellulose) forming a ‘sandwich’ around the antigen, to generate the characteristic red line, due to concentration of the gold nanoparticles. Glycans offer an alternative to these recognition units, and their study might bring new opportunities.¹⁴⁶ There are synthetic and ethical advantages to using glycans over antibodies: glycans can be cheaper to produce in large quantities synthetically or chemoenzymatically via animal-free routes and they are thermally stable. However, despite this, there are only few examples of glyco-LFDs. Damborský et al. demonstrated a lectin-based LFD to detect aberrantly glycosylated prostate-specific antigen.¹⁴⁷ Miura and co-workers developed a flow-through method for detecting Shiga Toxin-1, which was deposited on the nitrocellulose and binding was observed with galactosylated AuNPs.¹²⁴ This was taken further by using mannosylated-AuNPs to detect Con A.⁷⁹ In this case, the glyconanoparticles were used in the mobile phase and a rabbit anti-Con A antibody as the stationary phase test line with a further study varying to mannose density to optimisation of the readout.⁷⁸ Whilst not a full glycoLFD, this study clearly demonstrated that glycoLFDs are possible. Hernando et al. used glycan–BSA conjugates on gold nanoparticles to detect the ricin surrogate RCA₁₂₀.¹⁴⁸ Kim et al. exploited SARS-CoV-2's ability to bind glycosaminoglycans to develop GlycoGrip a glycopolymer capture line for SARS-CoV-2 which was used with anti-spike antibody coated AuNPs as the mobile phase.¹⁴⁹

Our team set out to make complete lateral flow glyco diagnostics¹⁴⁶ where the stationary and mobile phases comprised of glycans (Fig. 14A). In excess of 30 polymer-tethered glycosylated nanoparticles were optimised for chain length and nanoparticle core to avoid unwanted aggregation (which could lead to both false negatives and false positives) using galactosylated probes and glycosylated-BSA as the stationary phase.¹¹⁹ This set up allowed lectin detection at nM concentrations which was comparable to commercial antibody-LFDs (Fig. 14B).¹¹⁹ Further optimisation using glycosylated poly(vinyl pyrrolidone)amphiphilic anchors for the stationary phase enable construction of an “all glycopolymer” lateral flow device for lectin detection, showing how all biologic materials can be replaced with precision synthetic polymers.¹⁵⁰ A key advantage of antibody-LFDs is that they have been extensively optimised for use with complex media, such as saliva, which is far more challenging than proof of principal in buffer. To allow primary clinical swabs to be used, we used a flow-through assay (Fig. 14C), where by the clinical sample is dried as the stationary phase, and the mobile glyco-particles “flowed over”: in this set up SARS-CoV-2 diagnostics were shown with a panel of 100 clinical swabs, resulted in sensitivity of 85% and specificity of 93%,¹¹⁸ fully demonstrating that glyco-diagnostics can be rapidly assembled for pathogen detection (Fig. 14D).

Fig. 14 (A) Representative lateral flow glyco-assay and for SBA. Reproduced from ref. 146 with permission from American Chemical Society, copyright 2022. (B) Lateral flow dipsticks run with the indicated concentrations of SBA using Gal-PHEA₇₂@AuNP₁₆. Reproduced from ref. 119 with permission from Wiley-VCH GmbH, copyright 2022. (C) Representative flow-through glyco-assay for sensing SARS-CoV-2. Reproduced from ref. 146 with permission from American Chemical Society, copyright 2022 (D) results of flow-through device performance (hit or miss) as a function of Ct (PCR threshold cycles) for heat-inactivated primary patient swabs devices. Thresholds indicated are the sensitivity as a function of the Ct value. Confusion matrices. Sensitivity = TP/(TP + FN); specificity = TN/(TN + FP); PPV = TP/(TP + FP); NPV = TN/(TN + FN). TP = true positive; TN = true negative; FN = false negative; and FP = false positive. Reproduced from ref. 118 with permission from American Chemical Society, copyright 2021.

4. Conclusions

Here we have summarised the development process of polymer-tethered glyconanoparticles within our laboratory and their emerging role in glycobiology. The synthetic requirements for a useful polymer tether were covered in detail, including the chemistry and molecular weight requirements and the potential benefits of using RAFT-polymerisation chemistry to exploit the inevitable thio end groups for gold particle immobilisation. Glycan capture technologies, which now enable µg quantities of precious glycans to be conjugated to polymers were highlighted, with specific examples of fluorinated glycans used. The use of these in biosensing, from colourimetric aggregation assays to surface-coupled detection is discussed and comparisons drawn to established (printed) array technologies. The potential of these as the mobile phase of a new class of lateral flow glyco diagnostics is shown with specific examples including SARS-COV-2 detection. The challenges of translation are also shown, with particular focus on particle stability in complex media and non-specific binding. This technology is now at a stage where it is simple to use and can probe un-known binding events and is inherently compatible with automation strategies, in cases where sufficient glycan diversity is available.

As an outlook of this technology, the following challenges/opportunities remain to be addressed in the opinion of the authors:

(i) Full automation of the glycan addition, coating and running of assays to unlock high throughput screening, comparable to solid-phase microarrays.

(ii) Deployment of anisotropic particles to allow non-crosslinking targets to be detected and improved detection limits.

(iii) Reducing the non-specific binding which can bring unwanted ‘glyco-coronas’ to the particles.¹⁵¹

(iv) Overcoming the selectivity challenges in using glycans in real world diagnostics, which may include hybrid devices incorporating both antibodies and glycans.

(v) Using the synthetic versatility to introduce dynamic glycan presentation in response to stimuli for controlled and triggerable ‘revealing’ of specific glycans with spatio-temportal control.¹⁵²

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

This is a review-type article and contains no new data.

Acknowledgements

The authors thank the University of Manchester for funding. We also thank our previous and ongoing collaborators in this field and current/former group members for their contributions.

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83. N. S. Ieong, C. I. Biggs, M. Walker and M. I. Gibson, Comparison of RAFT Derived Poly(Vinylpyrolidone) Verses Poly(Oligoethyleneglycol Methacrylate) for the Stabilization of Glycosylated Gold Nanoparticles, J. Polym. Sci., Part A: Polym. Chem., 2017, 55(7), 1200–1208,  DOI:10.1002/pola.28481.
84. N. S. Ieong, K. Brebis, L. E. Daniel, R. K. O’Reilly and M. I. Gibson, The Critical Importance of Size on Thermoresponsive Nanoparticle Transition Temperatures: Gold and Micelle-Based Polymer Nanoparticles, Chem. Commun., 2011, 47(42), 11627–11629,  10.1039/C1CC15171A.
85. P. J. Roth, D. Kessler, R. Zentel and P. Theato, Versatile ω-End Group Functionalization of RAFT Polymers Using Functional Methane Thiosulfonates, J. Polym. Sci., Part A: Polym. Chem., 2009, 47(12), 3118–3130,  DOI:10.1002/pola.23392.
86. S.-J. Richards, A. N. Baker, M. Walker and M. I. Gibson, Polymer-Stabilized Sialylated Nanoparticles: Synthesis, Optimization, and Differential Binding to Influenza Hemagglutinins, Biomacromolecules, 2020, 21(4), 1604–1612,  DOI:10.1021/acs.biomac.0c00179.
87. J. Micallef, A. N. Baker, S.-J. Richards, D. E. Soutar, P. G. Georgiou, M. Walker and M. I. Gibson, Polymer-Tethered Glyconanoparticle Colourimetric Biosensors for Lectin Binding: Structural and Experimental Parameters to Ensure a Robust Output, RSC Adv., 2022, 12(51), 33080–33090,  10.1039/D2RA06265H.
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90. S.-J. Richards, M. W. Jones, M. Hunaban, D. M. Haddleton and M. I. Gibson, Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density, Angew. Chem., Int. Ed., 2012, 51(31), 7812–7816,  DOI:10.1002/anie.201202945.
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92. C. M. Jarvis, D. B. Zwick, J. C. Grim, M. M. Alam, L. R. Prost, J. C. Gardiner, S. Park, L. L. Zimdars, N. M. Sherer and L. L. Kiessling, Antigen Structure Affects Cellular Routing through DC-SIGN, Proc. Natl. Acad. Sci. U. S. A., 2019, 116(30), 14862–14867,  DOI:10.1073/pnas.1820165116.
93. P. G. Georgiou, A. N. Baker, S. J. Richards, A. Laezza, M. Walker and M. I. Gibson, Tuning Aggregative versus Non-Aggregative Lectin Binding with Glycosylated Nanoparticles by the Nature of the Polymer Ligand, J. Mater. Chem. B, 2020, 8(1), 136–145,  10.1039/C9TB02004G.
94. M. Ambrosi, A. S. Batsanov, N. R. Cameron, B. G. Davis, J. A. K. Howard and R. Hunter, Influence of Preparation Procedure on Polymer Composition: Synthesis and Characterisation of Polymethacrylates Bearing β-D-Glucopyranoside and β-D-Galactopyranoside Residues, J. Chem. Soc., Perkin Trans. 1, 2002, 45–52,  10.1039/B108421F.
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96. V. Ladmiral, G. Mantovani, G. J. Clarkson, S. Cauet, J. L. Irwin and D. M. Haddleton, Synthesis of Neoglycopolymers by a Combination of “Click Chemistry” and Living Radical Polymerization, J. Am. Chem. Soc., 2006, 128(14), 4823–4830,  DOI:10.1021/ja058364k.
97. G. Chen, S. Amajjahe and M. H. Stenzel, Synthesis of Thiol-Linked Neoglycopolymers and Thermo-Responsive Glycomicelles as Potential Drug Carrier, Chem. Commun., 2009, 1198–1200,  10.1039/B900215D.
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100. M. I. Gibson, E. Fröhlich and H.-A. Klok, Postpolymerization Modification of Poly(Pentafluorophenyl Methacrylate): Synthesis of a Diverse Water-Soluble Polymer Library, J. Polym. Sci., Part A: Polym. Chem., 2009, 47(17), 4332–4345,  DOI:10.1002/pola.23486.
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102. L. E. Wilkins, N. Badi, F. Du Prez and M. I. Gibson, Double-Modified Glycopolymers from Thiolactones to Modulate Lectin Selectivity and Affinity, ACS Macro Lett., 2018, 7(12), 1498–1502,  DOI:10.1021/acsmacrolett.8b00825.
103. T. Tanaka, H. Nagai, M. Noguchi, A. Kobayashi and S. Shoda, One-Step Conversion of Unprotected Sugars to β-Glycosyl Azides Using 2-Chloroimidazolinium Salt in Aqueous Solution, Chem. Commun., 2009, 3378–3379,  10.1039/B905761G.
104. D. Lim, M. A. Brimble, R. Kowalczyk, A. J. A. Watson and A. J. Fairbanks, Protecting-Group-Free One-Pot Synthesis of Glycoconjugates Directly from Reducing Sugars, Angew. Chem., Int. Ed., 2014, 53(44), 11907–11911,  DOI:10.1002/anie.201406694.
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106. S.-J. Richards, L. Otten and M. I. Gibson, Glycosylated Gold Nanoparticle Libraries for Label-Free Multiplexed Lectin Biosensing, J. Mater. Chem. B, 2016, 4(18), 3046–3053,  10.1039/C5TB01994J.
107. D. N. Crisan, O. Creese, R. Ball, J. L. Brioso, B. Martyn, J. Montenegro and F. Fernandez-Trillo, Poly(Acryloyl Hydrazide), a Versatile Scaffold for the Preparation of Functional Polymers: Synthesis and Post-Polymerisation Modification, Polym. Chem., 2017, 8(31), 4576–4584,  10.1039/C7PY00535K.
108. C. S. Mahon, M. A. Fascione, C. Sakonsinsiri, T. E. McAllister, W. B. Turnbull and D. A. Fulton, Templating Carbohydrate-Functionalised Polymer-Scaffolded Dynamic Combinatorial Libraries with Lectins, Org. Biomol. Chem., 2015, 13(9), 2756–2761,  10.1039/C4OB02587C.
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111. A. Laezza, P. G. Georgiou, S.-J. Richards, A. N. Baker, M. Walker and M. I. Gibson, Protecting Group Free Synthesis of Glyconanoparticles Using Amino–Oxy-Terminated Polymer Ligands, Bioconjugate Chem., 2020, 31(10), 2392–2403,  DOI:10.1021/acs.bioconjchem.0c00465.
112. X. Wang, O. Ramström and M. Yan, A Photochemically Initiated Chemistry for Coupling Underivatized Carbohydrates to Gold Nanoparticles, J. Mater. Chem., 2009, 19(47), 8944–8949,  10.1039/B917900C.
113. X. Wang, O. Ramström and M. Yan, Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and the Impact on Lectin Binding, Anal. Chem., 2010, 82(21), 9082–9089,  DOI:10.1021/ac102114z.
114. H. S. N. Jayawardena, X. Wang and M. Yan, Classification of Lectins by Pattern Recognition Using Glyconanoparticles, Anal. Chem., 2013, 85(21), 10277–10281,  DOI:10.1021/ac402069j.
115. L. Otten, D. Vlachou, S.-J. Richards and M. I. Gibson, Glycan Heterogeneity on Gold Nanoparticles Increases Lectin Discrimination Capacity in Label-Free Multiplexed Bioassays, Analyst, 2016, 141(14), 4305–4312,  10.1039/C6AN00549G.
116. S. Won, S.-J. Richards, M. Walker and M. I. Gibson, Externally Controllable Glycan Presentation on Nanoparticle Surfaces to Modulate Lectin Recognition, Nanoscale Horiz., 2017, 2(2), 106–109,  10.1039/C6NH00202A.
117. A. N. Baker, S.-J. Richards, C. S. Guy, T. R. Congdon, M. Hasan, A. J. Zwetsloot, A. Gallo, J. R. Lewandowski, P. J. Stansfeld, A. Straube, M. Walker, S. Chessa, G. Pergolizzi, S. Dedola, R. A. Field and M. I. Gibson, The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device, ACS Cent. Sci., 2020, 6(11), 2046–2052,  DOI:10.1021/acscentsci.0c00855.
118. A. N. Baker, S.-J. Richards, S. Pandey, C. S. Guy, A. Ahmad, M. Hasan, C. I. Biggs, P. G. Georgiou, A. J. Zwetsloot, A. Straube, S. Dedola, R. A. Field, N. R. Anderson, M. Walker, D. Grammatopoulos and M. I. Gibson, Glycan-Based Flow-Through Device for the Detection of SARS-COV-2, ACS Sens., 2021, 6(10), 3696–3705,  DOI:10.1021/acssensors.1c01470.
119. A. N. Baker, A. R. Muguruza, S.-J. Richards, P. G. Georgiou, S. Goetz, M. Walker, S. Dedola, R. A. Field and M. I. Gibson, Lateral Flow Glyco-Assays for the Rapid and Low-Cost Detection of Lectins–Polymeric Linkers and Particle Engineering Are Essential for Selectivity and Performance, Adv. Healthcare Mater., 2022, 11(4), 2101784,  DOI:10.1002/adhm.202101784.
120. A. Ahmad, P. G. Georgiou, A. Pancaro, M. Hasan, I. Nelissen and M. I. Gibson, Polymer-Tethered Glycosylated Gold Nanoparticles Recruit Sialylated Glycoproteins into Their Protein Corona, Leading to off-Target Lectin Binding, Nanoscale, 2022, 14(36), 13261–13273,  10.1039/D2NR01818G.
121. P. G. Georgiou, C. S. Guy, M. Hasan, A. Ahmad, S.-J. Richards, A. N. Baker, N. V. Thakkar, M. Walker, S. Pandey, N. R. Anderson, D. Grammatopoulos and M. I. Gibson, Plasmonic Detection of SARS-CoV-2 Spike Protein with Polymer-Stabilized Glycosylated Gold Nanorods, ACS Macro Lett., 2022, 11(3), 317–322,  DOI:10.1021/acsmacrolett.1c00716.
122. M. Eberhardt, R. Mruk, R. Zentel and P. Théato, Synthesis of Pentafluorophenyl(Meth)Acrylate Polymers: New Precursor Polymers for the Synthesis of Multifunctional Materials, Eur. Polym. J., 2005, 41(7), 1569–1575,  DOI:10.1016/j.eurpolymj.2005.01.025.
123. M. Eberhardt and P. Théato, RAFT Polymerization of Pentafluorophenyl Methacrylate: Preparation of Reactive Linear Diblock Copolymers, Macromol. Rapid Commun., 2005, 26(18), 1488–1493,  DOI:10.1002/marc.200500390.
124. S.-J. Richards, M. W. Jones, M. Hunaban, D. M. Haddleton and M. I. Gibson, Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density, Angew. Chem., Int. Ed., 2012, 51(31), 7812–7816,  DOI:10.1002/anie.201202945.
125. B. Martyn, C. I. Biggs and M. I. Gibson, Comparison of Systematically Functionalized Heterogeneous and Homogenous Glycopolymers as Toxin Inhibitors, J. Polym. Sci., Part A: Polym. Chem., 2019, 57(1), 40–47,  DOI:10.1002/pola.29279.
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