Synthetically Scalable Poly(ampholyte) Which Dramatically Enhances Cellular Cryopreservation.

The storage and transport of frozen cells underpin the emerging/existing cell-based therapies and are used in every biomedical research lab globally. The current gold-standard cryoprotectant dimethyl sulfoxide (DMSO) does not give quantitative cell recovery in suspension or in two-dimensional (2D) or three-dimensional (3D) cell models, and the solvent and cell debris must be removed prior to application/transfusion. There is a real need to improve this 50-year-old method to underpin emerging regenerative and cell-based therapies. Here, we introduce a potent and synthetically scalable polymeric cryopreservation enhancer which is easily obtained in a single step from a low cost and biocompatible precursor, poly(methyl vinyl ether-alt-maleic anhydride). This poly(ampholyte) enables post-thaw recoveries of up to 88% for a 2D cell monolayer model compared to just 24% using conventional DMSO cryopreservation. The poly(ampholyte) also enables reduction of [DMSO] from 10 wt % to just 2.5 wt % in suspension cryopreservation, which can reduce the negative side effects and speed up post-thaw processing. After thawing, the cells have reduced membrane damage and faster growth rates compared to those without the polymer. The polymer appears to function by a unique extracellular mechanism by stabilization of the cell membrane, rather than by modulation of ice formation and growth. This new macromolecular cryoprotectant will find applications across basic and translational biomedical science and may improve the cold chain for cell-based therapies.

Glycosylated gold nanoparticle libraries for label-free multiplexed lectin biosensing.

Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature. However, their measurement is complicated or limited by the analytical tools available. Most technologies require fluorescently labelled proteins (e.g. microarrays) or expensive infrastructure (such as surface plasmon resonance). This also limits their application in biosensing, especially for low-resource settings, where detection of pathogens based on glycan binding could speed up diagnosis. Here we employ a library-oriented approach to immobilise a range of monosaccharides onto polymer-stabilised gold nanoparticles to enable rapid and high-throughput evaluation of their binding specificities with a panel of lectins. The red to blue colour shift upon gold nanoparticle aggregation is used as the output, removing the need for labelled protein, enabling compatibility with 96-well microplates. Furthermore, we demonstrate the use of a flatbed scanner (or digital camera) to extract biophysical data, ensuring that only minimal resources are required. Finally, linear discriminant analysis is employed to demonstrate how the glyconanoparticles can be applied as a multiplexed biosensor capable of identifying pathogenic lectins without the need for any infrastructure and overcoming some of the issues of lectin promiscuity.

Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays.

The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents. Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature and are often the first site of adhesion/recognition during infection making them appealing targets for biosensors. Glycosylated gold nanoparticles have been developed that change colour from red to blue upon interaction with carbohydrate-binding proteins and may find use as biosensors, but are limited by the inherent promiscuity of some of these interactions. Here we mimic the natural heterogeneity of cell-surface glycans by displaying mixed monolayers of glycans on the surface of gold nanoparticles. These are then used in a multiplexed, label-free bioassay to create 'barcodes' which describe the lectin based on its binding profile. The increased information content encoded by using complex mixtures of a few sugars, rather than increased numbers of different sugars makes this approach both scalable and accessible. These nanoparticles show increased lectin identification power at a range of lectin concentrations, relative to single-channel sensors. It was also found that some information about the concentration of the lectins can be extracted, all from just a simple colour change, taking this technology closer to being a realistic biosensor.

Discrimination between bacterial species by ratiometric analysis of their carbohydrate binding profile.

Antibiotic resistance is a global health concern meaning there is an urgent need for new treatments and diagnostics. Here glycosylated surfaces are used to profile the binding patterns of a range of Gram-negative, Gram-positive and mycobacteria. This enables the creation of 'barcodes' to enable identification and discrimination between the strains, which could not be achieved by single-point glycan binding and offers a new concept in bacteria detection.

Gold nanoparticle-linked analysis of carbohydrate-protein interactions, and polymeric inhibitors, using unlabelled proteins; easy measurements using a 'simple' digital camera.

Traditional methods of measuring the affinity of lectins (or other carbohydrate-binding proteins) to their target carbohydrate ligand rely on the use of chemically/recombinantly modified proteins in sorbent assays, microarrays or the use of expensive label-free methods such as surface plasmon resonance spectrometry. In this work we exploit the extremely high extinction coefficient (i.e. colour) of gold nanoparticles as resolving agents in sorbent assays. The anionic nanoparticles adhere strongly to immobilized proteins, but not to the carbohydrate-surfaces allowing investigation of protein binding and screening of novel multivalent inhibitors. Furthermore, the use of a simple digital camera (or mobile phone) to obtain the data is shown, providing a simple ultra-low cost route to the detection of unmodified, carbohydrate-binding proteins.