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. Water is fundamental to all life on our planet, and despite it having a freezing point of 0˚C, Nature has evolved a series of unique adaptations to enable life to flourish in sub-zero climates, at high altitudes and at the Earth's poles. Such extremophiles include the wood frog (Lithobates sylvaticus) which can freeze solid over winter, tardigrades which can be desiccated and rehydrated and cold tolerant plants 1, 2 . The mechanisms of these cryoprotectants are varied, from enabling freeze-tolerance (being able to be frozen and then thawed) to freeze avoidance (preventing ice forming) and even freeze promotion (as a predatory mechanism) 3-6 .One particular adaptation is the production of macromolecular antifreezes (proteins and polysaccharides) which modulate ice formation and growth, and are found in freeze avoidant organisms. These can be broadly split into the antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs). AFGPs are highly conserved, with a relatively simple repeat tripeptide structure and a disaccharide on every third amino acid, but are produced in a range of chain lengths. Conversely, AFPs are far more diverse, with several subdivisions, and can assume different structures; from beta barrels to alpha helices and vary in size (Fig. 1). They all have a few core properties (discussed in detail below) including the ability to inhibit ice recrystallization, shape ice crystals into unusual morphologies and to depress the freezing point in a noncolligative manner. The relative magnitude of each effect varies between individual AF(G)Ps and the exact mechanisms, involving ice-face recognition, are still under investigation. What is clear, is that the ability to tune and modify ice growth and formation has the potential for huge industrial and societal impact. For example, ice adhesion limits the performance of wind farms by up to 50% 7 , is a major problem for aircraft 8 and even impacts our understanding of how biological components affect our climate 9, 10 .Some AFPs have already found application in improving the texture of ice cream products by preventing ice crystal growth, and other food uses are under investigation 4,11 . A key potential field where AF(G)Ps could be applied is in cell and tissue cryopreservation where ice

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.

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

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.The formation and growth of ice crystals presents problems in many fields from aerospace to cell cryopreservation to the automotive industry. Whilst ice formation is thermodynamically preferred below 0 °C, there exists a large barrier to this, such that pure water undergoes homogenous nucleation at ~ − 40 °C. For nucleation to occur above this temperature, a nucleator is necessary. In the environment, nucleators have been identified including dust,1 minerals, birch and conifer pollen2 and recently a species of fungus was found to be a potent nucleator.3 Ice nucleating proteins also exist in living species,4 for example Pseudomonas syringae produce ice nucleating proteins to promote frost formation on plant leaves, to release nutrients for feeding.5 However, the mechanisms of ice nucleation are not understood, and remain a significant challenge for modelling and theory.6,7 A key barrier to this understanding is the lack of sequentially modified materials to enable structure-activity relationships to be drawn. The few known nucleators are insoluble, inorganics such as kaolinite8 or bacterial proteins. Synthetic ice nucleators could play a key role in e.g. cellular cryopreservation, where control over the exact nucleation temperature could improve reproducibility in the cryopreservation process.9 In the past few years, significant progress has been made in the development of synthetic materials for ice recrystallization inhibition (IRI) -the growth of pre-formed ice crystals as mimics of antifreeze proteins (which sometimes, but not always, can influence ice nucleation also Considering the above, we hypothesised that the surface modification of base-washed graphene oxide would provide a versatile template to evaluate the potential of 2-dimensional carbon nanomaterials as ice-nucleating materials and also to provide a versatile scaffold to enable the role of surface chemistry to be probed. Base-washed GO (bwGO) was prepared using established methods16 to generate a 'clean' surface bearing epoxy groups available for orthogonal conjugation to thiols. For the surface modification, a small library of small molecule thiols was chosen, to give a range of hydrophilic/phobic functionalities. Watersoluble polymers with thiol-termini were also synthesised using RAFT (reversible addition fragmentation -transfer) polymerization. RAFT not only enables access to functional polymers of defined chain length and dispersity, but also introduces a thio-carbonyl at the ω-end-group, which can be reduced to a thiol. Using this method, poly(Nisopropylacrylamide), pNIPAM with degree of polymerization of 55 and 140 was synthesised, to use as a water soluble polymeric grafting age...

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.

Antifreeze proteins are site-specifically conjugated onto polymer-stabilised gold nanoparticles, resulting in hybrid materials capable of modulating ice growth processes.

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.

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.