Ice Nucleation Promotion Impact on the Ice Recrystallization Inhibition Activity of Polyols.

Heterogeneous ice nucleation occurs vis-à-vis nucleating agents already present in solution yet can occur within a rather broad range of temperatures (0 to ca. -38 °C). Controlling this temperature and the subsequent growth of resulting ice crystals is crucial for the survival of biological organisms (certain insects, fish, and plants that endure subzero temperatures), as well as in the context of medical cryopreservation and food science. In these environments, uncontrolled crystal shape and size can rupture the cell membrane causing irreversible and catastrophic damage. Antifreeze (AF) proteins and synthetic AF analogs address this issue to restrict crystal growth and to shape ice crystals. Yet, if the nucleation temperature is not controlled and occurs in a lower temperature range, nascent ice crystals will have grown to a significantly larger size before the AF agents can be active on their surface to halt or slow the Ostwald ripening process during recrystallization. At a higher nucleation temperature, diffusion of AF macromolecules is enhanced, and dynamic crystal shaping can start earlier, producing smaller crystals overall. While antifreeze proteins, the inspiration for these synthetic analogs, are always applied in a salt buffer aqueous environment (most typically phosphate-buffered saline (PBS) buffer), the heterogeneous nucleation events are stochastic and occur within a wide temperature range. Silver iodide (AgI), however, is a highly effective ice nucleation promoter as its crystal lattice structure is a 98% lattice match to the basal plane of hexagonal ice (Ih) crystals acting as a template for water molecule orientation and decreasing the interfacial free energy. Here, we expose the advantage of purposely seeding such nascent ice crystals with AgI at a defined and higher temperature (-7 °C) in ultrapure water (UPW) such that nucleation can only come from AgI (and also in AgI/PBS), resulting in the most potent synthetic IRI observed to date (at concentrations as low as 0.001 mg·mL-1).

An insect antifreeze protein from Anatolica polita enhances the cryoprotection of Xenopus laevis eggs and embryos.

Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. Xenopus laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze-thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.

Insights into Design of Biomimetic Glycerol-Grafted Polyol-Based Polymers for Ice Nucleation/Recrystallization Inhibition and Thermal Hysteresis Activity.

Many species living in colder regions of the world have adapted to the extreme climate by producing antifreeze (glycol) proteins (AF(G)P) which exhibit ice recrystallization inhibition (IRI), thermal hysteresis activity (THA), as well as other interactions with the freezing process of water. Although several synthetic approaches for the exploitation of these proteins have been investigated, challenges remain in the synthetic design of biomimetic polymers. Similar to biological antifreezes, poly(vinyl alcohol) (PVA) has potent IRI activity; however, by comparison, PVA has very little THA. In this study, we explored structural variations to polyol-based polymers to contrast with PVA as a control and identified several key structural elements for performance in IRI, THA, as well as in ice nucleation inhibition (INI). These structural features are bioinspired by the typical ice-binding plane of AFPs yet are surprisingly simple to produce with potency approaching that of typical AFPs. Key to the performance is positioning small organic functionalities with known antifreeze properties (such as ethylene glycol) pendent to a host polymer chain with consideration of their conformational freedom. To build systematic variations into both the backbone and side-chain structures, we used poly(vinyl alcohol), poly(isopropenyl acetate), poly(acrylic acid), and poly(methacrylic acid) parent polymers for such pendent modifications. One structure in particular, glycerol-grafted-PVA (G-g-PVA), shows potency rivaling that of AFPs at similar micromolar concentration. The findings in this study help guide the rational design of synthetic antifreeze polymers useful for applications such as anti-icing coatings through to cryopreservation methods for organ transport and cell preservation.

An integrated microfluidic platform for selective and real-time detection of thrombin biomarkers using a graphene FET.

Lab-on-a-chip technology offers an ideal platform for low-cost, reliable, and easy-to-use diagnostics of key biomarkers needed for early screening of diseases and other health concerns. In this work, a graphene field-effect transistor (GFET) functionalized with target-binding aptamers is used as a biosensor for the detection of thrombin protein biomarker. Furthermore, this GFET is integrated with a microfluidic device for enhanced sensing performances in terms of detection limit, sensitivity, and continuous monitoring. Under this platform, a picomolar limit of detection was achieved for measuring thrombin; in our experiment measured as low as 2.6 pM. FTIR, Raman and UV-Vis spectroscopy measurements were performed to confirm the device functionalization steps. Based on the concentration-dependent calibration curve, a dissociation constant of KD = 375.8 pM was obtained. Continuous real-time measurements were also conducted under a constant gate voltage (VGS) to observe the transient response of the sensor when analyte was introduced to the device. The target selectivity of the sensor platform was evaluated and confirmed by challenging the GFET biosensor with various concentrations of lysozyme protein. The results suggest that this device technology has the potential to be used as a general diagnostic platform for measuring clinically relevant biomarkers for point-of-care applications.

Inducing an LCST in hydrophilic polysaccharides via engineered macromolecular hydrophobicity.

Thermoresponsive polysaccharide-based materials with tunable transition temperatures regulating phase-separated microdomains offer substantial opportunities in tissue engineering and biomedical applications. To develop novel synthetic thermoresponsive polysaccharides, we employed versatile chemical routes to attach hydrophobic adducts to the backbone of hydrophilic dextran and gradually increased the hydrophobicity of the dextran chains to engineer phase separation. Conjugating methacrylate moieties to the dextran backbone yielded a continuous increase in macromolecular hydrophobicity that induced a reversible phase transition whose lower critical solution temperature can be modulated via variations in polysaccharide concentration, molecular weight, degree of methacrylation, ionic strength, surfactant, urea and Hofmeister salts. The phase separation is driven by increased hydrophobic interactions of methacrylate residues, where the addition of surfactant and urea disassociates hydrophobic interactions and eliminates phase transition. Morphological characterization of phase-separated dextran solutions via scanning electron and flow imaging microscopy revealed the formation of microdomains upon phase transition. These novel thermoresponsive dextrans exhibited promising cytocompatibility in cell culture where the phase transition exerted negligible effects on the attachment, spreading and proliferation of human dermal fibroblasts. Leveraging the conjugated methacrylate groups, we employed photo-initiated radical polymerization to generate phase-separated hydrogels with distinct microdomains. Our bottom-up approach to engineering macromolecular hydrophobicity of conventional hydrophilic, non-phase separating dextrans to induce robust phase transition and generate thermoresponsive phase-separated biomaterials will find applications in mechanobiology, tissue repair and regenerative medicine.

A surprisingly gentle approach to cavity containing spherocylindrical microparticles from ordinary polymer dispersions in flow.

Herein, we demonstrate a facile approach to fully transform spherical polymeric microparticles to elongated spherocylinders containing an internal cavity under ambient and mild stirring conditions. Critical to the process is to deform the amorphous and non-crosslinked particles under glassy conditions for an unusually long time; 120 hours for the poly(styrene-co-glycidyl methacrylate) microparticles discussed in greatest detail. Larger particles in the 5 micron and greater range were markedly more susceptible to the shear imposed by stirring the aqueous dispersion. The resulting morphology is robust and kinetically frozen yet reverts to the original spherical shape if annealed above the glass transition temperature with suitable temperature or plasticizer. The volume fraction of the internal void can be modulated by particle composition and process conditions and is irregular in shape we believe as a result of a cavitation event during plastic deformation.

Crosslinking in Semi-Batch Seeded Emulsion Polymerization: Effect of Linear and Non-Linear Monomer Feeding Rate Profiles on Gel Formation.

Waterborne latex is often called a product-of-process. Here, the effect of semi-batch monomer feed rate on the kinetics and gel formation in seeded emulsion polymerization was investigated for the copolymerization of n-butyl methacrylate (n-BMA) and ethylene glycol dimethacrylate (EGDMA). Strikingly, the gel fraction was observed to be significantly influenced by monomer feed rate, even while most of the experiments were performed under so-called starve-fed conditions. More flooded conditions from faster monomer feed rates, including seeded batch reactions, counterintuitively resulted in significantly higher gel fraction. Chain transfer to polymer was intentionally suppressed here via monomer selection so as to focus mechanistic insights to relate only to the influence of a divinyl monomer, as opposed to being clouded by contributions to topology from long chain branching. Simulations revealed that the dominant influence on this phenomenon was the sensitivity of primary intramolecular cyclization to the instantaneous unreacted monomer concentration, which is directly impacted by monomer feed rate. The rate constant for cyclization for these conditions was determined to be first order and 4000 s-1, approximately 4 times that typically observed for backbiting in acrylates. This concept has been explored previously for bulk and solution polymerizations, but not for emulsified reaction environments and especially for the very low mole fraction divinyl monomer. In addition, while gel fraction could be dramatically manipulated by variations in linear monomer feed rates, it could be markedly enhanced by leveraging non-linear feed profiles built from combination sequences of flooded and starved conditions. For a 2 h total feed time, a fully linear profile resulted in 30% gel while a corresponding non-linear profile with an early fast-feed segment resulted in 80% gel.

Hydroplasticization of polymers: model predictions and application to emulsion polymers.

The plasticization of a polymer by solvent has a dramatic impact on both its thermal and mechanical behavior. With increasing demand for zero volatile organic compound materials and coatings, water is often the sole solvent used both in the polymer synthesis and in formulation and application; latex colloids derived from emulsion polymerization are a good example. The impact of water on the glass transition temperature of a polymer thus becomes a critical physical property to predict. It has been shown here that in order to do so, one simply needs the dry state glass transition temperature (T(g)) of the (co)polymer, the T(g) of water, and the saturated weight fraction of water for the sample in question. Facile calculation of the later can be achieved using water sorption data and the group additivity method. With these readily available data, we show that a form of the Flory-Fox equation can be used to predict the hydroplasticized state of copolymers in exceptional agreement with direct experimental measurement. Furthermore, extending the prediction to include the impact of the degree of ionization for pH responsive components, only with extra knowledge of the pK(a), was also validated by experiment.

Selective Detection of Lysozyme Biomarker Utilizing Large Area Chemical Vapor Deposition-Grown Graphene-Based Field-Effect Transistor.

Selective and rapid detection of biomarkers is of utmost importance in modern day health care for early stage diagnosis to prevent fatal diseases and infections. Among several protein biomarkers, the role of lysozyme has been found to be especially important in human immune system to prevent several bacterial infections and other chronic disease such as bronchopulmonary dysplasia. Thus, real-time monitoring of lysozyme concentration in a human body can pave a facile route for early warning for potential bacterial infections. Here, we present for the first time a label-free lysozyme protein sensor that is rapid and selective based on a graphene field-effect transistor (GFET) functionalized with selectively designed single-stranded probe DNA (pDNA) with high binding affinity toward lysozyme molecules. When the target lysozyme molecules bind to the surface-immobilized pDNAs, the resulting shift of the charge neutrality points of the GFET device, also known as the Dirac voltage, varied systematically with the concentration of target lysozyme molecules. The experimental results show that the GFET-based biosensor is capable of detecting lysozyme molecules in the concentration range from 10 nM to 1 µM.

Dynamic Aggregation of Poly-N-Isopropylacrylamide Characterized Using Second-Order Scattering.

A second-order scattering (SOS) method is presented for the characterization of aqueous particle suspensions undergoing aggregation. Scattering intensities are measured at 90° by a standard fluorimeter and referenced against dynamic light scattering (DLS) measurements to determine particle size increase in a metal-promoted aggregation process for 0.05 mg/mL aqueous poly-N-isopropylacrylamide (PNIPAm), MW ∼10 k g/mol. Particle size increases monotonically from 30 nm to 210 nm at temperature 308 K. A further validation of the SOS method was performed using monodisperse polystyrene reference particles sized at 52 nm, 101 nm, 151 nm, and 206 nm, which demonstrated the technique's accuracy to within 6% and its versatility with respect to sample composition. The technique is ideal for monitoring colloidal stability and macromolecular assembly and it can be performed at lower concentrations than are typically used in DLS.