Immunosensors for C‑Reactive Protein Based on Ultrathin Films of Carboxylated Cellulose Nanofibrils

C-reactive protein (CRP) is an acute phase protein that has been widely used as a predictor of cardiovascular diseases. We report herein the synthesis of immunosensors based on carboxylated cellulose nanofibrils (CNF) for CRP detection, as demonstrated by quartz crystal microgravimetry (QCM). QCM sensors carrying ultrathin films of carboxylated CNF were prepared by using two protocols: (i) spin coating of CNF on the sensors followed by carboxylation via in situ oxidation with 2,2,6,6-tetramethylpiperidine 1-oxyl and (ii) carboxymethylation of CNF in aqueous dispersion followed by spin coating deposition on the sensors. Protein A was conjugated to the carboxylated CNF via <i>N</i>-(3-(Dimethylamino)­propyl)-<i>N</i>′-ethylcarbodiimide hydrochloride/<i>N</i>-hydroxysuccinimide and used as a ligand for oriented immobilization of anti C-reactive protein (anti-CRP). The different carboxyl group density of the two oxidized CNF surfaces influenced Protein A binding and, subsequently, the available immobilized anti-CRP molecules. The detection efficiency for CRP, specificity, and concentration range displayed by the carboxylated CNF-based immunosensors coupled with oriented and unoriented anti-CRP were determined and compared

Thermomechanical Properties of Lignin-Based Electrospun Nanofibers and Films Reinforced with Cellulose Nanocrystals: A Dynamic Mechanical and Nanoindentation Study

We produced defect-free electrospun fibers from aqueous dispersions of lignin, poly­(vinyl alcohol) (PVA), and cellulose nanocrystals (CNCs), which were used as reinforcing nanoparticles. The thermomechanical performance of the lignin-based electrospun fibers and the spin-coated thin films was improved when they were embedded with CNCs. Isochronal dynamic mechanical analysis (DMA) was used to assess the viscoelastic properties of the lignin:PVA electrospun fiber mats loaded with CNCs. DMA revealed that α relaxation processes became less prominent with an increased lignin content, an effect that correlated with the loss tangent (tan δ = E″/E′) and α peak (Tg) that shifted to higher temperatures. This can be ascribed to the restraint of the segmental motion of PVA in the amorphous regions caused by strong intermolecular interactions. The reinforcing effect and high humidity stability attained by addition of CNCs (5, 10, or 15 wt %) in the multicomponent fiber mats were revealed. Nanoindentation was performed to assess the elastic modulus and hardness of as-prepared and cross-section surfaces of spin-coated lignin:PVA (75:25) films loaded with CNC. The properties of the two surfaces differed, and only the trend in cross-section elastic modulus correlated with DMA results. After addition of 5 wt % CNCs, both the DMA and nanoindentation elastic modulus remained constant, while after addition of 15 wt % CNCs, both increased substantially. An indentation size effect was observed in the nanoindentation hardness, and the results provided insight into the effect of addition of CNCs on the microphysical processes controlling the yield behavior in the composites

Revisiting Cation Complexation and Hydrogen Bonding of Single-Chain Polyguluronate Alginate

Modifying the properties of bio-based materials has garnered increasing interest in recent years. In related applications, the ability of alginates to complex with metal ions has been shown to be effective in liquid-to-gel transitions, useful in the development of foodstuff and pharma products as well as biomaterials, among others. However, despite its ubiquitous use, alginate behavior as far as interactions with cations is not fully understood. Hence, this study presents a detailed comparison of alginate’s complexation with Na+ and Ca2+ and the involved intramolecular hydrogen bonding and biomolecular chain geometry. Using all-atom molecular dynamics simulations, we find that in contrast to accepted models, calcium cations strongly bind to alginate chains by disruption of hydrogen bonds between neighboring residues, stabilizing a left-hand, 3-fold helical chain structure that enhances chain stiffness. Hence, while present, the traditionally accepted egg-box binding mode was a minor subset of possible conformations. For a single chain, most of the cation binding occurred as single-cation interaction with a carboxyl group, without the coordination of other alginate oxygens. The monovalent Na+ ions were found to be mostly nonlocalized around alginate and therefore do not compete with intramolecular hydrogen bonding. The different binding modes observed for Na+ and Ca2+ contribute toward explaining the different solubility of sodium and calcium alginate

Lignin Particles for Multifunctional Membranes, Antioxidative Microfiltration, Patterning, and 3D Structuring

We introduce a new type of particle-based membrane based on the combination of lignin particles (LPs) and cellulose nanofibrils (CNF), the latter of which are introduced in small volume fractions to act as networking and adhesive agents. The synergies that are inherent to lignin and cellulose in plants are re-engineered to render materials with low surface energy (contact angle measurements) and can be rendered water-resistant with the aid of wet-strength agents (WSAs). Importantly, they are most suitable for antioxidative separation (ABTS•+ radical inhibition): membranes with uniform porous structures (air permeability and capillary flow porosimetry) allow effluent oxidation at 95 mL/cm2, demonstrating, for the first time, the use of unmodified lignin particles in flexible membranes for active microfiltration. Moreover, the membranes are found to be nonfouling (protein adhesion and activity rate). The inherent properties of lignin, including UV radiation blocking capacity (UV transmittance analysis) and reduced surface energy, are further exploited in the development of tailorable and self-standing architectures that are almost entirely comprised of nonbonding LP (solids content as high as 92 w/w%). Despite such composition, the materials develop high toughness (oscillatory dynamic mechanical analysis), owing to the addition of minor amounts of CNF. Multifunctional materials based on thin films (casting), 3D structures (molding), and patterned geometries (extrusion deposition) are developed as a demonstration of the potential use of lignin particles as precursors of new material generation. Remarkably, our observations hold for spherical LPs since a much poorer performance was observed after using amorphous powder, indicating the role of size and shape in related applications

Competing Effects of Hydration and Cation Complexation in Single-Chain Alginate

Alginic acid, a naturally occurring anionic polyelectrolyte, forms strong physically cross-linked hydrogels in the presence of metal cations. The latter engage in electrostatic interactions that compete with intra- and intermolecular hydrogen bonds, determining the gel structure and properties of the system in aqueous media. In this study, we use all-atom molecular dynamics simulations to systematically analyze the interactions between alginic acid chains and Na+ and Ca2+ counterions. The formed alginates originate from the competition of intramolecular hydrogen bonding and water coordination around the polyelectrolyte. In contrast to the established interpretation, we show that calcium cations strongly bind to alginate by disrupting hydrogen bonds within (1 → 4)-linked β-d-mannuronate (M) residues. On the other hand, Na+ cations enhance intramolecular hydrogen bonds that stabilize a left-hand, fourfold helical chain structure in poly-M alginate, resulting in stiffer chains. Hence, the traditionally accepted flexible flat-chain model for poly-M sequence is not valid in the presence of Na+. The two cations have a distinct effect on water coordination around alginate and therefore on its solubility. While Ca+ disrupts water coordination directly around the alginate chains, mobile Na+ cations significantly disrupt the second hydration layer

Formation and Antifouling Properties of Amphiphilic Coatings on Polypropylene Fibers

We describe the formation of amphiphilic polymeric assemblies via a three-step functionalization process applied to polypropylene (PP) nonwovens and to reference hydrophobic self-assembled <i>n</i>-octadecyltrichlorosilane (ODTS) monolayer surfaces. In the first step, denatured proteins (lysozyme or fibrinogen) are adsorbed onto the hydrophobic PP or the ODTS surfaces, followed by cross-linking with glutaraldehyde in the presence of sodium borohydride (NaBH₄). The hydroxyl and amine functional groups of the proteins permit the attachment of initiator molecules, from which poly (2-hydroxyethyl methacrylate) (PHEMA) polymer grafts are grown directly through “grafting from” atom transfer radical polymerization. The terminal hydroxyls of HEMA’s pendent groups are modified with fluorinating moieties of different chain lengths, resulting in amphiphilic brushes. A palette of analytical tools, including ellipsometry, contact angle goniometry, Fourier transform infrared spectroscopy in the attenuated total reflection mode, and X-ray photoelectron spectroscopy is employed to determine the changes in physicochemical properties of the functionalized surfaces after each modification step. Antifouling properties of the resultant amphiphilic coatings on PP are analyzed by following the adsorption of fluorescein isothiocyanate-labeled bovine serum albumin as a model fouling protein. Our results suggest that amphiphilic coatings suppress significantly adsorption of proteins as compared with PP fibers or PP surfaces coated with PHEMA brushes. The type of fluorinated chain grafted to PHEMA allows modulation of the surface composition of the topmost layer of the amphiphilic coating and its antifouling capability

Lignin Particles for Multifunctional Membranes, Antioxidative Microfiltration, Patterning, and 3D Structuring

We introduce a new type of particle-based membrane based on the combination of lignin particles (LPs) and cellulose nanofibrils (CNF), the latter of which are introduced in small volume fractions to act as networking and adhesive agents. The synergies that are inherent to lignin and cellulose in plants are re-engineered to render materials with low surface energy (contact angle measurements) and can be rendered water-resistant with the aid of wet-strength agents (WSAs). Importantly, they are most suitable for antioxidative separation (ABTS•+ radical inhibition): membranes with uniform porous structures (air permeability and capillary flow porosimetry) allow effluent oxidation at 95 mL/cm2, demonstrating, for the first time, the use of unmodified lignin particles in flexible membranes for active microfiltration. Moreover, the membranes are found to be nonfouling (protein adhesion and activity rate). The inherent properties of lignin, including UV radiation blocking capacity (UV transmittance analysis) and reduced surface energy, are further exploited in the development of tailorable and self-standing architectures that are almost entirely comprised of nonbonding LP (solids content as high as 92 w/w%). Despite such composition, the materials develop high toughness (oscillatory dynamic mechanical analysis), owing to the addition of minor amounts of CNF. Multifunctional materials based on thin films (casting), 3D structures (molding), and patterned geometries (extrusion deposition) are developed as a demonstration of the potential use of lignin particles as precursors of new material generation. Remarkably, our observations hold for spherical LPs since a much poorer performance was observed after using amorphous powder, indicating the role of size and shape in related applications

Formation and Antifouling Properties of Amphiphilic Coatings on Polypropylene Fibers

We describe the formation of amphiphilic polymeric assemblies via a three-step functionalization process applied to polypropylene (PP) nonwovens and to reference hydrophobic self-assembled <i>n</i>-octadecyltrichlorosilane (ODTS) monolayer surfaces. In the first step, denatured proteins (lysozyme or fibrinogen) are adsorbed onto the hydrophobic PP or the ODTS surfaces, followed by cross-linking with glutaraldehyde in the presence of sodium borohydride (NaBH₄). The hydroxyl and amine functional groups of the proteins permit the attachment of initiator molecules, from which poly (2-hydroxyethyl methacrylate) (PHEMA) polymer grafts are grown directly through “grafting from” atom transfer radical polymerization. The terminal hydroxyls of HEMA’s pendent groups are modified with fluorinating moieties of different chain lengths, resulting in amphiphilic brushes. A palette of analytical tools, including ellipsometry, contact angle goniometry, Fourier transform infrared spectroscopy in the attenuated total reflection mode, and X-ray photoelectron spectroscopy is employed to determine the changes in physicochemical properties of the functionalized surfaces after each modification step. Antifouling properties of the resultant amphiphilic coatings on PP are analyzed by following the adsorption of fluorescein isothiocyanate-labeled bovine serum albumin as a model fouling protein. Our results suggest that amphiphilic coatings suppress significantly adsorption of proteins as compared with PP fibers or PP surfaces coated with PHEMA brushes. The type of fluorinated chain grafted to PHEMA allows modulation of the surface composition of the topmost layer of the amphiphilic coating and its antifouling capability

Bioactive Cellulose Nanofibrils for Specific Human IgG Binding

Bioactive films were produced by conjugation of a short peptide onto modified cellulose nanofibrils (CNF). Specifically, a hydrophilic copolymer, poly­(2-aminoethyl methacrylate hydrochloride-<i>co</i>-2-hydroxyethylmethacrylate) (poly­(AMA-<i>co</i>-HEMA)), was grafted via surface initiated polymerization from an initiator coupled to CNF. The poly­(AMA-<i>co</i>-HEMA) was used as a spacer and support layer for immobilization of the peptide, acetylated-HWRGWVA, which has specific affinity with human immunoglobulin G (hIgG). Two methods for peptide grafting were compared: modification of CNF in aqueous suspension followed by assembly into a bioactive film and peptide grafting on a preformed CNF film. The CNF-based networks were examined on solid supports via atomic force microscopy (AFM) and extreme resolution imaging with ultralow electron landing energies (scanning low energy electron microscopy). The specific binding capability of hIgG and nonspecific protein resistance of the resultant peptide-modified CNF were evaluated by using quartz crystal microgravimetry (QCM). The effects of initiator concentration and thickness of poly­(AMA-<i>co</i>-HEMA) layer on hIgG adsorption were investigated in the developed systems, which exhibited high signal-to-noise response

Accounting for Substrate Interactions in the Measurement of the Dimensions of Cellulose Nanofibrils

Mechanically fibrillated cellulose nanofibrils (CNFs) have attracted special attention as building blocks for the development of advanced materials and composites. A correlation exists between CNF morphology and the properties of the materials they form. However, this correlation is often evaluated indirectly by process-centered approaches or by accessing a single dimensionality of CNFs adsorbed on solid supports. High-resolution imaging is currently the best approach to describe the morphological features of nanocelluloses; nevertheless, adsorption effects need to be accounted for. For instance, possible deformations of the CNFs arising from capillary forces and interactions with the substrate need to be considered in the determination of their cross-sectional dimensions. By considering soft matter imaging and adsorption effects, we provide evidence of the deformation of CNFs upon casting and drying. We determine a substantial flattening associated with the affinity of CNFs with the substrate corresponding to a highly anisotropic cross-sectional geometry (ellipsoidal) in the dried state. Negative-contrast scanning electron microscopy is also introduced as a new method to assess the dimensions of the CNFs. The images obtained by the latter, a faster imaging method, were correlated with those from atomic force microscopy. The cross-sectional area of the CNF is reconstructed by cross-correlating the widths and heights obtained by the two techniques