From Basic Principles of Protein-Polysaccharide Association to the Rational Design of Thermally Sensitive Materials

Abstract

Biology resolves design requirements toward functional materials by creating nanostructured composites, where individual components are combined to maximize the macroscale material performance. A major challenge in utilizing such design principles is the trade-off between the preservation of individual component properties and emerging composite functionalities. Here, polysaccharide pectin and silk fibroin were investigated in their composite form with pectin as a thermal-responsive ion conductor and fibroin with exceptional mechanical strength. We show that segregative phase separation occurs upon mixing, and within a limited compositional range, domains ∼50 nm in size are formed and distributed homogeneously so that decent matrix collective properties are established. The composite is characterized by slight conformational changes in the silk domains, sequestering the hydrogen-bonded β-sheets as well as the emergence of randomized pectin orientations. However, most dominant in the composite's properties is the introduction of dense domain interfaces, leading to increased hydration, surface hydrophilicity, and increased strain of the composite material. Using controlled surface charging in X-ray photoelectron spectroscopy, we further demonstrate Ca ions (Ca²⁺) diffusion in the pectin domains, with which the fingerprints of interactions at domain interfaces are revealed. Both the thermal response and the electrical conductance were found to be strongly dependent on the degree of composite hydration. Our results provide a fundamental understanding of the role of interfacial interactions and their potential applications in the design of material properties, polysaccharide-protein composites in particular.

Keywords: biomaterials; pectin; protein nanofibrils; self-assembly; silk protein; thermal induced conductivity.

Figure 1

Preparation of (a) pectin, (b) silk fibroin (fragment, N-terminal domain, PDB ID: 3UA0), and (c) pectin–silk fibroin composite thin films. Left: pictures of the source of (a) the pectin polysaccharide (i.e., fruit) and (b) fibroin protein (i.e., B. mori silkworm cocoons), with the insets showing the chemical structure of the pectin polysaccharide biopolymer (a) and molecular structure of fibroin chain (b). (c) Molecular structure of pectin and silk fibroin biopolymers in their composite form. Middle: graphical demonstration of each film type’s casting procedure. Right: SEM images of the corresponding films and a graphical demonstration of the molecular assembly, with each inset showing an enlarged image of the film morphology. The scale bar for the SEM images is 100 μm, and that for the inset images is 400 nm. Cartoon schematic insets for SEM images (bottom left): (a) pectin chain with Ca ions (Ca²⁺), (b) a fibrillar silk β-sheet-rich structure consisting of silk monomers (image was created with BioRender.com), and (c) a pectin–silk hybrid composite.

Figure 2

(a) Amide I FTIR spectra of silk fibroin (RSF), pectin, and pectin–fibroin composite films (RSF Pectin). (b) Comparative analysis of the secondary structure of (a) with the band positions of the β-sheets at 1610–1635 cm^–1, antiparallel β-sheets at 1690–1705 cm^–1, random coil and α-helixes at 1635–1665 cm^–1, and β-turns at 1665–1690 cm^–1. (c) XRD images of the studied films. Peak positions and corresponding distances are indicated. (d) WAXS of representative films.

Figure 3

(a–c) Contact angle measurements for the (a) pectin film—90.4°, (b) RSF film—94.6°, and (c) pectin–RSF composite film—42.7° (see the original data in Supporting Information, Figure S6), images were created with Autodesk Fusion 360; (d–f) Pictures of the (d) pectin, (e) fibroin (RSF), and (f) pectin–RSF composite films with an SEM image as an inset and an atomic force microscopy image of the respective film below each SEM image. The scale bars are 1 cm in the optical images, 20 μm in the SEM images (insets), and 1 μm in the AFM images.

Figure 4

(a) Schematic representation of the Approach 1 experimental setup for evaluating the electrical properties of the studied films as a function of the humidity changes. The setup consists of electrodes, clamps for film fixation, and a source. (b) Changes in the pectin film’s current response due to humidity changes (from 30 to 70% and then back to 30%) as a function of time at 23 °C. (c) Changes in the composite film’s current response due to humidity changes (humidity values: 30, 60, 30, 65, 30, and 70%) as a function of time at 20 °C. (d) Schematic representation of the Approach 2 experimental setup for evaluating the electrical properties of the studied films as a function of temperature changes. The setup consists of contact double-layered electrodes on the top and bottom, which enable uniform heating of the sample, clamps for the films’ fixation, and a source (see the Experimental Section). (e, f) Changes in the (e) pectin film’s and (f) the composite film’s current in response to the changes in temperature (from 23 to 60 °C and then back to 23 °C) as a function of time at a relative humidity of 30–33%. Images (a) and (d) were created with Autodesk AutoCAD.

Figure 5

(a–c) Time-dependent X-ray photoelectron spectroscopy (XPS) of the polysaccharide pectin and its composite with silk fibroin protein. (a, b) XPS spectra, normalized in order to visually emphasize changes in the line shape of (a) Ca in the pectin-only film and (b) Ca in the pectin–fibroin composite films. (c) Curve fitting for the O 1s XPS line in the pectin-Ca²⁺ film (O 1s, purple) showing a shoulder attributed to the egg-box oxygen atoms (COO-Ca, 531.4 eV, red) and a graphical representation of the electron density distribution around the two interacting groups: an electropositively charged calcium ion and two electronegative carboxylate groups (inset). (d) Quantitative evaluation of the beam-induced changes in the absolute atomic concentration of Ca, as revealed by XPS. t₁, t₂, and t₃ in (a), (b), and (d) are the time points during a long experiment, 24 h in total: 12 (t₁), 60 (t₂), and 120 (t₃) min. All time values refer to the beginning of the sample exposure to the X-ray + eFG irradiation, normalized to 75 W in the X-ray source power and averaged because of delays between C, O, and Ca, as dictated by serial scans. Differences between the pectin and the mixed sample should be noted, resulting from the longer scans of the Ca 2p line, which were essential in the case of a mixed sample. Note also the finite loss of OH groups and the related changes in the O 1s and C 1s spectra, described in Figure S2 in the Supporting Information.

Figure 6

(a) Representative stress–strain curves for pectin, RSF, and the composite. Average and standard deviation values of the (b) elastic modulus, (c) tensile strength, and (d) elongation at break.