Efficient Oxidation and Nano‑Dispersion of Bombyx mori and Antheraea pernyi Silk Fibers for High‑Performance Membranes

Abstract

We report a scalable, environmentally benign method that oxidizes Bombyx mori (BS) and Antheraea pernyi (AS) silk fibers with sodium hypochlorite, then sonicates the oxidized slurry to yield individual silk nanofibers (SNs) of ~105–112 nm diameter and >1 µm length. Casting these SNs produces optically transparent membranes (≥75 % light transmission) that are mechanically robust (≈4.5 GPa Young’s modulus) and exhibit enhanced hydrophilicity. By tuning the pH, the negatively charged SNs aggregate and can be re‑dispersed, allowing concentration up to ~20 wt %—a 100‑fold increase over the initial dispersion—facilitating storage, transport, and downstream engineering applications.

Introduction

Hierarchical materials in nature deliver superior properties by combining polymer chemistry with structural organization. Mimicking this strategy in synthetic materials demands processes that preserve native nanostructures while being cost‑effective and scalable. Traditional methods for isolating cellulose or chitin nanofibers—such as TEMPO‑mediated oxidation—rely on expensive or toxic reagents and typically produce low‑concentration dispersions, limiting practical use. Silk, produced by insects and spiders, also features a multi‑scale fibrous architecture, offering remarkable mechanical and biochemical performance. However, extracting individual silk nanofibers remains challenging due to the high crystallinity and strong inter‑fibril adhesion inherent to silk fibroin.

Our approach adapts the concept of selective oxidation without the need for TEMPO or NaBr catalysts. By introducing carboxyl groups through NaClO oxidation, we achieve electrostatic repulsion sufficient to disperse silk fibers into stable nanofibers, while retaining the β‑sheet crystalline core that confers strength. The resulting SNs demonstrate superior optical, mechanical, and wetting characteristics, and a unique pH‑driven aggregation‑dispersal behavior that enables high‑density packing.

Materials and Methods

Oxidation of Disassembled Silk Fibers

Raw BS and AS fibers (Xiehe Silk Co.) were boiled in 0.02 M Na₂CO₃ (1:400 w/w) for 30 min, washed, and dried. The degummed fibers were then immersed in 88 % formic acid (1:20 w/w) at room temperature for ≥1 h, followed by homogenization at 10 000 rpm for 3 min. Centrifugation at 8 000 rpm yielded solid disassembled fibers.

For oxidation, 1 g of disassembled fibers was added to 100 mL water. NaClO was introduced while NaOH maintained pH 10; the reaction was quenched with 0.5 M HCl to pH 7. The water‑insoluble fraction was collected by centrifugation (10 000 rpm), washed, and sonicated (19.5 kHz, 300 W, 20 min) in an ice bath to prevent overheating, producing the SN dispersion.

X‑ray Diffraction

Diffraction patterns were recorded on a Rigaku Ultima IV with Cu‑Kα (λ = 0.1542 nm). Deconvolution was performed using PeakFit 4.0, with peak positions fixed by second‑derivative analysis.

Morphology

Diluted SN dispersions (0.01 wt %) were deposited on silicon wafers or carbon‑coated Cu grids, dried, and imaged by JEOL JSM‑7600F SEM (5 kV) and FEI Titan 80‑300 TEM (80 kV). Particle dimensions were quantified with ImageJ.

Mechanical Testing

Membranes (~50 µm thick) of BS, AS, cellulose (CN), and chitin (ChN) nanofibers were cast by solvent evaporation. Strips (60–80 mm × 5 mm) were tested on an AG‑Xplus universal tester (1 mm/min, 20 mm initial span).

Optical and Wetting Properties

Transmittance (350–800 nm) was measured with an Ultrospec 2100 pro. Contact angles were determined by a drop meter (Kyowa Interface Science), analyzing 4 µL water droplets within 0.5 s.

Results and Discussion

Oxidation and Isolation of Silk Nanofibers

Our workflow (Figure 1) begins with formic acid pretreatment, yielding microfibers (5–20 µm). Subsequent NaClO oxidation (maintained at pH 10) introduces carboxyl groups by converting serine hydroxymethyl groups. Carboxyl concentration increased linearly with NaClO addition; 10 mM g⁻¹ protein provided ~0.72 mg g⁻¹ (BS) and ~0.84 mg g⁻¹ (AS) while preserving >75 % of the protein mass.

Sonication of the oxidized, water‑insoluble fraction produced SNs with diameters of 105 ± 27 nm (BS) and 112 ± 33 nm (AS) and lengths >1 µm, achieving a ~50 % yield relative to the oxidized input.

Crystallinity

XRD confirmed that β‑sheet crystallinity remained intact post‑oxidation. Deconvolution revealed an increase in crystallinity from 24.8 % to 41.3 % (BS) and 22.9 % to 39.2 % (AS) upon 10 mM g⁻¹ NaClO treatment, reflecting selective removal of amorphous regions without compromising the crystalline core.

Membrane Performance

SN membranes were optically clear (>75 % transmittance), mechanically strong (Young’s modulus 4.51 ± 0.71 GPa for BS, 4.43 ± 0.23 GPa for AS), and more hydrophilic than regenerated silk (contact angles 58.8° BS, 55.7° AS, 40.3° CN, 52.5° ChN).

pH‑Responsive Aggregation‑Dispersion

SNs carry a negative zeta potential (~−40 mV) at neutral pH. Lowering the pH shields carboxyl charges, causing aggregation; raising pH >7 re‑disperses the fibers. Aggregates can be recovered by centrifugation and re‑dispersed, achieving a ~100‑fold concentration (≈20 wt %) from the initial ~0.2 wt % dispersion, underscoring their suitability for drug delivery and convenient handling.

Conclusions

We demonstrate a simple, scalable route to individual BS and AS silk nanofibers via NaClO oxidation without toxic catalysts. The ~110 nm SNs possess high aspect ratio, maintain crystalline integrity, and form transparent, robust membranes. Their pH‑controlled aggregation‑dispersal and high‑density packing make them attractive for advanced material and biomedical applications.