Dual enzyme-powered chemotactic cross-β amyloid based functional nanomotors

The study addresses how to design soft, biocompatible nanomotors that can autonomously move and exhibit chemotaxis by leveraging orthogonal enzymatic processes, mimicking biological division of labor where energy consumption drives motility and sensing drives directionality. Biological machines such as enzymes can enhance diffusion and show chemotaxis toward substrate gradients. However, peptide-based chassis often lack robustness and persistence length needed for motility at low Reynolds number. The authors propose using cross β amyloid peptide assemblies derived from Aβ (1–42) fragments to form robust nanotubular morphologies capable of multi-enzyme loading. They hypothesize that integrating two enzymes with orthogonal substrates—urease for propulsion via self-diffusiophoresis and cytochrome C (CytC) for navigation toward pyrogallol—will realize chemotactic nanomotors and enable functional applications such as catalysis in biphasic media.

Prior work shows enzymes can act as nanomotors with enhanced diffusion and chemotaxis toward substrates, and synthetic systems have emulated chemotaxis using Janus particles, vesicles, and polymeric micromotors. Peptide and nucleic-acid supramolecular frameworks offer biocompatible chassis but suffer from structural plasticity impacting motility. Cross β amyloid nanotubes from Aβ fragments exhibit robustness, long persistence lengths, and high binding capacities for multi-enzyme loading. Urease-powered propulsion via ionic self-diffusiophoresis is established, and heme proteins like CytC can show enhanced diffusion in peroxide-containing media. The literature highlights the need to integrate separate processes for motility and directionality, akin to cell migration, which this work aims to achieve with dual enzymes on amyloid nanotubes.

• Nanotube chassis: Short peptide Ac-KLVFFAL (Ac-KL), from Aβ(1–42) nucleating core, self-assembled into nanotubes (mature: diameter 32 ± 2 nm, height 10 ± 1 nm, length 5–20 µm). CD/FTIR confirmed β-sheet structure. To reduce viscous drag, nanotubes were shortened by probe sonication (~30 min), yielding lengths 180–560 nm while retaining morphology (TEM/SEM/AFM). Surface characterization with negatively charged AuNPs indicated exposed lysines; hydrophobic dye binding (Nile red) indicated amphiphilic surfaces.
• Enzyme loading: Urease (pI 5.1, negatively charged) bound to cationic nanotubes. Loadings: 10.82 ± 2.5 µg nmol−1 (short) and 11.01 ± 1.7 µg nmol−1 (long). Localization confirmed with RITC-tagged urease (CLSM) and TEM. For dual-enzyme motors, CytC was co-loaded (FITC-labeled for imaging). AFM/TEM suggested patchy, inhomogeneous enzyme distribution on nanotubes.
• Motility assays: Time-lapse optical microscopy tracked urease-loaded nanotubes at varying urea (0–100 mM). Trajectories and MSDs were computed (n=20) to extract velocities. Long nanotubes (unsheared) served as high-drag controls. Effects of sonication time and enzyme loading on motility were also assessed.
• Simulation (particle-based model): Each amylobot modeled as a cylindrical particle undergoing overdamped Brownian dynamics, experiencing repulsive interactions, random forces, and a self-propulsion force from urease catalysis. Motility force parameter f_u varied from 0 to 24 × 10−6 Pa µm² (expressed in units of f_mot = 1 × 10−6 Pa µm²). 2D trajectories and MSDs were generated to compare with experiments. For chemotaxis, a diffusive pyrogallol field was imposed; motility forces set to f_u = 12 × 10−5 Pa µm² (urease) and f_c = 10 Pa µm² (CytC), with controls removing one engine at a time.
• Chemotaxis experiments: Custom setup with agarose reservoir creating a pyrogallol gradient. Population of dual-enzyme nanotubes was quantified at multiple positions over time; additional capillary assays used sealed-end glass capillaries containing pyrogallol (0–100 mM) immersed in a bath with nanomotors, urea, and H2O2. Confocal/DIC microscopy quantified localization near capillary openings.
• Controls to exclude artifacts: Tested (i) urease-only motors with pyrogallol gradient; (ii) dual-enzyme motors without pyrogallol gradient; (iii) addition of free CytC to urease-only motors; (iv) long equilibration to homogenize pyrogallol; (v) gel pre-mixed with CytC to test combined reaction effects; (vi) GOx replacing CytC; (vii) temperature and convection controls. Fluorescence assays tracked population changes (RITC-/FITC-tagged enzymes) to distinguish active (urease+CytC) vs passive (urease only) nanotubes.
• Biphasic catalysis test: Two-phase system (toluene with small percentage of buffer) with pyrogallol and H2O2 as substrates for CytC peroxidase. Amylobots with varying urea concentrations were added; purpurogallin formation rates were measured spectroscopically to assess motility-enhanced catalysis.
• Detailed Methods: Peptide synthesis via solid-phase Fmoc, acetylated N-terminus; assembly from HFIP-treated peptides in 40% acetonitrile/water with 0.1% TFA (1 month). Redispersion and centrifugation conditions specified. AuNP synthesis by citrate reduction and NaBH4; RITC labeling of urease and FITC labeling of CytC; Bradford assay for protein quantification; imaging by TEM/SEM/AFM/CLSM.

• Robust amyloid nanotube chassis formed from Ac-KL; mature nanotubes: 32 ± 2 nm diameter, 10 ± 1 nm height, 5–20 µm length. Sonication produced shortened nanotubes (180–560 nm) with preserved morphology and exposed lysines (AuNP binding).
• Urease loading achieved at ~11 µg nmol−1 for both short and long nanotubes; RITC-urease confirmed surface localization.
• Urea-dependent motility: Short urease-loaded nanotubes exhibited increased trajectory dispersion and MSD with urea concentration (0–100 mM), overcoming Brownian motion; long nanotubes showed negligible active motion due to drag. Simulations recapitulated passive-to-active transition as f_u increased (0 to 24 f_mot) and predicted velocities for given urea levels.
• Superdiffusive behavior quantified by MSD = 4DΔt^α; α and DA increased with urea concentration, indicating enhanced activity.
• Dual-enzyme (urease+CytC) amylobots displayed chemotactic migration toward a pyrogallol reservoir: significantly higher populations near the source in time-dependent assays; localization near capillary openings increased with pyrogallol concentration (0, 50, 100 mM). Multiple controls (no CytC, no gradient, free CytC, homogenized pyrogallol, GOx substitution) showed no directional bias, confirming CytC–pyrogallol-driven chemotaxis requiring urease-powered motility.
• Diffusivity metrics: In homogeneous media at 100 mM pyrogallol, only CytC-loaded nanotubes showed modest enhanced diffusion (D_cytc = 0.42 ± 0.017 µm²/s), while dual-enzyme nanotubes in urea had much higher diffusivity (D_urease/cytc = 6.99 ± 0.051 µm²/s). Dual-enzyme motors localized in ~15–20 min; in absence of urea, only-CytC motors showed subdued, slow chemotaxis (~1.5 h).
• Reaction rate enhancement at gradient interface: Purpurogallin generation was 7-fold faster at the gel–buffer interface with urea vs without urea, consistent with chemotactic accumulation elevating local substrate access.
• Biphasic catalysis (toluene/buffer): In presence of urea, amylobots showed 9-fold higher peroxidase activity (v1 = 35.1 ± 7.02 µM min−1) compared to control without urea (v1 = 3.9 ± 1.03 µM min−1), and ~2-fold higher than native CytC in buffer, demonstrating motility-induced catalytic augmentation across phases.
• Simulations of chemotaxis with substrate field reproduced directed accumulation with urease propulsion (f_u = 12 × 10−5 Pa µm²) and CytC navigation (f_c = 10 Pa µm²); removing urea yielded Brownian motion; removing CytC abolished directionality. MSD trends matched experimental chemotactic dynamics.

The findings validate the central hypothesis that integrating two orthogonal enzymatic processes on a robust amyloid nanotube chassis can decouple motility (urease-driven self-diffusiophoresis) from navigation (CytC-directed chemotaxis toward pyrogallol). Urease provides strong, fuel-dependent propulsion and superdiffusive motion, while CytC, despite lower catalytic proficiency, biases orientation and diminishes random rotational diffusion in a pyrogallol gradient, enabling net chemotactic migration. Extensive controls rule out convective, osmotic, or non-specific product effects. Particle-based simulations capture the transition from passive to active motion, the necessity of both engines for chemotaxis, and the temporal MSD signatures associated with gradient sensing. Functionally, chemotactic accumulation enhances access to substrate-rich regions, significantly boosting peroxidase turnover at interfaces and in biphasic organic media, illustrating how active transport can overcome mass-transfer limitations. This dual-enzyme strategy mirrors biological division of labor in cell migration and highlights how spatially inhomogeneous payload distribution can regulate active matter behavior.

The work introduces cross β amyloid peptide-based nanomotors (amylobots) that harness two enzymes to achieve autonomous chemotactic motility: urease for propulsion and cytochrome C for directional control toward pyrogallol. The system demonstrates fuel-dependent superdiffusion, CytC-mediated chemotaxis verified by microscopy and population assays, and computational corroboration via particle-based simulations. Practically, chemotactic motility translates into substantial catalytic augmentation, including a 9-fold peroxidase rate increase in a toluene/buffer biphasic system. These results establish a biofriendly, modular chassis with orthogonal bio-engines for smart catalysis and potential biomedical applications. Future research should dissect the mechanistic interplay and symmetry breaking between the two enzymatic activities, optimize enzyme spatial distribution on the chassis, and generalize the approach to other enzyme–substrate pairs and complex environments.

• Mechanistic details governing symmetry breaking and the coupling between urease-driven propulsion and CytC-mediated orientation remain unresolved and require further study.
• Enzyme distribution on nanotubes is patchy and inhomogeneous, potentially introducing variability in motility and chemotactic response.
• Chemotaxis driven by CytC alone is weak and slow, indicating reliance on urease-powered motility for timely navigation.
• Long nanotubes suffer from high viscous drag and limited motility, constraining chassis dimensions for effective operation.
• Experiments were performed in controlled in vitro setups; generalizability to complex biological fluids and in vivo conditions was not assessed.