Molecular engineering of piezoelectricity in collagen-mimicking peptide assemblies

Piezoelectric materials convert mechanical deformation into electrical energy but common inorganic and polymeric devices rely on toxic metals, complex synthesis, and have stability and sustainability issues. Biocompatible, flexible, durable green piezoelectrics are sought for health monitoring and regenerative medicine. Natural biomaterials such as bone, collagen, viruses, cellulose and chitosan exhibit piezoelectricity, typically 0.1–10 pm V−1, which is low for many applications. Collagen’s helical structure with aligned hydrogen bonds produces a macroscopic dipole and measurable piezoelectricity, potentially influencing bone growth. Hydroxyproline (Hyp), a post-translationally modified collagen residue, shows high piezoresponse but alone does not form collagen-like helices. Aromatic amino acids (e.g., Phe-Phe) can enhance electronic interactions and have been used in peptide-based energy harvesters, though most ultra-short peptides form β-sheets rather than helices. The research question is whether rational molecular and supramolecular engineering of ultra-short, collagen-mimicking helical peptides—by incorporating Hyp and aromatic Phe—can substantially enhance piezoelectric response to technologically relevant levels and enable high-performance, eco-friendly piezoelectric devices. The study integrates predictive modelling (DFT, MD) with experiments to design and validate helical tripeptides (Pro-Phe-Phe and Hyp-Phe-Phe) with enhanced electromechanical properties and device performance.

Prior work established piezoelectricity in biomaterials (bone, collagen, viruses) but with modest responses. Collagen fibrils show shear d14 ~12 pm V−1. Ultra-short peptide assemblies typically adopt β-sheets; Phe-Phe-based systems have been explored for green energy harvesting and enhanced conductivity. Hyp exhibits high piezoresponse among collagen amino acids, and helical packing with aligned H-bonds in collagen generates macroscopic polarization. DFT has been effectively used to predict piezoelectric tensors in inorganic crystals, polymers, amino acids, peptides and biominerals; PFM enables nanoscale electromechanical characterization, and MD captures kinetics and environmental effects. Recent advances produced ultra-short peptides mimicking collagen-like helical architectures and highlighted aromatic ‘steric zipper’ motifs conferring rigidity. However, deliberate design of ultra-short peptides to achieve collagen-like high piezoelectricity via helical supramolecular organization remained elusive. This study builds on that foundation, combining Hyp’s polarizability with Phe’s aromatic interactions in tripeptides to engineer enhanced piezoelectricity.

• Peptide design and assembly: Selected Pro-Phe-Phe as a helical, mechanically robust tripeptide; designed Hyp-Phe-Phe by substituting Pro with Hyp. Peptides (>95% purity) were dissolved in phosphate buffer (pH 7.4) at 1.5 mg ml−1, heated (90 °C) with vortexing, then incubated at 18 °C for 2 weeks with shaking for assembly.
• Structural characterization: FTIR on dried, D2O-exchanged films (Nicolet Nexus 470, 4 cm−1 resolution, 32 scans) to assess secondary structure (amide I region). Circular dichroism (Chirascan, 190–240 nm, 25 °C) to confirm helical signatures. AFM and TEM imaging to resolve supramolecular morphology; QNM-AFM (peakforce, Multimode 8, RTESPA 525 cantilevers) to map Young’s modulus along fibrils. Single-crystal X-ray diffraction to resolve molecular packing; crystals grown by vapor diffusion; data collected at 100 K (Rigaku XtaLabPro) and solved/refined with SHELXT/SHELXL.
• Computational modelling: • DFT (VASP, PBE, PAW): Periodic calculations on solvent-free XRD structures to compute elastic, dielectric and piezoelectric tensors using DFPT; stiffness via finite differences; plane-wave cutoff 1000 eV; k-point sampling 2×2×2 or 4×4×4 as appropriate; binding energies with Grimme-D3. Molecular dipoles by Gaussian16 (B3LYP/6-311++G**); crystal dipoles by CP2K (PBE, mixed Gaussian/plane waves; Berry phase; supercell 3×2×2). • MD simulations (GROMACS 2018.4, CHARMM36m, TIP3P water): Nanocrystal supercells (e.g., 14×10×6 replicas; ~7.1×9.5×12 nm) solvated and simulated at 300 K, 1 bar; equilibration and production in NPT ensemble with velocity-rescale thermostat and Parrinello–Rahman barostat; LINCS constraints; PME electrostatics. Hyp parameters from SwissParam; validation via CP2K supercell tests. Analyzed H-bond statistics and Phe–Phe ring contact distributions (distance/angle 2D histograms).
• Piezoresponse measurements: PFM point measurements due to small crystal size; probe held stationary on crystal, varying applied voltage; low frequency 21 kHz to minimize artifacts; stiff probes (5–6 N m−1) to mitigate electrostatic/flexoelectric effects; ambient 20 °C, 40% RH. Linear piezoresponse vs voltage assessed; controls included; statistical distributions of d33 and shear coefficients obtained.
• Device fabrication and testing: Coin-sized nanogenerator by tightly sandwiching densely packed peptide assemblies between two 0.7×0.7 cm2 Ag electrodes on 1.2×1.2 cm2 Si substrates; Kapton/PDMS support and encapsulation; PDMS damping layer; copper leads via carbon ink. Device mounted on linear motor for periodic compression; outputs measured as open-circuit voltage (SR560 preamp) and short-circuit current inside a Faraday cage. Force-dependent output, polarity switching tests, durability (1000 cycles), and load dependence assessed. A β-sheet Phe-Phe control device was fabricated and tested under identical conditions.

• Structure and mechanics: • Hyp-Phe-Phe forms uniform, high-aspect-ratio fibers (~500 nm diameter, microns long; L/D > 500) with helical-like molecular organization confirmed by FTIR (amide I ~1645 cm−1 with 1680 cm−1 shoulder) and CD (double negative maxima ~210 and 230 nm). • Single-crystal XRD shows head-to-tail H-bonded, elongated helical-like stacking and an aromatic ‘zipper’ of Phe–Phe side chains forming a dry interface. • QNM-AFM Young’s modulus along fibers: 60–90 GPa, indicating remarkable rigidity comparable to stiff biological materials; mechanical periodicity along fibrils with peak-to-peak spacing ~165 nm and periodicity ~84.9 nm.
• DFT predictions: • Dielectric constants similar (ε overall ~3.1–3.3); ε33 increased 25% in Hyp-Phe-Phe (4.0 vs 3.2 in Pro-Phe-Phe). • Hydroxylation lowers symmetry (monoclinic → triclinic), increases non-zero piezoelectric tensor components, raises eij (e33 ≈ 0.1 C m−2), and lowers shear stiffness, yielding larger d and g. • Maximum predicted piezoelectric strain constant in Hyp-Phe-Phe: d35 = −27.3 pm V−1; d33 ≈ 4.8 pm V−1; broad range 0.1–27.3 pm V−1. Pro-Phe-Phe: d up to ~3.1 pm V−1; significant voltage constants despite low ε (e.g., g22 up to 108 mV m N−1). • Hyp-Phe-Phe predicted voltage constant gmax ≈ g16 ≈ 1043 mV m N−1; estimated single-crystal voltage output under 4 µN load: ~39 mV vs ~7 mV for Pro-Phe-Phe. • Table 1: Pro-Phe-Phe molecule dipole 7.9 D, crystal dipole 2.8 D; longitudinal response 2.2 pm V−1; Hyp-Phe-Phe molecule dipole 6.7 D, crystal dipole 1.9 D; longitudinal 4.0 pm V−1; shear 16 pm V−1 permitted.
• Experimental piezoresponse (PFM): • Pro-Phe-Phe d33 = 2.15 ± 0.86 pm V−1. • Hyp-Phe-Phe d33 = 4.03 ± 1.96 pm V−1; effective shear coefficient d15 = 16.12 ± 2.3 pm V−1, exceeding LiNbO3 (~13), ZnO (~12), γ-glycine (~10), M13 bacteriophage (6–8), and collagen film (~1 pm V−1).
• MD insights: • Hydroxylation strengthens H-bond network while increasing conformational freedom of Phe–Phe rings; Hyp-Phe-Phe shows broader, shifted Phe–Phe contact distance/angle distributions; both variants retain stable aromatic zipper motif.
• Device performance: • Pro-Phe-Phe nanogenerator (F = 55 N): Voc ≈ 1.4 V; Isc ≈ 52 nA; outputs exceed several peptide/biomaterial alternatives. • Hyp-Phe-Phe (F = 23 N): Isc ≈ 39.3 nA; Voc ≈ 0.45 V; linear dependence on force with slopes ~15.08 mV N−1 (voltage) and ~1.33 nA N−1 (current); stable over >1000 cycles at 17 N with no degradation. • β-sheet Phe-Phe control (F = 23 N): Voc ≈ 0.14 V; Isc ≈ 3.9 nA; significantly lower than helical tripeptides.
• Overall: Simple hydroxylation (Pro→Hyp) and helical supramolecular design elevate piezoelectric response by ~order of magnitude in key tensor components, delivering high voltage constants and robust device outputs.

The study demonstrates that piezoelectricity in peptide biomaterials can be rationally amplified by engineering both primary sequence and supramolecular helical packing. Hydroxylation (Pro→Hyp) reduces crystal symmetry, increases the number and magnitude of non-zero piezoelectric tensor elements, and lowers shear stiffness, enabling larger ionic displacements and higher d and g coefficients. Strengthened H-bond networks and remodeled aromatic zipper interactions adjust unit-cell dipoles and facilitate stress-induced charge transfer. Despite similar dielectric constants, Hyp-Phe-Phe achieves markedly higher predicted shear d35 and voltage constants than Pro-Phe-Phe, rivaling or exceeding many inorganic crystals for specific tensor components. Experimental PFM validates the model, showing doubled d33 and high shear d15 for Hyp-Phe-Phe. The helical architecture, reminiscent of collagen, underpins enhanced macroscopic polarization compared with β-sheet assemblies, as confirmed by the weaker performance of β-sheet Phe-Phe devices. Device-level tests corroborate linear, durable electromechanical transduction, and polarity switching confirms true piezoelectric origin. The combined DFT tensor screening and device orientation strategy provides a route to optimize outputs by aligning crystals/assemblies along high-response axes.

This work establishes a computationally guided, molecular-engineering approach to create collagen-mimicking helical tripeptide assemblies with enhanced piezoelectricity. Incorporating Hyp into Phe-Phe-based helical tripeptides increases key piezoelectric tensor elements (predicted d35 ~−27 pm V−1) and voltage constants, validated by PFM (d33 ~4 pm V−1; shear ~16 pm V−1) and robust device performance (Voc up to 1.4 V; Isc >50 nA). The helical supramolecular organization and aromatic zipper motif deliver exceptional stiffness and stable transduction, while simple chemical modifications (C–H→C–OH) tune electromechanical responses. These insights highlight the importance of both sequence and packing symmetry for designing high-performance, eco-friendly piezoelectric biomaterials. Future work should focus on fabricating highly ordered, aligned arrays to maximize tensor-directed outputs, refining sequence chemistry (e.g., aromatic content and polar groups) to further enhance polarization, and integrating orientation control to scale device performance for practical nanotechnology and biointerface applications.

• Conventional PFM imaging (topography, amplitude, phase) was not feasible due to the small size and mobility of peptide single crystals; characterization relied on stationary point measurements.
• Molecular dynamics employed forcefield parameters (Hyp via SwissParam) and a crystal-in-water supercell approximation; although validated against DFT stability checks, such models carry typical forcefield and boundary limitations.
• Device outputs were demonstrated on randomly packed films; the authors note that performance could be further improved by fabricating highly ordered, aligned arrays, indicating current devices are not yet optimized for maximal output.
• Classical piezoelectric models do not fully capture hierarchical biomaterial organization, contributing to potential discrepancies between predicted and experimental responses at larger scales.