Femtosecond laser programmed artificial musculoskeletal systems

The study addresses the challenge of fabricating artificial musculoskeletal systems at the micro/nanoscale that combine rigid and soft materials in complex 3D architectures. Conventional microbots, often rigid and mechatronic, are limited by poor biocompatibility and flexibility. Soft microbots based on smart, low-modulus materials offer flexibility and diverse stimulus-responsiveness but often suffer from isotropic response, insufficient mechanical strength, and durability issues when built from a single material. Inspired by natural musculoskeletal systems, the authors aim to integrate a stiff skeleton (SU-8) with a soft, pH-responsive muscle (BSA) in 3D at submicron precision. They propose a successive on-chip two-photon polymerization (TPP) process enabling sequential structuring and in situ alignment of multiple photosensitive materials within a microfluidic environment. The purpose is to realize robust yet flexible 3D microbots capable of controlled actuation, demonstrated through a pH-responsive spider microbot and a 3D micro-gripper.

Successive on-chip two-photon polymerization (TPP) platform: A PDMS parapet bonded to a glass substrate forms an on-chip microfluidic chamber for reagent delivery and exchange (photoresists, developer, water). The workflow comprises: (i) TPP of the 3D SU-8 skeleton per a predesigned model; (ii) in situ development by flowing acetone to remove unexposed SU-8; (iii) secondary TPP to integrate a pH-responsive BSA hydrogel muscle into reserved vacancies; (iv) in situ development by flowing ultrapure water to remove unpolymerized BSA.

Materials: Skeleton material—SU-8 2025 negative photoresist. Muscle material—BSA hydrogel containing methylene blue (MB) photoinitiator (BSA 500 mg/mL; MB 0.6 mg/mL in ultrapure water). BSA solution was equilibrated at 4 °C for 24 h prior to use. FTIR confirmed material signatures and noncovalent SU-8/BSA interface characteristics.

Laser direct writing parameters: Femtosecond laser: 800 nm, 80 MHz repetition rate, 120 fs pulses. Focusing via oil-immersion objective (60×, NA 1.4). Average power before objective: ~10 mW for SU-8 writing, 20 mW for BSA writing. 3D scanning by galvo mirrors (XY) and piezo stage (Z). Step lengths (voxel spacing): SU-8—100, 200, or 300 nm; BSA—50, 100, 150, or 200 nm, chosen to tune stiffness (SU-8) and elasticity/swelling force (BSA). Post-exposure bake for SU-8: 95 °C, 30 min. SU-8 soft bake: 95 °C, 1 h before writing. In situ SU-8 development with acetone; BSA development with ultrapure water.

Device designs: (1) Spider microbot with SU-8 body and eight SU-8 legs, with reserved junction vacancies for BSA muscles; (2) Arm-muscle actuator: SU-8 arm (~35 µm length) with BSA joint; (3) Crab claw-muscle actuator: SU-8 zigzag pincers with BSA at one joint; (4) 3D micro-gripper with SU-8 skeleton and integrated BSA muscles, with laser step-length optimization for maximal folding.

Characterization and testing: SEM (JEOL JSM-7500F; samples gold-coated 3–5 nm) and optical microscopy (Motic BA400). pH control using calibrated meter (resolution 0.01). Mechanical property: SU-8 Young’s modulus measured by nanoindentation (Agilent G200, XP actuator, continuous stiffness measurement, Berkovich tip, 30 °C, RH 20%), yielding ~4.8 GPa for SU-8 at 200 nm step length. Actuation tests performed in aqueous solutions at varied pH (1–13), recording folding angle, response times, and cyclic stability. Force estimation: cantilever deflection method and COMSOL finite element analysis to back-calculate BSA muscle tensile force from measured tip displacements. Storage stability: samples stored at ~4 °C in ultrapure water (replaced every 3 days) and evaluated after 45 days.

• Multi-material 3D integration: A successive on-chip TPP process enabled in situ fabrication and alignment of SU-8 skeletons with BSA muscles into seamless, submicron-precision musculoskeletal microstructures (e.g., spider microbot). Interfaces were smooth and continuous in SEM; FTIR indicated noncovalent interactions at SU-8/BSA interface.
• pH-responsive behavior: BSA hydrogel exhibited minimal volume near its isoelectric point (pH ~5) and swelled at pH 1 and pH 13 due to electrostatic repulsion. Quantitatively, 10 µm × 10 µm BSA micro-squares showed a maximum area swelling ratio of ~1.44 at pH 13 relative to pH 5; SU-8 was inert over pH 1–13. BSA swelling/shrinking was stable over 200 pH-switching cycles (5→13→5→1).
• Arm-muscle actuator: Dynamic folding from ~2° to ~19° occurred within ~1.2 s when switching pH 5→13; recovery to ~2° in ~1.5 s upon 13→5. After 45 days storage, actuation performance remained comparable, indicating durability and robustness. SEM confirmed structural integrity over time.
• Crab claw-muscle actuator: Reversible opening/closing repeatedly over four consecutive cycles when alternating pH 5 and 13.
• Micro-gripper optimization: Tuning laser step length modulated BSA swelling and force generation and SU-8 stiffness. Free BSA blocks showed increasing swelling ratio with larger step length (max ~1.56 at 200 nm), but integrated gripper folding depended on swelling force and skeleton constraint. Optimal BSA step length was 100 nm, yielding a maximum folding angle of ~23°. SU-8 skeleton at 200 nm step length balanced bending range and stability (Young’s modulus ~4.8 GPa). Too rigid (100 nm) limited folding (~8°); too sparse (300 nm) led to deformation/collapse.
• Muscle force estimation: A BSA muscle ~25 µm long and ~2 µm wide produced a tensile force of ~1.4–1.5 µN on SU-8 cantilevers, causing 1.98–2.12 µm tip deflection (experimentally measured 2.05 µm; COMSOL simulation consistent). In separate load-capacity tests varying SU-8 cantilever widths, the setup indicated the muscle could drive cantilevers up to 6 µm width, corresponding to a maximum force of ~34 µN; 8 µm-width cantilevers could not be driven.
• Gripper response dynamics and endurance: The gripper reached maximum folding in ~1.6 s (pH 5→13) and recovered in ~2.3 s (13→5). Folding angle was tunable with pH (1–13). The device withstood at least 100 opening/closing cycles with reversible angles (~23°↔0°). Demonstrated precise capturing, transporting, and releasing of a ~10 µm SU-8 micro-cube target by pH control.
• Size advantage: The tens-of-micrometers 3D gripper exhibited high sensitivity and could perform complex deformations beyond 2D bilayer designs.

The work demonstrates that successive on-chip TPP can address the central challenge of integrating multiple, property-distinct materials into complex 3D micro/nanostructures, enabling artificial musculoskeletal systems that combine rigid support (SU-8 skeleton) with active, soft actuation (BSA muscles). The pronounced and reversible pH-driven deformation of BSA, alongside SU-8’s inertness and stiffness, yields robust, controllable actuators. The approach achieves rapid, repeatable actuation with long-term stability (≥45 days) and endurance (≥100 cycles), and mechanical outputs sufficient for micromanipulation tasks. The ability to program internal networks via voxel spacing allows fine control over elasticity and stiffness, optimizing device performance. Compared with 2D bilayer grippers, the fully 3D musculoskeletal architecture expands the deformation repertoire and sensitivity at small scales, positioning these microbots for applications in manipulation, assembly, and potential bio-related tasks when integrated with microfluidic platforms.

The study introduces a universal, femtosecond laser-based, successive on-chip TPP strategy for directly printing 3D multi-material microbots that mimic musculoskeletal systems. By integrating SU-8 skeletons with pH-responsive BSA muscles at submicron precision, the authors realize fast, reversible actuation in multiple prototypes (spider microbot, arm, crab claw, and a functional 3D micro-gripper). Mechanical properties and actuation can be tailored by laser scanning step length. The method is compatible with other micro/nanofabrication techniques (e.g., soft replication), offering a path to scalable, hierarchical devices. Future work could extend to other smart materials and stimuli, scale up fabrication, and integrate with microfluidic systems for robot-on-a-chip applications in cell handling, cargo transport, and minimally invasive procedures.

• Demonstrations focus on a single smart muscle material (BSA) and a single skeleton material (SU-8); generalization to other material pairs, while proposed, is not experimentally shown here.
• Actuation relies on pH modulation (including extremes such as pH 1 and 13), which may limit direct biological applicability without further optimization of operating ranges or materials.
• Mechanical optimization shows trade-offs: overly dense SU-8 (100 nm step) limits folding, while sparse SU-8 (300 nm step) compromises structural integrity; similarly, BSA voxel spacing modulates swelling force versus strength.
• Force capacity is bounded: the muscle could not drive wider (8 µm) SU-8 cantilevers under tested conditions.
• All tests are conducted in vitro under controlled laboratory conditions; performance in complex biological environments is not evaluated.