Geometry-Tunable Nanoneedle Arrays Reveal Membrane Penetration Mechanics for Intracellular Delivery

Nanoneedle arrays provide a promising interface for intracellular delivery, yet scalable control of array geometry and membrane penetration mechanics remains insufficiently understood. Here, we developed a rapid and scalable strategy for fabricating geometry-tunable silicon nanoneedle arrays. One-step SF6/O2 etching produced ordered arrays with center-to-center spacing of 1–5 μm, whereas pseudo-Bosch etching produced high-aspect-ratio (HAR) nanoneedles. Using a microwell-assisted cell-on-probe atomic force microscopy platform, we quantified the first penetration force, penetration probability, and number of penetration events at the single-cell level. For one-step-etching arrays, increasing spacing from 1 to 5 μm reduced the first penetration force from 34.87 ± 2.90 to 4.05 ± 0.30 nN and increased the penetration probability from 0.21 ± 0.03 to 0.87 ± 0.04. A phenomenological inverse-square model captured the force–spacing relationship, supporting an array-level load-sharing mechanism. Under identical vibration-assisted microfluidic conditions, the FITC-dextran-positive fraction increased from 22.8% for 1 μm arrays to 77.1% for 5 μm arrays, whereas 1 μm HAR arrays achieved 28.8%. These results identify array spacing as a key factor governing single-cell penetration and delivery and provide a mechanistic basis for nanoneedle-based biosensor and cell-interface design.