Researchers at the University of Maryland have demonstrated that nanoscale 3D printing can be used to fabricate high-aspect-ratio microneedles that eliminate clogging during embryo microinjection. The results, published in Nature Communications, show that additive manufacturing via two-photon laser writing enables precise, monolithic needle designs that outperform both conventional glass needles and printed controls in serial in vivo experiments.
Microinjection is a cornerstone technique in developmental biology and genetic engineering, enabling the delivery of cells, nucleic acids, or proteins into embryos and other biological targets. Standard microneedles, typically fabricated by thermally pulling glass capillaries, include a single opening aligned with the direction of insertion. This configuration leads to frequent clogging when cytoplasmic material becomes lodged in the tip during penetration, disrupting injections and compromising sensitive samples.
Additive manufacturing enables side-port, filter-integrated needle designs
To address these failure modes, the research team employed ex situ Direct Laser Writing (esDLW), a form of two-photon polymerization in which a femtosecond infrared laser initiates photopolymerization inside a photosensitive resin. The team used IP-L photoresist from Nanoscribe GmbH & Co. KG, a German manufacturer of high-resolution 3D printing systems, to print hollow microneedle tips directly atop fused silica capillaries.
Each microneedle measured 650 µm in height, with a 15 µm outer diameter and a 10 µm inner diameter. The 3D design incorporated a sealed fine-point tip, twenty side ports with 5 µm diameter, and an internal filter composed of 3.25 µm × 3.25 µm pores. Control microneedles shared the same dimensions and internal filter but used a traditional single top-port opening. The 3D needle took approximately ten minutes to print, while the simpler control variant required five minutes.
Side ports were positioned orthogonally to the insertion direction due to voxel geometry constraints: the esDLW voxel measures 0.6 µm laterally and 3.3 µm vertically, favoring horizontal feature resolution. This enabled consistent port formation without compromising tip sharpness or internal channel integrity.
Scanning electron microscopy confirmed full adhesion between the printed polymer and capillary base. Dimensional analysis showed fabrication repeatability within ±0.2 µm for printed microneedles, compared to ±4.0 µm in manually pulled glass tips—a roughly 40-fold improvement in geometric consistency.
To assess mechanical robustness and flow stability, the researchers conducted linear and cyclic burst-pressure tests using dyed aqueous solutions. Both control and 3D microneedles maintained structural and hydraulic integrity under input pressures up to 200 kPa and 100 consecutive pressure cycles at ≥100 kPa. Flow remained linear throughout, with no evidence of leak-induced resistance drops or tip detachment. These in vitro results indicate that both printed variants withstand forces typical of laboratory injection systems.
Because these tests used clean fluids, they did not capture failure modes induced by biological contaminants. Distinctions between the two printed designs emerged only in live embryo trials.
Live embryo injections demonstrate blockage prevention
The core experiment involved 100 serial microinjections per needle into live, dechorionated zebrafish embryos using rhodamine B–dyed water. Ten microneedles per type—glass, printed control, and 3D—were evaluated, totaling 3,000 injections. After every 20 injections, embryos were lysed and fluorescence intensity measured to quantify retained fluid volume.
3D microneedles delivered the highest volumes with the lowest variability. In the first 20 injections, 3D needles yielded fluorescence readings of 9.41 ± 1.87, compared to 4.52 ± 1.58 for glass needles and 5.73 ± 1.38 for control microneedles. Delivery remained consistent across all five 20-injection blocks for the 3D design, while the other groups showed declining or erratic output. By the 40-injection mark, glass needles averaged just 0.30 ± 0.37 of baseline delivery volume.
Between embryo injection segments, the team conducted pseudo-injections into oil droplets to measure delivery capacity and detect clogging. These pseudo-injections—performed before and after every 20 embryo injections—revealed multiple blockage events in glass (44 ± 26% rate) and control needles (26 ± 23%), but no complete blockages in the 3D design. All printed needles shared the same internal filter, confirming that side-port architecture was the key differentiator in avoiding obstruction.
Visual evidence further supported these results. Embryos injected with glass needles showed more fragmentation in post-injection micrographs, suggesting increased mechanical damage during insertion and withdrawal. This effect, noted in Supplementary Figure S7, was not quantified but is consistent with the hypothesis that the solid fine-point tip reduces coring and trauma.
Implications for biomedical tools and device design
Earlier efforts to reduce microinjection failure have explored silicon-based side-port needle designs fabricated via cleanroom microfabrication. These approaches, while functional, remain costly, labor-intensive, and geometrically constrained. By contrast, the esDLW process enables high-speed, flexible production of geometrically complex needles with integrated features—without adhesives, post-processing, or alignment steps.
The ability to print microneedles with aspect ratios approaching 40:1, sealed junctions, and multi-layered flow paths could extend to other microscale applications, including targeted drug delivery, intraocular injection, microsurgery, and membrane-integrated microfluidic systems. With ongoing improvements in laser power, scanning speeds, and high-throughput photoresists, print times could drop well below current levels.
In addition to resolving clogging, high dimensional fidelity could eliminate the need for per-needle calibration. Standard glass microneedles often vary in tip diameter and delivery behavior, requiring manual tuning. Printed needles with consistent geometry may enable automated or batch protocols with uniform performance.
While this study used a dye solution, the benefits of integrated filters may be more pronounced when injecting suspensions containing cells or particulates. Future work may investigate these scenarios, along with injection-induced injury, leakage, and post-delivery retention in other biological targets.
The study confirms that submicron 3D printing offers a practical, geometry-driven solution to persistent clogging problems in microinjection workflows. By shifting from manual fabrication to digitally controlled, additively manufactured architectures, researchers can improve precision, reduce variability, and expand the utility of microneedle-based delivery systems in both research and clinical environments.
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Experimental results for clogging phenomena associated with microinjections into live dechorionated zebrafish embryos for various microneedle types. Image via Nature Communications.
