High-throughput protein nanopatterning

Xiangyu Liu, Mohit Kumar, Annalisa Calo, Edoardo Albisetti, Xiaouri Zheng, Kylie B. Manning, Elisabeth Elacqua, Marcus Weck, Rein V. Ulijn, Elisa Riedo

Research output: Contribution to journalArticle


High-throughput and large-scale patterning of enzymes with sub-10 nm resolution, the size range of individual protein molecules, is crucial for propelling advancement in a variety of areas, from the development of chip-based biomolecular nano-devices to molecular-level studies of cell biology. Despite recent developments in bio-nanofabrication technology, combining 10 nm resolution with high-throughput and large-scale patterning of enzymes is still an open challenge. Here, we demonstrate a high resolution and high-throughput patterning method to generate enzyme nanopatterns with sub-10 nm resolution by using thermochemical scanning probe lithography (tc-SPL). First, tc-SPL is used to generate amine patterns on a methacrylate copolymer film. Thermolysin enzymes functionalized with sulfonate-containing fluorescent labels (Alexa-488) are then directly immobilized onto the amine patterns through electrostatic interaction. Enzyme patterns with sub-10 nm line width are obtained as evidenced by atomic force microscopy (AFM) and fluorescence microscopy. Moreover, we demonstrate large-scale and high throughput (0.13 × 0.1 mm2 at a throughput of 5.2 × 104 μm2 h-1) patterning of enzymes incorporating 10 nm detailed pattern features. This straightforward and high-throughput method of fabricating enzyme nanopatterns will have a significant impact on future bio-nanotechnology applications and molecular-level biological studies. By scaling up using parallel probes, tc-SPL is promising for implementation to scale up the fabrication of nano-biodevices.

Original languageEnglish (US)
Pages (from-to)33-43
Number of pages11
JournalFaraday discussions
StatePublished - 2019

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

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    Liu, X., Kumar, M., Calo, A., Albisetti, E., Zheng, X., Manning, K. B., Elacqua, E., Weck, M., Ulijn, R. V., & Riedo, E. (2019). High-throughput protein nanopatterning. Faraday discussions, 219, 33-43. https://doi.org/10.1039/c9fd00025a