Functionalized protein nanopores: Strategies and applications

Over the past years, significant progress has been made in the field of nanopores, particularly in single-molecule analysis, sequencing, and diagnostic applications, certainly underscoring the versatility of stochastic nanopore sensing. The subsequent advances in functionalizing protein nanopores with tailored properties or reactive handles have greatly expanded their detection capabilities, where customized computational design shows promise in driving nanopore functionalization and applications toward unprecedented flexibility.

A team of nanotechnology researchers led by Jia Geng and Shengli Zhang from Sichuan University and Shanghai Jiao Tong University, respectively, has recently provided a comprehensive overview of functionalization strategies and emerging applications of protein nanopores aimed at advancing the field. Beyond the expansion of applications driven by the discovery of new nanopores, major breakthroughs in stochastic nanopore sensing over the past decades have been largely enabled by the modification, reconstruction, and even de novo design of protein nanopores. Precise structural engineering of these nanopores has proven essential for enhancing the accuracy and versatility of single-molecule sensing, thereby opening new avenues for protein sequencing and sub-nanometer-scale analysis.

The team published their review in Nano Research on January 23, 2026.

"In this review, we offer a comprehensive overview of recent progress in protein nanopore modification, focusing on emerging strategies to optimize nanopore functionality. Although previous reviews have summarized some engineering approaches and corresponding sensing applications, our review systematically outlines evolving modification strategies for protein nanopores, organizing them from an application-oriented perspective. We aim to clarify key design principles and provide conceptual guidance to support future innovation in nanopore sensing. We also discuss ongoing challenges and promising opportunities that could shape the future of protein nanopores,” said Jia Geng, senior author of the review, Professor in the Department of Laboratory Medicine at the State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University. 

Protein nanopores are formed through the self-assembly of multiple subunits within lipid bilayers or synthetic block copolymer membranes. Their nano- to sub nanometer scale dimensions are defined with atomic precision by the underlying protein sequences, providing extensive opportunities for precise modification through genetic and chemical approaches.

The research team anticipates that this review will drive the development of innovative protein nanopore functionalization strategies, expanding the scope and depth of single-molecule sensing and particularly advancing next-generation point-of-care diagnostics and protein sequencing. “As nanopore modification methods continue to advance, the boundaries of nanopore sensing will keep expanding, and innovative functionalization strategies may ultimately enable nanopore technology to reach the “crown jewel” of protein sequencing in the post-genomic era.” Jia Geng said.

High-performance sensing architectures of nanopores are prerequisites for achieving single–amino acid resolution and accurate signal decoding in protein sequencing. Although artificial intelligence-based de novo methods appear to offer a promising approach for fabricating protease-nanopore systems, there still remain significant challenges. Overcoming this bottleneck will require more ingenious and refined functionalization for pores, like nanopores engineered with ultrathin constrictions or multiple reading heads analogous to DNA sequencing that enable residue-by-residue identification, and systems inspired by earlier successes in discriminating 20 amino acids through metal chelation or host–guest interactions, which suggest that strengthening specific interactions between engineered nanopore components and individual residues may be a promising route. "Of course, these customized modifications first need to be aligned with a specific sequencing route. The requirements for nanopores and even motor proteins differ markedly between the "chop-and-drop" and "thread-and-read" approaches. At present, pore resolution in the chop-and-drop route appears largely sufficient for amino acid sequencing, with future work mainly focused on building continuously processive motor systems. In contrast, the strand based sequencing route, which offers a true one pass solution, still has a long road ahead in terms of pore functionalization and will likely require another major wave of scientific inspiration." said Jia Geng.

Other contributors include Yuan Ju, Ke Sun, Mengze Wu, Xin Yu, Yue Li, Minghan Li from Sichuan University in Chengdu, China; and Jinna Wang from Shanghai Jiao Tong University in Shanghai, China.

This work is granted by the National Natural Science Foundation of China (32301258, 32372420), Natural Science Foundation of Sichuan, China (2024NSFSC1185), China Postdoctoral Science Foundation (2023TQ0230, GZB20230484, and 2024M752201), Post-Doctor Research Project, West China Hospital, Sichuan University (2024HXBH123).

DOI Link:

https://doi.org/10.26599/NR.2026.94908343

About Nano Research

Nano Research is a peer-reviewed, open access, international and interdisciplinary research journal, sponsored by Tsinghua University and the Chinese Chemical Society, published by Tsinghua University Press on the platform SciOpen. It publishes original high-quality research and significant review articles on all aspects of nanoscience and nanotechnology, ranging from basic aspects of the science of nanoscale materials to practical applications of such materials. After 18 years of development, it has become one of the most influential academic journals in the nano field. Nano Research has published more than 1,000 papers every year from 2022, with its cumulative count surpassing 8,000 articles. In 2025 InCites Journal Citation Reports, its 2025 IF is 9.4 (8.3, 5 years), and it continues to be the Q1 area among the four subject classifications. Nano Research Award, established by Nano Research together with TUP and Springer Nature in 2013, and Nano Research Young Innovators (NR45) Awards, established by Nano Research in 2018, have become international academic awards with global influence.