Glycans are essential for biological processes, disease diagnosis, and drug development. The diverse functions of oligosaccharides are intimately related to their highly complex structures. Therefore, the identification of oligosaccharide structures is essential for comprehending their roles in health and diseases. However, the low concentration and complicated structures and properties of natural oligosaccharides post a challenge to their analysis. Developing an ultrasensitive oligosaccharide sensor with a low detection limit is under urgent demand. Nanopore technology is a highly sensitive single-molecule and label-free technique, which has exhibited tremendous advantages in glycan sensing. Nevertheless, detecting neutral oligosaccharides with biological nanopore usually requires concentrations around 500 μM. Achieving both low-concentration detection and high spatial resolution remains challenging for biological nanopore sensing of oligosaccharides.
Professor Long and professor Ying’s group focuses on the single-molecule electrochemical nanopore sensing, establishing the correlation between nanopore stereo-structure and spatial resolution capability. By enhancing the lateral recognition field of the nanopore, highly sensitive identification of a series of peptide isomers was achieved (J. Am. Chem. Soc. 2022, 144, 33, 15072-15078; Nat. Nanotechnol. 2024, 19, 1693-1701). On this basis, they incorporated with the group in the Shanghai Institute of Materia Medica to develop a novel single-molecule oligosaccharide sensor based on OmpF nanopore. By virtue of the natural electroosmotic flow within the OmpF and its asymmetric confinement architecture, oligosaccharides at low concentrations could be sensitively determined in the label-free manner.
Figure 1. Oligosaccharide sensing based on OmpF nanopore.
Figure 2. Direct detection of oligosaccharides with the OmpF nanopore at low concentrations.
Through systematic study of the ion selectivity and stereo-structural characteristics of OmpF nanopore, the electroosmotic flow velocity of OmpF is calculated as 2.4 times that of the ion-selective N226Q/S228K aerolysin. providing a universal driving force for neutral oligosaccharides and reducing the detection limit by nearly two orders of magnitude. The robust native EOFwithin OmpF generates a robust driving force for unlabeled neutral oligosaccharides, reducing the detection concentration by nearly two orders of magnitude. With the assistance of multilayer perceptron (MLP) machine learning algorithms, the OmpF nanopore achieved a recognition accuracy of 99.9 % for natural human milk oligosaccharides differing in only one glycosidic bond. When further applied to oligosaccharide detection in cell lysates, this method could identify oligosaccharides at a low concentration of 10 μM and enabled accurate quantification of oligosaccharide isomer mixtures with the concentration ratio of 1:100. To summary, this nanopore sensor provides a highly sensitive analytical tool with a broad dynamic range and may support the development of nanopore-based glycan sequencing.
Figure 3. Discrimination of single glycosidic bond difference in oligosaccharide isomers by the OmpF.
Figure 4. OmpF identifying oligosaccharide isomers with polar groups.
The paper entitled Identification of Oligosaccharide Isomers Using Electrostatically Asymmetric OmpF Nanopore was online published on January 24, 2025 in Angewandte ChemieInternational Edition (https://onlinelibrary.wiley.com/doi/10.1002/anie.202422118). Ph.D. student Fan Gao from our department is the first author of the paper, and Professor Yi-Lun Ying from our department, Professor Liuqing Wen and Professor Bingqing Xia from the Shanghai Institute of Materia Medica are the co-corresponding authors. Professor Zhaobing Gao from the Shanghai Institute of Materia Medica is thanked for providing valuable guidance and assistance in oligosaccharide isomer sensing. This research was supported by the National Natural Science Foundation of China and the Shanghai Municipal Science and Technology Major Project.