Onderzoek naar moleculaire nanotechnologie voor de detectie van individuele moleculen met vaste-stof nanoporiën

Nanopores enable label-free single-molecule sensing with broad application potential in genomics, proteomics, drug discovery, and molecular data storage. Their exceptional sensitivity is exemplified by the successful commercialization of DNA sequencing using biological nanopores. This dissertation focuses on solid-state nanopores, which offer a distinct set of advantages over biological nanopores, including mechanical robustness, scalability through dense nanopore arrays, improved noise characteristics, and compatibility with CMOS integration for high-bandwidth measurements. Despite these benefits, fabricating solid-state nanopores with atomic-scale precision and sub-5 nm dimensions remains extremely challenging. This size mismatch between solid-state nanopores and the fine structural features of most biomolecular analytes limits their sensitivity. To overcome these limitations, this dissertation introduces novel strategies inspired by nature, using molecular nanotechnology to enhance single-molecule detection with solid-state nanopores. Specifically, the predictable folding of DNA nanostructures and the self-assembly of lipid entities are harnessed to design structural building blocks and functional components. Two central research questions are explored: (1) What are the minimal structural requirements for reliable detection of DNA nanostructures using solid-state nanopores? (2) Can programmable biomolecular components be used to engineer scalable hybrid nanopore systems? In the first part, DNA nanostructures were designed with large features, or labels, that match solid-state nanopore dimensions, enabling high-sensitivity readout. This approach addresses a fundamental limitation: solid-state nanopores are not yet capable of resolving the fine structural details of native DNA. The engineered DNA nanostructures with large labels, referred to as 'DNA structural barcodes', offer a direct route to scalable biosensing by avoiding the need for complex nanopore geometry tuning. This work presents the first systematic investigation of the minimum size requirements for barcode labels in membrane-based solid-state nanopore systems. Chapter 2 reveals that the physical characteristics of barcode labels influence their local translocation velocity, distinct from the DNA backbone, and significantly affect readout success. In the second part, two hybrid nanopore concepts are introduced to combine the strengths of biological and solid-state nanopores. The integration of the highly sensitive biological nanopores into solid-state platforms may ultimately enable the readout of fine structural features in biopolymers, like DNA. Existing hybrid strategies face technical challenges such as leakage, instability, and poor integration. To address these, programmable DNA and self-assembling lipids are used to develop engineerable components for hybrid nanopore formation. The first concept presents a DNA-lipid interposer that stabilizes biological nanopores in a lipophilic environment and enables guided docking onto solid-state nanopores. Chapters 3 and 4 detail the development and assembly of this interposer, resulting in the first successful integration of a lipid bicelle into a DNA origami structure. This novel DNA-lipid nanodisc marks a significant advancement in nanoscale membrane engineering. It provides a versatile platform for single-molecule studies of membrane proteins and was realized by two methodological innovations not previously reported: a detergent-free bicelle incorporation protocol and the use of DNA origami as a structural stabilizer. These approaches allow robust and tunable membrane integration within DNA nanostructures. Additionally, experimental evidence of biological nanopore monomer insertion represents a promising step toward realizing the envisioned hybrid nanopore platform. The second concept demonstrates, for the first time, the formation of pore-spanning supported lipid bilayers through the fusion of nanoscale lipid discs (i.e., bicelles). Chapter 5 establishes this method as a scalable and robust approach for hybrid nanopore assembly. Compared to liposome-based techniques, bicelles offer a more favorable geometry for spanning nanopores and enable high sealing resistance, which is essential for achieving high sensitivity. This streamlined strategy supports the development of high-density hybrid nanopore arrays with minimal fabrication complexity. Together, these contributions advance solid-state nanopore technology by introducing scalable and nature-inspired molecular strategies for improving the sensitivity. The use of DNA structural barcodes and hybrid nanopore platforms opens new possibilities for single-molecule biosensing and membrane protein studies in native-like environments.