Scanning ion-conductance spectroscopy and force microscopy of biomolecules

Single-molecule sensors have found widespread applications in probing biological processes at the fundamental scale and enabling detection at the ultimate sensitivity limit. In this work, a Scanning Ion Conductance Microscope (SICM) system is presented as a platform for single-molecule sensing and as a tool capable of extracting physical properties, such as stiffness.

This thesis is divided into two parts. The first part of the thesis focuses on the use of the Scanning Ion Conductance Spectroscopy (SICS) method, a nanopore-based single-molecule detector at the ultimate scale. SICS employs a glass capillary as a movable nanopore integrated into an SICM system. Unlike traditional nanopore experiments, in which analytes translocate through a fixed pore, here the analyte is immobilized on a surface while the nanopore approaches and scans across it. This allows for unprecedented control as the analytes can be repeatedly localized and measured. First, protocols and optimization steps for DNA carriers are presented, forming the foundation for the experiments described in the preceding chapter. Through these DNA carriers, molecules of interest can be positioned at programmable locations for repeated interrogation. This work presents the detection of DNA gaps, RNA structures, peptides, and protein-binding aptamers. To establish and demonstrate the resolution of this system, the size of single-stranded regions within a long double-stranded DNA construct (DNA gaps) is progressively reduced from 80, 40, 20, and 12 to a single missing base pair. By repeatedly sensing the same molecule (80b DNA gap) and averaging the measurements, a 20-fold reduction in RMS was observed compared to single-molecule SICS readout. This approach enabled the detection of a single missing base pair, representing the highest resolution achieved with a solid-state nanopore. The improved signal-to-noise ratio enabled protein detection using aptamer-based targets at lower ionic strength, reaching physiological conditions (150 mM KCl), and allows detection of the target proteins in human serum (protein mixture). This enabled extending the SICS platform as a diagnostic tool and improving its detection rate by multiplexing targets onto a single DNA carrier (3x compared with free translocation measurement). The system was also applied to short peptide sequences of varying charge, using the voltage-dependent force exerted by the nanopore to unfold peptides and maintain an extended conformation for more accurate sensing. Finally, RNA structure reconstruction was demonstrated by analyzing translocation signals to infer three-dimensional conformations. The second part of the thesis examines the topology and material properties of biomolecular condensates formed by the DEAD-box Dhh1 protein. Recent studies have shown that biomolecule condensate systems are sensitive to perturbations from fluorescence tags. First, available label-free techniques for probing biomolecular condensates were reviewed