Self-crowding and ligand-binding assisted single-molecule detection of small molecules by nanopipettes

About This Grant

Small molecules, including small biomolecules and bioactive molecules, such as adenosine triphosphate (ATP), hormones, neurotransmitters, and pharmaceutical drugs, play critical roles within biological systems. Detecting them and understanding their functions is important. However, compared to larger biomarker species, small molecules pose heightened detection challenges. Detecting and analyzing label-free small molecules under physiological conditions is even more challenging. This project will develop new sensing mechanisms and approaches for a nanopore sensor. The new sensor will be ultrasensitive and selective for various small molecules. It will be portable, inexpensive, easy to use, and applicable to a variety of areas such as protein analysis, pharmaceutical research, clinical molecular diagnostics, environment protection and preservation, and national defense and bioterrorism prevention. The knowledge and methods obtained from this project will benefit molecular biophysics and biochemistry, single-entity electrochemistry techniques and other stochastic sensors. The highly interdisciplinary nature of this research will provide excellent opportunities for academic training of undergraduate and graduate students and outreach STEM programs for K-12 students of Miami-Dade County. Nanopore biosensors have shown great promise as single-molecule sensors for a wide variety of biomedical applications. Glass nanopipettes, as a subtype of solid-state nanopore, have become increasingly popular due to their versatility, easy accessibility, and simple and cheap fabrication, but they are severely limited by their poor size resolution for the detection of small molecules. This project aims to use nanopipettes to develop a self-crowding and ligand-binding assisted ultrasensitive label-free electrical detection method for small biomolecules and bioactive small molecules at the single molecule level under physiological conditions based on their size, structure, charge, mobility and affinity. The project has two research tasks: (1) Understanding the unique ionic signals induced by self-crowding at the nanopipette tip and (2) Developing a small molecule sensing approach based on ligand binding. The proposed approach has the potential to solve the major challenges in nanopore sensing by slowing down the small molecules in the nanopipette sensing zone through self-crowding. The accumulation of small charged molecules in a confined nanoscale space also induces new nanofluidic phenomena, leading to new sensing mechanisms other than the volume exclusion mechanism (or Coulter Counter principle). The tunable crowding at the nanopipette tip and the enabled high sensitivity and structure resolution for molecular complexes also provide exciting opportunities to study the stoichiometry, binding strength and dynamics of small molecule involved interactions in a crowded environment at physiological conditions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.