Search

Understanding single-molecule multivalent ligand-receptor interactions is crucial for comprehending molecular recognition at biological interfaces. However, label-free identifications of these transient interactions during multistep binding processes remains challenging. Herein, we introduce a ligand-receptor-anchored nanopore that allows the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, we clarify two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent. Real-time kinetic analysis at the single-monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (-13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. Our single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.

Chirality is essential in nearly all biological organizations and chemical reactions but is rarely considered due to technical limitations in identifying L/D isomerization. Using OmpF, a membrane channel from Escherichia coli with an electrostatically asymmetric constriction zone, allows discriminating chiral amino acids in a single peptide. The heterogeneous distribution of charged residues in OmpF causes a strong lateral electrostatic field at the constriction. This laterally asymmetric constriction zone forces the sidechains of the peptides to specific orientations within OmpF, causing distinct ionic current fluctuations. Using statistical analysis of the respective ionic current variations allows distinguishing the presence and position of a single amino acid with different chiralities. To explore potential applications, the disease-related peptide β-Amyloid and its d-Asp 1 isoform and a mixture of the icatibant peptide drug (HOE 140) and its d-Ser 7 mutant have been discriminated. Both chiral isomers were not applicable to be distinguished by mass spectroscopy approaches. These findings highlight a novel sensing mechanism for identifying single amino acids in single peptides and even for achieving single-molecule protein sequencing.

Single-molecule measurements have greatly enhanced our understanding of living systems. Biological systems offer nanopores, a sub-class of membrane proteins, the well-defined confined space for accommodating a single molecule. The biological nanopore acts as a single-biomolecule interface for capturing and identifying a single molecule of interest, and thus it can be used as a single-molecule sensor. In this Perspective, we focus on biological nanopore-based single-biomolecule interfaces for single-biomolecule detection. First, we outline the design of the nanopore-based single-biomolecule interface, which provides rich stochastic information regarding each biomolecule. Next, we highlight future research directions beyond DNA sequencing, including detection of rare species, identification of hidden intermediates, spectral analysis of covalent/noncovalent interactions, and tracing of the dynamic pathways of single-biopolymer behaviors. The concept of a "single-molecule ionic spectrum" is discussed, which may allow mapping of noncovalent interactions at an atomic level in the future. We also discuss the challenges and goals for the future to make this measurement possible for addressing entirely new types of biological questions, which would be an exciting area of future research.

Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.