Exploring single-molecule interactions: heparin and FGF-1 proteins through solid-state nanopores.

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization-mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag13 (in the intact DNA host) to Ag10 (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine.

The development of novel methodologies that can detect biomarkers from cancer or other diseases is both a challenge and a need for clinical applications. This partly motivates efforts related to nanopore-based peptide sensing. Recent work has focused on the use of gold nanoparticles for selective detection of cysteine-containing peptides. Specifically, tiopronin-capped gold nanoparticles, trapped in the cis-side of a wild-type α-hemolysin nanopore, provide a suitable anchor for the attachment of cysteine-containing peptides. It was recently shown that the attachment of these peptides onto a nanoparticle yields unique current signatures that can be used to identify the peptide. In this article, we apply this technique to the detection of ovarian cancer marker peptides ranging in length from 8 to 23 amino acid residues. It is found that sequence variability complicates the detection of low-molecular-weight peptides (<10 amino acid residues), but higher-molecular-weight peptides yield complex, high-frequency current fluctuations. These fluctuations are characterized with chi-squared and autocorrelation analyses that yield significantly improved selectivity when compared to traditional open-pore analysis. We demonstrate that the technique is capable of detecting the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. We further demonstrate the detection of one of these LRG-1 peptides spiked into a sample of human female urine.

Real-time monitoring of Ti(IV) metal ion binding of transferrin using a solid-state nanopore.

Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin's enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal-protein interactions at the molecular level.

Selective Detection and Characterization of Small Cysteine-Containing Peptides with Cluster-Modified Nanopore Sensing.

It was recently demonstrated that one can monitor ligand-induced structure fluctuations of individual thiolate-capped gold nanoclusters using resistive-pulse nanopore sensing. The magnitude of the fluctuations scales with the size of the capping ligand, and it was later shown one can observe ligand exchange in this nanopore setup. We expand on these results by exploring the different types of current fluctuations associated with peptide ligands attaching to tiopronin-capped gold nanoclusters. We show here that the fluctuations can be used to identify the attaching peptide through either the magnitude of the peptide-induced current jumps or the onset of high-frequency current fluctuations. Importantly, the peptide attachment process requires that the peptide contains a cysteine residue. This suggests that nanopore-based monitoring of peptide attachments with thiolate-capped clusters could provide a means for selective detection of cysteine-containing peptides. Finally, we demonstrate the cluster-based protocol with various peptide mixtures to show that one can identify more than one cysteine-containing peptide in a mixture.

Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding.

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Single-molecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg2+). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1-10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.

Nanopore sensing: A physical-chemical approach.

Protein nanopores have emerged as an important class of sensors for the understanding of biophysical processes, such as molecular transport across membranes, and for the detection and characterization of biopolymers. Here, we trace the development of these sensors from the Coulter counter and squid axon studies to the modern applications including exquisite detection of small volume changes and molecular reactions at the single molecule (or reactant) scale. This review focuses on the chemistry of biological pores, and how that influences the physical chemistry of molecular detection.