monitoring-conformational-dynamics-single-unmodified-proteins-using

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

Nottingham Trent University

Resistive pulses generated by nanoparticles that translocate through a nanopore contain multi-parametric information about the physical properties of those particles. For example, non-spherical particles sample several different orientations during translocation, producing fluctuations in blockade current that relate to their shape. Due to the heterogenous distribution of electric field from the center to the wall of a nanopore while a particle travels through the pore, its radial position influences the blockade current, thereby affecting the quantification of parameters related to the particle's characteristics. Here, we investigate the influence of these off-axis effects on parameters estimated by performing finite element simulations of dielectric particles transiting a cylindrical nanopore. We varied the size, ellipsoidal shape, and radial position of individual particles, as well as the size of the nanopore. As expected, nanoparticles translocating near the nanopore wall produce increase current blockades, resulting in overestimates of particle volume. We demonstrated that off-axis effects also influence estimates of shape determined from resistive pulse analyses, sometimes producing a multiple-fold deviation in ellipsoidal length-to-diameter ratio between estimates and reference values. By using a nanopore with the minimum possible diameter that still allows the particle to rotate while translocating, off-axis effects on the determination of both volume and shape can be minimized. In addition, tethering the nanoparticles to a fluid coating on the nanopore wall makes it possible to determine an accurate particle shape with an overestimated volume. This work provides a framework to select optimal ratios of nanopore to nanoparticle size for experiments targeting free translocations.

Effects of off-axis translocation through nanopores on the determination of shape and volume estimates for individual particles.

Nanopore sensors provide a unique platform to detect individual nucleic acids, proteins, and other biomolecules without the need for fluorescent labeling or chemical modifications. Solid-state nanopores offer the potential to integrate nanopore sensing with other technologies such as field-effect transistors (FETs), optics, plasmonics, and microfluidics, thereby attracting attention to the development of commercial instruments for diagnostics and healthcare applications. Stable nanopores with ideal dimensions are particularly critical for nanopore sensors to be integrated into other sensing devices and provide a high signal-to-noise ratio. Nanopore fabrication, although having benefited largely from the development of sophisticated nanofabrication techniques, remains a challenge in terms of cost, time consumption and accessibility. One of the latest developed methods-controlled breakdown (CBD)-has made the nanopore technique broadly accessible, boosting the use of nanopore sensing in both fundamental research and biomedical applications. Many works have been developed to improve the efficiency and robustness of pore formation by CBD. However, nanopores formed by traditional CBD are randomly positioned in the membrane. To expand nanopore sensing to a wider biomedical application, controlling the localization of nanopores formed by CBD is essential. This article reviews the recent strategies to control the location of nanopores formed by CBD. We discuss the fundamental mechanism and the efforts of different approaches to confine the region of nanopore formation.

Localized Nanopore Fabrication via Controlled Breakdown.

Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or single-molecule approaches to interrogate labeled or modified proteins. Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo- and holo-ferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.

Optical Monitoring of  Iron Loading into Single, Native Ferritin Proteins.

Two-dimensional material nanochannels with molecular-scale confinement can be constructed by Van der Waals assembly and show unexpected fluid transport phenomena. The crystal structure of the channel surface plays a key role in controlling fluid transportation, and many strange properties are explored in these confined channels. Here, we use black phosphorus as the channel surface to enable ion transport along a specific crystal orientation. We observed a significant nonlinear and anisotropic ion transport phenomenon in the black phosphorus nanochannels. Theoretical results revealed an anisotropy of ion transport energy barrier on the black phosphorus surface, with the minimum energy barrier along the armchair direction approximately ten times larger than that along the zigzag direction. This difference in energy barrier affects the electrophoretic and electroosmotic transport of ions in the channel. This anisotropic transport, which depends on the orientation of the crystal, may provide new approaches to controlling the transport of fluids.

Nonlinear and Anisotropic Ion Transport in Black Phosphorus Nanochannels.

Single unmodified biomolecules in solution can be observed and characterized by interferometric imaging approaches; however, Rayleigh scattering limits this to larger proteins (typically >30 kDa). We observe real-time image tracking of unmodified proteins down to 14 kDa using interference imaging enhanced by surface plasmons launched at an aperture in a metal film. The larger proteins show slower diffusion, quantified by tracking. When the diffusing protein is finally trapped by the nanoaperture, we perform complementary power spectral density and noise amplitude analysis, which gives information about the protein. This approach allows for rapid protein characterization with minimal sample preparation and opens the door to characterizing protein interactions in real time.

Label-Free Tracking of Proteins through Plasmon-Enhanced Interference.

Confining DNA in nanochannels is an important approach to studying its structure and transportation dynamics. Graphene nanochannels are particularly attractive for studying DNA confinement due to their atomic flatness, precise height control, and excellent mechanical strength. Here, using femtosecond laser etching and wetting transfer, we fabricate graphene nanochannels down to less than 4.3 nm in height, with the length-to-height ratios up to 10. These channels exhibit high stability, low noise, and self-cleaning ability during the long-term ionic current recording. We report a clear linear relationship between DNA length and the residence time in the channel and further utilize this relationship to differentiate DNA fragments based on their lengths, ranging widely from 200 bps to 48.5 kbps. The graphene nanochannel presented here provides a potential platform for label-free analyses and reveals fundamental insights into the conformational dynamics of DNA and proteins in confined space.

Electrical Transport and Dynamics of Confined DNA through Highly Conductive 2D Graphene Nanochannels.

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. Understanding how various chemicals affect the structural behavior of ferritin is crucial for unravelling the origins of iron-related diseases in living organisms including humans. In particular, the influence of chemicals on ferritin's dynamics and iron release is barely explored at the single-protein level. Here, by employing optical nanotweezers using double-nanohole (DNH) structures, we examined the effect of ascorbic acid (reducing reagent) and pH on individual ferritin's conformational dynamics. The dynamics of ferritin increased as the concentration of ascorbic acid approached saturation. At pH 2.0, ferritin exhibited significant structural fluctuations and eventually underwent a stepwise disassembly into fragments. This work demonstrated the disassembly pathway and kinetics of a single ferritin molecule in solution. We identified four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer, and dimer subunits. Moreover, we present single-molecule evidence of the cooperative disassembly of ferritin. Interrogating ferritin's structural change in response to different chemicals holds importance for understanding their roles in iron metabolism, hence facilitating further development of medical treatments for its associated diseases.

Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers.

Nonlinear metasurfaces have experienced rapid growth recently due to their potential in various applications, including infrared imaging and spectroscopy. However, due to the low conversion efficiencies of metasurfaces, several strategies have been adopted to enhance their performances, including employing resonances at signal or nonlinear emission wavelengths. This strategy results in a narrow operational band of the nonlinear metasurfaces, which has bottlenecked many applications, including nonlinear holography, image encoding, and nonlinear metalenses. Here, we overcome this issue by introducing a new nonlinear imaging platform utilizing a pump beam to enhance signal conversion through four-wave mixing (FWM), whereby the metasurface is resonant at the pump wavelength rather than the signal or nonlinear emissions. As a result, we demonstrate broadband nonlinear imaging for arbitrary objects using metasurfaces. A silicon disk-on-slab metasurface is introduced with an excitable guided-mode resonance at the pump wavelength. This enabled direct conversion of a broad IR image ranging from >1000 to 4000 nm into visible. Importantly, adopting FWM substantially reduces the dependence on high-power signal inputs or resonant features at the signal beam of nonlinear imaging by utilizing the quadratic relationship between the pump beam intensity and the signal conversion efficiency. Our results, therefore, unlock the potential for broadband infrared imaging capabilities with metasurfaces, making a promising advancement for next-generation all-optical infrared imaging techniques with chip-scale photonic devices.

Broadband infrared imaging governed by guided-mode resonance in dielectric metasurfaces.

Single-molecule techniques to analyze proteins and other biomolecules involving labels and tethers have allowed for new understanding of the underlying biophysics; however, the impact of perturbation from the labels and tethers has recently been shown to be significant in several cases. New approaches are emerging to measure single proteins through light scattering without the need for labels and ideally without tethers. Here, the approaches of interference scattering, plasmonic scattering, microcavity sensing, nanoaperture optical tweezing, and variants are described and compared. The application of these approaches to sizing, oligomerization, interactions, conformational dynamics, diffusion, and vibrational mode analysis is described. With early commercial successes, these approaches are poised to have an impact in the field of single-molecule biophysics.

Optical scattering methods for the label-free analysis of single biomolecules.

Resolving the free energy landscapes that govern protein biophysics has been obscured by ensemble averaging. While the folding dynamics of single proteins have been observed using fluorescent labels and/or tethers, a simpler and more direct measurement of the conformational changes would not require modifications to the protein. We use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein, Bovine Serum Albumin (BSA), and quantify changes in the three-state conformation dynamics with temperature. A Markov model with Kramers' theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including mapping out the energy landscape of particularly challenging intrinsically disordered proteins.

Energy landscape of conformational changes for a single unmodified protein.

Metasurfaces, composed of artificial meta-atoms of subwavelength size, can support strong light-matter interaction based on multipolar resonances and plasmonics, hence offering the great capability of empowering nonlinear generation. Recently, owing to their ability to manipulate the amplitude and phase of the nonlinear emission in the subwavelength scale, metasurfaces have been recognized as ultra-compact, flat optical components for a vast range of applications, including nonlinear imaging, quantum light sources, and ultrasensitive sensing. This review focuses on the recent progress on nonlinear metasurfaces for those applications. The principles and advances of metasurfaces-based techniques for image generation, including image encoding, holography, and metalens, are investigated and presented. Additionally, the overview and development of spontaneous photon pair generation from metasurfaces are demonstrated and discussed, focusing on the aspects of photon pair generation rate and entanglement of photon pairs. The recent blossoming of the nonlinear metasurfaces field has triggered growing interest to explore its ability to efficiently up-convert infrared images of arbitrary objects to visible images and achieve spontaneous parametric down-conversion. This recently emerged direction holds promising potential for the next-generation technology in night-vision, quantum computing, and biosensing fields.

Advances in nonlinear metasurfaces for imaging, quantum, and sensing applications.

Nanofluidics is an interdisciplinary field of study that bridges hydrodynamics, statistical physics, chemistry, materials science, biology, and other fields to investigate the transport of fluids and ions on the nanometric scale. The progress in this field, however, has been constrained by challenges in fabricating nanofluidic devices suitable for systematic investigations. Recent advances in two-dimensional (2D) materials have revolutionized the development of nanofluids. Their ultrathin structure and photothermoelectric response make it possible to achieve the scale control, friction limitation, and regulatory response, all of which are challenging to achieve with traditional solid materials. In this review, we provide a comprehensive overview of the preparation methods and corresponding structures of three types of 2D material-based nanofluidic devices, including nanopores, nanochannels, and membranes. We highlight their applications and recent advances in exploring physical mechanisms, detecting biomolecules (DNA, protein), developing iontronics devices, improving ion/gas selectivity, and generating osmotic energy. We discuss the challenges facing 2D material-based nanofluidic devices and the prospects for future advancements in this field.

Two-Dimensional Material-Based Nanofluidic Devices and Their Applications.

Ion transport through atomically thin nano/subnanopores, such as those in monolayer graphene, presents challenges to traditional ion conduction models, primarily due to extreme confinement effects and hydration interactions. Under these conditions, existing models fail to account for conductance behaviors at the nano- and subnanometer scales. In this study, we perform a combined experimental and theoretical investigation of ion transport in monolayer graphene nano/subnanopores across varying salt concentrations. We introduce a conductance model that accurately predicts the observed scaling behavior by addressing the interaction between counterions and the edges of atomically thin pores, where counterion movement is constrained by the pore's structure. This model also quantifies the hydration energy barrier, highlighting the impact of the hydration shell structures on ion transport efficiency. Our findings reveal that hydrated potassium ions traverse these pores with higher efficiency than previously estimated, offering new insights into ion transport mechanisms under atomic-scale confinement.

Article	Year
Effects of off-axis translocation through nanopores on the determination of shape and volume estimates for individual particles. Nanotechnology\| PubMed ID: 35320779	2022
Localized Nanopore Fabrication via Controlled Breakdown. Nanomaterials (Basel, Switzerland)\| PubMed ID: 35889608	2022
Optical Monitoring of Iron Loading into Single, Native Ferritin Proteins. Nano letters\| PubMed ID: 37053043	2023
Nonlinear and Anisotropic Ion Transport in Black Phosphorus Nanochannels. Nano letters\| PubMed ID: 37338120	2023
Label-Free Tracking of Proteins through Plasmon-Enhanced Interference. ACS nanoscience Au\| PubMed ID: 38406310	2024
Electrical Transport and Dynamics of Confined DNA through Highly Conductive 2D Graphene Nanochannels. Nano letters\| PubMed ID: 38578031	2024
Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers. ACS nano\| PubMed ID: 38850556	2024
Broadband infrared imaging governed by guided-mode resonance in dielectric metasurfaces. Light, science & applications\| PubMed ID: 39256381	2024
Optical scattering methods for the label-free analysis of single biomolecules. Quarterly reviews of biophysics\| PubMed ID: 39443300	2024
Energy landscape of conformational changes for a single unmodified protein. Npj biosensing\| PubMed ID: 39524907	2024
Advances in nonlinear metasurfaces for imaging, quantum, and sensing applications. Nanophotonics (Berlin, Germany)\| PubMed ID: 39634716	2023
Two-Dimensional Material-Based Nanofluidic Devices and Their Applications. ACS nano\| PubMed ID: 39783262	2025
Scaling Behavior and Conductance Mechanisms of Ion Transport in Atomically Thin Graphene Nano/Subnanopores. Nano letters\| PubMed ID: 39835730	2025

Cuifeng Ying

Biography

JoVE Journal Publications

Other Publications