Single Biomolecules Research Articles

Inversion of Circularly Polarized Luminescence in the Left-Handed Chitosan-Templated Co-assemblies.

Circularly polarized luminescence (CPL) materials are attractive due to their unique applications in fields such as 3D displays, information encryption, and chiroptical switches. Natural biomolecules-based CPL materials are gaining plenty of attention due to their chiral diversity and sustainability. However, it is still challenging to construct CPL materials with opposite CPL signs from a single natural biomolecule due to its inherent chirality. Here, chiral assemblies with opposite CPL signs using chitosan oligosaccharide (COS) and achiral luminescent dyes are successfully prepared. It is found that COS can serve as a chiral template to induce the ordered assembly of the dyes along the polymer chain through electrostatic attraction interaction. It is demonstrated experimentally that the structural planarity of the dye molecules is crucial for the formation of chiral co-assemblies. Interestingly, the left-handed COS-templated co-assemblies can emit CPL with opposite handedness, which is controlled by the helicity degree of the co-assemblies. This study not only deepens the understanding of the complex assembly of natural biomacromolecules but also provides new insights into the design and construction of CPL materials.

Pretrained Deep Neural Network Kin-SiM for Single-Molecule FRET Trace Idealization.

Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a pivotal technique for probing biomolecular dynamics over time at nanometer scales. Quantitative analyses of smFRET time traces remain challenging due to confounding factors such as low signal-to-noise ratios, photophysical effects such as bleaching and blinking, and the complexity of modeling the underlying biomolecular states and kinetics. The dynamic distance information shaping the smFRET trace powerfully uncovers even transient conformational changes in single biomolecules both at or far from equilibrium, relying on trace idealization to identify specific interconverting states. Conventional trace idealization methods based on hidden Markov models (HMMs) require substantial a priori knowledge of the system under study, manual intervention, and assumptions about the number of states and transition probabilities. Here, we present a deep learning framework using long short-term memory (LSTM) to automate the trace idealization, termed Kin-SiM. Our approach employs neural networks pretrained on simulated data to learn high-order correlations in the multidimensional FRET trajectories. Without user input of Markovian assumptions, the trained LSTM networks directly idealize the FRET traces to extract the number of underlying biomolecular states, their interstate dynamics, and associated kinetic parameters. On benchmark smFRET data sets, Kin-SiM achieves a performance similar to conventional HMM-based methods but with less hands-on time and lower risk of bias. We further systematically evaluate the key training factors that affect network performance to define the correct hyperparameter tuning for applying deep neural networks to smFRET data analyses.

Aptamer-based biotherapeutic conjugate for shear responsive release of Von Willebrand factor A1 domain.

Smart polymers that mimic and even surpass the functionality of natural responsive materials have been actively researched. This study explores the design and characterization of a Single-MOlecule-based material REsponsive to Shear (SMORES) for the targeted release of A1, the platelet binding domain of the blood clotting protein von Willebrand factor (VWF). Each SMORES construct employs an aptamer molecule as the flow transducer and a microparticle to sense and amplify the hydrodynamic force. Within the construct, the aptamer, ARC1172, undergoes conformational changes beyond a shear stress threshold, mimicking the shear-responsive behavior of VWF. This conformational alteration modulates the bioavailability of its target, the VWF-A1 domain, ultimately releasing it at elevated shear. Through optical tweezer-based single-molecule force measurement, ARC1172s role as a force transducer was assessed by examining its unfolding under constant pulling force. We also investigated its refolding rate as a function of force under varied relaxation periods. These analyses revealed a narrow range of threshold forces (3-7 pN) governing the transition between folded and unfolded states. We subsequently constructed the SMORES material by conjugating ARC1172 and a microbead, and immobilizing the other end of the aptamer on a substrate. Single-molecule flow experiments on immobilized SMORES constructs revealed a peak A1 domain release within a flow rate range of (40-70 μL min-1). A COMSOL Multiphysics model translated these flow rates to total forces of 3.10 pN-5.63 pN experienced by the aptamers, aligning with single-molecule force microscopy predictions. Evaluation under variable flow conditions showed a peak binding of A1 to the platelet glycoprotein Ib (GPIB) within the same force range, confirming released payload functionality. Building on knowledge of aptamer biomechanics, this study presents a new strategy to create shear-stimulated biomaterials based on single biomolecules.

Development of MHz X-ray phase contrast imaging at the European XFEL.

We report on recent developments that enable megahertz hard X-ray phase contrast imaging (MHz XPCI) experiments at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL facility (EuXFEL). We describe the technical implementation of the key components, including an MHz fast camera and a modular indirect X-ray microscope system based on fast scintillators coupled through a high-resolution optical microscope, which enable full-field X-ray microscopy with phase contrast of fast and irreversible phenomena. The image quality for MHz XPCI data showed significant improvement compared with a pilot demonstration of the technique using parallel beam illumination, which also allows access to up to 24 keV photon energies at the SPB/SFX instrument of the EuXFEL. With these developments, MHz XPCI was implemented as a new method offered for a broad user community (academic and industrial) and is accessible via standard user proposals. Furthermore, intra-train pulse diagnostics with a high few-micrometre spatial resolution and recording up to 128 images of consecutive pulses in a train at up to 1.1 MHz repetition rate is available upstream of the instrument. Together with the diagnostic camera upstream of the instrument and the MHz XPCI setup at the SPB/SFX instrument, simultaneous two-plane measurements for future beam studies and feedback for machine parameter tuning are now possible.

Towards Point-of-Care Single Biomolecule Detection Using Next Generation Portable Nanoplasmonic Biosensors: A Review.

Over the past few years, nanoplasmonic biosensors have gained widespread interest for early diagnosis of diseases thanks to their simple design, low detection limit down to the biomolecule level, high sensitivity to even small molecules, cost-effectiveness, and potential for miniaturization, to name but a few benefits. These intrinsic natures of the technology make it the perfect solution for compact and portable designs that combine sampling, analysis, and measurement into a miniaturized chip. This review summarizes applications, theoretical modeling, and research on portable nanoplasmonic biosensor designs. In order to develop portable designs, three basic components have been miniaturized: light sources, plasmonic chips, and photodetectors. There are five types of portable designs: portable SPR, miniaturized components, flexible, wearable SERS-based, and microfluidic. The latter design also reduces diffusion times and allows small amounts of samples to be delivered near plasmonic chips. The properties of nanomaterials and nanostructures are also discussed, which have improved biosensor performance metrics. Researchers have also made progress in improving the reproducibility of these biosensors, which is a major obstacle to their commercialization. Furthermore, future trends will focus on enhancing performance metrics, optimizing biorecognition, addressing practical constraints, considering surface chemistry, and employing emerging technologies. In the foreseeable future, these trends will be merged to result in portable nanoplasmonic biosensors offering detection of even a single biomolecule.

PlQC based highly sensitive and reproducible novel SERS active substrate for biomolecule detection with high specificity

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for biomolecule sensing. When combined with a broadband plasmonic structure, label-free, highly sensitive detection of specific molecules is possible. It is non-invasive, sensitive, fast, and can be used for in-situ analysis, unlike enzyme-linked immunosorbent assay, fluorescence immunoassay, and radioimmunoassay. However, one of the challenges is to have an active SERS substrate that is uniform, sensitive, and specific to molecules of interest. In this work, we report plasmonic quasicrystal (PlQC) as a highly sensitive (enhancement factor ≈ 1014), uniform, reproducible, and stable (concerning time and ambient conditions) SERS active substrate. Herein, we present the label-free sensing of standard cotinine (up to 1 ng/mL), the ideal biomarker for nicotine exposure due to its long lifetime compared to nicotine. In addition, up to 1 nanogram level of cotinine has also been detected in synthetic urine and saliva employing PlQC as a SERS-active substrate. Our results on the principal component analysis of Rhodamine 6G and Cotinine demonstrate that broadband, dispersionless PlQC is suitable for label-free detection of single biomolecules.

Detecting and Controlling DNA Translocation through a Nanopore using a van der Waals Heterojunction Diode.

A long-unrealized goal in solid-state nanopore sensing is to achieve out-of-plane electrical sensing and control of DNA during translocation, which is a prerequisite for base-by-base ratcheting that enables DNA sequencing in biological nanopores. Two-dimensional (2D) heterostructures, with their capability to construct out-of-plane electronics with atomic layer precision, are ideal yet unexplored candidates for use as electrical sensing membranes. Here we demonstrate a nanopore architecture using a vertical 2D heterojunction diode consisting of p-type WSe2 on n-type MoS2. This diode exhibits rectified interlayer tunneling currents modulated by ionic potential, while the heterojunction potential reciprocally rectifies ionic transport through the nanopore. We achieve concurrent detection of DNA translocation using both ionic and diode currents and demonstrate a 2.3-fold electrostatic slowing of translocation speed. Encapsulation layers enable robust operation while preserving the spatial resolution of atomically sharp 2D heterointerface for sensing. These results establish a paradigm for out-of-plane electrical sensing and control of single biomolecules.

Controlled molecule injector for cold, dense, and pure molecular beams at the European x-ray free-electron laser.

A permanently available molecular-beam injection setup for controlled molecules (COMO) was installed and commissioned at the small quantum systems (SQS) instrument at the European x-ray free-electron laser (EuXFEL). A b-type electrostatic deflector allows for pure state-, size-, and isomer-selected samples of polar molecules and clusters. The source provides a rotationally cold (T ≈ 1 K) and dense (ρ ≈ 108 cm-3) molecular beam with pulse durations up to 100 µs generated by a new version of the Even-Lavie valve. Here, a performance overview of the COMO setup is presented along with characterization experiments performed both with an optical laser at the Center for Free-Electron-Laser Science and with x rays at EuXFEL under burst-mode operation. COMO was designed to be attached to different instruments at the EuXFEL, in particular, the SQS and single particles, clusters, and biomolecules (SPB) instruments. This advanced controlled-molecules injection setup enables x-ray free-electron laser studies using highly defined samples with soft and hard x-ray FEL radiation for applications ranging from atomic, molecular, and cluster physics to elementary processes in chemistry and biology.

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

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.

Modeling Conformational Transitions of Biomolecules from Atomic Force Microscopy Images using Normal Mode Analysis.

Observing a single biomolecule performing its function is fundamental in biophysics as it provides important information for elucidating the mechanism. High-speed atomic force microscopy (HS-AFM) is a unique and powerful technique that allows the observation of biomolecular motion in a near-native environment. However, the spatial resolution of HS-AFM is limited by the physical size of the cantilever tip, which restricts the ability to obtain atomic details of molecules. In this study, we propose a novel computational algorithm designed to derive atomistic models of conformational dynamics from AFM images. Our method uses normal-mode analysis to describe the expected motions of the molecule, allowing these motions to be represented with a limited number of coordinates. This approach mitigates the problem of overinterpretation inherent in the analysis of AFM images with limited resolution. We demonstrate the effectiveness of our algorithm, NMFF-AFM, using synthetic data sets for three proteins that undergo significant conformational changes. NMFF-AFM is a fast and user-friendly program that requires minimal setup and has the potential to be a valuable tool for biophysical studies using HS-AFM.

Large-enhancement nanoscale dynamic nuclear polarization near a silicon nanowire surface.

Dynamic nuclear polarization (DNP) has revolutionized the field of nuclear magnetic resonance spectroscopy, expanding its reach and capabilities to investigate diverse materials, biomolecules, and complex dynamic processes. Bringing high-efficiency DNP to the nanometer scale would open exciting avenues for studying nanoscale nuclear spin ensembles, such as single biomolecules, virus particles, and condensed matter systems. Combining pulsed DNP with nanoscale force-detected magnetic resonance measurements, we demonstrated a 100-fold enhancement in the Boltzmann polarization of proton spins in nanoscale sugar droplets at 6 kelvin and 0.33 tesla. Crucially, this enhancement corresponds to a factor of 200 reduction in the averaging time compared to measurements that rely on the detection of statistical fluctuations in nanoscale nuclear spin ensembles. These results substantially advance the capabilities of force-detected magnetic resonance detection as a practical tool for nanoscale imaging.

DNA origami-templated gold nanorod dimer nanoantennas: enabling addressable optical hotspots for single cancer biomarker SERS detection.

The convergence of DNA origami and surface-enhanced Raman spectroscopy (SERS) has opened a new avenue in bioanalytical sciences, particularly in the detection of single-molecule proteins. This breakthrough has enabled the development of advanced sensor technologies for diagnostics. DNA origami offers a highly controllable framework for the precise positioning of nanostructures, resulting in superior SERS signal amplification. In our investigation, we have successfully designed and synthesized DNA origami-based gold nanorod monomer and dimer assemblies. Moreover, we have evaluated the potential of dimer assemblies for label-free detection of a single biomolecule, namely epidermal growth factor receptor (EGFR), a crucial biomarker in cancer research. Our findings have revealed that the significant Raman amplification generated by DNA origami-assembled gold nanorod dimer nanoantennas facilitates the label-free identification of Raman peaks of single proteins, which is a prime aim in biomedical diagnostics. The present work represents a significant advancement in leveraging plasmonic nanoantennas to realize single protein SERS for the detection of various cancer biomarkers with single-molecule sensitivity.

Watching a Single Enzyme at Work Using Single-Molecule Surface-Enhanced Raman Scattering and DNA Origami-Based Plasmonic Antennas.

The detection of a single-enzyme catalytic reaction by surfaced-enhanced Raman scattering (SERS) is presented by utilizing DNA origami-based plasmonic antennas. A single horseradish peroxidase (HRP) was accommodated on a DNA origami nanofork plasmonic antenna (DONA) containing gold nanoparticles, enabling the tracing of single-molecule SERS signals during the peroxide reduction reaction. This allows monitoring of the structure of a single enzymatic catalytic center and products under suitable liquid conditions. Herein, we demonstrate the chemical changes of HRP and the appearance of tetramethylbenzidine (TMB), which works as a hydrogen donor before and after the catalytic reaction. The results show that the iron in HRP adopts Fe4+ and low spin states with the introduction of H2O2, indicating compound-I formation. Density functional theory (DFT) calculations were performed for later catalytic steps to rationalize the experimental Raman/SERS spectra. The presented data provide several possibilities for tracking single biomolecules in situ during a chemical reaction and further developing plasmon-enhanced biocatalysis.

High-precision high-speed nanopore ping-pong control system based on field programmable gate array.

"Molecular ping-pong," emerging as a control strategy in solid-state nanopore technology, presents a highly promising approach for repetitive measurements of single biomolecules, such as DNA. This paper introduces a high-precision, high-speed nanopore molecular ping-pong control system consisting of a home-built trans-impedance amplifier (TIA), a control system based on a Field Programmable Gate Array (FPGA), and a LabVIEW program operating on the host personal computer. Through feedback compensation and post-stage boosting, the TIA achieves a high bandwidth of about 200kHz with a gain of 100 MΩ, along with low input-referred current noise of 1.6 × 10-4pA2/Hz at 1kHz and 1.1 × 10-3pA2/Hz at 100kHz. The FPGA-based control system demonstrates a minimum overall response time (tdelay) of 6.5 μs from the analog input current signal trigger to the subsequent reversal of the analog output drive voltage signal, with a control precision of 1 μs. Additionally, a LabVIEW program has been developed to facilitate rapid data exchange and communication with the FPGA program, enabling real-time signal monitoring, parameter adjustment, and data storage. Successful recapture of individual DNA molecules at various tdelay, resulting in an improvement in capture rate by up to 2 orders of magnitude, has been demonstrated. With unprecedented control precision and capture efficiency, this system provides robust technical support and opens novel research avenues for nanopore single-molecule sensing and manipulation.

Lifting the Concentration Limit of Mass Photometry by PEG Nanopatterning.

Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to substantially lower surface binding, resulting in a 2 orders of magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate the facile tunability of degree of passivation, enabling measurements at increased analyte concentrations. These advances provide access to protein-protein interactions in the high nanomolar to low micromolar range, substantially expanding the application space of mass photometry.

GENESIS 2.1: High-Performance Molecular Dynamics Software for Enhanced Sampling and Free-Energy Calculations for Atomistic, Coarse-Grained, and Quantum Mechanics/Molecular Mechanics Models.

GENeralized-Ensemble SImulation System (GENESIS) is a molecular dynamics (MD) software developed to simulate the conformational dynamics of a single biomolecule, as well as molecular interactions in large biomolecular assemblies and between multiple biomolecules in cellular environments. To achieve the latter purpose, the earlier versions of GENESIS emphasized high performance in atomistic MD simulations on massively parallel supercomputers, with or without graphics processing units (GPUs). Here, we implemented multiscale MD simulations that include atomistic, coarse-grained, and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. They demonstrate high performance and are integrated with enhanced conformational sampling algorithms and free-energy calculations without using external programs except for the QM programs. In this article, we review new functions, molecular models, and other essential features in GENESIS version 2.1 and discuss ongoing developments for future releases.

Self-Assembled Block Copolymers as a Facile Pathway to Create Functional Nanobiosensor and Nanobiomaterial Surfaces.

Block copolymer (BCP) surfaces permit an exquisite level of nanoscale control in biomolecular assemblies solely based on self-assembly. Owing to this, BCP-based biomolecular assembly represents a much-needed, new paradigm for creating nanobiosensors and nanobiomaterials without the need for costly and time-consuming fabrication steps. Research endeavors in the BCP nanobiotechnology field have led to stimulating results that can promote our current understanding of biomolecular interactions at a solid interface to the never-explored size regimes comparable to individual biomolecules. Encouraging research outcomes have also been reported for the stability and activity of biomolecules bound on BCP thin film surfaces. A wide range of single and multicomponent biomolecules and BCP systems has been assessed to substantiate the potential utility in practical applications as next-generation nanobiosensors, nanobiodevices, and biomaterials. To this end, this Review highlights pioneering research efforts made in the BCP nanobiotechnology area. The discussions will be focused on those works particularly pertaining to nanoscale surface assembly of functional biomolecules, biomolecular interaction properties unique to nanoscale polymer interfaces, functionality of nanoscale surface-bound biomolecules, and specific examples in biosensing. Systems involving the incorporation of biomolecules as one of the blocks in BCPs, i.e., DNA-BCP hybrids, protein-BCP conjugates, and isolated BCP micelles of bioligand carriers used in drug delivery, are outside of the scope of this Review. Looking ahead, there awaits plenty of exciting research opportunities to advance the research field of BCP nanobiotechnology by capitalizing on the fundamental groundwork laid so far for the biomolecular interactions on BCP surfaces. In order to better guide the path forward, key fundamental questions yet to be addressed by the field are identified. In addition, future research directions of BCP nanobiotechnology are contemplated in the concluding section of this Review.

Review—Nanosystems-Enhanced Electrochemical Biosensors for Precision in One Health Management

One Health is a comprehensive and cohesive strategy that seeks to effectively manage and enhance the well-being of people, animals, and ecosystems in a sustainable manner. Electrochemical biosensors are indispensable vectors for One Health management, with diverse applications across industrial process control, environmental monitoring, healthcare, microbiology, and quality assurance. The integration of nanotechnology has propelled innovative sensing algorithms, enhancing the efficiency and precision of biosensors. Nanoscale material integration amplifies their ability to detect molecules and single biomolecules with nano-biosensors utilizing nanoparticles, nanowires, carbon nanotubes (CNTs), nanorods, and quantum dots. Nanomaterials, distinguished by surface functionalities, tunability of physical/chemical properties, high detection sensitivity, large surface area, stability, and thermal/electrical conductivity, are instrumental in architecting nano-biosensors. The main limitations in current times are scalability, cross-sensitivity, complex immobilization pathways, and the toxicity factor of nanosensors, which are hazardous for the ecosystem as well as humans. This review explores the fundamentals of nanotechnology-enabled electrochemical biosensors, encompassing their composition, manufacturing materials, and diverse applications, highlighting their necessity in advancing biosensor technology to control environmental contamination/human diseases in order to manage One Health.

MINSTED tracking of single biomolecules

Here we show that MINSTED localization, a method whereby the position of a fluorophore is identified with precisely controlled beams of a STED microscope, tracks fluorophores and hence labeled biomolecules with nanometer/millisecond spatiotemporal precision. By updating the position for each detected photon, MINSTED recognizes fluorophore steps of 16 nm within <250 μs using about 13 photons. The power of MINSTED tracking is demonstrated by resolving the stepping of the motor protein kinesin-1 walking on microtubules and switching protofilaments.

Single-protein optical holography

Light scattering by nanoscale objects is a fundamental physical property defined by their scattering cross-section and thus polarizability. Over the past decade, a number of studies have demonstrated single-molecule sensitivity by imaging the interference between scattering from the object of interest and a reference field. This approach has enabled mass measurement of single biomolecules in solution owing to the linear scaling of image contrast with molecular polarizability. Nevertheless, all implementations so far are based on a common-path interferometer and cannot separate and independently tune the reference and scattered light fields, thereby prohibiting access to the rich toolbox available to holographic imaging. Here we demonstrate comparable sensitivity using a non-common-path geometry based on a dark-field scattering microscope, similar to a Mach–Zehnder interferometer. We separate the scattering and reference light into four parallel, inherently phase-stable detection channels, delivering a five orders of magnitude boost in sensitivity in terms of scattering cross-section over state-of-the-art holographic methods. We demonstrate the detection, resolution and mass measurement of single proteins with mass below 100 kDa. Separate amplitude and phase measurements also yield direct information on sample identity and experimental determination of the polarizability of single biomolecules.