Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

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Quantifying protein-protein interactions by molecular counting with mass photometry

10.1101/2020.01.31.925156 ◽

2020 ◽

Author(s):

Fabian Soltermann ◽

Eric D.B. Foley ◽

Veronica Pagnoni ◽

Martin R. Galpin ◽

Justin L.P. Benesch ◽

...

Keyword(s):

Molecular Mass ◽

Single Molecule ◽

Protein Interactions ◽

Analytical Techniques ◽

Binding Affinities ◽

Label Free ◽

Protein Protein Interactions ◽

Single Molecule Level ◽

Minimal Invasiveness ◽

Universal Approach

AbstractInteractions between biomolecules control the processes of life in health, and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are particular desirable because of their simplicity and minimal invasiveness, but struggle to quantify tight interactions. Here, we show that we can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different un-labelled biomolecules and their complexes in mixtures at the single-molecule level by mass photometry. These measurements enable us to quantify binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as to determine the associated kinetics. Our results introduce mass photometry as a rapid, simple and label-free method for studying sub-μM binding affinities, with potential to be extended towards a universal approach for characterising complex biomolecular interactions.

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Cellular Lensing and Near Infrared Fluorescent Nanosensor Arrays to Enable Chemical Efflux Cytometry

10.21203/rs.3.rs-106483/v1 ◽

2020 ◽

Author(s):

Soo-Yeon Cho ◽

Xun Gong ◽

Volodymyr Koman ◽

Matthias Kuehne ◽

Sun Jin Moon ◽

...

Keyword(s):

High Throughput ◽

Single Molecule ◽

Near Infrared ◽

Microfluidic Channel ◽

Human Monocyte ◽

Cellular Heterogeneity ◽

Label Free ◽

Temporal Precision ◽

Single Molecule Level ◽

Chemical Cytometry

Abstract Nanosensor have proven to be powerful tools to monitor single biological cells and organisms, achieving spatial and temporal precision even at the single molecule level. However, there has not been a way of extending this approach to statistically relevant numbers of living cells and organisms. Herein, we design and fabricate a high throughput nanosensor array in a microfluidic channel that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). An array of nIR fluorescent single walled carbon nanotube (SWNT) nanosensors is integrated along a microfluidic channel through which a population of flowing cells is guided. We show that one can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR emission profiles and extract rich information on a per cell basis at high throughput. This unique biophotonic waveguide allows for quantified cross-correlation of the biomolecular information with physical properties such as cellular diameter, refractive index (RI), and eccentricity and creates a label-free chemical cytometer for the measurement of cellular heterogeneity with unprecedented precision. As an example, the NCC can profile the immune response heterogeneities of distinct human monocyte populations at attomolar (10-18 moles) sensitivity in a completely non-destructive and real-time manner with a rate of ~100 cells/frame, highest range demonstrated to date for state of the art chemical cytometry. We demonstrate distinct H2O2 efflux heterogeneities between 330 and 624 attomole/cell·min with cell projected areas between 271 and 263 µm2, eccentricity values between 0.405 and 0.363 and RI values between 1.383 and 1.377 for non-activated and activated human monocytes, respectively. Hence, we show that our nanotechnology based biophotonic cytometer has significant potential and versatility to answer important questions and provide new insight in immunology, cell manufacturing and biopharmaceutical research.

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A label-free approach to detect ligand binding to cell surface proteins in real time

10.1101/251629 ◽

2018 ◽

Author(s):

Verena Burtscher ◽

Matej Hotka ◽

Yang Li ◽

Michael Freissmuth ◽

Walter Sandtner

Keyword(s):

Ligand Binding ◽

Real Time ◽

Single Molecule ◽

Membrane Capacitance ◽

Surface Proteins ◽

Displacement Current ◽

High Temporal Resolution ◽

Label Free ◽

Single Molecule Level ◽

Electrophysiological Recordings

AbstractElectrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

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PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

10.1101/2022.01.09.475569 ◽

2022 ◽

Author(s):

Yizhe Zhang ◽

David A Weitz

Keyword(s):

Single Molecule ◽

High Specificity ◽

Label Free ◽

Hybrid Strategy ◽

Single Molecule Level ◽

Target Dna ◽

Label Free Detection ◽

Novel Method ◽

Picoliter Droplet

We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.

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A label-free approach to detect ligand binding to cell surface proteins in real time

eLife ◽

10.7554/elife.34944 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 10

Author(s):

Verena Burtscher ◽

Matej Hotka ◽

Yang Li ◽

Michael Freissmuth ◽

Walter Sandtner

Keyword(s):

Ligand Binding ◽

Real Time ◽

Single Molecule ◽

Membrane Capacitance ◽

Surface Proteins ◽

High Temporal Resolution ◽

Label Free ◽

Single Molecule Level ◽

Electrophysiological Recordings ◽

Displacement Currents

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

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Label-free tracking and mass measurement of single proteins on lipid bilayers

10.1101/2021.04.08.438951 ◽

2021 ◽

Author(s):

Eric Dylan Benjamin Foley ◽

Manish Kushwah ◽

Gavin Young ◽

Philipp Kukura

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Mass Measurement ◽

Label Free ◽

Single Molecule Level ◽

Dynamic Mass ◽

Membrane Affinity ◽

Fundamental Building Block ◽

Quantitative Studies ◽

Associated Proteins

We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

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A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

Research ◽

10.34133/2019/1050735 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Yi-Lun Ying ◽

Jie Yang ◽

Fu-Na Meng ◽

Shuang Li ◽

Meng-Ying Li ◽

...

Keyword(s):

Phosphatase Activity ◽

Single Molecule ◽

Critical Role ◽

Label Free ◽

Peptide Substrates ◽

Regulatory Pathways ◽

Single Molecule Level ◽

Cellular Processes ◽

One Step ◽

Targeted Drug Discovery

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

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Flexible SERS Sensors Based on Carbon Nanomaterials-Supported Au Nanostructures

Engineering Proceedings ◽

10.3390/i3s2021dresden-10075 ◽

2021 ◽

Vol 6 (1) ◽

pp. 7

Author(s):

Rong Yang ◽

Weichen Fang ◽

Xiao Zuo ◽

Igor M. De Rosa ◽

Wenbo Xin

Keyword(s):

Single Molecule ◽

High Performance ◽

Carbon Nanomaterials ◽

Label Free ◽

Single Molecule Level ◽

Low Concentrations ◽

Surface Enhanced ◽

Surface Enhanced Raman ◽

Enhanced Raman Scattering ◽

Au Nanowires

Surface-enhanced Raman scattering (SERS) is a powerful technique to detect analytes in a label-free and non-destructive way at extremely low concentrations, even down to the single-molecule level. In the present study, a series of anisotropic Au nanostructures are integrated onto the platforms of carbon nanomaterials, mainly including carbon nanotubes (CNTs) and graphene, in order to fabricate high-performance flexible SERS sensors. Sizes, dimensions, and shapes of Au nanostructures can be well controlled through this strategy, based on which Au nanowires, nanoribbons, nanoplates, nanobelts, and nanoframes are successfully deposited onto CNT films and graphene templates, respectively. Significantly enhanced plasmonic activity originates from these Au nanocrystals, which provide increased SERS signals of the analytes by many orders of magnitude, while CNT films or graphene substrates offer superior flexibility and accessibility. For instance, A flexible SERS sensor made of graphene supported Au nanoframes can detect the analyte R6G at the concentration as low as 10−9 M. The mechanism for the sensitivity enhancement could be attributed to the homogenous distribution of Au nanoframes on the graphene support as well as the strong molecule adsorption to the graphene nanoporous network.

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Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

2020 ◽

Author(s):

Nikolas Hundt

Keyword(s):

Single Molecule ◽

Cellular Systems ◽

Single Molecule Imaging ◽

Label Free ◽

Measurement Sensitivity ◽

Mass Distributions ◽

Quantitative Measurements ◽

Contrast Mechanism ◽

Cellular Components ◽

Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

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