Cellular Lensing and Near Infrared Fluorescent Nanosensor Arrays to Enable Chemical Efflux 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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Cellular lensing and near infrared fluorescent nanosensor arrays to enable chemical efflux cytometry

Nature Communications ◽

10.1038/s41467-021-23416-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Soo-Yeon Cho ◽

Xun Gong ◽

Volodymyr B. Koman ◽

Matthias Kuehne ◽

Sun Jin Moon ◽

...

Keyword(s):

Near Infrared ◽

Single Cells ◽

Microfluidic Channel ◽

Human Monocyte ◽

Cellular Heterogeneity ◽

Label Free ◽

Nanotube Array ◽

Chemical Cytometry ◽

Rich Information ◽

Non Destructive

AbstractNanosensors have proven to be powerful tools to monitor single cells, achieving spatiotemporal precision even at molecular level. However, there has not been way of extending this approach to statistically relevant numbers of living cells. Herein, we design and fabricate nanosensor array in microfluidics that addresses this limitation, creating a Nanosensor Chemical Cytometry (NCC). nIR fluorescent carbon nanotube array is integrated along microfluidic channel through which flowing cells is guided. We can utilize the flowing cell itself as highly informative Gaussian lenses projecting nIR profiles and extract rich information. This unique biophotonic waveguide allows for quantified cross-correlation of biomolecular information with various physical properties and creates label-free chemical cytometer for cellular heterogeneity measurement. As an example, the NCC can profile the immune heterogeneities of human monocyte populations at attomolar sensitivity in completely non-destructive and real-time manner with rate of ~600 cells/hr, highest range demonstrated to date for state-of-the-art chemical cytometry.

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Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2010-13039 ◽

2010 ◽

Author(s):

Kyungsuk Yum ◽

Sungsoo Na ◽

Yang Xiang ◽

Ning Wang ◽

Min-Feng Yu

Keyword(s):

Single Molecule ◽

Living Cells ◽

Endocytic Pathway ◽

Great Promise ◽

Biological Processes ◽

Temporal Precision ◽

Single Molecule Level ◽

Cellular Processes ◽

Cellular Environment ◽

Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

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

Biophysical Journal ◽

10.1016/j.bpj.2017.11.2108 ◽

2018 ◽

Vol 114 (3) ◽

pp. 381a

Author(s):

Nikolas Hundt ◽

Andrew Tyler ◽

Gavin Young ◽

Daniel Cole ◽

Adam J. Fineberg ◽

...

Keyword(s):

Single Molecule ◽

Label Free ◽

Actin Nucleation ◽

Single Molecule Level

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Single-molecule nanopore sensing of actin dynamics and drug binding

10.1101/829150 ◽

2019 ◽

Author(s):

Xiaoyi Wang ◽

Mark D. Wilkinson ◽

Xiaoyan Lin ◽

Ren Ren ◽

Keith Willison ◽

...

Keyword(s):

Protein Folding ◽

Single Molecule ◽

Eukaryotic Cell ◽

Molecular State ◽

Drug Binding ◽

Actin Dynamics ◽

Label Free ◽

Single Molecule Level ◽

Native Proteins ◽

Voltage Dependent

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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Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

10.1101/201079 ◽

2017 ◽

Author(s):

Carel Fijen ◽

Mattia Fontana ◽

Serge G. Lemay ◽

Klaus Mathwig ◽

Johannes Hohlbein

Keyword(s):

High Throughput ◽

Single Molecule ◽

Single Particle Tracking ◽

Biological Interactions ◽

Dynamic Heterogeneity ◽

High Throughput Analysis ◽

Equilibrium Studies ◽

Single Molecule Level ◽

Nanofluidic Devices ◽

And Performance

ABSTRACTSingle-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

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DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽

10.3390/applnano3010002 ◽

2022 ◽

Vol 3 (1) ◽

pp. 16-41

Author(s):

Aurimas Kopūstas ◽

Mindaugas Zaremba ◽

Marijonas Tutkus

Keyword(s):

High Throughput ◽

Single Molecule ◽

Dna Interaction ◽

Dna Interactions ◽

Ensemble Averaging ◽

Target Search ◽

Single Molecule Level ◽

Protein Dna Interactions ◽

Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

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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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Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

10.1101/2021.07.29.454136 ◽

2021 ◽

Author(s):

Sebastian Ferdinand Konrad ◽

Willem Vanderlinden ◽

Jan Lipfert

Keyword(s):

High Throughput ◽

Single Molecule ◽

Mode Of Action ◽

Chromatin Accessibility ◽

Wild Type ◽

Analysis Pipeline ◽

Data Set ◽

Single Molecule Level ◽

Afm Imaging ◽

Conformational Landscape

Nucleosomes are the basic units of chromatin and critical to the storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks of which post-translational modifications of histones play a key role. However, the mode of action and the structural implications on the single-molecule level of nucleosomes is often still poorly understood. Here, we apply a high-throughput AFM imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants H3K36me3, H3S10phos and H4K5/8/12/16ac. Our data set of >25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5 bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild type nucleosomes and additionally unwrap stochastically from both sides similar to CENP-A nucleosomes and in contrast to the highly anti-cooperative unwrapping of wild type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild type nucleosomes and also retain the same level of anti-cooperativity. Our findings help putting the mode of action of these modifications into context: While H3K36me3 likely partially acts by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

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Fluorescent Single-Walled Carbon Nanotubes for Protein Detection

Sensors ◽

10.3390/s19245403 ◽

2019 ◽

Vol 19 (24) ◽

pp. 5403 ◽

Cited By ~ 6

Author(s):

Adi Hendler-Neumark ◽

Gili Bisker

Keyword(s):

Carbon Nanotubes ◽

Single Molecule ◽

Near Infrared ◽

Protein Detection ◽

Single Walled Carbon Nanotubes ◽

Fluorescent Nanoparticles ◽

Single Molecule Level ◽

Single Walled Carbon ◽

Walled Carbon Nanotubes

Nanosensors have a central role in recent approaches to molecular recognition in applications like imaging, drug delivery systems, and phototherapy. Fluorescent nanoparticles are particularly attractive for such tasks owing to their emission signal that can serve as optical reporter for location or environmental properties. Single-walled carbon nanotubes (SWCNTs) fluoresce in the near-infrared part of the spectrum, where biological samples are relatively transparent, and they do not photobleach or blink. These unique optical properties and their biocompatibility make SWCNTs attractive for a variety of biomedical applications. Here, we review recent advancements in protein recognition using SWCNTs functionalized with either natural recognition moieties or synthetic heteropolymers. We emphasize the benefits of the versatile applicability of the SWCNT sensors in different systems ranging from single-molecule level to in-vivo sensing in whole animal models. Finally, we discuss challenges, opportunities, and future perspectives.

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High-throughput optical mapping of replicating DNA

10.1101/239251 ◽

2017 ◽

Cited By ~ 1

Author(s):

Francesco De Carli ◽

Nikita Menezes ◽

Wahiba Berrabah ◽

Valérie Barbe ◽

Auguste Genovesio ◽

...

Keyword(s):

Dna Replication ◽

High Throughput ◽

Single Molecule ◽

Large Cell ◽

High Coverage ◽

Single Molecule Level ◽

Single Dna Molecules ◽

Genome Wide ◽

Egg Extracts ◽

Living Organisms

AbstractDNA replication is a crucial process for the universal ability of living organisms to reproduce. Existing methods to map replication genome-wide use large cell populations and therefore smooth out variability between chromosomal copies. Single-molecule methods may in principle reveal this variability. However, current methods remain refractory to automated molecule detection and measurements. Their low throughput has therefore precluded genome-wide analyses. Here, we have repurposed a commercial optical DNA mapping device, the Bionano Genomics Irys system, to map the replication signal of single DNA molecules onto genomic position at high throughput. Our methodology (HOMARD) combines fluorescent labelling of replication tracks and nicking endonuclease (NE) sites with DNA linearization in nanochannel arrays and dedicated image processing. We demonstrate the robustness of our approach by providing an ultra-high coverage (23,311 x) replication map of bacteriophage λ DNA in Xenopus egg extracts. HOMARD opens the way to genome-wide analysis of DNA replication at the single-molecule level.

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