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

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.

scholarly journals High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices

Lab on a Chip ◽  
2019 ◽  
Vol 19 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Mattia Fontana ◽  
Carel Fijen ◽  
Serge G. Lemay ◽  
Klaus Mathwig ◽  
Johannes Hohlbein
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Dynamic Heterogeneity ◽  
Equilibrium Studies ◽  
Complex Samples ◽  
Powerful Means ◽  
Nanofluidic Devices ◽  
Detection Schemes ◽  
Non Equilibrium

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

scholarly journals Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Disha M. Bangalore ◽  
Hannah S. Heil ◽  
Christian F. Mehringer ◽  
Lisa Hirsch ◽  
Katherina Hemmen ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Efficiency ◽  
Excision Repair ◽  
Lesion Detection ◽  
Analysis Tool ◽  
Biological Interactions ◽  
Dna Glycosylases ◽  
High Throughput Analysis ◽  
Mutagenic Potential ◽  
Enhanced Sensitivity

Abstract Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

Nanophotonics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1993-2000 ◽  
Author(s):  
Chenshuo Ma ◽  
Chunyan Shan ◽  
Kevin Park ◽  
Aaron T. Mok ◽  
Paul J. Antonick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Excitation Power ◽  
Single Particle Tracking ◽  
Rare Earth Ions ◽  
High Excitation ◽  
Single Molecule Level ◽  
Collecting Efficiency ◽  
High Luminescence Intensity

AbstractUpconverting luminescent nanoparticles are photostable, nonblinking, and low chemically toxic fluorophores that are emerging as promising fluorescent probes at the single molecule level. High luminescence intensity upconversion nanoparticles (UCNPs) have previously been achieved by doping with high amounts of rare-earth ions using high excitation power (>2.5 MW/cm2). However, such particles are inadequate for in vitro live-cell imaging and single-particle tracking, as high excitation power can cause photodamage. Here, we compared UCNP luminescence intensities with different dopant concentrations and presented more efficient (about seven times) UCNPs at low excitation power by increasing the concentrations of Yb3+ and Tm3+ dopants (NaYF4: 60% Yb3+, 8% Tm3+) and adding a core-shell structure.

scholarly journals Nanoplastic Sizes and Numbers: Quantification by Single Particle Tracking

2020 ◽  
Author(s):  
Robert Molenaar ◽  
Swarupa Chatterjee ◽  
Mireille M. A. E Claessens ◽  
Christian Blum
Keyword(s):  
High Throughput ◽  
Particle Tracking ◽  
Single Particle ◽  
Environmental Samples ◽  
Single Particle Tracking ◽  
Water Bodies ◽  
High Throughput Analysis ◽  
Mixing Ratios ◽  
Almost Everywhere ◽  
Terrestrial Water

<p>Plastic particles have been found almost everywhere in the environment, in oceans, terrestrial water bodies, sediments and air. The extend of this unwanted contamination is difficult to fully capture. Existing quantification methods focus on the detection of millimeter to micrometer sized plastic particles, while plastic breakdown processes continue to smaller, nanometer sized, particles. For these nanoplastics methods that are inexpensive and can be (semi-) automated for high throughput analysis of dilute nanoplastic particle suspensions, are lacking. ​​​​​​​​​​​​​​Here we combine sensitive fluorescence video microsopy, NileRed staining of plastic particles, and Single Particle Tracking (SPT) to count and size nanoplastics. With this approach we show that particle diameters as low as 40 nm can be extracted, mixing ratios can be recovered, and number concentrations as low as 2·10<sup>6</sup> particles/ml can be determined. These results indicate that this approach is promising for the quantification of sizes and concentrations of nanoplastics in environmental samples.</p>

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

Applied Nano ◽  
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.

scholarly journals Supercoiling DNA optically

2019 ◽  
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J.L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Torque Control ◽  
Biological Interactions ◽  
Supercoiled Dna ◽  
Dna Structures ◽  
Single Molecule Level ◽  
Open Questions ◽  
Cellular Dna ◽  
Transcription And Replication

AbstractCellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures.1,2,3 For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic,4,5,6 angular-optical,7,8,9 micro-pipette,10 and magneto-optical tweezers.11 However, in order to address many open questions, there is a growing need for new techniques that can combine rapid torque control with both spatial manipulation and fluorescence microscopy. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. Supercoiled DNA formed in this way is amenable to fast buffer exchange, and can be interrogated with both force spectroscopy and fluorescence imaging of the whole DNA. We establish this method as a powerful platform to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

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

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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