2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

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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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A Novel Complex: A Quantum Dot Conjugated to an Active T7RNA Polymerase

Journal of Nanomaterials ◽

10.1155/2013/468105 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Mette Eriksen ◽

Peter Horvath ◽

Michael A. Sørensen ◽

Szabolcs Semsey ◽

Lene B. Oddershede ◽

...

Keyword(s):

Quantum Dot ◽

Rna Polymerase ◽

Single Molecule ◽

Rna Polymerases ◽

Fluorescent Signal ◽

Single Molecule Level ◽

Single Molecule Studies ◽

Novel Complex ◽

Luminescent Nanoparticle

To perform single-molecule studies of the T7RNA polymerase, it is crucial to visualize an individual T7RNA polymerase, for example, through a fluorescent signal. We present a novel complex combining two different molecular functions, an active T7RNA polymerase and a highly luminescent nanoparticle, a quantum dot. The complex has the advantage of both constituents: the complex can traffic along DNA and simultaneously be visualized, both at the ensemble and at the single-molecule level. The labeling was mediated through anin vivobiotinylation of a His-tagged T7RNA polymerase and subsequent binding of a streptavidin-coated quantum dot. Our technique allows for easy purification of the quantum dot labeled T7RNA polymerases from the reactants. Also, the conjugation does not alter the functionality of the polymerase; it retains the ability to bind and transcribe.

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Impact of Small Molecules on Intermolecular G-Quadruplex Formation

Molecules ◽

10.3390/molecules24081570 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1570 ◽

Cited By ~ 2

Author(s):

Prabesh Gyawali ◽

Keshav GC ◽

Yue Ma ◽

Sanjaya Abeysirigunawardena ◽

Kazuo Nagasawa ◽

...

Keyword(s):

Small Molecules ◽

Single Molecule ◽

The Other ◽

Single Molecule Level ◽

G Quadruplex ◽

Dna Strands ◽

Order Of Magnitude ◽

Single Molecule Studies ◽

Quadruplex Formation ◽

The Impact

We performed single molecule studies to investigate the impact of several prominent small molecules (the oxazole telomestatin derivative L2H2-6OTD, pyridostatin, and Phen-DC3) on intermolecular G-quadruplex (i-GQ) formation between two guanine-rich DNA strands that had 3-GGG repeats in one strand and 1-GGG repeat in the other (3+1 GGG), or 2-GGG repeats in each strand (2+2 GGG). Such structures are not only physiologically significant but have recently found use in various biotechnology applications, ranging from DNA-based wires to chemical sensors. Understanding the extent of stability imparted by small molecules on i-GQ structures, has implications for these applications. The small molecules resulted in different levels of enhancement in i-GQ formation, depending on the small molecule and arrangement of GGG repeats. The largest enhancement we observed was in the 3+1 GGG arrangement, where i-GQ formation increased by an order of magnitude, in the presence of L2H2-6OTD. On the other hand, the enhancement was limited to three-fold with Pyridostatin (PDS) or less for the other small molecules in the 2+2 GGG repeat case. By demonstrating detection of i-GQ formation at the single molecule level, our studies illustrate the feasibility to develop more sensitive sensors that could operate with limited quantities of materials.

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Single Molecule Studies of a 2,7-Bis-(Phenylethenyl)fluorenone: Implications for Green-Emission Bands in Fluorene-Based OLEDs

MRS Proceedings ◽

10.1557/proc-0965-s03-28 ◽

2006 ◽

Vol 965 ◽

Author(s):

Michael Y Odoi ◽

Nathan I Hammer ◽

Hemali Rathnayake ◽

Paul M Lahti ◽

Michael D Barnes

Keyword(s):

Single Molecule ◽

Fluorescence Emission ◽

Green Emission ◽

Peak Position ◽

Single Molecule Level ◽

Fluorene Derivative ◽

Single Molecule Studies ◽

Excimer Formation ◽

Red Luminescence ◽

Chain Interaction

ABSTRACTThe color purity of blue polyfluorene fluorescence emission is often adversely contaminated by green emission bands. The origin of this green emission has been attributed to various factors including on-chain oxidation of fluorene leading to monomeric fluorenone emission and inter/intra-chain interaction between segments of fluorenone units leading to excimer formation on the polyfluorene backbone. We report here emission properties at the bulk and single molecule level of a molecular fluorene derivative and compare them to those of its oxidative fluorenone derivative. Whereas the bulk fluorescence emission of 2,7-bis(3,4,5-trimethoxyphenylethenyl)-9,9-diethyl-9H-fluorene (OFPV) exhibits a blue to blue-green emission at about 480 nm, 2,7-bis(3,4,5-trimethoxyphenylethenyl)-9,9-diethyl-9H-fluorenone (OFOPV) shows red luminescence with a peak centered at about 630 nm. However, the peak position for OFOPV shifts to higher energies (∼540 nm) upon dilution or dispersion in polymer matrices like PMMA or Zeonex. Single molecule measurements of OFOPV show fluorescence spectra dominated by peaks around 540 nm, with a small minority at longer wavelengths that are attributed to emission from dimers or higher aggregates. This distribution indicates that monomeric emission of OFOPV is green, consistent with green emission bands seen in polyfluorenes.

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Characterizing microfluidic approaches for a fast and efficient reagent exchange in single-molecule studies

Scientific Reports ◽

10.1038/s41598-020-74523-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Julene Madariaga-Marcos ◽

Roberta Corti ◽

Silvia Hormeño ◽

Fernando Moreno-Herrero

Keyword(s):

Single Molecule ◽

Switching Time ◽

Flow Cell ◽

Liquid Environment ◽

Flow Rates ◽

Flow Cells ◽

Single Molecule Level ◽

Potential Applicability ◽

Single Molecule Studies ◽

Sequential Addition

Abstract Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level.

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