Proceeding: A Nanofluidic Mixing Device for High-Throughput Fluorescence Sensing of Single Molecules

K. Mathwig, C. Fijen, M. Fontana, S. G. Lemay and J. Hohlbein, Procedia Technology, 27, 141 2017, [link]

We introduce a nanofluidicmixing device
entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets. The fluorescently labeled molecules are tracked before, during and after they enter the mixing region, and their reactions with each other are observed by means of optical readout such as Förster Resonance Energy Transfer (FRET). Our method can be used for analyzing the kinetics of DNA annealing in a high-parallelized fashion.

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News: CHIPP proposal awarded

Good news! The proposal “Lipid Oxidation Control in Food Emulsions Enabled by Natural Strategies” submitted by Harry Gruppen (WUR) has been awarded a CHIPP grant by The Innovation Fund for Chemistry [link].

Our lab will head the work package titled “Localising oxidation: interfaces and transport” and a PhD position will be available in 2018.

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miCube: 3D super-resolution microscopy

Here the specs: 50k frames, 850k localisations, 6 min for data analysis. Many thanks to Sander Baas, Koen Martens and Arjen Bader, with cells provided by Gert-Jan Bakker and Ben Joosten (both Radboudumc, Nijmegen).

Posted in miCube

News: IPM proposal granted

Good news! Our proposal “Towards visualizing gene transfer at the single cell level using microfluidic devices” has been granted by the Innovation Program Microbiology, an initiative from the Laboratory of Microbiology at Wageningen University and Research.

In collaboration with Dr. Klaus Mathwig (Pharmacy, Groningen), Dr. Peter van Baarlen (Host-Microbe Interactions, Wageningen) and Simon van der Els (NIZO food research and HMI, Wageningen), we aim to monitor bacterial conjugation, in which DNA is transferred from a donor cell to a recipient cell either via conjugative plasmids or via integrative conjugative elements, in real time and at the single-cell level.

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News: (Update) miCube: Rethinking modular fluorescence microscopy

Hurrah, we finalised the design of our miCube (V0.1)! Details can be found [here] and include part numbers, CAD-drawings and STL files for CNC machining/milling or 3D printing published under a Creative Common license. Big shout out to Sander Baas and Koen Martens. If you have any questions, remarks or ideas, please feel to contact me.

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News: Congratulations to Shazia and welcome to Sander…

Congratulations to Shazia Farooq for successfully defending her PhD thesis titled “Studying fast dynamics in biological complexes: from photosynthesis in vivo to single DNA molecules in vitro” [link]. Well done and thanks to all the helpers for the great party afterwards!

Already a few weeks ago, Sander Baas joined our lab for his Master’s thesis to set up three-dimensional super-resolution microscopy. Looks very promising!

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Publication: A single-molecule FRET sensor for monitoring DNA synthesis in real time

C. Fijen, A. Montón Silva, A. Hochkoeppler and J. Hohlbein, Physical Chemistry Chemical Physics, 19, 4222-4230, 2017, [link]

fig1We developed a versatile DNA assay and framework for monitoring polymerization of DNA in real time and at the single-molecule level. The assay consists of an acceptor labelled DNA primer annealed to a DNA template that is labelled on its single stranded, downstream overhang with a donor fluorophore. Upon extension of the primer using a DNA polymerase, the overhang of the template alters its conformation from a random coil to the canonical structure of double stranded DNA. This conformational change increases the distance between the donor and the acceptor fluorophore and can be detected as a decrease in the Förster resonance energy transfer (FRET) efficiency between both fluorophores. Remarkably, the DNA assay does not require any modification of the DNA polymerase and albeit the simple and robust spectroscopic readout facilitates measurements even with conventional fluorimeters or stopped-flow equipment, single-molecule FRET provides additional access to parameters such as the processivity of DNA synthesis and, for one of the three DNA polymerases tested, the detection of binding and dissociation of the DNA polymerase to DNA. We furthermore demonstrate that primer extensions by a single base can be resolved.

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