ArticleNature protocols2013
Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes.
Article in Nature protocols, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 37 papers.
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Who cites it
37 citing papers in PubMed.
- Time-Resolved Single-Molecule FRET Reveals Length-Dependent Nucleosome Decompaction by Poly(ADP-ribose).bioRxiv : the preprint server for biology · 2026Article
- Quantitative analysis methods for free diffusion single-molecule FRET experiments.Current opinion in structural biology · 2025Review
- Advances in microfluidic mixers for time-resolved structural biology with X-rays.Biophysical reviews · 2025Review
- An Efficient 3D-Printed Gravity Mixer for Lab-on-a-CD Applications.Micromachines · 2024Article
- A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- Article
- An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics.Nature communications · 2023Article
- Rapid droplet-based mixing for single-molecule spectroscopy.Nature methods · 2023Article
- Article
- The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy.Essays in biochemistry · 2022Article
- Article
- Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins.Essays in biochemistry · 2021Review
- FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.eLife · 2021Review
- Biological applications of microchip electrophoresis with amperometric detection: in vivo monitoring and cell analysis.Analytical and bioanalytical chemistry · 2020Review
- A microfluidic platform integrating pressure-driven and electroosmotic-driven flow with inline filters for affinity separations.Analytical methods : advancing methods and applications · 2019Article
- High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins.Methods (San Diego, Calif.) · 2019Article
- Microfluidic Cell Trapping for Single-Cell Analysis.Micromachines · 2019Article
- Microfluidic Contact Lenses.Small (Weinheim an der Bergstrasse, Germany) · 2018Article
- Protein folding transition path times from single molecule FRET.Current opinion in structural biology · 2018Review
- Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer.Science (New York, N.Y.) · 2018Review
Corrections and comments
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Authors and funding
8 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Microfluidic mixing in combination with single-molecule spectroscopy allows the investigation of complex biomolecular processes under non-equilibrium conditions. Here we present a protocol for building, installing and operating microfluidic mixing devices optimized for this purpose. The mixer is fabricated by replica molding with polydimethylsiloxane (PDMS), which allows the production of large numbers of devices at a low cost using a single microfabricated silicon mold. The design is based on hydrodynamic focusing combined with diffusive mixing and allows single-molecule kinetics to be recorded over five orders of magnitude in time, from 1 ms to ∼100 s. Owing to microfabricated particle filters incorporated in the inlet channels, the devices provide stable flow for many hours to days without channel blockage, which allows reliable collection of high-quality data. Modular design enables rapid exchange of samples and mixing devices, which are mounted in a specifically designed holder for use with a confocal microscopy detection system. Integrated Peltier elements provide temperature control from 4 to 37 °C. The protocol includes the fabrication of a silicon master, production of the microfluidic devices, instrumentation setup and data acquisition. Once a silicon master is available, devices can be produced and experiments started within ∼1 d of preparation. We demonstrate the performance of the system with single-molecule Förster resonance energy transfer (FRET) measurements of kinetics of protein folding and conformational changes. The dead time of 1 ms, as predicted from finite element calculations, was confirmed by the measurements.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.