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Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes.

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.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Wunderlich B,Nettels D,Benke S,Clark J,Weidner S,Hofmann H,Pfeil SH,Schuler B

doi

10.1038/nprot.2013.082

subject

Has Abstract

pub_date

2013-08-01 00:00:00

pages

1459-74

issue

8

eissn

1754-2189

issn

1750-2799

pii

nprot.2013.082

journal_volume

8

pub_type

杂志文章

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