Microfluidics and Spectroscopy for Microsecond Folding Kinetics

Protein folding events occur on a wide range of timescales.  These are highlighted below with the various techniques used to probe them.  One of the limiting factors in protein folding studies has been access to the sub-millisecond timescale for kinetic measurements. Following the lead of the Rousseau, Eaton and Roder groups, we have

developed our own laser micromachined microsecond mixer for

protein folding studies.  We have developed two mixers with similar designs.

One of them is intended for fluorescence studies and has a dead-time of ~30 microseconds and the other has been adapted for use with SAXS and has a dead-time of ~80-100 microseconds.  The main limitation for SAXS measurements is the focusing of the x-ray beam and S/N (which necessitates a deeper flow channel) although with the implementation of CRLs at BioCAT and LiX the SAXS time-resolution will shortly be close to the tens of microseconds it is for fluorescence.   The continuous-flow SAXS work was done in collaboration with the NIH-funded BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory. 

The basic idea behind chaotic/turbulent mixers to mix faster is to reduce the dimensions of the mixing chamber to <100 microns.  An overview is given in the picture below:

Our more recent mixers utilize an all-quartz fabrication process using the femtoetch process developed by our industrial partner, Translume and are based on computational fluid dynamics simulations in collaboration with Venkatesh Inguva and J. Blair Perot at UMass-Amherst.   Some newer design ideas and simulation strategies can be found in a recent paper.  The chips are made by etching channels in a substrate and then fusing a window onto it.  We have had 3 and 5-layer versions made for various purposes and also some with coverslip bottoms for continuous-flow TCSPC applications utilizing a confocal.  Here’s a pic of one of our chips with an etched central window for SAXS measurements: 

The overall idea is similar to our earlier PEEK based mixers:

Here’s the arrangement of the pumps and sample loops we use:

 

The Isco syringe pumps are great to have but we also use Harvard PHD4400 pumps (without sample loops) and they work quite well with a smaller footprint. The experimental arrangement of the CF-TCSPC apparatus is shown below.  We can collect two channels, which are useful for anisotropy experiments.  The resulting data, shown in the inset, yields two timescales: one for the nanosecond excited state decay (which has the distance distribution information in a FRET experiment) and the other for the kinetics (which has the folding timescale ranging from microseconds to tens of ms).  Data acquisition is fully automated with an xy stage, pump and valve control and polarizer and shutter control.  Laser power is also logged for all experiments.  The experiment produces a very rich data set that can be analyzed using global analysis and/or matrix decomposition methods such as SVD.  I should add that, if the alignment is done properly, there is only a small variation of the excitation intensity as the laser is scanned along the channel (actually, the mixer is moved instead of the laser)