1Department of Physics and Astronomy, Michigan State University, 2Department of Mechanical Engineering, Hong Kong University of Science and Technology, 3Center for Biophotonics, University of California, Davis
Waldauer, S. A., Wu, L., Yao, S., Bakajin, O., Lapidus, L. J. Microfluidic Mixers for Studying Protein Folding. J. Vis. Exp. (62), e3976, doi:10.3791/3976 (2012).
The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re ≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.
1. Fabrication of Microfluidic Mixing Chips
Figure 3 shows the basic fabrication steps.
2. Mounting and Loading Chips
3. Data Collection
Figure 5 shows the basic optical instrument layout.
4. Representative Results
Figure 6 shows a contour plot of the intensity in the mixing region measured by the photon counter. The background in the exit channel outside of the jet should be close to the noise floor of the detector and is typically not subtracted. A higher background may indicate poor alignment or that the protein is sticking to the walls of the channel. A jet that looks kinked or crooked, especially near the mixing region, indicates a poor etch of the nozzles.
Figure 7 shows the time resolved spectra from the protein acyl-coenzyme A-binding protein (ACBP) labeled with Alexa 488 and Alexa 647. This data has been background subtracted to remove the dark charge and laser signal. The background signal was taken with the center channel pressure set to 0 PSI.
The mixing time can be measured by measuring a rapid reaction that is much faster than mixing such as Trp fluorescence quenching by KI or collapse of single stranded DNA, labeled with FRET dyes, by the addition of NaCl. Because the jet is smaller than the optical resolution of the microscope, there is a large intensity drop in the mixing region as the jet forms. This instrument response can be removed by making a control measurement in which no solution conditions change. Figure 8 shows the ratio of mixing (into 400 mM KI) and non-mixing experiments of N-acetyl tryptophan amide fluorescence. The line shows the predicted concentration of KI from COMSOL simulations. The mixing time, as measured as the time for the concentration to decrease 80%, is 8 μs.
Since data is collected in 100 μm increments along the 500 μm-long exit channel, data must be pasted together from multiple views. There are occasionally small offsets in signal between these views, likely due to small changes in the focus as the chip is moved by the microscope stage. By moving the stage 80 or 90 μm per view, these offsets can be eliminated by examining overlapping data. Typically data is collected with at least two flow rates and the data is combined to confirm any signal changes are due to real spectroscopic changes in the protein, rather than optical or flow effects. Figure 9 shows the FRET changes in the protein ACBP after dilution of denaturant from 6 M to 0.06 M. This data does not need a control experiment since the FRET is already a ratio and the instrument response is already removed. The rapid jump near T=0 represents a rapid change in FRET signal within the mixing time.
Figure 1. Layout of mixing region measured by electron microscopy.
Figure 2. Schematic of hydrodynamic mixing to study protein folding. The protein, dissolved in high denaturant to unfold it, flows down the center channel towards the mixing region where it meets buffer flowing ~100 times faster. Laminar flow forces the protein stream into a narrow jet from which denaturant molecules can rapidly diffuse. As the jet flows down the exit channel, the protein can be observed with high spatial and time resolution using a confocal microscope.
Figure 3. Fused silica fabrication. 1) The process starts with a highly polished 500 μm thick fused silica substrate (a) with a layer of polysilicon (poly) deposited on the top and bottom surfaces (b) and spin-coated with a layer of photoresist (c). 2) A photomask (e) is brought into hard contact with the wafer and the photoresist is exposed to UV light (d). 3) The wafer is placed in a developer bath and the pattern is revealed in the photoresist. 4) The wafer is placed in a DRIE and the features are etched into the top poly coating. The photoresist is then removed. 5) The poly then acts as a mask when the wafer is placed in an oxide etcher and the features are etched 10 - 40 μm into the fused silica substrate. 6) The poly coating is then removed in a XeF2 etcher. 7) The wafer is bonded to a sacrificial glass plate with a heat-activated sealant (g), features side down, and a diamond drill bit (f) abrasively drills through-holes from the backside. 8) A surface profiler (h) then directly measures the etched feature depths. 9) A 170 μm thick fused silica coverslip wafer is directly bonded on the top surface of the wafer. 10) The wafer is diced into individual microfluidic chips.
Figure 4. The mixer manifold. The microfluidic-chip is secured to the solution manifold with a machined aluminum faceplate and four o-rings under the ~200 μL reservoirs. A large hole is machined through the center of the manifold directly above the mixing region of the affixed microfluidic-chip, allowing excess UV light to escape without backscattering, inhibiting any auto-fluorescence from the manifold itself and allowing direct illumination of the chip by eye or camera for alignment. The air manifold seals each of the reservoirs such that the air pressure in each can be controlled via an external pressure control box.
Figure 5. Schematic of optical confocal microscope.
Figure 6. Contour plot of tryptophan fluorescence in the microfluidic mixer. The mixing region is located at ~90 μm on the y-axis.
Figure 7. Time dependent fluorescent spectra collected within the mixer. The green and red rectangles show the wavelengths designated as donor and acceptor channels, respectively.
Figure 8. Mixing time measured by fluorescence quenching of tryptophan by potassium iodide (points and right axis) and calculated by finite element analysis (line and left axis).
Figure 9. FRET changes measured during the folding of the protein ACBP. The rapid rise near t=0 represents a burst phase within the mixing time and the slower rise represents a gradual accumulation of structure as the protein folds. The data was taken at two different flow rates and overlaid, leading to different densities of measurements at different times.
Rapid mixing has been a development goal for the field of protein folding for many years because it has long been recognized that molecular processes of biomolecules occur over time scales ranging from picoseconds to seconds. Conventional stopped-flow mixers have dead times of 1-5 ms which is primarily limited by turbulence. Turbulent continuous flow mixers with mixing times of 30-300 μs have been developed over the past 15 years by a small number of groups but generally require high flow rates to achieve these mixing times and therefore use a lot of sample6-8.
This protocol describes the use of microfluidic mixing chips to achieve rapid dilution in the laminar flow regime. There are multiple advantages to working within this regime, but a primary one is the ability to simulate the entire mixing process with high precision which allows one to modify the mixing response. T-mixers developed by other groups with simple geometries have demonstrated mixing times of 100-200 μs that were primarily limited by the size of the channels9-10. By scaling the size of the channels to 10 μm Knight reduced the mixing time to ~10 μs11. Yao and Bakajin showed that small changes in geometry could lead to significant improvements in the mixing time4. However, that work showed that acceleration of mixing can also lead to slower phases as well. The design used in this work12-13 is a compromise between the two best designs in reference4 to minimize the slower exponential decay in denaturant concentration and also to make the feature more reproducible in DRIE etching.
There has been some controversy in the field of ultrarapid mixing over the definition of the mixing time. It is important to recognize the difference between "mixing time" and "dead time". The dead time is defined in a turbulent mixer as the time during which a measurement cannot be made and may in fact be significantly longer than the actual mixing time. It is typically determined by making several measurements of a pseudo-first order bimolecular reaction (such as quenching of tryptophan fluorescence by N bromosuccinate) at various concentrations. The observed exponential decays are fitted to converge at a single value assumed to be t=0 s and the time between that point and the first measured point is the dead time. For laminar flow mixers, measurements can be made during the mixing process so there is no dead time, only a mixing time. The mixing time is simply the time to combine two solutions until sufficient uniformity is reached. We have previously defined the mixing time to be a 90/10 time, the time for the concentration of denaturant to decrease from 90% to 10% of the unmixed value. However, the shape of the mixing curve is not perfectly symmetric with the tail of the decay having a small exponential component. Furthermore, protein folding has a highly non-linear dependence on denaturant concentration so that folding can often commence even if the denaturant is only reduced by two fold. Therefore we have more recently defined the mixing time as the time to reduce the denaturant concentration by 80%. Certainly other definitions can be used depending on the requirements of the experiment.
The use of this mixer to study protein folding has consistently revealed surprising results. Folding of cytochrome c and apomyoglobin have long been studied to have multiple folding steps and early results with continuous flow mixers showed collapse to occur on the ~100 μs timescale10,14-15. Our measurements with this mixer showed there to be at least 2 steps on this timescale, a very fast, likely non-specific, collapse (as measured by Trp fluorescence spectral shift) within the mixing time of the mixer followed by a slower phase (as measured by the quenching of total Trp emission) that is likely the first formation of native structure5. We have also observed a 50 μs process in the B1 domain of protein L, overturning the conventional interpretation that this protein is a 2-state folder13.
An advantage of this rapid mixer is that it can probe the folding of the fastest folding proteins that have usually only been measureable by nanosecond T-jump instruments. T-jump typically requires observation of folding/unfolding relaxation near the melting temperature of the protein and therefore never follows the folding of the entire population. With this mixer we have looked at the folding of γ-repressor (λ6-86) with both Trp total emission and spectral shift and have found evidence that the protein has no barrier to folding under strong folding conditions that were not accessible to previous T-jump measurements12. Finally we have measured the folding of the villin headpiece HP-35, one of the fastest folders yet measured and found that the folding rate measured after mixing is ~5 times slower than measured by T-jump under the same conditions16. This suggests that folding kinetics depends on the starting conditions, overturning a major assumption in the field.
We have nothing to disclose.
This work is supported by National Science Foundation FIBR (NSF EF-0623664) and IDBR (NSF DBI-0754570). This work was partially supported by funding from National Science Foundation FIBR Grant 0623664 administered by the Center for Biophotonics, an NSF Science and Technology Center, managed by the University of California, Davis, under Cooperative Agreement PHY 0120999. The research of Lisa Lapidus, Ph.D. is supported in part by a Career Award at the Scientific Interface from the Burroughs Welcome Fund.
|AZ P4110 Photoresist||AZ Electronic Materials|
|AZ 400 K Developer||AZ Electronic Materials|
|Baker PRS 2000 photoresist stripper||Avantor Performance Materials|
|AquaBond||AquaBond Technologies||AquaBond 55|
|500 μm cover wafer||SENSOR Prep Services||: 100 mm ± 0.5 mm
Thickness: 0.5 mm ± 0.025 mm
Standard Tolerances (Unless Noted)
Surface Finish: SI: 4-8Å; SII: 8-12Å
Flats: One Primary SEMI-STD; One Scribe
Clean and Polished, Both Sides
|170 μm cover wafer||SENSOR Prep Services||7980 2G Wafers||: 100 mm ± 0.5 mm
Thickness: 0.17 mm ± 0.025 mm
Standard Tolerances (Unless Noted)
Surface Finish: SI: 4-8Å; SII: 8-12Å
Flats: One Primary SEMI-STD; One Scribe
Clean and Polished, Both Sides
|Deep reactive ion etcher for oxides||ULVAC||ULVAC NL-6000|
|Argon Ion Laser||Cambridge Laser Laboratories||Lexel 95-SHG||λ=258 nm|
|Argon Ion Laser||Melles Griot||λ=488 nm|
|Microscope Objective||Thorlabs Inc.||OFR LMU-40X-UVB 0.5 NA||λ= 258 nm|
|Microscope Objective||Olympus Corporation||UPLSAPO 60XW 1.2 NA||λ=488 nm|
|Nanopositioner||Mad City Labs||Nano-LP100|
|Motorized Translation Stage||Semprex Corp||KL-Series 12-6436|
|GaAsP Photon Counter||Hamamatsu Corp.||H7421-40|
|Monochrometer||Horiba Instruments Inc||MicroHR|
|CCD camera||Andor||iDus 420A-BU|
|Guanidine hydrochloride (GuHCl)||Sigma-Aldrich||G4505|