Continuous-flow Mixing Research Articles

Microfluidic serial dilution ladder

Serial dilution is a fundamental procedure that is common to a large number of laboratory protocols. Automation of serial dilution is thus a valuable component for lab-on-a-chip systems. While a handful of different microfluidic strategies for serial dilution have been reported, approaches based on continuous flow mixing inherently consume larger amounts of sample volume and chip real estate. We employ valve-driven circulatory mixing to address these issues and also introduce a novel device structure to store each stage of the dilution process. The dilution strategy is based on sequentially mixing the rungs of a ladder structure. We demonstrate a 7-stage series of 1 : 1 dilutions with R(2) equal to 0.995 in an active device area of 1 cm(2).

Investigation of Phasing on Synthetic Jet Actuator Arrays

Unsteady computations of laminar flow have been performed for two-dimensional configurations of micro-channel equipped with two synthetic jets. The effect of phasing has been investigated at in-phase and 180̊ out-of-phase of the synthetic jet actuators at a fixed operating frequency and oscillating amplitudes. It was shown that the 180̊ out-of-phase configuration of the synthetic jets promotes better and more continuous flow mixing within the channel during the oscillation. This was due to the discrete pattern of vortex forming which disrupts the main channel flow. The 180̊ out-of-phase jet configuration exhibits higher cooling performance compared to the in-phase jet configuration in terms of the reduction in the maximum temperature in the silicon wafer.

Using tomography to visualize the continuous-flow mixing of biopolymer solutions inside a stirred tank reactor

The vast majority of non-Newtonian fluids are naturally opaque; therefore, visualizing the flow field of such fluids inside a reactor is a challenging task. To identify non-ideal flows, such as dead volumes and channeling, in a continuous-flow mixing system, the flow field inside a stirred tank reactor was visualized using electrical resistance tomography (ERT), an efficient non-intrusive measurement technique. The key objective of this study was to employ the ERT technique in order to explore the effects of the inlet and outlet locations (four configurations: top inlet–bottom outlet, bottom inlet–top outlet, bottom inlet–bottom outlet, and top inlet–top outlet), fluid rheology (0.5–1.5% xanthan gum concentration), jet velocity (0.317–1.660ms−1), feed flow rate (5.3×10−5–2.36×10−4m3s−1), impeller type (the Rushton turbine and Maxblend impellers), and impeller speed (54–250rpm) on the flow patterns generated in the continuous-flow mixing of the xanthan gum solution, which is a pseudoplastic fluid exhibiting yield stress. Using 2D and 3D tomography images, this article effectively presents a competent method to visualize the flow of opaque fluids in laminar and transitional regions inside a reactor. In this study, the existence of non-ideal flows in a stirred tank reactor for opaque fluids was identified using ERT. The quantitative results showed that the tracer distribution (or mixing quality) inside stirred vessel was enhanced by decreasing the fluid yield stress, increasing the impeller speed, increasing the jet velocity, and using the close clearance impeller. The results also showed that the location of the inlet and outlet streams has a significant effect on the mixing quality. To improve the design of the continuous-flow mixing of non-Newtonian fluids, the findings of this study can be integrated into the design criteria to achieve optimal mixing results.

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-time-resolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.

X-ray scattering experiments with high-flux X-ray source coupled rapid mixing microchannel device and their potential for high-flux neutron scattering investigations

Small-angle X-ray scattering provides global, shape-sensitive structural information about macromolecules in solution. Its extension to time dimension in the form of time-resolved SAXS investigations and combination with other time-resolved biophysical methods contributes immensely to the study of protein dynamics. TR-SAXS can also provide unique information about the global structures of transient intermediates during protein dynamics. An experimental set-up with low protein consumption is essential for an extensive use of TR-SAXS experiments on protein dynamics. In this direction, a newly developed 20-microchannel microfluidic continuous-flow mixer was combined with SAXS. With this set-up, we demonstrate ubiquitin unfolding dynamics after rapid mixing with the chaotropic agent Guanidinium-HCl within milliseconds using only ∼ 40 nanoliters of the protein sample per scattering image. It is suggested that, in the future, this new TR-SAXS platform will help to increase the use of time-resolved small-angle X-ray scattering, wide-angle X-ray scattering and neutron scattering experiments for studying protein dynamics in the early millisecond regime. The potential research field for this set-up includes protein folding, protein misfolding, aggregation in amyloidogenic diseases, function of intrinsically disordered proteins and various protein-ligand interactions.

Advances in turbulent mixing techniques to study microsecond protein folding reactions

Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps, and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10's of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer, Circular Dichroism, Small-Angle X-ray Scattering, Hydrogen Exchange followed by Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy, Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy, has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of the CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, which are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

The effect of shear rate on aggregate size distribution and structure at steady state: a comparison between a Taylor–Couette reactor and a mixing tank

The paper deals with the dependence of aggregate properties on the shear rate ( G ) at steady state. Aggregation of natural raw water and ferric sulphate was carried out in a laboratory Taylor–Couette reactor (TC) and continuous flow mixing tank (MT) with a paddle stirrer. Shear rates in the range of 20–350 s −1 were used. Methods of image and fractal analyses were used to determine the aggregate size and structure, respectively. It was found that the aggregate size decreased with increasing shear rate. There was a small difference in sizes of aggregates formed in TC and MT at G −1 , but at higher shear rates, almost no difference between TC and MT was observed. The D 2 fractal dimension increased with increasing shear rate indicating that aggregates became less porous and more compact. Moreover, a very close match in D 2 values was attained for both mixing devices. The D pf fractal dimension decreased with increasing shear rate meaning that aggregates were more regular at higher shear rates, but jagged on the surface with irregular shape at lower shear rates. In contrast to D 2 , aggregates formed in MT were much more irregular (jagged) than those formed in TC.

Editorial on the Topical Issue “Neutron Biological Physics”

This special issue brings together a number of original research papers focused on recent developments in biological membrane and membrane model studies by a variety of state-of-the-art biophysical methods. Neutron scattering is especially well represented. Our knowledge of biological macromolecules and their interactions is based on the application of physical methods, ranging from classical thermodynamics to recently developed techniques for the detection and manipulation of single molecules. The role of neutron diffraction and spectroscopy techniques in improving this knowledge has been outstanding and the potential for future developments is remarkable. Many challenges remain for neutron techniques to answer the questions raised by molecular and cellullar biology. The following pages give a flavour of the progress in the field. A report [1] describes time-resolved small angle scattering of light-induced changes in chloroplast thylakoid membranes. In the context of surface functionalization, the structure in different ionic conditions of sodium hyaluronan grafted at a solid-liquid interface was observed by neutron reflectivity [2]. A review of membrane protein studies [3] addresses the difficulties encountered by providing information on the properties of surfactants used to solubilise membrane proteins for contrast variation studies by neutron small angle scattering, as well as on membrane protein deuterium labelling strategies. The thought-provoking Zaccai neutron resilience and site-specific hydration dynamics in a globular protein are discussed in [4]. Membrane raft model structures with tuneable compositions were characterised by neutron diffraction and reflectivity [5], while raft dynamics in large uni-lamellar vesicles in interaction with beta amyloid peptide was observed by the neutron spin echo method combined with small angle scattering [6]. The bending stiffness of biological membranes as determined by neutron spin echo is also presented [7]. Two contributions [8, 9] report on undulation dynamics and shape fluctuations, respectively, in lipid vesicles as a function of lipid composition and other parameters observed by neutron spin echo in combination with other methods like small angle scattering and light scattering. Elastic incoherent neutron scattering was applied to compare dynamics in natural membranes extracted from hyperthermophile and mesophile bacteria in the context of their different biological activities [10]. A molecular dynamics simulation study discusses macromolecular resilience as was defined from neutron spectroscopy experiments. The underlying mechanisms of bioprotection by sugars were explored by using data from Infrared Spectroscopy, Molecular Dynamics simulations, Small Angle X-ray Scattering and Calorimetry [11]. Time resolved small angle X-ray scattering in combination with other biophysical methods can contribute immensely to the study of protein dynamics and a paper presents a new set-up, including a microfluidic continuous-flow mixer, which provided results on unfolding dynamics. And the project of a neutron dynamics data bank is presented in a Perspectives article [12]. The aim of the bank will be to make neutron dynamics data on biological systems widely available to the scientific community.

Characterization of the continuous-flow mixing of non-Newtonian fluids using the ratio of residence time to batch mixing time

Continuous-flow mixing of pseudoplastic fluids possessing yield stress is a complex phenomenon exhibiting non-ideal flows within the stirred vessels. Electrical resistance tomography (ERT), a non-intrusive technique, was employed to measure the mixing time in the batch mode while dynamic tests were performed to study the mixing system in the continuous mode. This study attempts to explore the effects of the operating conditions and design parameters on the ratio of the residence time (τ) to the mixing time (θ) for the continuous-flow mixing of non-Newtonian fluids. To achieve these objectives, the effects of impeller types (four axial-flow impellers: A310, A315, 3AH, and 3AM; and three radial-flow impellers: RSB, RT, and Scaba), impeller speed (290–754rpm), fluid rheology (0.5–1.5%, w/v), impeller off-bottom clearance (H/2.7–H/2.1, where H is the fluid height in the vessel), locations of inlet and outlet (configurations: top inlet-bottom outlet and bottom inlet-top outlet), pumping directions of an axial-flow impeller (up-pumping and down-pumping), fluid height in the vessel (T/1.06–T/0.83, where T is the tank diameter), residence time (257–328s), and jet velocity (0.317–1.66ms−1) on the ratio of τ to θ were investigated. The results showed that the extent of the non-ideal flows (channeling and dead volume) in the continuous-flow mixing approached zero when the value of τ/θ varied from 8.2 to 24.5 depending on the operating conditions and design parameters. Thus, to design an efficient continuous-flow mixing system for non-Newtonian fluids, the ratio of the residence time to the mixing time should be at least 8.2 or higher.

Snapshots of a protein folding intermediate

We have investigated the folding dynamics of Thermus thermophilus cytochrome c(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.

Using a tracer technique to identify the extent of non-ideal flows in the continuous mixing of non-Newtonian fluids

The identification of non-ideal flows in a continuous-flow mixing of non-Newtonian fluids is a challenging task for various chemical industries: plastic manufacturing, water and wastewater treatment, and pulp and paper manufacturing. Non-ideal flows such as channelling, recirculation, and dead zones significantly affect the performance of continuous-flow mixing systems. Therefore, the main objective of this paper was to develop an identification protocol to measure non-ideal flows in the continuous-flow mixing system. The extent of non-ideal flows was quantified using a dynamic model that incorporated channelling, recirculation, and dead volume in the mixing vessel. To estimate the dynamic model parameters, the system was excited using a frequency-modulated random binary input by injecting the saline solution (as a tracer) into the fresh feed stream prior to being pumped into the mixing vessel. The injection of the tracer was controlled by a computer-controlled on-off solenoid valve. Using the trace technique, the extent of channelling and the effective mixed volume were successfully determined and used as mixing quality criteria. Such identification procedures can be applied at various areas of chemical engineering in order to improve the mixing quality.

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics

We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

Characterization of a temperable mixer with the Acetal Cleavage Method

The ‘Acetal Cleavage Method’ (aka 4th Bourne reaction or Walker scheme) is a competing chemical reaction scheme for the characterization of mixing processes in stirred laboratory tanks and continuous flow mixers. Due to the overall exothermicity of the reactions, in addition to mixing phenomena, temperature effects are represented in the experimental results. The characterization of a temperable mixer manufactured at the Institute for Micro Process Engineering is reported in this contribution. Furthermore, this examination method is applied to a selection of continuous flow mixers, which have been previously examined with the Iodide Iodate Reaction Method and the Bromination Reaction Method. A comparison of devices and methods is given.Additionally, an overview of earlier literature on this characterization method is presented. Details of the UV analytics are discussed.

Online conductivity measurement of residence time distribution of thin film flow in the spinning disc reactor

Spinning disc reactors provide effective continuous flow mixing in highly sheared liquid films flowing under the action of high centrifugal forces generated as a result of the disc rotation. Although the flow is theoretically characterised to be laminar on the basis of the low Reynolds numbers, the film surface is normally covered with numerous ripples which can change the film hydrodynamics and velocity distribution and thereby affect its residence time distribution (RTD) on the rotating disc. The aim of this study is to determine the experimental conditions for which near plug flow behaviour prevails on the spinning disc. Our findings indicate that the higher the disc speed and liquid flow rate and the lower the viscosity of the processing liquid, the closer conditions within the liquid film on the disc approximate to plug flow behaviour. These effects are attributed to the greater degree of turbulence induced in the liquid film as a result of an increased intensity of surface waves, promoting transverse mixing across the film thickness and thus a more uniform velocity profile at any given radial position. The centrifugal force directing the liquid radially to the disc periphery may also play a part in reducing radial dispersion. The texture of the disc surface is also an influential parameter in achieving plug flow on the rotating disc. With flow on a grooved disc, the number of tanks-in-series increased quite significantly under identical operating conditions as those for a smooth disc, confirming that radial dispersion is lowered. One of the explanations for this effect is that the constantly changing topography enabled by the series of concentric grooves across the disc surface offers the opportunity for repeated film detachment and re-attachment which cause induced recirculation or vortices within the film, giving better transverse mixing and eliminating velocity gradients.

Interconnection of Salt-induced Hydrophobic Compaction and Secondary Structure Formation Depends on Solution Conditions

What happens in the early stage of protein folding remains an interesting unsolved problem. Rapid kinetics measurements with cytochrome c using submillisecond continuous flow mixing devices suggest simultaneous formation of a compact collapsed state and secondary structure. These data seem to indicate that collapse formation is guided by specific short and long range interactions (heteropolymer collapse). A contrasting interpretation also has been proposed, which suggests that the collapse formation is rapid, nonspecific, and a trivial solvent related compaction, which could as well be observed by a homopolymer (homopolymer collapse). We address this controversy using fluorescence correlation spectroscopy (FCS), which enables us to monitor the salt-induced compaction accompanying collapse formation and the associated time constant directly at single molecule resolution. In addition, we follow the formation of secondary structure using far UV CD. The data presented here suggest that both these models (homopolymer and heteropolymer) could be applicable depending on the solution conditions. For example, the formation of secondary structure and compact state is not simultaneous in aqueous buffer. In aqueous buffer, formation of the compact state occurs through a two-state co-operative transition following heteropolymer formalism, whereas secondary structure formation takes place gradually. In contrast, in the presence of urea, a compaction of the protein radius occurs gradually over an extended range of salt concentration following homopolymer formalism. The salt-induced compaction and the formation of secondary structure take place simultaneously in the presence of urea.

Achroma: a software strategy for analysing (a-)typical mass-spectrometric data

Today, (bio-)analytical researchers use various software tools for improving data analysis and the evaluation of their experimental results. Bioinformaticians typically program these software tools; however, analysts and bioinformaticians have distinct views on these data. Achroma is a software tool for the analysis of spectrometric data; it is our hope that explaining the software strategy and the working modules behind Achroma may give analysts a better understanding of how (bio-)informaticians work out software solutions, thus facilitating the interaction between these two expert groups. Achroma is based on a model-view-controller (MVC) software architecture. Furthermore, Achroma has a modular structure and each module has its own MVC architecture. Typically, each module delivers just one specific function to the user. Finally, every module is embedded within Achroma as a small “stand alone” software application. Analytically, Achroma software is a tool to handle typical and untypical mass spectrometric data. Achroma was originally programmed to circumvent problems with mass spectrometric vendor software in the analysis of data from new experimental strategies. Specifically, existing software enables the data analysis from continuous-flow mixing systems monitoring enzyme–inhibitor reactions only manually and indirectly. This data-handling bottleneck is resolved in Achroma. The principles behind this implementation are described in detail for two modules—the ‘chromatogram comparison’ and ‘signal recognition’ modules. Further specific examples of data analysis are presented, such as signal recognition, chromatogram smoothing and signal area calculation. We regard such understanding between the analysts' and programmers' worlds as essential for future improvements in analytical software.

Improving the dynamic performance of continuous-flow mixing of pseudoplastic fluids possessing yield stress using Maxblend impeller

The strategic approach of this article is to characterize the continuous-flow mixing of pseudoplastic fluids possessing yield stress in a stirred reactor with the Maxblend impeller. Dynamic experiments were carried out through the frequency-modulated random binary input of a brine solution to determine the extent of non-ideal flows. Mixing quality was determined on the basis of the extent of channeling and fully mixed volume. The effects of important parameters such as impeller speed (25–500rpm), absence of baffles, fluid rheology (0.5–1.5%), fluid flow rate (3.20–14.17Lmin−1), and the locations of inlet/outlet on the dynamic performance of the continuous-flow mixing vessel were explored. The performance of the Maxblend impeller was then compared to the performances of various types of impellers such as close-clearance (an anchor), axial-flow (a Lightnin A320), and radial-flow (a Scaba 6SRGT) impellers. It was found when the channeling approached zero and the fully mixed volume approached the total fluid volume in the vessel, the power drawn by the A320 impeller and the Scaba impeller were about 2.9 and 4.3 times greater than that of the Maxblend impeller. Thus, the Maxblend impeller was able to drastically improve the performance of continuous-flow mixing with huge power savings. The mixing quality was further improved by optimizing the impeller speed, decreasing the fluid flow rate, decreasing the fluid concentration, and using bottom inlet- top outlet configuration. The flow non-ideality of the mixing system increased in the absence of the baffles. Thus, better mixing quality and more energy savings can be achieved by employing the findings of this study.

Dynamic Performance of Continuous-Flow Mixing of Pseudoplastic Fluids Exhibiting Yield Stress in Stirred Reactors

The core objectives of this work were to characterize and optimize the continuous-flow mixing of pseudoplastic fluids exhibiting yield stress in stirred reactors. To achieve these objectives, the effects of impeller type (for the seven axial-flow impellers A100, A200, A310, A315, A320, 3AH, and 3AM and the four radial-flow impellers R500, RSB, RT, and Scaba), impeller speed (50–800 rpm), impeller diameter (T/3.2–T/1.6, where T is the tank diameter), impeller off-bottom clearance (H/3.4–H/1.7, where H is the fluid height in the vessel), inlet and outlet locations (for the four configurations top inlet–top outlet, top inlet–bottom outlet, bottom inlet–bottom outlet, and bottom inlet–top outlet), pumping directions for axial-flow impellers (upward and downward pumping), fluid height in the vessel (T/1.06–T/0.83), residence time (257–328 s), and jet velocity (0.317–3.24 m s–1) on the dynamic performance of the mixing vessel were explored. To identify nonideal flows, dynamic tests were conducted using the frequency-modulated random binary input of a brine solution with the feed. The mixing quality in the vessel was substantially improved by increasing the impeller diameter, increasing the residence time, optimizing the impeller off-bottom clearance, decreasing the fluid height, optimizing the jet velocity, and using the up-pumping axial-flow impeller. Applying these findings will lead to improved quality of products and more efficient use of power in continuous-flow mixing of yield-pseudoplastic fluids.

Microsecond Subdomain Folding in Dihydrofolate Reductase

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼ 550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR—in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse—emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.

Minireview: Structural insights into early folding events using continuous‐flow time‐resolved small‐angle X‐ray scattering

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.