Microfluidic mixers for studying protein folding software

Probing microsecond reactions with microfluidic mixers and. Among these, fluid mixing within microchannels is one of the most common and important functions since many biological and chemical processes, such as protein folding, enzyme reaction, protein ligand interaction, cell activation, and chemical synthesis require complete mixing of the reagents. Francesca volpetti, jose garciacordero, and sebastian j. A microfluidic platform for highthroughput multiplexed. A microfluidic device can be confirmed the fact that it has one or more channels and at least one size less than 1 mm. An electrowettingbased lineararray droplet mixer has previously. A microfluidic mixer was coupled to a confocal microscope to measure the fret efficiencies.

Microfluidic mixing and its use in labonachip devices by zheng xia august, 2008 chair. Furthermore by integrating the concept of microfluidic mixer with magnetic beads, make feasible to study the nextgeneration sequencing. Jun 15, 2018 in this work, we consider a microfluidic mixer that uses hydrodynamic diffusion stream to induce the beginning of the folding process of a certain protein. In microfluidics, when re is below or on the order of 1 and fast mixing is required, so far. The uflux microfluidics system consists of a highly stable passive pressure system, automated fluidic regulation and a laminar flow cell. Microfluidics is a technology where application span the biomedical field and beyond. A microfluidic mixer for studying protein folding with single. A microfluidic platform for highthroughput multiplexed protein quantitation. The ability to thoroughly mix two fluids is a fundamental need in microfluidics. After labeling the csp with donor and acceptor dyes at key residues, they probed the conformation. Thus a microfluidic mixer has become a crucial component in these applications.

Microfluidic devices have since been regularly used to study protein folding at the singlemolecule level. Computer design of microfluidic mixers for protein rna folding studies kinetic studies of biological macromolecules increasingly use microfluidic mixers to initiate and monitor reaction progress. Two and threedimensional modeling and optimization. However, by slowing down the fluid flows in such mixers to 110. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. The new design also provides more uniform mixing across streamlines that enter the mixer. Apr 10, 2012 the use of this mixer to study protein folding has consistently revealed surprising results. We describe the minimization problem and constraints.

Microfluidicbased singlemolecule detection broke ground early in protein folding with a study in 2003 by lipman and colleagues in which a laminar flow mixer was used to study nonequilibrium states in the denaturing of a cold shock protein csp. Transient misfolding dominates multidomain protein folding. Improvements in mixing time and mixing uniformity in. Optimization of a microfluidic mixer for studying protein folding kinetics article pdf available in analytical chemistry 78. They labeled a substrate protein, rhodanese, with donor and acceptor fluorophores in order to characterize the folding pathway with and without binding inside the cavity of the groelgroes chaperonin. For each application, our modelingsimulation are validated by posteriori. As volumes approach the subnanoliter regime, the mixing of liquids is hindered by laminar flow conditions. Waldauer 1, ling wu 1, shuhuai yao 2, olgica bakajin 3, lisa j. Some applications, such as smallangle xray scattering, also require large 10 micron sampling areas to ensure high signaltonoise ratios and to. However, various discrete dropletbased mixing platforms have also been proposed.

Finding the optimal design of a passive microfluidic mixer. We present a design of a microfluidic mixer based on hydrodynamic focusing which is used to initiate the folding process i. Reagents are shunted, stored and reacted in flow channels that are gated by valves formed by. Pdf microfluidic mixers for studying protein folding. Many mixers have been proposed to facilitate this task over the past 10 years. We focus on small ultrafast folding proteins and peptide models to facilitate quantitative comparison of experiment with the predictions of folding theory and simulations. Nonequilibrium single molecule protein folding in a. A method to measure fluid speeds on the order of 10,000 mms in microchannels is presented.

The folding process is initiated by quickly diluting a local denaturant concentration, and we define mixing time as the time advecting proteins experience a specified to achieve a local drop in denaturant concentration. Micropillars mixing in microfluidic devices by andy aditya flow in a cross by adam field. Improvements in mixing time and mixing uniformity in devices. Nih r01 gm53640019, fast events in protein folding, dyer, p. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. 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. As the field grows, the need for more versatile mixers grows with it. A combined singlemolecule fluorescence resonance energy transfer smfret and microfluidic mixing study reveals that the. Femtomole mixer for microsecond kinetic studies of protein. Lapidus 1 1 department of physics and astronomy, michigan state university, 2 department of mechanical engineering, hong kong university of science and technology, 3 center for biophotonics, university of california. In the past, they applied microfluidic mixers to trigger and monitor protein and rna folding using saxs. The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. Unlike a stoppedflow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. These devices can be used in studying of other biological systems.

Morshedd materials processing research centre, dublin city university, dublin 9, ireland. A useful method for studying proteinfolding dynamics is singlemolecule spectroscopy, using forster resonance energy transfer fret. We present a highthroughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low reynolds numbers determined by the channels hydraulic diameter, flow velocity, and solutions kinetic viscosity. Sdl offers rapid prototyping and iterative design services for your labonachip device, with scaleup capabilities. 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.

Ultrafast microfluidic mixer with threedimensional flow. Hydrodynamic focusing owen oconnor, svilen kolev, lily. Together, they offer the potential to obtain structural information on biomacromolecules, nanoparticles and condensed matter. Microfluidic mixers for studying protein folding steven a.

Optimization of a microfluidic mixer for studying protein. The group has also developed a microfluidic cell to trigger and measure lightdependent protein conformational changes. Keywords microfluidic, electrostatic actuation, micro mixer, ansys i. There has been a lot of research done on the kinetics of protein folding but mixing times have limited research done on proteins with folding times with less than 10 microseconds. One such scheme involves the use of air pressure to form, actuate and mix two liquid droplets in a hydrophobic microcapillary valve device. Development of a fast microfluidic mixer for studies of.

In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. Raleigh, use of the novel fluorescent amino acid pcyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the nterminal domain of the ribosomal protein l9 and provides evidence for twostate folding. We have applied an optimization method in conjunction with numerical simulations to minimize the mixing time of a microfluidic mixer developed for protein folding studies. Microfluidic mixing and its use in labonachip devices.

Microfluidics for biological measurements with single. A motivation for using microfluidic mixers is to reduce sample consumption and decrease mixing time to microseconds. We designed and built a computer 10channel gas mixer and used it together with several microfluidic and multiwell plate setups to study cellular behavior and gene expression as a function of oxygen tension in the medium at a high throughput. Four lamination layers in a shallow channel reduce the diffusion. The mixing of analytes and reagents for a biological or chemical labonachip is an important, yet difficult, microfluidic operation. Nov 17, 2015 rapid mixing experiments were performed with a microfluidic mixing device fabricated by replica moulding in polydimethylsiloxane pdms described in refs 29, 30.

Polymeric microfluidic continuous flow mixer combined with. Fair department of electrical engineering, duke university, durham, north carolina 27708, usa. Common liquid used to microfluidic devices include whole blood samples, bacterial cells suspension, protein or antibody. By using a highly efficient passive micromixer based on multilaminar flow, mixing times in the low millisecond range are reached. Nov, 2004 we have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8. Microfluidics take a tour of the possibilities microfluidics is an exciting new technology with a number of promising applications. The initial concentration at y 0 is shown on the top. Microfluidic mixer designed for performing singlemolecule. Two and threedimensional modeling and optimization applied. Specifically, it allows for screening crystallization conditions together with onchip xray analysis of crystals formed, and can be further used for relevant functional studies of proteins via. Quantitative analysis of molecular interaction in a. Mixing in microfluidic devices and enhancement methods. Computer design of microfluidic mixers for proteinrna. While a variety of different microfluidic mixers have been designed by researchers, it remains unknown which if any of these mixers are optimal that is, which designs provide the most thorough mixing with the smallest possible fluidic resistance across the mixer.

In motion a spot undergoes the taylor dispersion active mixing using chaos micromixer staggered herringbone. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. Microfluidics for studying protein folding diseases. Obtaining these kinetics requires perturbation methods, such as rapid changes in solution conditions or temperature. With this highly efficient 3d microfluidic mixer, we further characterized the early folding kinetics of human telomere gquadruplex under molecular crowding conditions, and unravelled a new folding process within 550. In this work we developed a microfluidic mixer to measure single molecule fret under nonequilibrium. We have recently developed a microfluidic mixer that dilutes denaturant similar to 100fold in similar to 8 mu s2. Such ensemble mixers attempt to resolve proteinfolding intermediates along the temporal reaction coordinate alone. Both fields are highly versatile and find use in multiple scientific disciplines. There can be turbulence in microfluidics at low reynolds number. A mems microfluidic mixer with electrostatic actuation. The microfluidic crystallization platform developed in this work serves to overcome issues with current methods in protein structure determination.

Rapid threedimensional microfluidic mixer for high viscosity. Microfluidics for smallangle xray scattering intechopen. Accordingly, this paper commences by providing a high level overview of the field of microfluidic mixing devices before describing some of the more significant proposals for active and passive mixers. The optimization method uses a semideterministic algorithm to find the global minimum of the mixing time by varying the mixer geometry and flow conditions. Modelling and optimization applied to the design of fast. Rapid droplet mixers for digital microfluidic systems. Video article microfluidic mixers for studying protein folding. The confinement of a 3dfocused sample to a narrow stream near the middle of a microchannel renders its velocity nearly uniform and makes it possible to monitor the reaction kinetics without exclusion of any parts of the sample. International journal for numerical methods in engineering. Rnaprotein binding kinetics in an automated microfluidic. Here we describe the design and operation of an ultrafast microfluidic mixer with threedimensional 3d flow focusing.

We rely on the proteins to keep us healthy and they assemble themselves by folding. Here we propose a new design of a passive microfluidic mixer that utilizes the theories of chaotic advection to enhance mixing. Mechanical engineering because of the difficulty to achieve convective mixing in laminar flows, microfluidic mixers have been developed in this work to rapidly homogenized fluids in labonachip devices. Universal curve for mixing with laminar flow in microfluidic devices.

Here, we report a method to obtain submillisecond temporal resolution and molecular structural information of protein mis folding events by using a microfluidic continuousflow mixer mcfm in combination with fourier transform infrared ftir imaging. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. Smallangle xray scattering is a wellestablished biophysical technique, whilst microfluidics is proving to be a convenient technology for creating miniaturised multifunctional devices. We have recently developed a microfluidic mixer that dilutes denaturant 100fold in 8. The vast majority of mixers in the literature are designed specifically for one type. The devices are fabricated out of fused silica, so that they are transparent in the uv. To perform these molecular changes, the concentration of the denaturant, which is introduced into the mixer together with the protein, has to be diminished until a given value in a short period of time, known as mixing time. To perform these molecular changes, the concentration of the denaturant, which is introduced into the mixer together with the protein, has to be diminished until a given value in a short. Samples are quantitated in each unit cell by four independent mitomi detection areas, allowing four samples to be analyzed in. Rapid droplet mixers for digital microfluidic systems phil paik, vamsee k.

The microfluidic mixers presented above are all considered for the mixing of continuous bulk liquids. Folding refers to the way human protein folds in the cells that make up your body. To follow such reactions fast mixing of reagents enzyme and substrate is crucial. The riboreactor microfluidic chip prepares complex reactions using a commonring approach developed for the microfluidic formulator chip. In this work, we consider a microfluidic mixer that uses hydrodynamic diffusion stream to induce the beginning of the folding process of a certain protein. Finlayson1, andy aditya1, vann brasher1, lisa dahl1, ha quan dinh1, adam field1, jordan flynn1, curtis jenssen1, daniel kress, 1 anna moon1, francis ninh1, andrew nordmeier1, ho hack song1, and cindy yuen1 1university of washington corresponding author. Daniel deponte of the lcls sample environment department at slac national laboratory we are developing rapid microfluidic mixers for a variety of assays including studies of the kinetics of protein folding and enzymatic reaction rates.

The folding time of proteins may vary from nanoseconds to seconds or even longer. We describe the minimization problem and constraints and give a. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein 1. We consider a microfluidic mixer based on hydrodynamic focusing, which is used to initiate the folding process of individual proteins. Wang,ab fang yanga and wei zhaoa turbulence is commonly viewed as a type of macroflow, where the reynolds number re has to be sufficiently high. The mixers are designed to rapidly initiate protein folding reaction through the dilution of denaturant. Optimization of a fast microfluidic mixer for studying.

The optimized mixer is the fastest reported continuous flow mixer for protein folding. Previous literatures have demonstrated the study of folding kinetics by coupling microfluidic mixers with various detection methods such. Microfluidic mixing has developed into an effective tech nique to study biochemical kinetics, synthesis 46, and protein folding 710. Single molecule fret can be used to quantitatively analyse the conformational distributions. Fret is a well established phenomena where energy transfer occurs over small distances where energy is transferred from a donor fluorosphere to an acceptor molecule. Kinetic studies of biological macromolecules increasingly use microfluidic mixers to initiate and monitor reaction progress. We have demonstrated a microfluidic mixer device to characterized and study the macromolecule dynamics such as kinetics of protein folding, dnarna sequencing, single molecule study and detection etc. Microfluidic mixers for studying protein folding protocol. Rapid threedimensional microfluidic mixer for high. Optimization of a microfluidic mixer for studying protein folding kinetics.

Modeling and optimization applied to the design of fast. Mar 01, 2016 it was the fastest micromixer for high viscosity solutions compared with previous reports. Conventional stoppedflow mixers have allowed measurement of folding kinetics starting at about 1 ms. Characterizing homogeneous chemistry using wellmixed microeddies. When studying the kinetics of protein folding, hydrodynamic focusing may be used. The folding process is initiated by diluting from 90% to 30% the local denaturant concentration initially 6 m gdcl solution in a short time interval we refer to as mixing time.