We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8. Optimization of a microfluidic mixer for studying protein folding. A motivation for using microfluidic mixers is to reduce sample consumption and decrease mixing time to microseconds. Transient misfolding dominates multidomain protein folding. Two and threedimensional modeling and optimization. Microfluidic approaches for the analysis of protein. In protein folding, internal friction may play a more. Conventional stoppedflow mixers have allowed measurement of folding kinetics starting at about 1. Pdf applications of micromixing technology researchgate.
Video article microfluidic mixers for studying protein folding. Unlike a stoppedflow mixer, this mixer operates in. To simplify the study of fast protein dynamics, we have developed an inexpensive continuousflow microfluidic mixer, requiring no specialized equipment or techniques. 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. Kinetic studies of biological macromolecules increasingly use microfluidic mixers to initiate and monitor reaction progress. Microfluidic mixers for studying protein folding ncbi. Pdf single molecule protein folding kinetics in a co. Optimization of a microfluidic mixer for studying protein. The first folding study to employ a microfluidic mixer studied. Microfluidic mixers for studying protein folding core. 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. 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. In this paper, we discuss the design and optimization of the mixer using modeling of convective. Unlike a stoppedflow mixer, this mixer operates in the laminar flow regime in.
Development of a fast microfluidic mixer for studies of. Protein hydrophobic collapse and early folding steps observed in a micro. 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. Optimization of a fast microfluidic mixer for studying. David hertzog 2005 optimization of a fast microfluidic mixer for studying protein folding kinetics david hertzog,1,2 benjamin ivorra,3 bijan mohammadi. Ultrafast microfluidic mixer with threedimensional flow. A method to measure fluid speeds on the order of 10,000 mms in microchannels is presented. A tool for protein folding unfolding study pc3267 updated in jan. In this study, we show that by combining highthroughput microfluidic screening with wholegenome sequencing of the selected clones from yeast libraries we can identify and map the mutations associated with significantly improved protein. We have recently developed a microfluidic mixer that dilutes denaturant 100fold in 8. Optimization of a microfluidic mixer for studying protein folding kinetics david e. Since then, the mixer has been optimized and used intensively in the. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood.
Optimization of a fast microfluidic mixer for studying protein folding. Micromachines free fulltext complete procedure for. Microfluidic emulsification through a monolithic integrated glass micronozzle suspended inside a flowfocusing geometry appl. Femtomole mixer for microsecond kinetic studies of protein. Furthermore by integrating the concept of microfluidic mixer with magnetic beads, make feasible to study the nextgeneration sequencing applications like genomic dna. We describe the minimization problem and constraints and give a. Design sensitivity and mixing uniformity of a micro. Here we describe the design and operation of an ultrafast microfluidic mixer with threedimensional 3d flow focusing. Optimization of a microfluidic mixer for studying protein folding kinetics article pdf available in analytical chemistry 78. A microfluidic mixer based on hydrodynamic focusing made from silicon was first introduced in 1996 by brody. People have instead studied the folding of pure protein. Microfluidics for studying protein folding diseases.
Hertzogs phone number, address, insurance information, hospital affiliations and more. During this study, the optimized mixer was demonstrated to be. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. Investigating fast folding of rna pseudoknot vpk with an ultrafast microfluidic mixer by andreas john meindl despite advances in understanding the theory behind rna folding, ab initio prediction of the folding process has not been achieved yet. The mixers are designed to rapidly initiate protein folding. We have recently developed a microfluidic mixer that dilutes denaturant 100 fold in 8. Microsecond protein folding events revealed by time. Unlike a stoppedflow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur.
Microsecond protein folding events revealed by timeresolved fluorescence resonance energy transfer in a micro. We consider a particular hydrodynamic focusing microfluidic mixer used to initiate the folding process of individual proteins, which has been designed in a previous work and exhibited a mixing time of 0. In protein folding, internal friction may play a more significant role than previously thought 24 april 2012 this is an amino acid chain folding into a three. Modelling and optimization applied to the design of fast. We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 ms that is widely accessible because it is made of commercially available parts. Lapidus 1 1 department of physics and astronomy, michigan state. Microfluidic mixer designed for performing singlemolecule. Complete procedure for fabrication of a fused silica. Highspeed velocimetry in microfluidic protein mixers. The csar mixer can work in a wide range of reynolds number 34. In contrast, conventional stoppedflow mixers have dead times of 15 ms, largely. Since then, the mixer has been optimized and used intensively in the field of protein folding due to its extremely low mixing time, which is on the order of microseconds 2,3,4,5,6,7,8,9,10,11. In clinical medicine and biological studies, microfluidic systems have been. Cytochrome c, in particular, has become a benchmark for development of fast folding techniques.
Conventional stoppedflow mixers have allowed measurement of folding kinetics starting at about 1 ms. 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. Microfluidic mixers for studying protein folding steven a. These devices can be used in studying of other biological systems. Santiago, department of mechanical engineering, stanford university, stanford, california 94305, chemistry and materials science. Abstract this paper details the making, characterization, and use of a simple and versatile capillarybased coaxial singlemolecule mixing device which has a response time of 510 milliseconds and which can be used to monitor bioconformational.
Polymeric microfluidic continuous flow mixer combined with. Microfluidics for biological measurements with single. 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. Protein hydrophobic collapse and early folding steps. Due to this complexity, the study of protein folding is extremely limited in vivo. Microfluidic devices have since been regularly used to study protein folding at the singlemolecule level.
Improvements in mixing time and mixing uniformity in. The optimization method uses a semideterministic algorithm to find the. Observations of a small protein show the evolution of the. We have recently developed a microfluidic mixer that dilutes denaturant similar to 100fold in similar to 8 mu s2. Computer design of microfluidic mixers for proteinrna. Hertzog,yz emily tubman, and olgica bakajiny department of physics and astronomy, michigan state university, east lansing, michigan. Microfluidic mixers for the investigation of rapid protein folding. In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminarflow mixer to a confocal optical system. Computer design of microfluidic mixers for proteinrna folding studies. Microfluidic mixers for studying protein folding protocol. 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.
The optimization method uses a semideterministic algorithm to find the global minimum of the mixing time by varying the mixer geometry and flow conditions. More rapid and sampleefficient mixing is achieved in laminar flow in a microfluidic device, in which the sample is twodimensionally 2d focused to a thin sheet. Waldauer 1, ling wu 1, shuhuai yao 2, olgica bakajin 3, lisa j. Pdf microfluidic mixers for studying protein folding. Some applications, such as smallangle xray scattering, also require large 10 micron sampling areas to ensure high signaltonoise ratios and to.
The objectiv e in this example is to capture protein folding, the process by. The use of this mixer to study protein folding has consistently revealed surprising results. Numerous experimental studies have revealed the structure of chaotic. 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. Protein hydrophobic collapse and early folding steps observed in. We describe the minimization problem and constraints and give a brief overview of the optimization algorithm. They labeled a substrate protein, rhodanese, with donor and acceptor fluorophores in. Mixing enhancement of a novel csar microfluidic mixer.
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