Contact Information:

 Department of Chemistry

 Office: CIS E315

 Email:  r-ismagilov@uchicago.edu

 Voice: 773 702 5816

 Fax:    773 834 3544

 Mailing Address: 929 East 57th Street,

 Chicago, IL 60637 Directions

POSTDOCTORAL POSITIONS AVAILABLE

Research Interests:

Our research goal is to control and understand complex chemical and biological systems in space and time using microfluidics.  Our research is highly interdisciplinary, involving components of Chemistry, Physics, Biology, and Engineering.  We work towards our goal in three directions.

I.  Developing new microfluidic technology:
Microfluidic systems use networks of channels to manipulate small volumes of reagents, and they are becoming essential for chemical and biological analysis and synthesis.  Microfluidics has the potential to be a great tool for studying complex chemical networks, but both time and space control is made difficult by slow mixing and high dispersion of reagents transported through microchannels.  We are developing microfluidic networks that rely on pL-sized droplets to mix the reagents rapidly (less than 1 ms) and to transport them with no dispersion. These microfluidic networks use fluid flow to convert spatial evolution of chemical systems into temporal evolution (convert distance into time).  To develop this technology we go all the way from synthesizing new molecules to understanding details of physics of multiphase fluid flow.

Movies of plug transport and mixing of reagents:

straight channels

winding channels

(Flash Player 6 required)

II.  Validating technology by applying it to functional and structural characterization of proteins.
We use our microfluidic technology to develop simple tools that would help thousands of researchers in proteomics.  These tools are attractive because they consume tiny amounts of sample and allow conceptually new and exciting experiments to be performed.

II.a Droplet-based microfluidic system for evaluation protein crystallization conditions with on-chip x-ray diffraction.

Growing high-quality crystals of proteins is a crucial aspect of modern structural biology.  Despite theoretical investigations into crystallization phenomena, crystallizations of structurally unresolved proteins are generally empirical processes.  Crystallization assays are typically performed by pipetting protein, precipitant, and other additives together by hand, in which case solutions can be accurately dispensed on the order of 1 µL, or they may be pipetted robotically, in which case solutions may be accurately dispensed on the order of 10-100 nL.  Hand-pipetting is a labor intensive process, while robotic dispensing systems are financially unfeasible for many individual laboratories.  Microfluidic technology, however, offers the opportunity to make high-throughput fluid handling available to any laboratory at an affordable level. 

 A microfluidic method was developed to screen protein crystallization conditions in nL-sized droplets, each droplet representing a different crystallization condition.  By systematically changing the flow rates of aqueous reagent streams and flowing these streams into a flow of immiscible fluorinated oil on a microfluidic chip, hundreds of trials contained in droplets could be performed in a single experiment.  After the formation of a droplets containing assays, the droplets could be flowed directly into a glass capillary where precise control over evaporation allowed the experimenter to perform microfluidic crystallizations analogous to either large-scale microbatch or vapor diffusion experiments.  Crystals formed in glass capillaries could be diffracted directly “on-chip”. 

 We are now collaborating on a structural genomic project with the goal of integrating several promising instrumental, methodological, and software technologies, including our high-throughput microfluidic screening, into a program for low-cost structure determination. 

For more information, please see Accelerated Technologies Center for Gene to 3D structure (ATCG3D) -- New approach to protein crystallization:  www.atcg3d.org

II. b  Millisecond Kinetics on a Microfluidic Chip Using Nanoliters of Reagents

We are also developing a system for kinetic measurements in which a complete ms-resolved reaction profile can be obtained in a few seconds from a single spatially resolved image using < 100 nL of solutions.  We use this system as a research tool for studying time-dependent processes -- especially those that involve complex networks and autocatalytic reactions -- in chemistry, biochemistry and biophysics. 

III.  Using this technology to understand the dynamics of complex chemical systems.

In the first set of projects, we develop and implement using microfluidics  "minimal functional models" of complex biochemical networks (10-100 reactions).  We aim to reproduce the function of the network using a few (2-4) carefully chosen chemical (non-biological) reactions with proper kinetics; these chemical networks serve as the basis of biomimetic chemical systems.  We are applying this philosophy to hemostasis and cell differentiation. In the second set of projects, we apply the technology and the knowledge from minimal models to study the biochemical networks themselves, e.g. hemostasis and developmental pathways.

III.a Dynamics of Drosophila Embryonic Patterning Network Perturbed in Space and Time with Microfluidics

The sequencing of genomes such as that of the fruit fly, Drosophila melanogaster, has afforded a wealth of information regarding genes that regulate important biological processes.  However, how do chemical reactions involving these genes lead to crucial biological processes?  Essentially, a developing fruit fly is life’s beaker with thousands of chemicals (genes, proteins, and other small molecules) reacting in specific places at specific times.  The difference between chemical reactions in a beaker and chemical reactions in living organisms is that living organisms control where and when reactions occur.  How networks of chemical reactions in living organisms are orchestrated in space and time remains a difficult question in biology.  One such challenge is understanding how the chemical network that patterns the Drosophila embryo is orchestrated.  This patterning is critical in determining which part of the embryo will become which part of the adult body.   

The first step in patterning the embryo is determining the middle of the embryo.  This is accomplished through precise expression of the protein Hunchback in half of the embryo.  Amazingly, the embryo can determine its middle precisely under different environmental conditions such as temperature.  This is not surprising since the embryo experienced these conditions during evolution and developed a compensation mechanism to correct for its varying environment.  However, the critical compensation event in the network which sets the middle of the embryo remains unknown.

The reductionism approach – looking at a single component of a network of reactions – has been important in determining which genes and proteins are present in the network which determines the middle of the embryo.    Looking at the network as a whole can provide information on where and when critical events occur.  We used the approach of looking at this developmental network in Drosophila embryos as a whole. 

We used microfluidics to control development of wild-type Drosophila embryos in space and time.  Two streams of fluid flow, one at a lower temperature and one at a warmer temperature, were used to cool one half of the embryo and warm the other half of the embryo (Figure 1a).  Using this system, we asked three questions:  1) Will the embryo feel the effect of the unnatural environment, 2) Will the middle of the embryo still be correctly determined under these extremely unnatural conditions, and 3) Can we learn something about the mechanism by which the embryo determines its middle? 

Embryos developed with one half cool and one half warm (Figure 1a) felt the effect of the temperature step – the warm half of the embryo developed more rapidly than the cool half. 

Surprisingly, these embryos still determined the correct middle.  The network responsible for determining the middle still functioned under these conditions not seen during evolution.

However, one specific perturbation in space and time (Figure 1a-c) disrupted the developmental network.  In these experiments, first the embryo was developed with its anterior warm and its posterior cool from 0-65 minutes (Figure 1a).  Then, the temperatures were reversed and the embryo developed with its anterior cool and its posterior warm from 65-100 minutes (Figure 1b).  Finally, the temperatures were reversed back and the embryo developed with its anterior warm and its posterior cool from 100 minutes to the conclusion of the experiment (Figure 1c).  Embryos from these experiments did not always determine the correct middle.  These experiments suggested that the time at which the compensation mechanism performs its critical function is between 65 and 100 minutes in development.

Ultimately, to determine both how the chemical network is orchestrated in space and time and the molecular players involved in the compensation mechanism, the reductionism approach will have to be combined with the approach of looking at the entire network.  Microfluidic technology could prove to be a powerful tool in combining these two approaches for both this developmental network and other biochemical networks.

III. b Predicting the dynamics of blood clotting using a minimal functional model

Hemostasis, the complex reaction network that regulates blood clotting, is another system into which we are gaining insight through a simple functional model.  To design this model, we represented the ~80 reactions of hemostasis as three interacting modules (see rate plot in figure below).  The functional chemical model consists of three reactions with kinetics matching those of the modules.  We have shown that this simple chemical system can both reproduce and predict non-trivial behavior of blood clotting (Angew. Chem. Int. Edit. 200443:1541-1536 and PNAS 2006 103:15747-15752, see "publications" for full text).  Microfluidics is utilized to probe the chemical model and human blood plasma under conditions that mimic the geometry of blood vessels and the surface chemistry of vascular damage.

Using microfluidics and this simple chemical model for the blood clotting network, we have predicted that initiation of clotting on surfaces of clotting stimuli will only occur if the surface patch of stimuli is larger than a threshold size, and we verified this prediction with human blood plasma (see fluorescence images in figure below).  The chemical model both correctly predicted the value of the critical patch size for human blood plasma and that clotting depends on the actual size of the patch and not the total area of surface stimuli.  These experiments explain how blood can remain in a fluid state, where clotting occurs only with significant damage to our vascular system even though there is always constant exposure of blood to low amounts of stimuli.

Using the chemical model we have also generated a testable hypothesis for how clotting can remain localized to regions of damage after clotting has initiated.  The chemical model predicts that vessel geometry and flow rates are regulators of clot propagation, and these predictions are currently being tested with human blood plasma.