Microfluidic Applications Research
We pursue a variety of projects in which microfluidic chips are designed for specific applications. In all of these we rely on the ability to control transport phenomena with great precision in microfluidic chips. At times, these efforts purely focus 'merely' on downscaling similar processes traditionally performed at much larger scales to reduce sample needs, increase throughput, and/or increase precision. At other times, we exploit the different properties of the microscale (e.g. laminar flow) to accomplish a certain task that cannot be performed in a similar way at the macroscale. These are the current areas of our research:
Microreactors for Biocatalysis
Microreactors for High-throughput Biological Assays
Microreactors for Biocatalysis
Collaboration: Dr. Theodore Tzedakis, Universite´ Paul Sabatier, Toulouse, France
Funding: University of Illinois - CNRS, France Exchange Program
The development of environmentally friendly and stereoselective enzymatic transformations (biocatalytic processes) in pharmaceutical and fine chemical industries is an area of high current interest. In many instances, cofactors ('helper molecules/ions') are essential to promote rapid and efficient biocatalysis, and are capable of performing complex chemistry and catalyzing a large number of synthetically useful reactions. However, the cofactors are too expensive to be used in stoichiometric quantities, and in situ regeneration of cofactors provides the most promising route towards the creation of efficient and cost-effective biocatalysis platforms. Yet, the development of methods for competent cofactor regeneration is one of the long-standing challenges that has prevented the more widespread use of biocatalysis. In our research group, we exploit the controlled laminar flow characteristics and fast diffusive mixing at small scales for regeneration of cofactors for biocatalysis in a continuous process.
For cofactor regeneration in a microfluidic reactor (microreactor), two liquid streams (buffer and substrate) are guided into a Y-shaped microchannel, where the ratio of the flow rates of the streams (Q2/Q1) controls the relative widths (w1 and w2) of the two streams. The ability of a multi-stream laminar flow to focus a stream of reactants close to the electrode enables reversal of normally unfavorable reaction equilibrium, which is essential for enzyme/cofactor regeneration. COMSOL simulations (finite element analysis) and preliminary experiments with standard redox chemistry are used to determine the optimum operating conditions of the microreactors for cofactor regeneration experiments.
To demonstrate the effectiveness of microfluidics for cofactor regeneration, we have addressed the regeneration of nicotinamide adenine dinucleotide (NADH), using FAD (flavin adenine dinucleotide) as a mediator via electrochemical reactions. The enzymes that synthesize and use NADH are important in both, current pharmacology and research into future treatments for diseases. Using UV-vis spectroscopy, we calculated a maximum conversion of 31 % NADH, which is significantly better than any cofactor conversion efficiency reported previously. We also used the microreactor for in situ conversion of an achiral substrate (pyruvate) into a chiral product (L-lactate), using lactate dehydrogenase (LDH) as an enzyme. The 'turnover number' (TN, moles of product formed per mole of cofactor per unit of time) for this biocatalytic experiment is an order of magnitude higher than that reported in literature. The TN also can be significantly increased within the microreactor by engineering of the depletion concentration boundary layer formed at the electrode. While further research and development is required to utilize this cofactor regeneration technique in actual biocatalytic processes, the challenge has now shifted from the long-standing problem of cofactor regeneration to a more tangible engineering challenge of integration of a large number of these microreactors (parallel operation) in a recirculating system for biocatalytic synthesis in larger quantities (i.e. kilograms).
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| (a) Schematic diagram of the electrochemical microreactor for cofactor regeneration and subsequent biocatalytic substrate conversion. In gray: reaction depletion zones (not to scale). (b) Adjustment of the width or focusing of the substrate stream by changing the ratio of volumetric flow rates, Q2/Q1. Insets: Optical micrographs of two differently dyed aqueous streams in laminar flow for different ratio of flow rates. |
- Laminar Flow-Based Electrochemical Microreactor for Efficient Regeneration of Nicotinamide Cofactors for Biocatalysis. S.K. Yoon, E.R. Choban, C. Kane, T. Tzedakis, P.J.A. Kenis, J. Am. Chem. Soc., 2005, 127, 10466-10467. [Supplemental Material]
- Active Control of the Depletion Boundary Layer in Microfluidic Electrochemical Reactors. S.K. Yoon, G. Fichtl, P.J.A. Kenis, Lab on a Chip, 2006, 6, 1516-1524.
Microreactors for High-throughput Biological Assays
Collaboration: Dr. Brian T. Cunningham, Electrical and Computer Engineering, University of Illinois at Urbana-Champaign
Funding: National Science Foundation (NSF)
To address the demand for high-throughput assays for drug screening and systems biology, macro-scale tools, such as multi-well plates and robotics, are adopted for large-scale screening. However, these methods suffer from limitations of poor small-volume liquid handling ability, large consumption of expensive and not-easily-available reagents and high cost of operation. On the other hand, microfluidic chips can overcome these problems and provide potentially inexpensive and efficient tools for high-throughput biological assays. Previous efforts for combinatorial mixing and analysis (high-throughput processing) have focused on continuously flowing microfluidic systems, which are limited to nanoliter volumes and simple reaction schemes due to the serial nature of mixing and analysis. The advent of microfluidic networks with vast arrays of valves has enabled massively parallel chemical synthesis and biological studies in very small volumes (nano- to picoliters). However, these microfluidic systems require a continuous connection to an external power source that restricts the portability of the system, which is not desirable for high-throughput analytical techniques. To address the above problems, we develop microfluidic chips or microreactors with non-conventional components for high-throughput biological assays.
One of the non-conventional microfluidic components in our high-throughput microreactors are Actuate-to-Open (AtO) valves. Conventionally, fluids are manipulated in microfluidic chips using Actuate-to-Close (AtC) valves that are pneumatically actuated at high pressures to actively close off fluid lines. However, the AtC valves require to be continuously connected to an external pneumatic pump to hold the liquids in their compartments, which limits the portability of the chips, e.g. between a filling station for the liquids and detection ancillaries for analysis. On the other hand, AtO valves are closed in rest state (preferred state for most of the time), which increases the portability of the chips. Additionally, the relatively simple integration of the valves in microfluidic chips enables for massive scaling for combinatorial mixing and screening capabilities.
As proof-of-concept, we created a 4 X 4 array of microfluidic well plates (200 picoliter compartments) with combinatorial mixing and sensing capabilities as enabled by the use of AtO valves (figure 2, bottom right). The wells contain patterned photonic crystal (PC) biosensors to enable on-chip, in situ detection of biomolecular binding events. Reagents are introduced into the microfluidic network by actuation with a negative pressure, and when the AtO valves are relaxed (rest state), all compartments are sealed off, so that the chip can be transported for further analysis. The central picture in figure 3 is an optical micrograph of the wells; the 4 rows and 4 columns are each filled with an aqueous solution of different color to show the combinatorial generation of 16 different combinations of reagents. We investigated protein/antibody binding assay using PC biosensors for different combination of antibodies and solution concentrations. The top right and bottom left pictures in figure 3 are spectrographs of the PC biosensors. This microfluidic array chip is thus a promising candidate for chemical synthesis and combinatorial screening applications where multiple steps have to be carried out in parallel using minimal reagent volumes.
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| Figure 2. 4 X 4 Array Microfluidic Well Plate with integrated Photonic Crystal Biosensors. |
- Microfluidic chip for combinatorial mixing and screening of assays. B. R. Schudel, C. J. Choi, B. T. Cunningham, P. J. A. Kenis, Lab on a Chip, 2009, 9 (12), 1676-1680.