Electrophoretic Band Crossing
For Measurements of Biomolecular Binding Kinetics
Motivation
Micro total analysis systems (mTAS) are devices that aim to perform key chemical assay functions on-chip including sampling, sample pre-treatment (filtration, concentration, reaction, separation, etc.) and detection [2]. Kinetic measurements play an essential role in the study of biomolecular interactions, such as immuno-based and enzyme assays. To date, there have been several on-chip reaction kinetics studies [3, 4]. However, the typical mixing method employed is simple diffusive mixing of reactive species (e.g, as in small-scale stopped-flow reaction chambers). Diffusive mixing can severly limit the time resolution of these studies.
Project Description
We are developing an on-chip electrophoretic band-crossing system with a temporal resolution of order 100 ms. The idea is to simultaneously introduce reactive species into a microchannel and mix them using electromigration, by overspeeding one species through the other (Figure 1). The time scale for the crossing is dependent on the band width and the electrophoretic mobilities of reactants and products.
We have demonstrated non-reactive band crossing of BODIPY over Fluorescein (Figure 2), and reactive band crossing of Streptavidin-BODIPY conjugate over Biotin-Fluorescein conjugate (Figure 3). We are currently working toward integration of numerical simulation and experimental data to yield binding kinetics [5].
References
For Measurements of Biomolecular Binding Kinetics
Motivation
Micro total analysis systems (mTAS) are devices that aim to perform key chemical assay functions on-chip including sampling, sample pre-treatment (filtration, concentration, reaction, separation, etc.) and detection [2]. Kinetic measurements play an essential role in the study of biomolecular interactions, such as immuno-based and enzyme assays. To date, there have been several on-chip reaction kinetics studies [3, 4]. However, the typical mixing method employed is simple diffusive mixing of reactive species (e.g, as in small-scale stopped-flow reaction chambers). Diffusive mixing can severly limit the time resolution of these studies.
Project Description
We are developing an on-chip electrophoretic band-crossing system with a temporal resolution of order 100 ms. The idea is to simultaneously introduce reactive species into a microchannel and mix them using electromigration, by overspeeding one species through the other (Figure 1). The time scale for the crossing is dependent on the band width and the electrophoretic mobilities of reactants and products.
Figure 1. Schematic of band crossing
We have demonstrated non-reactive band crossing of BODIPY over Fluorescein (Figure 2), and reactive band crossing of Streptavidin-BODIPY conjugate over Biotin-Fluorescein conjugate (Figure 3). We are currently working toward integration of numerical simulation and experimental data to yield binding kinetics [5].
Figure 2. Non reactive band crossing 0.1 mM Fluorescein, 0.2 mM BODIPY, 10 mM Borate Buffer
Figure 3. Reactive band crossing 20
mM Biotin-Fluorecein (1:1), 70 mM Streptavidin-BODIPY (1:2.9), 10 mM Borate BufferReferences
- 1) A. Manz, N. Graber, H. M. Widmer, 1990, “Miniaturized total chemical analysis systems: a novel concept for chemical sensing”, Sensors and Actuators B1, 244-248.
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2) G. H. M. Sanders, A. Manz, 2000, “Chip-based microsystems for genomic and proteomic analysis”, Trends in Analytical Chemistry, Vol. 9, No. 6, 364-378. -
3) A. G. Hadd, D. E. Raymond, J. W. Halliwell, S. C. Jacobson, J. M. Ramsey, 1997, “Microchip device for performing enzyme assays”, Analytical Chemistry, 69, 3407-3412. -
4) N. H. Chiem, D. J. Harrison, 1998, “Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination”, Clinical Chemistry, 44:3, 591-598. -
5) C. H. Chen, J. C. Mikkelsen, J. G. Santiago, 2000, “Electrophoretic band crossing for measurements of biomolecular binding kinetics”, presented at 2000 International Forum on Biochip Technologies, Beijing, China