Proteomics
We are presently interested in developing microfluidic devices for addressing proteomics problems and automated single cell assays. Proteomics is a term used to describe the understanding of the full complement of proteins expressed by an organism. As a given type of cell will contain several thousand proteins the task of identifying all proteins is a significant challenge. Traditional approaches to solving this problem involve the use of milligram quantities of protein and chemical separations that unfold over a period of days. We are pursuing microfabricated fluidics approaches to implementing multidimensional liquid phase separations that utilize nanogram to picogram quantities of materials with separations unfolding over a period of tens of minutes. The ability to perform electrospray ionization from microchips is also being developed to allow structural analysis via mass spectrometry of the isolated proteins.
Automated microfluidic devices are also being developed to analyze single non-adherent cells. Individual cells will be characterized by flow cytometry techniques. Cells of interest will then be lysed and their contents chemically analyzed using various chemical separation techniques. This automated approach allows high-throughput characterization at the single cell level so that statistics can be rapidly generated on cell heterogeneity. Through collaboration with Prof. Nancy Allbritton (UNC), single cell kinase activity is being assessed using these devices to study signaling transduction pathways. Moreover, through collaborations with Prof. Andrew Green (Medical College of Wisconsin) and Prof. Lloyd Smith (University of Wisconsin) technology is being developed to perform proteome studies of single cells.
Microchip ESI MS
Proteomics relies on the use of mass spectrometry detection for the identification of whole proteins or peptide fragments. Microfluidic technology offers many capabilities for sample processing and separation which could improve upon current proteomics applications. This project is working towards utilizing those capabilities by interfacing microfluidic devices with mass spectrometry detection via electrospray ionization. The microchip electrospray interface uses electroosmotic pumping to force liquid flow out of the corner of the chip. Figure 1 shows a schematic and image of one electrospray microchip design. In this design the side channel is left uncoated while all of the other channels are coated with a polyamine to reverse the surface charge. The EOF in both the separation channel and the side channel flows toward the ESI orifice, generating pressure which drives the liquid out of the chip. An ESI voltage of ∼3.5-kV generates stable and sensitive electrospray. The polyamine coating repels peptides and proteins in acidic buffers allowing for highly efficient electrophoretic separations. Figure 2 shows a movie where the generated electrospray plume is illuminated by a green laser. Future work will optimize the separation parameters and electrospray performance for single cell proteomic analysis.
2D Separations
Two dimensional separation techniques are frequently used for complex biological sample analyses. The advantage of 2D separation techniques is that high peak capacities are achievable if the two techniques are based on fundamentally different separation techniques. We have previously demonstrated a 2D separation using MEKC and CE to analyze a BSA digest capable of generating peak capacities of ∼4000 (Ramsey, J. D. et al., Anal Chem 2003, 75, 3758-3764). Adjacent image shows the 2D chip design and the 2D separation.
Our current research efforts are focused on improving the first dimension separation capability with an ultimate goal of coupling the 2D separation technique with a mass spectrometer as described in the CE-ESI-MS section.
Single Cell Analysis
In collaboration with Nancy Allbritton’s laboratory (UNC), we are developing methods for integrating single cell analysis on microchips. Analysis on the single cell level allows for assessment of the heterogeneity in cellular responses. This heterogeneity is obscured by current high throughput techniques which collect the average cellular response. The goal is to study how enzymatic proteins function and how they are regulated within cellular signaling transduction pathways. This information can be used in developing predictive models and therapeutic tools to target elements in the network.
Some of the methods currently under development are the coupling of cell lysis and capillary electrophoresis of single cells. The images below show a single cell being pulled hydrodynamically through a channel. The cell is lysed when it encounters the electric field applied along the analysis channel. At 0.000 s, the cell enters the frame. At 1.000 s, the cell is focused toward the bottom of the channel by flow from the focusing channel. At 1.233 s, the cell is lysed from exposure to the electric field with the lysate being injected into the analysis channel at 1.267 s. The separation of the lysate occurs at 1.367 seconds and the membrane debris can be seen moving towards a cell waste reservoir at 1.500 s.