Instrument Development

 

For more than three decades, The Dovichi Group has developed highly sensitive instruments for biological applications. If you are interested in learning about our current projects, check back soon for more information or contact a lab member for details!

 

Ultra-Sensitive LIF

 

Laser-induced fluorescence (LIF) is the most sensitive method for detection in capillary electrophoresis.  The Dovichi group pioneered the use of a sheath flow cuvette that hydrodynamically focuses the sample stream at the tip of the capillary and dramatically lowers scatter.  The focused sample stream then passes through a focused laser and any emitted fluorescence is then sent to an avalanche photodiode for detection.  This setup lowers the limit of detection to 100 molecules or less.  Further adaptations can allow for even single molecule detection.  The exquisite sensitivity of our CE-LIF systems allows us to obtain unprecedented biological information from single cells. 

Our lab coined the term “metabolic cytometry” to describe the use of CE-LIF to monitor metabolism in single cells.  In this technique, we incubate cells with a fluorescently labeled substrate.  The cell’s natural machinery makes a more complex structure (anabolism) or breaks down the substrate into a simpler molecule (catabolism).  As long as the fluorophore tag remains intact, all metabolic products will be detected.  The single cell is then aspirated and lysed inside of a capillary, with all metabolic products being separated and detected using CE-LIF. 

Multicolor CE-LIF instrumentation allows us to simultaneously probe distinct metabolic pathways in single cells.  We have used this scheme to monitor the heterogeneity of glycolipid metabolism in single primary neurons.  Glycolipids constitute over 80% of the conjugated sugar content in the central nervous system and are involved in a variety of processes including neuronal differentiation, cellular signaling, oncogenesis, and pathogen binding.  Below we show capillary electrophoresis analysis of seven cerebellar granule neurons incubated with both GM1-TMR and LacCer-BODIPY-FL. 

Left:  Data shown for the TMR spectral channel - (A) full scale and (B) expanded scale.  Right:  Data shown for the BODIPY-FL spectral channel - (A) full scale and (B) expanded scale. Unknown components are marked with “?”.

 

CE-MS

 

Capillary electrophoresis-mass spectrometry (CE-MS) is widely used for large and small molecular characterization. However, it is still hampered by the interface due to serious sample dilution. Our group has developed an electrokinetically driven sheath flow interface for high sensitive detection of large molecules (i.e. proteins/peptides). The interface has several advantages, including high sensitivity due to reduced sample dilution (low amole detection limit for peptides), no requirement of an additional pump for sheath flow, and compatibility with both basic and acidic separation buffers. The interface has been used for one and multidimensional CE-MS systems for bimolecular identification and quantitation in our group. Different CE modes (capillary zone electrophoresis and capillary isoelectric focusing) have been coupled to different mass spectrometers (LCQ, LTQ, LTQ-Orbitrap Velos, Q-TOF (Bruker), QqQ (AB SCIEX), Q-Exactive, Q-Exactive HF and Oribtrap Fusion) with the interface at present. Our goal is to characterize the protein expression in large numbers of cells and in a single cell with the highly sensitive CE-MS system. A commercialized version of the interface is now available for purchase through CMP Scientific.