Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays.
High-quality single-cell data are required for a quantitative systems biology description of cellular function. However, data of this type are difficult and time-consuming to collect using traditional techniques. We present a robust and simple microfluidic method for trapping single cells in large arrays to address this problem. Ordered single-cell isolation arrays allow for high-density microscopic analysis with simplified image processing. Moreover, for fluorescent assays, on-chip sample preparation (e.g., fluorescent labeling, washing) can be performed, as opposed to manual intensive operations of incubation, centrifugation, and resuspension in previous techniques-saving time and reagents. This technology was applied to determine novel single-cell enzyme kinetics for three different cell types (HeLa, 293T, Jurkat). A kinetic model of this process predicted this varied response was due to variation in the concentration of carboxylesterase between cell types. Nordihydroguaiaretic acid (NDGA) was also characterized as an inhibitor of carboxylesterases. For HeLa cells, 20 nM of the 50 nM total carboxylesterases was unaffected by NDGA. This type of analysis could be directly applied to quantify a variety of intracellular enzymes with available fluorogenic substrates.
Di Carlo
Dino
D
Biomolecular Nanotechnology Center, Berkeley Sensor and Actuator Center, Department of Bioengineering, University of California, Berkeley, California 94720, USA.
Aghdam
Nima
N
Lee
Luke P
LP
D043182:Carboxylesterase / Q000378:metabolism*; D002460:Cell Line; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009637:Masoprocol; D046228:Microarray Analysis / Q000295:instrumentation* / Q000379:methods*; D015394:Molecular Structure
D009637:Masoprocol; D043182:Carboxylesterase