Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator.

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.

[1]  Sung Jae Kim,et al.  Concentration polarization and nonlinear electrokinetic flow near a nanofluidic channel. , 2007, Physical review letters.

[2]  Helen Song,et al.  Reactions in droplets in microfluidic channels. , 2006, Angewandte Chemie.

[3]  R. Ismagilov,et al.  Protein crystallization using microfluidic technologies based on valves, droplets, and SlipChip. , 2010, Annual review of biophysics.

[4]  Jongyoon Han,et al.  Increase of reaction rate and sensitivity of low-abundance enzyme assay using micro/nanofluidic preconcentration chip. , 2008, Analytical chemistry.

[5]  A. Abate,et al.  High-throughput injection with microfluidics using picoinjectors , 2010, Proceedings of the National Academy of Sciences.

[6]  Jongyoon Han,et al.  Microfluidic concentration-enhanced cellular kinase activity assay. , 2009, Journal of the American Chemical Society.

[7]  Sindy K. Y. Tang,et al.  Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets. , 2010, Angewandte Chemie.

[8]  Rustem F Ismagilov,et al.  Using microfluidics to observe the effect of mixing on nucleation of protein crystals. , 2005, Journal of the American Chemical Society.

[9]  Kinneret Keren,et al.  Dynamic imaging of protease activity with fluorescently quenched activity-based probes , 2005, Nature chemical biology.

[10]  Liang Li,et al.  Using a multijunction microfluidic device to inject substrate into an array of preformed plugs without cross-contamination: comparing theory and experiments. , 2007, Analytical chemistry.

[11]  D. Wild The Immunoassay Handbook , 2001 .

[12]  Se-kwon Kim,et al.  Creation of stepwise concentration gradient in picoliter droplets for parallel reactions of matrix metalloproteinase II and IX. , 2011, Analytical chemistry.

[13]  Jongyoon Han,et al.  Non-linear and linear enhancement of enzymatic reaction kinetics using a biomolecule concentrator. , 2011, Lab on a chip.

[14]  Sung Jae Kim,et al.  Amplified electrokinetic response by concentration polarization near nanofluidic channel. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[15]  Amy E Herr,et al.  Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography. , 2010, Analytical chemistry.

[16]  Petra Schwille,et al.  A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices , 2005, Chembiochem : a European journal of chemical biology.

[17]  Jongyoon Han,et al.  Pre-binding dynamic range and sensitivity enhancement for immuno-sensors using nanofluidic preconcentrator. , 2008, Lab on a chip.

[18]  Yiqiong Zhao,et al.  Compartmentalization of chemically separated components into droplets. , 2009, Angewandte Chemie.

[19]  W. Stetler-Stevenson,et al.  Quantitative zymography: detection of picogram quantities of gelatinases. , 1994, Analytical biochemistry.

[20]  R. Probstein Physicochemical Hydrodynamics: An Introduction , 1989 .

[21]  Sung Jae Kim,et al.  Direct seawater desalination by ion concentration polarization. , 2010, Nature nanotechnology.

[22]  Douglas A Lauffenburger,et al.  Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities. , 2011, Integrative biology : quantitative biosciences from nano to macro.

[23]  W. Stetler-Stevenson,et al.  Quantitative reverse zymography: analysis of picogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms. , 1997, Analytical biochemistry.

[24]  Wilhelm T S Huck,et al.  Generation of picoliter droplets with defined contents and concentration gradients from the separation of chemical mixtures. , 2010, Analytical chemistry.

[25]  J. S. Johnson,et al.  Biocompatible surfactants for water-in-fluorocarbon emulsions. , 2008, Lab on a chip.

[26]  A. L. Stevens,et al.  Million-fold preconcentration of proteins and peptides by nanofluidic filter. , 2005, Analytical chemistry.

[27]  Helene Andersson-Svahn,et al.  Detection and analysis of low-abundance cell-surface biomarkers using enzymatic amplification in microfluidic droplets. , 2009, Angewandte Chemie.

[28]  Rustem F Ismagilov,et al.  Microfluidic stochastic confinement enhances analysis of rare cells by isolating cells and creating high density environments for control of diffusible signals. , 2010, Chemical Society reviews.

[29]  Yong-Ak Song,et al.  Multiplexed proteomic sample preconcentration device using surface-patterned ion-selective membrane. , 2008, Lab on a chip.

[30]  Daniel Bratton,et al.  Development of quantitative cell-based enzyme assays in microdroplets. , 2008, Analytical chemistry.

[31]  Liang Li,et al.  Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins , 2006, Proceedings of the National Academy of Sciences.

[32]  Liang Li,et al.  Simple host-guest chemistry to modulate the process of concentration and crystallization of membrane proteins by detergent capture in a microfluidic device. , 2008, Journal of the American Chemical Society.

[33]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[34]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[35]  Christoph A. Merten,et al.  Drop-based microfluidic devices for encapsulation of single cells. , 2008, Lab on a chip.

[36]  A. deMello,et al.  Quantitative detection of protein expression in single cells using droplet microfluidics. , 2007, Chemical communications.

[37]  Sung Jae Kim,et al.  Stabilization of ion concentration polarization using a heterogeneous nanoporous junction. , 2009, Nano letters.

[38]  M. Stack,et al.  Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion , 2007, Nature Cell Biology.

[39]  Liang Li,et al.  Laterally mobile, functionalized self-assembled monolayers at the fluorous-aqueous interface in a plug-based microfluidic system: characterization and testing with membrane protein crystallization. , 2009, Journal of the American Chemical Society.