Combining microfluidic networks and peptide arrays for multi-enzyme assays
Abstract
This paper reports the use of microfluidic networks (μFNs) to both prepare peptide microarrays and carry out label-free enzyme assays on self-assembled monolayers (SAMs) of alkanethiolates on gold. A poly(dimethylsiloxane) (PDMS) stamp fabricated with microchannels is used to immobilize a linear array of cysteine-terminated peptides onto SAMs presenting maleimide groups. The stamp is then reapplied to the SAM in a perpendicular direction to introduce enzyme solutions so that each solution can interact with an identical linear array of immobilized peptides. The μFNs enable multiple enzyme−substrate interactions to be simultaneously evaluated at a submicroliter scale, while the use of SAMs enables the use of MALDI mass spectrometry (MS) to analyze the enzyme activities. This paper demonstrates applications of this system for assaying multiple kinases and for profiling the activities of kinases and phosphatases in human K562 cell extracts. The combination of μFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples.
Additional Information
Copyright © 2005 American Chemical Society. Published In Issue: May 25, 2005. Received March 3, 2005. This work was supported by the NSF-MRSEC and used the microfluidics and protein expression facilities. Supporting Information Available: Details for fabrication of μFNs, preparation of SAMs, enzyme assays, mass spectrometric analysis, and cell extract preparation. This material is available free of charge via the Internet at http://pubs.acs.org.Attached Files
Supplemental Material - ja051371osi20050417_102001.pdf
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Additional details
- Eprint ID
- 40877
- DOI
- 10.1021/ja051371o
- Resolver ID
- CaltechAUTHORS:20130821-160733236
- NSF
- Created
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2013-08-28Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field