Combining microfluidic networks and peptide arrays for multi-enzyme assays.

Abstract

This paper reports the use of microfluidic networks (muFNs) 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 muFNs 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 muFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples.

• Authors: Jing Su, Michelle R Bringer, Rustem F Ismagilov, Milan Mrksich
• Journal: Journal of the American Chemical Society (J Am Chem Soc) Vol. 127 Issue 20 Pg. 7280-1 (May 25 2005) ISSN: 0002-7863 [Print] United States
• PMID: 15898754 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Enzymes
• Peptides
• Protein Kinases
• Phosphoric Monoester Hydrolases

Topics

• Amino Acid Sequence
• Enzymes (analysis, metabolism)
• Microfluidic Analytical Techniques (methods)
• Molecular Sequence Data
• Peptides (chemistry, metabolism)
• Phosphoric Monoester Hydrolases (analysis, metabolism)
• Protein Kinases (analysis, metabolism)