J. Lee, S.A. Soper, K.K. Murray, “Development of an efficient on-chip digestion system for protein analysis using MALDI-TOF MS,” Analyst. 134 (2009) 2426–2433. doi:10.1039/b916556h.

Abstract

(a) Assembled tryptic digestion microfluidic chip; chip components including PMMA substrate and cover slip, inlet and outlet connectors, capillary and stainless steel tubes. The sample solution was electrokinetically infused through the bioreactor and the matrix solution was loaded hydrodynamically with a syringe pump. Coaxial tubes mixed the bioreactor output with a matrix solution for deposition on a MALDI target. (b) Schematic top view of the fluid connection between the micropost bioreactor and the capillary tube interface to the deposition system. Two Pt electrodes were inserted into the sample inlet and the end of the bioreactor to electrokinetically drive the sample through the bioreactor.

A solid-phase trypsin microreactor was constructed and operated with electrokinetically-driven flow for the digestion of proteins and coupled off-line with MALDI-TOF MS. The bioreactor was fabricated from poly(methyl methacrylate), PMMA, by hot embossing using a mold master prepared by micro-milling. The solid-phase bioreactor consisted of a 4 cm long, 200 microm wide, and 50 µm deep microfluidic channel that was populated with an array of 50 µm diameter micropost structures with a 50 µm inter-post spacing. The bioreactor was prepared by covalently attaching the proteolytic enzyme, trypsin, to the UV-modified surface of the PMMA microstructures using the appropriate coupling reagents. The performance of the system was evaluated using a set of proteins. The bioreactor provided efficient digestion of cytochrome c at a field strength of 375 V/cm, producing a reaction time of approximately 20 s to produce 97% sequence coverage for protein identification. Bovine serum albumin (BSA), phosphorylase b, and beta-casein were also assessed and the sequence coverages were 46, 63, and 79%, respectively, using the same reactor residence time. Furthermore, Escherichia coli was used as a model to demonstrate the feasibility of fingerprint analysis for intact cells using this solid-phase bioreactor.