Multilayer-Assembled Microchip for Enzyme Immobilization as Reactor Toward Low-Level Protein Identification

A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization timeof- flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayerassembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATRIR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was 600 mM/min íg, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as model substrates for the tryptic digestion. The standard proteins were identified at a low femtomole per analysis at a concentration of 0.5 ng/íL with the digestion time