Multilayer-Assembled Microchip for Enzyme Immobilization as Reactor Toward Low-Level Protein Identification | doi.page

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

Analytical Chemistry2005Vol. 78(3), pp. 801–808

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Yun Liu, Haojie Lu, Wei Zhong, Pengyu Song, Jilie Kong, Pengyuan Yang, Hubert H. Girault, Baohong Liu

Abstract

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 time-of-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled 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, ATR-IR, 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 approximately 600 mM/min mug, 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/muL with the digestion time <5s. This simple technique may offer a potential solution for low-level protein analysis.

Citations Over TimeTop 10% of 2005 papers

Abstract

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