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Microscale enzyme reactors comprising gold nanoparticles with immobilized trypsin for efficient protein digestion.

Microscale sample processing devices for application in bioanalysis and proteome related studies have undergone rapid advancements during the recent years. Several techniques for the preparation of chip-based and capillary-based microfluidic systems and their modification with desired functionalities have been reported. The use of microfluidic devices allow easy processing of extremely small sample quantities with low reagent consumption and offer high surface areas and surface-to-volume ratios. Microscale sample processing devices find application in various research fields such as chemical analysis, kinetic studies, and bioanalytical research. In MS-based proteomics, enzymatic digestion is one of the key steps in the identification of proteins and their posttranslational modifications. However, this is usually the slowest step in the analysis workflow. Thus, the development of suitable devices and methods capable of performing efficient protein digestion more rapidly has become an area of considerable interest. The immobilization of proteolytic enzymes on different kinds of stationary phases, such as monoliths, microparticles and nanoparticles and inner surface of sample transfer capillaries has shown promising results. On the other hand, problems associated with the fabrication and applicability of different stationary phases may pose some challenges, such as monolithic supports involve time consuming fabrication procedures. Similarly, particle-based supports usually involve multistep synthesis and require extended incubation times for protein digestion. Hence, in order to accelerate digestion, other techniques such as microwave irradiation have been employed, which will further complicate the experimental setup. Alternatively, open channel microreactors comprising immobilized enzymes have appeared as a promising alternative, offering minimal restriction over the flow rates. Krenkova et al. prepared open tubular enzyme-immobilized reactors by coupling of trypsin and pepsin to the inner wall of a fused silica capillary. Stigter et al. reported the development of an open tubular trypsin reactor based on amino-modified and carboxyl-modified dextran. Later, the same group prepared a pepsin-immobilized reactor in a dextran-modified fused silica capillary for online protein digestion. The coating of the capillary wall with dextran hydrogel was carried out in order to load higher amounts