Microscale enzyme reactors comprising gold nanoparticles with immobilized trypsin for efficient protein digestion Muhammad Safdar, a Jens Sproß b and Janne Jänis a * Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: immobilization; digestion; microreactor; gold nanoparticle; FT-ICR MS Dear Editor, 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. [1,2] The use of microfluidic devices allow easy pro- cessing 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. [3–5] In MS-based proteomics, enzymatic digestion is one of the key steps in the identification of proteins and their posttranslational modifications. [6] 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. [7–9] On the other hand, problems associated with the fabrication and applicability of different sta- tionary phases may pose some challenges, such as monolithic supports involve time consuming fabrication procedures. Simi- larly, particle-based supports usually involve multistep synthesis and require extended incubation times for protein digestion. [10] Hence, in order to accelerate digestion, other techniques such as microwave irradiation have been employed, [11] 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. [2] Stigter et al. reported the development of an open tubular trypsin reactor based on amino-modified and carboxyl-modified dextran. [9] Later, the same group prepared a pepsin-immobilized reactor in a dextran-modified fused silica capillary for online protein digestion. [12] The coating of the capillary wall with dextran hydrogel was carried out in order to load higher amounts of trypsin, thus providing better enzyme/support interactions as compared to the flat surfaces. Because of their unique features, such as high surface area and surface-to-volume ratios, nanoparticles offer a great potential for various applications. Gold nanoparticles (GNPs), in particular, can serve as a stable and nontoxic support for drug delivery, biosensing and bioimaging purposes. [13] The use of GNPs in separation science is also growing and has been reviewed recently. [14] For the purpose of protein digestion, Liu et al. have reported fabrication of a microfluidic enzyme reactor by coating the microchannel of a polyethylene terephthalate chip with poly(diallyldimethylammonium chloride)/GNP multilayer films containing trypsin, for protein digestion. [15] Similarly, Hinterwirth and coworkers reported bioconjugation of trypsin onto GNPs by using different spacers of varying lengths. [16] The effect of differ- ent variables on the enzymatic activity was studied, such as the size of the GNPs and the nature and length of the spacer. The protein samples were incubated with trypsin-conjugated GNPs for different time intervals to accomplish proteolysis. We report here, for the first time, the preparation of open tubular microreactors comprising GNPs with immobilized trypsin, capable of performing protein digestion in a flow through fashion. Two different strategies were followed to obtain trypsin immobilized GNPs attached to the inner wall of a fused silica capillary. Fused-silica capillaries (50 μm i.d. and 360 μm o.d. with 100 cm length) were washed with water and treated with 1 M NaOH solution for 2 h at a flow rate of 1 μL/min. After washing the capillaries with water for 30 min, 0.1 M HCl solution was pumped through for 1h at the same flow rate. Subsequently, the capillaries were washed with water and acetone for 30 min each and dried under a stream of nitrogen gas for 2 h. A solution of 3-(trimethoxysilyl)-1-propanethiol and methanol (1 : 4, v/v) was flushed through the capillaries for 4 h at 1 μL/min and allowed to stand overnight. Next, the capillaries were washed with metha- nol and dried under a stream of nitrogen gas. These capillaries were further used for the preparation of microreactors. In the first strategy, 2.5 mg of trypsin was dissolved in 250 μL of 100 mM sodium phosphate buffer (pH 7.0) containing 50 mM * Correspondence to: Janne Jänis, Department of Chemistry, University of Eastern Finland, Yliopistokatu 7, FI-80101 Joensuu, Finland. E-mail: janne.janis@uef.fi J. Mass Spectrom. 2013, 48, 1281–1284 Copyright © 2013 John Wiley & Sons, Ltd. JMS letters Received: 12 July 2013 Revised: 11 October 2013 Accepted: 15 October 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3297 1281