Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing.
Hydrogels are networks of hydrophilic polymer chains that are swollen with water, and they are useful for a wide range of applications because they provide stable niches for immobilizing proteins and cells. We report here the marriage of hydrogels with digital microfluidic devices. Until recently, digital microfluidics, a fluid handling technique in which discrete droplets are manipulated electromechanically on the surface of an array of electrodes, has been used only for homogeneous systems involving liquid reagents. Here, we demonstrate for the first time that the cylindrical hydrogel discs can be incorporated into digital microfluidic systems and that these discs can be systematically addressed by droplets of reagents. Droplet movement is observed to be unimpeded by interaction with the gel discs, and gel discs remain stationary when droplets pass through them. Analyte transport into gel discs is observed to be identical to diffusion in cases in which droplets are incubated with gels passively, but transport is enhanced when droplets are continually actuated through the gels. The system is useful for generating integrated enzymatic microreactors and for three-dimensional cell culture. This paper demonstrates a new combination of techniques for lab-on-a-chip systems which we propose will be useful for a wide range of applications.
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