Hydrogel discs for digital microfluidics.

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.

Digital microfluidic hydrogel microreactors for proteomics.

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.

Digital microfluidics for automated proteomic processing.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.

A digital microfluidic approach to proteomic sample processing.

A common characteristic for proteomic analyses is the need for extensive biochemical processing. Digital microfluidics (DMF), a technique characterized by the manipulation of discrete microdroplets (100 nL-10 microL) on an open array of electrodes, is a good match for carrying out rapid, automated solution-phase reactions. Here, we report a DMF-based method integrating several common processing steps in proteomics, including reduction, alkylation, and enzymatic digestion. Fluorogenic assays were used to quantitatively evaluate the kinetics and reproducibility of each reaction step, and MALDI-MS was used for qualitative confirmation. The method is fast, facile, and reproducible, and thus has the potential to be a useful new tool in proteomics.

A world-to-chip interface for digital microfluidics.

Digital microfluidics (DMF) is a fluid handling technique that enables manipulation of discrete droplets on an array of electrodes. There is considerable enthusiasm for this method because of the potential for array-based screening applications. A limitation for DMF is nonspecific adsorption of reagents to device surfaces. If a given device is used to actuate multiple reagents, this phenomenon can cause undesirable cross-contamination. A second limitation for DMF (and all other microfluidic systems) is the "world-to-chip" interface; it is notoriously difficult to deliver reagents and samples to such systems without compromising the oft-hyped advantages of rapid analyses and reduced reagent consumption. We introduce a new strategy for digital microfluidics, in which a removable plastic "skin" is used to (a) eliminate cross-contamination and (b) bridge the world-to-chip interface. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots. This new method has the potential to transform DMF from being a curiosity for aficionados into a technology that is useful for biochemical applications at large.

Pluronic additives: a solution to sticky problems in digital microfluidics.

Digital microfluidics (DMF) is a promising technique for carrying out miniaturized, automated biochemical assays in which discrete droplets of reagents are actuated on the surface of an array of electrodes. A limitation for DMF is nonspecific protein adsorption to device surfaces, which interferes with assay fidelity and can cause droplets to become unmovable. Here, we report the results of a quantitative analysis of protein adsorption on DMF devices by means of confocal microscopy and secondary ion mass spectrometry. This study led us to a simple and effective method for limiting the extent of protein adsorption: the use of low concentrations of Pluronic F127 as a solution additive. This strategy has a transformative effect on digital microfluidics, facilitating the actuation of droplets containing greater than 1000-fold higher protein concentrations than is possible without the additive. To illustrate the benefits of this new method, we implemented a DMF-driven protein digest assay using large concentrations (1 mg/mL) of protein-substrate. The use of Pluronic additives solves a sticky problem in DMF, which greatly expands the range of applications that are compatible with this promising technology.