Integration of protein processing steps on a droplet microfluidics platform for MALDI-MS analysis.

A droplet-based (digital) microfluidics platform has been developed to prepare and purify protein samples for measurement by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Liquid droplets are moved in air by sequentially applying an electric potential to an array of electrodes patterned beneath a hydrophobic dielectric layer. We show that a complete integrated sequence of protein processing steps can be performed on this platform, including disulfide reduction, alkylation, and enzymatic digestion, followed by cocrystallization with a MALDI matrix and analysis of the sample in situ by MALDI-MS. Proteins carbonic anhydrase, cytochrome c, and ubiquitin were used to demonstrate the digestion and postdigestion steps; insulin, serum albumin, and lysozyme were used to illustrate the complete sequence of protein processing steps available with the platform. Several functional improvements in the platform are reported, notably, the incorporation of acetonitrile in the protein droplets to facilitate movement, and patterning the device surfaces to optimize sample crystallization. The method is fast, simple, repeatable, and results in lower reagent consumption and sample loss than conventional techniques for proteomics sample preparation.

Electromechanical model for actuating liquids in a two-plate droplet microfluidic device.

Both conducting and insulating liquids can be actuated in two-plate droplet ("digital") microfluidic devices. Droplet movement is accomplished by applying a voltage across electrodes patterned beneath the dielectric-coated top and bottom plates. This report presents a general electromechanical model for calculating the forces on insulating and conducting liquids in two-plate devices. The devices are modeled as an equivalent circuit in which the dielectric layers and ambient medium (air or oil) are described as capacitors, while the liquid being actuated is described as a resistor and capacitor in parallel. The experimental variables are the thickness and dielectric constant of each layer in the device, the gap between plates, the applied voltage and frequency, and the conductivity of the liquid. The model has been used to calculate the total force acting on droplets of liquids that have been studied experimentally, and to explain the relative ease with which liquids of different conductivities can be actuated. The contributions of the electrowetting (EW) and dielectrophoretic (DEP) forces to droplet actuation have also been calculated. While for conductive liquids the EW force dominates, for dielectric liquids, both DEP and EW contribute, and the DEP force may dominate. The general utility of the model is that it can be used to predict the operating conditions needed to actuate particular liquids in devices of known geometry, and to optimize the design and operating conditions to enable movement of virtually any liquid.

Droplet-based microfluidics with nonaqueous solvents and solutions.

In droplet-based ("digital") microfluidics, liquid droplets in contact with dielectric surfaces are created, moved, merged and mixed by applying AC or DC potentials across electrodes patterned beneath the dielectric. We show for the first time that it is possible to manipulate droplets of organic solvents, ionic liquids, and aqueous surfactant solutions in air by these mechanisms using only modest voltages (<100 V) and frequencies (<10 kHz). The feasibility of moving any liquid can be predicted empirically from its frequency-dependent complex permittivity, epsilon*. The threshold for droplet actuation in air with our two-plate device configuration is /epsilon*/>8x10(-11). The mechanistic implications of these results are discussed, along with the greatly expanded range of applications for digital microfluidics that these results suggest are now feasible.

Fluorinated liquid-enabled protein handling and surfactant-aided crystallization for fully in situ digital microfluidic MALDI-MS analysis.

A droplet (digital) microfluidic device has been developed that enables complete protein sample preparation for MALDI-MS analysis. Protein solution dispensing, disulfide bond reduction and alkylation, tryptic digestion, sample crystallization, and mass spectrometric analysis are all performed on a single device without the need for any ex situ sample purification. Fluorinated solvents are used as an alternative to surfactants to facilitate droplet movement and limit protein adsorption onto the device surface. The fluorinated solvent is removed by evaporation and so does not interfere with the MALDI-MS analysis. Adding a small amount of perfluorooctanoic acid to the MALDI matrix solution improves the yield, quality and consistency of the protein-matrix co-crystals, reducing the need for extensive 'sweet spot' searching and improving the spectral signal-to-noise ratio. These innovations are demonstrated in the complete processing and MALDI-MS analysis of lysozyme and cytochrome c. Because all of the sample processing steps and analysis can be performed on a single digital microfluidic device without the need for ex situ sample handling, higher throughput can be obtained in proteomics applications. More generally, the results presented here suggest that fluorinated liquids could also be used to minimize protein adsorption and improve crystallization in other types of lab-on-a-chip devices and applications.