MALDI EWOD

One of the requirements in MALDI-MS analysis is the use of a liquid matrix. The electrowetting-on-dielectric (EWOD) method has been used to move and mix droplets containing proteins and peptides with the liquid matrix, all of which were situated at specific locations on an array of electrodes. With this method, insulin (1.75 pM), insulin chain B (2 pM), cytochrome c (1.85 pM), and myoglobin (1.45 pM) have been analyzed [518]. 

Also the use of an EWOD system for the automated sample preparation of peptides and proteins for matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) was reported. In that work, standard MALDI-MS reagents, analytes, concentrations, and recipes have been demonstrated to be compatible with the EWOD technology, and mass spectra comparable to those collected by conventional methods were obtained [271]. Also a PCR assay has been realized on the platform by temperature cycling of a droplet at rest [272]. Additional informations about the EWOD platform can be found in a comprehensive review [273]. 

EWOD-based digital microfluidics have already been combined with MALDI-TOF-MS by Wheeler et al.13 However, the Wheeler et al. device would have several drawbacks when used in pre-steady state kinetic studies, including lower throughput - for reasons explained in the Section 12.3 - and the absence of a mixing element. Further, while Paik et al.5 have demonstrated droplet mixers for EWOD-based systems, their system is simply not fast enough to be of use in pre-steady state kinetics. 

The coupling of microfabricated on-chip SPE devices to MALDI-MS has previously been presented by our group. Another approach to on-chip SPE—the microfluidic compact disk Gyro Lab MALDI SPl (Gyros AB, Sweden)— has been presented and commercialized by Gyros AB, where centrifugal force is used to transport liquids for the purpose of SPE before MALDI-MS. EWOD and electrocapture are techniques that can be used instead of SPE for purification and concentration before MALDI-MS. 

Recently, EWOD actuation chips were developed into a multiplexed device that was used to simultaneously cleanup four samples. A sequence of seven actuation steps were performed for each sample (1) generation of sample droplets (0.02 p.L), (2) transport and drying of sample droplets, (3) generation of rinsing droplets, (4) transport of rinsing droplets to the sample sites for selective dissolution of urea, (5) transport and disposal of the rinsing droplets, (6) generation of MALDI matrix solution droplets, and (7) delivery of matrix droplets to the dried peptide spots. 

Figure 7 illustrates an in-line sample purification method for MALDI-MS which relies on an EWOD-based technique. In this study, air was... 

In MALDI-MS, impurities at low to intermediate concentrations form adducts with analyte molecules that can obscure the relevant data and reduce the overall signal intensity. Figure 7b and c contain representative EWOD-MALDl-MS spectra for rinsed and nominsed samples of angiotensin n prepared with 20 mM sodium phosphate. In the spectrum of the nmuinsed sample (Fig. 7b), peaks at M + 23, M + 45, and A/ + 67 corresponded to the mmiosodium, disodium, and trisodium adducts of the parent molecule. These adduct peaks are barely visible in the spectrum of the rinsed sample. The intensities of the molecular-ion peaks in the rinsed spectra were about twice as great as those observed in the cmitrol spectra [12]. 

Digital Microfluidics, Figure 7 (a) EWOD-driven sample purification and micrographs of dried spots. Insets the rinsed sample (lefi) appears to be primarily composed of DHB crystals, while the nonrinsed sample (right) is dominated by urea MALDI-MS spectra of (b) rinsed and (c) control spots... 

Figure 7 illustrates an in-line sample purification method for MALDI-MS which relies on an EWOD-based technique. In this study, air was used as the filler medium instead of silicone oil because the oil hindered the dropletdrying process. A droplet containing peptides and impurities was moved by EWOD to a specific location on the array and then dried to form a spot. Subsequently, an EWOD-driven droplet of deionized water was moved onto the spot after visual confirmation that the impurities had dissolved, the droplet was moved away from the spot. After this rinsing step, a droplet containing a MALDI matrix was moved by EWOD to the same spot and dried finally, the spot was analyzed by MALDI-MS. Control... 

---