Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS.
Localized heating of droplets on an electrowetting-on-dielectric (EWOD) chip has been implemented and shown to accelerate trypsin digestion reaction rates, sample drying, and matrix crystallization for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Achieving this involved extending the functionality of previous EWOD droplet-based techniques by developing a multifunctional electrode with closed-loop temperature control, while minimizing overall system complexity and addressing challenges associated with rapid evaporation. For the EWOD chip design, we discuss the performance of multifunctional surface electrodes for actuation, localized Joule heating, and thermistic temperature sensing. Furthermore, a hydrophilic pattern is formed in the multifunctional electrode to control the location of an evaporating droplet on the electrode. To demonstrate the capabilities and limitations of this technique, we performed three experiments and measured the results using MALDI-MS: (i) insulin disulfide reductions in dithiothreitol (DTT) over a range of heater temperatures (22-70 °C) to show how reaction rates can be affected by thermal control, (ii) insulin disulfide reductions at 130 °C in dimethyl sulfoxide (DMSO) to demonstrate a reaction in a high boiling point solvent, and (iii) tryptic digestions of cytochrome c at 22 and 40 °C to show that heated droplets can yield reasonably higher peptide sequence coverage than unheated droplets. Although they do not decouple the effects of changing temperatures and concentrations, these experiments verified that thermal cycling by EWOD electrodes accelerates reaction rates in liquid droplets in air.
Mechanical and Aerospace Engineering Department, 420 Westwood Plaza, Engineering IV Room 37-129, University of California, Los Angeles (UCLA), Los Angeles, California 90095, USA.
Wyatt C
WC
Nelson
Ivory
I
Peng
Geun-An
GA
Lee
Joseph A
JA
Loo
Robin L
RL
Garrell
Chang-Jin C J
CJ
Kim
D045304:Cytochromes c / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004566:Electrodes; D058874:Electrowetting / Q000379:methods; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D007328:Insulin; D045304:Cytochromes c; D014357:Trypsin; D004121:Dimethyl Sulfoxide