A Digital Microfluidic Approach to Proteomic Sample Processing

Abstract

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL -10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.iii Methods for implementing proteomic multi-step solution-phase processes (reduction, alkylation, and digestion) on DMF were developed. This was accompanied by the development of heterogenous enzymatic microreactors for efficient protein digestion. Strategies for reducing non-specific adsorption were developed. Finally, in proof-of-concept work, a combination of protein extraction and protein processing was demonstrated on DMF devices.iv ACKNOWLEDGEMENTS

Droplet Actuation by Electrowetting-on-Dielectric (EWOD): A Review

“…Demonstrated examples for specific applications include variable-focus liquid lenses [15], optical displays [16,17], mirrors [18], electrical [19] and thermal [20] switches, a tensiometer [21], rheometers [22,23], and many digital (droplet) microfluidic devices for bioanalysis [5,7,8]. The development of these technologies in the last decade has been spurred by the ability to construct sub-millimeter scale components using micro-electromechanical systems (MEMS) manufacturing methods.…”

Droplet dispensing in digital microfluidic devices: Assessment of long-term reproducibility

“…3,13,[15][16][17][18] Comparing the studies presented here to those in Table I, it is clear that introducing feedback control 13,16 and off-chip pressure sources 16,18 greatly affects droplet dispensing. Most previous work makes use of these and other "aids to dispensing" (such as walls around the reservoir 17,18 and double reservoir electrodes to increase backpressure 16 ).…”

“…Each droplet can be individually controlled in both space and time and used as a miniaturised reaction vessel. The versatility of digital microfluidic technology is evident from the breadth of reported application areas, which range from proteomics 3 and point-of-care diagnostics 4 to chemical synthesis, 5 textiles with electrowetting capabilities, 6 variable focus microlenses, 7 and display technologies. 8 This breadth also demonstrates the adaptability of DMF devices, since they can be configured to suit a given application.…”

“…In particular, the geometric layout of the electrodes used for droplet manipulation creates an environment eminently suited for assay-based applications 9 and for the performance of sequential sample preparation steps. 3,10 To function as an alternative to macroscale laboratory techniques, DMF devices must be able to perform a basic set of common operations. This "droplet toolkit" consists of droplet dispensing (creating daughter droplets from a larger "reservoir" drop), droplet movement, droplet merging, and droplet splitting ( Figure 2).…”

Accurate, consistent, and fast droplet splitting and dispensing in electrowetting on dielectric digital microfluidics

“…In addition, direct manipulation of discrete droplets enables fabrication and operation of highly automated microfluidics systems with more flexibility and higher efficiency [6][7][8][9][10][11][12]. Due to these unique advantages, EWOD digital microfluidics has been used for tremendous applications such as medical [13][14][15][16][17][18][19], display [20,21], optics [22,23], and cooling [24,25]. In addition, there are many recent EWOD studies on engineering applications such as optofluidics and solar energy [26,27].…”