Digital Microfluidic Method for Protein Extraction by Precipitation

Jebrail, Mais J.; Wheeler, Aaron R.

doi:10.1021/ac8021554

Cited by 102 publications

(82 citation statements)

References 34 publications

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“…[49][50][51][52] In DMF, discrete microdroplets are manipulated electrostatically on an array of electrodes coated with a hydrophobic, dielectric insulator. 53,54 The classical characterization of droplet movement in DMF is based on a thermodynamic approach using the Young-Lippman equation (Eq 1), 55,56 where change in contact angle, , is a function of applied voltage, V. Here,   is the contact angle when the electric field across interfacial layer is zero, ε r is the relative permittivity of the dielectric, ε o is the permittivity of free space,  is the is the liquid-filler media surface tension, and d is the dielectric thickness.…”

Section: Digital Microfluidicsmentioning

confidence: 99%

“…DMF-driven reduction and digestion reactions are 90% complete within 15 -45 min and 10 -50 min, respectively. This suggests that a full sample processing workup (including protein extraction by precipitation 50 ) would require approximately 2 h to complete. This time frame is fast relative to conventional methods, 13 but we note that longer times will likely be required for more complex samples (e.g., serum or cell lysate).…”

Section: Processing Timementioning

confidence: 99%

“…[49][50][51][52]128,163 But until now, all enzyme digestion methods implemented by digital microfluidics have been homogeneous (i.e., solution only). As noted in the introduction, there are significant benefits to Gel disc microreactor optimization.…”

Section: Comparison Of In-gel and In-solution Digestion Qualitymentioning

confidence: 99%

“…DMF is a fluid-handling technique in which liquid samples are manipulated on the surface of an array of electrodes by electrostatic forces, 122 and has been applied to a variety of applications related to proteomics including sample purification by precipitation, 50 reduction of disulfides (chapter 3), 49,52 alkylation of thiols (chapter 3), 49,51,52 proteolytic digestion (chapters 2 and 3), 49,51,52,128 and combinations thereof. 163 These previous reports represent important steps towards the goal of integrated proteome profiling, but they are limited by the challenges inherent to homogeneous digestions described above.…”

Section: Introductionmentioning

confidence: 99%

“…DMF is a fluid-handling technique in which discrete droplets of samples and reagents are manipulated (i.e., dispensed from reservoirs, split, merged and mixed) on an open surface by applying a series of electrical potentials to an array of electrodes. 122,123 DMF has previously been used to (1) extract, clean-up and resolubilize proteins from biological fluids, 50 as well as to (2) carry out solution phase reduction, alkylation and digestion step (chapter 3) 49,52 . Here, we report the integration of steps (1) and (2) onto a single platform, as an important step towards achieving reliable, multiplexed biomarker diagnostic assays.…”

Section: Introductionmentioning

confidence: 99%

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A Digital Microfluidic Approach to Proteomic Sample Processing

2009

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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

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