Editing Digital microfluidics (section)

=== Cell culture ===
Connecting the DMF chip to use in the field or world-to-chip interfaces have been accomplished by means of manual pumps and reservoirs which deliver microbes, cells, and media to the device.<ref name="Moazami_2019">{{cite journal | vauthors = Moazami E, Perry JM, Soffer G, Husser MC, Shih SC | title = Integration of World-to-Chip Interfaces with Digital Microfluidics for Bacterial Transformation and Enzymatic Assays | journal = Analytical Chemistry | volume = 91 | issue = 8 | pages = 5159–5168 | date = April 2019 | pmid = 30945840 | doi = 10.1021/acs.analchem.8b05754 | s2cid = 93000574 | url = https://figshare.com/articles/Integration_of_World-to-Chip_Interfaces_with_Digital_Microfluidics_for_Bacterial_Transformation_and_Enzymatic_Assays/7952468 | url-access = subscription }}</ref> The lack of extensive pumps and valves allow for elaborate multi step applications involving cells performed in a simple and compact system.<ref name="Digital Microfluidic Cell Culture"/> In one application, microbial cultures have been transferred onto the chip and allowed to grow with the use of sterile procedures and temperature required for microbial incubation. To validate that this was a viable space for microbial growth, a [[Transformation (genetics)|transformation assay]] was carried out in the device.<ref name="Moazami_2019"/> This involves exposing [[Escherichia coli|E.coli]] to a vector and heat shocking the bacteria until they take up the DNA. This is then followed by running a [[Agarose gel electrophoresis|DNA gel]] to assure that the wanted [[Plasmid vector|vector]] was taken up by the bacteria. This study found that the DNA indeed was taken up by the bacteria and expressed as predicted.

Human cells have also been manipulated in Digital Microfluidic [[Immunocytochemistry]] in Single Cells (DISC) where DMF platforms were used to culture and use antibodies to label phosphorylated proteins in the cell.<ref name="Ng_2015">{{cite journal | vauthors = Ng AH, Dean Chamberlain M, Situ H, Lee V, Wheeler AR | title = Digital microfluidic immunocytochemistry in single cells | journal = Nature Communications | volume = 6 | issue = 1 | pages = 7513 | date = June 2015 | pmid = 26104298 | pmc = 4491823 | doi = 10.1038/ncomms8513 | bibcode = 2015NatCo...6.7513N }}</ref> Cultured cells are then removed and taken off chip for screening. Another technique synthesizes hydrogels within DMF platforms. This process uses electrodes to deliver reagents to produce the [[hydrogel]], and delivery of cell culture reagents for absorption into the gel.<ref name="Javed_2014" /><ref name="George_2015"/> The [[hydrogels]] are an improvement over 2D cell culture because 3D cell culture have increased cell-cell interactions and cel-extracellular matrix interactions.<ref name="George_2015"/> Spherical cell cultures are another method developed around the ability of DMF to deliver droplets to cells. Application of an electric potential allows for automation of droplet transfer directly to the hanging cell culture.<ref name="Javed_2014" />[[Digital microfluidics#cite note-:13-71|<sup><nowiki>]</nowiki></sup>]]<ref name="Aijian_2015">{{cite journal | vauthors = Aijian AP, Garrell RL | title = Digital microfluidics for automated hanging drop cell spheroid culture | journal = Journal of Laboratory Automation | volume = 20 | issue = 3 | pages = 283–95 | date = June 2015 | pmid = 25510471 | doi = 10.1177/2211068214562002 | s2cid = 23720265 | url = https://escholarship.org/uc/item/74v4329w | doi-access = free }}</ref> This is beneficial as 3 dimensional cell culture and [[spheroid]]s better mimic in vivo tissue by allowing for more biologically relevant cultures that have cells growing in an extracellular matrix similarly resembling that in the human body.<ref name="Aijian_2015"/> Another use of DMF platforms in cell culture is its ability to conduct ''in vitro'' cell-free cloning using single molecule [[Polymerase chain reaction|PCR]] inside droplets.<ref name="Ben_Yehezkel_2016">{{cite journal | vauthors = Ben Yehezkel T, Rival A, Raz O, Cohen R, Marx Z, Camara M, Dubern JF, Koch B, Heeb S, Krasnogor N, Delattre C, Shapiro E | display-authors = 6 | title = Synthesis and cell-free cloning of DNA libraries using programmable microfluidics | journal = Nucleic Acids Research | volume = 44 | issue = 4 | pages = e35 | date = February 2016 | pmid = 26481354 | pmc = 4770201 | doi = 10.1093/nar/gkv1087 }}</ref> [[Polymerase chain reaction|PCR]] amplified products are then validated by transfection into yeast cells and a Western blot protein identification.<ref name="Ben_Yehezkel_2016" />

Problems arising from cell culture applications using DMF include protein [[adsorption]] to the device floor, and [[cytotoxicity]] to cells. To prevent adsorption of protein to the platform's floor, a [[surfactant]] stabilized Silicon oil or hexane was used to coat the surface of the device, and droplets were manipulated atop of the oil or hexane.<ref name="Ng_2015" /> Hexane was later rapidly evaporated from cultures to prevent a toxic effect on cell cultures.<ref>{{cite journal | vauthors = Fan SK, Hsu YW, Chen CH | title = Encapsulated droplets with metered and removable oil shells by electrowetting and dielectrophoresis | journal = Lab on a Chip | volume = 11 | issue = 15 | pages = 2500–8 | date = August 2011 | pmid = 21666906 | doi = 10.1039/c1lc20142e }}</ref> Another approach to solve protein adhesion is the addition of [[Pluronic]] additives to droplets in the device.<ref>{{Cite journal|date=March 2004|title=Millipore and HyClone form bioprocessing alliance|journal=Membrane Technology|volume=2004|issue=3|pages=1|doi=10.1016/s0958-2118(04)00087-4|issn=0958-2118}}</ref> Pluronic additives are generally not cytotoxic but some have been shown to be harmful to cell cultures.<ref name="Barbulovic-Nad_2008" />

Bio-compatibility of device set up is important for biological analyses. Along with finding Pluronic additives that are not cytotoxic, creating a device whose voltage and disruptive movement would not affect cell viability was accomplished. Through the readout of live/dead assays it was shown that neither [[voltage]] required to move droplets, nor the motion of moving cultures affected cell viability.<ref name="Barbulovic-Nad_2008" />

==== Biological extraction ====
Biological separations usually involve low concentration high volume samples. This can pose an issue for digital microfluidics due to the small sample volume necessary.<ref name="Shah_2009" /> Digital microfluidic systems can be combined with a macrofluidic system designed to decrease sample volume, in turn increasing analyte concentration.<ref name="Shah_2009" /> It follows the same principles as  the magnetic particles for separation, but includes pumping of the droplet to cycle a larger volume of fluid around the magnetic particles.<ref name="Shah_2009" />
Extraction of drug analytes from dried urine samples has also been reported. A droplet of extraction solvent, in this case methanol, is repeatedly flowed over a sample of dried urine sample then moved to a final electrode where the liquid is extracted through a capillary and then analyzed using mass spectrometry.<ref name="Kirby_2014">{{cite journal | vauthors = Kirby AE, Lafrenière NM, Seale B, Hendricks PI, Cooks RG, Wheeler AR | title = Analysis on the go: quantitation of drugs of abuse in dried urine with digital microfluidics and miniature mass spectrometry | journal = Analytical Chemistry | volume = 86 | issue = 12 | pages = 6121–9 | date = June 2014 | pmid = 24906177 | doi = 10.1021/ac5012969 }}</ref>

==== Immunoassays ====
The advanced fluid handling capabilities of digital microfluidics (DMF) allows for the adoption of DMF as an [[immunoassay]] platform  as DMF devices can precisely manipulate small quantities of liquid reagents. Both heterogeneous immunoassays (antigens interacting with immobilized antibodies) and homogeneous immunoassays (antigens interacting with antibodies in solution) have been developed using a DMF platform.<ref>{{cite journal | vauthors = Ng AH, Uddayasankar U, Wheeler AR | title = Immunoassays in microfluidic systems. Analytical and bioanalytical chemistry | journal = Analytical and Bioanalytical Chemistry | date = June 2010 | volume = 397 | issue = 3 | pages = 991–1007 | doi = 10.1007/s00216-010-3678-8 | pmid = 20422163 | s2cid = 30670634 }}</ref> With regards to heterogeneous immunoassays, DMF can simplify the extended and intensive procedural steps by performing all delivery, mixing, incubation, and washing steps on the surface of the device (on-chip). Further, existing immunoassay techniques and methods, such as magnetic bead-based assays, [[ELISA]]s, and electrochemical detection, have been incorporated onto DMF immunoassay platforms.<ref name="Vergauwe_2011">{{cite journal | vauthors = Vergauwe N, Witters D, Ceyssens F, Vermeir S, Verbruggen B, Puers R, Lammertyn J | title = A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays. | journal = Journal of Micromechanics and Microengineering | date = April 2011 | volume = 21 | issue = 5 | pages = 054026 | doi = 10.1088/0960-1317/21/5/054026 | bibcode = 2011JMiMi..21e4026V | s2cid = 111122895 }}</ref><ref name="Sista_2008">{{cite journal | vauthors = Sista R, Hua Z, Thwar P, Sudarsan A, Srinivasan V, Eckhardt A, Pollack M, Pamula V | title = Development of a digital microfluidic platform for point of care testing | journal = Lab on a Chip | volume = 8 | issue = 12 | pages = 2091–104 | date = December 2008 | pmid = 19023472 | pmc = 2726010 | doi = 10.1039/b814922d }}</ref><ref name="Ng_2012">{{cite journal | vauthors = Ng AH, Choi K, Luoma RP, Robinson JM, Wheeler AR | title = Digital microfluidic magnetic separation for particle-based immunoassays | journal = Analytical Chemistry | volume = 84 | issue = 20 | pages = 8805–12 | date = October 2012 | pmid = 23013543 | doi = 10.1021/ac3020627 }}</ref><ref name="Shamsi_2014">{{cite journal | vauthors = Shamsi MH, Choi K, Ng AH, Wheeler AR | title = A digital microfluidic electrochemical immunoassay | journal = Lab on a Chip | volume = 14 | issue = 3 | pages = 547–54 | date = February 2014 | pmid = 24292705 | doi = 10.1039/c3lc51063h }}</ref>

The incorporation of magnetic bead-based assays onto a DMF immunoassay platform has been demonstrated for the detection of multiple analytes, such as human insulin, [[Wikipedia:Interleukin 6|IL-6]], cardiac marker Troponin I (cTnI), thyroid stimulating hormone (TSH), sTNF-RI, and 17β-estradiol.<ref name="Ng_2012" /><ref name="Sista_2008b">{{cite journal | vauthors = Sista RS, Eckhardt AE, Srinivasan V, Pollack MG, Palanki S, Pamula VK | title = Heterogeneous immunoassays using magnetic beads on a digital microfluidic platform | journal = Lab on a Chip | volume = 8 | issue = 12 | pages = 2188–96 | date = December 2008 | pmid = 19023486 | pmc = 2726047 | doi = 10.1039/b807855f }}</ref><ref name="Tsaloglou_2014">{{cite journal | vauthors = Tsaloglou MN, Jacobs A, Morgan H | title = A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device | journal = Analytical and Bioanalytical Chemistry | volume = 406 | issue = 24 | pages = 5967–76 | date = September 2014 | pmid = 25074544 | doi = 10.1007/s00216-014-7997-z | s2cid = 24266593 }}</ref><ref name="Huang_2016">{{cite journal | vauthors = Huang CY, Tsai PY, Lee IC, Hsu HY, Huang HY, Fan SK, Yao DJ, Liu CH, Hsu W | title = A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay | journal = Biomicrofluidics | volume = 10 | issue = 1 | pages = 011901 | date = January 2016 | pmid = 26858807 | pmc = 4714987 | doi = 10.1063/1.4939942 }}</ref> For example, a magnetic bead-based approached has been used for the detection of cTnI from whole blood in less than 8 minutes.<ref name="Sista_2008b" /> Briefly, magnetic beads containing primary antibodies were mixed with labeled secondary antibodies, incubated, and immobilized with a magnet for the washing steps. The droplet was then mixed with a chemiluminescent reagent and detection of the accompanying enzymatic reaction was measured on-chip with a [[photomultiplier]] tube.

The ELISA template, commonly used for performing immunoassays and other enzyme-based biochemical assays, has been adapted for use with the DMF platform for the detection of analytes such as IgE and IgG.<ref name="Zhu_2012">{{cite journal | vauthors = Zhu L, Feng Y, Ye X, Feng J, Wu Y, Zhou Z | title = An ELISA chip based on an EWOD microfluidic platform. | journal = Journal of Adhesion Science and Technology | date = September 2012 | volume = 26 | issue = 12–17 | pages = 2113–24 | doi = 10.1163/156856111x600172 | s2cid = 136668522 }}</ref><ref name="pmid21057776">{{cite journal | vauthors = Miller EM, Ng AH, Uddayasankar U, Wheeler AR | title = A digital microfluidic approach to heterogeneous immunoassays | journal = Analytical and Bioanalytical Chemistry | volume = 399 | issue = 1 | pages = 337–45 | date = January 2011 | pmid = 21057776 | doi = 10.1007/s00216-010-4368-2 | s2cid = 2809777 }}</ref> In one example,<ref name="Vergauwe_2011" /> a series of bioassays were conducted to establish the quantification capabilities of DMF devices, including an ELISA-based immunoassay for the detection of IgE. Superparamagnetic nanoparticles were  immobilized with anti-IgE antibodies and fluorescently labeled aptamers to quantify IgE using an ELISA template. Similarly, for the detection of IgG, IgG can be immobilized onto a DMF chip, conjugated with horseradish-peroxidase (HRP)-labeled IgG, and then quantified through measurement of the color change associated with product formation of the reaction between HRP and tetramethylbenzidine.<ref name="Zhu_2012" />

To further expand the capabilities and applications of DMF immunoassays beyond [[Colorimetry|colorimetric]] detection (i.e., ELISA, magnetic bead-based assays), electrochemical detection tools (e.g., microelectrodes) have been incorporated into DMF chips for the detection of analytes such as TSH and rubella virus.<ref name="Shamsi_2014" /><ref name="Rackus_2015">{{cite journal | vauthors = Rackus DG, Dryden MD, Lamanna J, Zaragoza A, Lam B, Kelley SO, Wheeler AR | title = A digital microfluidic device with integrated nanostructured microelectrodes for electrochemical immunoassays | journal = Lab on a Chip | volume = 15 | issue = 18 | pages = 3776–84 | date = 2015 | pmid = 26247922 | doi = 10.1039/c5lc00660k }}</ref><ref name="Dixon_2016">{{cite journal | vauthors = Dixon C, Ng AH, Fobel R, Miltenburg MB, Wheeler AR | title = An inkjet printed, roll-coated digital microfluidic device for inexpensive, miniaturized diagnostic assays | journal = Lab on a Chip | volume = 16 | issue = 23 | pages = 4560–4568 | date = November 2016 | pmid = 27801455 | doi = 10.1039/c6lc01064d | url = https://authors.library.caltech.edu/71665/4/c6lc01064d.pdf }}</ref> For example, Rackus et al.<ref name="Rackus_2015" /> integrated microelectrodes onto a DMF chip surface and substituted a previously reported chemiluminescent IgG immunoassay<ref name="Ng_2015b">{{cite journal | vauthors = Ng AH, Lee M, Choi K, Fischer AT, Robinson JM, Wheeler AR | title = Digital microfluidic platform for the detection of rubella infection and immunity: a proof of concept | journal = Clinical Chemistry | volume = 61 | issue = 2 | pages = 420–9 | date = February 2015 | pmid = 25512641 | doi = 10.1373/clinchem.2014.232181 | doi-access = free }}</ref> with an electroactive species, enabling detection of rubella virus. They coated magnetic beads with rubella virus, anti-rubella IgG, and anti-human IgG coupled with alkaline phosphatase, which in turn catalyzed an electron transfer reaction that was detected by the on-chip microelectrodes.