Editing Digital microfluidics (section)

== Applications ==

=== Laboratory automation ===
In research fields such as [[synthetic biology]], where highly iterative experimentation is common, considerable efforts have been made to automate workflows.<ref>{{cite journal | vauthors = Sparkes A, Aubrey W, Byrne E, Clare A, Khan MN, Liakata M, Markham M, Rowland J, Soldatova LN, Whelan KE, Young M, King RD | display-authors = 6 | title = Towards Robot Scientists for autonomous scientific discovery | journal = Automated Experimentation | volume = 2 | issue = 1 | pages = 1 | date = January 2010 | pmid = 20119518 | pmc = 2813846 | doi = 10.1186/1759-4499-2-1 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Meng F, Ellis T | title = The second decade of synthetic biology: 2010-2020 | journal = Nature Communications | volume = 11 | issue = 1 | pages = 5174 | date = October 2020 | pmid = 33057059 | pmc = 7560693 | doi = 10.1038/s41467-020-19092-2 | bibcode = 2020NatCo..11.5174M }}</ref><ref>{{cite journal | vauthors = Carbonell P, Radivojevic T, García Martín H | title = Opportunities at the Intersection of Synthetic Biology, Machine Learning, and Automation | journal = ACS Synthetic Biology | volume = 8 | issue = 7 | pages = 1474–1477 | date = July 2019 | pmid = 31319671 | doi = 10.1021/acssynbio.8b00540 | hdl = 20.500.11824/998 | s2cid = 197664634 | doi-access = free | hdl-access = free }}</ref> Digital microfluidics is often touted as a laboratory automation solution, with a number of advantages over alternative solutions such as [[Liquid handling robot|pipetting robots]] and [[Droplet-based microfluidics|droplet microfluidics]].<ref name="Kothamachu_2020">{{cite journal | vauthors = Kothamachu VB, Zaini S, Muffatto F | title = Role of Digital Microfluidics in Enabling Access to Laboratory Automation and Making Biology Programmable | language = English | journal = SLAS Technology | volume = 25 | issue = 5 | pages = 411–426 | date = October 2020 | pmid = 32584152 | doi = 10.1177/2472630320931794 | s2cid = 220062017 | doi-access = free }}</ref><ref name="Husser_2018">{{cite journal | vauthors = Husser MC, Vo PQ, Sinha H, Ahmadi F, Shih SC | title = An Automated Induction Microfluidics System for Synthetic Biology | journal = ACS Synthetic Biology | volume = 7 | issue = 3 | pages = 933–944 | date = March 2018 | pmid = 29516725 | doi = 10.1021/acssynbio.8b00025 }}</ref><ref name="Ruan_2020">{{cite journal | vauthors = Ruan Q, Ruan W, Lin X, Wang Y, Zou F, Zhou L, Zhu Z, Yang C | display-authors = 6 | title = Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics | journal = Science Advances | volume = 6 | issue = 50 | pages = eabd6454 | date = December 2020 | pmid = 33298451 | pmc = 7725457 | doi = 10.1126/sciadv.abd6454 | bibcode = 2020SciA....6.6454R }}</ref> These stated advantages often include a reduction in the required volume of experimental reagents, a reduction in the likelihood of contamination and cross-contamination, potential improvements in reproducibility, increased throughput, individual droplet addressability, and the ability to integrate with sensor and detector modules to perform end-to-end or even closed loop workflow automation.<ref name="Kothamachu_2020" /><ref name="Husser_2018" /><ref name="Ruan_2020" /><ref>{{cite journal | vauthors = Liu D, Yang Z, Zhang L, Wei M, Lu Y | title = Cell-free biology using remote-controlled digital microfluidics for individual droplet control | journal = RSC Advances | volume = 10 | issue = 45 | pages = 26972–26981 | date = July 2020 | pmid = 35515808 | pmc = 9055536 | doi = 10.1039/d0ra04588h | bibcode = 2020RSCAd..1026972L }}</ref>

==== Reduced experimental footprint ====
One of the core advantages of digital microfluidics, and of microfluidics in general, is the use and actuation of picoliter to microliter scale volumes. Workflows adapted from the bench to a DMF system are miniaturized, meaning working volumes are reduced to fractions of what is normally required for conventional methods. For example, Thaitrong et al. developed a DMF system with a [[Capillary electrophoresis|capillary electrophoresis (CE)]] module with the purpose of automating the process of [[Next-generation sequencing|next generation sequencing (NGS)]] library characterization. Compared to an [[Agilent Technologies|Agilent]] BioAnalyzer (an instrument commonly used to measure sequencing library size distribution), the DMF-CE system consumed ten-fold less sample volume.<ref>{{cite journal | vauthors = Thaitrong N, Kim H, Renzi RF, Bartsch MS, Meagher RJ, Patel KD | title = Quality control of next-generation sequencing library through an integrative digital microfluidic platform | journal = Electrophoresis | volume = 33 | issue = 23 | pages = 3506–3513 | date = December 2012 | pmid = 23135807 | doi = 10.1002/elps.201200441 | s2cid = 205802837 }}</ref> Reducing volumes for a workflow can be especially beneficial if the reagents are expensive or when manipulating rare samples such as circulating tumor cells and prenatal samples.<ref name="Ruan_2020" /> Miniaturization also means a reduction in waste product volumes.

==== Reduced probability of contamination ====
DMF-based workflows, particularly those using a closed configuration with a top-plate ground electrode, have been shown to be less susceptible to outside contamination compared to some conventional laboratory workflows. This can be attributed to minimal user interaction during automated steps, and the fact that the smaller volumes are less exposed to environmental contaminants than larger volumes which would need to be exposed to open air during mixing. Ruan et al. observed minimal contamination from exogenous nonhuman DNA and no cross-contamination between samples while using their DMF-based digital whole genome sequencing system.<ref name="Ruan_2020" />

==== Improved reproducibility ====
Overcoming issues of [[reproducibility]] has become a topic of growing concern across scientific disciplines.<ref>{{Cite journal | vauthors = Baker M |date=2016-05-01 |title=1,500 scientists lift the lid on reproducibility |journal=Nature |language=en |volume=533 |issue=7604 |pages=452–454 |doi=10.1038/533452a |pmid=27225100 |bibcode=2016Natur.533..452B |s2cid=4460617 |issn=1476-4687|doi-access=free }}</ref> Reproducibility can be especially salient when multiple iterations of the same experimental protocol need to be repeated.<ref>{{cite journal | vauthors = Jessop-Fabre MM, Sonnenschein N | title = Improving Reproducibility in Synthetic Biology | journal = Frontiers in Bioengineering and Biotechnology | volume = 7 | pages = 18 | date = 2019 | pmid = 30805337 | pmc = 6378554 | doi = 10.3389/fbioe.2019.00018 | doi-access = free }}</ref> Using liquid handling robots that can minimize volume loss between experimental steps are often used to reduce error rates and improve reproducibility. An automated DMF system for [[CRISPR gene editing|CRISPR-Cas9]] genome editing was described by Sinha et al, and was used to culture and genetically modify [[H1299]] lung cancer cells. The authors noted that no variation in [[Gene knockout|knockout efficiencies]] across loci was observed when cells were cultured on the DMF device, whereas cells cultured in well-plates showed variability in upstream loci knockout efficiencies. This reduction in variability was attributed to culturing on a DMF device being more homogenous and reproducible compared with well plate methods.<ref>{{cite journal | vauthors = Sinha H, Quach AB, Vo PQ, Shih SC | title = An automated microfluidic gene-editing platform for deciphering cancer genes | journal = Lab on a Chip | volume = 18 | issue = 15 | pages = 2300–2312 | date = July 2018 | pmid = 29989627 | doi = 10.1039/C8LC00470F }}</ref>

==== Increased throughput ====
While DMF systems cannot match the same throughput achieved by some liquid handling pipetting robots, or by some droplet-based microfluidic systems, there are still throughput advantages when compared to conventional methods carried out manually.<ref name="Digital Microfluidic Cell Culture">{{cite journal | vauthors = Ng AH, Li BB, Chamberlain MD, Wheeler AR | title = Digital Microfluidic Cell Culture | journal = Annual Review of Biomedical Engineering | volume = 17 | issue = 1 | pages = 91–112 | date = 2015-12-07 | pmid = 26643019 | doi = 10.1146/annurev-bioeng-071114-040808 }}</ref>

==== Individual droplet addressability ====
DMF allows for droplet level addressability, meaning individual droplets can be treated as spatially distinct [[microreactor]]s.<ref name="Kothamachu_2020" /> This level of droplet control is important for workflows where reactions are sensitive to the order of reagent mixing and incubation times, but where the optimal values of these parameters may still need to be determined. These types of workflows are common in [[Cell-free system|cell-free biology]], and Liu et al. were able to demonstrate a proof-of-concept DMF-based strategy for carrying out remote-controlled [[Cell-free protein synthesis|cell-free protein expression]] on an OpenDrop chip.<ref name="Liu_2020">{{cite journal | vauthors = Liu D, Yang Z, Zhang L, Wei M, Lu Y | title = Cell-free biology using remote-controlled digital microfluidics for individual droplet control | journal = RSC Advances | volume = 10 | issue = 45 | pages = 26972–26981 | date = July 2020 | pmid = 35515808 | pmc = 9055536 | doi = 10.1039/D0RA04588H | bibcode = 2020RSCAd..1026972L }}</ref>

==== Detector module integration for end-to-end and closed-loop automation ====
An often cited advantage DMF platforms have is their potential to integrate with on-chip sensors and off-chip detector modules.<ref name="Kothamachu_2020" /><ref name="Liu_2020" /> In theory, real-time and end-point data can be used in conjunction with [[machine learning]] methods to automate the process of parameter optimization.

=== Separation and extraction ===
Digital [[microfluidics]] can be used for separation and extraction of target analytes. These methods include the use of magnetic particles,<ref name = "Wang_2007">{{cite journal| vauthors = Wang Y, Zhao Y, Cho SK |title=Efficient in-droplet separation of magnetic particles for digital microfluidics|journal=Journal of Micromechanics and Microengineering|date=1 October 2007|volume=17|issue=10|pages=2148–2156|doi=10.1088/0960-1317/17/10/029|bibcode=2007JMiMi..17.2148W|s2cid=135789543 }}</ref><ref name = "Vergauwe_2014">{{cite journal| vauthors = Vergauwe N, Vermeir S, Wacker JB, Ceyssens F, Cornaglia M, Puers R, Gijs MA, Lammertyn J, Witters D | display-authors = 6 |title=A highly efficient extraction protocol for magnetic particles on a digital microfluidic chip|journal=Sensors and Actuators B: Chemical|date=June 2014|volume=196|pages=282–291|doi=10.1016/j.snb.2014.01.076| bibcode = 2014SeAcB.196..282V }}</ref><ref name = "Seale_2016">{{cite journal | vauthors = Seale B, Lam C, Rackus DG, Chamberlain MD, Liu C, Wheeler AR | title = Digital Microfluidics for Immunoprecipitation | journal = Analytical Chemistry | volume = 88 | issue = 20 | pages = 10223–10230 | date = October 2016 | pmid = 27700039 | doi = 10.1021/acs.analchem.6b02915 }}</ref><ref name = "Shah_2009">{{cite journal| vauthors = Shah GJ, Kim CJ |title=Meniscus-Assisted High-Efficiency Magnetic Collection and Separation for EWOD Droplet Microfluidics|journal=Journal of Microelectromechanical Systems|date=April 2009|volume=18|issue=2|pages=363–375|doi=10.1109/JMEMS.2009.2013394|s2cid=24845666}}</ref><ref name = "Jebrail_2014">{{cite journal | vauthors = Jebrail MJ, Sinha A, Vellucci S, Renzi RF, Ambriz C, Gondhalekar C, Schoeniger JS, Patel KD, Branda SS | display-authors = 6 | title = World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood | journal = Analytical Chemistry | volume = 86 | issue = 8 | pages = 3856–3862 | date = April 2014 | pmid = 24479881 | doi = 10.1021/ac404085p }}</ref><ref name = "Hung_2015">{{cite journal| vauthors = Hung PY, Jiang PS, Lee EF, Fan SK, Lu YW |title=Genomic DNA extraction from whole blood using a digital microfluidic (DMF) platform with magnetic beads|journal=Microsystem Technologies|date= April 2015|volume=23|issue=2|pages=313–320|doi=10.1007/s00542-015-2512-9|s2cid=137531469}}</ref><ref name = "Choi_2013">{{cite journal | vauthors = Choi K, Ng AH, Fobel R, Chang-Yen DA, Yarnell LE, Pearson EL, Oleksak CM, Fischer AT, Luoma RP, Robinson JM, Audet J, Wheeler AR | display-authors = 6 | title = Automated digital microfluidic platform for magnetic-particle-based immunoassays with optimization by design of experiments | journal = Analytical Chemistry | volume = 85 | issue = 20 | pages = 9638–9646 | date = October 2013 | pmid = 23978190 | doi = 10.1021/ac401847x }}</ref><ref name = "Choi_2016">{{cite journal | vauthors = Choi K, Boyacı E, Kim J, Seale B, Barrera-Arbelaez L, Pawliszyn J, Wheeler AR | title = A digital microfluidic interface between solid-phase microextraction and liquid chromatography-mass spectrometry | journal = Journal of Chromatography A | volume = 1444 | pages = 1–7 | date = April 2016 | pmid = 27048987 | doi = 10.1016/j.chroma.2016.03.029 }}</ref> [[liquid-liquid extraction]],<ref name = "Wijethunga_2011">{{cite journal | vauthors = Wijethunga PA, Nanayakkara YS, Kunchala P, Armstrong DW, Moon H | title = On-chip drop-to-drop liquid microextraction coupled with real-time concentration monitoring technique | journal = Analytical Chemistry | volume = 83 | issue = 5 | pages = 1658–1664 | date = March 2011 | pmid = 21294515 | doi = 10.1021/ac102716s }}</ref> [[optical tweezers]],<ref name = "Shah_2009b">{{cite journal | vauthors = Shah GJ, Ohta AT, Chiou EP, Wu MC, Kim CJ | title = EWOD-driven droplet microfluidic device integrated with optoelectronic tweezers as an automated platform for cellular isolation and analysis | journal = Lab on a Chip | volume = 9 | issue = 12 | pages = 1732–1739 | date = June 2009 | pmid = 19495457 | doi = 10.1039/b821508a }}</ref> and [[Fluid dynamics|hydrodynamic effects]].<ref name = "Nejad_2015">{{cite journal| vauthors = Nejad HR, Samiei E, Ahmadi A, Hoorfar M |title=Gravity-driven hydrodynamic particle separation in digital microfluidic systems |journal=RSC Adv. |date=2015 |volume=5 |issue=45 |pages=35966–35975 |doi=10.1039/C5RA02068A|bibcode=2015RSCAd...535966N }}</ref>

==== Magnetic particles ====
For magnetic particle separations a droplet of solution containing the analyte of interest is placed on a digital microfluidics [[electrode array]] and moved by the changes in the charges of the electrodes. The droplet is moved to an electrode with a magnet on one side of the array with magnetic particles functionalized to bind to the analyte. Then it is moved over the electrode, the magnetic field is removed and the particles are suspended in the droplet. The droplet is swirled on the electrode array to ensure mixing. The magnet is reintroduced and the particles are immobilized and the droplet is moved away. This process is repeated with wash and elution buffers to extract the analyte.<ref name="Wang_2007" /><ref name="Vergauwe_2014" /><ref name="Seale_2016" /><ref name="Shah_2009" /><ref name="Jebrail_2014" /><ref name="Hung_2015" /><ref name="Choi_2013" /><ref name="Choi_2016" />

Magnetic particles coated with antihuman [[serum albumin]] antibodies have been used to isolate human serum albumin, as proof of concept work for immunoprecipitation using digital microfluidics.<sup>5</sup> DNA extraction from a whole blood sample has also been performed with digital microfluidics.<sup>3</sup> The procedure follows the general methodology  as the magnetic particles, but includes pre-treatment on the digital microfluidic platform to [[Lysis|lyse]] the cells prior to DNA extraction.<ref name="Seale_2016" />

==== Liquid-liquid extraction ====
[[Liquid-liquid extraction]]s can be carried out on digital microfluidic device by taking advantage of immiscible liquids.<sup>9</sup> Two droplets, one containing the analyte in aqueous phase, and the other an immiscible ionic liquid are present on the electrode array. The two droplets are mixed and the ionic liquid extracts the analyte, and the droplets are easily separable.<ref name="Wijethunga_2011" />

==== Optical tweezers ====
[[Optical tweezers]] have also been used to separate cells in droplets. Two droplets are mixed on an electrode array, one containing the cells, and the other with nutrients or drugs. The droplets are mixed and then optical tweezers are used to move the cells to one side of the larger droplet before it is split.<ref>{{cite journal | vauthors = Neuman KC, Block SM | title = Optical trapping | journal = The Review of Scientific Instruments | volume = 75 | issue = 9 | pages = 2787–809 | date = September 2004 | pmid = 16878180 | pmc = 1523313 | doi = 10.1063/1.1785844 | bibcode = 2004RScI...75.2787N }}</ref><ref name="Shah_2009b" /> For a more detailed explanation on the underlying principles, see [[Optical tweezers]].

==== Hydrodynamic separation ====
Particles have been applied for use outside of magnetic separation, with hydrodynamic forces to separate particles from the bulk of a droplet.<ref name="Nejad_2015" /> This is performed on electrode arrays with a central electrode and ‘slices’ of electrodes surrounding it. Droplets are added onto the array and swirled in a circular pattern, and the hydrodynamic forces from the swirling cause the particles to aggregate onto the central electrode.<ref name="Nejad_2015" />

=== Chemical synthesis ===
Digital Microfluidics (DMF) allows for precise manipulation and coordination in small-scale chemical synthesis reactions due to its ability to control micro scale volumes of liquid reagents, allowing for overall less reagent use and waste.<ref name="Geng_2017">{{cite journal | vauthors = Geng H, Feng J, Stabryla LM, Cho SK | title = Dielectrowetting manipulation for digital microfluidics: creating, transporting, splitting, and merging of droplets | journal = Lab on a Chip | volume = 17 | issue = 6 | pages = 1060–1068 | date = March 2017 | pmid = 28217772 | doi = 10.1039/c7lc00006e }}</ref> This technology can be used in the synthesis compounds such as [[peptidomimetic]]s and [[Positron emission tomography|PET]] tracers.<ref name="Jebrail_2012">{{Cite journal| vauthors = Jebrail MJ, Assem N, Mudrik JM, Dryden MD, Lin K, Yudin AK, Wheeler AR |date=2012-08-01|title=Combinatorial Synthesis of Peptidomimetics Using Digital Microfluidics|journal=Journal of Flow Chemistry|volume=2|issue=3|pages=103–107|doi=10.1556/JFC-D-12-00012 |bibcode=2012JFlCh...2..103J |s2cid=34049157}}</ref><ref name="Chen_2014">{{cite journal | vauthors = Chen S, Javed MR, Kim HK, Lei J, Lazari M, Shah GJ, van Dam RM, Keng PY, Kim CJ | display-authors = 6 | title = Radiolabelling diverse positron emission tomography (PET) tracers using a single digital microfluidic reactor chip | journal = Lab on a Chip | volume = 14 | issue = 5 | pages = 902–10 | date = March 2014 | pmid = 24352530 | doi = 10.1039/c3lc51195b | s2cid = 40777981 | url = http://www.escholarship.org/uc/item/44r9z66x }}</ref><ref name="Javed_2014">{{cite journal | vauthors = Javed MR, Chen S, Kim HK, Wei L, Czernin J, Kim CJ, van Dam RM, Keng PY | display-authors = 6 | title = Efficient radiosynthesis of 3'-deoxy-3'-18F-fluorothymidine using electrowetting-on-dielectric digital microfluidic chip | journal = Journal of Nuclear Medicine | volume = 55 | issue = 2 | pages = 321–8 | date = February 2014 | pmid = 24365651 | pmc = 4494735 | doi = 10.2967/jnumed.113.121053 }}</ref> [[Positron emission tomography|PET]] tracers require nanogram quantities and as such, DMF allows for automated and rapid synthesis of tracers with 90-95% efficiency compared to conventional macro-scale techniques.<ref name="Chen_2014" /><ref name="Keng_2012">{{cite journal | vauthors = Keng PY, Chen S, Ding H, Sadeghi S, Shah GJ, Dooraghi A, Phelps ME, Satyamurthy N, Chatziioannou AF, Kim CJ, van Dam RM | display-authors = 6 | title = Micro-chemical synthesis of molecular probes on an electronic microfluidic device | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 3 | pages = 690–5 | date = January 2012 | pmid = 22210110 | pmc = 3271918 | doi = 10.1073/pnas.1117566109 | bibcode = 2012PNAS..109..690K | doi-access = free }}</ref>

Organic reagents are not commonly used in DMF because they tend to wet the DMF device and cause flooding; however synthesis of organic reagents can be achieved through DMF techniques by carrying the organic reagents through an ionic liquid droplet, thus preventing the organic reagent from flooding the DMF device.<ref name="Dubois_2006">{{cite journal | vauthors = Dubois P, Marchand G, Fouillet Y, Berthier J, Douki T, Hassine F, Gmouh S, Vaultier M | display-authors = 6 | title = Ionic liquid droplet as e-microreactor | journal = Analytical Chemistry | volume = 78 | issue = 14 | pages = 4909–17 | date = July 2006 | pmid = 16841910 | doi = 10.1021/ac060481q | url = https://figshare.com/articles/Ionic_Liquid_Droplet_as_e_Microreactor/3070660 | url-access = subscription }}</ref> Droplets are combined together by inducing opposite charges thus attracting them to each other.<ref name="Um_2016">{{cite journal | vauthors = Um T, Hong J, Im do J, Lee SJ, Kang IS | title = Electrically Controllable Microparticle Synthesis and Digital Microfluidic Manipulation by Electric-Field-Induced Droplet Dispensing into Immiscible Fluids | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 31901 | date = August 2016 | pmid = 27534580 | pmc = 4989170 | doi = 10.1038/srep31901 | bibcode = 2016NatSR...631901U }}</ref> This allows for automated mixing of droplets. Mixing of droplets are also used to deposit [[Metal-organic framework|MOF]] crystals for printing by delivering reagents into wells and evaporating the solutions for crystal deposition.<ref name="Witters_2012">{{cite journal | vauthors = Witters D, Vergauwe N, Ameloot R, Vermeir S, De Vos D, Puers R, Sels B, Lammertyn J | display-authors = 6 | title = Digital microfluidic high-throughput printing of single metal-organic framework crystals | journal = Advanced Materials | volume = 24 | issue = 10 | pages = 1316–20 | date = March 2012 | pmid = 22298246 | doi = 10.1002/adma.201104922 | bibcode = 2012AdM....24.1316W | s2cid = 205244275 }}</ref> This method of [[Metal-organic framework|MOF]] crystal deposition is relatively cheap and does not require extensive robotic equipment.<ref name="Witters_2012" />

Chemical synthesis using digital microfluidics (DMF) has been applied to many noteworthy biological reactions. These include [[polymerase chain reaction]] (PCR), as well as the formation of [[DNA]] and [[peptide]]s.<ref name="Dubois_2006" /><ref name="Jebrail_2010">{{cite journal | vauthors = Jebrail MJ, Ng AH, Rai V, Hili R, Yudin AK, Wheeler AR | title = Synchronized synthesis of peptide-based macrocycles by digital microfluidics | journal = Angewandte Chemie | volume = 49 | issue = 46 | pages = 8625–8629 | date = November 2010 | pmid = 20715231 | doi = 10.1002/anie.201001604 }}</ref> Reduction, alkylation, and enzymatic digestion have also shown robustness and reproducibility utilizing DMF, indicating potential in the synthesis and manipulation of [[proteomics]].<ref>{{cite journal | vauthors = Luk VN, Wheeler AR | title = A digital microfluidic approach to proteomic sample processing | journal = Analytical Chemistry | volume = 81 | issue = 11 | pages = 4524–4530 | date = June 2009 | pmid = 19476392 | doi = 10.1021/ac900522a | hdl-access = free | hdl = 1807/34790 }}</ref> Spectra obtained from the products of these reactions are often identical to their library spectra, while only utilizing a small fraction of bench-scale reactants.<ref name="Jebrail_2012" /> Thus, conducting these syntheses on the microscale has the benefit of limiting money spent on purchasing reagents and waste products produced while yielding desirable experimental results. However, numerous challenges need to be overcome to push these reactions to completion through DMF. There have been reports of reduced efficiency in chemical reactions as compared to bench-scale versions of the same syntheses, as lower product yields have been observed.<ref name="Jebrail_2010" /> Furthermore, since picoliter and nanoliter size samples must be analyzed, any instrument used in analysis needs to be high in sensitivity. In addition, system setup is often difficult due to extensive amounts of wiring and pumps that are required to operate microchannels and reservoirs.<ref name="Jebrail_2010" /> Finally, samples are often subject to solvent evaporation which leads to changes in volume and concentration of reactants, and in some cases reactions to not go to completion.<ref>{{cite journal | vauthors = Sadeghi S, Ding H, Shah GJ, Chen S, Keng PY, Kim CJ, van Dam RM | title = On chip droplet characterization: a practical, high-sensitivity measurement of droplet impedance in digital microfluidics | journal = Analytical Chemistry | volume = 84 | issue = 4 | pages = 1915–1923 | date = February 2012 | pmid = 22248060 | doi = 10.1021/ac202715f | s2cid = 9113055 | url = http://www.escholarship.org/uc/item/4tk0p54d }}</ref>

The composition and purity of molecules synthesized by DMF are often determined utilizing classic analytical techniques. [[Nuclear magnetic resonance]] (NMR) spectroscopy has been successfully applied to analyze corresponding intermediates, products, and reaction kinetics.<ref name="Jebrail_2012" /><ref>{{cite journal | vauthors = Wu B, von der Ecken S, Swyer I, Li C, Jenne A, Vincent F, Schmidig D, Kuehn T, Beck A, Busse F, Stronks H, Soong R, Wheeler AR, Simpson A | display-authors = 6 | title = Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform | journal = Angewandte Chemie | volume = 58 | issue = 43 | pages = 15372–15376 | date = October 2019 | pmid = 31449724 | doi = 10.1002/anie.201910052 | s2cid = 201728604 }}</ref> A potential issue that arises through the use of NMR is low mass sensitivity, however this can be corrected for by employing [[microcoil]]s that assist in distinguishing molecules of differing masses.<ref name="Jebrail_2012" /> This is necessary since the [[signal-to-noise ratio]] of sample sizes in the microliter to nanoliter range is dramatically reduced compared to bench-scale sample sizes, and microcoils have been shown to resolve this issue.<ref>{{cite journal | vauthors = Peck TL, Magin RL, Lauterbur PC | title = Design and analysis of microcoils for NMR microscopy | journal = Journal of Magnetic Resonance, Series B | volume = 108 | issue = 2 | pages = 114–124 | date = August 1995 | pmid = 7648010 | doi = 10.1006/jmrb.1995.1112 | bibcode = 1995JMRB..108..114P }}</ref> [[Mass spectrometry]] (MS) and [[high-performance liquid chromatography]] (HPLC) have also been used to overcome this challenge.<ref name="Dubois_2006" /><ref name="Jebrail_2012" /> Although MS is an attractive analytical technique for distinguishing the products of reactions accomplished through DMF, it poses its own weaknesses. [[Matrix-assisted laser desorption/ionization|Matrix-assisted laser desorption ionization]] (MALDI) and [[electrospray ionization]] (ESI) MS have recently been paired with analyzing microfluidic chemical reactions. However, crystallization and dilution associated with these methods often leads to unfavorable side effects, such as sample loss and side reactions occurring.<ref name="Kirby_2013" /> The use of MS in DMF is discussed in more detail in a later 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.

=== Mass spectrometry ===
The coupling of digital microfluidics (DMF) and [[Mass spectrometry|Mass Spectrometry]] can largely be categorized into indirect off-line analysis, direct off-line analysis, and in-line analysis<ref name="Kirby_2013">{{cite journal | vauthors = Kirby AE, Wheeler AR | title = Digital microfluidics: an emerging sample preparation platform for mass spectrometry | journal = Analytical Chemistry | volume = 85 | issue = 13 | pages = 6178–84 | date = July 2013 | pmid = 23777536 | doi = 10.1021/ac401150q }}</ref> and the main advantages of this coupling are decreased solvent and reagent use, as well as decreased analysis times.<ref name="Wang_2015">{{cite journal | vauthors = Wang X, Yi L, Mukhitov N, Schrell AM, Dhumpa R, Roper MG | title = Microfluidics-to-mass spectrometry: a review of coupling methods and applications | journal = Journal of Chromatography A | volume = 1382 | pages = 98–116 | date = February 2015 | pmid = 25458901 | pmc = 4318794 | doi = 10.1016/j.chroma.2014.10.039 | series = Editors' Choice IX }}</ref>

Indirect off-line analysis is the usage of DMF devices to combine reactants and isolate products, which are then removed and manually transferred to a mass spectrometer. This approach takes advantage of DMF for the sample preparation step but also introduces opportunities for contamination as manual intervention is required to transfer the sample. In one example of this technique, a [[Grieco three-component condensation]] was carried out on chip and was taken off the chip by micropipette for quenching and further analysis.<ref name="Dubois_2006"/>

Direct off-line analysis is the usage of DMF devices that have been fabricated and incorporated partially or totally into a mass spectrometer. This process is still considered off-line, however as some post-reaction procedures may be carried out manually (but on chip),  without the use of the digital capabilities of the device. Such devices are most often used in conjugation with [[Matrix-assisted laser desorption/ionization|MALDI-MS]]. In MALDI-based direct off-line devices, the droplet must be dried and recrystallized along with matrix – operations that oftentimes require vacuum chambers.<ref name="Kirby_2013" /><ref>{{cite journal | vauthors = Chatterjee D, Ytterberg AJ, Son SU, Loo JA, Garrell RL | title = Integration of protein processing steps on a droplet microfluidics platform for MALDI-MS analysis | journal = Analytical Chemistry | volume = 82 | issue = 5 | pages = 2095–101 | date = March 2010 | pmid = 20146460 | doi = 10.1021/ac9029373 | url = https://figshare.com/articles/Integration_of_Protein_Processing_Steps_on_a_Droplet_Microfluidics_Platform_for_MALDI_MS_Analysis/2788444 | url-access = subscription }}</ref> The chip with crystallized analyte is then placed in to the MALDI-MS for analysis. One issue raised with MALDI-MS coupling to DMF is that the matrix necessary for MALDI-MS can be highly acidic, which may interfere with the on-chip reactions<ref>{{cite journal | vauthors = Küster SK, Fagerer SR, Verboket PE, Eyer K, Jefimovs K, Zenobi R, Dittrich PS | title = Interfacing droplet microfluidics with matrix-assisted laser desorption/ionization mass spectrometry: label-free content analysis of single droplets | journal = Analytical Chemistry | volume = 85 | issue = 3 | pages = 1285–9 | date = February 2013 | pmid = 23289755 | doi = 10.1021/ac3033189 }}</ref>

Inline analysis is the usage of devices that feed directly into mass spectrometers, thereby eliminating any manual manipulation. Inline analysis may require specially fabricated devices and connecting hardware between the device and the mass spectrometer.<ref name="Kirby_2013" /> Inline analysis is often coupled with [[electrospray ionization]]. In one example, a DMF chip was fabricated with a hole that led to a microchannel<ref>{{cite journal | vauthors = Jebrail MJ, Yang H, Mudrik JM, Lafrenière NM, McRoberts C, Al-Dirbashi OY, Fisher L, Chakraborty P, Wheeler AR | display-authors = 6 | title = A digital microfluidic method for dried blood spot analysis | journal = Lab on a Chip | volume = 11 | issue = 19 | pages = 3218–24 | date = October 2011 | pmid = 21869989 | doi = 10.1039/c1lc20524b }}</ref> This microchannel was, in turn, connected to an electrospray ionizer that emitted directly into a mass spectrometer. Integration ambient ionization techniques where ions are formed outside of the mass spectrometer with little or no treatment pairs well with the open or semi-open microfluidic nature of DMF and allows easy inline couping between DMF and MS systems. Ambient Ionization techniques such as Surface Acoustic Wave (SAW) ionization generate surface waves on a flat piezoelectric surface that imparts enough acoustic energy on the liquid interface to overcome surface tension and desorb ions off the chip into the mass analyzer.<ref>{{cite journal | vauthors = Yeo LY, Friend JR | title = Ultrafast microfluidics using surface acoustic waves | journal = Biomicrofluidics | volume = 3 | issue = 1 | pages = 12002 | date = January 2009 | pmid = 19693383 | pmc = 2717600 | doi = 10.1063/1.3056040 }}</ref><ref name="Kirby_2013" /> Some couplings utilize an external high-voltage pulse source at the physical inlet to the mass spectrometer <ref>{{cite journal | vauthors = Heron SR, Wilson R, Shaffer SA, Goodlett DR, Cooper JM | title = Surface acoustic wave nebulization of peptides as a microfluidic interface for mass spectrometry | journal = Analytical Chemistry | volume = 82 | issue = 10 | pages = 3985–9 | date = May 2010 | pmid = 20364823 | pmc = 3073871 | doi = 10.1021/ac100372c }}</ref> but the true role of such additions is uncertain.<ref>{{cite journal | vauthors = Ho J, Tan MK, Go DB, Yeo LY, Friend JR, Chang HC | title = Paper-based microfluidic surface acoustic wave sample delivery and ionization source for rapid and sensitive ambient mass spectrometry | journal = Analytical Chemistry | volume = 83 | issue = 9 | pages = 3260–6 | date = May 2011 | pmid = 21456580 | doi = 10.1021/ac200380q }}</ref>

A significant barrier to the widespread integration of DMF with mass spectrometry is biological contamination, often termed bio-fouling.<ref name="Kirby_2013" /> High throughput analysis is a significant advantage in the use of DMF systems,<ref name="Wang_2015" /> but means that they are particularly susceptible to cross contamination between experiments. As a result, the coupling of DMF with mass spectrometry often requires the integration of a variety of methods to prevent cross contamination such as multiple washing steps,<ref>{{Cite journal| vauthors = Zhao Y, Chakrabarty K |date=June 2010|title=Synchronization of washing operations with droplet routing for cross-contamination avoidance in digital microfluidic biochips|url=https://ieeexplore.ieee.org/document/5523385|journal=Design Automation Conference|pages=635–640}}</ref><ref name="Shih_2012">{{cite journal | vauthors = Shih SC, Yang H, Jebrail MJ, Fobel R, McIntosh N, Al-Dirbashi OY, Chakraborty P, Wheeler AR | display-authors = 6 | title = Dried blood spot analysis by digital microfluidics coupled to nanoelectrospray ionization mass spectrometry | journal = Analytical Chemistry | volume = 84 | issue = 8 | pages = 3731–3738 | date = April 2012 | pmid = 22413743 | doi = 10.1021/ac300305s }}</ref> biologically compatible surfactants,<ref>{{cite journal | vauthors = Aijian AP, Chatterjee D, Garrell RL | title = Fluorinated liquid-enabled protein handling and surfactant-aided crystallization for fully in situ digital microfluidic MALDI-MS analysis | journal = Lab on a Chip | volume = 12 | issue = 14 | pages = 2552–2559 | date = July 2012 | pmid = 22569918 | doi = 10.1039/C2LC21135A }}</ref> and or super hydrophobic surfaces to prevent droplet adsorption.<ref>{{cite journal | vauthors = Samiei E, Tabrizian M, Hoorfar M | title = A review of digital microfluidics as portable platforms for lab-on a-chip applications | journal = Lab on a Chip | volume = 16 | issue = 13 | pages = 2376–2396 | date = July 2016 | pmid = 27272540 | doi = 10.1039/C6LC00387G | url = https://escholarship.mcgill.ca/concern/articles/vt150p551 }}</ref><ref>{{cite journal | vauthors = Lapierre F, Piret G, Drobecq H, Melnyk O, Coffinier Y, Thomy V, Boukherroub R | title = High sensitive matrix-free mass spectrometry analysis of peptides using silicon nanowires-based digital microfluidic device | journal = Lab on a Chip | volume = 11 | issue = 9 | pages = 1620–1628 | date = May 2011 | pmid = 21423926 | doi = 10.1039/C0LC00716A }}</ref> In one example, a reduction in cross contaminant signal during the characterization of an amino acid required 4-5 wash steps between each sample droplet for the contamination intensity to fall below the limit of detection.<ref name="Shih_2012" />

==== Miniature Mass Spectrometers ====
Conventional mass spectrometers are often large as well as prohibitively expensive and complex in their operation which has led to the increased attractiveness of miniature mass spectrometers (MMS) for a variety of applications. MMS are optimized towards affordability and simple operation, often forgoing the need for experienced technicians, having a low cost of manufacture, and being small enough in size to allow for the transfer of data collection from the laboratory into the field.<ref>{{cite journal | vauthors = Ouyang Z, Cooks RG | title = Miniature mass spectrometers | journal = Annual Review of Analytical Chemistry | volume = 2 | issue = 1 | pages = 187–214 | date = 2009-07-19 | pmid = 20636059 | doi = 10.1146/annurev-anchem-060908-155229 | bibcode = 2009ARAC....2..187O }}</ref> These advantages often come at the cost of reduced performance where MMS resolution, as well as the limits of detection and quantitation, are often barely adequate to perform specialized tasks. The integration of DMF with MMS has the potential for significant improvement of MMS systems by increasing throughput, resolution, and automation, while decreasing solvent cost, enabling lab grade analysis at a much reduced cost. In one example the use of a custom DMF system for urine drug testing enabled the creation of an instrument weighing only 25&nbsp;kg with performance comparable to standard laboratory analysis.<ref>{{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 = 6121–6129 | date = June 2014 | pmid = 24906177 | doi = 10.1021/ac5012969 }}</ref>

===Nuclear magnetic resonance spectroscopy===

[[Nuclear magnetic resonance spectroscopy|Nuclear magnetic resonance (NMR) spectroscopy]] can be used in conjunction with digital microfluidics (DMF) through the use of NMR microcoils, which are electromagnetic conducting coils that are less than 1&nbsp;mm in size. Due to their size, these microcoils have several limitations, directly influencing the sensitivity of the machinery they operate within.

Microchannel/microcoil interfaces, previous to digital microfluidics, had several drawbacks such as  in that many created large amounts of solvent waste and were easily contaminated.<ref name="Swyer">{{cite journal | vauthors = Swyer I, Soong R, Dryden MD, Fey M, Maas WE, Simpson A, Wheeler AR | title = Interfacing digital microfluidics with high-field nuclear magnetic resonance spectroscopy | journal = Lab on a Chip | volume = 16 | issue = 22 | pages = 4424–4435 | date = November 2016 | pmid = 27757467 | doi = 10.1039/c6lc01073c }}</ref><ref name="Lei_2015">{{cite journal | vauthors = Lei KM, Mak PI, Law MK, Martins RP | title = A palm-size μNMR relaxometer using a digital microfluidic device and a semiconductor transceiver for chemical/biological diagnosis | journal = The Analyst | volume = 140 | issue = 15 | pages = 5129–37 | date = August 2015 | pmid = 26034784 | doi = 10.1039/c5an00500k | bibcode = 2015Ana...140.5129L | doi-access = free }}</ref> In this way, the use of digital microfluidics and its capability to manipulate singlet droplets is promising.

The interface between digital microfluidics and NMR [[relaxometry]] has led to the creation of systems such as those used to detect and quantify the concentrations of specific molecules on microscales<ref name="Lei_2015" /> with some such systems using two step processes in which DMF devices guide droplets to the NMR detection site.<ref>{{cite journal | vauthors = Lei KM, Mak PI, Law MK, Martins RP | title = NMR-DMF: a modular nuclear magnetic resonance-digital microfluidics system for biological assays | journal = The Analyst | volume = 139 | issue = 23 | pages = 6204–13 | date = December 2014 | pmid = 25315808 | doi = 10.1039/c4an01285b | bibcode = 2014Ana...139.6204L | doi-access = free }}</ref> Introductory systems of high-field NMR and 2D NMR in conjunction with microfluidics have also been developed.<ref name="Swyer" />  These systems use single plate DMF devices with NMR microcoils in place of the second plate. Recently, further modified version of this interface included pulsed field gradients (PFG) units that enabled this platform to perform more sophisticated NMR measurements (e.g. NMR diffusometry, gradients encoded pulse measurements).<ref name="Swyer_2019">{{cite journal | vauthors = Swyer I, von der Ecken S, Wu B, Jenne A, Soong R, Vincent F, Schmidig D, Frei T, Busse F, Stronks HJ, Simpson AJ, Wheeler AR | title = Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring | journal = Lab on a Chip | volume = 19 | issue = 4 | pages = 641–653 | date = January 2019 | pmid = 30648175 | doi = 10.1039/C8LC01214H | s2cid = 58600090 }}</ref> This system has been successfully applied into monitoring rapid organic reactions.<ref name="Wu_2019">{{cite journal | vauthors = Wu B, von der Ecken S, Swyer I, Li CL, Jenne A, Vincent F, Schmidig D, Kuehn T, Beck A, Busse F, Stronks HJ, Soong R, Wheeler AR, Simpson AJ | title = Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform | journal = Angewandte Chemie International Edition | volume = 58 | issue = 43 | pages = 15372–15376 | date = October 2019 | pmid = 31449724 | doi = 10.1002/anie.201910052 | s2cid = 201728604 }}</ref>