Digital microfluidics

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Digital microfluidics (DMF) is a platform for lab-on-a-chip systems that is based upon the manipulation of microdroplets. Droplets are dispensed, moved, stored, mixed, reacted, or analyzed on a platform with a set of insulated electrodes. Digital microfluidics can be used together with analytical analysis procedures such as mass spectrometry, colorimetry, electrochemical, and electrochemiluminescense. works much the same way as traditional bench-top protocols, only with much smaller volumes and much higher automation. Thus a wide range of established chemical procedures and protocols can be seamlessly transferred to a nanoliter droplet format. Electrowetting, dielectrophoresis, and immiscible-fluid flows are the three most commonly used principles, which have been used to generate and manipulate microdroplets in a digital microfluidic device.

A digital microfluidic (DMF) device set-up depends on the substrates used, the electrodes, the configuration of those electrodes, the use of a dielectric material, the thickness of that dielectric material, the hydrophobic layers, and the applied voltage.

[[File:DMF open and closed system.png|alt=|right|frameless|577x577px|<nowiki>Aqueous droplet sitting on top of an open and closed digital microfluidic system with a cross section view. This shows the movement of the droplet once an electrode is activated. Device design can be manipulated to fit user's needs (modified electrodes, electrode pattern, materials used, etc.).[3][4]]]</nowiki>]]

A common substrate used in this type of system is glass. Depending if the system is open or closed, there would be either one or two layers of glass. The bottom layer of the device contains a patterned array of individually controllable electrodes.

Modifications to this foundation can also be fabricated into the basic design structure. One example of this is the addition of electrochemiluminescence detectors within the indium tin oxide layer (the ground electrode in a closed system) which aid in the detection of luminophores in droplets. In general, different materials may also be used to replace basic components of a DMF system such as the use of PDMS instead of glass for the substrate. Liquid materials can be added, such as oil or another substance, to a closed system to prevent evaporation of materials and decrease surface contamination. Also, DMF systems can be compatible with ionic liquid droplets with the use of an oil in a closed device or with the use of a catena (a suspended wire) over an open DMF device. The photoelectrowetting effect can also be used to achieve droplet transport on a silicon wafer without the necessity of patterned electrodes.

Working principle

Droplets are formed using the surface tension properties of a liquid. For example, water placed on a hydrophobic surface such as wax paper will form spherical droplets to minimize its contact with the surface. Differences in surface hydrophobicity affect a liquid's ability to spread and 'wet' a surface by changing the contact angle. As the hydrophobicity of a surface increases, the contact angle increases, and the ability of the droplet to wet the surface decreases. The change in contact angle, and therefore wetting, is regulated by the Young-Lippmann equation. Both processes are only known to work in closed devices, though this often is not a problem as the top plates of DMF devices are typically removable,

The conventional method This difference is caused by local perturbations due to the rapid mass transport. such as immunoassays and DNA amplification. To overcome the limitation of the conventional method, an existing droplet can be split by gradually changing the potential of the electrodes at the splitting region instead of simply switching them on and off. In contrast to splitting, though, dispensing droplets in this manner is inconsistent in scale and results. There is no reliable distance liquid will need to be pulled from the reservoir for the neck to collapse, if it even collapses at all. Because this distance varies, the volumes of dispensed droplets will also vary within the same device. using a continuous and replenishable electrowetting channel,

Droplet transportation

Discrete droplets can be transported in a highly controlled way using an array of electrodes. The purpose of this method is to transfer the droplet from a lower planar surface to an upper parallel planar surface and back down via electrostatic forces. The physics behind such particle actuation and perpendicular movement can be understood from early works of N. N. Lebedev and I. P. Skal'skaya. In their research, they attempted to model the Maxwell electrical charge acquired by a perfectly round conducting particle in the presence of a uniform magnetic field caused by a perfectly-conducting and infinitely-stretching surface. By enabling three-dimensional movement within DMF, the technology can be used even more extensively in biological applications, as it could more accurately mimic 3-D microenvironments. A large benefit of employing this type of method is that it allows for two different environments to be accessible by the droplet, which can be taken advantage of by splitting the microfluidic tasks among the two surfaces. For example, while the lower plane can be used to move droplets, the upper plate can carry out the necessary chemical and/or biological processes. This also allows for the chip to be smaller, and to give researchers more freedom in designing platforms for microdroplet analysis. Unlike traditional microfluidics platform, which are generally restricted to planar and horizontal surfaces, ATDA enables droplet manipulation over curved, non-horizontal, and inverted surfaces. This device works very well with many liquids, including aqueous buffers, solutions of proteins and DNA, and undiluted bovine serum. A drawback of a setup like this is accelerated droplet evaporation.

Implementation

In one of various embodiments of EWOD-based microfluidic biochips, investigated first by Cytonix in 1987 and subsequently commercialized by Advanced Liquid Logic, there are two parallel glass plates. The bottom plate contains a patterned array of individually controllable electrodes and the top plate is coated with a continuous grounding electrode. A dielectric insulator coated with a hydrophobic is added to the plates to decrease the wet-ability of the surface and to add capacitance between the droplet and the control electrode. The droplet containing biochemical samples and the filler medium, such as the silicone oil, a fluorinated oil, or air, are sandwiched between the plates and the droplets travel inside the filler medium. In order to move a droplet, a control voltage is applied to an electrode adjacent to the droplet, and at the same time, the electrode just under the droplet is deactivated. By varying the electric potential along a linear array of electrodes, electrowetting can be used to move droplets along this line of electrodes.

Applications

Laboratory automation

In research fields such as synthetic biology, where highly iterative experimentation is common, considerable efforts have been made to automate workflows. Digital microfluidics is often touted as a laboratory automation solution, with a number of advantages over alternative solutions such as pipetting robots and droplet microfluidics. 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.

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 (CE) module with the purpose of automating the process of next generation sequencing (NGS) library characterization. Compared to an Agilent BioAnalyzer (an instrument commonly used to measure sequencing library size distribution), the DMF-CE system consumed ten-fold less sample volume. 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. Reproducibility can be especially salient when multiple iterations of the same experimental protocol need to be repeated. 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-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 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.

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.

Individual droplet addressability

DMF allows for droplet level addressability, meaning individual droplets can be treated as spatially distinct microreactors.

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. liquid-liquid extraction, optical tweezers, and hydrodynamic effects.

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. This technology can be used in the synthesis compounds such as peptidomimetics and PET tracers. 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.

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. Droplets are combined together by inducing opposite charges thus attracting them to each other. This allows for automated mixing of droplets. Mixing of droplets are also used to deposit MOF crystals for printing by delivering reagents into wells and evaporating the solutions for crystal deposition. This method of MOF crystal deposition is relatively cheap and does not require extensive robotic equipment. Reduction, alkylation, and enzymatic digestion have also shown robustness and reproducibility utilizing DMF, indicating potential in the synthesis and manipulation of proteomics. 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.

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. A potential issue that arises through the use of NMR is low mass sensitivity, however this can be corrected for by employing microcoils that assist in distinguishing molecules of differing masses. Mass spectrometry (MS) and high-performance liquid chromatography (HPLC) have also been used to overcome this challenge. The lack of extensive pumps and valves allow for elaborate multi step applications involving cells performed in a simple and compact system. 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. This is beneficial as 3 dimensional cell culture and spheroids 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. PCR amplified products are then validated by transfection into yeast cells and a Western blot protein identification. Another approach to solve protein adhesion is the addition of Pluronic additives to droplets in the device. Pluronic additives are generally not cytotoxic but some have been shown to be harmful to cell cultures.

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. 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, ELISAs, and electrochemical detection, have been incorporated onto DMF immunoassay platforms.

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, IL-6, cardiac marker Troponin I (cTnI), thyroid stimulating hormone (TSH), sTNF-RI, and 17β-estradiol. For example, a magnetic bead-based approached has been used for the detection of cTnI from whole blood in less than 8 minutes. In one example, For example, Rackus et al. 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 can largely be categorized into indirect off-line analysis, direct off-line analysis, and in-line analysis and the main advantages of this coupling are decreased solvent and reagent use, as well as decreased analysis times.

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. 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

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. 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. but the true role of such additions is uncertain.

A significant barrier to the widespread integration of DMF with mass spectrometry is biological contamination, often termed bio-fouling. biologically compatible surfactants, and or super hydrophobic surfaces to prevent droplet adsorption. 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. 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 kg with performance comparable to standard laboratory analysis.

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 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. 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 Introductory systems of high-field NMR and 2D NMR in conjunction with microfluidics have also been developed. This system has been successfully applied into monitoring rapid organic reactions.