We would like to acknowledge the contribution of our colleagues from the Microfluidic &#38; Microengineering Research Group for their work on the mechanical design and system integration. The authors would like to thank Dstl Porton Down for their invaluable support and financial contribution, to past and ongoing projects that further develop the DMF technology and its applications.

1. Preliminary steps necessary for the assay
NOTE (IMPORTANT): All preliminary steps must be conducted in a sterile environment to avoid contaminations. Sodium azide should not be used for storage as it would inhibit the activity of the horseradish peroxidase (HRP) enzyme.
<ol>
	<li>Prepare running buffer (100 mM HEPES, pH 7.5), using the (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, and add Tween 80 to a concentration of 0.01% (v/v).</li>
	<li>Immobilize the primary antibodies (capture antibodies) onto the surface of the NHS-activated magnetic microbeads by following the protocol provided by the supplier. Briefly, the Magnetic IP/Co-IP Kit (Table of Materials) protocol encompasses the following steps.
	<ol>
		<li>Aliquot 0.2 mL of beads (2 mg) into a plastic tube and remove the supernatant.</li>
		<li>Wash the beads by adding 1 mL of ice-cold 1 mM HCl.</li>
		<li>Bind the selected antibody (Anti-Human Serum Albumin [15C7], Rb anti-BG polyclonal, Rb anti-E.coli MRE 162 polyclonal or Goat anti-MS2 polyclonal), 40 &#956;g/mL in 67 mM Borate Buffer, covalently to the beads for 1 h with shaking at 37 &#176;C. Table 1 contains the list of all antibodies used with the current protocol and the antigen pathogens6.</li>
		<li>Wash the unbound antibodies twice with 0.8 mL of Elution Buffer.</li>
		<li>Quench the reaction with 1 mL of Quenching Buffer for 1 h.</li>
		<li>Wash the beads once with Modified Borate Buffer and once with IP Lysis/Wash Buffer.</li>
		<li>Resuspend the beads in 0.5 mL of IP Lysis/Wash Buffer and store at 4 &#176;C until required for use. 
		NOTE: For best results, a fresh batch of microbeads are coupled to the antibodies the day before the EWOD-isolation and ELISA on-chip. However, the microbeads coupled to the primary antibody can be stored at 4 &#176;C up to one month. Should agglomeration occur, tap the vial to break the precipitate and to resuspend the beads in the solution.</li>
		<li>Block the microbeads with the coupled antibody overnight, using final concentration 4 mg/mL for the microbeads, in a Blocker Casein (1% w/v) in 100 mM phosphate-buffered saline (PBS). 
		NOTE: The blocking step is always conducted the day before the biodetection assay run.</li>
		<li>Separate the beads from the Blocker Casein using a magnet and remove the supernatant.</li>
		<li>Resuspend in 1 mL of running buffer and mix for 1 min.</li>
		<li>Separate the beads using the magnet and remove the supernatant.</li>
		<li>Repeat the above washing steps (1.2.10 and 1.2.11) twice. 
		NOTE: Three washing steps are sufficient to reduce the adhesion of the beads to the surface and to facilitate free movement of the droplet.</li>
		<li>Resuspend the beads in the running buffer at a concentration of 2.5 mg/mL. This solution of microbeads is ready to use with the EWOD chip.</li>
	</ol>
	</li>
	<li>Prepare a solution of the neutravidin conjugated horseradish peroxidase (HRP) and the secondary biotinylated antibody to a final concentration for each of 1 &#956;g/mL in Running Buffer (used for BG detection6). 
	NOTE: To target the antigens (Table 1) various concentrations of secondary biotinylated antibody, ranging from 0.5 to 4.0 &#956;g/mL were tested successfully.</li>
	<li>Mix equal volumes of the Luminol with hydrogen peroxide solution just before running the assay 
	NOTE: Luminol is used to quantify the number of binding events based on the HRP enzyme that is bound covalently to the secondary antibody which targets the antigen (e.g., pathogen). However, different reporting molecule and strategies can be used for detection17 instead of the chemiluminescence and Luminol.</li>
</ol>
2. Manufacturing and surface treatment of the EWOD chip components
NOTE: The EWOD chip consists of an actuation plate with patterned chromium electrodes to alternate the apparent contact angle of a droplet and a cover plate to define the height of droplets.
<ol>
	<li>Draw the design of the electrodes and connectors in 2D using standard CAD software. Include also the waste collection pad with dimensions of 5 x 5 mm2 to store the used solvents (Figure 1). 
	NOTE: To manipulate the droplets we use 47 electrodes, each with dimensions of 1.7 x 1.7 mm2. This electrode size accommodates droplet volumes ranging from 1.5 &#181;L to 3 &#181;L (for a 500 &#181;m gap). From experience, when working with a 500 &#181;m gap, 1.5 &#181;L is the minimum droplet size that can be actuated. It corresponds roughly to the droplet (projected) contour being circumscribed to the square pad. However, there is not any theoretical limit in size other than caused by the resistance (friction) of the body of fluid. Nonetheless, it is recommended that the volume does not exceed 3 &#181;L for reliable actuation using a 500 &#181;m gap. The electrodes are addressed by 48-channel electronics. 
	NOTE: The design dimensions and sizes of the pads can vary depending on the intended volumes and the laboratory unit operations (LUOs) that comprise the protocol.</li>
	<li>Send the design drawing to a mask manufacturing service for printing the chromium mask onto a glass substrate. The thickness of the chromium layer is 100 nm. 
	NOTE: The chromium layer on the photomask used as a substrate for the EWOD chip is, by definition, opaque. The design of each of our electrodes includes a grid layout to ensure semi-transparency.</li>
	<li>Cut the plate to size of 56 x 56 mm2 with a precision CNC Dicing/Cutting Saw.</li>
	<li>Stick masking tape over the electrical contacts in order to isolate them during the two coating steps.</li>
	<li>Coat the plate chromium-glass plate with a dielectric layer by depositing 6 &#181;m Parylene-C onto its surface. The Gorham process18 is used with 7.4 g of DPX-C in a Parylene Deposition System (Table of Materials).</li>
	<li>Spin-coat amorphous fluoropolymers (Table of Materials) using a spin-coater at 1500 rpm for 30 s on top of the plate and bake it at 140 &#176;C for 30 min. 
	NOTE: This renders the surface hydrophobic. One can validate whether the coating has been deposited successfully by placing a droplet of water onto the surface. The contact angle between the droplet and the plate must be in the region of 110&#186;.</li>
	<li>Remove the masking tape from the electrical contacts.</li>
	<li>Spin-coat amorphous fluoropolymers (Table of Materials) using a spin-coater at 1500 rpm for 30 s on top of the 4-in silicon wafer and bake it at 140 &#176;C for 30 min. 
	NOTE: It is important that both the surfaces of the actuation and cover plates are hydrophobic in order to facilitate smooth movement of the discrete droplets during the assay.</li>
</ol>
3. Loading, assembly and operation of the EWOD chip on the DMF platform
NOTE: The EWOD chip operates using parallel-plate configuration with a precisely defined gap 0.5 mm between the actuation plate and the conductive, grounded cover plate. This sandwich assembly is described in the current section.
<ol>
	<li>Remove the lid from the DMF platform6 and place it onto the bench. 
	NOTE: A dark Faraday cage chamber, is necessary to prevent stray light and electromagnetic interference during the detection. There is no interlock on the lid, hence, the platform allows us to observe droplets&#39; movement.</li>
	<li>Place a clean actuation plate onto the rotating stage, chromium facing upwards. The plate needs to be aligned with the upper left corner of the recessed stage (Figure 2A).</li>
	<li>Clamp the actuation plate from the top using the panel with the 47 contact pins. This secures the plate into place and facilitates alignment with the contact pins.</li>
	<li>Lay the 0.5 mm shim and the 2 mm polymethylmethacrylate (PMMA) separator onto the rotating stage, in order to provide a controlled gap between the actuation and the cover plates. 
	NOTE: Optionally, wicking paper can be placed onto the waste disposal pad prior to aliquoting the droplets before the next step. As the assay proceeds, the waste is directly absorbed into the paper that can be removed at the end of the assay.</li>
	<li>Load the droplets on to the proposed loading pads (Figure 1).
	<ol>
		<li>Aliquot four 2.5-&#181;L droplets from the Running buffer onto the B-, A-, R-, E-denoted pads, one droplet on each pad.</li>
		<li>Aliquot 2.5 &#181;L of Luminol:H2O2 (1:1, v/v) solution onto the D-denoted pad.</li>
		<li>Aliquot 2.5 &#181;L of Neutravidin conjugated to HRP (1 &#956;g/mL) onto the F-denoted pad.</li>
		<li>Aliquot 2.5 &#181;L of Biotinylated secondary antibody (1 &#956;g/mL) onto the G-denoted pad.</li>
		<li>Aliquot 2.5 &#181;L of Microbeads with conjugated primary antibody (2.5 mg/mL) go onto I-denoted pad.</li>
		<li>Aliquot 2.5 &#181;L of the unknown sample onto C-denoted pad. 
		NOTE: The proposed loading pattern is only one example of an experimental layout, however, the loading pattern can be changed to match the users&#39; needs, as long as those changes match the sequence defined in the software (Supplementary File 1).</li>
	</ol>
	</li>
	<li>Place the cover plate on the surface of the rig, besides the round recess area, and slide it laterally into the recess and on top of the actuation plate (Figure 2B).</li>
	<li>Put the permanent magnet on top of the cover plate and secure it by sliding the two latches (Figure 2C).</li>
	<li>Rotate the stage by 180&#176; and inspect visually if the loaded droplets are still in place (Figure 1C). 
	NOTE: One should be able to eyeball the position and the shape of droplets through the transparent stage and the back of the actuation plate (Figure 2D). The assay is ready to run if round droplets could be seen on top of the loading electrode pads. In case, a droplet is displaced one can remove the magnet and the cover plate, then recover the displaced droplet with a clean pipette and place it again on the loading pad.</li>
	<li>Check that the loading position for each droplet matches the programmed actuation sequence in the software (see Supplementary File 1 for details on the software). 
	NOTE: To be able to visually check the position of the droplets, the photodetector needs to be dismounted (Figure 2D).</li>
	<li>Position the photodetector screened &#34;can&#34; into the slot of the rotating stage. 
	NOTE: The photodetection system pivots around a photodiode, which has a large collection area (10 x 10 mm2) to maximize light collection without additional optics, plus a trans-impedance amplifier to minimize noise6. However, the system is extremely sensitive and can collect minute signals. To reduce the noise level, a number of electronics-based strategies were implemented (e.g., the photodetection system was protected by putting it in a Faraday cage, a metal casing known as a screened &#34;can&#34;).</li>
	<li>Connect the cable to the photodetector screened &#34;can&#34;. 
	NOTE: Once the EWOD chip is aligned with the photodetector, the DMF platform is completely assembled and is now ready to operate.</li>
	<li>Place the lid over the DMF platform and start the program sequence using the software interface developed at the University of Hertfordshire. 
	NOTE: Additional software is used for reading and recording the luminescence from the droplet as a function of time in a CSV file (see Supplementary File 2).
	<ol>
		<li>Ensure that the programmed sequence (see Supplementary File 1) prompt messages appear at the interface to inform the operator that &#34;The luminol droplet is ready to collect the extracted magnetic beads&#34; or &#34;The detection droplet is ready to be moved to the detection site.&#34; In both cases, confirmation from the operator is required to proceed with the sequence.</li>
	</ol>
	</li>
</ol>
4. Operating in visual mode (Optional for optimization of protocols)
NOTE: If desired, in order to visualize every droplet-based operation, the assay can be operated by skipping steps 3.10-3.12 and replacing them by the following operations.
<ol>
	<li>Start the program sequence using the software interface. 
	NOTE: The droplet movement can be observed while operating in visual mode, which is useful during protocol optimization. Firstly, to check the reproducibility of the magnetic separation operation. For instance, if the amount of beads used is too low, the magnetic separation will not occur, vice versa, if the amount of beads is too high, the droplet may be immobilized by the bead pellet. Secondly, to ascertain the reliability of a new assay as some droplet formulation may cause actuation impairment that can be detected by observation.</li>
	<li>Mount the photodetector screened &#34;can&#34; into the slit of the rotating stage, when prompted.</li>
	<li>Connect the cable to the photodetector screened &#34;can&#34;, by inserting the pins in the socket.</li>
	<li>Place the lid over the DMF platform and resume the assay. 
	NOTE: Use the additional software for reading and recording the luminescence from the droplet as a function of time in a CSV file (see Supplementary File 2)</li>
</ol>
5. Removing liquid waste and cleaning the chip
CAUTION: Make sure the equipment is switched off and disconnected from power sources (computer, main) prior to cleaning. Wear gloves, a lab coat and goggles protective glass (PPE) when removing biological samples from the chip!
<ol>
	<li>To access the electrodes and the used solvents on the actuation plate, open the DMF platform lid and rotate the stage 180&#176;.</li>
	<li>Unhinge the magnet casing, remove the magnet from the rotating stage and place it onto the bench.</li>
	<li>Remove the cover plate, silicon wafer, from the slit with a pair of tweezers, rinse it with DI water, dry it with compressed air and place it in a Petri dish, where the wafer can be stored and reused.</li>
	<li>Use a micropipette to move the liquid waste from the pad without touching the surface.</li>
	<li>Clean the surface by wicking off the liquid from the actuation plate using absorbent paper (filter material). 
	NOTE: Keeping the surface intact will increase the longevity of the actuation plate, that allows multiple uses.</li>
	<li>Clean the actuation plate by sweeping gently the surface of the electrodes with a clean DI water droplet using a clean pipette. Then remove the droplet with wicking paper (filter material). 
	CAUTION: Dispose of the tissues, papers, pipettes tips and gloves that are contaminated with biological material into the bio-waste bin.</li>
	<li>Use the actuation plate for a different assay or remove it from the DMF platform for storage or recycling.</li>
</ol>

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The authors have nothing to disclose.

The EWOD immunoassay protocol is flexible and can include a various number of laboratory unit operations (e.g., capture antigen, mixing, incubation, bead extraction, washing) depending on the reagent type, stability and usage requirements defined by the assay protocol. As a proof of principle, in the current article, two immunoassay protocols are considered showing the implementation of eight or ten LUOs (Figure 4) with the EWOD chip described. Such miniaturization merits from the microliter, discrete volumes of reagents/analyte that increase the efficacy of the ELISA by reducing both the consumption of reagents, the time required per operation, essentially, the total experimental time (6 to 10 min). Furthermore, the assay is automated with timed manipulation of the droplets which decreases variations and improves the precision of the immunoassay17. In its present format, the experiment involves manual handling of droplets at the beginning of each assay, which is a point for further discussion in the next section.
One critical step in the current DMF method is dispensing the droplets onto the surface of the EWOD chip. Typically, a micropipette with a disposable tip is used to measure the exact volume and to load it. However, it can become challenging to immobilize the droplet on the hydrophobic surface of the actuation plate because of interactions between the droplet and the charged surface of the disposable tip. As a result, the droplet can shoot up following the outer surface of the tip instead of remaining onto the plate. To avoid this, the micropipette must be held in an upright position, perpendicular to the chip surface, without touching it, then the droplet can be dispensed on to the loading pad by bringing it in contact with the surface. Should the droplet stick to the pipette tip, return it to the stock solution, exchange the tip and redeposit a fresh droplet. In a further development of the current proof-of-concept system, automatic delivery of droplet can be envisaged.
Another critical step, before running the assay, is closing the lid of the parallel plate assembly. As stated earlier in the protocol, the lid must be slid on top of the actuation plate. The hydrophobic surface of the lid prevents the distortion and displacement of the droplets sitting on the actuation plate. To guarantee the smooth movement of the droplet, it is highly recommended to use pristine actuation plate, correct loading of the droplets and chip assembly. Reusability of the actuation plates is possible; however, the number of cycles depends on the actuation voltages (Figure 3) and the analyte/reagent deposition onto the surface, aka biofouling. The presented platform utilized chrome printed EWOD chip, which could be reused reliably for consecutive measurements up to four times at operating voltage of 120 V and intermediate plate cleaning after each experiment. Plates were recycled, to reduce the cost per experiment, by decontaminating (brushing the surface with undiluted cleaning agent before thoroughly rinsing) the biofouled amorphous fluoropolymers (Table of Materials) coating and spin-coating a fresh one on top of the plate. However, actuation plate recycling requires manual handling, costly reagents (amorphous fluoropolymers (Table of Materials)) and specialized equipment (spin-coater). Alternative EWOD chips are investigated successfully with cost-efficient substrates such as paper19, acetate films or printed circuit boards (PCBs)20,21. Such disposable consumables can facilitate reliable and affordable use of the DMF platform and can provide means to sidestep the biofouling issue.
Biofouling is the main limitation of EWOD for biological applications22,23. Earlier studies on DMF have identified two mechanisms that contribute to biofouling, namely, passive adsorption due to hydrophobic interactions, and an electrostatically driven adsorption manifesting when an electric field is applied24. The findings in the current article are consistent with this theory as it was documented the actuation plate reusability decreases at high actuation voltages. One possible explanation is that proteins adsorb readily on Fluoropolymer-coated (Teflon-like) surfaces and they aggregate faster on fouled in comparison to pristine surfaces24. As a consequence, protein-related assays on DMF are hard to quantify and may experience loss of analyte, cross-contamination and diminished precision17. The worst-case scenario is when a critical amount of protein adsorbs thus rendering the device useless. To minimize the biofouling, various approaches have been investigated from minimizing the residence time of the droplet on the chip, through coatings23, to additives (i.e., surfactants or pluronic acid) into the biomaterial-laden droplets6,22. Hence an important aspect of the immunoassay assay on EWOD is to choose anti-biofouling strategies that are compatible with the specific protocol at hand.
The automated DMF platform is designed to perform a single sandwich ELISA test per run while using microliter volumes for both reagents and analyte. When it is required, conventional sandwich ELISA kits exist based on pre-coated 96-well or 384-well plates that in combination with auxiliary laboratory equipment result in higher throughput per run; based on reagents price only, the approximate cost per assay/well is 6.04 USD (580 USD/96) and 0.33 USD (2&#215;580 USD/384) respectively. This renders the conventional ELISA methods ideal for a large number of samples processed typically by trained technical personnel at centralized laboratory facilities. However, in remote locations, the detailed cost analysis of ELISA for environmental monitoring showed that when capital costs (i.e., laboratory operating costs, recurrent costs, sample transportation, supplies and personnel) were included the actual price per ELISA was 60 USD of which 34 USD were for supplies per sample25. In contrast, the proposed DMF platform is portable, requires minimum training to operate and with pre-coated beads can provide sample-to-answer analysis in minutes. Hence, the presented technology can be deployed to point-of-need locations and complement analyses otherwise available in centralized laboratories.
In the representative results section, the automated DMF immunoassay platform was used for direct detection of pathogens for defense application. Other possible applications for the DMF platform encompass but are not limited to, biodiagnostic, continuous monitoring and automated sampling. Potentially the DMF could impact diverse sectors from point-of-care for personalized healthcare, as well as controlled environment monitoring for the protection of patients from airborne Hospital Acquired Infection, to crop monitoring system for farming and food production.

The proposed method aims to facilitate automated sample preparation for ELISA with quantitative detection of antigens using EWOD-based approach with digital microfluidics (DMF) and magnetophoretic separation. It has been demonstrated for multiple biological applications that DMF in combination with magnetophoresis is an interesting alternative to liquid handling applications1. More specifically, the detection of pathogens is an implicit aspect in many sectors, ranging from healthcare2 to agriculture and the environment3,4 to national security5. A detection technology capable of addressing the threats from pathogens must feature high-throughput (e.g. short assay time), efficiency (low Limit of Detection &#8211; LoD &#8211; and high sensitivity) and specificity (to the target pathogen type) for it to be functional6.
Previously, EWOD-based DMF has been implemented successfully for Reverse Transcription Polymerase Chain Reaction (RT-PCR), detection of an antibiotic-resistant pathogen (Methicillin-resistant Staphylococcus aureaus or MRSA), M.pneumonia and C.albicans using a low-budget, printed-circuit-board chip and magnetophoresis7. The technique was applied also for the detection of deoxyribonucleic acid (DNA) mutations through pyrosequencing and chemiluminescent detection8. EWOD-based platforms also expand their functionality towards immunoassay applications, thereby enabling simultaneous sample recovery and detection all within a single, integrated platform. For instance, a single EWOD-chip design was demonstrated successfully with a DMF platform for point-of-care testing for both bead-based immunoassays of cardiac troponin I from a whole blood sample and as a separate experiment RT-PCR for MRSA detection2. That chip utilizes oil filler, which prevents evaporation of the droplets and facilitates the reliable automated manipulation of nanoliter volumes. Versatile bioapplications were investigated with the implementation of similar DMF approaches covering quantitative homogeneous and heterogeneous immunoassays9,10 including design of experiments (DoE) studies for assay parameter optimization11.
Despite its obvious merits to process intensification due to miniscule working volumes, an oil-filled DMF platform can be challenging and requires a certain level of expertise to operate. Oil-filled systems, because they require sealed component, are not ideal for certain in-field application where system transportability is important. In addition, an oil-based system would be very difficult if not impossible to use for some specific applications taking advantage of dry material collection on a surface such as proposed by Zhao and Cho12, J&#246;nsson-Niedzi&#243;&#322;ka et al.13, and Foat et al.14. In contrast, oil-free systems are simple to integrate and have the advantage of providing easy chip-to-chip sample translation. For these reasons, the proposed method was developed to provide an EWOD-based immunoassay on DMF which would not require oil, effectively simplifying the device operation.
In this contribution, we report on using a bespoke, free-standing, fully automated DMF platform for immunoassays, and we elaborate on the protocol for the rapid detection of biomolecules, namely: proteins, vegetative bacteria, bacterial spores and viruses. Combination of EWOD-chip with magnetic particles for automated sample preparation and immunoprecipitation has been demonstrated already with an additional off-line MS measurement15. Recently, in-field diagnostic against measles and rubella IgG has been demonstrated in remote Northwestern Kenya&#39;s population by the Wheeler group16. Both Wheeler&#39;s and our system, being transportable, self-contained, fully automated with included on-chip, real-time chemiluminescent measurements are arguably among the most advanced DMF biodetection systems available.
The two systems have been designed with very different applications in mind. Wheeler&#39;s system targets biomarker to allow biomedical diagnostics on patients whereas our biodetection system is built around defense requirement for direct detection of pathogen previously sampled from air. The similarity between the two is the underlying principle of droplet actuation, which demonstrates the broad range of life-influencing sectors that the EWOD-based technology can impact. Namely, the DMF-based detection platform and associated EWOD system could find key implication in health (biomedical diagnostic); military and civilian protection (threat detection); Agri-tech (crop monitoring) and work safety (controlled environment monitoring)
The performance of our DMF platform is assessed against fully automated detection of human serum albumin (HSA, a globular protein), Escherichia coli (E. coli, a vegetative bacteria), Bacillus atrophaeus (BG, a bacterial spore) and MS2 (a bacteriophage virus). More importantly, the proposed DMF-method is extremely versatile in the sense that the capture antibodies could be exchanged to target the detection of other antigens different from the four that are considered in this article. Sidestepping the antibody-based sensing entirely, the DMF platform could build to a potential application based on aptamer biosensing, where the magnetic beads carry specific aptamers for capture and/or detection of nucleotides. The design and realization of the different components constituting the integrated, completely self-contained DMF platform, including the high voltage waveform generator and drive electronics is disclosed elsewhere6.

<table><tbody><tr><td>(4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, HEPES</td><td>Sigma-Aldrich</td><td>H9897</td><td></td></tr><tr><td>Anti-Human Serum Albumin [15C7]</td><td>Abcam</td><td>ab10241</td><td></td></tr><tr><td>Anti-Human Serum Albumin [1A9] (HRP)</td><td>Abcam</td><td>ab24438</td><td></td></tr><tr><td>B. atrophaeus (BG) spores</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Biotinylated Rb anti-BG polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Biotinylated Rb anti-E. coli MRE 162 polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Biotinylated Rb anti-MS2 polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Blocker Casein</td><td>Thermo Scientific</td><td>TFS 37582</td><td></td></tr><tr><td>CNC Dicing/Cutting Saw</td><td>MTI Corp, USA</td><td>SYJ-400</td><td></td></tr><tr><td>Cytop</td><td>AGC, Japan</td><td>CTL-809M</td><td>Amorphous fluoropolymers. This is a two component coating.</td></tr><tr><td>E. coli MRE 162</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Goat anti-MS2 polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Hamamatsu photodiode</td><td>Hamamatsu, Japan</td><td>S9270</td><td></td></tr><tr><td>Hidrochloric acid (32%)</td><td>Sigma-Aldrich</td><td>W530574</td><td></td></tr><tr><td>Mask manufacturing service</td><td>Compugraphics, Scotland, UK</td><td>N/a</td><td></td></tr><tr><td>MS2 virus</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Parylene-C, DPX-C</td><td>Specialty Coating System, USA</td><td>CAS No.: 28804-46-8</td><td></td></tr><tr><td>Pierce Direct Magnetic IP/Co-IP Kit</td><td>Thermo Scientific</td><td>88828</td><td>Contains all buffers and reagents required for enzyme immobilisation. Store at 4 &amp;deg;C.</td></tr><tr><td>Rb anti-BG polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Rb anti-E. coli MRE 162 polyclonal</td><td>Dstl, UK</td><td>N/a</td><td></td></tr><tr><td>Recombinant Human Serum Albumin protein, HAS</td><td>Abcam</td><td>ab201876</td><td></td></tr><tr><td>SCS Parylene Deposition System</td><td>Specialty Coating System, USA</td><td>2010</td><td></td></tr><tr><td>Silicon wafer, 4&#039;&#039;, p-type, &amp;lt;100&gt;, 1&amp;ndash;10 &amp;Omega;cm</td><td>Pi Kem Ltd</td><td>N/a</td><td></td></tr><tr><td>Spin Coater</td><td>S&amp;Uuml;SS MicroTec AG, Germany</td><td></td><td></td></tr><tr><td>SuperSignal ELISA Femto Maximum Sensitivity Substrate</td><td>Thermo Scientific</td><td>37075</td><td>It contains 50 mL of Luminol/ Enhancer and Stable Peroxide solutions. Store at 4 &amp;deg;C.</td></tr><tr><td>Tween 80</td><td>Thermo Scientific</td><td>28328</td><td>The manifacturer is Surfact-Amps Detergent Solution.</td></tr></tbody></table>

electrowetting-based digital microfluidics platform for automated enzyme-linked immunosorbent assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Actuation voltage impact was investigated in order to elucidate what the optimal conditions were to perform the assays. A droplet from the buffer was driven at various actuation voltages and its motion was recorded. The findings demonstrated (Figure 3) a correlation existed between the root mean square actuation voltage (Vrms) and the average velocity. However, the longevity of an actuation plate was reduced when high values for Vrms were used. Based on these results, 105 Vrms was chosen as the standard actuation voltage, 120 Vrms was found to work best for the H2O2/Luminol droplet and 165 Vrms was implemented for the extraction LUO. These voltages were included in the automated programming sequence (Supplementary File 1).
Two immunoassays (Figure 4) were tested successfully using the EWOD chip with the DMF platform for four different pathogens (Table 1). The EWOD chip facilitated the consecutive movement of the droplets from the loading pads to the mixing region and finally to the waste. There were two basic LUOs that were repeated throughout the protocol to complete ELISA. The first was the extraction LUO; briefly described here, the droplet containing the suspended beads was driven to the separation site in the middle of the mixing zone, the magnet was activated automatically to approach the chip and to pool the magnetic beads into a pellet (Figure 5). Next, the droplet was moved towards the waste pad, leaving the beads onto the actuation plate, thus concluding the extraction LUO. Mixing was the next key LUO to take place on the EWOD chip. The analyte sample with an unknown concentration of pathogens was moved onto the beads by electrowetting. Then the beads were resuspended by moving the droplet with the clumped beads over the mixing area (10 pads in total). These two LUOs were essential as they facilitated a miniaturized, rapid and reproducible sample processing with consecutive detection of the pathogens in 6 to 10 min. Figure 6 shows the complete sequence of LUOs from an immunoassay accomplished with the EWOD chip.
To meet the desired levels of automation, variations in the protocol could be introduced. For instance, the beads were separated from the antigen depleted droplet, which was then transferred to the waste pad, repeating the basic extraction LUO. At this stage, the protocol could branch depending on whether the detection antibody was conjugated to the HRP already, effectively using eight LUOs in total for the detection of the different antigens (Figure 7A-C). In these cases, the droplet with the detection antibody was brought to the beads and then mixed by actuation. Alternatively, binding the detection antibody to the Neutravidin-HRP conjugate could be performed sequentially in situ on the EWOD chip, as it was demonstrated for the quantification of E. coli (Figure 7D). Both protocols, the eight- and ten-step ELISA (Figure 4), yielded reproducible detection of antigens.
Incubation times and conjugate concentrations were varied to find experimentally the optimum conditions for the assay (Figure 7A). It was found that the incubation time of 160 s and conjugate concentration of 2 &#956;g/mL achieved the best signal to noise ratio with a 36% increase of signal strength and virtually no change in the background noise levels. All of the figures and data used in the representative results section were modified from an earlier work6.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Primary / Capture antibody</td>
			<td>Detection antibody</td>
			<td>Antigen</td>
		</tr>
		<tr>
			<td>Anti-Human Serum Albumin [15C7] (anti-HSA, Abcam ab10241)</td>
			<td>Horse Radish Peroxidase (HRP) tagged anti-HSA [1A9] (Abcam ab24438)</td>
			<td>Human Serum Albumin (HSA, Abcam)</td>
		</tr>
		<tr>
			<td>Rb anti-BG polyclonal</td>
			<td>Biotinylated Rb anti-BG polyclonal</td>
			<td>B. globigii (BG) spores</td>
		</tr>
		<tr>
			<td>Rb anti-E.coli MRE 162 polyclonal</td>
			<td>Biotinylated Rb anti-E.coli 162 MRE polyclonal</td>
			<td>E. coli MRE 162</td>
		</tr>
		<tr>
			<td>Goat anti-MS2 polyclonal</td>
			<td>Biotinylated Rb anti-MS2 polyclonal</td>
			<td>MS2 bacteriophage virus</td>
		</tr>
	</tbody>
</table>
Table 1: Antigens and antibodies tested with this protocol. Four types of pathogen antigens were used to demonstrate the capabilities of the EWOD chip with the DMF platform.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60489/60489fig1a.jpg" /> 
Figure 1: Design of the EWOD plate. (a) Schematic notation of the EWOD actuation plate with connectors (squares, Top) that are linked (lines) to the electrodes (squares, Bottom). Each pad is assigned a number and can be addressed from the software code (Supplementary File 1). The loading electrode pads are marked by arrows and denoted by a capital letter above or below each pad. A key feature for the DMF platform is the mixing zone comprised of ten pads (No. 31, 32, 33, 36, 37, 42, 43, 44, 46, 47). As a visual guide, the mixing zone is marked with a red rectangle. (b) Micrograph of the pads&#39; microgrid design. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60489/60489fig2.jpg" /> 
Figure 2: Components and key stages for the digital microfluidic system (DMF) assembly. (A) Fix the EWOD actuation plate, place the shim onto the rotating stage and load the droplets. (B) Position the cover plate. (C) Mount the magnet case, fasten the latches and rotate the stage 180&#176;. (D) The automated magnet is pointing downwards. Inspect the position and shape of the droplets, check that the printed circuit board (PCB) pins are aligned with the contacts on the EWOD chip, connect the photodetector and place it into the photodetector slot. After connecting the control electronics to a computer, the system is ready to run the assay. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60489/60489fig3large.jpg" /> 
Figure 3: Repetitive usage of the actuation plates and the impact on actuation voltage. The average velocity of a droplet from the running buffer is plotted as a function of the actuation voltage (blue circles) and the standard deviation from three independent measurements (N = 3). Here the number of assays per plate (grey bars) indicates enhanced decay of the surface at higher voltages. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60489/60489fig4a.jpg" /> 
Figure 4: Diagram of the immunoassays tested with EWOD. Each circle in this diagram represents a volume of 2.5 &#956;L loaded onto the EWOD chip. The first protocol (on the left-hand side) shows eight LUOs using premixed antibody-HRP conjugate; whilst, the second protocol encompasses ten LUOs, separately adding of the biotinylated detection antibody, bead extraction and consecutive binding of Neutravidin-HRP conjugate. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60489/60489fig5.jpg" /> 
Figure 5: Magnetic bead extraction. This process is broken down into (a-c) actuating the droplet with the suspended magnetic beads to the magnetic separation site in the middle of the mixing zone (pad No. 33), (d, e) the magnet is moving into position focusing the beads, (f, g, h) beads are held in place by the magnetic force while the droplet is actuated away by EWOD towards the waste pad (pad No. 41). <a href="https://www.jove.com/files/ftp_upload/60489/60489fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60489/60489fig6.jpg" /> 
Figure 6: Complete immunoassay sequence using EWOD, showing the reagents, sample loading and laboratory unit operations. Each row contains a sequence of sample images from the characteristic operations on a droplet. The operations are divided into columns. Mixing is not performed for the beads in suspension, presented by a black broken line. Droplet directions are indicated by blue arrows, the beads are highlighted in one of the images by an orange arrow. The grey box (bottom right corner) separates the two images that represent movement and position in the detection area, the broken-line circle highlights the detection area. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig6alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60489/60489fig7a.jpg" /> 
Figure 7: Calibration curves from immunoassays conducted on EWOD chip with the DMF platform. As previously reported6 the output voltages (mV) versus concentrations are shown for: (A) Human serum albumin, which is used to study the effect of the conjugate antibody concentration [C] and the incubation time, tinc, measured from the mixing of the beads with known analyte until the extraction LUO, (B) B. atrophaeus (BG) spores showing the reproducibility of the immunoassay, (C) MS2 bacteriophage immunoassay, and (D) ten-LUOs protocol results for E. coli. Abbreviations: colony forming units (cfu), plaque-forming units (pfu), number of independent experiments (N), laboratory unit operation (LUO). Figure modified from previous publication6. <a href="https://www.jove.com/files/ftp_upload/60489/60489fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Complete sequence to run the DMF platform for automated ELISA assay with Neutravidin-HRP as conjugate. <a href="https://www.jove.com/files/ftp_upload/60489/APPENDIX_A.docx" target="_blank">Please click here to download this file.</a>
Supplementary File 2: The GUI for the chemiluminescence measurement and an example from a measurement with the software are shown. <a href="https://www.jove.com/files/ftp_upload/60489/APPENDIX_B.docx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay at JoVE.com

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

electrowetting-based-digital-microfluidics-platform-for-automated

Research

JoVE Journal

Bioengineering

9.4K Views.  University of Hertfordshire. Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Video: Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

Engineering

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.