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

Automating the activation of individual droplets allow complex sequences of a laboratory unit operation to be brought into one chip. Our digital microfluidic platform tackles rapid specific in-field detection of virus pathogens. This method is based on quantitative detection of specific antigens using EWOD-based digital microfluidics in combination with magnetic immunoprecipitation.

In this contribution, the method is assessed against samples containing various concentrations of bacteria, spores, viruses, and proteins. This process is fully automated, sequential, scalable, and versatile. The sequence of and the droplet volume can be modified to match the requirements of a particular protocol.

Sidestepping the antibody-based sensing entirely, digital microfluidics platform could build to a potential application based on aptamer biosensing where the magnetic beads carry specific aptamers for capture and detection of nucleotide sequences. When trying this technique for the first time, it is important to consider the type of surfactant and how it interacts with your choice of biochemistry and the hydrophobic polymer coating. Begin by removing the magnet from the digital microfluidics or DMF platform and placing it on the bench.

Place a clean actuation plate on the rotating stage with the chromium facing upward, aligning the plate with the upper left corner of the recessed stage. Clamp the actuation plate from the top using the panel with the 47 contact pins, which will secure the plate into place and facilitate alignment with the contact pins. Then plate the 0.5 millimeter shim and the two millimeter PMMA separator onto the rotating stage to provide a controlled gap between the actuation and cover plates.

To load the droplets on the proposed loading pads, aliquot four 2.5 microliter droplets from the running buffer onto the B, A, R, and E denoted pads using one droplet per pad. Then aliquot 2.5 microliters of luminol hydrogen peroxide solution onto the E denoted pad. Next, aliquot 2.5 microliters of Neutravidin conjugated to HRP biotinylated secondary antibody and microbeads onto the F, G, and I denoted pads respectively.

Finally, aliquot 2.5 microliters of the unknown sample onto the C denoted pad. Place the cover plate on the surface of the rig beside the round recess area and slide it laterally into the recess and on top of the actuation plate. Put the permanent magnet on top of the cover plate and secure it by sliding the two latches, then rotate the stage by 180 degrees and inspect it visually to make sure the loaded droplets are still in place.

Check that the loading position for each droplet matches the programmed actuation sequence in the software. Position the photodetector screened can into the slot of the rotating stage and connect the cable. Place the lid over the DMF platform and start the program sequence using the software interface.

Droplet location is recorded via capacitive sensing and can then be observed on the user interface. The program sequence will prompt messages that appear at the interface to inform the operator that the luminol droplet is ready to collect the extracted magnetic beads or the detection droplet is ready to be moved to the detection site. In both cases, confirmation from the operator is required to proceed.

The light intensity produced by chemiluminescent reaction is read by photodetector and recorded in real time. To operate in visual mode for optimization of protocols, start the program sequence using the software interface. The automated droplet actuation can be visualized on the chip for each of the critical assay steps, such as magnetic extraction of the beads, beads resuspension and mixing.

When prompted, mount the photodetector screened can into the slit of the rotating stage. Connect the cable of the photodetector screened can by inserting the pins in the socket. Place the lid over the DMF platform and resume the assay.

Droplets are monitored via capacitive sensing and can be observed on the user interface. To clean the equipment, open the DMF platform lid and rotate the stage 180 degrees. Unhinge the magnet casing and remove the magnet from the rotating stage.

Remove the silicon wafer from the slip with a pair of tweezers and rinse it with distilled water. Then dry it with compressed air and place it in a Petri dish where the wafer can be stored and reused. Use a micropipette to remove the liquid waste from the pad without touching the surface and clean the surface by wicking the liquid from the actuation plate using absorbent paper.

To investigate actuation voltage impact, a droplet from the buffer was driven at various actuation voltages and its motion was recorded. A correlation between Vrms and the average velocity was demonstrated and a velocity plateau was observed after a certain Vrms value. The longevity of the actuation plate was reduced when high values for Vrms were used.

For the extraction laboratory unit operation, the droplet containing the suspended beads was driven to the separation site in the middle of the mixing zone. Then the magnet was activated automatically to approach the chip and to focus the beads. Next, the droplet was moved towards the waste pad, leaving the beads.

The extraction and mixing laboratory unit operations on the EWOD chip facilitated a miniaturized rapid and reproducible sample processing with consecutive detection of the pathogens in 6 to 10 minutes. Incubation times and conjugate concentrations were varied to find the optimum conditions for the assay. It was found that the incubation time of 160 seconds and conjugate concentration of two micrograms per milliliter achieved the best signal-to-noise ratio.

Variations in the protocol can be introduced to achieve desired levels of automation. The eight-step ELISA was used to detect different antigens such as Bacillus atrophaeus spores and the MS2 bacteriophage. Meanwhile, the 10-step protocol was used to quantify E.coli.

It is important to make sure that the applied voltages, the concentrations of analytes and reagents are optimal for the activation of the droplets and successful magnetic separation on chip. To achieve precise measurement, bead binding capacity should be considered and serial dilutions of the analytes might be needed. By exchanging the type of antibody, one could detect different pathogens from the one reported in the results section.

The method could also be applied for instance for biomarker detection or point-of-care diagnostics. In addition to the applications already presented, the system has been developed with in-field detection of airborne pathogen in mind. DMF is an ideal platform for this application because the droplet volume matches the output of other sampler that we have recently developed.