Combining Electrowetting and Magnetic Bead Manipulations for Use in Microfluidic Immunoassays

Amanda Barbour Ronit Langer [email protected] [email protected]

Tiffany Li Katherine Tsang [email protected] [email protected]

Rose Yin [email protected]

Abstract afford to lose large amounts of blood.1 It This experiment investigated reducing the also takes 2 days of incubation time, which cost of microfluidic immunoassays. places a risk on patients with urgent health Magnetic microbeads are used in these concerns.1 Technologies in microfluidics assays to increase the efficiency of have created a different type of immunoassays, but they also raise the cost. immunoassay that uses magnetic This experiment aims to reduce the number microbeads. It can be performed within of magnetic beads necessary by using minutes with only 5-10 µL of blood, making electrowetting in conjunction with the beads. it safer for patients who cannot afford to lose Single and parallel plate electrowetting blood, and more efficient for doctors.2 devices were tested individually and the However, this process is very expensive due magnetic microbeads. The devices did not to the large number of microbeads needed. succeed in separating the beads from the During the assay, the microbeads need to be droplets, but the results show many separated from each sample and pulled promising options for further investigation. through microchannels into a new solution. In order to separate, the microbeads need to 1. Introduction break the surface tension of the sample, and 3 With the increasing pace of today’s society, this requires thousands of microbeads. current medicine requires fast and accurate This project aims to create a method diagnoses for patients. One way of making of performing the immunoassay with fewer diagnoses is through immunoassays, which microbeads, therefore reducing the price. are becoming very prevalent. An The microbeads cost $852 for a 10 mL vial, immunoassay is a type of test that involves so the current method of testing is screening biological samples to determine economically unfeasible for widespread the concentrations of various antigens. use.4 To reduce this cost, it is proposed that These allow doctors to make more informed magnetic actuation be combined with decisions regarding the patient’s treatment. electrowetting. Electrowetting is a technique ELISA (enzyme-linked immunosorbent that manipulates liquid using electricity to assay) is a common type of immunoassay. It reduce the contact angle between fluid and a requires a blood sample size on the scale of solid surface, thus reducing the surface mL, which is unsuitable for infants and tension. One of the roles of the magnetic immunocompromised patients who cannot beads in the immunoassay is to break the

1 surface tension of the sample fluid. antigen-specific monoclonal primary Reducing the surface tension with antibody and placed in a well containing a electrowetting means that a smaller force is blood sample so the primary antibodies bind required, and therefore fewer beads are with the target antigen. Then, the beads are needed to complete the same task. The pulled with a magnet through a successful combination of magnetic microchannel containing oil into a different actuation and electrowetting can lead to a well. The new well contains a secondary smaller microbead requirement and a more antibody that binds to a different epitope of affordable microfluidic immunoassay. the target antigen. The secondary antibody is then bound to a fluorescent or colorimetric 2. Background tag that is used to detect the concentration of 2.1 ELISA antigens found in the blood sample.6 ELISA (enzyme-linked immunosorbent assay) is a test used to identify and quantify specific antigens in a sample, and it is often used to monitor antigen concentrations in blood samples. The mechanism for ELISA utilizes antibodies specific to the desired antigen.5 Primary antibodies in wells are immersed in the sample, and the antigen in the sample binds to this antibody. The wells are then Fig. 1. Diagram of microbead in washed, and a solution containing a microfluidic immunoassay 7 secondary antibody is added, and the This type of antigen capture and detection is secondary antibody binds to a different very similar to the process used in a epitope of the target antigen. This secondary traditional ELISA, but the microfluidic antibody has a fluorescent or a colorimetric method uses only 5-10 µL of blood, which is enzyme tag that is used to determine the ideal for infants and immunocompromised concentration of antigens in the sample. This patients that cannot afford to lose large tag is easily measurable and correlates to the quantities of blood.6 The microbeads also concentration of the substance of interest. create a large surface area-to-volume ratio to ELISA is traditionally performed in a 96 ensure maximum antigen capture.8 In well plate, which requires 50 µL of blood addition, their magnetic properties allow a per well and a minimum of two days in a 6 separation system in which magnets can pull laboratory to process the test. the microbeads from one sample to another through microchannels. This process creates 2.2 Immunoassays and Microfluidics a series of incubations within the device and Microfluidics is the study, design, therefore it increases the speed of the wash and creation of systems dealing with the 1 steps necessary in an ELISA. This means flow of small volumes of liquid. In recent that the immunoassay can be performed at years, microfluidic techniques have been much faster speeds, which is more practical combined with magnetic beads to create a for use in surgery or other urgent situations.6 new method of performing immunoassays. In addition, there is a possibility of These tests reduce the amount of blood and 6 developing a continuous flow test. It will time required for medical procedures. First, produce results at even faster speeds, and it magnetic microbeads are conjugated with an will require very little blood from the

2 patient. One application of this type of typical paramagnetic substances. This means immunoassay is surgery. A continuous flow that the superparamagnetic beads react test will allow surgeons to monitor quickly when a magnetic field is applied, biomarkers continuously, which is important and in addition, they do not agglomerate for procedures such as cardiopulmonary after the magnetic field is removed.9 In bypasses (CPB). The ability to detect addition, the addition of microspheres to a inflammatory biomarkers in the blood will solution reduces the viscosity of the overall alert surgeons to problems earlier, which solution, since the fluid molecules interact will reduce complications and increase more with the beads than with the walls of recovery speed.6 the microchannels. A low viscosity fluid requires less energy to flow, which is ideal for small systems. The magnetic beads in this experiment are composed of a magnetite 9 (Fe3O4) core, and a streptavidin coating. In microfluidic immunoassays, thousands of beads are required to break the surface tension of the fluid and eject a bead droplet from the sample, as seen in Fig. 2. Since the microbeads are too costly, this number needs to be reduced to make the test 4 economical.

Fig. 2. Diagram of slide used to conduct the 2.4 Electrowetting microfluidic immunoassays. First, the beads Electrowetting-on-dielectric is the are ejected from the reservoir with a process of changing the wetting properties magnet, then they are transported through of a surface by applying an external electric the channels, and then deposited in the 11 3 field. If a droplet of conductive liquid is adjacent well. exposed to an external electric field, the distribution of ions within the droplet changes, and as a result the shape of the droplet adjusts to maintain equilibrium. As a higher charge is applied, the droplet becomes more wettable, and therefore the contact angle of the droplet decreases. The contact angle of a droplet is described by Young’s equation. � − � ���� = !" !" Fig.3. Magnetic beads being pulled through ! � a channel in a microfluidic immunoassay 9 !" In this equation, θY is the contact angle between the droplet and the solid surface, 2.3 Magnetic Beads σsv is the surface tension between the solid The magnetic microbeads used in and the surrounding vapor, σsl is the surface these microfluidic devices are tension between the solid and the liquid, and superparamagnetic. Superparamagnetic σlv is the surface tension between the liquid substances have magnetization that and the vapor. When a voltage is applied, randomly flips as temperature varies, the change in contact angle due to leading to a higher magnetic sensitivity than electrowetting is described by the Young-

3 Lippmann equation. This is beneficial, because since PBS is isotonic with blood, converting from water � � ! ! ! to a biological fluid for use in an ���� = ����! + � 2��!" immunoassay will not be difficult. In order to actuate a droplet, it has to In this equation, θ is the new contact angle, bridge two or more electrodes. For a εd is the dielectric constant of the insulator, parallel-plate configuration, a second plate is ε0 is the dielectric constant of the liquid, d is placed on top of the first one and either air the distance between the electrodes and the or an immiscible liquid surrounds the water droplet, and U is the voltage applied. droplet. This configuration makes the As the voltage is increased, θ decreases, droplet thinner, further increasing the which decreases the surface tension between control over the droplet’s surface tension14 the liquid and the solid, making it easier to 12 and consequently the liquid’s ability to actuate the droplet. move, split, and merge.15

3. Procedure 3.1 Device Fabrication The template for the electrode pattern necessary for the microfluidic device was created using AutoCAD, a computer

Fig. 4. Diagram of droplets being aided design program, and patterned onto a manipulated by electrowetting13 glass microscope slide through photolithography, a process that transfers a Electrowetting is performed on a design onto a substrate. The design created prepared plate embedded with electrodes. In in CAD was printed onto a film mask, which order for the electrowetting to work would later be used in the exposure phase of properly, there must be an insulation layer photolithography. The resulting plate between the electrodes and the liquid to formed the basis of the microfluidic device. The process of photolithography prevent hydrolysis. In addition, it must be 16 hydrophobic, so the contact angle at zero required 4 steps. First, a glass microscope voltage is as large as possible. In addition, slide was soaked in acetone, isopropanol, the insulation layer should be as thin as and deionized water for ten minutes each to possible, so the voltage can actuate the remove contaminants. The slide was then droplets.12 Cytop is an amorphous baked to assure that the slide was fluoropolymer that is often used in completely dry. In the next step, a positive electrowetting because it is hydrophobic, photoresist, which degrades when exposed insulating, and able to be applied in to UV light, was spin coated onto the slide. thicknesses on the order of µm.11 The third step consisted of the softbake, in Electrowetting uses a hydrophobic which the plate was heated to cure the solid surface with a hydrophilic liquid, but it photoresist and prevent bubbling. In the next depends very little on the type of step, a photolithography system was used to hydrophilic liquid used. Although the expose the photoresist to UV light in the movement is caused by a reorganization of pattern defined by the AutoCAD mask. charges within the droplet, both PBS Then, the slide was placed in a developing (phosphate buffered saline) and deionized solution, causing the areas that were water display electrowetting properties.12 exposed to the UV light to degrade.

4 Fig. 6. Process of fabricating the electrode plates In order to perform electrowetting, After the photolithography process, the slides were coated with Cytop, an the photoresist was hardened with the amorphous fluoropolymer that forms a postbake. Next, hydrofluoric acid (HF) was hydrophobic barrier between the electrodes used to etch the exposed glass so that the and the water droplet. First, the slides were platinum used to make the electrodes can be placed in the plasma generator at 100 W for effectively deposited and bonded to the 60 seconds in order to activate the surface to glass. The HF does not affect the improve bonding. Next, 250 µL of Cytop photoresist, so it only etches onto the was pipetted onto the slide and spun using exposed glass in the shape of the electrodes. the spin coater for 30 seconds at 3000 rpm. Then, platinum was sputtered onto the slides In order to cure the Cytop, the slides were using a physical vapor deposition system. then placed on a hotplate at 100ºC for 90 The platinum only binds permanently to the seconds and placed in a 150ºC oven for 2 places where the glass was etched with HF. hours. Finally, the slide was rinsed with acetone to Single plate electrowetting only remove the photoresist and the excess requires the electrode slide, but in order to platinum, so that only the platinum perform parallel plate electrowetting, a electrodes remained in the glass slide.17 separate device must be built. The electrodes that were used in the single-plate electrowetting formed the base of the device. Then, a gasket was created out of PDMS (polydimethylsiloxane). The PDMS and a hardener were mixed in a ratio of 10:1, and then 1.5 ml was poured onto a clean petri dish. Then, the dish was placed in a desiccator for two hours to remove the air Fig. 5. Top: Photolithography mask created bubbles from the solution. in CAD Bottom: Resulting slide after the photoresist developed

5 was used. In this procedure, 2 µL, 4 µL, and 6 µL droplets of deionized water were tested. In addition, phosphate-buffered saline (PBS) was tested in the same volumes. The droplet being tested was placed so that it bridged two electrodes, and wires connected to a DC power supply were touched to the corresponding electrodes in order to make the droplet move from one electrode plate to another. Applying a positive charge to one electrode and Fig. 7. PDMS pipetted onto a clean petri grounding an adjacent one made the water dish to make the gasket droplet move towards the positive voltage. After it hardened, the PDMS layer was cut The DC voltages were tested at 40 V, 60 V, so that when it was placed on the electrode and 80 V. slide, the electrodes were still exposed. Single plate electrowetting was also Finally, an ITO (Indium tin oxide) slide was tested using a non-conductive top plate to coated with Cytop using the same procedure reduce the contact angle of the droplet. A as before. ITO is a conductive material that PDMS gasket was placed on top of the is spread onto a glass slide, and it is used to original electrode plate so that it did not apply ground to the top of the water droplet. cover the electrodes, but so it created an As with the platinum electrodes, the ITO elevated platform for the top slide. The also needs a layer of Cytop to insulate the water droplet was placed between the two water droplet from the conductive material. slides, which flattened the droplet and After the ITO slide is coated, it is placed on reduced the contact angle. The top of the PDMS gasket, leaving the wire- electrowetting was then conducted using the connected electrodes uncovered. An edge of same procedure as the single-plate the ITO slide was also left uncovered, and electrowetting without the top slide. then the layers were secured with tape.

Fig. 9. Voltage applied to single plate device Fig. 8. Parallel Plate Device with ITO to perform electrowetting coated top slide. 3.3 Parallel Plate Electrowetting 3.2 Single Plate Electrowetting Parallel plate electrowetting is a process In order to test the effectiveness of used to split droplets. This is useful for the electrodes, single-plate electrowetting microfluidic immunoassays because the

6 beads have to separate from the sample droplets in order to go through the channel to the next well. The parallel plate device consists of an electrode slide, a PDMS gasket to support the top slide, and an ITO slide that covers a water droplet. In order to test this technique, 3 wire leads were needed. The ground lead was attached with an alligator clip to the ITO top slide. The water droplet was placed in the center of the electrode plate, and the two voltage leads Fig. 10. Manipulations of magnetic beads in were touched to each adjacent electrode. a water droplet The droplet will be pulled towards each positively charged electrode, and with 4. Results sufficient elongation, the surface tension 4.1 Single Plate Electrowetting will break and the droplet will split in half. The 2 µL droplet was too small to

bridge the electrodes used, so it could not 3.4 Electrowetting and Magnetic Beads move. The 4 µL droplet was the best volume Testing Magnetic Bead Manipulations for this experiment. It was big enough to A solution for testing the effectiveness of bridge the electrode plates and small enough the microbeads was made with 4 parts water to move quickly with the applied voltage. and 1 part bead solution. The small vial The 6 µL droplet was also big enough to containing the solution was first placed in a bridge the plates, but since it had a larger vortex mixer to combine the beads with the volume, it took more energy to move it water solution, and to excite the microbeads. easily. The deionized water and the PBS A 4 µl droplet of the solution was tested on worked equally well. For the voltages tested, a single plate that was elevated, and a the higher voltages yielded more movement magnet was placed under the plate so that it from the droplets, but they also raised the could move and affect the magnetic beads. risk of inducing hydrolysis as the Cytop A 10 lb. pull strength magnet and a 5 lb. pull coating failed and the water droplets came strength magnet were both used. Next, the into direct contact with the electrodes. A magnets were tested in conjunction with voltage of 40 V was too small to induce any single-plate electrowetting. A 4 µL droplet movement from the droplets because it did of the bead solution was placed on an not reduce the contact angle enough. A elevated electrode slide, and a magnet was voltage of 100 V was too large, as it created used to aggregate the beads on one side of a lot of movement, but hydrolysis occurred the droplet. At the same time, the procedure within a very short time after testing began. for single-plate electrowetting was used to Both 60 V and 80 V worked well, providing pull the droplet in the opposite direction, enough voltage to reduce the contact angle with the goal of separating the magnetic and move the droplet while not inducing beads and the water droplet. almost immediate hydrolysis. The non-conductive top plate helped reduce the contact angle between the droplet and the slide by flattening the droplet. It was able to actuate the water droplet with lower

7 voltages since the contact angle at zero volts was also lower.

Fig. 12. Hydrolysis of a water droplet on the parallel plate device

Fig. 11. Electrowetting 4 µL droplet on a The second device used two single plate electrode slides lying on top of each other with the electrodes aligned. On the top plate, 4.2 Parallel Plate Electrowetting ground was applied on the center electrode, Parallel plate electrowetting did not and on the bottom slide, voltage was applied work successfully, as the water droplets on the second and fourth electrode. This would not split. The electrodes used for device also failed; the two normal electrode testing were known to work, as they were slides on top of each other did not induce used for single plate electrowetting; hydrolysis, but they did not move the however, the ITO slides used for the top droplets either. 40 V had no effect on the conductive plate did not bind effectively droplet, and at 80 V the droplet only with the Cytop, causing hydrolysis and twitched. The area of conductivity on the top failure. plate was too narrow to provide enough A few slides were tested to ground, so the droplet could not be split. determine the effectiveness of parallel plate The devices were first tested with a 2 electrowetting. The first slide was tested mm thick PDMS gasket and then switched using a 4 µL droplet at 60V and 80V. At 60 to a thinner 500 µm gasket. The 500 µm V and 80 V, elongation was induced, gasket, which lowered the contact angle of showing that the level of voltage applied the droplet, induced more movement. At 40 was proportional to the amount of droplet V, neither device induced significant droplet movement. In both cases, though, the deformation. At 60 V, only the device with droplet was still not split. Also, at both the 500 µm gasket caused any deformation. voltages, the ITO slides failed. The Cytop At 80 V, both devices induced deformation was not bonded effectively to the ITO, and but the device with the 500 µm gasket had as it peeled off, it exposed the conductive significantly more movement. Neither ITO slide to the water droplets, which device was able to deform the droplets caused hydrolysis. None of the baking enough to split without inducing hydrolysis, procedures tested had any effect on the but the 500 µm gasket was superior to the 2 ability of the Cytop to bind to the ITO. The mm one. hydrolysis not only evaporated the water droplet but also destroyed the slides. Only 4.3 Magnetic Bead Manipulations voltages of 40 V or below prevented The 10 lb. pull strength magnet was hydrolysis, but they were not able to induce effective in attracting the magnetic splitting either. microbeads to one side of the droplet, but it was too strong and caused the beads to

8 magnetize. This caused clumping as the droplet could not be split or moved. At low beads attracted each other, and this voltages, there was not sufficient elongation, interfered with further movement. The 5 lb. and at high voltages, the droplet elongated pull strength magnet performed better, as it but hydrolyzed as well. Hydrolysis occurs was strong enough to attract the magnetic when the droplets come in direct contact microbeads, and it was weak enough to with a conductive layer. The electricity prevent the beads from congregating, causes the water to split into hydrogen and allowing the microbeads to disperse again oxygen gas. The hydrolysis was caused by a after the magnet was removed from the poor ITO-Cytop bond. The Cytop is the vicinity. This is important because in an protective barrier between the droplet and immunoassay, the beads must have the the conductive ITO, so when it fails, the highest surface area possible to interact with water and the ITO can touch. This not only the antigens. ruins the droplet, but it also destroys the ITO While coupled with the slide, and it cannot be used in further testing. electrowetting, however, the magnets were In the future, a better bonding agent not successful in splitting from the droplets. such as hexamethyldisilazane, HMDS, could The magnets were able to move the beads be used in order to improve the bonding within the droplet, and the voltage leads between the Cytop and ITO so hydrolysis were able to move the droplet, but the beads does not interrupt the splitting process. It were not able to break through the solution was clear that the ITO slide had potential to and separate from the water. split the droplets effectively, because at 60 V, the droplet elongated significantly. If the 5. Conclusions Cytop had not failed, the voltage could have Microfluidic immunoassays show been increased and eventually, splitting potential as fast and efficient alternatives to would have likely occurred. traditional immunoassays. Unfortunately, The use of single plate the magnetic microbeads used in such electrowetting in conjunction with the models are too expensive. This experiment magnetic bead manipulations did not work. tested a combination of the electrowetting The lowered concentration of the technique with the magnetic beads to reduce microbeads within the water was too low to the number of beads required. break the surface tension of the droplet. In The single plate electrowetting addition, the single plate electrowetting was worked well. It was able to actuate the incapable of splitting the droplet. In the droplets consistently, and it showed that the future, once a better top slide is fabricated, electrode plates worked. The procedure used parallel plate electrowetting should be is effective for both moving and merging combined with the magnetic bead droplets. Also, the addition of the manipulations. The parallel plate reduces the nonconductive top plate further increased contact angle of the droplet, and the the success. Since electrowetting is able to grounded top slide is able to help elongate actuate a droplet by lowering its contact the droplet to the point where it will split. angle, beginning with a flattened droplet is When combined with the magnetic beads, beneficial. This means that lower voltages this procedure should be able to effectively can be used to produce the same effect, separate the beads from the rest of the fluid making it a better procedure. sample. The parallel plate electrowetting Another future test could use a procedure did not work as expected. The stronger magnet to move the beads more

9 quickly. To counteract the effect of a very experiment and Rutgers University for strong magnet, a surfactant could be added hosting the Governor’s School of to the microbead solution to prevent the Engineering and Technology (GSET). beads from being permanently magnetized, Finally, we thank all of the sponsors who which causes clumping. In order to be useful support the program, including Rutgers in a microfluidic immunoassay, the beads University, the State of New Jersey, Morgan need to remain dispersed to maximize the Stanley, Lockheed Martin, Silver Line surface area exposed to the antigens. Windows and Doors, South Jersey Another way to potentially reduce Industries, The Provident Bank Foundation, the cost of the microfluidics immunoassay Novo Nordisk, and the GSET alumni would be to use FTO (fluorine-doped tin community. oxide) instead of ITO. FTO is also a conductive substance that can be spread onto References a glass slide, but it is less expensive, and this 1Sasso, Lawrence A., Kiana Aran, Yulong would be beneficial in making the test more Guan, Akif Ündar, and Jeffrey D. Zahn. economically feasible. "Continuous Monitoring of Inflammation Although this experiment as a whole Biomarkers During Simulated was unable to combine electrowetting and Cardiopulmonary Bypass Using a magnetic bead manipulations effectively, Microfluidic Immunoassay Device-A Pilot individual tests showed potential for future Study." Artificial Organs 37.1 (2013): E9- successes. Electrowetting is a conceivable E17. Web. supplement to current microfluidic devices that will reduce the cost of the microfluidic 2Casquillas, Guilhem V. "Microfluidics immunoassay and make it economically and Microfluidic Devices: A Review." feasible. Elveflow. N.p., n.d. Web. 19 July 2014.

Acknowledgements 3M. Shikida, K. Takayanagi, K. Inouchi, H. We gratefully acknowledge the Honda, K. Sato, Sensors and Actuators B assistance of Eamon Collins, our mentor and 113 (2006) 563–569 Residential Teaching Associate (RTA), whose knowledge about microfluidics 4"Streptavidin Coated COMPEL™ provided the basis of our report. We would Magnetic (CM01N/11374)." Bangs also like to thank Dr. Jeffrey Zahn, our Laboratories, Inc. N.p., 2014. Web. 20 July mentor and the principal investigator of the 2014. Rutgers BioMEMS and Microfluidics Laboratory, for providing a wealth of 5Vorvick, Linda. "ELISA: MedlinePlus resources and knowledge in support of our Medical Encyclopedia." U.S National project. We also extend our gratitude to all Library of Medicine. U.S. National Library of the other RTAs who guided us through of Medicine, 14 Oct. 2012. Web. 05 July the program, Ilene Rosen, the director of 2014. GSET, Jean Patrick Antoine, the assistant director of GSET, and the Governor’s 6Campell, M. "ELISA (Enzyme-Linked School Board of Overseers for organizing ImmunoSorbant Assay)." ELISA Method. the program. Additionally, we thank the Davidson College, 2002. Web. 05 July 2014. Rutgers Department of Engineering for providing a lab and resources to conduct the

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