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Combining Electrowetting and Magnetic Bead Manipulations for Use in Microfluidic Immunoassays

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Combining Electrowetting and Magnetic Bead Manipulations for Use in Microfluidic Immunoassays 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.
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