Development of a flow-free magnetic actuation platform for an automated microfluidic ELISA

There is a need to create an easily deployable and point-of-care (POC) diagnostic platform for disease outbreaks and for monitoring and maintenance of chronic illnesses. Such platforms are useful in regions where access to clinical laboratories may be limited or constrained using cost-effective solutions to quickly process high numbers of samples. Using oil and water liquid–liquid interphase separation, immunoassays developed for microfluidic chips can potentially meet this need when leveraged with electromagnetic actuation and antibody-coated superparamagnetic beads. We have developed a microfluidic immunoassay detection platform, which enables assay automation and maintains successful liquid containment for future use in the field. The assay was studied through a series of magnetic and fluid simulations to demonstrate these optimizations, and an optimized chip was tested using a target model for HIV-1, the p24 capsid antigen. The use of minimal reagents further lowers the cost of each assay and lowers the required sample volume for testing (<50 μL), that can offer easy turnaround for sample collection and assay results. The developed microfluidic immunoassay platform can be easily scaled for multiplex or multi-panel specific testing at the POC.

Circuit Schematic and System Diagram S1: System diagram of the magnetic actuator; To facilitate rapid chip development, the system was designed as a collection of modules with well-defined interfaces.

S2:
Circuit schematic for the actuator hardware Platform Control Diagram S3: Diagram for actuator control over serial. The Arduino begins in S0 and waits for a serial connection or manual input. If a connection is present, the algorithm waits for a newlineterminated command, dispatches the command to the hardware, verifies that the command has been executed, and signals to the driver that it is ready for the next command.

Magnetic Force Simulations
Supplemental Equation 1 is the conventional expression for the force experienced by a magnetic particle in an applied magnetic field.
(1) F is the force exerted by the field on the particle (newton), V is the particle volume (m^3), X v is the dimensionless magnetic volume susceptibility, μ 0 = 4π * 10^-7, and B is the magnetic flux density (tesla).
X v was obtained from the magnetization curve provided by the nanoparticle manufacturer using a standard method. V was directly specified by the manufacturer.
Supplemental equation 1 was implemented in COMSOL using the partial differential equation (PDE) module. A stationary simulation was performed on a model of the permanent neodymium magnet (K & J Magnetics) using COMSOL's magnetic-fields-no-current interface. The results were used with the PDE module to obtain the partial spatial derivatives of the convective operation in Equation 1. Results were exported and post-processed in Python to obtain the final force results for the beads.

Fluid Simulations
The pressure difference at the interface of two immiscible fluids can be found using Supplemental equation 2 when there is no fluid flow. Since our microfluidic chips do not use any flow and our simulations run until steady-state conditions are achieved, Equation 2 is an accurate description of the pressure differences on either side of the oil-water interface.
(2) -= ∇ ⋅ p and are the absolute pressure values on either side of the interface, σ is the surface tension ĉ oefficient, and is the curvature, where n is the unit normal vector to the interface and ∇ is ∇ ⋅ used as shorthand for the divergence operation.
We computed the pressure fields and steady-state interface locations for our microfluidic chip using the level set method in COMSOL. We used the laminar flow interface and set the initial conditions to the well loading volumes used for the assay.

Direct anti-p24 ELISA: p24 antigen Target Validation
Using a recombinant p24 antigen protein (Abcam), a series of seven (7) two-fold dilutions were used respectively in Rows A-H: 20 micrograms/mL, 10 micrograms/mL, 5 micrograms/mL, 2.5 micrograms/mL, 1.25 micrograms/mL, 0.625 micrograms/mL, 0.3125 micrograms/mL. Each antigen target was diluted in a carbonate buffer (pH 10) (Fisher Scientific) then loaded into the 96-well plate. The plate was covered with parafilm wax and left to incubate overnight at 4 degrees C. The plate was washed with Phosphate-Buffered Saline (PBS) (pH 7.0) 3 times and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. The plate was then washed with PBS (pH 7.0) three (3) times, and tapped on between each rinse to empty the wells completely. The plate 30 had 100 microliters of blocking solution (Thermofisher) added to each well, and the plate was covered with parafilm wax, and left to incubate overnight at 4 degrees C. The plate was washed with PBS (pH 7.0) four (4) times, and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. The anti-p24 conjugate (Abcam) was diluted to 0.2 micrograms/mL (1:5000), and 100 microliters was added to each well, and incubated at room temperature for 1 hour. The plate was washed with PBS (pH 7.0) three (3) times, and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. The plate was developed with the TMB substrate (Thermofisher) with 100 microliters to each well, and stopped 100 microliters with 2M H2SO4 after five minutes to each well. The plate was read by spectrophotometer at 450 nm (SpectraMax) S5: The Anti-p24 Direct ELISA;

Indirect anti-p24 ELISA: anti-p24 Capture Antibody Validation
Using the same recombinant p24 antigen protein (Abcam), a series of seven (7) two-fold dilutions were used respectively in Rows A-H: 20 micrograms/mL, 10 micrograms/mL, 5 micrograms/mL, 2.5 micrograms/mL, 1.25 micrograms/mL, 0.625 micrograms/mL, 0.3125 micrograms/mL, Blank. Each antigen target was diluted in a carbonate buffer (pH 10) (Fisher Scientific) then loaded into the 96-well plate. The plate was covered with parafilm wax and left to incubate overnight at 4 degrees C. The plate was washed with Phosphate-Buffered Saline (PBS) (pH 7.0) 3 times and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. The plate was then washed with PBS (pH 7.0) three (3) times, and tapped on between each rinse to empty the wells completely. The plate had 100 microliters of blocking solution (Thermofisher) added to each well, and the plate was covered with parafilm wax, and left to incubate overnight at 4degreesC. The plate was washed with PBS (pH 7.0) four (4) times, and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. The anti-p24 antibody (Abcam) was diluted to 0.2 micrograms/mL (1:5000), and 100 microliters was added to each well, and incubated at room temperature for 1 hour. The plate was washed with PBS (pH 7.0) three (3) times, and tapped on Kim Wipes (Fisher Science) between each rinse to empty the wells completely. 100 microliters of diluted anti-mouse conjugate (Abcam) (1:1000) to each well, and incubated for 1 hour. The plate was washed 4 times with a permanent magnet using PBS-T and discarded the supernatant. The plate was developed with the TMB substrate (Thermofisher) with 100 microliters to each well, and stopped 100 microliters with 2M H2SO4 after five minutes to each well. The plate was read by spectrophotometer at 450 nm (SpectraMax) S6: The Anti-p24 Indirect ELISA;

Anti-p24 Western Blot for Antibody Validation
The recombinant p24 antigen protein (Abcam) was used to make serial dilutions of 0.25 micrograms/microliter, 0.125 micrograms/microliter and 0.0625 micrograms/microliter. Each antigen was diluted of PBS (pH 7.4) (Fisher Scientific) then lysed with the lysis buffer. After vortexing for 30 seconds it was placed on the heater for 5 minutes at 95 degrees C. Then it was centrifuged at 13000 rpm for 10 mins and then the samples are ready to load. The glass plates were then set up on the rack of the electrophorator (Amersham Biosciences) following by preparing 13% running gel using ddH2O (10.4ml), 40% acrylamide (Thermofishcer) 8.1 ml, 1.5 M Tris (pH 8.8) 6.25 ml, 10 % SDS 250 microliter, 10% ammonium persulfate 250 microliter and TEMED (Thermofisher) 40 microliter. After pouring the running gel on the glass plate 2 ml of Butanol (Thermofisher) was added to make the top layer of gel straight. Once the TEMED was added, the gel starts polymerizing and after 25 mins the top layer was rinsed with water and make dried using blot paper. The comb was set up and stacking gel was poured on top of the running gel. The stacking gel was prepared using ddH2O (3.13 ml), 40% acrylamide (Thermofisher) 0.625 ml, 1.5 M Tris (pH 6.8) 125 ml, 10% ammonium persulfate 50 microliter and TEMED (Thermofisher) 12-15 microliter.