A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Take a microfluidic device affixed to a microscope stage, comprising an inlet for dispensing reagents, interconnected channels for fluid flow, and outlets for purging the fluids.

The main channel contains a central staggered restriction with trapped aggregated iron microparticles.

The channels are incubated with a blocking solution to prevent non-specific antibody binding.

Flow a wash buffer, removing excess blocking solution.

Introduce an immunocomplex suspension at a defined flow rate. The immunocomplex consists of magnetic nanoparticles, coated with antigens bound to primary antibodies, which are further bound to peroxidase-conjugated secondary antibodies.

Apply an external magnetic field, magnetizing the aggregated microparticles and transforming them into a magnetic trap for capturing the immunocomplexes.

Introduce a fluorogenic substrate that is converted by the peroxidase into a fluorescent product and flows through the trap.

Using the microscope, compare the fluorescence before and after the trap, confirming the capture of immunocomplexes.

Fill the channels with distilled water using a syringe. Immerse the device in an ultrasonic bath for 10 minutes. Then, empty the water inside the device channels and use a syringe to introduce 5% BSA solution.

Prepare a suspension of iron microparticles of diameter 7.5 micrometers in 5% BSA. Incubate the chip and the microparticle suspension with the blocking solution for at least 1 hour at room temperature. Next, insert the microparticles into the chip with a syringe needle through the side channel outlet hose.

Place the chip vertically; then, rotate the chip in two steps of 90 degrees such that the microparticles target and compact at the 5-micrometer restriction. Seal all hoses of the acrylic device with heat. Cut the inlet hose until only a few millimeters are left.

Fill the dispensing needle with wash buffer and insert it into the cut hose. Allow the solution to drip, and then connect the needle to the device. Cut the outlet hose from the lateral channel, then connect to the syringe pump. Next, repeat the same procedure for the main channel outlet hose, then attach the chip to the magnet.

For immunodetection, keep the wash buffer flowing for 10 minutes at 50 microliters per hour. Remove the remaining wash buffer from the dispensing needle with a micropipette and add 50 microliters of the nanoparticle suspension. Flow the nanoparticle suspension for 7 minutes at a flow rate of 100 microliters per hour. Then, change the flow rate to 50 microliters per hour and continue the flow for another 15 minutes.

Change the dispensing needle, and flow the wash buffer for 10 minutes at the same rate. Remove the remaining wash buffer from the dispensing needle with a micropipette and add 100 microliters of the fluorogenic substrate. Adjust the flow rate and time parameters for substrate input, fluorescence measurement, and wash step.

Activate the input flow of the fluorogenic substrate for 6 minutes at 50 microliters per hour. 15 seconds before the substrate flow stops, turn on the fluorescence of the microscope. Start the image capture with the software of the microscope camera 10 seconds before the substrate stops with an exposure time of 1,000 milliseconds.

Click on the start button of the desired flow rate parameter immediately after the substrate wash stops. Perform imaging for 6 minutes at one frame per second. Click on the start button of the wash flow immediately after the selected measurement flow stops.