Our protocol describes a high-resolution micromilling method to fabricate an acrylic microfluidic device for quantitative immunodetection of analyte concentrations comparable to the gold standard ELISA technique. The highlight is the fabrication of a simple and low-cost thermoplastic microfluidic device without a clean room, which allows for shorter assay times and good detection limits in a quantitative manner. Begin by surface grinding.
Cut 9 x 25 millimeter rectangles of 1.3 millimeter thick PMMA with the 800 micrometer end mill bit. Attach one of these rectangles carefully with double-sided adhesive tape to the piezoelectric platform. Connect and place the Z-sensor on the surface of the PMMA rectangle.
Select the detection pin and move it over the sensor surface. Lower the pin manually without contacting the sensor. Activate the Z-zero sensing mode.
Spin the 200 micrometer end mill bit at 14, 500 rpm. Slowly lower it to the origin coordinate on the z-axis. Then reset the z-axis 30 micrometers below the origin.
Set this coordinate as the new Z-origin. Click on the Cut button in the micromilling machine software to activate the cut panel. Click on the Add button and select the TXT file with a previously created code for the grinding of the acrylic surface.
Click on the Output button to start the process. For milling of the five micrometer restriction, first, set the speed of rotation of the end mill bit to 11, 000 rpm. Then, raise the platform by 6.5 micrometers with the interface of the piezoelectric platform.
Move the end mill bit along the y-axis by 500 micrometers. Return the piezoelectric platform to its initial value on the z-axis with the control interface. Next, perform milling of microchannels by opening the previously created design file from the design software.
Click on the Print button, access the properties menu and click on the color window corresponding to the layer containing the design to be machined. Set the fabrication parameters in the tool panel. For milling of holes, switch to the 800 micrometer end mill bit.
Activate the design layer of the 1.2 millimeter diameter holes by clicking on the corresponding color window and selecting the corresponding fabrication parameters. Machine two additional holes on contralateral corners of the rectangle for the alignment of the acrylic in an inverted manner on a new platform. Flip the acrylic and tape it with double-sided adhesive tape over the adapter with the machined pillars.
Open the file with the design of the holes for the opposite face from the design software. Mill the remaining half of the reagent inlet and outlet holes with a diameter of 1.5 millimeters and a depth of 0.7 millimeters. Clean both acrylic rectangle sheets with isopropyl alcohol and rinse with distilled water.
Immerse the acrylic in an ultrasonic bath for 10 minutes. Dry both acrylic sheets and tape them to the inside of a glass Petri dish lid with double-sided tape. Then place the base of the glass Petri dish inside a bigger glass Petri dish.
Pour one milliliter of chloroform into the base of the Petri dish and quickly place the lid with the acrylic sheets. Immediately add distilled water to the base of the bigger Petri dish up to the level of the Petri dish lid. Allow exposure of the acrylic to chloroform gas for one minute.
Then tilt the Petri dish to break the water seal and immediately uncover the Petri dish. For bonding, align both acrylics with the chloroform exposed sides face-to-face and form a sandwich. Place the acrylics in the press for two intervals of two minutes, changing the alignment of the acrylic.
Then attach two to three centimeters of hose to each of the holes of the device with instant drying liquid adhesive. 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 at 7.5 micrometers in 5%BSA. Incubate the chip and the microparticle suspension with the blocking solution for at least one 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 five 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 seven 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 six 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 six minutes at one frame per second.
Click on the Start button of the wash flow immediately after the selected measurement flow stops. Lysozyme conjugated nanoparticles and horse radish peroxidase conjugated secondary antibody were used for the immunoassay. An increase in fluorescence intensity for different concentrations of primary antibody was observed comparing regions before and after the trap, showing that the change in substrate fluorescence is directly proportional to the concentration of primary antibody.
For a given concentration of primary antibody, the fluorescence intensity was plotted as a function of time at different flow rates of the fluorogenic substrate. The conversion capacity of the substrate by the HRP enzyme was inversely proportional to the flow rate, a maximum intensity was obtained for a flow rate of one microliter per hour. For the different flow rates for various primary antibody concentrations, curves of the fluorescence differences after and before immunoreaction showed that for a concentration of 1, 000 nanograms per milliliter, the fluorescence saturates for all the evaluated flow rates.
A calibration curve was prepared using the maximum value of the differences in fluorescence intensity obtained with respect to the concentration of primary antibody for each flow rate. The high variability and high fluorescence levels at one microliter per hour suggested that the rate does not favor the flow of the reacting substrate and tends to accumulate just after the trap. Special attention should be paid to the sealing steps of the microchannels since exposure to chloroform is very sensitive to temperature.
For reproducible results, the temperature must be always the same. Our system helps to understand where compactness and size of microparticles, nanoparticle size, antigens, detection antibody and substrate are determining factors for the limit of detection in nanoparticle-based microfluidic immunoassay.
