The overall goal of this procedure is to improve the detection sensitivity of ELISA using natural spun nanofibers. This video demonstrates how to achieve femtomolar detection sensitivity of proteins in 10 microliter of whole blood samples within 30 minutes. In a lab-on-a-disc platform, the centrifugal force is used to transfer the liquid through chambers, and the total process is fully automated.

The detection sensitivity could be highly enhanced by the integration of nanofibers. However, the problem was that the nanofiber is very fragile and difficult to handle. In this video, we introduce a simple process to attach fragile titanium dioxide nanofiber mats on a plastic disc.

To fabricate titanium dioxide nanofiber mats on silicone substrate by electrospinning, the silicone substrate is first silanized, coated with PDMS, and precured. A nanofiber mat is then transferred to this precured PDMS and this product is punched to produce a circular piece for attachment. Coat the reaction chamber with a drop of PDMS as an adhesive, and attach the circular piece to the bottom of the chamber.

Then, cure the PDMS in the oven. Finally, assemble the disc layers into one complete device for ELISA. This nanofiber transfer technique allows us to utilize the high surface area of nanofibers for bioanalysis.

The centrifugal microphylitic device offers the full integration and automation of all the process required in ELISA. The main advantage of this technique over other existing methods is that it could detect femtomolar concentrations of proteins from only 10 microliter of whole blood. Prepare the precursor solutions as described in the accompanying test protocol.

Then load the solution in the electrospinner and fabricate the nanofibers by electrospinning, using the optimal conditions. When the polymer nanofiber mats are prepared, calcinate them in a furnace under vacuum. After calcination, the titanium dioxide nanofiber mats are finally prepared.

Prepare the microphylitic layout by using 3D designing software such as SOLIDWORKS, and transfer the design to the CNC milling machine. Operate the CNC milling machine to fabricate the disc with channels and chambers. The disc and nanofiber mat is now ready to be integrated.

Prepare a vacuum chamber, silicone substrate and fluorosilane. Silanize the silicone substrate with fluorosilane under vacuum for 30 minutes. Prepare the guest PDMS by preparing prepolymer with a curing agent in a 10 to one ratio, and spin coat onto the silanized silicone substrate at 650 RPM for 60 seconds.

Cure the PDMS coated on the silanized wafer at 65 degrees Celsius for 10 minutes to make it adhesive. Now transfer those nanofiber mats onto the PDMS-coated silicone by applying appropriate pressure using a flat object such as a glass slide. Bake the sample at 80 degrees Celsius for one hour to completely cure the adhesive PDMS.

Dispense a few drops of ethanol on the nanofiber mats attached on the PDMS layer, and make a six millimeter hole in diameter using a PunchIt. Then peel of the punched nanofiber mats attached on the PDMS from the silicone substrate. Next, coat the binding reaction chamber in the disc with about 20 microliters of PDMS mixture, and place the nanofibers attached on the PDMS into the chambers.

Then, incubate the nanofiber mat integrated disc at 80 degrees Celsius for one hour. Prepare 1%volume per volume solution of GPDES in ethanol. Then, treat the nanofibers in the disc with oxygen plasma.

Dispense 100 microliters of GPDES solution on each nanofiber mat, and incubate at room temperature for two hours in a sealed container. After the incubation, remove the GPDES solution by tapping the disc on a wiper. Wash with ethanol, remove, and bake at 80 degrees Celsius for one hour.

After baking, wash with ethanol and tap to remove. Repeat this process and blow dry with a nitrogen stream. For antibody immobilization, dispense diluted captured antibody solution.

Keep the discs in a humidified chamber, and incubate at 37 degrees for four hours. Wash off the remaining solution with washing buffer. Now, assemble the disc using a double-sided adhesive tape.

Apply reasonable pressure to assemble the layers into a complete disc. Fill the binding reaction chambers with blocking buffer and incubate at 37 degrees Celsius for one hour. Wash twice with washing buffer and store the disc at 40 degrees Celsius until use.

Load the reagents, washing buffers and 10 microliter of whole blood in the reagent and sample chambers. Place the disc in a custom built visualization machine for automated immunoassay. The visualization system contains a rotor connected to a computer for automated disc operation together with a strobe light and a CCD camera for taking images.

Spin the disc at 3, 600 RPM for 60 seconds to separate the red blood cells. Mix plasma with the detection antibodies. Transfer the mixture to the reaction chamber integrated with titanium dioxide nanofiber mat.

After immunoreaction, wash the nanofibers by transferring the washing buffer to the reaction chamber. Then, transfer the chemiluminescent substrate solution to the binding reaction chamber, and incubate in a mixing mode for one minute. Finally, transfer the reacted substrate solution to the detection chamber.

When all the reaction is finished, transfer the disc to a detection system and read the chemiluminescent signal by using custom-built software. The disc operation and the detection system can be integrated in a single platform. By optimizing voltage and the fluorate, uniform nanofiber mats were obtained as shown in this SEM image.

In order to optimize the PDMS curing time, the adhesion force was measured. If it was too short, the nanofiber mat was embedded in PDMS, or peeled off if too long. After optimization, the nanofiber mat was then stably attached on the PDMS.

The linear range for C-reactive protein detection from two platforms is shown in this graph. Using the nanofiber integrity lab on the disc, we were able to achieve highly enhanced detection sensitivity of C-reactive protein from only 10 microliters of serum within 30 minutes. Currently available technologies, such as 96-well plate-based ELISA require a larger sample volume and a longer detection time.

Overall, nanofiber integrated disc-based ELISA showed a broad, dynamic range of six orders of magnitude with a detection limit of six femtomole of C-reactive protein. This detection sensitivity aided by high surface area provided by nanofiber integrated in the disc exceeded that of the conventional ELISA, which was limited at 2, 000 femtomoles. After watching this video, we hope you have a good understanding of how to prepare centrifugal microphylitic device, how to integrate phasal nanofibers, and how to perform onto protein detection using ELISA.

This technique can be useful to detect low bonded proteins in a very small amount of biological samples.