Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

The overall goal of this protocol is to demonstrate our process for fabricating three-dimensional paper-based devices, which we use as a platform to develop point of care immunoassays. This method provides a paper-based microfluidics platform with a reliable manufacturing process, such that time and effort can be directed to developing assays instead of designing devices. The main advantage of our technique is that it allows many devices to be prepared in parallel and at a quantity that is desirable for academic research projects.

Generally, new users will struggle with this method because there are many variables to consider when fabricating devices, like proper alignment of layers. Mistakes can results in devices that malfunction. A visual demonstration of this method is critical because assembly can be difficult to envision using only the details provided in manuscripts.

First, prepare layers of qualitative filter paper by cutting a stock sheet of paper into a standard paper size to facilitate patterning using a wax printer. Load a cut sheet of paper into the printer tray. Then print previously designed layers.

Next, cut a stock row of nylon membrane into sheets using a tabletop paper cutter, taking great care in handling the nylon membrane to maintain its integrity and protect against ripping. Store any unused material in a desiccator cabinet, as nylon membranes are moisture sensitive. Using a wax printer, print a capture layer pattern onto a piece of copy paper, and tape it to a light box to serve as a guide for positioning of the nylon membrane.

Following this, place a clean sheet of copy paper onto the previously printed sheet of copy paper. Tape the clean sheet of paper to the light box but do not tape the two sheets together. Now, place a cut sheet of nylon membrane onto the clean sheet of copy paper, making sure that the membrane covers the printed area of the bottom layer of copy paper.

Tape all four sides of the nylon membrane to the clean sheet of copy paper. Load a sheet of nylon membrane supported by the copy paper affixed to it into the manual feed printer tray. Then print one sheet of nylon membrane at a time.

Tape the printed layers onto an acrylic frame for even heating above and below the layer when placed in a gravity convection oven. Now, place the layers in the oven at 150 degrees Celsius for 30 seconds until the wax melts into the thickness of the paper. After removing the paper from the oven, confirm that the wax has permeated the thickness of the paper by turning it over and checking for imperfections in the design.

Remove the paper and nylon membrane from the acrylic frame. Then remove the nylon membrane from the support sheet of copy paper using a paper cutter. Pattern double-sided sheets of adhesive films using a robotic knife plotter with previously prepared design files.

Following this, tape a pattern layer of paper that needs to be backed with adhesive onto a light box with the printed side down. Peel one side of protective liner from the pattern sheet of adhesive. Press the pattern sheet of adhesive and layer of paper together.

Then place the partially assembled device into a protective slip. Next, pass the resulting two layer assembly through an automated laminator to completely press the adhesive and paper together, removing any pockets of air from the adjoined layers. Tape a conjugate layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.

Add 2.5 microliters of BSA and one X PBS to the hydrophilic zone on the conjugate layer. After allowing the sample to dry at room temperature for two minutes, dry it at 65 degrees Celsius for five minutes. Following this, add five microliters of five OD colloidal gold nanoparticle conjugated to anti-beta hCG antibody, then repeat the drying process.

Tape a lateral channel layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame. Add 10 microliters of blocking agent to treat the lateral channel, then repeat the same drying process used for the conjugate layer. Next, tape a capture layer onto an acrylic frame such that the hydrophilic zone to be treated is suspended and not in contact with the frame.

Treat the capture layer with five microliters of anti-alpha hCG antibody. After allowing the sample to dry at room temperature for two minutes, dry it at 65 degrees Celsius for eight minutes. Add two microliters of blocking agent, then repeat the same drying process used for the capture layer.

Tape the wash layer to the light box with the printed side facing upwards. If alignment holes are used, remove them from subsequent layers using a handheld hole punch tool. Remove the protective film on the back of the capture layer to expose the adhesive.

Align the capture layer above the wash layer using the alignment holes as a guide, then press the two layers together and avoid touching the hydrophilic zones to minimize contamination or damage to the device. Following this, remove the protective film on the back of the incubation layer to expose the adhesive. Align the incubation layer above the capture layer and press them together.

Continue adding layers in this manner until all active layers are assembled. Now, place the partially assembled device into a protective slip and firmly affix the layers together using a laminator. Remove the protective film on the back of the wash layer and affix the blot layer to the bottom of the device.

After laminating to complete the assembly of the three-dimensional paper-based microfluidic device, cut the desired number of devices from sheets of fully assembled devices using scissors. Add 20 microliters of a positive control sample of hCG buffer to the hydrophilic zone on top of the device. Once the sample has wicked completely into the device, add 15 microliters of wash buffer.

After the first aliquot of wash buffer has wicked completely into the device, add a second 15 microliter aliquot of wash buffer. To reveal the results of the assay, peel away the three top layers of the device using tweezers to expose the capture layer. The wax printing method can be used to form hydrophobic barriers within paper-based microfluidic devices, and produces fluidic pathways with reproducible dimensions, which is critical for assays with repeatable performances and duration times.

The performance of the hCG paper-based immunoassay was demonstrated by performing 35 positive and 35 negative assays in parallel. The coefficient of variation for each dataset was determined to be 1%for assays performed using negative samples and 3%for assays performed using positive samples. A misalignment between layers comprising the incubation channel and capture zone can cause the development of an irregular pattern in the positive signal, which may result in a misinterpretation of the qualitative signal.

If the wax is not printed in a sufficient amount or not allowed to melt completely through the thickness of the paper, then the integrity of the resulting hydrophobic barriers may be compromised and lead to leaks within the device. Assays that take longer than expected to complete may indicate a malfunction in the fabrication of a device. Paper-based microfluidic devices provide researchers with a versatile platform to develop low cost, point of care analytical tests.

While a number of applications exist, the approach we demonstrate here results in a general device architecture to perform immunoassays, which are critical in healthcare. While attempting this procedure, it's important to remember to check for imperfections during the fabrication process and before treating the layers with expensive biochemical reagents. Once mastered, this method, from printing layers to assembling treated layers, can be used to prepare a sheet of functional immunoassays in two hours.

After watching this video, you should have a good understanding of how to fabricate three-dimensional paper-based devices, and be comfortable enough to tailor this platform to other assay types of interest.