The overall goal of this protocol is to fabricate an analytic device from an easily-sourced material, cotton, to use for your analysis. The main advantage of this technique is that the device is easy to fabricate. It's very low-cost, can be used for multiple assays, and is quick to assemble.
The analytical method with the device is sensitive to detecting sample and reaction contamination. Demonstrating the procedure will be Shang-Chi Lin, who was instrumental in innovating the method. Begin with preparing the test strip devices.
First, determine the hydrophobicity of the cleansing cotton for the test strips using a standard contact angle measurement with droplets of deionized water. Then, start fabricating the cotton-based analytical device by cutting cleansing cotton into 5.5 centimeter by one centimeter pieces. Use a paper cutter.
The next step is to drill sets of three 0.5-centimeter diameter holes in a sheet of lamination film. Drill the holes in rows with one centimeter between the holes. Each set of holes makes a test strip, and up to 36 strips can be fit on a standard sheet of lamination film.
Next, sandwich the cleansing cotton between the hole-drilled sheet of lamination film and an un-drilled sheet of lamination film. Then, use a standard laminator to package the device. Now, cut individual 5.5 by one centimeter test strip devices from the assembly.
To make standard curves of three test solutions, 48 strips are needed. On each test area, use a sharp blade to make a small channel from the center of each test area to the hydrophilic layer of the longitudinal device. Be careful to only cut halfway through the device.
Now, insert half-centimeter diameter chromatography paper discs into each of the three holes. The lamination film should have a small lip under which the pad edges can be tucked. For a urinalysis strip, coat the nitrite assay pads with nitrite indicator solution.
Coat the protein assay pad with BSA indicator solution. And coat the urobilinogen assay pad with urobilinogen indicator. Allow the strips to dry completely at ambient temperatures before proceeding.
For each standard prepared, dip the absorptive end of a prepared device into the standard solution for ten seconds. Each strip should soak up 200 to 300 microliters of solution. Prepare three test strips for each standard concentration being tested.
Wait ten minutes for the colorimetric reaction to complete at room temperature. Now, scan the devices to analyze each test pad's color. Use a desktop scanner in RGB mode and at 600 dpi.
Analyze the scanned image using standard software such as ImageJ. After opening the scanned image files, select the image in the menu commands and select Type to convert RGB images to 8-bit grayscale. Then, use the oval tool in the toolbar to select the test pad analytical area.
Next, select Analyze in the menu commands, and select Measure to analyze color intensity. From the mean intensity results, make the standard curves for the assays. In this case, fit the standard curve for the nitrite assay using a quadratic model, and fit the standard curves for total protein and urobilinogen using linear regression models.
On the hydrophobic interface of the fabricated device, the contact angle at the exterior cotton was measured 127 degrees, give or take 5 degrees. Using the devices, standard curves were made to establish the limits of detections for each assay. For nitrites, the limit of detection was 0.147 millimolar.
For bovine serum albumin, the protein test solution, the limit of detection was 3.7 micromolar. For urobilinogen, the limit of detection was 4.9 milligrams per liter. After watching this video, you should have a good understanding of how to fabricate cotton diagnostic devices with the hydrophobicity of the external layer that helps avoid contamination.
Once mastered, this technique can be done in one or two hours, if it is performed properly. While attempting this procedure, it's important to determine the hydrophobicity of the external layer of the cleansing cotton by contact angle measurement.
