A Paper-Based Immunoassay for the Detection of Immunoglobulin G

1. Grafting Amine Functional Groups on Cellulose Paper Discs

1. Prepare one piece of square paper with a dimension of 1 cm × 1 cm, and 100 paper discs made from grade No. 1 cellulose paper with a diameter of 6.0 mm (medium-flow filter paper) using a hole punch.
2. To derive -NH₂ groups on the paper discs, mix 1 ml 3-aminopropyltrimethoxysilane (APS) and 10 ml acetone in a 50 ml glass bottle in the fume hood. Add paper discs to the freshly prepared APS reagent mixture, and incubate for 5 hr with orbital stirring (200 rpm) at room temperature. 
   CAUTION: Handle APS and acetone in the fume hood.
3. Decant excess solution from the 50 ml glass bottle into an organic waste container.
4. Add 10 ml of acetone to the glass bottle, mix well and decant completely to remove any unreacted APS and other impurities. Repeat this step two times.
5. Spread the paper discs on the paper towel and place in a 110 °C oven for 3 hr. Allow the paper discs to cool. Store the discs in a 50 ml centrifuge tube at room temperature.
6. Use Fourier transform infrared spectroscopy (FTIR) to check the grafting of amine groups on the cellulose square paper, as described below (Figure 1A).
   1. Turn on the computer and open the FTIR spectroscopy instrument.
   2. Open the software for FTIR spectroscopy.
   3. Go to 'Measurement → Initialize'. The rectangles for 'BS: KBr', 'Lamp: Infrared' and 'Laser' will turn green when the initialization is finished.
   4. Choose 'Data' below the rectangles, and select '%Transmittance', 'Happ-Genzel', '45', '4.0', and 'Min: 400, Max: 4000' for 'Measurement Mode', 'Apodization', 'No. of Scans', 'Resolution', and 'Range (cm-1)'.
   5. Click 'Measure'.
   6. Select 'Data file' for the background data. Write down the comments.
   7. Click 'BKG' to get the baseline for the background.
   8. Fix the square paper on the film sample holder.
   9. Select 'Data file' for the sample data. Write down the comments.
   10. Click 'Sample' to obtain the spectra for the sample.
   11. Close the FTIR spectroscopy application and turn off the computer.
7. Repeat the above steps (steps 1.1 to 1.6) to prepare amine-functionalized grade No. 113 cellulose square paper and discs (fast-flow filter paper), and obtain the FTIR spectra for the grade No. 113 square paper (Figure 1B).

2. Paper-based enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) Detection

1. Add 5 ml of 0.05% glutaraldehyde solution (prepared in 50 mM PBS buffer, pH 7.4) to a 20 ml glass bottle. Immerse 15 amine-functionalized medium-flow filter paper discs in this solution and keep for 1 hr with shaking at room temperature.
   1. Concurrently, repeat Step 2.1 to prepare another 15 aldehyde functionalized fast-flow filter paper discs.
      CAUTION: Handle glutaraldehyde in the fume hood.
2. To remove unreacted glutaraldehyde from the paper discs, place the 15 medium-flow filter paper discs in a 15 ml centrifuge tube, and the 15 fast-flow filter paper discs in another 15 ml centrifuge tube. Add 5 ml of DI water to each tube and shake the tubes for 10 sec. Remove the water by aspirating with a pipette. Repeat two times to remove any unreacted glutaraldehyde.
3. Dry the paper discs in a 37 °C oven.
4. Add 5 µl and 8 µl of 0.025 mg/ml mouse IgG-Fc fragment antibodies to each of the medium-flow and fast-flow filter paper discs, respectively, and incubate for 20 min.
5. Add 10 µl of 50 mM PBS (pH 7.4) to each paper disc without removing the antibodies and incubate for 40 min for the amine aldehyde reaction.
6. Wash the paper discs with 0.2 ml of washing buffer on top of a paper towel. Repeat the wash three times.
7. Dry the paper discs in an oven at 37 °C.
8. Block the paper discs with 15 µl of blocking buffer for 10 min at room temperature.
9. Wash each paper disc with 0.2 ml of washing buffer on top of a paper towel. Repeat the wash three times.
10. Run IgG standards.
    1. Load 10 µl of various IgG concentrations (e.g., 0, 10, 125, 250, and 500 ng/ml in PBS) onto each disc in triplicate. Incubate for 1 hr at room temperature.
11. Wash the paper discs with 0.2 ml of washing buffer on top of a paper towel. Repeat the wash three times.
12. Load 10 µl of HRP conjugated mouse IgG-Fc fragment antibodies (1:10,000, 10 mM PBS, pH 7.4), and incubate for 1 hr at room temperature.
13. Wash the paper discs with 0.2 ml of washing buffer on top of a paper towel. Repeat the wash three times.      
    NOTE: It is not necessary to remove the washing buffer as the results are not affected by the presence of buffer.
14. Load a 10 µl mixture of TMB and hydrogen peroxide onto each disc.
15. Take images of all paper discs with a digital camera or smart phone after 5 min of incubation.
    NOTE: In Figure 2A, '0' stands for paper discs treated with capture antibody immobilization, and the antibody-HRP/TMB solution without IgG serum.
16. Analyze the intensity of each paper disc in the image by Image J.
    1. Convert the images taken in step 3.15 to '.tif' format.
    2. Open 'Image J' software.
    3. Go to 'File → Open', choose the image to analyze.
    4. Choose the shape button 'Oval'.
    5. Go to 'Image → Type → 32 bit'.
    6. Go to 'Edit → Invert'.
    7. Go to 'Analyze → Measure'.
    8. Copy and analyze the data in a spreadsheet.

Figure 1. Fourier transform infrared (FTIR) spectra of untreated and APS-treated medium-flow filter square paper (A) and fast-flow filter square paper (B). A. The spectra for untreated medium-flow filter square paper was similar to that of APS treated medium-flow filter square paper. The increase in intensities at bands of 902-1,170 cm^-1 and 1,210-1,500 cm^-1 for the APS-treated square paper belonged to Si-O-cellulose and C-H deformations of SiOC-H groups, respectively. The increment of the bands at 1,650 cm^-1 and 2,885 cm^-1 belongs to the bending of -NH₂ and CH₂ vibrations from the saline propyl moiety, respectively. B. The characteristic peaks in the 972 to 1,180 cm^-1 range were attributed to the Si-O-Si and S-O-cellulose bonds. The peak at 1,003 cm^-1 was caused by the overlap of the Si-O-Si bond and the C-O stretching of cellulose. All of the results show that APS was successfully grafted onto the cellulose square papers.

Figure 2. Calibration curves for the determination of IgG by paper-based ELISA. #1 represents grade No. 1, medium-flow filter paper disc, and #113 represents grade No. 113 fast-flow filter paper disc. The upper panel A presents the color readout for medium-flow (#1) and fast-flow (#113) cellulose paper discs with different concentration of IgG. The bottom panel B presents the ELISA result for different concentration of IgG. Triplicates were used to determine the standard deviation (SD).