Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

Podsumowanie

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

Streszczenie

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

Wprowadzenie

The point-of-care testing (POCT) diagnostic study is important for the development of new strategies for therapeutics, personalized medicine, and home care¹. Cellulose papers are widely used as platforms in immunoassays, as they are cheap, accessible, and familiar to users². In addition, the porous structure of cellulose paper possesses the power to drive liquid flow without additional energy impact. Records of paper-based bioanalysis can be found as early as the 20^th century, when paper chromatography was first invented in 1952. The most prevalent example is immunochromatographic tests³, such as pregnancy and diabetes test strips. These tests provide relatively fast assay times and inexpensive analysis⁴. Due to their simplicity, these conventional paper strip tests have been widely used in POCT diagnostics⁵.

Detection methods including colorimetric⁶, electrochemical⁷, and electrochemiluminescence⁸ methods have been reported to measure targets in biological samples. In addition to these quantitative methods, a reliable method for immobilizing antibodies on cellulose paper is also important for the development of diagnostic devices. Non-specific adsorption is the main strategy for modifying antibodies on the surface of the paper-based devices^{9, 10} to ensure maximum binding capacity to their targets after immobilization. However, a previous study showed that antibodies that are adsorbed onto cellulose paper can desorb from the fibers¹¹ by 40%. Thus, direct adsorption of antibodies onto cellulose may not provide reproducible results¹². Covalent immobilization of antibodies that are grafted on the paper surfaces is an alternative method of developing effective paper-based bioassays¹³. Various methods have been reported for the modification of cellulose^{14, 15}. Ideally, antibodies should maintain their original functionality after immobilization¹². Carbonyldiimidazole combined with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate¹⁶; 1-fluoro-2-nitro-4-azidobenzene through a UV-based activation strategy^{17, 18}; a chemoenzymatic strategy based on xyloglucan modification¹⁹; a 1,4-phenylenediisothiocyanate linking agent²⁰; heteropolysaccharide oxidation²¹ click chemistry²²; and cationic porphyrins²³ have been used to covalently immobilize biomolecules on cellulose paper. Chitosan modified paper has been used to develop paper based immunodevices^24-26 since it is abundant and biocompatible²⁷. Chitosan is cationic and adheres strongly to anionic cellulose²⁷. The capture antibodies are immobilized on the paper through chitosan coating and glutaraldehyde cross-linking. Periodate oxidation is another method for grafting the capture antibodies on the cellulose paper²⁸. In this method, sodium periodate is spotted on the paper to convert 1,2-dihydroxyl (glycol) groups in cellulose directly to aldehyde groups. The aldehyde groups are then used to form covalent bonds between polysaccharides and antibodies²⁸. Although the fabrication is simple, it is difficult to completely wash out sodium periodate. The unwashed sodium periodate can cause further oxidation of the antibodies that are immobilized on the cellulose paper, affecting the activity and stability of the antibodies. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide cross linkers are also used to covalently immobilize antibodies on electrospun poly-L-lactic acid and cellulose acetate nanofibers for the development of nanofiber-based assays²⁹.

In this study, a silane coupling technique was used to graft amine functional groups on cellulose paper discs. This technique helps to retain the original pore size, wicking, and filtration rate of the cellulose filter papers, allowing maximum vertical flow-through in immunoassays. The silane coupling technique has been widely used in biosensors to functionalize substrate surfaces with secondary amine groups, followed by further modification using biomolecules. The grafting of amine groups on the matrix surface comprises a condensation reaction between -OH groups of the organofunctional silane agents and matrix substrate³⁰. The cellulose paper discs were functionalized with amine groups by silane coupling through 3-aminopropyltrimethoxysilane (APS)³¹. This was followed by covalently immobilizing capture antibodies using two different methods. The first method involved binding of periodate oxidized capture antibodies to the amine functionalized cellulose paper discs. The second method used glutaraldehyde as a cross-linking agent to attach the capture antibodies to the amine group-functionalized cellulose paper discs. The presence of capture antibodies was confirmed by rabbit anti-human IgG-fluorescein isothiocyanate (FITC), using a fluorescence molecular imager. The binding activity of rabbit anti-human IgG-FITC to goat anti-rabbit IgG was also evaluated by peroxidase substrate. The effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies were investigated. The application test of the immobilized capture antibody was successfully performed through the detection of IgG serum.

Protokół

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 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 temperature³².
   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 3A).
   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 3B).

2. Covalent Immobilization of Antibodies on Amine-functionalized Cellulose Paper Discs

Figure 1. Covalent immobilization of antibodies by two different methods. A. Antibodies immobilized on amine-functionalized cellulose paper discs through periodate oxidation. The carbohydrate residues were oxidized by sodium periodate to produce aldehyde functional groups. Then, the oxidized antibodies were loaded onto amine-functionalized cellulose paper discs. B. The antibodies were then immobilized on amine-functionalized cellulose paper discs through glutaraldehyde. The amine functionalized cellulose paper discs were immersed in 0.05% glutaraldehyde solution to introduce aldehyde groups to the paper discs. After washing, antibodies were loaded onto the aldehyde functionalized paper discs. Please click here to view a larger version of this figure.

1. Immobilize antibodies on amine-functionalized cellulose paper discs through periodate oxidation (Figure 1A).
   1. Mix 1 µl of 2.5 mM sodium periodate with 2 µl of 0.1 mg/ml rabbit anti-human IgG-FITC and 7 µl of 100 mM pH 5.5 acetate buffer in a 1.5 ml tube, and incubate the mixture in the dark for 30 min.
      NOTE: Follow this volume ratio to prepare more oxidized antibodies if necessary. Change the volume ratio to optimize the concentration of sodium periodate and rabbit anti-human IgG-FITC.
   2. Dilute the above antibody solution with 30 µl of 50 mM phosphate buffer saline (PBS; pH 7.4) to a final volume of 40 µl.
   3. Prepare three amine-functionalized medium-flow filter paper discs and three amine-functionalized fast-flow paper discs (as described in Section 1).
      1. Load 5 µl of the sodium periodate oxidized rabbit anti-human IgG-FITC onto each medium-flow filter paper disc and 8 µl onto each fast-flow filter paper disc. Keep these paper discs in the dark for one hour at room temperature.
   4. Wash each of the paper disc with 0.2 ml of washing buffer (50 mM Tris buffer with 0.15 M NaCl and 0.05% surfactant, pH 7.4). Repeat the wash three times.
   5. Photograph the fluorescence images through a fluorescence molecular imager to identify the presence of antibodies on each cellulose paper disc³². Use blank paper discs (treated with the same concentration of antibodies in the absence of sodium periodate) as a control (Figure S1 to Figure S3).
      NOTE: For experimental optimization, change the concentration of one parameter that needs to be optimized and fix the concentrations of all other parameters. A high fluorescence intensity increases the amount of capture antibodies that are immobilized on the cellulose paper discs.
2. Immobilize antibodies on amine functionalized cellulose paper discs through glutaraldehyde (Figure 1B).
   1. Add the three APS treated medium-flow filter paper discs and the three fast-flow filter paper discs (described in Section 1) to 2 ml of 50 mM PBS (pH 7.4) that contains 0.05% glutaraldehyde for 1 hr, with orbital stirring at room temperature.
      Caution: Handle glutaraldehyde in the fume hood.
   2. Place three discs each in two 1.5 ml centrifuge tubes. Add 1 ml of deionized (DI) water to each tube and shake the tubes for 10 sec. Remove the water by aspirating with a pipette. Repeat two more times to remove any unreacted glutaraldehyde.
   3. Load 5 µl of 25 µg/ml rabbit anti-human IgG-FITC (capture antibody) onto each aldehyde-functionalized medium-flow filter paper disc, and add 8 µl onto each aldehyde-functionalized fast-flow filter paper disc. Incubate in the dark for approximately 20 min at room temperature. Then, 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.
   4. Wash the paper discs with 0.2 ml of washing buffer on top of a paper towel. Repeat the wash two times.
   5. Photograph the fluorescence images through a fluorescence molecular imager to check for the presence of antibodies on each cellulose paper disc³³. Use blank paper discs as a control.
      NOTE: In Figure 4, '0' stands for the blank paper disc that was treated with the same concentration of FITC-antibody in the absence of glutaraldehyde; in Figure 5A, the blank paper discs were treated with glutaraldehyde, but no FITC-antibodies were loaded onto the paper discs.
3. Dry the paper discs (from Sections 2.1 and 2.2) at 37 °C for 10 min.
4. Block the paper discs with 15 µl of blocking buffer (10% skimmed milk powder in 50 mM Tris buffer, pH 7.4, with 0.15 M NaCl) for 10 min at room temperature.
5. Load 5 µl and 8 µl of peroxidase conjugated goat anti-rabbit IgG in PBS (1:10,000) onto medium-flow and fast-flow filter paper discs, respectively. Incubate for 30 min in the dark at room temperature.
6. 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 buffer.
7. Load a 10 µl mixture of 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide solution onto each disc.
8. Take images of the paper discs with a digital camera or smart phone after 5 min of incubation.
   NOTE: In Figure 5B, '0' stands for the paper discs that were treated with glutaraldehyde, followed by loading antibody-HRP (horse radish peroxidase) conjugate in the absence of FITC-antibody.

3. Paper-based ELISA for IgG Detection

Figure 2. Schematic representation of the paper-based ELISA for IgG detection. Capture antibodies were covalently immobilized on the aldehyde-functionalized cellulose paper discs through glutaraldehyde. The cellulose paper discs were blocked with blocking buffer. Target IgG was then added to the discs, followed by the loading of HRP-conjugated signal antibodies. Finally, the TMB and hydrogen peroxide mixture solution was loaded onto each paper disc for the color readout. Please click here to view a larger version of this figure.

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 3.1 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 6A, '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.

Wyniki

Figure 3. 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...

Dyskusje

Direct coating of affinity purified goat anti-Mouse IgG-Fc capture antibody on unmodified cellulose paper discs was performed to detect IgG concentrations. The results indicated that, further fixation of the capture antibodies is required for reproducibility. The silane technique was successfully used to introduce amine functional groups to the cellulose paper discs³⁴. The concentration of APS affects the immobilization of antibodies. Therefore, the amount of APS in acetone was also optimized. 1 ml of APS in 1...

Materiały

Name	Company	Catalog Number	Comments
Cellulose filter paper, Grade1 (medium flow filter paper)	GE Healthcare Pte Ltd Singapore	1001 110
Cellulose filter paper, Grade113 (Fast flow filter paper)	Sigma-Aldrich, Singapore	1113-320
3-​Aminopropyl​trimethoxysilane	Sigma-Aldrich, Singapore	T1255	>96%
Glutaraldehyde	Sigma-Aldrich, Singapore	G6257	Grade II, 25% in H2O
Surfactant	Tween-20, Sigma-Aldrich, Singapore	P2287
Bovine serum album	Sigma-Aldrich, Singapore	A2153
Skimmed milk powder	Louis François		Packed by Kitchen Capers, Singapore
Tris base	Promega	H5135
Sodium periodate	Merck	106597
Na2HPO4	Merck	106585
KH2PO4	Merck	104873
NaCl	CALBIOCHEM	567441
NaOH	Merck	106462
HCL	Merck	100317
phosphate buffer saline (PBS)	N/A	N/A	PBS, containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.0 mmol/L Na2HPO4 and 1.5 mmol/L KH2PO4, is prepared with water and adjusted to pH 7.4 with 0.1 mol/L NaOH or 0.1 mol/L HCl
Acetone	Tee Hai Chem Pte Ltd Singapore	9005-68
Mixture of TMB and hydrogen peroxide solution	1-Step ultra TMB-ELISA solution , Thermo Scientific Pierce	34029	1 L
Rabbit anti-human IgG-FITC	TWC/Bio Pte Ltd Singapore	sc-2278
Peroxidase conjugated goat anti-rabbit IgG	TWC/Bio Pte Ltd Singapore	sc-2030
Affinity purified goat anti-Mouse IgG-Fc coating antibody	Bethyl Laboratories, Inc	A90-131A
Mouse reference serum	Bethyl Laboratories, Inc	RS10-101-5	9.5 mg/mL
HRP conjugated goat anti-mouse IgG-Fc detection antibody	Bethyl Laboratories, Inc	A90-131P
Equipment
Fourier transform infrared spectrophotometer	Shimadzu IR Prestige-21	N/A
Fluorescence molecular imager	Pharos FXTM plus molecular imager, Bio-Rad, Singapore	N/A
Oven	NUVE FN500	N/A
Turbo mixer VM-2000	MYC LTD	N/A
Image J	RGB, free download	N/A