2.1 Materials and chemicals
Paper materials used as a platform device in this study were chromatography filter paper (CHR, Whatman, UK), and nitrocellulose membrane with laminated backing support (NC, FF180HP, Merck, USA). Gloss laminate pouch with 80-µm thickness (GBC, Australia) were used to make paper hydrophobic. For immunological assay, Rabbit monoclonal anti-human CD9 (Rabbit Anti-CD9, ab92726, Abcam, UK), Goat anti-rabbit IgG (Anti-rabbit IgG, ab6721, Abcam, UK), Mouse monoclonal Anti-human CD63 conjugated with horseradish peroxidase (Anti-CD63-HRP, NBP2-42225H, Novus, USA) were used for coating and detecting antibody. HRP-conjugation kit (ab102890, Abcam, UK) was used to conjugate HRP onto an antibody. For blocking paper-based assay, Bovine Serum Albumin (BSA, A1595, Sigma Aldrich, USA) was used to block the assay to minimize background signal due to non-specific binding. 3,3’,5,5’-tetramethylbenzidine substrate solution (TMB, 002023, Thermo Fisher Scientific, Germany) was used to employ colorimetric immunological assay, employed by HRP/TMB reaction. For general chemical usage, phosphate buffer saline solution (PBS) was prepared by dissolving phosphate buffer saline tablet (10388739, Fisher Scientific, USA) in deionized water (DI water, Milli-Q, Merck, USA). Tween-20 (655205, Merck, USA) as a surfactant was mixed into PBS for preparing PBST (0.05% Tween-20 in PBS).
For exosome preparation, SKOV3 (ovarian cancer cells, ATCC, USA), OVCAR3 (ovarian cancer cells, ATCC, USA), HEK293 (epithelial human cells, ATCC, USA) and Met5a (epithelial human cells, ATCC, USA) were used as a cell line to produce exosome. RPMI-1640 media (11875-093, Gibco, Thermo Fisher Scientific, Germany) served as the base medium. Foetal Bovine Serum (FBS, A3160901, Gibco, Thermo Fisher Scientific, Germany) and Exosome-depleted Foetal Bovine Serum (Exosome-free FBS, A25904DG, Gibco, Thermo Fisher Scientific, Germany) were used as a main composition for cell culture medium. To prevent bacterial and fungal contamination, 1% Penicillin and Streptomycin (PenStrep, 15140-122, Gibco, Thermo Fisher Scientific, Germany) was mixed into the cell culture medium. TrypLE (12604-021, Gibco, Thermo Fisher Scientific, Germany) was used for trypsinization process for subculturing. General buffer for washing cells was sterile HBSS (14175-095, Gibco, Thermo Fisher Scientific, Germany). After exosome isolation, exosome sample was purified by using Total Exosome Isolation Kits (4478359, Invitrogen, Thermo Fisher Scientific, Germany). MCF7 standard exosome (MCF7, ab239691, Abcam, UK) was used to validate the exosome detection assay.
2.2 Paper-based device fabrication
The designed pattern on paper was drawn by CorelDRAW software (CorelDRAW2019, Corel Corporation Inc, Canada), and then the paper was cut with laser cutting machine (Rayjet 50 Laser Engraver, Rayjet, Australia). In this study, we fabricated three types of paper devices for different experiments. First, NC without lamination, a paper platform which is NC only was directly cut by laser cutting machine. Second, with lamination device (Figure 1), CHR paper were cut with 5-mm diameter piece. A laminated film was cut with the hole of 4-mm diameter. After the paper was aligned into the centre of laminated film hole, the aligned component was fed into a laminator (JL330T, PFEC, Australia) at 130oC and at a speed of 10 mm/s. Finally, the paper device was cut into individual device.
2.3 Exosome sample preparation
SKOV3, OVCAR3, HEK293 and Met5a were cultured with RPMI-1640 containing 10% FBS and 1% PenStrep. After reaching 80% confluence, the media was changed to exosome-free media which is RPMI-1640 consisting of 10% exosome-depleted FBS. After cells were incubated with exosome-free media for 60 hours, the media was collected and centrifuged at 2,000 g for 30 min to remove dead cells and other debris. The exosome isolation kit was used to precipitate the exosome to increase the exosome concentration from the collected media. Before obtaining exosome pellet by centrifugation at 10,000 g for 1 hour, the mixture was incubated overnight. After the centrifugation process, the exosome pellet was acquired by discarding the supernatant, resuspended with PBS and kept in -80oC for long-term storage.
2.4 Colorimetric assay for exosome detection
In this study, we employed sandwich ELISA assay on a paper-based platform using TMB/HRP colorimetric reactions. Briefly, the primary antibody specific to tetraspanin family on exosome membrane e.g. CD9 or CD63 was firstly immobilized onto the paper. Then, the paper was blocked by BSA or NFM to minimize non-specific binding. Subsequently, exosome sample was loaded and incubated onto the paper to allow to be captured by primary antibody using affinity between primary antibody and specific protein on exosome membrane. Subsequently, the secondary antibody which is tagged with HRP was loaded to attach onto exosome membrane. Next, TMB substrate will be added onto the paper to employ TMB/HRP reaction. In the presence of HRP, TMB was oxidized by the HRP and generated blue colour from colourless solution. On the other hand, in the absence of HRP, TMB was inert and left solution to be colourless.
2.5 Equipment setup and data acquisition
As the experimental results are evaluated based on colour change from TMB/HRP reaction, the paper device was observed under 1.3 megapixels TOUPCAM camera (UCMOS01300KPA, Touptek, China) with 1/3” Aptina CMOS sensor attached with 6.2 mm focal lens (58428, Edmund Optics Inc, USA) for image acquisition (Figure 2) under LED strip box made from PMMA for light control. Camera setup was connected via USB and controlled by Toupview software (Version 4.8, Touptek, China). Image processing was quantified with programming code in computing software (MATLAB R2018b, The MatkWorks Inc, USA)
2.6 Experimental design and assay optimization
2.6.1 Blocking efficiency test
Most colorimetric assays involve with enzyme incubation, so blocking and washing are important steps to prevent non-specific binding. We evaluated the blocking capability by coating different BSA concentrations. A volume of 5 µL of blocking solution was added to incubate for 10 min and subsequently dry for 10 min. Next, we dropped Anti-CD63-HRP for 3 minutes onto the paper and washed it subsequently. Finally, we incubated TMB substrate and observed colour change over time. It is anticipated that the best condition for blocking could result in the lowest intensity of blue colour change. The first configuration is to incubate NC paper without lamination into different BSA concentrations from 1–10% for 30 min in dipping configuration. In this case, we used only NC paper because we take advantage of high protein binding of NC materials. Moreover, we also evaluated the blocking capability by dropping and incubating BSA onto laminated CHR paper with wash-through configuration.
After getting optimal blocking condition, we investigated antibody binding capability. We dropped 5 µL of 0.1 mg/mL rabbit anti-human CD9 on a laminated paper and incubate for 10 minutes, then dry at 37oC for 10 minutes. Next, coated antibody laminated paper was blocked with BSA. In the presence of rabbit anti-human CD9, we expected that after loading goat anti-rabbit IgG conjugated with HRP, TMB could change from colourless to blue colour due to in the presence of rabbit anti-human CD9 on paper. However, In the absence of rabbit anti-human CD9, TMB could remain colourless as there is no binding of goat anti-rabbit IgG conjugated with HRP.
2.6.2 Analytical performance assay
After obtaining optimal condition for blocking and paper type for the paper-based device, we carried out the analytical performance assay. Assay preparation including antibody concentration and operation procedure were adapted from the previous study (Figure 3a).  We dropped 5 µL of 0.1 mg/mL rabbit anti-human CD9 on a laminated paper and incubate for 10 min, then drird at 37oC for 10 minutes. Next, we dropped 5 µL of 2% BSA on the laminated paper. Subsequently, we incubated exosome sample on the paper for 10 minutes. In the absence of exosomes in the sample, PBS was used as control. Subsequently, we loaded 3 µL of 1 µg/mL Anti-CD63-HRP for 3 min. Finally, we added TMB substrate and placed under the camera and captured photos at 1st, 3rd, 5th, 10th, 15th, and 20th minute.