No Cellular Contamination in Serum Samples

Supplementary Information

Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles

5 Chihchen Chena*, Bo-Ren Lina, Hsi-Kai Wanga, Shu-Ting Fana, Min-Yen Hsub, and Chao-Min Chenga*

a Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan. Fax: +886-3-574-5454; Tel:+886-3-516-2403; 10E-mail: chihchen @ mx.nthu.edu.tw; [email protected] b Taichung Veterans General Hospital, Taichung 407, Taiwan.

Characterization of Surface Modification Protocols We used fluorescence-based readouts to assess the effectiveness of surface modification methods. For modifying paper devices using the chemical conjugation approach, paper devices were first treated with 15oxygen plasma (100 mW, 1% oxygen, 30 s) in a PX-250 plasma chamber (March Instruments, Concord, MA), and then immediately incubated in a 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol for 30 min, followed by an incubation of 0.01 mol/mL N--maleimidobutyryloxy succinimide ester (GMBS) in ethanol for 15 min. Next, paper devices were incubated with 10 μg/mL NeutrAvidin solution in PBS for 1 h at 4ºC. Lastly, 10 L of PBS containing 1% (w/v) BSA was added to circular test zones of 5 mm in diameter 20patterned in filter paper for 3  10 min before 10 L 20 g/mL R-phycoerythrin conjugated biotin (PE-biotin, Invitrogen, Camarillo, CA) in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide was spotted on the paper test zone to react with NeutrAvidin for 3  10 min. Unbound PE-biotin molecules were rinsed off using PBS buffer. All steps were performed at room temperature unless otherwise specified. 10 L 20 g/mLPE-biotin in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide was directly 25spotted on the paper test zone for 3  10 min, followed by PBS rinse, in the physisorption protocol. Modified paper devices (“crosslinker-dye”) along with unprocessed paper devices (“pristine paper”), paper devices modified with the same protocol without the application of PE-biotin molecules (“crosslinker”) and paper devices with physisorbed PE-biotin molecules (“physisorbed-dye”) were scanned using an image-record system (GeneSys G:BOX Chemi-XX8, J&H Technology Co.; set to 540-560 nm excitation, 575-LP emission), 30and the data was saved in the form of 16-bit files and analyzed using imageJ software. Protocols for each test condition were summarized in Table S1.

Table S1. Surface modification protocols

step vol. time pristine paper crosslinker crosslinker- physicsorbed- dye dye oxygen plasma - 30 s - + + - 3-mercaptopropyl trimethoxysilane 10 L 30 min - + + - ethanol rinse 10 L 2 min3 - + + - GMBS 10 L 15 min - + + - ethanol rinse 10 L 2 min3 - + + - NeutrAvidin 10 L 60 min - + + - PBS rinse 10 L 2 min3 - + + + PE-biotin 10 L 10 min3 - - + + PBS rinse 10 L 2 min3 + - + +

1 Fig. S1. Fluorescently labeled biotin molecules are immobilized onto filter paper through chemical conjugation process. Fluorescence intensity profiles of paper devices with different surface modification protocols were 52,470±112 for “pristine paper”, 3,214±431 for “crosslinker”, 21,093±5,955 for “crosslinker-dye”, and 3,087±180 for “physisorbed-dye”, respectively. N = 10 for each groups and protocols for each test condition were summarized in Table S1. ***p < 10-7 (Student’s t-test), indicating significant differences compared with the “crosslinker-dye” group. Insets show representative light and fluorescent scanned images of paper test zones.

2 Few Extracellular Vesicles Were Captured on the Paper Device Non-specifically Paper devices were coated with anti-CD63 antibodies, anexin V molecules, or PBS containing 1% (w/v) BSA (PBS/BSA) respectively using protocols described in Section 4.3. Paper devices coated with PBS/BSA were used as a control to assess the extent of non-specific capturing of extracellular vesicles to the paper devices. 72 5µL serum from a healthy donor was spotted onto each paper test zone followed with rinsing and fixation for SEM inspection. There were very few objects that could be found under SEM on paper devices without the coating of vesicle-capturing molecules (Fig. S2a, b). In contrast, many vesicles were observed on paper devices coated with anti-CD63 antibodies (Fig. S2c), or anexin V molecules (Fig. S2d), suggesting the capturing of vesicles was specific. Few environmental dusts and impurities that did not bear specific antigens 10seemed to retain on the paper device.

Fig. S2. Representative SEM images showing little non-specific capturing of extracellular vesicles in the serum sample to functionalized filter paper devices. 72 µL serum from a healthy donor was spotted onto each paper test 15zone. There were very few vesicle-like objects on paper devices coated with PBS containing 1% (w/v) BSA (a, b), while there were different appearing vesicle-like objects on paper devices coated with anti-CD63 antibodies (c), or annexin V molecules (d), suggesting the capturing of extracellular vesicles on the paper device was specific (scale bars: 400 nm).