A Three-Dimensional Origami Paper-Based Device for Potentiometric Biosensing

Materials
2-Nitrophenyl octyl ether (o-NPOE), heptakis(2,3,6-tri-o-methyl)-β-cyclodextrin
(β-CD), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), poly
(methyl methacrylate-co-butyl methacrylate), high molecular weight poly(vinyl
chloride) (PVC) were purchased from Fluka AG (Buchs, Switzerland).
Butyrylthiocholine iodide was obtained from Aladdin (Shanghai, China). Bovine
serum albumin (BSA), butyrylcholine chloride and 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB) were obtained from Sigma. Butyrylcholinesterase (BuChE, 5.64 U mg-1) was purchased from Sangon (Shanghai, China). Methyl parathion (99%) was obtained
from Best Reagent (Chengdu, China). Carbon and Ag/AgCl inks (dispersed in vinisol)
were purchase from Bohui New Materials Tech. Co. Ltd (Jiangsu, China). All other
reagents used were purchased from Sinopharm Chemical Reagent and of analytical
grade purity or better. Freshly deionized water (18.2 MΩ cm specific resistance) was obtained with a Pall Cascada laboratory water system.

Membrane preparation
The butyrylcholine selective membranes contained 1.0 wt% NaTFPB, 1.0 wt% β-CD,
65.0 wt% o-NPOE and 33.0 wt% PVC.1 200 mg of the membrane components were
dissolved in 1.0 mL of THF. The reference electrode membrane cocktail was prepared
as described before with some modifications. 200 mg of the copolymer, 68.0 mg of
KCl, and 5.0 mg of ETH 500 were dispersed in 1.0 mL of THF.2 Each deposited diskmembrane had a diameter of 4 mm. ETH 500, as a kind of ionic liquid, was used to decrease the membrane resistance.

The stability of the reference membrane electrode was tested against a
commercial double-junction silver/silver chloride (3 M KCl) electrode, and an amount of 20 μL reference membrane cocktail was coated onto a silver/silver chloride ink screen-printed electrode on a ceramic substrate.

Chip fabrication
The fabrication process was performed following by our previously described
procedures.3 Briefly, a XEROX Phaser 8560DN wax inkjet printer purchased from
Fuji XEROX Company was used to pattern wax-based solid ink on the Whatman
chromatography paper (Whatman chromatography paper, GE) with highest
resolution 2400 dpi. The chip design was implemented using drawing software
(Adobe Illustrator). After printing, the paper was kept in an oven at 150 °C for 30 second to let the wax to penetrate through the paper compley to form hydrophobicwax barriers and then cooled to 20 °C. The static water contact angle was measured at room temperature by a contact angle system JGW-360B (Chengde, China).Measurements were repeated on at least three drops.

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微流控實驗濾膜選擇上海必泰生物科技有限公司銷售經營的whatman的3001-861尺寸是20cm*20cm