Hfb24004

Multiwalled carbon nanotubes (MWCNTs, SN2302, purity>95%) were purchased from Nanomaterial Store (Fremont, CA, https://www.nanomaterialstore.com/), FeCl₂·4H₂O (purity>99%) was purchased from Acros Organics BVBA (Geel, Belgium, http://www.acros.com/), FeCl₃·6H₂O, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), 2-(4-Morpholino) ethanesulfonic acid (MES), streptavidin, sucrose, Tween 20, bovine serum albumin (BSA), Hexadecyltrimethylammonium bromide (CTAB) and phosphate buffer saline (0.01 M, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com/). Glass fibers (GFCP000800), cellulose fiber (CFSP001700), nitrocellulose membranes (HF090MC100, HFB18004 and HFB24004) and laminated cards (HF000MC100) were purchased from Millipore (Billerica, MA, http://www.merckmillipore.com/). CA 19-9 protein, Mouse monoclonal CA 19-9 antibody 10-CA19A (Ab₁) and 10-CA19B (Ab₂) were purchased from Fitzgerald Industries International (Acton, MA, https://www.fitzgerald-fii.com/). Goat anti-mouse IgG (Ab₃) were purchased from ThermoFisher Scientific (Rockford, IL, https://www.thermofisher.com/).
All the chemicals used in this study were analytical reagent grade. Solutions were prepared with ultrapure (Z18 MΩ) water from Millipore Milli-Q water purification system (Billerica, MA, http://www.merckmillipore.com).

A schematic description of the NALFA is presented in Fig. 1. The strip is about 6.0 cm long; its development involved a backing card (used to hold all the pads), a glass fiber conjugate pad (1.0 × 30 cm, GFCP203000, Millipore), a cellulose absorbent pad (CFSP223000), a nitrocellulose membrane (Millipore HFB 24004) containing a capture and a control region and a cellulose absorbent pad facilitating liquid flow. To prepare the capture and control probes, 10 μl of 1 nM biotinylated primers was added onto 38.3 μl of streptavidin-PBS solution and 41.7 μl of PBS. The mixture was incubated for 1 hour at room temperature and then 10 μl of ethanol was added for a final concentration of 100 μM for both the capture and control probes. Both capture and control probe solutions were manually applied to the nitrocellulose membrane in the capture and control regions, respectively. Using a micropipette, the AuNP conjugates were manually added to the conjugate pad and then the strips were incubated at −20 °C for 15 min before being stored in a dry place at room temperature.