ELISA chip fabrication and preparation
The chips were designed in AutoCAD (Autodesk), exported as “STL” files, and printed with a stereolithography 3D printer with the LED wavelength of 405 nm (Pr 110, Creative CADworks, Concord, Canada) using a monocure 3D rapid clear resin (Monocure 3D, NSW, Australia) with the following printing parameters: exposure time per layer: 2.5 s (10 s for the base layer); transition buffer layers: 2; layer thickness: 20 µm; printing delay: 1 min; and gap adjustment: 0.1 mm. Once printed, the chips were cleaned with isopropanol (Fisher Scientific, Saint-Laurent, Quebec, Canada), dried under a stream of pressurized nitrogen gas, cured for 1 min in a UV lamp (CureZone; Creative CADWorks; Concord; Canada), plasma treated for 10 sec at 100% power (PE50 plasma chamber, Plasma Etch, Carson City, USA), and sealed with a microfluidic diagnostic tape (catalog number: 9795R; 3M Science. Applied to Life.™, Ontario, Canada).
A strip of Whatman CF4 paper (Cytiva, Marlborough, Massachusetts, United States) was clamped between 2 absorbent pads (Electrophoresis and Blotting Paper, Grade 320, Ahlstrom-Munksjo Chromatography, Helsinki, Finland) from the back end to collectively serve as the capillary pump. For the main capillary pump, a strip of Vivid™ 120 lateral flow nitrocellulose membrane (Catalog number: VIV1202503R; Pall Corporation, Port Washington, USA) was clamped between the same absorbent pads from the back end and to a G041 glass fiber conjugate pad (Millipore Sigma, Oakville, Ontario, Canada) from the front end to facilitate connection to the chip.
The strips of vivid™ 120 lateral flow nitrocellulose membranes were designed in AutoCAD with the dimensions of 5.2 mm wide and 12 mm long and cut using the Silhouette Portrait paper cutter (Silhouette, Lindon, USA). Membranes were stripped with a 5 mm-wide test line of SARS-CoV-2 N protein mouse monoclonal antibody (catalog number: 40143-MM08; Sino Biological, Inc., Beijing, China) at the concentration of 1 mg/mL and a 5 mm-wide control line of bovine serum albumin (BSA)-biotin solution at the concentration of 50 µg/mL, both delivered using a programmable inkjet spotter (sciFLEXARRAYER SX, Scienion, Berlin, Germany). The membranes were dried for 1 h at 37 °C and blocked by dipping into the blocking buffer solution (1% BSA and 0.1% Tween 20 in PBS) until completely wet, followed by shaking on a rocker for 60 min at 75 rpm. The membranes were then retrieved, dried in an oven for 1 h at 37 °C, and stored with a desiccant at 4 °C until use on the next day.
SARS-CoV-2 N protein ELISA
The sample solutions were prepared by spiking SARS-CoV-2 N protein (catalog number: 40588-V08B; Sino Biological, Inc., Beijing, China) at the concentrations of 0, 1, 5, 10, 50, 102, 103 104 105, 106 pg/mL in either the ELISA buffer solution (0.1% BSA and 0.05% Tween 20 in PBS) or 4×-diluted pooled saliva solution. Fresh saliva specimens were collected using oral cotton swabs (Salivette, Sarstedt, Numbrecht, Germany), pooled, filtered through a 0.22-micron filter, and diluted by a factor of 4 in the ELISA buffer solution. The biotinylated SARS-CoV-2 N protein rabbit monoclonal antibody (catalog number: 40143-R004-B; Sino Biological, Inc.; Beijing, China) and streptavidin poly-HRP (Pierce; catalog number: 21140; ThermoFisher; Ottawa, Canada) solutions were prepared in the ELISA buffer solution both with the concentration of 7.5 µg/mL. The substrate solution was prepared by dissolving SIGMAFAST™ DAB tablets (catalog number: D4293-50SET; Sigma-Aldrich; Oakville, Canada) in Mili-Q water. The washing buffer solution was the same as the ELISA buffer solution.
For benchmarking, the developed SARS-CoV-2 N protein assay was compared with the SARS-CoV-2 (2019-nCoV) Nucleocapsid Detection ELISA Kit (catalog number: KIT40588; Sino Biological, Inc.; Beijing, China) and the RayBio® COVID-19 / SARS-COV-2 Nucleocapsid Protein ELISA Kit (catalog number: ELV-COVID19N; RayBiotech Life, Inc.; Peachtree Corners, United States).
Nitrocellulose membranes image analysis and LOD calculation
After completion of the ELISA, the nitrocellulose strips were removed from the ELISA chip, left to dry at room temperature, and scanned at 1200 dpi in TIFF format (Epson Perfection V600) (see Supplementary Fig. S4). The images were imported in Photoshop (Version: CS5 ME) and superposed with guide structures to locate the region of interest (2.5 × 0.4 mm) for the test line as well as the bottom background and top background, each located 1.5 mm below and above the test line respectively. The superposed images were then imported in Fiji to measure the gray value of the three regions of interest for each nitrocellulose membrane (see Supplementary Fig. S5). For each concentration and the negative control, the local signal intensity of the test line was calculated by subtracting the gray value of the test line from the average gray value of the top and bottom local backgrounds. The relative signal intensity was then calculated by subtracting the local signal intensity of the test line from the average of the local signal intensity of the negative controls.
The experimental data were fitted using a 4-parameter logistic regression with the following equation:35