We established a simple imaging rig to systematically determine the performance of a range of digital cameras for imaging colorimetric and fluorometric assays within microfluidic devices. To allow us to control the microfluidic device characteristics and directly compare camera performances, we filled microcapillary films with colorimetric and fluorescent dye solutions. Di-amino Phenazine (DAP) is the yellow dye product produced by the horseradish peroxidase enzyme, commonly used in colorimetric immunoassays, acting upon the o-Phenylenediamine dihydrochloride (OPD) substrate often used in colorimetric immunoassays. Fluorescein is used both directly for fluorescent detection and as the product of alkaline phosphatase conversion of the substrate fluorescein di-phosphate (FDP), and is spectrally similar to other alkaline phosphatase substrates such as Attophos™ (Promega). Following this systematic comparison using dyes, selected cameras were used to image full immunoassays that simulated the measurement of antibodies against important viral antigens, to confirm the findings could be applied to clinically relevant diagnostics bioassays.
For the detection of fluorescent and colorimetric signals, we used a wide range of cameras and phones, and professional imaging systems. We used a DSLR camera EOS 1300D with a Canon EF-S 60mm f/2.8 Macro USM Lens (Canon), a compact camera Powershot S120 (Canon), a compact waterproof and shock-resistant WG4 camera (Ricoh) and a toy camera (Sakar). We used smartphones including iPhones 6S and 4S (Apple) and Alba SIM Free 5'' Android and mobile phones such as CAT B30 Phone (Caterpillar) and Alcatel 2008G phone (Alcatel) purchased from Argos. The industrial machine vision camera USB 3 uEye® XC with a Macro Lens (AE00126; IDS Imaging Development Systems, Obersulm, Germany), a camera module v1 powered by Rasperry Pi 3 model B+ W, an Epson Perfection V600 Photo scanner and the laboratory imaging systems G:BOX (Syngene) and Typhoon (Amersham) were also used to detect fluorescent and colorimetric signals.
To improve the image quality and resolution of some imaging systems by allowing closer focusing, three type of lenses were used: an injection-molded plastic magnifying lens, a smartphone clip-on macro lens (Amazon UK) and an industrial machine vision macro lens (IDS). These were held directly onto the front of the digital camera lens during imaging.
For the colorimetric signal, the images were taken under a white light. For the fluorescent signal, the images were taken using a LED Transilluminator (IO Rodeo) to provide blue light excitation in a dark room with the amber emission filter held between the camera and the MCF.