Paper discs in a 3D printed microplate hybrid microfluidic device for low-cost, rapid, and ultrasensitive paper-based bioluminescence detection of human epidermal growth factor receptor 2 (HER2) breast cancer biomarker

Breast cancer is the most common cancer type in women and it has the highest probability of developing into invasive cancer. Early detection of breast cancer is crucial to reduce the disease burden and decrease the mortality rate. Detection of cancer biomarkers is an attractive non-invasive way to implement early diagnosis and follow-up. Colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most common techniques used for the detection of cancer biomarkers. However, it requires a long incubation time and a large reagent volume, and it has low sensitivity. Here, we propose use of a paper disc in a 3D printed microplate hybrid microfluidic device for ultrasensitive paper-based bioluminescence ELISA for detection of HER2 breast cancer biomarker. Chromatographic paper discs are good substrates for fast immobilization of capture antibody without making any surface modification and they can be replaced with new discs to reuse the 3D printed microplate. The 3D printed microplate has microvalves in the bottom of the wells, so it can stop flow of the reagents for the desired incubation time and it allows the washing solution to flow vertically and drain onto an adsorption pad which increases the washing efficiency. NanoLuc luciferase was used as a label for the detection antibody to achieve the highest sensitivity. Bioluminescence sandwich ELISA for HER2 detection was performed using the hybrid device in just 20 min and the limit of detection was 1.3 fg/mL which is more than 10⁴-fold better than commercial ELISA kits for HER2.