A multistep immunoassay on a 3D-printed capillarity-driven microfluidic device for point-of-care diagnostics

Although efficient diagnostic testing often relies on centralized laboratories, rapid diagnostics at the point-of-care (POC) are increasingly investigated to provide an affordable and easy-to-use alternative that requires minimal external equipment. Implementing complex assays on such POC devices is challenging, and existing methods such as paper-based analytical devices suffer from laborious and heterogeneous fabrication. 3D printing is a promising technology to enable the single-step fabrication of such diagnostic devices. We previously reported monolithic, 3D-printed microfluidics driven by capillarity, and used the platform to implement a proof-of-concept enzyme-linked immunosorbent assay (ELISA) for immunoglobulin E (IgE) detection. In this study, we aimed to improve the time-to-result and detection limit of this approach by (i) designing a second-generation 3D-printed device and (ii) further optimizing the IgE ELISA. Herein, we selected horseradish peroxidase for faster and more sensitive signal amplification compared to the first-generation device, for which we adapted the microfluidic device design to allow sequential flow while using separated reagent channels for each assay step. To implement the ELISA on this new design, we decreased reagent volumes and incubation times to be compatible with POC, and then we translated this assay stepwise from microtiter plates to the completely 3D-printed, second-generation device. Overall, this work gives additional insight into the biocompatibility of these 3D-printed devices, as well as demonstrates the huge potential of powder bed 3D printing to fabricate POC diagnostic devices.