3D-printed autonomous microfluidic chip for multistep point-of-care bioassays

Here, we present a radically novel method for fabricating 3D passive microfluidics in one piece that is capable of accommodating multistep bioassays and actuating fluids within the channels without any external devices.Furthermore, this 3D printed (3DPed) platform allows on-chip pre-treatment of sample and the incorporation of semi-quantitative readout, next to being easily scalable and cost-effective.As such, it represents a very attractive concept for point-of-care (POC) testing in low resource settings that comply with the WHO guidelines to be affordable, sensitive, specific, user-friendly, rapid, equipment-free and delivered to those who need it (ASSURED) [1].POC testing is crucial for healthcare in developing countries, but remains difficult to achieve.Lateral flow devices (LFD) persist as the most promising[2],[3], although they offer solutions only partially since they fail to accommodate complex bioassays that are often needed to reach desired sensitivity [4].Sparked by the success of LFDs, many attempts were made to automate a timed assay sequence in 'passive microfluidics' driven solely by capillary flow.These approaches are based on defining 3D flow paths within stacks of porous membranes that are individually functionalized with hydrophobic/hydrophilic patterns and assay reagents.[5-7] The resulting interlayer contact issues led to unpredictable or defective devices.[7] To fabricate monolithic passive microfluidics with a 3DP process based on digital data, a polymer powder was combined with liquid binders (Fig. 1A).The 3DPed material was characterized using scanning electron microscopy, SEM(Fig.1B), revealing considerable macroporosity that resulted from remaining interparticle space.For testing the biocompatibility of the 3DPed platform, a sandwich ELISA targeting IgE was used as a model system (Fig. 2A).The ELISA was performed in different formats, being microtiter plates, 3DPed discs, PDMS chips containing 3DPed plugs and fully 3DPed channels (the latter depicted in Fig. 2B).A standard ELISA protocol was successfully downscaled to comply with requirements for microfluidics POC platform by substantially reducing incubation time and reagent volumes of different steps (Fig. 2A).The modified ELISA (total assay time 30 min) was eventually implemented in 3DPed channels (Fig. 2C), demonstrating (1) biocompatibility of the build material with the bioassay (i.e.low non-specific interaction) and (2) compatibility of selected ELISA with the flow system.Finally, a completely integrated microfluidics device was designed after extensive characterization of flow rates for all buffers (Fig. 2D), resulting in a 3DPed microfluidics device (Fig. 2E) that can accommodate complex bioassays in completely autonomous fashion and as such can fill the gap between lateral flow tests and laboratory based ELISA.