Novel 3D-printed microfluidic chip for automated real-time multi-route in vivo nanoplastics toxicity testing

Plastics are one of the most pervasive pollutants in marine environments, with smaller-sized plastics, such as nanoplastics (NPLs), posing significant but understudied threats to marine organisms. Traditional bulk and end-point testing methods offer limited capability for real-time, in situ assessment of nanotoxicity. In this study, we present a fully 3D-printed, automated microfluidic chip designed for multi-route in vivo nanotoxicity testing, which addresses the limitations of conventional methods by offering automated and dynamic exposure conditions alongside real-time monitoring through fluorescence imaging and oxygen sensing. Additionally, this platform simplifies the fabrication process, avoiding the time-consuming soft-lithography typically used for microfluidic chips and increasing accessibility to users without specialized expertise. As a proof of concept, the chip was used to assess nanotoxicity in model marine organisms across different trophic levels: Artemia franciscana (instar II and III stages) and a mixture of five microalgae. Polystyrene nanoplastics, one of the most prevalent plastics in marine environments, were used as the model NPLs and tested in two exposure routes: waterborne and foodborne. The results revealed distinct uptake dynamics; in Artemia, NPL-associated fluorescence initially increased and subsequently declined during waterborne exposure, whereas it continued to increase over the measured period during foodborne exposure. Oxygen-consumption responses varied by organism and exposure route: waterborne exposure produced a higher trend in Artemia and a significant increase in algae, whereas foodborne exposure produced a response comparable to its control. Together, continuous oxygen sensing and fluorescence imaging demonstrated the capability of the chip to monitor complementary physiological and NPL-uptake responses under multiple exposure routes in real time.