Development of 3D printed capillaric circuit interfacing with silver nanoparticle embedded membrane for simultaneous analysis of duplex samples via fluorescence sandwich immunoassays.

Capillaric microfluidic circuits provide the capability to miniaturize immunoassays, reduce assay time, and perform precise liquid handling without the need for external equipment. While capillaric circuits have been well studied and have often been combined with paper substrates, most circuits require complex fabrication processes and are limited to the analysis of a single sample. In this work, we evaluated the effects of surface treatment on capillary flow, controlled sample volume through altering the number of reservoirs used for sample delivery, evaluated the effects of inkjet microarray printing on various immunoassay formats, and enabled the simultaneous detection of duplex samples within a single device using fluorescence sandwich immunoassays. We demonstrated that plasma treatment provides inconsistency in fluid flow compared to untreated circuits that use Tween-20 additive in reagents for flow control. Furthermore, due to the stress that molecules undergo during inkjet microarray printing, sandwich immunoassays exhibited resilience and reliability compared to reverse-phase immunoassays. In this work, we controlled sample volume through utilizing multiple channels to deliver sample to the circuit, offering flexibility based on sample availability. Using a silver nanoparticle embedded membrane to provide metal enhanced fluorescence, we enabled the simultaneous detection of the EGFR protein from two samples with a unique 2-sample circuit design, offering detection limits of 0.98 ng/mL in PBS-T and 0.13 ng/mL in spiked normal human plasma within 1 h. This technology offers potential scalability of capillaric circuits for the automation of bioassays for various samples with reduced time and cost, suitable for rapid disease diagnostics.