Pump-free and high-throughput generation of monodisperse hydrogel beads by microfluidic step emulsification for dLAMP-on-a-chip

Abstract

Step emulsification (SE), which generates droplets by a sharp change in confinement, has emerged as a potential alternative to flow-focusing technology. Water/dispersed phase is continuously pumped through a shallow inlet channel into a deep chamber pre-filled with the oil/continuous phase. The need for one or more pumps to maintain a continuous flow for droplet generation, and the consequent use of high sample volumes, limit this technique to research labs. Here, we report a pump-free SE technique for rapid and high-throughput generation of monodisperse hydrogel (agarose) beads using <40 μl sample volume. Instead of using syringe pumps, we sequentially pipetted oil and liquid agarose into a microfluidic SE device to generate between 20000 and 80000 agarose beads in ~ 2 min. We also demonstrated the encapsulation of loop-mediated isothermal amplification mixture inside these beads at the time of their formation. Finally, using these beads as reaction chambers, we amplified nucleic acids from P. falciparum and SARS-CoV-2 inside them. The pump-free operation, tiny sample volume, and high-throughput generation of droplets by SE make our technique suitable for point-of-care diagnostics.

Novel polymer-based hydrogels of recent research in drug delivery for disease treatment related to SARS-CoV-2 virus

“…Hydrogels can be functionalized with specific probes, such as DNA or RNA sequences, antibodies, or antigens, to identify viral genetic material or specific biomarkers associated with SARS-CoV-2 infection [142,143]. These hydrogels are adaptable to various diagnostic techniques, including polymerase chain reaction (PCR) [139], loop-mediated isothermal amplification (LAMP) [171,172], or nucleic acid hybridization assays [155]. They can also be engineered to capture and detect viral antigens, like the spike protein or nucleocapsid protein of SARS-CoV-2.…”

“…They can also be engineered to capture and detect viral antigens, like the spike protein or nucleocapsid protein of SARS-CoV-2. Frequently, these hydrogels are coupled with immunological assays such as enzyme-linked immunosorbent assays (ELISA), lateral flow assays, or immunofluorescence assays, enabling rapid and precise detection of viral antigens in patient samples [45,155,[171][172][173]. Hydrogels serve as sensitive platforms in biosensors for the detection of viral particles or biomarkers [174].…”