*Article* **Picoliter Droplet Generation and Dense Bead-in-Droplet Encapsulation via Microfluidic Devices Fabricated via 3D Printed Molds**

**Tochukwu D. Anyaduba 1,2 , Jonas A. Otoo <sup>1</sup> and Travis S. Schlappi 1,\***


**\*** Correspondence: travis\_schlappi@kgi.edu

**Abstract:** Picoliter-scale droplets have many applications in chemistry and biology, such as biomolecule synthesis, drug discovery, nucleic acid quantification, and single cell analysis. However, due to the complicated processes used to fabricate microfluidic channels, most picoliter (pL) droplet generation methods are limited to research in laboratories with cleanroom facilities and complex instrumentation. The purpose of this work is to investigate a method that uses 3D printing to fabricate microfluidic devices that can generate droplets with sizes <100 pL and encapsulate single dense beads mechanistically. Our device generated monodisperse droplets as small as ~48 pL and we demonstrated the usefulness of this droplet generation technique in biomolecule analysis by detecting Lactobacillus acidophillus 16s rRNA via digital loop-mediated isothermal amplification (dLAMP). We also designed a mixer that can be integrated into a syringe to overcome dense bead sedimentation and found that the bead-in-droplet (BiD) emulsions created from our device had <2% of the droplets populated with more than 1 bead. This study will enable researchers to create devices that generate pL-scale droplets and encapsulate dense beads with inexpensive and simple instrumentation (3D printer and syringe pump). The rapid prototyping and integration ability of this module with other components or processes can accelerate the development of point-of-care microfluidic devices that use droplet-bead emulsions to analyze biological or chemical samples with high throughput and precision.

**Keywords:** microfluidics; picoliter droplets; rapid prototyping; bead encapsulation; 3D printing
