Parallel DLD microfluidics for chloroplast isolation and sorting.

Chloroplasts are characteristic organelles of plant cells, essential for photosynthesis and various other metabolic processes, including amino acid, lipid, and hormone biosynthesis. Beyond their classical functions, chloroplasts have emerged as promising targets in biotechnology, particularly in therapeutic applications and biofuel production. However, their isolation remains technically challenging due to the limitations of conventional methods, which typically require complex protocols, specialized equipment, and trained personnel. Here, we present a microfluidic-based platform that enables size-based chloroplast separation using deterministic lateral displacement (DLD). Our device integrates four parallel DLD arrays, each with a distinct critical diameter (CD). This configuration enables bandpass filtering and allows the simultaneous isolation of chloroplasts of various sizes within a single device. Shared inlets and uniform flow conditions across all arrays enhance reproducibility compared to conventional techniques. Unlike traditional sucrose density gradients, which lack precise size-based separation, our system achieves separation efficiencies of 50-85% for chloroplasts ranging from 3 to 8 μm, with recovered fractions having purities of 17-66%. This platform provides a rapid, automated, and scalable solution for chloroplast isolation, with significant potential applications in plant research, biotechnology, and synthetic biology.