Hydrogel Beads for Enhanced Nucleic Acid Analysis in Complex Fluid Matrices

Abstract

We present a functionalized hydrogel bead platform designed to capture cell-free DNA (cfDNA), which is released from tumor cells into the bloodstream, and the use of this cfDNA as a biomarker for cancer detection. Hydrogel beads with covalently incorporated probes (HBP) are generated via photo-cross-linking in a two-phase microfluidic system. The precursor solutions from which the beads are generated are comprised of a photoreactive copolymer, magnetic nanoparticles, Cy3-labelled oligonucleotides serving as a barcode for bead identification, and a specific probe designed to bind the target DNA. From these solutions, droplets are generated in the microfluidic system and photocross-linked through C, H-insertion cross-linking (CHic). As a demonstration case, the hydrogel beads are used to detect a mutation (c.1633G>A) in MCF-7 cells, which is known to promote breast cancer progression by activating oncogenic signalling pathways. The HBPs were successfully employed to detect a fluorescently labelled mutation sequence (92 bp) in phosphate-buffered saline (PBS), serum, and whole blood using a fluorescent reader, followed by amplification through polymerase chain reaction (PCR). The sensitivity of the described cfDNA detection method demonstrates a detection limit as low as 0.36 ng/mL, which is lower than the detection limits reported in the literature so far. The sensitivity of the hydrogel bead platform can be further strongly enhanced as the beads, containing the captured DNA, can be directly subjected to PCR for the amplification of the target sequence without any additional purification steps. The simplicity of production, modification, and functionalization of the hydrogel beads, coupled with their high sensitivity in detecting cfDNA, makes this platform a promising approach for diagnosing a range of diseases.