Integrated microfluidic cell lysis and plasmonic assay for detecting circulating tumor genetic biomarkers in liquid biopsy

Liquid biopsy is a groundbreaking advancement in cancer diagnostics, offering a non-invasive alternative to traditional tissue biopsies. Liquid biopsies can be done with minimal discomfort, allowing for repeated tests without significant risk to patients, and have shown promise in detecting cancers at earlier stages, greatly improving treatment outcomes. This innovative method uses body fluids to identify cancer-related biomarkers, enabling early diagnosis and ongoing monitoring of disease progression. Technology mainly focuses on circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and other genetic materials, which provide insights into tumor heterogeneity and treatment responses. Current methods for analyzing liquid biopsy samples often involve complex procedures, such as pre-analytical processing and target isolation. These steps usually require a polymerase chain reaction to amplify sequences for genomic DNA analysis. Liquid biopsy employs microfluidics to simplify these processes. Microfluidics improves the analysis of circulating tumor cells (CTCs) and tumor-derived ucleic acids, enabling efficient separation and enrichment of these biomarkers from blood. This provides comprehensive insights into tumor biology and atient-specific treatment responses. Combining microfluidics with advanced imaging and sequencing technologies has greatly enhanced the accuracy and usefulness of liquid biopsies. To achieve this, we developed a microfluidic device for handling liquid biopsy samples, enhancing the processing of circulating tumor cells. The device features cellular lysis through two inlet channels for the sample and lysis reagent. It includes a serpentine zone for initial homogenization and a long channel for completion. The channel's width and depth—500 µm and 80 µm—allow processing of a few tens of microliters of sample. Lysis optimization used human wild-type (All-RAS WT) HT-29 and mutated (KRAS p. G13D) LoVo cell lines, isolated from colorectal adenocarcinoma tissues, expressing a mutation in exon 2 of the K-RAS oncogene. Genomic DNA from these cells was analyzed using optical fluorescence microscopy with dsDNA-specific intercalating dyes. gDNA detection was performed via a plasmonic assay based on surface plasmon resonance imaging, through target capture by functionalized uperparamagnetic beads.