Colorectal cancer (CRC) remains a major global health challenge, with an increasing incidence of early-onset cases among young adults. Targeted analysis of cell-free DNA (cfDNA) methylation in blood has emerged as a promising minimally invasive diagnostic approach. While digital PCR (dPCR) offers high sensitivity and low turnaround times, conventional bisulfite-based dPCR assays require large plasma volumes due to cfDNA degradation, limiting clinical feasibility. To overcome this limitation, we developed a bisulfite-free, low-plasma-volume assay by coupling cell-free methylated DNA immunoprecipitation (cfMeDIP) with multiplexed dPCR for methylation detection. Assays were designed for CRC targets based on publicly available bisulfite-based plasma data and optimized for native, bisulfite-untreated cfDNA. The cfMeDIP-dPCR assays were first developed and optimized on circulating tumor DNA surrogates derived from HCT116 cells and subsequently validated in a pilot study, including 32 early-onset CRC (EO-CRC) patients and 29 non-CRC individuals. Methylation ratios, defined as the proportion of methylated to total cfDNA copies per marker, served as a diagnostic indicator. Three out of four selected markers (SEPT9, KCNQ5, and C9orf50) were successfully adapted, with significantly higher methylation ratios (p ≤ 0.001) in the EO-CRC cohort. KCNQ5 demonstrated the highest diagnostic performance, achieving an 85% sensitivity at a 90% specificity, with methylation ratios correlating with the tumor stage. This study presents the first cfMeDIP-dPCR approach, demonstrating its potential as a sensitive liquid biopsy assay. Requiring only 0.5 mL of plasma, i.e., more than 20 times less than a sensitivity-matched bisulfite-based assay, cfMeDIP-dPCR facilitates clinical implementation for CRC and other diseases with epigenetic signatures.

Background: The growing demand for versatile laboratory automation is exemplified in the context of liquid biopsy, where multi-analyte approaches are increasingly recognised for their potential to enhance diagnostic sensitivity in oncology. However, current practice often necessitates the use of dedicated instruments and workflows for the extraction of each analyte, posing financial and logistical barriers for automated multi-analyte liquid biopsy. Methods: Here, we present Robotic Centrifugal Microfluidics (RoCM), an all-in-one platform that combines the versatility of centrifugal microfluidics and operational flexibility of robotic liquid handling. This combination enables the automation of complex micro- and macrofluidic protocols, realised through the use of (1). exchangeable microfluidic cartridges and (2). programmable robotic operations such as in-rotation liquid supply, magnetic bead manipulation, or microfluidic valving. In-rotation robotic liquid manipulation maintains fluid control under centrifugal forces and reduces the cartridge footprint associated with pre-loaded liquid reservoirs. Platform applicability was demonstrated using two exemplary liquid biopsy workflows: the extraction of cell-free DNA (cfDNA) from blood plasma using RoCM-cfDNA slices and the extraction of extracellular vesicles (EVs) from blood plasma using RoCM-EV slices. Results: In a pilot study with patient samples from different cancer entities, the RoCM-cfDNA slices yielded comparable variant allele frequencies to a commercial bead-based instrument, while the RoCM-EV slices achieved a recovery of a greater diversity of EV subpopulations than semi-automated size-exclusion chromatography. Conclusions: By simply exchanging cartridges, RoCM enables the extraction of diverse analytes within a single automated system. Its application can be extended to further analytes, such as circulating tumour cells (CTCs), or to applications beyond liquid biopsies, where versatile micro- and macrofluidic protocols benefit from implementation in a single automation instrument.

Background: The growing demand for versatile laboratory automation is exemplified in the context of liquid biopsy, where multi-analyte approaches are increasingly recognised for their potential to enhance diagnostic sensitivity in oncology. However, current practice often necessitates the use of dedicated instruments and workflows for the extraction of each analyte, posing financial and logistical barriers for automated multi-analyte liquid biopsy. Methods: Here, we present Robotic Centrifugal Microfluidics (RoCM), an all-in-one platform that combines the versatility of centrifugal microfluidics and operational flexibility of robotic liquid handling. This combination enables the automation of complex micro- and macrofluidic protocols, realised through the use of (1.) exchangeable microfluidic cartridges and (2.) programmable robotic operations such as in-rotation liquid supply, magnetic bead manipulation, or microfluidic valving. In-rotation robotic liquid manipulation maintains fluid control under centrifugal forces and reduces the cartridge footprint associated with pre-loaded liquid reservoirs. Platform applicability was demonstrated using two exemplary liquid biopsy workflows: the extraction of cell-free DNA (cfDNA) from blood plasma using RoCM-cfDNA slices and the extraction of extracellular vesicles (EVs) from blood plasma using RoCM-EV slices. Results: In a pilot study with patient samples from different cancer entities, the RoCM-cfDNA slices yielded comparable variant allele frequencies to a commercial bead-based instrument, while the RoCM-EV slices achieved a recovery of a greater diversity of EV subpopulations and up to one magnitude higher recovery than semi-automated size exclusion chromatography. Conclusion: By simply exchanging cartridges, RoCM enables the extraction of diverse analytes within a single automated system. Its application can be extended to further analytes, such as circulating tumor cells (CTCs), or to applications beyond liquid biopsies, where versatile micro- and macrofluidic protocols benefit from implementation in a single automation instrument.