Complex DNA mixtures from trace biological evidence remain a major challenge in forensic genetics, particularly in sexual assault cases where minor contributors (i.e. perpetrators) are often masked by major donor (i.e. victims). Conventional bulk DNA profiling approaches have limited power to resolve such mixtures, motivating development of single-cell strategies that enable contributor-specific analysis. However, existing single-cell isolation methods often require large amounts of cellular material and are labor-intensive, low-throughput or insufficiently flexible for routine forensic applications.

In this study, we followed a novel single-cell encapsulation approach using semi-permeable capsules (SPCs) generated by the FLUX® microfluidic platform from Atrandi Biosciences, that enables high-throughput capture of individual cells within a controlled microenvironment, while preserving cell integrity and compatibility with downstream molecular analyses.

The workflow comprised several steps, including cell culture (Jurkat cell line), cell counting, cell encapsulation using SPCs on the FLUX® instrument, SPC evaluation and washing, direct cell lysis and nucleic acid extraction within SPCs, followed by SPC lysis and removal. Method performance was evaluated in three stages: (i) implementation under standard conditions at the lowest manufacturer-recommended input (6,250 cells); (ii) sensitivity assessment across decreasing cell inputs (3,125, 1,560, and 780 cells) to determine the limit of detection; and (iii) application to different cell types, including sperm cells and mixtures, to assess robustness in realistic scenarios. Encapsulation efficiency, defined as the proportion of SPCs containing a cell, was measured by imaging and counting, and cell recovery was estimated by qPCR-based genomic DNA quantification using the Quantifiler® Trio assay (Thermo Fisher Scientific). Experiments were performed in duplicate with negative controls.

The workflow was successfully implemented, with consistent replicate measurements and no evidence of contamination. Encapsulation efficiency remained within expected range (1.2–2.1%) and was stable across input levels. Genomic DNA was recovered across all tested inputs, including low-input conditions, with recovery rates of approximately 10–20% that increased with higher cell inputs. Application to forensically relevant samples demonstrated successful encapsulation and isolation of diverse cell types within SPCs. Challenges were identified, including time-consuming manual SPC evaluation and cell/DNA loss during washing, guiding ongoing optimization.

In conclusion, this study provides the first insights into the performance of SPC-based single-cell encapsulation using the FLUX® platform. By retaining and protecting DNA from individual donors, each SPC functions as a discrete analytical unit for forensic single-cell profiling. This approach has the potential to enable cellular-level deconvolution of complex DNA mixtures and improve contributor resolution in challenging forensic casework.

Authors

• Camille Ropert (Department of Clinical Genetics, Maastricht University Medical Center; Department of Genetics & Cell Biology, GROW Institute, Maastricht University, Netherlands)
• Bram Bekaert (KU Leuven, Forensic Biomedical Sciences, Department of Imaging & Pathology, KU Leuven, LISCO, The KU Leuven Institute for Single Cell Omics, UZ Leuven, Laboratory of Forensic Genetics, Belgium)
• Athina Vidaki (Department of Clinical Genetics, Maastricht University Medical Center; Department of Genetics & Cell Biology, GROW Institute, Maastricht University, Netherlands)