Microfluidics pertains the controlled, on-chip manipulation of small volumes in channels with dimensions of tens to hundreds of micrometres. Droplet microfluidics herein specifically involves the generation and manipulation of discrete droplets through the encapsulation of one fluid in a second immiscible carrier fluid. By consequence, each droplet poses as an individual reaction chamber with a nano- to picoliter volume. Major advantages of such systems include limited reagent and sample volume requirements, disposability of the microfluidic devices, and the ability to perform complex liquid manipulations on chip (e.g., mixing, pico-injection, etc.). 

In this proof-of-concept study, these benefits have been exploited to address limitations associated with differential lysis (DL) for the analysis of sexual assault casework samples. DL is a multi-step lysis procedure that takes advantage of the difference in sperm and non-sperm cell membrane stability to generate two respective fractions. Although DL remains the standard method for sperm cell separation, it often fails to produce male DNA profiles when the epithelial-to-sperm cell ratio is unfavourable. This is in part due to carry-over of non-sperm DNA in the sperm fraction and is exacerbated in samples with a low sperm content. 

Here, we present a novel approach towards sperm cell separation by droplet encapsulation, detection, and fluorescence-activated sorting on an integrated microfluidic device. Following a mild alkaline lysis step to eradicate the majority of the non-sperm cells, a dual staining strategy is performed using DAPI and CellTraceTM Far Red. The former dye serves as a positive marker for intact cells, which are predominantly sperm, while the latter enables differentiation from residual non-sperm components. The sperm-enriched, stained lysate is then introduced on the microfluidic chip for sorting of sperm-containing droplets, which are subsequently pooled and disrupted for downstream non-sperm DNA digestion, sperm lysis, PCR, and capillary electrophoresis. 

This method has been experimentally validated using assembled mock casework samples with varying sperm-to-epithelial cell ratios and post-coital samples collected by volunteers. DNA profile quality parameters (i.e., allele drop in/out, peak height, etc.) and microscopic image analysis of the sorted and waste droplet populations were used as performance metrics. With this work, we demonstrate the potential of integrating droplet microfluidics into advanced sample processing workflows in forensics.

Authors

• Kristina Fokias (KU Leuven, Belgium)
• Iene Rutten (KU Leuven, Belgium)
• Karen Ven (KU Leuven, Belgium)
• Bram Bekaert (UZ/KU Leuven, Belgium)
• Jeroen Lammertyn (KU Leuven, Belgium)