Rapid Microchip Electrophoretic Separation of Novel Transcriptomic Body Fluid Markers for Forensic Fluid Profiling

Abstract

Initial screening of criminal evidence often involves serological testing of stains of unknown composition and/or origin discovered at a crime scene to determine the tissue of origin. This testing is presumptive but critical for contextualizing the scene. Here, we describe a microfluidic approach for body fluid profiling via fluorescent electrophoretic separation of a published mRNA panel that provides unparalleled specificity and sensitivity. This centrifugal microfluidic approach expedites and automates the electrophoresis process by allowing for simple, rotationally driven flow and polymer loading through a 5 cm separation channel; with each disc containing three identical domains, multi-sample analysis is possible with a single disc and multi-sample detection per disc. The centrifugal platform enables a series of sequential unit operations (metering, mixing, aliquoting, heating, storage) to execute automated electrophoretic separation. Results show on-disc fluorescent detection and sizing of amplicons to perform comparably with a commercial ‘gold standard’ benchtop instrument and permitted sensitive, empirical discrimination between five distinct body fluids in less than 10 min. Notably, our microfluidic platform represents a faster, simpler method for separation of a transcriptomic panel to be used for forensically relevant body fluid identification.

On the Application of Microfluidic-Based Technologies in Forensics: A Review

“…Body fluid identification (BFID): current methods for BFID, e.g., spectroscopic-based techniques (Raman or FT-IR), require trained personnel, expensive instruments, and are time-consuming [150]. Emerging methods based on molecular biology, such as transcriptomics, are a promising confirmatory method for BFID since RNA profiling and recovery can be performed on body fluid stains without compromising or consuming the valuable DNA (see confirmatory tests for blood (Section 2.1.1), number 6) [151]. Various types of RNA, e.g., mRNA [152], microRNA [153,154], and circular RNA [155,156] have been investigated.…”

“…Recently, Layne et al developed a microfluidic platform (centrifugal microelectrophoresis Disc (µEDisc)) to separate mRNA amplicons in BFs using electrophoresis. A low-cost prototyping technique, namely "print-cut-laminate" [158], was used to fabricate the layered microfluidic platform using various polymers (Figure 4) [151]. PCR-amplified fragments were detected using laser-induced fluorescence.…”