Rapid, inexpensive fabrication of electrophoretic microdevices for fluorescence detection.

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis. A series of separation channel designs and centrifugation conditions that provided successful loading of sieving polymer in less than 3 min was described. Separation of a 400-base DNA sizing ladder provided calculated base resolution between 3 and 4 bases, a greater than 18-fold improvement over separations on similar substrates. Finally, the accuracy and precision capabilities of these devices were demonstrated by separating and sizing DNA fragments of 147 and 167 bases as 148.62 ± 2 and 166.48 ± 3 bases, respectively.

Microwave-assisted extraction, separation, and chromogenic detection of laced marijuana for presumptive point-of-interdiction testing.

Presumptive drug screening enables timely procurement of search and arrest warrants and represents a crucial first step in crime scene analysis. Screening also reduces the burden on forensic laboratories which often face insurmountable backlogs. In most scenarios, on-site presumptive drug screening relies on chemical field tests for initial identification. However, even when used appropriately, these test kits remain limited to subjective colorimetric analysis, produce false positive or negative results with excessive sample quantities, and are known to cross-react with numerous innocuous substances. Previous efforts to develop microfluidic devices that incorporate these chromogenic indicator reagents address only a few of the many challenges associated with these kits. This is especially true for samples where the drug of interest is present as a lacing agent. This work describes the development of a centrifugal microfluidic device capable of integrating facile sample preparation, by way of a 3D printed snap-on cartridge amenable to microwave assisted extraction, followed by chromatographic separation and chromogenic detection on-disc. As cannabis is among the most widely used controlled substance worldwide, and displays strong interference with these indicator reagents, mock samples of laced marijuana are used for a proof-of-concept demonstration. Post extraction, the microdevice completes high throughput metering just prior to simultaneous reaction with four of the most commonly employed microchemical tests, followed by objective image analysis in CIELAB (a device-independent color model). Separation and recovery of a representative controlled substance with 93% efficiency is achieved. Correct identification, according to hierarchical cluster analysis, of three illicit drugs (e.g., heroin, phencyclidine, and cocaine) in artificially laced samples is also demonstrated on-disc. The cost effective microdevice is capable of complete automation post-extraction, with a total analysis time (including extraction) of <8 min. Finally, sample consumption is minimized, thereby preventing the complete destruction of forensic evidence.

Taking the microfluidic approach to nucleic acid analysis in forensics: Review and perspectives.

Forensic laboratories are universally acknowledged as being overburdened, underfunded, and in need of improved analytical methods to expedite investigations, decrease the costs associated with nucleic acid (NA) analysis, and perform human identification (HID) at the point of need (e.g., crime scene, booking station, etc.). In response, numerous research and development (R&D) efforts have resulted in microfluidic tools that automate portions of the forensic genetic workflow, including DNA extraction, amplification, and short tandem repeat (STR) typing. By the early 2000 s, reports from the National Institute of Justice (NIJ) anticipated that microfluidic 'swab-in-profile-out' systems would be available for use at the crime scene by 2015 and the FBI's 2010 'Rapid DNA' Initiative, approved by Congress in 2017, directed this effort by guiding the development and implementation of maturing systems. At present, few fully-automated microfluidic DNA technologies are commercially available for forensic HID and their adoption by agencies interested in identification has been limited. In practice, the integration of complex laboratory processes to produce one autonomous unit, along with the highly variable nature of forensic input samples, resulted in systems that are more expensive per sample and not comparable to gold-standard identification methods in terms of sensitivity, reproducibility, and multiplex capability. This Review and Perspective provides insight into the contributing factors to this outcome; namely, we focus on the complications associated with the tremendous undertaking that is developing a sample-in-answer-out platform for HID. For context, we also describe the intricate forensic landscape that contributes to a nuanced marketplace, not easily distilled down to cases of simple supply and demand. Moving forward and considering the trade-offs associated with developing methods to compete, sometimes directly, with conventional ones, we recommend a focus shift for microfluidics developers toward the creation of innovative solutions for emerging applications in the field to increase the bandwidth of the forensic investigative toolkit. Likewise, we urge case working personnel to reframe how they conceptualize the currently available Rapid DNA tools; rather than comparing these microfluidic methods to gold-standard procedures, take advantage of their rapid and integrated modes for those situations requiring expedited identifications in an informed manner.

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

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.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.