Acoustic trapping of sperm cells from mock sexual assault samples.

We report the successful separation of sperm cells from a relevant composition of mock sexual assault samples using a novel acoustic differential extraction (ADE) technology. A multi-layer microfluidic device fabricated in a non-photolithographic process from glass and polydimethylsiloxane (PDMS) was capable of interfacing with custom-built instrumentation to exploit a standing acoustic wave for the trapping of individual sperm cells in a sample containing an abundance of epithelial cells. Samples were generated from buccal and vaginal swabs to mimic post-coital vaginal swabs, and processed through the ADE system followed by DNA extraction of the captured cells with amplification of DNA using a custom short tandem repeat (STR) chemistry. The prototype acoustic trapping technology was fully capable of isolating intact sperm cells from mock samples with disparate masses of male and female DNA. Other biological components were evaluated for adverse effects on sperm cell trapping, including blood, yeast, and bacteria (E. coli), and these had negligible effects on observed sperm cell trapping. Finally, we demonstrate the successful capture of sperm cells from mock samples containing a 40-fold excess in female epithelial cells over sperm cells. The effectiveness of sperm cell purification was ascertained with polymerase chain reaction (PCR) amplification of STR loci from the male fraction post separation with an 18-plex amplification kit, which resulted in male-only profiles.

Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis.

Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis. Primer concentrations were adjusted specifically for microdevice amplification, resulting in well-balanced short tandem repeat (STR) profiles. Furthermore, studies were performed to push the limits of the DNA polymerase to achieve rapid, multiplexed PCR on various substrates, including transparent and black polyethylene terephthalate (Pe), and with two distinct adhesives, toner and heat sensitive adhesive (HSA). Rapid STR-based multiplexed PCR amplification is demonstrated in 15 min on a Pe microdevice using a custom-built system for fluid flow control and thermocycling for the full 10-plex, and in 10 min for a smaller multiplex consisting of six core CODIS loci plus Amelogenin with amplicons shorter than 200bp. Lastly, preliminary studies indicate the capability of this PCR microdevice platform to be integrated with both upstream DNA extraction, and downstream microchip electrophoresis. This, coupled to the use of reagents that are compatible with lyophilization (lyo-compatible) for PCR, represents the potential for a fully integrated rotationally-driven microdevice for complete forensic DNA analysis.

A centrifugal microfluidic device with integrated gold leaf electrodes for the electrophoretic separation of DNA.

Current conventional methods utilized for forensic DNA analysis are time consuming and labor-intensive requiring large and expensive equipment and instrumentation. While more portable Rapid DNA systems have been developed, introducing them to a working laboratory still necessitates a high cost of initiation followed by the recurrent cost of the devices. This has highlighted the need for an inexpensive, rapid and portable DNA analysis tool for human identification in a forensic setting. In order for an integrated DNA analysis system such as this to be realized, device operations must always be concluded by a rapid separation of short-tandem repeat (STR) DNA fragments. Contributing to this, we report the development of a unique, multi-level, centrifugal microdevice that can perform both reagent loading and DNA separation. The fabrication protocol was inspired by the print, cut and laminate (PCL) technique described previously by our group, and in accordance, offers a rapid and inexpensive option compared with existing approaches. The device comprises multiple polyester-toner fluidic layers, a cyclic olefin copolymer separation domain and integrated gold leaf electrodes. All materials are commercially-available and complement the PCL process in a way that permits fabrication of increasingly sought after single-use devices. All reagents, including a viscous sieving matrix, are loaded centrifugally, eliminating external pneumatic pumping, and the sample is separated in <5 minutes using an effective separation length of only 4 cm (reagent loading to completed separation, is <37 minutes). The protocol for gold leaf electrode manufacture yielded up to 30 electrodes for less than $3 (cost of a 79 mm × 79 mm gold leaf sheet) and when using a device combining these electrodes and centrifugal reagent/polymer loading, the electrophoretic separation of STR fragments with two base resolution was demonstrated. This exemplary performance makes the device an ideal candidate for further integration and development of an inexpensive, portable and rapid forensic human identification system.

Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng µL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification.

Metformin induced expression of Hsp60 in human THP-1 monocyte cells.

Metformin is in widespread clinical use for the treatment of diabetes mellitus in patients. It has been shown to inhibit mitochondrial bioenergetic functions by inhibiting complex I of the electron transport chain. The expression of mitochondrial-specific molecular stress protein Hsp60 is a key consequence of mitochondrial impairment. Since this protein has important immune-modulatory properties, we have investigated the expression of Hsp60 in human THP-1 monocyte cells exposed to metformin. In this study, we demonstrate significant up-regulation of Hsp60 at both mRNA and protein levels when these cells were exposed to metformin at therapeutic dosage levels. Interestingly, there was also an increase in expression of CD14 mRNA in these cells. This suggested a possible modulation of the differentiation rates of the THP-1 cells during exposure to metformin. As monocyte differentiation marks a critical step in atherosclerosis, these observations suggest that long-term exposure to metformin could have important implications for the diabetic patient.

Prevalence and numbers of coliphages and Campylobacter jejuni bacteriophages in New Zealand foods.

Vegetable samples were tested for the presence of coliphages. None of the 55 samples contained these phages at concentrations greater than 10 g(-1) (the limit of detection). Spiking and recovery experiments indicated that the method was efficient at detecting coliphage T4 added to the food, and so it was concluded that phage titres were not being falsely underestimated. In addition 51 samples of chicken skin from retail portions were tested for the presence and numbers of coliphages and for presence only of Campylobacter jejuni phages. Coliphages were isolated from 46 samples (90.2% positive), at up to 2570 PFU 10 g sample(-1) but no C. jejuni phages were isolated. Several other methods were used to isolate C. jejuni phages from retail chicken but none was successful. However, when pooled whole chicken rinses from 39 flocks were tested for the presence of C. jejuni phages, 11 (28.2%) of the flocks were positive. It is possible that phages present on birds at the start of processing were either inactivated or simply diluted out during spin chilling. These data add to the body of information indicating that phages can readily be isolated from certain foods and indicate that consumers are exposed to them on a regular basis.