Rapid antibiotic susceptibility testing using resazurin bulk modified screen-printed electrochemical sensing platforms.

Urinary tract infections (UTIs) are one of the most common types of bacterial infection. UTIs can be associated with multidrug resistant bacteria and current methods of determining an effective antibiotic for UTIs can take up to 48 hours, which increases the chances of a negative prognosis for the patient. In this paper we report for the first time, the fabrication of resazurin bulk modified screen-printed macroelectrodes (R-SPEs) demonstrating them to be effective platforms for the electrochemical detection of antibiotic susceptibility in complicated UTIs. Using differential pulse voltammetry (DPV), resazurin was able to be detected down to 15.6 µM. R-SPEs were utilised to conduct antibiotic susceptibility testing (AST) of E. coli (ATCC® 25922) to the antibiotic gentamicin sulphate using DPV to detect the relative concentrations of resazurin between antibiotic treated bacteria, and bacteria without antibiotic treatment. Using R-SPEs, antibiotic susceptibility was determined after a total elapsed time of 90 minutes including the inoculation of the artificial urine, preincubation and testing time. The use of electrochemistry as a phenotypic means of identifying an effective antibiotic to treat a complicated UTI offers a rapid and accurate alternative to culture based methods for AST with R-SPEs offering an inexpensive and simpler alternative to other AST methods utilising electrochemical based approaches.

The preparation of microfluidic architecture with monolithic materials using a dual porous silica structure.

A microfluidic device (MD) has been developed which features a porous silica (PS) monolithic disk synthesized from tetramethyl orthosilicate, incorporated into the device post-fabrication and sealed in place with a second PS monolithic layer, synthesized from potassium silicate. This dual porous silica (DPS) structure provides a pathway for sample introduction to the MD and offers an ideal platform for solid phase extraction (SPE) methodologies which can be rapidly and efficiently integrated into a chip-based format. All silica disk manufacture and functionalization was carried out in batch to provide a readily scalable method of production. Application of this design for processing samples was demonstrated using two alternative nucleic acid purification chemistries, yielding polymerase chain reaction amplifiable DNA extracted from 150 µL of human urine in less than 35 min. It is proposed that this DPS system could be further developed for a diverse range of chip-based SPE applications, providing an interface facilitating sample delivery and enabling SPE on-chip. Furthermore, to the author's knowledge it is the first reporting of two different types of PS amalgamated in a single MD.

Genetic identification of three CITES-listed sharks using a paper-based Lab-on-a-Chip (LOC).

Threatened shark species are caught in large numbers by artisanal and commercial fisheries and traded globally. Monitoring both which shark species are caught and sold in fisheries, and the export of CITES-restricted products, are essential in reducing illegal fishing. Current methods for species identification rely on visual examination by experts or DNA barcoding techniques requiring specialist laboratory facilities and trained personnel. The need for specialist equipment and/or input from experts means many markets are currently not monitored. We have developed a paper-based Lab-on-a-Chip (LOC) to facilitate identification of three threatened and CITES-listed sharks, bigeye thresher (Alopias superciliosus), pelagic thresher (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus) at market source. DNA was successfully extracted from shark meat and fin samples and combined with DNA amplification and visualisation using Loop Mediated Isothermal Amplification (LAMP) on the LOC. This resulted in the successful identification of the target species of sharks in under an hour, with a working positive and negative control. The LOC provided a simple "yes" or "no" result via a colour change from pink to yellow when one of the target species was present. The LOC serves as proof-of-concept (PoC) for field-based species identification as it does not require specialist facilities. It can be used by non-scientifically trained personnel, especially in areas where there are suspected high frequencies of mislabelling or for the identification of dried shark fins in seizures.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device.

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Non-thermal plasma-based inactivation of bacteria in water using a microfluidic reactor.

Failure of conventional water treatment systems may lead to the contamination of water sources, which can cause outbreaks of waterborne healthcare associated infections. Advanced oxidation processing by non-thermal plasma has the potential to treat water without the addition of chemicals. Antibiotic resistant Pseudomonas aeruginosa and Escherichia coli were chosen to investigate the use of non-thermal plasma generated in a microfluidic reactor to disinfect bacteria contaminated water. The microfluidic reactor used in this study utilized a dielectric barrier discharge, in a gas-liquid phase annular flow regime. Microbiological analysis of water inoculated with P. aeruginosa and E. coli was carried out before and after plasma treatment. Using air as the carrier gas, effective disinfection of water was achieved. At the lowest flow rate (35 µL/min), P. aeruginosa and E. coli viability were drastically reduced, with an approximate 8 log maximum decrease in viability following an estimated residence time of 5 s of plasma treatment. Scanning electron microscopy indicated changes in cell morphology due to the plasma treatment. Live/Dead assays revealed that the membranes of the cells had been damaged after plasma treatment. This work demonstrated that non-thermal plasma has the potential to disinfect against microbial contamination in water.

On-site genetic analysis for species identification using lab-on-a-chip.

This paper presents a microfluidic device capable of performing genetic analysis on dung samples to identify White Rhinoceros (Ceratotherium simum). The development of a microfluidic device, which can be used in the field, offers a portable and cost-effective solution for DNA analysis and species identification to aid conservation efforts. Optimization of the DNA extraction processes produced equivalent yields compared to conventional kit-based methods within just 5 minutes. The use of a color-changing loop-mediated isothermal amplification reaction for simultaneous detection of the cytochrome B sequence of C. simum enabled positive results to be obtained within as little as 30 minutes. Field testing was performed at Knowsley Safari to demonstrate real-world applicability of the microfluidic device for testing of biological samples.

Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling.

A microwave heating system is described for performing polymerase chain reaction (PCR) in a microfluidic device. The heating system, in combination with air impingement cooling, provided rapid thermal cycling with heating and cooling rates of up to 65 degrees C s(-1) and minimal over- or under-shoot (+/-0.1 degrees C) when reaching target temperatures. In addition, once the required temperature was reached it could be maintained with an accuracy of +/-0.1 degrees C. To demonstrate the functionality of the system, PCR was successfully performed for the amplification of the Amelogenin locus using heating rates and quantities an order of magnitude faster and smaller than current commercial instruments.

Investigating cat predation as the cause of bat wing tears using forensic DNA analysis.

Cat predation upon bat species has been reported to have significant effects on bat populations in both rural and urban areas. The majority of research in this area has focussed on observational data from bat rehabilitators documenting injuries, and cat owners, when domestic cats present prey. However, this has the potential to underestimate the number of bats killed or injured by cats. Here, we use forensic DNA analysis techniques to analyze swabs taken from injured bats in the United Kingdom, mainly including Pipistrellus pipistrellus (40 out of 72 specimens). Using quantitative PCR, cat DNA was found in two-thirds of samples submitted by bat rehabilitators. Of these samples, short tandem repeat analysis produced partial DNA profiles for approximately one-third of samples, which could be used to link predation events to individual cats. The use of genetic analysis can complement observational data and potentially provide additional information to give a more accurate estimation of cat predation.

Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device.

A silica monolith used to support both electro-osmotic pumping (EOP) and the extraction/elution of DNA coupled with gel-supported reagents is described. The benefits of the combined EOP extraction/elution system were illustrated by combining DNA extraction and gene amplification using the polymerase chain reaction (PCR) process. All the reagents necessary for both processes were supported within pre-loaded gels that allow the reagents to be stored at 4 degrees C for up to four weeks in the microfluidic device. When carrying out an analysis the crude sample only needed to be hydrodynamically introduced into the device which was connected to an external computer controlled power supply via platinum wire electrodes. DNA was extracted with 65% efficiency after loading lysed cells onto a silica monolith. Ethanol contained within an agarose gel matrix was then used to wash unwanted debris away from the sample by EOP (100 V cm(-1) for 5 min). The retained DNA was subsequently eluted from the monolith by water contained in a second agarose gel, again by EOP using an electric field of 100 V cm(-1) for 5 min, and transferred into the PCR reagent containing gel. The eluted DNA in solution was successfully amplified by PCR, confirming that the concept of a complete self-contained microfluidic device could be realised for DNA sample clean up and amplification, using a simple pumping and on-chip reagent storage methodology.

Characterizing wing tears in common pipistrelles (Pipistrellus pipistrellus): investigating tear distribution, wing strength, and possible causes.

Bats have large, thin wings that are particularly susceptible to tearing. Anatomical specializations, such as fiber reinforcement, strengthen the wing and increase its resistance to puncture, and an extensive vasculature system across the wing also promotes healing. We investigated whether tear positioning is associated with anatomy in common pipistrelles (Pipistrellus pipistrellus). Wing anatomy was described using histological techniques, imaging, and material testing. Tear information, including type, position, time in rehabilitation, and possible causes, was collected from rehabilitators of injured bats across the United Kingdom. Results suggest that the position of the plagiopatagium (the most proximal wing section to the body), rather than its anatomy, influenced the number, location, and orientation of wing tears. While material testing did not identify the plagiopatagium as being significantly weaker than the chiropatagium (the more distal sections of the wing), the plagiopatagium tended to have the most tears. The position of the tears, close to the body and toward the trailing edge, suggests that they are caused by predator attacks, such as from a cat (Felis catus), rather than collisions. Consistent with this, 38% of P. pipistrellus individuals had confirmed wing tears caused by cats, with an additional 38% identified by rehabilitators as due to suspected cat attacks. The plagiopatagium had the lowest number of blood vessels and highest amounts of elastin fibers, suggesting that healing may take longer in this section. Further investigations into the causes of tears, and their effect on flight capabilities, will help to improve bat rehabilitation.

Combining Electro-Osmotic Flow and FTA® Paper for DNA Analysis on Microfluidic Devices.

FTA® paper can be used to protect a variety of biological samples prior to analysis, facilitating ease-of-transport to laboratories or long-term archive storage. The use of FTA® paper as a solid phase eradicates the need to elute the nucleic acids from the matrix prior to DNA amplification, enabling both DNA purification and polymerase chain reaction (PCR)-based DNA amplification to be performed in a single chamber on the microfluidic device. A disc of FTA® paper, containing a biological sample, was placed within the microfluidic device on top of wax-encapsulated DNA amplification reagents. The disc containing the biological sample was then cleaned up using Tris-EDTA (TE) buffer, which was passed over the disc, via electro-osmotic flow, in order to remove any potential inhibitors of downstream processes. DNA amplification was successfully performed (from buccal cells, whole blood and semen) using a Peltier thermal cycling system, whereupon the stored PCR reagents were released during the initial denaturing step due to the wax barrier melting between the FTA® disc and PCR reagents. Such a system offers advantages in terms of a simple sample introduction interface and the ability to process archived samples in an integrated microfluidic environment with minimal risk of contamination.

Sex identification of ancient DNA samples using a microfluidic device.

Ancient DNA is the name given to the degraded, fragmented, and chemically damaged biomolecules that can be recovered from archaeological remains of plants, animals, and humans. Where ancient human DNA has survived at archaeological sites, it can give valuable information and is especially useful for its potential to identify kinship, population affinities, pathogens, and biological sex. Here, we describe the operation of a microfluidic device for the sex identification of ancient DNA samples using an efficient sample handling process. DNA is extracted from powdered bone samples and abasic sites labeled with biotin. Streptavidin-coated superparamagnetic particles are used to isolate the labeled DNA prior to amplification of the Amelogenin sex marker.

Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device.

The evaluation of a micro fluidic system with an integrated silica monolith for performing DNA extraction from limited biological samples has been carried out. Low DNA target concentrations usually require the addition of carrier RNA to ensure desired extraction efficiencies. Here, we demonstrate a micro fluidic extraction system with increasingly efficient extraction performances for biological samples containing <15 ng of total DNA without the need of adding carrier nucleic acids. All extracted DNA showed successful amplification via the polymerase chain reaction demonstrating both the effectiveness of the proposed system at removing potential inhibitors and yielding good quality DNA. The work presented here beneficially identifies reduced sample volumes/concentrations as suitable for processing with respect to downstream analysis by enabling pre-concentration of the biological sample, particularly important when dealing with clinical or forensic specimens.

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths.

DNA extraction was carried out on silica-based monoliths within a microfluidic device. Solid-phase DNA extraction methodology was applied in which the DNA binds to silica in the presence of a chaotropic salt, such as guanidine hydrochloride, and is eluted in a low ionic strength solution, such as water. The addition of poly-A carrier RNA to the chaotropic salt solution resulted in a marked increase in the effective amount of DNA that could be recovered (25ng) compared to the absence of RNA (5ng) using the silica-based monolith. These findings confirm that techniques utilising nucleic acid carrier molecules can enhance DNA extraction methodologies in microfluidic applications.

Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device.

A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification.