Thiamine deficiency disorders: diagnosis, prevalence, and a roadmap for global control programs.

Thiamine is an essential micronutrient that plays a key role in energy metabolism. Many populations worldwide may be at risk of clinical or subclinical thiamine deficiencies, due to famine, reliance on staple crops with low thiamine content, or food preparation practices, such as milling grains and washing milled rice. Clinical manifestations of thiamine deficiency are variable; this, along with the lack of a readily accessible and widely agreed upon biomarker of thiamine status, complicates efforts to diagnose thiamine deficiency and assess its global prevalence. Strategies to identify regions at risk of thiamine deficiency through proxy measures, such as analysis of food balance sheet data and month-specific infant mortality rates, may be valuable for understanding the scope of thiamine deficiency. Urgent public health responses are warranted in high-risk regions, considering the contribution of thiamine deficiency to infant mortality and research suggesting that even subclinical thiamine deficiency in childhood may have lifelong neurodevelopmental consequences. Food fortification and maternal and/or infant thiamine supplementation have proven effective in raising thiamine status and reducing the incidence of infantile beriberi in regions where thiamine deficiency is prevalent, but trial data are limited. Efforts to determine culturally and environmentally appropriate food vehicles for thiamine fortification are ongoing.

A novel portable filtration system for sampling and concentration of microorganisms: Demonstration on marine microalgae with subsequent quantification using IC-NASBA.

This paper presents a novel portable sample filtration/concentration system, designed for use on samples of microorganisms with very low cell concentrations and large volumes, such as water-borne parasites, pathogens associated with faecal matter, or toxic phytoplankton. The example application used for demonstration was the in-field collection and concentration of microalgae from seawater samples. This type of organism is responsible for Harmful Algal Blooms (HABs), an example of which is commonly referred to as "red tides", which are typically the result of rapid proliferation and high biomass accumulation of harmful microalgal species in the water column or at the sea surface. For instance, Karenia brevis red tides are the cause of aquatic organism mortality and persistent blooms may cause widespread die-offs of populations of other organisms including vertebrates. In order to respond to, and adequately manage HABs, monitoring of toxic microalgae is required and large-volume sample concentrators would be a useful tool for in situ monitoring of HABs. The filtering system presented in this work enables consistent sample collection and concentration from 1 L to 1 mL in five minutes, allowing for subsequent benchtop sample extraction and analysis using molecular methods such as NASBA and IC-NASBA. The microalga Tetraselmis suecica was successfully detected at concentrations ranging from 2 × 105 cells/L to 20 cells/L. Karenia brevis was also detected and quantified at concentrations between 10 cells/L and 106 cells/L. Further analysis showed that the filter system, which concentrates cells from very large volumes with consequently more reliable sampling, produced samples that were more consistent than the independent non-filtered samples (benchtop controls), with a logarithmic dependency on increasing cell numbers. This filtering system provides simple, rapid, and consistent sample collection and concentration for further analysis, and could be applied to a wide range of different samples and target organisms in situations lacking laboratories.

Open-Source Potentiostat for Wireless Electrochemical Detection with Smartphones.

This paper describes the design and characterization of an open-source "universal wireless electrochemical detector" (UWED). This detector interfaces with a smartphone (or a tablet) using "Bluetooth Low Energy" protocol; the smartphone provides (i) a user interface for receiving the experimental parameters from the user and visualizing the result in real time, and (ii) a proxy for storing, processing, and transmitting the data and experimental protocols. This approach simplifies the design, and decreases both the size and the cost of the hardware; it also makes the UWED adaptable to different types of analyses by simple modification of the software. The UWED can perform the most common electroanalytical techniques of potentiometry, chronoamperometry, cyclic voltammetry, and square wave voltammetry, with results closely comparable to benchtop commercial potentiostats. Although the operating ranges of electrical current and voltage of the UWED (±1.5 V, ±180 µA) are more limited than most benchtop commercial potentiostats, its functional range is sufficient for most electrochemical analyses in aqueous solutions. Because the UWED is simple, small in size, assembled from inexpensive components, and completely wireless, it offers new opportunities for the development of affordable diagnostics, sensors, and wearable devices.

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings.

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/µL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

Sliding-strip microfluidic device enables ELISA on paper.

This article describes a 3D microfluidic paper-based analytical device that can be used to conduct an enzyme-linked immunosorbent assay (ELISA). The device comprises two parts: a sliding strip (which contains the active sensing area) and a structure surrounding the sliding strip (which holds stored reagents-buffers, antibodies, and enzymatic substrate-and distributes fluid). Running an ELISA involves adding sample (e.g. blood) and water, moving the sliding strip at scheduled times, and analyzing the resulting color in the sensing area visually or using a flatbed scanner. We demonstrate that this device can be used to detect C-reactive protein (CRP)-a biomarker for neonatal sepsis, pelvic inflammatory disease, and inflammatory bowel diseases-at a concentration range of 1-100ng/mL in 1000-fold diluted blood (1-100µg/mL in undiluted blood). The accuracy of the device (as characterized by the area under the receiver operator characteristics curve) is 89% and 83% for cut-offs of 10ng/mL (for neonatal sepsis and pelvic inflammatory disease) and 30ng/mL (for inflammatory bowel diseases) CRP in 1000-fold diluted blood respectively. In resource-limited settings, the device can be used as a part of a kit (containing the device, a fixed-volume capillary, a pre-filled tube, a syringe, and a dropper); this kit would cost ~ $0.50 when produced in large scale (>100,000 devices/week). This kit has the technical characteristics to be employed as a pre-screening tool, when combined with other data such as patient history and clinical signs.

Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification.

Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card.

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform.

The widespread dissemination of CTX-M extended spectrum ß-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

A fluorogenic heterogeneous immunoassay for cardiac muscle troponin cTnI on a digital microfluidic device.

We describe a fluorogenic two-site noncompetitive heterogeneous immunoassay with magnetic beads on a low-voltage digital microfluidic platform using closed electrowetting-on-dielectric (EWOD). All the steps of an enzyme-linked immunosorbent assay (ELISA) were performed on the device using 9H-(1, 3-dichloro-9, 9-dimethylacridin-2-one-7-yl) phosphate as the fluorogenic substrate for the enzyme alkaline phosphatase. The performance of the system was demonstrated with cardiac marker Troponin I (cTnI) as a model analyte in phosphate-buffered saline samples. cTnI was detected within the diagnostically relevant range with a limit of detection of 2.0 ng/mL (CV = 6.47 %). Washing of magnetic beads was achieved by movement through a narrow region of buffer bridging one drop to another with minimal fluid transfer. More than 90 % of the unbound reagents were removed after five washes. Further experiments testing human blood serum on the same platform demonstrated a sample-to-answer time at ∼18.5 min detecting 6.79 ng/mL cTnI.

Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform.

Quantitation of specific RNA sequences is a useful technique in marine biology that can elucidate cell abundance, speciation and viability, especially for early detection of harmful algal blooms. We are thus developing an integrated microfluidic system for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system is based on a microfluidic cartridge, or "lab-card", using a low-cost injection moulded device, with a laminated lid. Here we present real-time isothermal RNA amplification using reagent master-mixes preserved on-chip in a gel at 4 °C for up to eight months. We demonstrate quantitation by reference to an internal control in a competitive assay with 500 cell equivalents of the toxic microalga Karenia brevis. Annealing of primers, amplification at 41 °C and real-time fluorescence detection of the internal control and target using sequence-specific molecular beacons were all performed on-chip.

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus.

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V(max), depending on the lipid concentration and vesicle composition, but no change in K(m). Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.