We have developed a simple and direct method to fabricate paper-based microfluidic devices that can be used for a wide range of colorimetric assay applications. With these devices, assays can be performed within minutes to allow for quantitative colorimetric analysis by use of a widely accessible iPhone camera and an RGB color reader application (app) to measure color intensity. In the described laboratory experiment, students design and create their own microfluidic devices with common laboratory supplies such as Kimwipes, Parafilm, and a thermal laminator, and gain hands-on experience in the analysis of Fe 2+ and Cu 2+ by colorimetric determination.
Current colorimetric presumptive identification of illicit drugs for determining illegal possession of controlled substances by law enforcement relies solely on the subjective interpretation of color change using drug- or class-specific reactions. Here, we describe the use of inexpensive polyester-toner, rotation-driven microfluidic devices with a smartphone as a potential alternative for current presumptive colorimetric field-testing of illicit drugs, allowing for an objective and user-friendly image analysis technique for detection. The centrifugal microfluidic platform accommodates simultaneous presumptive testing of material from a single input to multiple reaction chambers, enabling rapid screening. Hue and saturation image analysis parameters are used to define threshold values for the detection of cocaine and methamphetamine as proof-of-principle with 0.25 and 0.75 mg/mL limits of detection, respectively, with nonvolatile reagents stored on-board and smartphone for detection. Reported LODs are lower than those concentrations used in the field. Additionally, the developed objective detection method addresses the testing of drugs with various common cutting agents, including those known to produce false negative and positive results. We demonstrate the effectiveness of the method by successfully identifying the composition of 30 unknown samples.
Networks of neurons control feeding and activity patterns by integrating internal metabolic signals of energy balance with external environmental cues such as time-of-day. Proper circadian alignment of feeding behavior is necessary to prevent metabolic disease, and thus it is imperative that molecular players that maintain neuronal coordination of energy homeostasis are identified. Here, we demonstrate that mice lacking the p75 neurotrophin receptor, p75NTR, decrease their feeding and food anticipatory behavior (FAA) in response to daytime, but not nighttime, restricted feeding. These effects lead to increased weight loss, but do not require p75NTR during development. Instead, p75NTR is required for fasting-induced activation of neurons within the arcuate hypothalamus. Indeed, p75NTR specifically in AgRP neurons is required for FAA in response to daytime restricted feeding. These findings establish p75NTR as a novel regulator gating behavioral response to food scarcity and time-of-day dependence of circadian food anticipation.
The use of overhead transparencies as substrates for the fabrication of microfluidic devices brings new capabilities in the creation of fully-integrated systems.
WR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human dhfr have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of 1 and 2; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed 1 as WR99210 and 2 as a dihydrotriazine regioisomer. Induced fit, computational docking models showed that 1 binds tightly and specifically in the P. falciparum DHFR active site whereas 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.
ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human dhfr have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of 1 and 2; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed 1 as WR99210 and 2 as an isomeric dihydrotriazine. Induced fit, computational docking models showed that 1 binds tightly and specifically in the P. falciparum DHFR active site whereas 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of isomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that can promote isomerization. Absorption spectroscopy may serve for assays of the unrearranged and rearranged triazines.
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