Biomedical subjects

Alison M Skelley

Publications and source records attributed to Alison M Skelley.

6 recordsLinked to original sources

PubMed2006

Microfluidic serial dilution circuit.

In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipeting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

Dimethylpolysiloxanes↗

PubMed2006

Rapid on-column analysis of glucosamine and its mutarotation by microchip capillary electrophoresis.

A novel electrophoretic microchip method for analyzing alpha- and beta-d-glucosamine and their interconversion in solution is presented. d-Glucosamine is labeled with fluorescamine and analyzed by capillary electrophoresis in under 2 min revealing its pH-dependent mutarotation between the alpha- and beta-anomers. The forward interconversion rates for the labeled sugars, based on an iterative analysis of the plateau heights between the peaks, are 0.72+/-0.09, 1.3+/-0.1, and 2.2+/-0.3 x 10(-3)s(-1) at pH 8.99, 9.51 and 10.01, respectively. In a separate experiment, the mutarotation of the unlabeled alpha-d-anomer was followed; the relative intensities of the alpha- and beta-peaks as a function of reaction time at pH 9.51 give a forward rate constant of 0.6+/-0.1 x 10(-3)s(-1). These results demonstrate that fast microchip separations, previously exploited for amine, amino acid, and nucleobase analysis, can also be used to analyze amino sugars and their mutarotation.

Electrophoresis, Microchip↗

PubMed2006

Application of the Mars Organic Analyzer to nucleobase and amine biomarker detection.

The Mars Organic Analyzer (MOA), a portable microfabricated capillary electrophoresis instrument being developed for planetary exploration, is used to analyze a wide variety of fluorescamine-labeled amine-containing biomarker compounds, including amino acids, mono and diaminoalkanes, amino sugars, nucleobases, and nucleobase degradation products. The nucleobases cytosine and adenine, which contain an exocyclic primary amine, were effectively labeled, separated, and detected at concentrations <500 nM. To test the general applicability of the MOA for biomarker detection, amino acids and mono- and diamines were extracted from bacterial cells using both hydrolysis and sublimation followed by analysis. The extrapolated limit of detection provided by the valine biomarker was approximately 4 x 10(3) cells per sample. Products of an NH(4)CN polymerization that simulate a prebiotic synthesis were also successfully isolated via sublimation and analyzed. Adenine and alanine/serine were detected with no additional sample cleanup at 120 +/- 13 microM and 4.1 +/- 1 microM, respectively, corresponding to a reaction yield of 0.04% and 0.0003%, respectively. This study demonstrates that the MOA provides sensitive detection and analysis of low levels of a wide variety of amine-containing organic compounds from both biological and abiotic sources.

Adenine↗

PubMed2005

Development and evaluation of a microdevice for amino acid biomarker detection and analysis on Mars.

The Mars Organic Analyzer (MOA), a microfabricated capillary electrophoresis (CE) instrument for sensitive amino acid biomarker analysis, has been developed and evaluated. The microdevice consists of a four-wafer sandwich combining glass CE separation channels, microfabricated pneumatic membrane valves and pumps, and a nanoliter fluidic network. The portable MOA instrument integrates high voltage CE power supplies, pneumatic controls, and fluorescence detection optics necessary for field operation. The amino acid concentration sensitivities range from micromolar to 0.1 nM, corresponding to part-per-trillion sensitivity. The MOA was first used in the lab to analyze soil extracts from the Atacama Desert, Chile, detecting amino acids ranging from 10-600 parts per billion. Field tests of the MOA in the Panoche Valley, CA, successfully detected amino acids at 70 parts per trillion to 100 parts per billion in jarosite, a sulfate-rich mineral associated with liquid water that was recently detected on Mars. These results demonstrate the feasibility of using the MOA to perform sensitive in situ amino acid biomarker analysis on soil samples representative of a Mars-like environment.

Amino Acids↗

PubMed2003

Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration.

Chiral separations of fluorescamine-labeled amino acids are characterized and optimized on a microfabricated capillary electrophoresis (CE) device. A standard mixture of acidic and neutral amino acids is labeled with fluorescamine in less than 5 min and the hydroxypropyl-beta-cyclodextrin (HPbetaCD) concentration, temperature, and pH are optimized (15 mM HPbetaCD, 6 degrees C, pH < 9) to achieve high-quality and low background chiral separations in less than 200 s. All four stereoisomers formed in the labeling reaction of the chiral dye with the chiral amino acids are typically resolved. At pH > 9, isomerization of the dye chiral center is observed that occurs on the time scale of the chip separation. Typical limits of detection are approximately 50 nM. These results demonstrate the feasibility of combining fluorescamine labeling of amino acids with microfabricated CE devices to develop low-volume, high-sensitivity apparatus and methods for extraterrestrial exploration.

Electrophoresis, Capillary↗

PubMed2003

Integrated hydrogenated amorphous Si photodiode detector for microfluidic bioanalytical devices.

Hydrogenated amorphous silicon (a-Si:H) PIN photodiodes have been developed and characterized as fluorescence detectors for microfluidic analysis devices. A discrete a-Si:H photodiode is first fabricated on a glass substrate and used to detect fluorescent dye standards using conventional confocal microscopy. In this format, the limit of detection for fluorescein flowing in a 50-microm deep channel is 680 pM (S/N = 3). A hybrid integrated detection system consisting of a half-ball lens, a ZnS/YF3 multilayer optical interference filter with a pinhole, and an annular a-Si:H photodiode is also developed that allows the laser excitation to pass up through the central aperture in the detector. Using this integrated detection device, the limit of detection for fluorescein is 17 nM, and DNA fragment sizing and chiral analysis of glutamic acid are successfully performed. The a-Si:H detector exhibits high sensitivity at the emission wavelengths of commonly used fluorescent dyes and is readily microfabricated and integrated at low cost making it ideal for portable microfluidic bioanalyzers and emerging large scale integrated microfluidic technologies.

Bacteriophage phi X 174↗