Tracy L. Paxon

Analysis of biogenic amine variability among individual fly heads with micellar electrokinetic capillary chromatography-electrochemical detection.

Neurochemical variability among individual Drosophila heads has been examined with the sensitivity of electrochemical detection and the selectivity of micellar electrokinetic capillary chromatography. Homogenization of single Drosophila heads in volumes as small as 100 nL has been accomplished. Here we demonstrate reproducible separations for single fly heads in 250-nL volumes providing a 4-fold increase in sensitivity without overloading the electrochemical detector. This increase in sensitivity allows detection of previously undetected analytes, such as N-acetyltyramine (naTA) and octopamine (OA). Analytes including L-3,4-dihydroxyphenylalanine, N-acetyl octopamine, N-acetyldopamine, naTA, N-acetylserotonin, OA, dopamine, tyramine, and serotonin also have been consistently identified in single-head homogenates and observed with homogenates representing populations of Drosophila. Neurochemical variation between individual flies as well as the consistency within a population indicates varying amounts of neurotransmitter turnover. The inception, design, and fabrication of a miniature tissue homogenizer has enabled the separation of biogenic amines and metabolites from these severely volume-limited single Drosophila head homogenates.

Improved Specific Biodetection with Ion Trap Mobility Spectrometry (ITMS): A 10-min, Multiplexed, Immunomagnetic ELISA

Enabling trace chemical detection equipment utilized in the field to transduce a biodetection assay would be advantageous from a logistics, training, and maintenance standpoint. Described herein is an assay design that uses an unmodified, commercial off-the-shelf (COTS) ion trap mobility spectrometer to analyze an immunomagnetic enzyme-linked immunosorbant assay (ELISA). The assay, which uses undetectable enzymatic substrates and ELISA-generated detectable products, was optimized to quantitatively report the amount of target in the sample. Optimization of this ELISA design retained the assay specificity and detection limit (approximately 10(3) E. coli per assay) while decreasing the number of user steps and reducing the assay time to 10 min (>9-fold decrease as compared to past studies). Also discussed are previously undescribed, independent substrate/enzyme/product combinations used in the immunomagnetic ELISA. These discoveries allow for the possibility of a quantitative, multiplexed, 10-min assay that is analyzed by the ion trap mobility spectrometer trace chemical detector.

Matrix Effects by Specific Buffer Components in the Analysis of Metabolites with Ion Trap Mobility Spectrometry

The impact of buffer conditions on the ion trap mobility spectrometer (ITMS) signal is investigated through a series of statistically driven design of experiments (DOEs). A growing number of new ion mobility spectrometry (IMS) and ITMS applications are being performed with a physiological sample matrix, which is vastly different from the particulate and vapor matrixes that have been traditionally analyzed. Currently, there have been no efforts to globally examine the possibility of matrix suppression or enhancement of the IMS signal by these various components within physiological matrixes. This investigation consists of an effort to gauge the effects of common physiological buffer components and concentrations on two analytes of interest for ITMS analysis: o-nitrophenol and ephedrine. We show that, among the factors investigated, for a specific analyte and instrumental detection mode (i.e., negative/positive) the solution pH, presence of a protein, the buffer identity, and buffer concentration should be considered as they will enhance or suppress the ITMS signal while factors such as surfactant and salt concentration may play less of a role in impacting detectable ITMS signal. These observations are supported through statistical analysis of the DOE-derived data set.

Identifying biological agents with surface-enhanced Raman scattering

The anthrax attacks which afflicted the United States in 2001 underscore the necessity of a quick response to biological terrorism agents. Optimal organization and capacity are critical to saving lives at minimal cost.1 Important to this effort is rapid biological agent identification aided by a field-ready analytical protocol. Given its capacity for unique molecular identification, Raman spectroscopy is uniquely poised to contribute to this need. Although Raman spectroscopy readily detects chemical species, direct identification of biological materials is often difficult due to spectral complexity and low signal intensity. Several direct detection methods are available, including extraction and detection of a specific molecular marker, as well as surfaceenhanced Raman spectroscopy (SERS) spectral generation by adsorbing an intact biological species onto a roughened gold or silver surface.2–10 Several indirect SERS detection methods are also available, such as labeling the analyte with a Ramanactive dye and bringing it to the SERS substrate for readout.11, 12 Nanometer-scale SERS-active tags conjugated with specific biological recognition moieties, thereby effecting direct binding to the analyte, is another possibility.13–19 We developed an indirect biological detection system compatible with the Raman-based StreetLab Mobile. It employs SERS tags as unique labels for each target of interest in a sandwich immunoassay format.20, 21 Unique spectroscopic signatures are generated with SERS tags consisting of individual glass-encapsulated gold nanoparticles and surface-bound Raman active reporter molecules, as depicted in Figure 1. These SERS tags are bound to a specific antibody and provide a strong, spectroscopically-consistent label. Superparamagnetic particles conjugated to the antibodies capture and concentrate the SERSlabeled complex at the focal point of the Raman laser using a Figure 1. Diagram of the silica-encapsulated surface-enhanced Raman spectroscopy (SERS) tag.