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Elevated glutamate is implicated in the pathology of PDR. The ability to rapidly assess the glutamate and amino acid content of vitreous provides a more complete picture of the chemical changes occurring at the diabetic retina and may lead to a better understanding of the pathology of PDR. Vitreous humor was collected following vitrectomies of patients with PDR and control conditions of macular hole or epiretinal membrane. A capillary electrophoresis method was developed to quantify glutamate and arginine. The analysis is relatively fast (

The determination of the presence of nitric oxide metabolites in the rat vitreous cavity in a regioselective manner is complicated by the size and shape of the eye as well as the diffusivity of the molecule and its metabolites. In this work, in vivo low-flow push-pull perfusion sampling was utilized with a rapid capillary electrophoretic assay to monitor levels of the major NO metabolite, nitrate, at the vitreoretinal interface (VRI) of normal and aged rat models. The sampling probe tips were placed in three different positions in the posterior chamber through a 29-gauge guide needle. Sampling was performed along the VRI over the optic nerve head and regions peripheral to the optic nerve head. Additionally, samples were collected from the middle vitreous region to compare to VRI sampling. A significant (P < 0.05) difference in the perfusate nitrate concentration was observed in each location, which may be due to the source of NO production or the clearance mechanism of the molecule from the vitreous cavity. Infusion of L-NAME with physiological saline led to a significant decrease (35%) in the observed nitrate level. LFPPP was then utilized to observe nitrate levels after an average of 4.5 months of aging. A 3-fold increase in the mean level of nitrate over the optic nerve head was observed in mature animals compared to younger control animals. Precise measurement of NO metabolites along the VRI may provide insights into the function of NO in maintaining homeostatic conditions and the molecular changes at the diseased retina.

Byline: David E. Featherstone (Department of Biological Sciences, University of Illinois at Chicago, def@uic.edu); Scott A. Shippy (Department of Chemistry, University of Illinois at Chicago) Keywords: Glutamate; Synaptic; Synapse; Glutamate [...]

One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.

A Hadamard transform (HT) detection method for microchip capillary electrophoresis with laser-induced fluorescence and a charge-coupled device (CCD) is described and compared to signal-averaged detection. A low-noise CCD camera is used to image a section of a separation channel where each camera pixel can be thought of as a unique detector. For signal averaging, electropherograms corresponding to individual pixels can be averaged for improved S/N. HT detection is performed on each pixel electropherogram to generate a contour plot electropherogram. The multiple injections required for HT provides an enhancement at the cost of longer times for the pseudorandom injection sequences. A short sample injection length of 0.25 s is used to reduce the overall analysis time and improve sensitivity compared to previously published results. An injection sequence is performed on the microchip that is based on a cyclic S-matrix of 513 elements that generates an 8-fold improvement in S/N compared to a single injection. This spatially resolved HT detection method is also capable of performing a multi-component separation. Signal-averaged HT and single-injection data are compared to experimental HT and single-injection results. The unique capabilities of each method are described.

A competitive immunoassay with near-infrared (NIR) fluorescence detection to analyze microliter biological samples with an amol limit of detection (LOD) is described. An important feature about this technique is that the immunoreaction and fluorescence detection are separated into two distinct steps, allowing for independent optimization. In the immunoreaction step, NIR fluorescence-labeled antigen (Ag*) competes with the unlabeled analyte (Ag) for antibodies (Ab) immobilized on the surface of paramagnetic beads. A magnet is then used to separate the bound antigen from the free in the supernatant. As the amount of Ag in the sample increases, there is less binding between Ag* and immobilized Ab; therefore, the amount of Ag* in the supernatant is proportionally related to the amount of Ag in the sample. In the fluorescence detection step, aliquots of the supernatant are concentrated onto a protein binding membrane by a capillary blotting technique with an optimized 33 nL/min flow rate. The fluorescence of the blotted spots is detected with a NIR sensitive photon counting system that is optimized to an instrumental LOD of 30 000 fluorophore molecules. This competitive assay demonstrates a sample LOD of 400 pg/mL of unlabeled rabbit immunoglobulin G spiked into bovine serum. This design features low sample volumes and reagent consumption.

This paper describes an improved format for Shah convolution Fourier transform (SCOFT) detection that utilizes the spatial resolution of a charge-coupled device (CCD) rather than a fixed optical mask to perform a Shah or sine convolution over a fluorescence signal. The laser-induced fluorescence from a 9-mm section of microfabricated channel is collected with a CCD at 28 Hz. Each image frame is multiplied by a convolution function to modulate the collected signal through space. Each frame is then summed to generate an intensity-versus-time data set for Fourier analysis. The fluorescence signal oscillates at a frequency dependent upon both the convolution function multiplied across each data frame and the velocity of fluorescent microspheres of a plug of fluorescent dye flowing through the channel. This SCOFF technique affords more flexibility over formats that employ a physical mask and provides data that can be optimized for signal-to-noise (S/N) or resolution information. A 1000-fold improvement in S/N is demonstrated for a plug of fluorescein dye. Detection of fluorescent beads exhibited frequency signals that were dependent upon the bead size distribution, the electric field, and the electrophoresis buffer concentration. Data are presented demonstrating the quantitation of fluorescent microspheres.

Elevated glutamate is implicated in the pathology of PDR. The ability to rapidly assess the glutamate and amino acid content of vitreous provides a more complete picture of the chemical changes occurring at the diabetic retina and may lead to a better understanding of the pathology of PDR. Vitreous humor was collected following vitrectomies of patients with PDR and control conditions of macular hole or epiretinal membrane. A capillary electrophoresis method was developed to quantify glutamate and arginine. The analysis is relatively fast (<6 minutes) and utilizes a poly(ethylene)oxide and sodium dodecylsulfate run buffer. Both amino acid levels show significant increases in PDR patients versus controls and are comparable to other reports. The levels of vitreal glutamate vary inversely with the degree of observed hemorrhage. The results demonstrate a rapid method for assessment of a number of amino acids to characterize the chemical changes at the diabetic retina to better understand tissue changes and potentially identify new treatments. [ABSTRACT FROM AUTHOR]

A competitive immunoassay with near-infrared (NIR) fluorescence detection to analyze microliter biological samples with an amol limit of detection (LOD) is described. An important feature about this technique is that the immunoreaction and fluorescence detection are separated into two distinct steps, allowing for independent optimization. In the immunoreaction step, NIR fluorescence-labeled antigen (Ag*) competes with the unlabeled analyte (Ag) for antibodies (Ab) immobilized on the surface of paramagnetic beads. A magnet is then used to separate the bound antigen from the free in the supernatant. As the amount of Ag in the sample increases, there is less binding between Ag* and immobilized Ab; therefore, the amount of Ag* in the supernatant is proportion- ally related to the amount of Ag in the sample. In the fluorescence detection step, aliquots of the supernatant are concentrated onto a protein binding membrane by a capillary blotting technique with an optimized 33 nL/min flow rate. The fluorescence of the blotted spots is detected with a NIR sensitive photon counting system that is optimized to an instrumental LOD of 30 000 fluorophore molecules. This competitive assay demonstrates a sample LOD of 400 pg/mL of unlabeled rabbit immunoglobulin G spiked into bovine serum. This design features low sample volumes and reagent consumption. [ABSTRACT FROM AUTHOR]