Analysis of several fluorescent detector molecules for protein microarray use

Abstract

The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE-antibody complex. The two array architectures tested consisted of an array of murine Fc-biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc-biotin array, detector signal intensity (background adjusted) was as follows: RPE-antibody complex > fluospheres > RPE > liposomes > Alexa 546: for the IgG array: RPE/antibody complex > RPE > fluospheres > Alexa546 > liposomes. The RPE-antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc-biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE-antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE-antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays.

Sensitive Chemiluminescence Detection of Prion Protein on a Membrane by Using a Peroxidase-Labeled Dextran Probe

“…1 It allows fast and simultaneous detection of a number of addressable proteins in a single experiment. 2,3 Protein-microarray technology uses surface chemistry for facile immobilization of the proteins onto a solid support for recognizing target proteins; also, a specific protein-protein interaction can be detected by fluorescence 4 or chemiluminescence (CL)-triggering enzyme-labeled probes, [5][6][7] such as horseradish peroxidase (HRP)-labeled streptavidin (HRP-SA), biotin-labeled HRP (b-HRP) and biotin-labeled alkaline phosphatase (b-ALP). Therefore, in a CL detection system utilizing HRP-catalyzed oxidation of luminol, one of the chemiluminogenic compounds has been widely applied to immunoassays due to high sensitivity of the detection, radioactivity-free handling and inexpensive instrumentation.…”

Silica-coated liposomes loaded with quantum dots as labels for multiplex fluorescent immunoassay

“…Loaded liposomes were successfully applied in different immunassays, such as the microtiter-plate sorbent assay [8][9][10][11], flow-injection analysis [12][13][14], lateral flow on-site tests [15], chemiluminescent [16] and electrochemical [17] biosensors and also microarray [18].…”

Optical Detection of Single Nanoparticles and Viruses

“…Furthermore, specially engineered particles such as nanoshells are employed for photothermal tumor ablation and for cancer therapies [12]. Polymer nanoparticles are used as calibration standards and, in functionalized form, also as probes in biological imaging [13]. Therefore, the ability to detect and characterize single nanoparticles is beneficial for a broad range of applications.…”