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.