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. Copyright © 2002 John Wiley & Sons, Ltd.