Photo- and Biophysical Studies of Lectin-Conjugated Fluorescent Nanoparticles: Reduced Sensitivity in High Density Assays Yaqi Wang Jeffrey C. Gildersleeve Amit Basu Matthew B. Zimmt 10.1021/jp101854m.s002 https://acs.figshare.com/articles/dataset/Photo_and_Biophysical_Studies_of_Lectin_Conjugated_Fluorescent_Nanoparticles_Reduced_Sensitivity_in_High_Density_Assays/2711932 Lectin-conjugated, fluorescent silica nanoparticles (fNP) have been developed for carbohydrate-based histopathology evaluations of epithelial tissue biopsies. The fNP platform was selected for its enhanced emissive brightness compared to direct dye labeling. Carbohydrate microarray studies were performed to compare the carbohydrate selectivity of the mannose-recognizing lectin Concanavalin A (ConA) before and after conjugation to fluorescent silica nanoparticles (ConA−fNP). These studies revealed surprisingly low emission intensities upon staining with ConA−fNP compared to those with biotin−ConA/Cy3−streptavidin staining. A series of photophysical and biophysical characterizations of the fNP and ConA−fNP conjugates were performed to probe the low sensitivity from fNP in the microarray assays. Up to 1200 fluorescein (FL) and 80 tetramethylrhodamine (TR) dye molecules were incorporated into 46 nm diameter fNP, yielding emissive brightness values 400 and 35 times larger than the individual dye molecules, respectively. ConA lectin conjugated to carboxylic acid surface-modified nanoparticles covers 15−30% of the fNP surface. The CD spectra and mannose substrate selectivity of ConA conjugated to the fNP differed slightly compared to that of soluble ConA. Although, the high emissive brightness of fNP enhances detection sensitivity for samples with low analyte densities, large fNP diameters limit fNP recruitment and binding to samples with high analyte densities. The high analyte density and nearly two-dimensional target format of carbohydrate microarrays make probe size a critical parameter. In this application, fNP labels afford minimal sensitivity advantage compared to direct dye labeling. 2010-11-18 00:00:00 emissive brightness values 400 46 nm diameter fNP fNP diameters limit fNP recruitment dye molecules Carbohydrate microarray studies mannose substrate selectivity ConA FL emissive brightness silica nanoparticles TR epithelial tissue biopsies analyte densities