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We have discovered a phenomenon where the electrophoretic mobility of a QD-DNA nanoassembly can be precisely and predictably tuned by the level of surface DNA conjugation. By using streptavidin-coated quantum dots (QDs) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis. Based on this unique property, we have developed a quantification nanoassay termed as quantum dot electrophoretic mobility assay, or QEMSA. QEMSA demonstrates ultrahigh resolution in quantification, capable of differentiating ~9% difference in DNA quantity. In this report, we further present a new analytical model that better describe the QEMSA process by taking into account the intrinsic charges carried by the quantum dot. We also extend the applications of QEMSA to measure the enzyme activity of exonuclease I.