Quantum dot (QD)-assembled silica nanoparticles bearing a polydiacetylene (PDA) supramolecule on their surface (SiO2@QDs@PDA NPs) were developed for label-free and multiplexed detection of biological molecules. Two types of QD-assembled silica NPs (SiO2@QDs NPs) were prepared and coated with the PDA supramolecule via photo-induced polymerization of 10,12-pentacosadiynoic acid. One of the SiO2@QDs NPs was embedded with blue-QDs, and the other was embedded with green-QDs for encoding. The resulting SiO2@QDs@PDA NPs showed discrete QD photoluminescence for encoding as well as PDA fluorescence for sensing a target without interference or overlap. Under heating stress of the SiO2@QDs@PDA NPs, the color of the PDA changed from blue to red, which allowed us to observe the fluorescence emitted from red PDA. The mixture of two different SiO2@QDs@PDA NPs, SiO2@QDs@blue-PDA NPs not emitting the fluorescence of PDA and SiO2@QDs@red-PDA NPs where stress was brought onto turn on the PDA fluorescence, was effectively imaged and readily distinguished via fluorescence microscopy, indicating their potential for label-free and multiplexed detection of target molecules. (C) 2012 Elsevier Inc. All rights reserved.