Fluorescent semiconductor nanoparticles or quantum dots have become a promising platform for the engineering of biofunctional probes for a variety of biomedical applications ranging from multicolor imaging to single-molecule tracking to traceable drug delivery. for transferring hydrophobic nanoparticles into physiologically-relevant aqueous buffers. Taken together hydrophobic nanoparticle platforms and polymer encapsulation should offer great flexibility for implementation of novel probe designs. However the success of the encapsulation and purification depends on many factors often overlooked in the scientific literature such as close match between nanoparticle and polymer physicochemical properties and dimensions slow dynamics of polymer arrangement on the nanoparticle surface and the size and charge similarity of resultant polymer-coated quantum dots and empty byproduct polymer micelles. To make this general hydrophobic nanoparticle modification IkBKA antibody strategy accessible by a broad range of biomedical research groups we focus on the important technical aspects of nanoparticle polymer encapsulation purification bioconjugation and characterization. Keywords: quantum dot polymer encapsulation bioconjugation fluorescence nanoparticle 1 Introduction Advances in bio-nanotechnology are introducing novel nanoscale PIK-293 materials with unique chemical and physical features potentially useful for advancing PIK-293 existing and creating new biomedical applications. Quantum dots (QDots) fluorescent semiconductor nanoparticles introduced to biomedical research nearly two decades ago [1] have catalyzed development of such directions as single-cell molecular profiling [2 3 real-time PIK-293 molecule tracking [4] in vivo molecular imaging [5] and traceable drug delivery.[6 7 This rich functionality stems from a number of unique photo-physical and chemical properties possessed by QDots. Most notably narrow size-tunable emission profiles featured by nanoparticles of the same composition efficient light absorption over a broad spectral range outstanding photostability and relatively small size comparable to that of large proteins make QDots a versatile and resourceful imaging probe for examination of biological systems.[8] Despite a number of attractive features and innovative proof-of-concept studies published to date QDot technology has made little impact on biomedical discoveries. One factor contributing to the lack of technology adoption is complexity of QDot probe engineering and preparation. A number of water-soluble QDots currently available from commercial sources offer a simple off-the-shelf solution to this issue but only cover basic imaging and detection applications and often prove sub-optimal for implementation of custom probe designs and development of novel methodologies. In this regard high-quality QDots synthesized via organometallic procedure[9] in non-polar solvents and stabilized with hydrophobic surface ligands represent a more versatile platform. The hydrophobic nature makes such nanoparticles incompatible with biologically-relevant assay conditions and requires further surface modification to render nanoparticles PIK-293 water-soluble. One approach polymer encapsulation [10 11 provides a desirable probe design flexibility as custom hydrophilic coatings can be tailored to specific parameters and applications. However many important aspects of QDot probe preparation have not been well described. In particular non-intuitive size and charge similarity between polymer-encapsulated QDots PIK-293 and byproduct empty polymer micelles complicates probe purification and downstream application. Given the lack of expertise working with nanoparticles in the biomedical research community further discussion is warranted. To facilitate implementation of novel QDot probes by a broad range of biomedical research groups we highlight critical steps in probe preparation purification bioconjugation characterization and purity control which are often overlooked in the scientific literature. 2 Results and Discussion 2.1 Preparation of Water-Soluble QDots Hydrophobic QDots were rendered hydrophilic via encapsulation with an amphiphilic polymer poly(maleic anhydride-alt-1-tetradecene) (PMAT MW=9 0 Da) a robust nanoparticle polymer encapsulation procedure described by Pellegrino et al.[11] The general procedure consisted of three main steps (Figure 1): polymer encapsulation of hydrophobic QDots with PMAT cross-linking of a portion of the maleic anhydrides in the polymer shell and rendering particles hydrophilic via hydrolysis of the remaining maleic anhydride.