Water-Stable Biphasic Nanocolloids with Potential Use as Anisotropic Imaging Probes Kyung-Ho Roh, Mutsumi Yoshida, and Joerg Lahann* Macromolecular Science and Engineering Center, Department of Chemical Engineering, and Department of Materials Science and Engineering, The UniVersity of Michigan, 48109 Ann Arbor, Michigan ReceiVed August 1, 2006. In Final Form: NoVember 8, 2006 Electrified co-jetting of two aqueous polymer solutions followed by a thermal cross-linking step was used to create water-stable biphasic nanocolloids. For this purpose, aqueous solution mixtures of poly(acrylamide-co-acrylic acid) and poly(acrylic acid) were employed as jetting solutions. When the biphasic nanocolloids created by side-by-side electrified co-jetting were thermally treated, a cross-linking reaction occurred between amide groups and carboxylic groups to form stable imide groups. Infrared spectroscopy was employed to monitor the reaction. The quality and the integrity of the resulting biphasic nanocolloids were confirmed by confocal laser scanning microscopy, flow cytometry analysis, and dynamic light scattering. Selective encapsulation of two biomolecules in each phase of the biphasic colloids was maintained even after thermal reaction and suspension in aqueous environment. Well-dispersed spherical colloids with stable dye loadings in each hemisphere were kept intact without aggregation or dissolution for several weeks. Finally, biphasic nanocolloids were selectively surface-modified with a biotin-dextran resulting in water-stable particles to ensure binding of proteins only to a single hemisphere. Introduction Recent advances in the field of nano-biotechnology are pointing toward the design of functional probes that are able to self-orient relative to a cell surface, thereby encodingsand ultimately revealingsuseful biological information. 1 In this concept, the controlled distribution of matter or “patchiness” 2,3 is important for creating anisotropic building blocks and introduces an additional design parametersin addition to particle geometry. 4 A series of methods have been recently reported on the fabrication of anisotropic particles, such as Janus particles. 5 Janus particles have been made by partial masking, 6-8 selective deposition, 9,10 and microcontact printing. 11 Two-sided particles with different material compositions in each side have also been created by a range of methods including template-assisted self-assembly of particles, 12,13 rearrangement or recrystallization of materials from precursor core-shell particles, 14,15 phase separation in emulsion polymerization, 15 and partial or selective surface nucleation 17-19 and microfluidic co-flow. 20,21 We recently pursued an alternative route toward the design and synthesis of polymer-based nanoparticles with two or multiple distinct phases, which exploits electrified co-jetting. 22,23 We demonstrated that individual phases of the Janus particles can be independently loaded with biomolecules or selectively modified with model ligands. 22,23 Prior to our work, electrified jetting, a process to generate liquid jets by use of electrohy- drodynamics, was used for the creation of aerosol, macromo- lecular ions, nanospheres, and nanofibers. 24-26 When side-by- side dual capillaries 27,28 are used for processing of two parallel polymer solutions pumped at a controlled flow rate, a biphasic liquid droplet is formed at the outlet of the capillaries. If an electric potential above a certain value (generally several kilovolts, depending on the jetting liquids) is applied between the conductive capillaries and the substrate, an electrostatically charged liquid droplet forms in a cone shape (Taylor cone) 29 and an ultrathin liquid thread is ejected out of the apex of the droplet, where the two aqueous jetting liquids interface with each other. The anisotropic material distribution originally stemming from the liquid regimen is maintained throughout the jet formation, vigorous jet extension, jet breakdown, and solvent evaporation, finally resulting in solid Janus-type nanocolloids. 22 In the co- jetting process, the use of water-based jetting solutions has various advantages, such as the secure working environments, which simplifies scale-up of the manufacturing process, the compatibility with biomolecules, or the appropriate volatility of water for the generation of nanocolloids under ambient conditions. 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