Clickable Multifunctional Dumbbell Particles for in Situ Multiplex Single-Cell Cytokine Detection Peng Zhao, † Justin George, † Bin Li, † Nooshin Amini, # Janet Paluh, # and Jun Wang* ,†,‡ † Multiplex Biotechnology Laboratory, Department of Chemistry, University at Albany, State University of New York, Albany, New York 12222, United States # College of Nanoscale Science and Engineering, State University of New York Polytechnic Institute, Albany, New York 12203, United States ‡ Cancer Research Center, University at Albany, State University of New York, Rensselaer, New York 12144, United States * S Supporting Information ABSTRACT: We report a novel strategy for fabrication of multifunctional dumbbell particles (DPs) through click chemistry for monitoring single-cell cytokine releasing. Two different types of DPs were prepared on a large scale through covalent bioorthogonal reaction between methyltetrazine and trans-cyclooctene on a microchip under a magnetic field. After collection of the DPs, the two sides of each particle were further functionalized with different antibodies for cell capturing and cytokine detection, respectively. These DPs labeled with different fluorescent dyes have been used for multiplex detection and analysis of cytokines secreted by single live cells. Our results show that this new type of DPs are promising for applications in cell sorting, bioimaging, single-cell analysis, and biomedical diagnostics. KEYWORDS: microspheres, dumbbell, microarray, single cell, cytokine 1. INTRODUCTION Janus particles possessing distinct surfaces with different chemical compositions have attracted enormous attention. 1 The asymmetry of the surfaces drastically imparts the particles directionality and new physical/chemical properties, such as optical activity, mechanical strength, and electronic and magnetic properties, which are inconceivable for homogeneous symmetric particles. The compartmentalization of Janus particles is highly dependent on synthesis approaches, which are normally based on modification of immobilized particles on a surface, microfluidic solidification, and phase separation. 2-5 Although a variety of Janus particles have been introduced in the past decade, exploration of their biomedical applications has only been emerging recently. 6,7 The major challenge that hinders the adoption of Janus particles in biomedical applications is the lack of easy-to-follow strategy for generating the nano- to micro-sized anisotropic particles, which possess practically useful functionalities superior to their homogeneous counterparts. 8 For example, the phase separation synthesis methods rely on the interaction energy between precursors and therefore require special fabrication conditions. 9,10 Surface immobilization approaches often result in limited quantities of produced particles that are not enough for molecular and cellular analyses. This is mainly because a high quality of monolayer must be ensured but adversely is difficult to scale up. 11 Other synthesis methods such as microfluidic techniques, seed polymerization technol- ogy, and gel trapping, 10-12 are either not amenable to scale up or not convenient for further functionalization and applications. The other related issue about the utility of Janus particle is to find a niche where the special functionality of the particles converges with real biomedical needs. Thus, the chemical composition of the particles should be tailored to biomolecule conjugation. Conjugation of antiepidermal growth factor receptor antibody on one side of dumbbell shape Au-Fe 3 O 4 nanoparticles (NPs) facilitates the engulfment of particles while the Au compartment enables optical visualization of cancer cells. 13 The similar strategy has been used for multimodal dark- field, surface-enhanced Raman spectroscopy, two-photon and fluorescence imaging as well as MRI imaging. 14,15 Many complicated Janus particles have been used as barcoding device for multiplexed sensing of DNA and proteins. 16-18 These conjugations have not been integrated into fabrication process, or are not as convenient as click chemistry. The bioorthogonal reaction between a tetrazine (Tz) and a trans-cyclooctene (TCO) has been used for small molecular labeling. 19 The [4 + 2] cycloaddition is fast, chemoselective, and does not require a catalyst. This clickable reaction permits conjugation of virtually Received: June 10, 2017 Accepted: September 8, 2017 Published: September 8, 2017 Research Article www.acsami.org © XXXX American Chemical Society A DOI: 10.1021/acsami.7b08338 ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX