Encoded Capsules DOI: 10.1002/anie.200906498 Inwards Buildup of Concentric Polymer Layers: A Method for Biomolecule Encapsulation and Microcapsule Encoding** Jianhao Bai, Sebastian Beyer, Wing Cheung Mak, Raj Rajagopalan, and Dieter Trau* The phenomenon of polyelectrolytes being able to form complexes with each other have allowed researchers to achieve fabrication of hydrogel particles from these polyelec- trolyte complexes, [1] gene delivery, [2] and fabrication of ultra- thin polymeric multilayers. [3] Fabrication of these polyelec- trolyte multilayers by the layer-by-layer (LbL) technique is now used regularly for the encapsulation of biomolecules within microcapsules. [4] Encapsulation of biomolecules, such as proteins [5] and DNA, [6] within microcapsules can be used for many biomedical applications, which include but are not limited to biosensors, [7] bioreactors, [8] and cell targeting [9] and release applications. [10] Although many microcapsule fabrica- tion techniques have been developed for the encapsulation of biomolecules, to our knowledge, none of these techniques demonstrate the simultaneous capability of encoding. Herein we present the inwards buildup of concentric colored polymeric layers for the fabrication of striated multicolored spherical shells within agarose microbeads. These shells can simultaneously encapsulate biomolecules within and encode the microbeads. Using a hydrogel as microcapsule core material can provide a favorable environ- ment for biomolecules and maintain the spherical shape of microcapsules. Encoding of the microcapsules is achieved through color and layer thickness permutation, thereby providing up to two levels of encoding for each microcapsule. These concentric polymer layers form in both agarose and alginate microbeads (Supporting Information, Figure S1) dispersed in 1-butanol. However, for the ease of comparing encoding and encapsulation, in the following, only those results obtained from agarose microbeads will be described. The general steps for the inwards buildup of concentric colored polymer layers into the matrices of agarose microbeads are shown in Scheme 1. Agarose microbeads containing the desired biomolecules to be encapsulated are first dispersed in 1-butanol. An organic solvent was used so as to minimize loss of pre-loaded biomolecules [11] during the fabrication process that forms the striated shells. Next, fluorescence-labeled non- ionic (free base) poly(allylamine) (niPA) in 1-butanol is added to the microbead suspension and the first concentric colored layer is formed. The microbeads are then washed with 1-butanol and incubated with another fluorescence-labeled niPA in 1-butanol to form the second concentric colored layer. This incubation and washing process is repeated until the desired number and permutation of concentric colored layers for encoding purpose is obtained (non-fluorescently labeled niPA can also be used for the color encoding, which leads to a non-colored layer). Interestingly, discrete multiple polymer layers can be observed to build up inwards into each agarose microbead matrix by repeated incubation of niPA. As a final step, the striated multicolored polymeric shells are stabilized by cross-linking with disuccinimidyl suberate (DSS) before transferring into 0.01  PBS (phosphate buffered saline). Overlayed bright-field and confocal images of agarose microbeads in 1-butanol, with different numbers of concentric Scheme 1. The inwards buildup of concentric colored polymer layers into the matrices of agarose microbeads for the encapsulation of biomolecules and encoding. The polymer used is non-ionized poly- (allylamine) (niPA). [*] Prof. R. Rajagopalan, Dr. D. Trau Department of Chemical & Biomolecular Engineering National University of Singapore Engineering Drive 1, 117576 (Singapore) Fax: (+ 65) 6872-3069 E-mail: bietrau@nus.edu.sg Homepage: http://www.biosingapore.com J. Bai, S. Beyer, Dr. D. Trau Division of Bioengineering National University of Singapore Engineering Drive 1, 117574 (Singapore) S. Beyer NUS Graduate School for Integrative Sciences and Engineering National University of Singapore 28 Medical Drive, 117456 (Singapore) Dr. W. C. Mak Department of Chemistry Hong Kong University of Science and Technology Hong Kong SAR (P.R. China) [**] This work was supported by Research Grant R-397-000-077-112 from the National University of Singapore (NUS). We thank Colin Sheppard and Lu Fa Ke of the Bioimaging Laboratory, NUS, for assistance and use of the confocal microscope. We are grateful for the comments raised by the anonymous referees that allowed us to improve this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.200906498. A ngewandte Chemi e 5189 Angew. Chem. Int. Ed. 2010, 49, 5189 –5193  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim