Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy Bong-Hyun Jun a,b , Mi Suk Noh c , Gunsung Kim d , Homan Kang c , Jong-Ho Kim a,1 , Woo-Jae Chung a,2 , Min-Soo Kim b , Yong-Kweon Kim b,c , Myung-Haing Cho c,e, * , Dae Hong Jeong c,d, * , Yoon-Sik Lee a,c, * a School of Chemical and Biological Engineering, Seoul National University, Seoul 151-747, Republic of Korea b School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-747, Republic of Korea c Interdisciplinary Program in Nano-Science and Technology, Seoul National University, Seoul 151-747, Republic of Korea d Department of Chemistry Education, Seoul National University, Seoul 151-747, Republic of Korea e College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul 151-747, Republic of Korea article info Article history: Received 11 February 2009 Available online 9 May 2009 Keywords: Silver nanoparticles Optically active materials Magnetic materials Polymeric materials Surface-enhanced Raman scattering abstract This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8 lm diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling mol- ecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-amino- thiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology. Ó 2009 Elsevier Inc. All rights reserved. Magnetic microparticles are widely used in the biomedical field as a solid support for immunoassays, DNA sequencing, and cell analysis. The magnetic microparticle-based bioassay has the advantages of quick, easy, and gentle separation of biological com- pounds by using an external magnetic field gradient [1–6]. For tak- ing advantage of magnetic properties, many multifunctional materials have been reported for use in bioapplications [7–12]. Re- cently, some magnetic bead-based materials have been developed for microfluidic immunoassays [13]. However, current magnetic bead-based microfluidic immunoassays have limited capability when used in multiplexed detection in comparison with flow cyto- metric assays. Flow cytometry is one of most commonly used methods for assaying suspended particles in a high-throughput manner. Flow cytometry provides high accuracy in relative quantification (per- centage abundance) of cells or particles among diverse mixed samples. Moreover, flow cytometry can separate each particle or cell based on a statistical difference of any one of 10 to 20 variables [14–16]. However, multiplexing is limited by encoding numbers that can be generated by both encoding and decoding strategies when fluorescence dyes with broad spectral bands are used. Recently, quantum dot-encoded beads were used for multiplex as- say, which demonstrated many advantages over traditional dyes due to improved narrower emission peaks ( 30 nm in peak width) with higher intensity [17–20]. Surface-enhanced Raman spectroscopy (SERS) 3 encoding meth- ods have also emerged as another class of labeling methods. SERS encoding methods have advantages such that a single laser line is 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.005 * Corresponding authors. Fax: +82 2 876 9625 (Y.-S. Lee); fax: +82 2 873 1268 (M.-H. Cho); fax: +82 2 889 0749 (D.H. Jeong). E-mail addresses: mchotox@snu.ac.kr (M.-H. Cho), jeongdh@snu.ac.kr (D.H. Jeong), yslee@snu.ac.kr (Y.-S. Lee). 1 Present address: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 2 Present address: Department of Bioengineering, University of California, Berkeley, CA 94720, USA. 3 Abbreviations used: SERS, surface-enhanced Raman spectroscopy; NP, nanoparticle; M-SERS bead, SERS-encoded magnetic bead; PS–DVB, polystyrene–divinylbenezene; PVP-40, polyvinylpyrrolidone-40; AIBN, 2,2 0 -azobis-isobutyronitrile; DBP, dibutyl phthalate; SDS, sodium dodecyl sulfate; PVA, poly(vinyl alcohol); BPO, dibenzoyl peroxide; THF, tetrahydrofuran; TFA, trifluoroacetic acid; FE–SEM, field emission scanning electron microscopy; TEM, transmission electron microscopy; EDX, energy dispersive X-ray; ATP, 4-aminothiophenol; 4-MT, 4-methylbenzenethiol; 2-NT, 2- naphthalenethiol; BT, benzenethiol; MPTS, 3-mercaptopropyltrimethoxysilane; TEOS, tetraethylorthosilicate; CCD, charge-coupled device; APTS, 3-aminopropyltriethoxysi- lane; DMF, N,N-dimethylformamide; Fmoc–ACA–OH, Fmoc-e-aminocaproic acid; BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; HOBT, 1-hydroxybenzotriazole; DIEA, N,N-diisopropylethylamine; PBS, phosphate buffer solution; BSA, bovine serum albumin; CLSM, confocal laser scanning microscopy. Analytical Biochemistry 391 (2009) 24–30 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio