Silver-Nanoparticle-Conjugated Polypeptide Brushes for Surface-Enhanced Raman Scattering Di-Yan Wang, † Tzu-Shen Teng, †,‡ Yi-Chou Wu, † Yi-Cheng Lee, ‡ Kuei-Hsien Chen, § Chung-Hsuan Chen, ‡ Ying-Chih Chang,* ,‡ and Chia-Chun Chen* ,†,§ Department of Chemistry, National Taiwan Normal UniVersity, Taipei, Taiwan, Taipei 116, Taiwan, The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, and Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan ReceiVed: April 21, 2009; ReVised Manuscript ReceiVed: June 10, 2009 A soft surface-enhanced Raman scattering (SERS) substrate was fabricated based on a three-dimensional (3D) structure of biocompatible end-tethered poly(L-lysine) (“t-PLL”) with a brushlike configuration conjugated with silver nanoparticles (Ag NPs) (Ag NP-t-PLL film). The conjugation procedures were carefully adjusted to generate the films with different interval widths (W) between Ag NPs and diameters (D) of Ag NPs. The resulting film was then characterized by zeta potential, CD spectropolarimeter, and scanning electron microscopy. Furthermore, the studies of SERS enhancements using Ag NP-t-PLL film as a substrate were performed. The significant increases of SERS enhancements have been obtained as W/D was decreased from 0.9 to 0.2. Our results not only afford a facile fabrication of a 3D soft substrate for SERS with high sensitivity and biocompatibility but also offer great potentials for the development of new biosensors. 1. Introduction Surface-enhanced Raman scattering (SERS) of organic mol- ecules adsorbed on metal NPs has been intensively explored in experimental and theoretical aspects recently. 1-9 Many great efforts have been made to increase the detection limit of SERS signal of analyte concentration. 5,6 For instance, the SERS sensitivity of Rhodamine 6G (R6G) molecules adsorbed on silver NPs (Ag NPs) has been reached to ∼1 × 10 -15 M. 5 In comparison to fluorescence spectra, SERS spectra can provide not only the optical properties of adsorbed molecules but also their structural fingerprints. 6,10 Therefore, one of the important SERS applications is for the identification on trace biological molecules without fluorescence signal such as DNA and RNA. 11,12 Recent studies have shown that detection DNA or RNA have been performed dye-labeled oligonucleotide se- quences in a quantitative manner 13,14 and resolved label-free direct identification of mononucleotides in low concentration aqueous solution. 11 Furthermore, SERS techniques have exhib- ited great potentials for the understanding on some biological activities such as rapid and ultrasensitive determination of enzyme activities, 15 characterization of a number of species and strains of bacteria, 16 and even direct detection of cancer cells as a potential diagnostic maker. 17 New biological applications of SERS are being explored. A soft substrate is required in order to study biological activities of living cells or species by SERS. The technical challenge for the fabrication of an adaptable substrate is to load metal NPs properly onto the biocompatible materials on the surface. Two main factors should be controlled carefully in order to reach the optimization, reliability and reproducibility of Raman signal. First, the growth of metal NPs directly on a soft substrate has increased the difficulty in controlling the sizes and shapes of resulting NPs. Thus, the maximized SERS signal is difficult to obtain because the signal enhancements have exhibited dependencies on the size 18,19 and shape 20,21 of NPs. Second, to reach the maximized SERS enhancement, it is necessary to adjust the ratio (W/D) of nanoparticle diameter (D) and interval width (W) between NPs on substrates. The great SERS enhancement has been reported while significant near- field interaction occurs between adjacent Ag nanorods with their W reaching half the value of their diameter. 9 Of recent, different types of SERS substrates have been fabricated by the controlled growth, conjugation or assembly of metal NPs on the surface. For examples, the porous anodic aluminum oxide (AAO) deposited with Ag NPs has been utilized as a SERS substrate. 22 The W and D of the NPs have been controlled through the hole and wall thickness of AAO during the conjugation of Ag. The monolayers of aligned Ag nanowires are assembled by using the Langmuir-Blodgett technique on a silicon wafer to generate a suitable SERS substrate. 23 Also, the silicon nanotips with Ag NPs on the tip for SERS measurements is fabricated by ion sputtering technique. 24 Besides the metal NPs on an inorganic substrate, they can be also directly grown and deposited onto biocompatible materials such as polymers, 25-31 polypeptides, 1,5 and mononucleotides 13 on the substrate surface. For example, dendrimer/metallic NPs are deposited on the surface to form a layer-by-layer film as a SERS substrate. 25 A high-density nanoparticle film has been fabricated for SERS measurements using polymers as a template. 26 In this study, the spacing between NPs is adjusted by generating the polymer swelling and shrinkage through the control of temperature. In spite of the polymers, 3-aminepropyl-triethoxysilane (APS) dispersed on a glass slide has usually been exploited as a SERS substrate for probing biological molecules on the unique gold nanoparticle aggregates. 32 In addition, the studies of SERS detection for micro-organisms have also been reported using different types of substrates such as Au NP-coated SiO 2 , 16 metalized nano- structured poly(p-xylylene) films, 28 and codeposited bacteria and Ag on an inert surface. 33 But practical SERS probes for * Corresponding author. E-mail: cjchen@ntnu.edu.tw (C.-C.C); yingchih@ gate.sinica.edu.tw (Y.-C.C.). † National Taiwan Normal University. ‡ The Genomics Research Center, Academia Sinica. § Institute of Atomic and Molecular Sciences, Academia Sinica. J. Phys. Chem. C 2009, 113, 13498–13504 13498 10.1021/jp903664u CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009