Gold Nanorods Functionalized with Self-assembled Glycopolymers for Ultrasensitive Detection of Proteins Hidenori Otsuka,* 1,2 Yuki Muramatsu, 2 and Daisuke Matsukuma 1 1 Department of Applied Chemistry, Faculty of Science Division I, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601 2 Department of Chemical Science and Technology, Graduate School of Chemical Science and Technology, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601 (E-mail: h.otsuka@rs.kagu.tus.ac.jp) New, stable, and glyoco-derivatized gold nanorods (GNRs) were prepared using developed amphiphilicblock glycopoly- mers poly[(lactose) m -b-(pyridine) n ] to engineer self-organized, densely packed glycopolymeric recognition layers and generate a multivalent binding cavity onto the gold surface. We report a rapid and ultrasensitive biosensing for the weak affinity of proteincarbohydrate binding employing a combination of the longitudinal SPR of GNRs and the cluster glycoside effect. Thus, a very small amount oflectin (100 pg mL ¹1 = 8.3 © 10 ¹13 M) was detected by the aggregation of GNRs. Plasmonic metal nanoparticles have great potential for chemical and biological sensor applications, due to their sensitive spectral response to the local environment of the nanoparticle surface and ease of monitoring the light signal due to their strong scattering or absorption. 13 Polymer grafts can be used as molecular spacers to control the metalnanoparticle separation distances during the assembly process. By thought- fully tailoring the length of the graft chain, inter-nanoparticle junctions can be tuned to control the degree of plasmonic coupling between nanoparticles. Smaller spacings give rise to a red shift in the observed LSPR (localized surface plasmon resonance) wavelength due to increased plasmon coupling. 4,5 Utilizing these capabilities in biological sensing, we have previously reported that lactose-conjugated gold nanoparticles using a PEG spacer exhibited selective aggregation when exposed to Recinus communis agglutinin (RCA 120 ), a bivalent lectin specifically recognizing the ¢-D-galactose residue, induc- ing significant changes in the absorption spectrum with assay sensitivity appreciablyhigh enough to detect lectin concentra- tion to ¥l ¯g mL ¹1 (1 ppm), comparable to that ofimmunolog- ical assay methods such as ELISA. 6 In this way, spherical gold nanoparticles have extensively been employed as analytical probes inbiotechnological systems such as diagnostic and biologicalimaging, as well as cancer therapy. 1,79 Nevertheless, enhanced assay sensitivity is still the next challenge. Theoretical consideration on the surface plasmon resonance condition revealed that the spectral sensitivity, defined as the relative shift in resonance wavelength with respect to the refractive index change of surrounding materials, has two controlling factors: first the bulkplasma wavelength, a property dependent on the metal type, and second the aspect ratioof the nanoparticles from spheres to rods, which is a geometrical parameter. 1012 It is observed that the sensitivity does not depend on the type of the metal but depends largely on the aspect ratio of nanorods. For example, increase in the aspect ratioof gold nanorods (GNRs) enhances the plasmon sensitivity. The analyte- induced aggregation of GNRs has been utilized for an analysis of the specific antibodyantigen binding process by employing the LSPR wavelength shift. 13 Although most of the results show that a very small amount of protein, h-IgG (60 ng mL ¹1 ) as an example, was detected by the red shiftof LSPR, the assayed reaction is usually known for the strong affinities between antibody and antigen. The studies of proteincarbohydrate interactions have been challenged due to sensing limitations resulting from the inherent structure complexity of carbohydrates and the typically weak affinities of the carbohydrate binding with proteins. 14 These disadvantages have been overcome by multivalent interactions, i.e., simultaneous contact between the clustered carbohydrates on the cell surface and protein receptors that contain multiple carbohydrate recognition domains. This is accepted as the “cluster glycoside effect.” 15,16 It has been reported that multi- valent forms of synthetic ligands, either polymers or dendrimers, often have amplified inhibitory effects over the monovalent counterparts. 1719 We recently described the amphiphilicblock copolymers (poly(Lac m -b-Py n )) consisting of hydrophilic lactose glycopolymers (Lac) as ligands for lectinbinding and hydro- phobic pyridines (Py) as anchor groups on the gold surface; m and n denote the respective polymerization degree. 20 Our interest was to combine the self-assembly strategy with the specially designed functional glycopolymers to engineer self-organized, densely packed glycopolymeric recognition layers which could be chemoadsorbed onto the gold surface and generate a multi- valent binding cavity. Herein, we report a rapid and ultrasensitive biosensing for the weak affinity of proteincarbohydrate binding using the LSPR of GNRs modified by a preferentially oriented carbohydrate brush layer of poly(Lac m -b-Py n ). A very small amount oflectin (100 pg mL ¹1 ) was detected by the aggregation of GNRs through the cluster glycoside effect. To the best of our knowledge, this is the first report on pg protein assay (in pg mL ¹1 unit) using plasmonic nanoparticles. Comparing with the previous immunoassay methods, including colorimetric assay, 21 photonic crystal assay, 22 enzyme immunoassay (EIA), and fluoroimmunoassay (FIA), this novel method issimple and sensitive and may provide broad potential applications inviral infection assay, disease diagnosis, and immunoassay. The GNRs were chemically synthesized by the seed- mediated growth method, 23 and the resultof TEM analysis was described in the Supporting Information with experimental details. The prepared GNRs with average aspect ratio 4.0 were fairly uniform in shape and highlydispersed in water due to the positive charge of the CTAB bilayer capped on the gold surface. The surface modification of GNRs with poly(Lac 23 -b-Py 36 ) has been then carried out. The aqueous medium was removed after CL-140943 Received: October 12, 2014 | Accepted: October 27, 2014 | Web Released: November 1, 2014 132 | Chem. Lett. 2015, 44, 132–134 | doi:10.1246/cl.140943 © 2015 The Chemical Society of Japan