Resonant Rayleigh light scattering response of individual Au nanoparticles to antigen–antibody interaction† Cuong Cao and Sang Jun Sim * Received 13th February 2009, Accepted 14th May 2009 First published as an Advance Article on the web 20th May 2009 DOI: 10.1039/b901327j A proof-of-concept study was reported on analysis of antigen– antibody recognition based on resonant Rayleigh scattering response of single Au nanoparticles in an imaging chamber. As benefited by a traditional dark-field microscope and a spectrograph, individual Au nanoparticles (30 nm) were observed with high signal- to-noise ratio and they were effectively utilized to monitor changes in refractive index induced by specific binding of the adsorbates. Using PSA antigen as a model, a LSPR l max shift of about 2.85 nm was recorded for a molecular binding corresponding to 0.1 pg ml 1 of the protein biomarker. This result successfully demonstrates a non- labeling detection system for proteins as well as thousands of different chemical or biological species, and it possesses a great potential as a sensitive, on-chip and multiplexing detection. In conjunction with optical transduction based on excitation of surface plasmons for planar gold surface (SPR) for non-labeling analysis of biomolecular interaction, recently there has been an increasing interest in surface plasmon resonance for nanometer-sized metallic structure. When the nanometer-sized metallic structures are excited by light, polarization of the free electrons relative to the ionic cluster will be induced by the electric field of the incoming light. As a result, the collective electrons of the nanoparticles intrinsically oscillate with the incident photon frequency which leads to so-called localized surface plasmon resonance (LSPR). Based on the unique properties of noble metallic nanoparticles materials such as silver and gold, several research groups have explored alternative strategies for the development of optical biosensors and chemosensors. 1,2 More- over, these extraordinary optical features make the nanostructures useful for surface-enhanced spectroscopy, 3,4 plasmonic devices, 5 and sensors. 6–8 It is now well-known that optical excitation of the LSPR of silver or gold nanoparticles results in not only a strong UV-Vis absorption band, which is not present in the spectrum of the bulk metal, with extremely large molar extinction coefficients of 3  10 11 M 1 cm 1 but also a resonant Rayleigh light scattering with an efficiency equivalent to that of 10 6 fluorophors, or strong enhancement of the local electromagnetic fields near the nanoparticle surface. 1,9 Furthermore, the maximum peak of extinction or resonant Rayleigh scattering wavelength (l max ), intensity, and spectral bandwidth of these LSPR spectra are strongly dependent on their size, 10 shape, 11–14 interparticle spacing, 15 and local dielectric environment. 15–17 With a view to applications based on fundamental research, Van Duyne’s group has shown that the LSPR spectrum of noble metal nano- particles is very sensitive to adsorbate-induced changes in the dielectric constant of the surrounding nanoenvironment, which provides several improvements over existing array- or cluster-based techniques; the resonant Rayleigh scattering pattern of a single silver nanoparticle could be significantly differentiated when about 60 000 molecules (or 100 zeptomoles) of 1-hexadecanethiol or <100 strep- tavidin molecules were adsorbed on its surface. 1 Raschke has devel- oped a method for biomolecular recognition using light scattering of a single gold nanoparticle functionalized with biotin. Addition of streptavidin and subsequent specific binding events altered the dielectric environment of the nanoparticle, resulting in a spectral shift of the particle plasmon resonance. Spectral shifts as low as 2 meV could be detected when single nanoparticles were used as sensing counterparts. 2 Hereafter, a proof-of-concept of using the resonant Rayleigh light scattering response of individual Au nanoparticles for analyzing an antigen–antibody interaction is introduced. The LSPR sensitivity of the single Au nanoparticles induced by binding of adsorbates on the surface of single gold nanoparticles was proven to be an effective tool for quantification of a target protein biomarker, prostate specific antigen (PSA–ACT complex). When the target antigen reacts with its capture antibody, the immune reaction created an increase in refractive index surrounding the particle nanoenvironment which could be consequently recorded by the Rayleigh light scattering microspectroscopy as LSPR maximum wavelength (LSPR l max ) shift. An amount of 0.1 pg ml 1 for the detection of PSA–ACT complex could be recorded indicating that the detection platform is very sensitive and vanquishes all detection limits of commercial tests for PSA so far. Correlatively, it is totally comparable to those obtained from the famous DNA biobarcode assay utilizing PCR amplification. 18 To observe the scattered light of single nano-metallic particles, a transmission configuration for the resonant Rayleigh light scat- tering microspectroscopy was established by using a dark-field microscope (Eclipse TE2000-U, Nikon) to exploit advantages of a traditional microscope setup (Fig. 1). For spectral investigations, the microscope is equipped with a spectrograph (Microspec 2300i, Roper Scientifics) and a highly sensitive CCD camera (PIXIS:400B, Princeton Instruments). A color camera (D50, Nikon) was attached to the front port of the microscope. A 100 W tungsten lamp and a dark-field condenser (dry condenser, NA ¼ 0.80–0.95, Nikon) were used to illuminate the nanoparticles. The scattered light was collected with a 100  objective (CFI Plan Fluor ELWD, NA ¼ 0.6) and directed to the spectrograph in order to be dispersed into mono- chromatic light. Intensity of the monochromatic lights was measured Nano-optics and Biomolecular Engineering National Laboratory, Department of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746, Korea. E-mail: simsj@skku.edu; Fax: +82-31-290-7272; Tel: +82-31-290-7341 † Electronic supplementary information (ESI) available: color photograph of the system setup. See DOI: 10.1039/b901327j 1836 | Lab Chip, 2009, 9, 1836–1839 This journal is ª The Royal Society of Chemistry 2009 COMMUNICATION www.rsc.org/loc | Lab on a Chip