Biosensors and Bioelectronics 26 (2010) 1009–1015 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G Jing Neng a,1 , Mark H. Harpster b,c,1 , Hao Zhang b , James O. Mecham c , William C. Wilson c , Patrick A. Johnson a,b,∗ a Molecular and Cellular Life Sciences Program, University of Wyoming, Laramie, WY 82071, United States b Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY 82071, United States c Arthropod-Borne Animal Diseases Research Unit, USDA/ARS, Manhattan, KS 66502, United States article info Article history: Received 22 May 2010 Received in revised form 23 July 2010 Accepted 12 August 2010 Available online 19 August 2010 Keywords: SERS Immunoassay Protein A/G Malachite green QCM-D abstract A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite green (MG)-conjugated protein A/G (MG-pA/G) as a bi-functional Raman tag/antibody binding reporter. Upon incubation of these reagents with serum collected from rabbits inoculated with E antigen, laser interro- gation of the sandwiched immunocomplex revealed a SERS signaling response diagnostic for MG. The intensification of signature spectral peaks is shown to be proportionate to the concentration of added serum and the limit of antibody detection is 2 ng/ml of serum. To assess assay performance relative to more a traditional immunoassay, indirect enzyme-linked immunosorbent assays conducted using the same concentrations of reagents were found to be >400-fold less sensitive. Quartz crystal microbalance with dissipation (QCM-D) monitoring of immunocomplex film deposition on solid Au surfaces also con- firmed the formation of antigen–antibody–protein A/G trilayers and provided quantitative measurements of film thickness which likely position MG within the sensing distance of laser-elicited, enhanced elec- tromagnetic fields. The sensitivity and inherent versatility of the assay, which is provided by the binding of pA/G to a broad spectrum of immunoglobulins in different mammalian species, suggest that it could be developed as an alternative immunoassay format to the ELISA. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Research developments in biosensor applications for surface enhanced Raman spectroscopy (SERS) have demonstrated a broad range of solutions for the sensitive and selective detection of nucleic acids (Cao et al., 2002; Hering et al., 2008; Harpster et al., 2009), proteins (Xu et al., 2005; Porter et al., 2008; Han et al., 2009a), protein–DNA interactions (Bonham et al., 2007) and in vivo tumor tissue (Qian et al., 2008). Although technical strate- gies can vary widely, SERS-based assays for analyte detection are largely predicated on the interdependence of target molecule cap- ture and positioning of an appropriate Raman scattering label, at, or in close proximity to roughened noble metal surfaces. The subsequent interrogation of the reaction sample with an incident laser light which is tuned to the plasmon resonance wavelength ∗ Corresponding author at: Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, Wyoming 82071, USA. Tel.: +1 307 766 6524. E-mail address: pjohns27@uwyo.edu (P.A. Johnson). 1 Both these authors contributed equally to work. of the metal surface establishes an enhanced electromagnetic field in the regional space occupied by analyte capture and provides a “fingerprint” Raman spectrum profile unique to the Raman label (Moskovits, 1985). Despite a lag in the commercial development and availability of SERS-based assays, SERS affords several advan- tages over current fluorescence detection technologies. These include a reduced susceptibility to photobleaching, which is a prob- lematic feature of fluorophore-based assays and limits the capacity for signal averaging, and Raman spectral band widths which are significantly narrower than fluorescent band emissions. The high information content of Raman spectra and the availability of a large number of suitable Raman tags creates the potential for develop- ing an enhanced capacity for multiplexing that greatly exceeds the limits for fluorescence-based multiplex detection assays. The development of SERS-based immunoassays has focused heavily on sandwich assay formats consisting of solid surface SERS- active substrates which immobilize target analyte and an adlayer of extrinsic Raman labels (ERL) consisting of immuno-recognition molecules and Raman labels on Au or Ag nanoparticles. In an early example of this assay, Grubisha et al. (2003) reported a detection limit of 1 pg/ml for prostate specific antigen (PSA) in human serum. 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.08.015