Biosensors and Bioelectronics 25 (2009) 674–681 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios SERS detection of indirect viral DNA capture using colloidal gold and methylene blue as a Raman label Mark H. Harpster a,1 , Hao Zhang b,1 , Ajaya K. Sankara-Warrier c , Bryan H. Ray d , Timothy R. Ward c , J. Pablo Kollmar c , Keith T. Carron d , James O. Mecham a , Robert C. Corcoran c , William C. Wilson a , Patrick A. Johnson b,∗ a Arthropod-Borne Animal Diseases Research Laboratory, USDA/ARS, Laramie, WY 82071, United States b Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY 82071, United States c Department of Chemistry, University of Wyoming, Laramie, WY 82071, United States d DeltaNu, Inc., 5452 Old Highway 130, Laramie, WY 82070, United States article info Article history: Received 15 May 2009 Accepted 19 May 2009 Available online 27 May 2009 Keywords: Surface enhanced Raman scattering DNA West Nile Virus Indirect nucleic acid capture Methylene blue Quartz crystal microbalance-dissipation abstract An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The subsequent elicitation of surface enhanced plasmon resonance by laser exci- tation provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB. Detection sensitivity is in the submicromolar range and was shown to be highest for thiol, and less so for amino, modifications at the 5 ′ terminus of the capture oligonucleotide. Finally, using Quartz Crystal Microbalance-Dissipation as a tool for modeling ternary complex binding to Au surfaces, quantitative measurements of surface mass coverage on Au plated sensor crystals established a positive correlation between levels of ternary complex adsorption and their correspondent levels of SERS signal intensification. Adapted to a compact Raman spectrometer, which is designed for analyte detection in cap- illary tubes, this assay provides a rapid, mobile and cost effective alternative to expensive spectroscopic instrumentation, which is often restricted to analytical laboratories. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Recent advances in the field of surface enhanced Raman scatter- ing (SERS) spectroscopy have demonstrated the utility of this tech- nology in diverse research disciplines ranging from surface chem- istry and electrochemistry to forensics and the development of protein/nucleic acid sensor technologies. In principle, SERS is based on the finding that the light scattering cross-sections of molecules adsorbed on roughened noble metal surfaces, typically Au and Ag, and to a lesser extent Cu, are significantly enhanced upon laser excitation. Enhancements typically range from 10 5 to 10 6 , but have been reported to be as high as 10 14 –10 15 for single molecule detec- tion (Nie and Emory, 1997). Overall, the recorded SERS, or spectral ∗ Corresponding author. Tel.: +1 307 766 6524. E-mail address: pjohns27@uwyo.edu (P.A. Johnson). 1 Both these authors contributed equally to this work. peak, profile that is diagnostic for a Raman scattering analyte is dependent on two phenomena, a weakly enhancing short-range chemical enhancement mechanism (CE) and a stronger long-range electromagnetic enhancement mechanism (EM). CE is attributed to the charge–transfer interactions of a chemiadsorbed scatterer on a metal surface (i.e. SERS substrate) (Guzonas et al., 1990) and EM to the spatial positioning of a physiadsorbed scatterer within an enhanced electromagnetic field arising from light excitation of surface metal electron oscillations, or plasmon resonance (Zeman and Schatz, 1987). The highest levels of SERS enhancement have been reported for EM “hot spots”, which are defined as the intersti- tial, or fractal space, junctions between two or more SERS substrates (Michaels et al., 2007). Although it has been shown that the sensing distance of a Raman scatterer from a SERS substrate declines expo- nentially with a decay length of 2 nm (Schatz and Van Duyne, 2002), varying experimental conditions are likely to impact the dynamic range of detection, which has been reported to be as low as 3–5 nm (Murray et al., 1982) and as high as 30–45 nm (Liu et al., 2006). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.05.020