Biosensors and Bioelectronics 24 (2008) 178–183 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios A new protein A assay based on Raman reporter labeled immunogold nanoparticles Chi-Chang Lin a , Ying-Mei Yang a , Yan-Fu Chen a , Tzyy-Schiuan Yang b,∗ , Hsien-Chang Chang a,c,d,∗∗ a Institute of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan, ROC b Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi 621, Taiwan, ROC c Institute of Nanotechnology and Microsystems Engineering, Tainan 701, Taiwan, ROC d Center for Micro/Nano Technology Research National Cheng Kung University, Tainan 701, Taiwan, ROC article info Article history: Received 2 January 2008 Received in revised form 18 March 2008 Accepted 25 March 2008 Available online 4 April 2008 Keywords: Protein A Immunoassay Surface-enhanced Raman scattering Molecular probe Au nanoparticle abstract A unique, sensitive, highly specific, and photobleaching-resistant immunoassay system utilizing gold nanoparticles and surface-enhanced Raman scattering (SERS) is described. This new system, featuring a capability of bifunctional analysis, is manufactured by chemisorption of antibody immunoglobulin G (IgG) on gold nanoparticles (AuNP), followed by coupling the Raman-active reporter molecule, 5,5 ′ -dithiobis(2- nitrobenzoic acid) (DTNB) to the surface of IgG–AuNP. The adsorbed DTNB molecules exhibit strong Raman signals via both electromagnetic and chemical enhancement. The narrow spectral widths and high pho- tostability assure the system to be an excellent detection label. This SERS-based immunoassay is applied to the detection of protein A, which is a specific surface antigen of Staphylococcus aureus. A working curve is obtained by plotting the intensity of the SERS signal of symmetric NO 2 stretching of DTNB at 1333 cm -1 versus the concentration of the analyte (antigen). A dynamic range of two to three orders of magnitude and a detection limit of 1 pg/mL of protein A are achieved. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Specific biological recognitions, such as antigen–antibody, protein–ligand, and avidin–biotin interactions, lead to the devel- opment of immunosensors for food, environmental pollutants, pharmaceutical chemicals, and clinical diagnostics (Terry et al., 2005; Rowe et al., 1999; Gonz´ alez-Mart´ ınez et al., 1999). Nowa- days, enzyme-linked immunosorbent assay is the most common and widespread method to detect the presence of an antigen or its complementary antibody. Once the specific binding between antigen–antibody or protein–substrate is formed, the readout tools can be fluorescence, surface plasmon resonance, atomic force microscopy, a microcantilever system, quartz crystal microbalance, and electrochemical detection (Oh et al., 2003; Li et al., 2002, 2006; Nakanishi et al., 1996; Chen et al., 2006; Chang et al., 1996). Among them, fluorescence has long been used in immunoassay labeling ∗ Corresponding author. Tel.: +886 5 2720411x66415; fax: +886 5 2721040. ∗∗ Corresponding author at: Institute of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan, ROC. Tel.: +886 6 2757575x63426; fax: +886 6 2343270. E-mail addresses: chetsy@ccu.edu.tw (T.-S. Yang), hcchang@mail.ncku.edu.tw (H.-C. Chang). for their inherent sensitivity (Duan et al., 1994; Chan et al., 1998; Bruchez et al., 1998; Yakovleva et al., 2002). However, photobleach- ing of fluorophores complicates data interpretation and the broad spectral width of fluorescence limits the application to simultane- ous multiple-detections. Therefore, new optical methods resistant to photobleaching and with improved sensitivity are in demand for ultradilute immunoassay. Raman scattering, in contrast to broad featureless fluorescence, provides rich structural as well as quantitative and qualitative molecular information through the sharp and distinguishable vibrational bands. One of the greatest advantage of Raman spec- troscopy over infrared vibrational spectroscopy is that the aqueous samples under the physiological condition can be easily analyzed with negligible interference of water signals. Nevertheless, con- ventional Raman spectroscopy usually lacks sufficient sensitivity required for a readout method in immunoassay because of its weak intensity. Surface-enhanced Raman scattering (SERS) overcomes this difficulty. Many researchers have demonstrated that SERS can be used to detect pico- to femto-molar amount of material adsorbed on various types of roughened metal surfaces. The surface enhancement factor ranges from 10 3 up to 10 14 (Kneipp et al., 1999). The conformations of adsorbed molecules such as oligonucleotides can also be studied by SERS (Wu et al., 2006). The major concerns in application of SERS to the immunoassay are the 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.03.035