One-step fabrication of sub-10-nm plasmonic nanogaps for reliable SERS sensing of microorganisms Jing Chen a , Gaowu Qin a,n , Jiansheng Wang a , Jiangyu Yu b , Bo Shen a , Song Li a , Yuping Ren a , Liang Zuo a , Wen Shen c , Biswajit Das c,nn a Key Laboratory for Anisotropy and Texture of Materials (Ministry of Education), Northeastern University, Shenyang 110819, China b College of Materials and Metallurgy, Northeastern University, Shenyang 110819, China c Nevada Nanotechnology Center, Howard R. Hughes College of Engineering, University of Nevada, Las Vegas, NV 89154-4026, USA article info Article history: Received 5 November 2012 Received in revised form 31 December 2012 Accepted 21 January 2013 Available online 29 January 2013 Keywords: Biosensor High pressure sputtering Microorganisms Sub-10-nm nanogaps Surface enhanced Raman scattering abstract Nanoscale gaps in noble metal films can produce intense electromagnetic enhancement. When Raman- active molecules are positioned in these regions, their surface-enhanced Raman scattering (SERS) signals can be dramatically enhanced. However, the lack of convenient and reliable fabrication methods with ultrasmall nanogaps ( o10 nm) severely block the application of SERS. Here, we propose a cost- effective and reproducible technique to fabricate the large-area Ag SERS-active substrates which are full of the high-density, sub-10-nm nanogaps by high pressure sputtering, and the enhancement factor (EF) is testified to improve by 10 3 times compared to the continuous Ag film with a smooth surface (the roughness is 0.5 nm) and without nanogaps. Since there are no chemicals used during fabrication, this substrate has a clean surface, which is crucial for acquiring reliable SERS spectra. This SERS-active substrate has then been applied to identify a series of microorganisms, and excellent, reproducible SERS spectra were obtained. Finally, a set of piecewise-linear equations is provided according to the correlation between SERS intensity and rhodamine 6G (R6G) concentration, and the detection limit is calculated to be 0.2  10 8 M. These results suggest that the high pressure sputtering is an excellent, reliable technique for fabricating sub-10-nm plasmonic nanogaps, and the SERS-based methodology is very promising for being used in biological sensing field. & 2013 Elsevier B.V. All rights reserved. 1. Introduction It is now well accepted that the surface-enhanced Raman scatter- ing (SERS) primarily resulted from the intense electromagnetic enhancement arising from surface plasmon resonances of metallic nanoparticles (usually of silver or gold) (Le Ru and Etchegoin, 2009; Lim et al., 2011). In the regions normally less than 10 nm between metallic nanoparticles, plasmon will be created when the incident light with a proper frequency interacts with the metal (Smith, 2008). Then the Raman-active molecules trapped in these regions will interact with the created plasmon, and their Raman signals will be enhanced drastically. (Smith, 2008; Lal et al., 2008; Liberman et al., 2010; Alexander et al., 2010) To date, SERS can enhance the normal Raman signals of analytes up to 14 orders of magnitude under the optimum conditions, which can fulfill the detection of single mole- cule (Nie and Emory, 1997; Kneipp et al., 1997). As SERS enhance- ment capability is strongly related to the interparticle spacing, how to precisely adjust the interparticle distance, and to create as many hot spots as possible to fabricate reliable, ultrasensitive SERS substrates is a big challenge at present. Chemical method is a traditional approach to fabricate SERS-active substrates. By this means, the sub-10-nm interparticle distance is easy to adjust by using different surfactants capped on the particle surface. However, the remaining surfactants derived from the chemical reaction are difficult to remove afterwards, which will severely interfere with SERS spectra and may have an adverse influence on the high selectivity of SERS detection (Smith, 2008; Huang et al., 2011). In recent years, lithography has attracted great attention in the research field of SERS substrates fabrication and lots of substrates with periodic and ordered structure have been made by this technology, sometimes combined with other techniques, such as the angle evaporation, and the atomic layer deposition (ALD) technique (Duan et al., 2011; Theiss et al., 2010; Yu et al., 2008; Im et al., 2010). However, lithography is costly, time- consuming, labor intensive, and not appropriate for extending to large dimensions. An alternative route that employs colloidal crystals as templates, which is known as nanosphere lithography, has also been widely exploited (Haynes and Van Duyne, 2001; Ye and Qi, 2011). This is a less-expensive and efficient approach. But the SERS substrates Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.01.038 n Corresponding author. Tel.: þ86 24 83691565; fax: þ86 24 83691570. nn Corresponding author. E-mail addresses: qingw@smm.neu.edu.cn (G. Qin), dasb@unlv.nevada.edu (B. Das). Biosensors and Bioelectronics 44 (2013) 191–197