Colloids and Surfaces B: Biointerfaces 83 (2011) 10–15 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Mapping the placement of oligonucleotide molecules using scanning probe microscopy Robert D. Boyd ∗ , Laurie Winkless, Alexandre Cuenat, Olga Kazakova The National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK article info Article history: Received 17 May 2010 Received in revised form 30 September 2010 Accepted 11 October 2010 Available online 15 October 2010 Keywords: Atomic force microscopy Force Oligonucleotide Functionalisation Templates Patterned substrates abstract The successful development of novel bio-inspired devices requires the ability to place specific biomolecules on a substrate with nanometre precision, in such a way so that their bioactivity is retained. A method is required that can verify this bio-modification. Scanning probe microscopy (SPM) can image and probe a surface in a liquid environment with nanometre resolution. Using short chain complementary oligonucleotides as the bioactive molecules we have modified continuous and patterned gold substrates and SPM probes. We demonstrated that the attached oligonucleotides retained their biological activity after surface attachment with a hybridization interaction force that varies between 50 and 400 pN as measured by SPM force measurements. Finally, the position of the attached oligonucleotides was deter- mined with nanometre resolution. Thus we have demonstrated the capabilities of SPM in the application of the development of substrates and templates suitable for forming the basis of novel and innovative devices. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Specific interactions, where a single molecule will only react with its counterpart, are commonplace in biology and include those between antibody and antigens, ligand and receptors and com- plementary oligonucleotide chains. New bionanotechnology-based devices which use such interactions are currently gaining much interest [1]. For example, antibody–antigen interactions are being exploited in existing and emerging biosensors [2,3] and devices, such as nanoactuators [4]. Molecular self-assembly often occurs in various biological processes to produce a range of complex structures, including extracellular and globular proteins, cells and bacterial colonies [5]. Use of these self-assembly processes is one of the few practical strategies for the production of nano- and microstructures [5], especially as traditional pick and place meth- ods become exponentially more difficult when the size of individual components decrease below 100 m [6]. Full exploitation of these specific biological interactions in novel devices requires the ability to attach molecules to a suitable sub- strate or template in such a way that their bioactivity is retained. Whilst there are many potentially routes to achieve this, they all suffer from several common problems. These include having com- plete and reliable homogenous attachment of the biomolecules ∗ Corresponding author at: Tel.: +44 208 943 8779. E-mail address: Robert.boyd@npl.co.uk (R.D. Boyd). in the correct location [7], correct orientation of the attached biomolecule [8] and changes in the surface topography [9], all of which will affect how bioactive species interact with their envi- ronment. Traditional methods of determining bio-activity, such as enzyme linked immunosorbent assay (ELISA) [10] and electro- chemical methods [11], only offer an average indication of activity providing no information on either the homogeneity or factors affecting the molecular activity. On the other hand, fluorescence resonance energy transfer is a relatively new technique that can achieve 10 nm-resolution of specific ligand receptor interactions [12]. However, this is a very complicated research-focused equip- ment, which is generally not suitable for routine analysis. One technique with the potential to meet both biological and metrolog- ical challenges is scanning probe microscopy (SPM) [13]. SPM has been extensively applied to biological systems [14], including the study of protein structures [15]. By modifying the SPM probe with a biological active agent, both the location and activity of specific biomolecules can be measured [16,17]. The majority of the work done to date has concentrated on ideal systems, which hold little practical significance. Although a recent paper has demonstrate the principle of using SPM combined with DNA oligomers to assemble biomolecular structures [18]. In this paper we demonstrate applicability of the SPM technique to substrates and templates that hold a real possibility of forming the basis of practical bio-inspired devices. A test system of thiol- modified oligonucleotide chains was initially selectively attached to gold substrates. An important requirement of new bioinspired 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.022