Colloids and Surfaces B: Biointerfaces 80 (2010) 12–17 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Probing microbubble targeting with atomic force microscopy V. Sboros a,∗ , E. Glynos b,1 , J.A. Ross d , C.M. Moran a , S.D. Pye c , M. Butler a , W.N. McDicken a , S.B. Brown e , V. Koutsos b a Medical Physics, School of Clinical Sciences and Community Health, University of Edinburgh, Edinburgh, UK b Institute for Materials and Processes, School of Engineering, Centre for Materials Science and Engineering, University of Edinburgh, Edinburgh, UK c Medical Physics, Royal Infirmary of Edinburgh, Edinburgh, UK d Clinical and Surgical Sciences, University of Edinburgh, Edinburgh, UK e MRC/University of Edinburgh Centre for Inflammation Research, Edinburgh, UK article info Article history: Received 27 August 2009 Received in revised form 11 May 2010 Accepted 11 May 2010 Available online 20 May 2010 Keywords: Targeted microbubbles Atomic force microscope Microbubble adhesion Microbubble-cell avidity CD31 PECAM abstract Microbubble science is expanding beyond ultrasound imaging applications to biological targeting and drug/gene delivery. The characteristics of molecular targeting should be tested by a measurement system that can assess targeting efficacy and strength. Atomic force microscopy (AFM) is capable of piconew- ton force resolution, and is reported to measure the strength of single hydrogen bonds. An in-house targeted microbubble modified using the biotin–avidin chemistry and the CD31 antibody was used to probe cultures of Sk-Hep1 hepatic endothelial cells. We report that the targeted microbubbles provide a single distribution of adhesion forces with a median of 93 pN. This interaction is assigned to the CD31 antibody–antigen unbinding event. Information on the distances between the interaction forces was obtained and could be important for future microbubble fabrication. In conclusion, the capability of single microbubbles to target cell lines was shown to be feasible with AFM. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Microbubbles are being developed as a targeting tool for gene/drug delivery with the capacity for simultaneous therapy and monitoring of pathology. As the field develops, more tar- geting strategies will cover the range of pathologies that require such protocols [1]. Microbubble researchers have investigated the behaviour of single microbubbles both optically [2–6] and acoustically [7–10]. The information gained is pivotal to such a multidisciplinary effort, as it offers statistics from multiple single microbubble events and thus provides a direct test of microbub- ble engineering. The majority of experimental techniques that are used to test microbubble features, such as attachment or detach- ment assays, assess them qualitatively and in bulk. The success of these techniques is mainly in the detection of binding events and they are less successful in specifying binding forces [11]. Previous experimental approaches have not provided a clear separation of ∗ Corresponding author at: Medical Physics, School of Clinical Sciences and Com- munity Health, The University of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB, UK. Tel.: +44 131 242 6292; fax: +44 131 242 6314. E-mail address: Vassilis.Sboros@ed.ac.uk (V. Sboros). 1 Present address: Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA. individual processes, namely cell resistance, rigidity, and binding, and so have limited value in deciphering binding processes [11]. Techniques such as Scatchard analysis, Biacore, and shear plates have generated some thermodynamic and macroscopic values, pro- viding, at best, what are termed the affinity, binding, or association constants [12–18]. This information is satisfactory up to a point, but it does not provide a satisfactory measurement of the forces of individual molecular interactions. The macroscopic nature of such techniques does not allow a meaningful understanding of the development of adhesion, and the unambiguous measurement of affinity and avidity which is a nanometer scale phenomenon. Cell micromanipulation techniques using aspiration are of microscopic nature [19] and do not allow the observation of individual bond detachment events. Cell adhesion events are of wide biological sig- nificance, being involved in cell–cell and cell–matrix interactions. These events form an important feature of the tissue architecture and allow cells to survive, function or communicate with adja- cent cells. Today, scientists are increasingly exploring cell surface molecules involved in adhesion in order to achieve specific molecu- lar targeting for diagnostic and therapeutic purposes. Microbubbles that carry targeting moieties appear to be a strong candidate to fulfil this dual role. The atomic force microscope (AFM) provides a spatial and force resolution in the order of Angstroms and sub-nanonewtons respectively [20,21]. The AFM has provided the first repro- 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.05.022