© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 www.advmat.de www.MaterialsViews.com wileyonlinelibrary.com COMMUNICATION Katarzyna Makyla, Christoph Müller, Samuel Lörcher, Thomas Winkler, Martin G. Nussbaumer, Michaela Eder, and Nico Bruns* Fluorescent Protein Senses and Reports Mechanical Damage in Glass-Fiber-Reinforced Polymer Composites Fiber-reinforced composites are prime examples of strong and lightweight polymeric materials. Due to high fiber tensile strength, the composites are highly resistant to forces acting along the length of fiber. They are thus suitable for high per- formance applications, e.g. in the aircraft and automotive industries, as rotor blades of wind turbines or as sporting equipment. Easy and early detection of emergent (microscopic) damage in these materials caused by the impact of objects or other mechanical loading is highly desirable. [1,2] Any micro- scopic damage such as delamination, fiber fracture, or inter- facial debonding of the polymer from the fibers causes stress concentrations when the material is under load. This may lead to an extension of the damage. Therefore, sites of microdamage can represent a severe safety problem when undetected. Con- ventional methods to detect such barely visible damage rely on dye penetrants applied to surfaces [2] or the use of nonde- structive evaluation techniques, such as X-rays, ultrasound, or spectroscopic methods. [2] Living organisms, on the other hand, immediately sense and report injured tissue to prevent further injury and to initiate healing. This is accomplished by nerves signaling pain. Furthermore, optical signals such as the dark red color of a bleeding wound provide essential information that tissue is damaged. These intrinsic mechanisms are una- vailable to man-made polymeric materials, but would be highly desirable, e.g. as a safety feature in load-bearing components. Self-reporting materials that mimic Nature’s ability to signal damage or deformation autonomously within their bulk are just starting to emerge in the literature, [3,4] often in the con- text of self-healing materials. [5] Examples include polymers that embed dye-filled micro capsules, [6] hollow glass fibers, [1] or microvascular networks. [7,8] Furthermore, mechanoresponsive dye-aggregates [9,10] and fluorescence resonance energy transfer (FRET) systems [11] have been explored. Mechanophores are mol- ecules that undergo reactions in response to mechanical forces. Some mechanophores change their spectral properties, such as color, fluorescence or FRET efficiency, under load. [3,4] When incorporated in polymeric materials, they become useful probes that enable detection of micron-scale damage or render stress distributions visual. Examples are modified spiropyrans, [12,13] functionalized cyclopropane, [14] a protein cage, [15] as well as cyclobutane-and cyclooctane-type dimers. [16,17] Fluorescent proteins [18] have been discussed as molecular force sensors in biological applications, [19–21] because they unfold under mechanical stress and their fluorescence is linked to their native structure. [22] Although hybrid materials of poly- mers and fluorescent proteins have been synthesized in the past, the mechano-responsive property of these biomolecules was not exploited. [23] Compared to other mechanophores, fluo- rescent proteins feature some advantages. Genetic engineering allows tailoring their mechanical stability. [19–22] Their three- dimensional structure is a spacer that separates the fluorophore from the surrounding material, thus reducing quenching of flu- orescence due to energy transfer to the material. Furthermore, they are available in all colors of the rainbow, [18] which allows their emission characteristics to match the optical properties of the material. Here, we present the first implementation of a fluores- cent protein as a mechanophore in a polymeric material. We used enhanced yellow fluorescent protein (eYFP) as a force- sensitive link between glass surfaces and epoxy resin in glass- fiber-reinforced composites ( Scheme 1). We assumed that force- transfer from material to protein would be most efficient at the glass-polymer interface. Separation of the resin from the fiber would result in mechanical unfolding of the eYFP, and the protein could report barely visible impact damage such as fiber- matrix interfacial debonding by loss of its yellow fluorescence. To synthesize protein-containing composite material, eYFP was first immobilized on glass surfaces. The protein-coated glass was then embedded in an epoxy resin. This strategy, which will be described in detail in the following paragraphs, circumvents the problem that eYFP is insoluble in the hydro- phobic polymer resin. The chemical nature of a surface can exert a profound influ- ence on the stability and fluorescence of immobilized fluores- cent proteins. [24] Therefore, two methods to immobilize eYFP on glass were investigated, using cover slips as planar model surfaces ( Figure 1 and Scheme S1). eYFP was non-covalently physisorbed on amino glass (SiO 2 / γ-APS), which was obtained by treatment of glass with (3-aminopropyl)triethoxysilane ( γ-APTES). For covalent protein immobilization, amino glass was further treated with the homobifunctional linker disuccin- imidyl terephtalate (DST) in order to obtain activated glass sur- faces (SiO 2 / γ-APS/DST). [25] Due to the rigidity of DST, only one of its functional groups can react with the amino groups on the Dr. K. Makyla, C. Müller, S. Lörcher, T. Winkler, M. G. Nussbaumer, Dr. N. Bruns Department of Chemistry University of Basel Klingelbergstrasse 80, 4056 Basel, Switzerland E-mail: nico.bruns@unibas.ch Dr. M. Eder Department of Biomaterials Max-Planck-Institute of Colloids and Interfaces 14424 Potsdam, Germany DOI: 10.1002/adma.201205226 Adv. Mater. 2013, DOI: 10.1002/adma.201205226