Eur. Phys. J. D 16, 317–320 (2001) T HE EUROPEAN P HYSICAL JOURNAL D c  EDP Sciences Societ` a Italiana di Fisica Springer-Verlag 2001 Formation and manipulation of regular metallic nanoparticle arrays on bacterial surface layers: an advanced TEM study M. Mertig 1, a , R. Wahl 1 , M. Lehmann 2 , P. Simon 2 , and W. Pompe 1, b 1 Institut f¨ ur Werkstoffwissenschaft, Technische Universit¨at Dresden, D-01069 Dresden, Germany 2 Institut f¨ ur Angewandte Physik (IAPD), Technische Universit¨at Dresden, D-01069 Dresden, Germany Received 6 December 2000 Abstract. The template-directed formation of regular nanoparticle arrays on two-dimensional crystalline protein layers after their treatment with metal salt complexes was studied by transmission electron mi- croscopy. For these investigations, bacterial surface layers (S layers), recrystallized in vitro into sheets and tube-shaped protein crystals with typical dimensions in the micrometer range, were used as the template. As identified by electron holography and scanning force microscopy, the S-layer tubes form alternating double layers when deposited onto a solid substrate surface. Two distinct pathways for the metal particle formation at the templates have been found: the site-specific growth of metal clusters by chemical reduction of the metal salt complexes, and the electron-beam induced growth of nanoparticles in the transmission electron microscope. Both mechanisms lead to regular arrays with particle densities > 6 × 10 11 cm -2 . Nanoparticle formation by electron exposure takes exclusively place in the flat-lying double-layered pro- tein tubes, where a sufficient amount of metal complexes can be accumulated during sample preparation. PACS. 82.30.Nr Association, addition, insertion, cluster formation – 68.37.Lp Transmission electron microscopy (TEM) (including STEM, HRTEM, etc.) – 82.35.Pq Biopolymers, biopolymerization 1 Introduction With progressive miniaturisation of electronic and opti- cal devices there is an increasing interest in the develop- ment of advanced methods for the bottom-up construc- tion of functional nanostructures. In particular, the use of biological molecules and structures constitutes a very promising approach for the organisation of inorganic ma- terials at the nanometer length scale [1]. Due to the well defined chemical, physical and structural properties of the templates, the method of biomolecular templating allows parallel fabrication of desired inorganic nanostructures of different morphology [2–7]. Especially, regular S layers are found to be well suited as templates for the predefined fabrication of ordered metallic and semiconducting cluster arrays [8–10]. S layers are regular quasi two-dimensional protein crystals with a thickness of 5 to 15 nm. They con- stitute the outermost component of the cell envelope of many bacteria and archaea [11–13], exhibit different lat- tice symmetries with lattice constants ranging from 3 to 30 nm and possess nanopores of well-defined size. S lay- ers are composed of periodically-arranged identical sub- a e-mail: mertig@tmfs.mpgfk.tu-dresden.de b The authors wish to thank S. Selenska-Pobell and J. Raff for stimulating discussions concerning the interaction of tran- sition metals with proteins and for purification of the protein, and H. Lichte for a critical reading of the manuscript. units leading to a precise spatial arrangement of physico- chemical affinity sites at the protein surface, that can be used to accomplish a site-specific chemistry. These partic- ular properties allow to employ S layers for the template- directed parallel fabrication and manipulation of metal clusters with a narrow size distribution [10]. Moreover, the possibility to reconstitute S layers in vitro into large-area two-dimensional crystals [14] makes them an almost ideal biomolecular template for supramolecular engineering. Here, we report two different methods of the forma- tion of regular cluster arrays on the S layer of Bacillus sphaericus NCTC 9602: the chemical deposition of metal- lic clusters, and the template-directed synthesis of Pt and Pd nanoparticle arrays in the transmission electron micro- scope. In the latter method, the simultaneous growth of ordered nanoparticles is solely initiated by electron ex- posure. That is, the metal clusters are manipulated in parallel. Furthermore, advanced transmission electron mi- croscopy (TEM) methods like electron holography [15] are exploited for the sample characterisation. 2 Experimental The S-layer protein was isolated from Bacillus sphaeri- cus NCTC 9602 cells. The cell cultivation conditions and protein purification are described elsewhere [16,17]. The obtained S-layer preparation was washed, resuspended in