Biosensors DOI: 10.1002/smll.200500265 Selective Loading of Kinesin-Powered Molecular Shuttles with Protein Cargo and its Application to Biosensing** Sujatha Ramachandran, Karl-Heinz Ernst, George D. Bachand, Viola Vogel, and Henry Hess* Molecular shuttles, nanoscale biomolecular-motor-driven transport systems, are a bioinspired alternative to pressure- driven fluid flow or electroosmotic flow in micro- and nano- fluidic systems. [1] Significant progress has been made in di- recting the movement of such shuttles, [2–6] in controlling their activation, [7–9] and in loading some types of cargo. However, a generalized approach to selectively bind nano- scale cargo, such as proteins, viruses, or inorganic nano- particles, to a molecular shuttle is a pressing concern for researchers interested in the technological applica- tions of active transport, since it enables the design of a variety of analytical devi- ces. Here, we demonstrate that selective binding and subsequent transport of target proteins can be achieved by assembling a multilayer structure consisting of streptavidin and biotinylated antibod- ies on a biotinylated microtubule, which can be transported by surface-immobilized kinesin motor proteins. Taxol-stabilized biotinylated microtubules, tubular struc- tures with a diameter of 25 nm and a length of several mi- crometers, have been previously used to transport a variety of streptavidin-coated cargoes, including microspheres, [7] DNA, [10] and quantum dots. [11,12] The critical drawback of this approach is that the cargo has to be tagged prior to cap- ture and transport with streptavidin or at least biotin, if a streptavidin bridge is employed. [13] We have overcome this drawback by using streptavidin to immobilize commercially available biotinylated antibodies onto the biotinylated mi- crotubules, thus permitting the capture of a wide range of targets. This technique complements the recently reported conjugation of cyclodextrin to microtubules via streptavidin, which permits the capture of selected chemical agents. [14] Moreover, the resulting antibody-coated microtubule be- comes a motile platform for established biosensing techni- ques, such as the double-antibody sandwich assay (Figure 1). The challenge in this approach lies primarily in the as- sembly of a large supramolecular structure from more than ten thousand individual proteins, which are capable of cross- linking with each other in a variety of ways. While attempts to successively link streptavidin and antibodies to micro- tubules in solution proved unfruitful due to the difficulty of preventing unwanted crosslinking between microtubules and the difficulty of repeatedly purifying intact microtubules from a protein solution in high yield, we succeeded by ex- ploiting the inherent affinity of microtubules to kinesin- coated surfaces. By immobilizing microtubules onto kinesin- coated surfaces, we were able to successively expose them to up to ten distinct solutions containing streptavidin, biotin- ylated antibodies, or buffer, and to observe the progress of the binding of proteins from solution to the microtubules in real time. In the following, details for the assembly steps and a demonstration of the selective binding of myoglobin (an important cardiac marker) are presented. [15] The initial coating of the kinesin-bound, biotinylated mi- crotubules with streptavidin has to fill the available biotin binding sites completely in order to avoid unwanted cross- linking of microtubules with each other. [16] Fluorescence measurements of the binding of streptavidin labeled with Alexa 594 as a function of time and initial streptavidin con- Figure 1. Biotinylated antibodies can be connected to biotinylated, taxol-stabilized microtubules via a streptavidin bridge, enabling specific capture of target antigens. A fluorescently labeled antibody can be used for double-antibody sandwich detection of antigens. [*] S. Ramachandran Department of Bioengineering, University of Washington Seattle, WA 98195 (USA) Dr. K.-H. Ernst Molecular Surface Science Group Swiss Federal Institute for Materials Science and Technology (EMPA) Dübendorf (Switzerland) Dr. G. D. Bachand Biomolecular Materials and Interfaces Department and Center for Integrated Nanotechnologies Sandia National Laboratories, Albuquerque, NM 87185 (USA) Prof.V.Vogel Department of Materials, ETH Zurich Hçnggerberg, 8093 Zürich, (Switzerland) Prof. H. Hess Department of Bioengineering, University of Washington Seattle, WA 98195 (USA) and Department of Materials Science and Engineering Rhines Hall 100, University of Florida Gainesville, FL 32611 (USA) Fax:(+ 1)206-685-4434 E-mail: hhess@mse.ufl.edu [**] We gratefully acknowledge financial support from the DARPA Bio- molecular Motors Program, and the US Department of Energy, Office of Basic Energy Sciences, under grant DE-FG02- 05ER46193. 330 # 2006 Wiley-VCH Verlag GmbH&Co. KGaA, D-69451 Weinheim small 2006,2,No.3,330–334 communications