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The inexpensive fabrication and intrinsic optical and filtering properties of porous silicon (PSi) make optical PSi biosensors ideally suited for diagnostic testing. Long term goals of a diagnostic sensor would benefit from reversible binding chemistry to allow retesting of patient samples and collection of captured target for analysis. This work illustrates a versatile method to immobilize hexahistidine (His-6) tagged molecular probes and utilizes reversible competitive chelating chemistry to reuse the porous silicon sensor device. Reuse of this Ni–His-6 chemistry in a PSi affinity-based biosensor was demonstrated with two different His-6 tagged proteins to give consistent probe loading values within mimimal error over 4 cycles of use. Advantages of utilizing any site-directed immobilization method include the homogeneity of surface bound probe molecule orientation, which we also illustrate resulted in a 2-fold increase in detection capability of a secondary target antibody. As the current sensitivity range of macroporous PSi sensors (diameters of 30–100 nm) pose limitations to their use in medical applications; this work identifies the ability to improve target capture through the development of an efficient and versatile linking chemistry. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)