Reusable linking chemistry for His‐6 tagged proteins in an affinity‐based porous silicon biosensor

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)