We demonstrate a localized protein immobilization method based on controlled physical adsorption on the three-phase boundary of an aqueous phase, a gas phase, and a polymeric material. By imprinting micrometer and sub-micrometer pillars onto a polymeric foil, superhydrophobic surfaces are fabricated. Those structures force the fluid locally into the Cassie–Baxter state and generate an artificial three-phase boundary at the edges of the imprinted pillars. First, fluorescence-labeled bovine serum albumin (BSA) and streptavidin dissolved in various buffer solutions are utilized to investigate protein adsorption on the structured surfaces. A stable adsorption of the respective protein on the three-phase boundary is observed. The following experiments use streptavidin adsorbed on the pillars to immobilize biotinylated antibodies for analyte detection. The pillars are passivated with an excess concentration of BSA to reduce nonspecific protein adsorption. Implemented in a lab-on-a-chip device, the proposed immobilization method is utilized in a sandwich assay to detect the inflammation marker C-reactive protein in human serum, showing the potential of this immobilization method for diagnostic applications. The method overcomes laborious procedures to immobilize proteins on thermoplastic materials, which enables the fast transfer of point-of-care applications from research to commercial scale.
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The authors would like to thank T. Robinson for capturing the laser scanning microscopy images. Thanks go to P. Abaffy and G. Papagno for capturing the SEM micrographs and L. Zimmermann and M. Röhrig for supplying the hot embossing mold inserts. N. Steidle would like to thank E. Rubiu and P. Tritschler for their help preparing the samples. This work is supported by the Bürkert Technology Center (BTC), a cooperation between Bürkert Werke GmbH & Co. KG and the Institute of Microstructure Technology (IMT) at the Karlsruhe Institute of Technology (KIT). This work was carried out with the support of the Karlsruhe Nano Micro Facility (KNMF), a Helmholtz Research Infrastructure at KIT.
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Steidle, N.E., Hahn, T., Bader, C. et al. Localized protein immobilization on microstructured polymeric surfaces for diagnostic applications. Microfluid Nanofluid 20, 30 (2016). https://doi.org/10.1007/s10404-015-1696-8
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