Bioactive protein nanoarrays on nickel oxide surfaces formed by dip-pen nanolithography.

Abstract

Biologically functional protein arrays are important for chipbased protein detection assays and proteomic profiling experiments. Nanoscale arrays allow for smaller chips with more reaction sites, smaller test sample volumes, potentially higher sensitivity and speed, and direct feature analysis with a scanning probe instrument. Several promising routes to protein nanoarrays with submicrometer and even sub-100-nm features have been reported. The activity of the immobilized proteins in some of the arrays generated by dip-pen nanolithography (DPN) has been confirmed by fluorescence labeling studies and direct imaging by atomic force microscopy (AFM). Nickel is a commonly used substrate for biological arrays because the oxidized Ni surface has a high affinity for polyhistidine residues, and this specific interaction, in principle, can provide control over the uniformity of protein binding and presentation to the analyte solution. The histidine tag allows for protein adsorption without direct contact between the active area of the protein and the substrate surface. The deposition of histidine-tagged peptides and proteins on Ni substrates by using electrochemical DPN was recently reported; however, it was concluded that peptide and protein transport could not be effected without an applied field, and the biological activities of the generated nanofeatures were not studied. The requirement of an applied field is limiting with respect to chemical compatibility of the protein inks and protein denaturation under such conditions ( 2 to 3 V) and the complexity of the hardware used to effect such a process. Herein, we report a methodology based upon DPN and conditions that allow one to generate biologically active protein nanoarrays with feature sizes as small as approximately 80 nm on Ni surfaces without the need for an applied field (Figure 1). To facilitate ink wetting and transport, AFM tips were coated with a thin layer of Ni (ca. 5 nm) by thermal