Molecular patterning on carbon based surfaces through photobiotin activation

Abstract

We have demonstrated the site-specific adhesion of photobiotin as a method of producing protein micropatterns. These patterns were created by the selective UV irradiation of a thin film of deposited photobiotin. The UV activated areas of photobiotin were then developed using fluorescently labelled avidin. The size of pattern produced is an order of magnitude smaller than those previously reported by this method. The patterns were characterised, using atomic force microscopy (AFM) to determine their microstructure. It was found that the AFM could discriminate between the areas of protein immobilised to the surface through the activated photobiotin, and the bare substrate surface where the inactivated photobiotin had been removed during the washing process. The potential of these patterns as sensing surfaces is demonstrated through the creation of a spatially patterned immunosensing surface. In this case, a biotinylated antibody was bound to the surface and the pattern developed using a second antibody specific to the immobilised biotinylated antibody. This technique could thus provide a simple and efficient method of producing high density immunoassay systems.

Covalent modification of graphitic carbon substrates by non-electrochemical methods

“…In their second method, UV laser light (325 nm) was focused on the photobiotin-coated GC surface and the GC was moved in the controlled manner to 'write' covalently-attached biotin features on the surface, with line widths of 5 -10 µm [82]. A similar approach to biotin patterning has also been reported based on irradiation of the photobiotin surface through a mask [83]. The biotin-derivatised surfaces react with avidin-tagged enzymes, providing a general and versatile method for enzyme immobilization on carbon surfaces.…”

Enhanced electrochemical activity of redox-labels in multi-layered protein films on indium tin oxide nanoparticle-based electrode

“…This requires careful and precise surface immobilization technique, which is difficult to achieve. Alternatively, a rough surface can be used to deliberately prevent dense packing of proteins, as demonstrated already on carbon electrodes [25,26]. We chose to create a rough ITO surface by depositing a thick film of nanoparticles on sputtered ITO glass substrate.…”

“…Because the small molecule diffuses freely in solution, its electrode reaction is usually not a problem, unless surface immobilization of proteins prevents it from getting close to the electrode. To overcome this potential problem, separate micro-and nano-sized domains for protein immobilization and electron transfer reaction were fabricated on carbon electrodes [25][26][27]. Many DNA assays employ redox-active transition metal complexes or organic dyes as labels [28,29].…”

Three-dimensional biomolecule patterning

“…Micro-patterned surfaces have become increasingly important to biosensor systems, where patterned arrays of antibodies, DNA and enzymes have been proposed to monitor the functions of biomolecules in situ [2]. The construction of arrays with micrometer dimension allows the measurement of many different biological analytes using samples of very small volume.…”