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Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C′-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic–inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture. Biotechnol. Bioeng. 2012; 109:1120–1130. © 2011 Wiley Periodicals, Inc.

Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics.