The facile preparation of arrays of plasmonic nanoparticles over a square centimeter surface area is reported. The developed method relies on tailored laser interference lithography (LIL) that is combined with dry etching and it offers means for the rapid fabrication of periodic arrays of metallic nanostructures with well controlled morphology. Adjusting the parameters of the LIL process allows for the preparation of arrays of nanoparticles with a diameter below hundred nanometers independently of their lattice spacing. Gold nanoparticle arrays were precisely engineered to support localized surface plasmon resonance (LSPR) with different damping at desired wavelengths in the visible and near infrared part of the spectrum. The applicability of these substrates for surface enhanced Raman scattering is demonstrated where cost-effective, uniform and reproducible substrates are of paramount importance. The role of deviations in the spectral position and the width of the LSPR band affected by slight variations of plasmonic nanostructures is discussed.

We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin α v β 3 via high-throughput screening of partially randomized libraries. Peptide libraries (672 different compounds) comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop and a randomized sequence XXX (X being one of 18 canonical L-amino acids) in the second loop, both enclosed by either an L-or D-Cys residue, were converted to bicyclic peptides via reaction with 1,3,5-tris(bromomethyl)benzene (T3). Screening of first-generation libraries yielded lead bicyclic inhibitors displaying submicromolar affinities for integrin α v β 3 (e.g., C T3 HEQc T3 RGDc T3 , IC 50 = 195 nM). Next generation (second and third) libraries were obtained by partially varying the structure of the strongest lead inhibitors and screening for improved affinities and selectivities. In this way, we identified the highly selective bicyclic α v β 3 -binders C T3 HPQc T3 RGDc T3 (IC 50 = 30 nM), C T3 HPQC T3 RGDc T3 (IC 50 = 31 nM), and C T3 HSQC T3 RGDc T3 (IC 50 = 42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC 50 = 38 nM) and outstanding selectivities over integrins α v β 5 (IC 50 > 10000 nM) and α 5 β 1 (IC 50 > 10000 nM). Affinity measurements using surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded K d values of 0.4 and 0.6 nM for the Cy5-labeled bicycle C T3 HPQc T3 RGDc T3 and RGD "knottin" peptide, respectively. In vitro staining of HT29 cells with Cy5labeled bicycles using confocal microscopy revealed strong binding to integrins in their natural environment, which highlights the high potential of these peptides as markers of integrin expression.

We report the identification of high-affinity and selectivity integrin α5β1-binding bicyclic peptides via “designed random libraries”, that is, the screening of libraries comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop in combination with a randomized sequence (XXX) in the second loop. Screening of first-generation libraries for α5β1-binding peptides yielded a triple-digit nanomolar bicyclic α5β1-binder (C T3 RGDc T3 AYGC T3 , IC50 = 406 nM). Next-generation libraries were designed by partially varying the structure of the strongest first-generation lead inhibitor and screened for improved affinities and selectivities for this receptor. In this way, we identified three high-affinity α5β1-binders (C T3 RGDc T3 AYJC T3 , J = d-Leu, IC50 = 90 nM; C T3 RGDc T3 AYaC T3 , IC50 = 156 nM; C T3 RGDc T3 AWGC T3 , IC50 = 173 nM), of which one even showed a higher α5β1-affinity than the 32 amino acid benchmark peptide knottin-RGD (IC50 = 114 nM). Affinity for α5β1-integrin was confirmed by SPFS analysis showing a K d of 4.1 nM for Cy5-labeled RGD-bicycle C T3 RGDc T3 AYJC T3 (J = d-Leu) and a somewhat higher K d (9.0 nM) for Cy5-labeled knottin-RGD. The α5β1-bicycles, for example, C T3 RGDc T3 AYJC T3 (J = d-Leu), showed excellent selectivities over αvβ5 (IC50 ratio α5β1/αvβ5 between <0.009 and 0.039) and acceptable selectivities over αvβ3 (IC50 ratios α5β1/αvβ3 between 0.090 and 0.157). In vitro staining of adipose-derived stem cells with Cy5-labeled peptides using confocal microscopy revealed strong binding of the α5β1-selective bicycle C T3 RGDc T3 AWGC T3 to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for α5β1-integrin expression.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmonenhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.