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1. DF Illumination And SPR Collection Au-coated glass slide Right prism SPR measurement TIR Illumination 26 nm 22 nm 18 nm 14 nm 10 nm 6 nm 2 nm 0 nm Image/Specimen Plane 60-nm Au NP SLM 45-nm Au film glass slide polyelectrolyte layer 100x 1.25 NA oil immersion objective Fourier Lens cover glass H2O ELP Au-coated glass slide dove prism Image Phase Hologram Reconstructed Image NIRT: Hierarchical Bionanomanufacturing PI: Robert L. Clark1. Co-PIs: Ashutosh Chilkoti2, Eric Toone3, and Stefan Zauscher1 Mechanical Engineering and Materials Science1, Biomedical Engineering2, Chemistry3, Duke University Abstract II) Localized Actuation of Thermally Responsive Polymers and Polypeptides III) Fabrication of Bioconjugated and Hybrid Polymeric Nanostructures by Field-Induced Scanning Probe Lithography Here we report on progress on our NIRT on hierarchical nanomanufacturing. I) First we report on a holographic technique we adopted that provides a noninvasive laser-based approach for adaptable, real-time nanofabrication and nanomodification of (bio)-polymeric materials substrates. II) We then discuss a technique we are developing in which optically addressable metal nanoparticles display large spectral shifts as a function of subtle changes in distance from conductive films, and hence, represent a promising way to measure angstrom-scale distances in potential biosensor applications. III) Finally we report on a process we developed for the chemical modification of protein resistant polymer brushes for the fabrication of protein arrays by field-induced scanning probe lithography. We are currently developing a novel and highly sensitive method of sensing nanometer-scale distances between single nanoparticles (NPs) and conductive surfaces. Once characterized, this method will be used to indicate thermally responsive polymer actuation that is localized to specific regions in-between metal nanoparticles and a conductive film.  Here, the high-energy focusing capabilities of nanoparticles in proximity to metal films upon laser irradiation will cause a temperature increase, and likely trigger polymer actuation, in a highly localized manner. Highly controlled patterning of polymeric and biomolecular nanostructures on surfaces is a critical step in the fabrication of biomolecular devices and sensors. We use field-induced scanning probe lithography (FISPL) to chemically modify polymer brushes to allow conjugation of biomolecules. Surface-confined, non-fouling (i.e., protein resistant) poly(oligo(ethylene glycol) methyl methacrylate) (p(OEGMA)) brushes were prepared on silicon substrates by surface-initiated atom transfer radical polymerization (SI-ATRP) in a “grafting-from” approach. These p(OEGMA) brushes were then patterned directly on the nanoscale by FISPL, generating chemical functionalities that allowed for subsequent protein conjugation (Schematic and AFM images below). Although our approach works, we do not yet have a complete physical-chemical understanding of the process. We hypothesize that -CH3 groups are oxidized in the presence of high electric fields and converted to -COOH groups. After chemical modification, protein conjugation is achieved on the patterned areas via biotin-streptavidin coupling. With this approach we were able to create periodic BSA protein arrays with a feature width of ~130 nm. Non-specific adsorption of protein was dramatically reduced due to the non-fouling nature of the polymer. Au Au-coated glass I) Three-dimensional Dynamic Mask-Less Holographic Lithography Spectroscopy of Nanoparticles near Gold Films A method is presented for dynamic, computer-controlled, maskless beam-steering, by spatial light modulators (SLMs), to address specific locations on arrays with large spatial and temporal selectivity. The dynamic maskless holographic lithography (DMHL) approach is ideally suited to trigger and direct nanofabrication in the optical near-field through easily controllable far-field, broad beam illumination sources. The beam from the laser is centered on the computer-controlled SLM, reflected into the microscope and directed upwards through the objective to illuminate the specimen plane. The optical system ensures the plane of the SLM is imaged to and completely fills the rear aperture of the microscope objective. Beam propagation from the rear aperture through the objective results in reconstruction of the original image on the micro/nanoscale. Single NP, TIR Illumination (SPP + LSP) SPP and LSP Deconvolution We also found that both (a) raised features and (b) trenches can be created on the p(OEGMA) brushes by controlling the contact force between the tip and the substrate. While the feature size of the patterns could be controlled by adjusting the patterning parameters such as applied voltage, relative humidity and tip velocity. We note that if a negative tip bias or no bias is applied with respect to the substrate, no appreciable changes are observed. This highlights the directionality of the electrochemical process and shows that physical interactions between the tip and the sample alone cannot be responsible for the formation of the patterns. Layer-by-layer (LBL) assembly of oppositely charged polyelectrolytes (PELs) was used to control the distance between NPs and gold films. The surface plasmon polariton (SPP) resonance propagating along the gold film red shifts as the number of PEL layers increases (left plot, green trend). Single NP spectra can be acquired using two different methods of illumination: i) dark field (DF) or ii) total internal reflectance (TIR) illumination. Dark field illumination primarily excites the localized surface plasmon (LSP) resonance of the NP. Single NP spectra, obtained using DF illumination, are increasingly blue-shifted as the NPs are spaced further away from the high-dielectric gold film (left plot, blue trend). While TIR illumination primarily excites the SPP resonance of the gold film, LSP resonances of NPs in close proximity to the film are also excited via an evanescent field. For this reason, single NP spectra, obtained using TIR illumination, show a convoluted dependence on layer height that is dominated by the blue-shifting LSP when NPs are less than 14 nm away from the gold film, and by red-shifting (SPP-dominated) when NPs are further than 14 nm from the gold film (right plot). These data suggest that the SPR of NPs is shifted by ~12 nm for every 1‑nm change in distance from 0 – 6 nm and ~7 nm for every 1-nm change in distance from 6 – 14 nm. With a high resolution grating this experimental design is potentially capable of sensing distance changes on the order of Ångstroms! a) b) Localized Optothermal ELP Actuation We have made use of the chemical changes on the brush surface that occur due to the oxidative nature of the electrochemical patterning process and demonstrate that they can be used to create bioconjugated nanostructures. Our unique patterning approach can potentially form the basis for the fabrication of a large range of novel polymeric and biomolecular nanostructures that may find application as biosensors or substrates for the precise presentation of biomolecular queues to cells. We are currently developing the tools and procedures to expand the lithographic approach into a massively parallel, anodization stamping process. Micro/Nano Patterning with Photopolymer Initial system testing was performed using an optically responsive epoxy (Norland Photopolymer 63). (a) CAD-generated pattern (b) SEM image taken normal to the substrate surface showing 880 nm to 2.5 m feature widths. Note the letter ‘L’ has fallen over due to post-processing after patterning. (c) SEM images taken at a 40˚ and (d) 60˚ from the surface show the height of the features ranging between 3-5 microns. With an optimized system and improved feedback control, resolution could be improved to at least the diffraction limit of ~ 250 nm. a) b) Acknowledgments We acknowledge support through NSF-NIRT 0609265. We also acknowledge the substantial contributions to the optothermal NP interrogation by Prof. David Smith and Jack Mock at Duke University. c) d) Preliminary experiments are aimed at localized actuation of elastin-like polypeptides (ELP, is a thermally responsive polypeptide) sandwiched between single NPs and a gold film, using laser heating. The schematic experimental setup is shown above; the plot (above right) shows a shift of ~30 nm in the SPR response of a single nanoparticle after 1 min of laser exposure. Although a spectral shift of this magnitude would indicate a change in distance between the NP and the gold film, it cannot yet be concluded that this shift is due to thermally induced changes in ELP conformation. Current studies are focused on characterizing spectral properties of NPs in close proximity to gold films with water as the surrounding medium to provide a distance calibration for localized ELP actuation experiments.