Covalent immobilization of molecularly imprinted polymer nanoparticles on a gold surface using carbodiimide coupling for chemical sensing.

One challenging task in building (bio)chemical sensors is the efficient and stable immobilization of receptor on a suitable transducer. Herein, we report a method for covalent immobilization of molecularly imprinted core-shell nanoparticles for construction of robust chemical sensors. The imprinted nanoparticles with a core-shell structure have selective molecular binding sites in the core and multiple amino groups in the shell. The model Au transducer surface is first functionalized with a self-assembled monolayer of 11-mercaptoundecanoic acid. The 11-mercaptoundecanoic acid is activated by treatment with carbodiimide/N-hydroxysuccinimide and then reacted with the core-shell nanoparticles to form amide bonds. We have characterized the process by studying the treated surfaces after each preparation step using atomic force microscopy, scanning electron microscopy, fluorescence microscopy, contact angle measurements and X-ray photoelectron spectroscopy. The microscopy results show the successful immobilization of the imprinted nanoparticles on the surface. The photoelectron spectroscopy results further confirm the success of each functionalization step. Further, the amino groups on the MIP surface were activated by electrostatically adsorbing negatively charged Au colloids. The functionalized surface was shown to be active for surface enhanced Raman scattering detection of propranolol. The particle immobilization and surface enhanced Raman scattering approach described here has a general applicability for constructing chemical sensors in different formats.

Photoconjugation of Molecularly Imprinted Polymer Nanoparticles for Surface-Enhanced Raman Detection of Propranolol.

We report a simple and versatile method to covalently immobilize molecularly imprinted polymer (MIP) nanoparticles on a Raman active substrate (Klarite) using a disulfide-derivatized perfluorophenylazide (PFPA-disulfide). Gold-coated Klarite was functionalized with PFPA-disulfide via a gold-sulfur bond. Upon light radiation, the available azido groups were converted to highly reactive singlet perfluorophenyl nitrene that undergoes a CH insertion reaction and form covalent bonds with the MIP nanoparticles. The resulting surfaces were characterized using scanning electron microscopy and surface enhanced Raman spectroscopy to study the morphology and template affinity of the surfaces, respectively. The Raman measurements clearly show a dose-responsive signal when propranolol binds to the MIP surface. Because the MIP particles were covalently attached to the Raman active substrate, the sensing surface was stable and could be reused after regeneration in acetic acid solution. The MIP-based Raman sensor was used successfully to detect propranolol in urine samples (7.7 × 10(-4) M). Our results show that the high selectivity of MIPs and the fingerprint Raman identification can be integrated into a compact sensing unit using high-efficiency photoconjugation. Thus, the method proposed is reliable, efficient and fast for fabricating label-free chemical sensors.

Scalable, high performance, enzymatic cathodes based on nanoimprint lithography.

Here we detail high performance, enzymatic electrodes for oxygen bio-electroreduction, which can be easily and reproducibly fabricated with industry-scale throughput. Planar and nanostructured electrodes were built on biocompatible, flexible polymer sheets, while nanoimprint lithography was used for electrode nanostructuring. To the best of our knowledge, this is one of the first reports concerning the usage of nanoimprint lithography for amperometric bioelectronic devices. The enzyme (Myrothecium verrucaria bilirubin oxidase) was immobilised on planar (control) and artificially nanostructured, gold electrodes by direct physical adsorption. The detailed electrochemical investigation of bioelectrodes was performed and the following parameters were obtained: open circuit voltage of approximately 0.75 V, and maximum bio-electrocatalytic current densities of 18 µA/cm(2) and 58 µA/cm(2) in air-saturated buffers versus 48 µA/cm(2) and 186 µA/cm(2) in oxygen-saturated buffers for planar and nanostructured electrodes, respectively. The half-deactivation times of planar and nanostructured biocathodes were measured to be 2 h and 14 h, respectively. The comparison of standard heterogeneous and bio-electrocatalytic rate constants showed that the improved bio-electrocatalytic performance of the nanostructured biocathodes compared to planar biodevices is due to the increased surface area of the nanostructured electrodes, whereas their improved operational stability is attributed to stabilisation of the enzyme inside nanocavities.

Implementation of molecularly imprinted polymer beads for surface enhanced Raman detection.

Molecularly imprinted polymers (MIPs) have a predesigned molecular recognition capability that can be used to build robust chemical sensors. MIP-based chemical sensors allow label-free detection and are particularly interesting due to their simple operation. In this work we report the use of thiol-terminated MIP microspheres to construct surfaces for detection of a model organic analyte, nicotine, by surface enhanced Raman scattering (SERS). The nicotine-imprinted microspheres are synthesized by RAFT precipitation polymerization and converted into thiol-terminated microspheres through aminolysis. The thiol groups on the MIP surface allow the microspheres to be immobilized on a gold-coated substrate. Three different strategies are investigated to achieve surface enhanced Raman scattering in the vicinity of the imprinted sites: (1) direct sputtering of gold nanoparticles, (2) immobilization of gold colloids through the MIP's thiol groups, and (3) trapping of the MIP microspheres in a patterned SERS substrate. For the first time we show that large MIP microspheres can be turned into selective SERS surfaces through the three different approaches of assembly. The MIP-based sensing surfaces are used to detect nicotine to demonstrate the proof of concept. As synthesis and surface functionalization of MIP microspheres and nanoparticles are well established, the methods reported in this work are handy and efficient for constructing label-free chemical sensors, in particular for those based on SERS detection.

Covalent immobilization of molecularly imprinted polymer nanoparticles using an epoxy silane.

Molecularly imprinted polymers (MIPs) can be used as antibody mimics to develop robust chemical sensors. One challenging problem in using MIPs for sensor development is the lack of reliable conjugation chemistry that allows MIPs to be fixed on transducer surface. In this work, we study the use of epoxy silane to immobilize MIP nanoparticles on model transducer surfaces without impairing the function of the immobilized nanoparticles. The MIP nanoparticles with a core-shell structure have selective molecular binding sites in the core and multiple amino groups in the shell. The model transducer surface is functionalized with a self-assembled monolayer of epoxy silane, which reacts with the core-shell MIP particles to enable straightforward immobilization. The whole process is characterized by studying the treated surfaces after each preparation step using atomic force microscopy, scanning electron microscopy, fluorescence microscopy, contact angle measurements and X-ray photoelectron spectroscopy. The microscopy results show that the MIP particles are immobilized uniformly on surface. The photoelectron spectroscopy results further confirm the action of each functionalization step. The molecular selectivity of the MIP-functionalized surface is verified by radioligand binding analysis. The particle immobilization approach described here has a general applicability for constructing selective chemical sensors in different formats.

Nanowire-based electrode for acute in vivo neural recordings in the brain.

We present an electrode, based on structurally controlled nanowires, as a first step towards developing a useful nanostructured device for neurophysiological measurements in vivo. The sensing part of the electrode is made of a metal film deposited on top of an array of epitaxially grown gallium phosphide nanowires. We achieved the first functional testing of the nanowire-based electrode by performing acute in vivo recordings in the rat cerebral cortex and withstanding multiple brain implantations. Due to the controllable geometry of the nanowires, this type of electrode can be used as a model system for further analysis of the functional properties of nanostructured neuronal interfaces in vivo.

Self-organization of motor-propelled cytoskeletal filaments at topographically defined borders.

Self-organization phenomena are of critical importance in living organisms and of great interest to exploit in nanotechnology. Here we describe in vitro self-organization of molecular motor-propelled actin filaments, manifested as a tendency of the filaments to accumulate in high density close to topographically defined edges on nano- and microstructured surfaces. We hypothesized that this "edge-tracing" effect either (1) results from increased motor density along the guiding edges or (2) is a direct consequence of the asymmetric constraints on stochastic changes in filament sliding direction imposed by the edges. The latter hypothesis is well captured by a model explicitly defining the constraints of motility on structured surfaces in combination with Monte-Carlo simulations [cf. Nitta et al. (2006)] of filament sliding. In support of hypothesis 2 we found that the model reproduced the edge tracing effect without the need to assume increased motor density at the edges. We then used model simulations to elucidate mechanistic details. The results are discussed in relation to nanotechnological applications and future experiments to test model predictions.

Efficient methods of nanoimprint stamp cleaning based on imprint self-cleaning effect.

Nanoimprint lithography (NIL) is a nonconventional lithographic technique that promises low-cost, high-throughput patterning of structures with sub-10 nm resolution. Contamination of nanoimprint stamps is one of the key obstacles to industrialize the NIL technology. Here, we report two efficient approaches for removal of typical contamination of particles and residual resist from stamps: thermal and ultraviolet (UV) imprinting cleaning-both based on the self-cleaning effect of imprinting process. The contaminated stamps were imprinted onto polymer substrates and after demolding, they were treated with an organic solvent. The images of the stamp before and after the cleaning processes show that the two cleaning approaches can effectively remove contamination from stamps without destroying the stamp structures. The contact angles of the stamp before and after the cleaning processes indicate that the cleaning methods do not significantly degrade the anti-sticking layer. The cleaning processes reported in this work could also be used for substrate cleaning.

Molecularly imprinted nanostructures by nanoimprint lithography.

Simultaneous imprinting on two length scales (nanometer and ångström) delivers highly specific molecular recognition sites in polymer patterns.

Nanofluidics in hollow nanowires.

We present a novel scheme for producing nanotube membranes using free-standing hollow nanowires, with easily controllable dimensions. GaAs-AlInP core-shell nanowires were grown by metal-organic vapor phase epitaxy and were partially embedded in a polymer film. The GaAs core and substrate were etched selectively, leaving tubes with open access to both sides of the membrane. Electrophoretic transport of T4-phage DNA through the hollow nanowires was demonstrated using epifluorescence microscopy.

Fifteen-piconewton force detection from neural growth cones using nanowire arrays.

We used epitaxially grown monodisperse nanowire arrays to measure cellular forces with a spatial resolution of 1 mum. Nerve cells were cultured on the array and cellular forces were calculated from the displacement of the nanowire tips. The measurements were done in situ on live cells using confocal microscopy. Forces down to 15 pN were measured on neural growth cones, showing that this method can be used to study the fine details of growth-cone dynamics.

Nanowire biocompatibility in the brain--looking for a needle in a 3D stack.

We investigated the brain-tissue response to nanowire implantations in the rat striatum after 1, 6, and 12 weeks using immunohistochemistry. The nanowires could be visualized in the scar by confocal microscopy (through the scattered laser light). For the nanowire-implanted animals, there is a significant astrocyte response at week 1 compared to controls. The nanowires are phagocytized by ED1 positive microglia, and some of them are degraded and/or transported away from the brain.

Attovial-based antibody nanoarrays.

Antibody array-based technology is a powerful emerging tool in proteomics, but to enable global proteome analysis, antibody array layouts with even higher density has to be developed. To this end, we have further developed the first generation of a nanoarray platform, based on attoliter-sized vials, attovials, which we have characterized and used for the detection of complement factor C1q in human serum samples. Finally, we demonstrated proof-of-concept for individual functionalization of the attovials with a recombinant antibody.

Rectifying and sorting of regenerating axons by free-standing nanowire patterns: a highway for nerve fibers.

We present an EBL-defined nanowire pattern that can sort axons coming from different directions on a substrate. The pattern defines tracks for left-bound traffic and right-bound traffic, which opens up new possibilities for designing neural networks on a chip.

Diffusion dynamics of motor-driven transport: gradient production and self-organization of surfaces.

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.

Axonal guidance on patterned free-standing nanowire surfaces.

We demonstrate high-fidelity guidance of axons using rows of nanowires. The axons are prevented from crossing the rows, making it possible to guide and sort a large number of axons as opposed to when chemical patterns are used. Focal adhesion forms at the nanowires establishing a possible site of information transfer between the surface and the cells. Rows of gallium phosphide (GaP) nanowires were epitaxially grown on GaP(111) substrates in patterns defined by electron beam lithography.

Gallium phosphide nanowires as a substrate for cultured neurons.

Dissociated sensory neurons were cultured on epitaxial gallium phosphide (GaP) nanowires grown vertically from a gallium phosphide surface. Substrates covered by 2.5 microm long, 50 nm wide nanowires supported cell adhesion and axonal outgrowth. Cell survival was better on nanowire substrates than on planar control substrates. The cells interacted closely with the nanostructures, and cells penetrated by hundreds of wires were observed as well as wire bending due to forces exerted by the cells.