We present a scanning probe lithography technique that allows for patterning of adsorbed, water-soluble polymers on functionalized oxide surfaces. SiO₂ and borosilicate glass surfaces were functionalized with a negatively charged carboxyl-terminated silane monolayer. A ca. 2 nm poly-L-lysine layer was then deposited over the silane film. An atomic force microscope (AFM) probe was used to scribe away lines and areas of the positive poly-L-lysine layer, exposing the negatively charged silane underlayer. The AFM scribing experiments were performed both in air and in water. Regions were scribed and then backfilled with a fluorescently tagged polymer. Characterization of the scribing was done with tapping mode AFM. Optical fluorescence microscopy was used to image backfilled regions. AFM height and phase mode data showed lines and spaces with half pitch features as small as 12 nm created with a scribing force of 0.3 μN.

RF plasma treated nanocrystalline hybrid chitosan/TiO₂ matrix was used to develop high sensitivity electrochemical immnunosensor. The observed interfacial charge transfer resistance (R_CT) and double layer capacitance (C_d) of plasma modified matrixes were decreased. An improved strength of amide I and II groups in FTIR spectra were observed which was associated with enhancement in immobilized rabbit antibodies (IgGs) on plasma altered surfaces. Electrochemically q uantitative detection of Ochratoxin-A (OTA) concentrations varying in buffer solution was carried out on IgGs immobilized plasma treated and untreated ITO/CS/TiO₂ electrodes. Plasma modified electrodes had showed very good sensitivity at very low OTA concentrations whereas the untreated electrodes sensitivity was deprived.

We have studied the Fe 2p X-ray magnetic circular dichroism (XMCD) of individual magnetosomes - biomineralized ferrimagnetic nano-crystals in magnetotactic bacteria (MTB ) - using scanning transmission X-ray microscopy(STXM). Magnetosomes are intracellular magnetite (Fe₃O₄) or greigite (Fe₃S₄) nano-crystals (typically 30-60 nm in size), enclosed in a lipid membrane. A chain of magnetosomes is used by MTBs to orient relative to the earth’s field, and guide motion to optimal living environments. Our initial goal, which has been achieved, was to demonstrate that the STXM has the capability to investigate magnetic properties of sub-50 nm areas in biological systems. The Fe 2p XMCD of individual Fe₃O₄ magnetosomes of MV-1, a marine vibrio species of magnetotactic bacteria, was measured with the sample at 30 degrees relative to the beam to sense the in-plane magnetic component. This is the first such measurement of the XMCD of a single magnetosome to our knowledge. Evidence for multiple domains was found in some magnetosomes. In addition we have begun to explore the associated biochemistry by STXM spectromicroscopy at high spatial resolution (30 nm) at the C 1s and O 1s absorption edges. The combined XMCD and biochemical imaging will help further the understanding of biomineralization processes present in MTB and other environmental organisms.

Research funded by NSERC. The Canadian Light Source is supported by NSERC, NRC, CIHR, and the University of Saskatchewan. Some measurements were also made at STXM 11.0.2 at the Advanced Light Source, which is supported by the Division of Basic Energy Sciences of the U.S. Department of Energy.

Dentin is a natural composite material consisting of highly mineralized tubules (peritubular dentin, PTD) embedded in an intertubular matrix (ITD) consisting predominantly of collagen. Although the mechanics of dentin has been studied for over a century, only recently has the advent of nanomechanical testing allowed investigation of its microscopic characteristics. In particular, the role of the PTD in overall dentin mechanics can now be explored. By selecting small enough volumes of dentin, the orientational effect of the tubules can be examined. In this study, micron-sized pillars were fashioned by a novel femtosecond laser ablation technique, which avoids the material damage induced when milling is performed by a high energy ion beam or ablation by slower laser pulses. Testing of these structures was performed in a nanoindenter by recording force vs. deformation curves under constant strain rate (until failure) while compressing the pillar with a flat punch tip. These data provided both the modulus and strength of the sample. The small size of the pillars, approximately 20 x 20 microns in cross-section and 100 microns height, guarantee that the tubular orientation is well-defined within a single pillar compression experiment. A statistical correlation was observed between the tubule orientation and measured modulus, with a higher modulus being recorded when the tubule axis was oriented along or near the axis of compression. Such studies allow correlations between the local tubular orientation and biomechanical function. The new method described in this work does not expose the sample to dehydration in vacuum or high energy ions, and does not require coating with conductive materials. It is generally applicable to a variety of biological specimens.

Recently, we developed a novel complex material with combined optical and biological functionality [1, 2]. It consists of dielectric nanoparticles, which are adsorbed onto a plain gold surface and subsequently metallized by deposition of gold colloid prior to electroless plating. Upon reflection of white light, the layers exhibit pronounced extinction peaks which shift to higher wavelengths when molecules adsorb onto the surface. For simple alkanethiols a significantly higher red-shift of the extinction maximum was observed than reported for conventional surface plasmon resonance. To detect biomolecular interactions in array format it is crucial to guarantee homogenous optical response of the nanoparticle layers on macroscopic scales. We will, therefore, discuss the impact of different coating procedures on the optical properties of the films. To optimize sensitivity, effects of particle layer density, dielectric inter­layers and plating time have been investigated. We also compared the response of core-shell nanoparticle layers to the optical properties of surface adsorbed gold colloid films. The final goal is to incorporate high-density peptide arrays into the optically responsive nano­particle films by combinatorial laser printer synthesis [3] to facilitate label-free high-throughput screening of biomolecular interactions for biomedical and pharmaceutical applications. For this purpose, the peptide probes are embedded into a protein resistant matrix based on poly(ethylene glycol)methacrylate (PEGMA). The stability of both nanoparticle layers and PEGMA coating has been optimized with respect to the chemical and physical requirements of the biomolecular coupling reactions.

[1] R. Dahint, E. Trileva, H. Acunman, U. Konrad, M. Zimmer, V. Stadler, M. Himmelhaus, Biosens. Bioelectron., 22, 3174-3181 (2007)

[2] P. Buecker, E. Trileva, M. Himmelhaus, R. Dahint, Langmuir, 24, 8229-8239 (2008)

[3] V. Stadler, T. Felgenhauer, M. Beyer, S. Fernandez, K. Leibe, S. Güttler, M. Gröning, K. König, G. Torralba, M. Hausmann, V. Lindenstruth, A. Nesterov, I. Block, R. Pipkorn, A. Poustka, F. R. Bischoff, F. Breitling, Angew. Chem. Int. Ed ., 47, 7132-7135 (2008)

Surface patterning is often used to immobilize bioactive molecules including proteins, oligonucleotides and small ligands, to localize surface reactions for bioassays and to provide desired cell and bacterial adhesion. This study reports extensive surface analysis of a commercial PEG-based surface chemistry with active ester (NHS)-activity in patterned films. The study followed sequential immobilization and masking reactions on photolithographic patterns used to immobilize peptides, proteins, and cultured cells to specific patterned regions of NHS-reactive or de-activated chemistry.^[1] Biotin and peptide patterns were correlated to patterned reactive NHS surface chemistry using high-resolution time-of-flight secondary ion mass spectrometry (ToF-SIMS) for each species. Cell growth and patterning in 15-day serum cultures followed peptide patterns. In other patterned samples, mixed protein (streptavidin and HaloTag™) solutions produced spontaneous self-recognized, bound patterns on photolithographically surface-patterned affinity ligands for each (i.e., biotin and chloroalkane, respectively). The approach uses high-affinity protein-surface self-selection onto patterned PEG-NHS surfaces that exhibit intrinsically low non-specific adsorption background. Fluorescence images and ToF-SIMs imaging of the resulting protein surface selection from mixtures support highly specific interactions of proteins with their respective ligands patterned on the surface.^[2] On-going work comparing ToF-SIMs imaging of antibody F_c and F_ab fragments supports some ability to produce different whole antibody orientations on neighboring patterns spontaneously. Use of principal component analysis (PCA) helps to increase the ToF-SIMS image contrast and provide protein orientational details based on amino acid compositions.^[3]

References:

^[1]Takahashi, H., Emoto, K., Dubey, M., Castner, D. G., Grainger, D. W., Adv. Func. Matls. 2008. 18(14): p.2079-2088.

^[2]Dubey M., Emoto K., Takahashi H., Castner D.G., Grainger, D.W., submitted, 2009.

^[3] Dubey, M., Takahashi, H., Lew, F., Emoto, K., Castner, D.G., Grainger, D.W., submitted, 2009.

Though surface coatings of Poly-Ethylene Glycol (PEG) has been recognised for decades as a particularly effective non-fouling surface, recent advances in polymer brush fabricated thin film Poly-Oligio(Ethylene Glycol) Methyl Methacrylate (POEGMA) has presented a myriad of novel applications. The capability to easily tune brush height and still maintain a high surface grafting density has been shown to prepare surfaces which essentially eliminate the non-specific adsorption of both proteins and cells. Here we present the effects of selectively modifying the surface of polymer brush surfaces, such as POEGMA, via Electric Field Nanolithography (EFN).

EFN, utilizing a spatially localized potential bias to produce chemical modifications sites on a wide range of surfaces, has proven capable of serially modifying the chemical and conformational structure of a variety of polymer-brush film surfaces such as POEGMA, PolyAcrylic Acid, and PolyMethyl Methacrylate surfaces. Such work, however, has presented an interesting and novel bias voltage dependence previously unreported in literature.

Traditionally, EFN has been utilized to produce oxide-rich regions available for further reaction sites processing. Integration of a responsive, non-fouling, polymer brush surface, however, severely alters the voltage modification dependence from the traditional negative tip bias requirement to the now positive tip bias dependence.

Each polymer thin film studied presents a different surface energy landscape, hydrodynamic interaction characteristic, and intramolecular interaction. Presented results, in addition to the contrasted effects seen on spun-coated polymer thin films, will further illuminate the mechanism and effects of EFN integration with polymer brush thin films. In addition to topographical and chemical effects of these thin films, an elevated, film-thickness dependent, threshold bias voltage is reported. Films have been characterized using Xray Photonelectron Spectroscopy, Atomic Force Microscopy, and Contact angle measurements.

In furthering the understanding of how EFN interacts with polymer thin films, it will become possible to produce selective deposition of biological arrays and assays for next generation sensing applications.

Two dimensional (2D)-controlled adsorption is a versatile tool for creating well-defined arrays of biological molecules on surfaces. Such surfaces hold potential for a wide range of future applications, including for the development of biosensors and biomolecular screening technologies. Functionalizing a surface with some periodical structure (or a network) is one promising way to spatially control the adsorption process. Hydrogen-bonded networks are reported to be well-ordered structures, presenting periodical 2D-pores suitable for the adsorption of different guest molecules, and thus may provide a reasonable template for 2D-controlled biomolecular adsorption.