Robert E. Ducker

Zwitterionic poly(amino acid methacrylate) brushes.

A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 °C. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micropatterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nanopatterned PCysMA brushes were obtained using interference lithography, and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.

Polymeric and biomacromolecular brush nanostructures: progress in synthesis, patterning and characterization

A significant scientific and engineering challenge of recent years has been the fabrication of patterned polymeric and biomacromolecular brush nanostructures on surfaces. These structures provide researchers with a rich platform on which to exploit and observe nanoscale phenomena. In this review we present an overview of the field and highlight, through selected examples, recent advances in the nanostructuring of polymer and biomacromolecular brushes. This includes a brief overview of polymer brush synthesis techniques and how these are integrated with nanolithographic and templating approaches. We discuss the characterization of polymeric nanostructures and its associated difficulties, and we provide some perspective of how we see the future direction of the field evolving.

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents

Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.

Fabrication of submicrometer biomolecular patterns by near-field exposure of plasma-polymerized tetraglyme films.

Plasma-polymerized tetraglyme films (PP4G) have been modified by exposure to ultraviolet (UV) light from a frequency-doubled argon ion laser (244 nm) and characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS data indicated that the ether component of the C 1s spectrum declined after UV exposure, while components due to carbonyl and carboxylate groups increased. The film was physically eroded by UV exposure: after 100 s the rate of erosion reached a steady state of 0.05 nm s(-1). The coefficient of friction, measured by friction force microscopy (FFM), increased substantially following exposure to UV light, reaching a limiting value after 10 min exposure, in agreement with the time taken for the ether and carboxylate components in the C 1s spectrum to reach a limiting value. Samples exposed to UV light through a mask yielded excellent frictional contrast. When immersed in solutions of proteins and protein-functionalized nanoparticles labeled with fluorescent markers, selective adsorption occurred onto the exposed regions of these samples. Excellent fluorescence contrast was obtained when samples were characterized by confocal microscopy, indicating that the exposed areas become adhesive toward proteins, while the masked areas remain resistant to adsorption. Submicrometer structures have been formed by exposing PP4G films to UV light using a scanning near-field optical microscope coupled to a UV laser. Structures as small as 338 nm have been formed and used to immobilize proteins. Again, excellent contrast difference was observed when labeled proteins were adsorbed and characterized by confocal microscopy, suggesting a simple and effective route to the formation of submicrometer scale protein patterns.

Fabrication of Two-Component, Brush-on-Brush Topographical Microstructures by Combination of Atom-Transfer Radical Polymerization with Polymer End-Functionalization and Photopatterning.

Poly(oligoethylene glycol methyl ether methacrylate) (POEGMEMA) brushes, grown from silicon oxide surfaces by surface-initiated atom transfer radical polymerization (SI-ATRP), were end-capped by reaction with sodium azide leading to effective termination of polymerization. Reduction of the terminal azide to an amine, followed by derivatization with the reagent of choice, enabled end-functionalization of the polymers. Reaction with bromoisobutryl bromide yielded a terminal bromine atom that could be used as an initiator for ATRP with a second, contrasting monomer (methacrylic acid). Attachment of a nitrophenyl protecting group to the amine facilitated photopatterning: when the sample was exposed to UV light through a mask, the amine was deprotected in exposed regions, enabling selective bromination and the growth of a patterned brush by ATRP. Using this approach, micropatterned pH-responsive poly(methacrylic acid) (PMAA) brushes were grown on a protein resistant planar poly(oligoethylene glycol methyl ether methacrylate) (POEGMEMA) brush. Atomic force microscopy analysis by tapping mode and PeakForce quantitative nanomechanical mapping (QNM) mode allowed topographical verification of the spatially specific secondary brush growth and its stimulus responsiveness. Chemical confirmation of selective polymer growth was achieved by secondary ion mass spectrometry (SIMS).

Photo-deprotection patterning of self-assembled monolayers

Photo-deprotectable self-assembled monolayers (SAMs) provide a versatile platform for creating functional patterned surfaces. In this study, we present nanoscale photo-patterning, multi-component patterning, and a method for producing molecular gradients using photo-deprotectable SAMs. Nanoscale patterning of photo-deprotectable SAMs was achieved by coupling a UV laser (365 nm) through a scanning near field probe to produce nanoscale lines of ∼40 nm, i.e. λ/9. Multi-component patterning was achieved by a two-stage method combining both microcontact printing and soft-UV photo-patterning. The example demonstrated in this study produced a three-component patterned surface with regions of CF3, CH3 and COOH/CF3 functionality. The versatility of these photocleavable SAMs is further demonstrated by creating linear molecular gradients of two functionalities along a distance of ∼25 mm. The use of ‘soft’ UV gives several advantages including the ability to pattern SAMs with micron-scale features over large areas quickly, with greater control over the photochemical reactions, and compatibility with existing lithographic facilities thus offering an effective alternative to other patterning methods such microcontact-printing or deep UV patterning.

Versatile thiol-based reactions for micrometer- and nanometer-scale photopatterning of polymers and biomolecules

Thiol-based chemistry provides a mild and versatile tool for surface functionalization. In the present work, mercaptosilane films were patterned by utilizing UV-induced photo-oxidation of the thiol to yield sulfonate groups via contact and interferometric lithography (IL). These photo-generated sulfonic acid groups were used for selective immobilization of amino-functionalized molecules after activation with triphenylphosphine ditriflate (TPPDF). Moreover, protein-resistant poly(oligoethyleneglycolmethacrylate) (POEGMA) brushes were grown from the intact thiol groups by a surface-induced polymerization reaction. Exploiting both reactions it is possible to couple amino-labelled nitrilotriacetic acid (NH2-NTA) to sulfonate-functionalized regions, enabling the site-specific binding of green fluorescent protein (GFP) to regions defined lithographically, while exploiting the protein-resistant character of POEGMA brushes to prevent non-specific protein adsorption to previously masked areas. The outstanding reactivity of thiol groups paves the way towards novel strategies for the fabrication of complex protein nanopatterns beyond thiol-ene chemistry.

From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups

We show that sequential protein deposition is possible by photodeprotection of films formed from a tetraethylene-glycol functionalized nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (NPEOC-APTES). Exposure to near-UV irradiation removes the protein-resistant protecting group, and allows protein adsorption onto the resulting aminated surface. The protein resistance was tested using proteins with fluorescent labels and microspectroscopy of two-component structures formed by micro- and nanopatterning and deposition of yellow and green fluorescent proteins (YFP/GFP). Nonspecific adsorption onto regions where the protecting group remained intact was negligible. Multiple component patterns were also formed by near-field methods. Because reading and writing can be decoupled in a near-field microscope, it is possible to carry out sequential patterning steps at a single location involving different proteins. Up to four different proteins were formed into geometric patterns using near-field lithography. Interferometric lithography facilitates the organization of proteins over square cm areas. Two-component patterns consisting of 150 nm streptavidin dots formed within an orthogonal grid of bars of GFP at a period of ca. 500 nm could just be resolved by fluorescence microscopy.

Fabrication of sub-diffraction-limit molecular structures by scanning near-field photolithography

Using a scanning near-field optical microscope coupled to a UV laser, an approach we term scanning near-field photolithography (SNP), structures as small as 9 nm (ca. λ/30) may be fabricated in self-assembled monolayers of alkanethiols on gold surfaces. Selective exposure of the adsorbate molecules in the near field leads to photoconversion of the alkylthiolate to a weakly bound alkylsulfonate which may be displaced readily be a contrasting thiol, leading to a chemical pattern, or used as a resist for the selective etching of the underlying metal. A novel ultra-mild etch for gold is reported, and used to etch structures as small as 9 nm. Photopatterning of oligo(ethylene glycol) (OEG) terminated selfassembled monolayers facilitates the fabrication of biomolecular nanostructures. Selective removal of the protein-resistant OEG terminated adsorbates created regions that may be functionalized with a second thiol and derivatized with a biomolecule. Finally, the application of SNP to nanopatterning on oxide surfaces is demonstrated. Selective exposure of monolayers of phosphonic acids adsorbed onto aluminum oxide leads to cleavage of the P-C bond and desorption of the adsorbate molecule. Subsequent etching, using aqueous based, yields structures as small as 100 nm.