Brad J. Berron

Functionalized nanoporous gold leaf electrode films for the immobilization of photosystem I.

Plants and some types of bacteria demonstrate an elegant means to capitalize on the superabundance of solar energy that reaches our planet with their energy conversion process called photosynthesis. Seeking to harness Natures optimization of this process, we have devised a biomimetic photonic energy conversion system that makes use of the photoactive protein complex Photosystem I, immobilized on the surface of nanoporous gold leaf (NPGL) electrodes, to drive a photoinduced electric current through an electrochemical cell. The intent of this study is to further the understanding of how the useful functionality of these naturally mass-produced, biological light-harvesting complexes can be integrated with nonbiological materials. Here, we show that the protein complexes retain their photonic energy conversion functionality after attachment to the nanoporous electrode surface and, further, that the additional PSI/electrode interfacial area provided by the NPGL allows for an increase in PSI-mediated electron transfer with respect to an analogous 2D system if the pores are sufficiently enlarged by dealloying. This increase of interfacial area is pertinent for other applications involving electron transfer between phases; thus, we also report on the widely accessible and scalable method by which the NPGL electrode films used in this study are fabricated and attached to glass and Au/Si supports and demonstrate their adaptability by modification with various self-assembled monolayers. Finally, we demonstrate that the magnitude of the PSI-catalyzed photocurrents provided by the NPGL electrode films is dependent upon the intensity of the light used to irradiate the electrodes.

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

We report the first use of a polymerization‐based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual‐mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization‐based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization‐based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx‐avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme‐mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non‐specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor‐beta. Biotechnol. Bioeng. 2011; 108:1521–1528.

Sensitive Immunofluorescent Staining of Cells via Generation of Fluorescent Nanoscale Polymer Films in Response to Biorecognition

Immunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerization-based amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA’s unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques.

Antigen-responsive, microfluidic valves for single use diagnostics

The growing need for medical diagnostics in resource limited settings is driving the development of simple, standalone immunoassay devices. A capillary flow device using polymerization based amplification is capable of blocking a microfluidic channel in response to target biomaterials, enabling multiple modes of detection that require little or no supplemental instrumentation.

Surface-initiated growth of ionomer films from pt-modified gold electrodes.

The ability to chemically wire ionomer films to electrode surfaces can promote transport near interfaces and impact a host of energy-related applications. Here, we demonstrate proof-of-concept principles for the surface-initiated ring-opening metathesis polymerization (SI-ROMP) of norbornene (NB), 5-butylnorbornene (NBH4), and 5-perfluorobutylnorbornene (NBF4) from Pt-modified gold substrates and the subsequent sulfonation of olefins along the polymer backbones to produce ultrathin sulfonated polymer films. Prior to sulfonation, the films are hydrophobic and exhibit large barriers against ion transport, but sulfonation dramatically reduces the resistance of the films by providing pathways for proton diffusion. Sulfonated films derived from NBF4 and NBH4 yield more anodic potentials for oxygen reduction than those derived from NB or unfunctionalized electrodes. These improvements are consistent with hydrophobic structuring by the fluorocarbon or hydrocarbon side groups to minimize interfacial flooding and generate pathways for enhanced O(2) permeation near the interface. Importantly, we demonstrate that the sulfonated polymer chains remain anchored to the surface during voltammetry for oxygen reduction whereas short-chain thiolates that do not tether polymer are removed from the substrate. This approach, which we extend to unmodified gold electrodes at neutral pH, presents a method of cleaning the ionomer/electrode interface to remove molecular components that may hamper the performance of the electrode.

Thiol-Yne Adsorbates for Stable, Low-Density, Self-Assembled Monolayers on Gold

We present a novel approach toward carboxylate-terminated, low-density monolayers on gold, which provides exceptional adsorbate stability and conformational freedom of interfacial functional groups. Adsorbates are synthesized through the thiol-yne addition of two thiol-containing head groups to an alkyne-containing tail group. The resulting monolayers have two distinct phases: a highly crystalline head phase adjacent to the gold substrate, and a reduced density tail phase, which is in contact with the environment. The ellipsometric thickness of 27 Å is consistent with the proposed structure, where a densely packed decanedithiol monolayer is capped with an 11 carbon long, second layer at 50% lateral chain density. The Fourier transform infrared peak at 1710 cm(-1) supports the presence of the carbonyl group. Further, the peaks associated with asymmetric and symmetric methylene stretching are shifted toward higher wavenumbers compared to those of well-packed self-assembled monolayers (SAMs), which shows a lower average crystallinity of the thiol-yne monolayers compared to a typical monolayer. Contact angle measurements indicate an intermediate surface energy for the thiol-yne monolayer surface, owing to the contribution of exposed methylene functionality at the surface in addition to the carbonyl terminal group. The conformational freedom at the surface was demonstrated through remodeling the thiol-yne surface under an applied potential. Changes in the receding contact angle in response to an external potential support the capacity for reorientation of the surface presenting groups. Despite the low packing at the solution interface, thiol-yne monolayers are resistant to water and ion transport (R(f) ~ 10(5)), supporting the presence of a densely structured layer at the gold surface. Further, the electrochemical stability of the thiol-yne adsorbates exceeded that of well-packed SAMs, requiring a more reductive potential to desorb the thiol-yne monolayers from the gold surface. The thiol-yne monolayer approach is not limited to carboxylate functionality and is readily adapted for low-density monolayers of varied functionality.

Characterization of Molecular Transport in Ultrathin Hydrogel Coatings for Cellular Immunoprotection

PEG hydrogels are routinely used in immunoprotection applications to hide foreign cells from a host immune system. Size-dependent transport is typically exploited in these systems to prevent access by macromolecular elements of the immune system while allowing the transport of low molecular weight nutrients. This work studies a nanoscale hydrogel coating for improved transport of beneficial low molecular weight materials across thicker hydrogel coatings while completely blocking transport of undesired larger molecular weight materials. Coatings composed of PEG diacrylate of molecular weight 575 and 3500 Da were studied by tracking the transport of fluorescently labeled dextrans across the coatings. The molecular weight of dextran at which the transport is blocked by these coatings are consistent with cutoff values in analogous bulk PEG materials. Additionally, the diffusion constants of 4 kDa dextrans across PEG 575 coatings (9.5 × 10(-10)-2.0 × 10(-9) cm(2)/s) was lower than across PEG 3500 coatings (5.9-9.8 × 10(-9) cm(2)/s), and these trends and magnitudes agree with bulk scale models. Overall, these nanoscale thin PEG diacrylate films offer the same size selective transport behavior of bulk PEG diacrylate materials, while the lower thickness translates directly to increased flux of beneficial low molecular weight materials.

Interfacial polymerization for colorimetric labeling of protein expression in cells.

Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

Protective Polymer Coatings for High-Throughput, High-Purity Cellular Isolation

Cell-based therapies are emerging as the next frontier of medicine, offering a plausible path forward in the treatment of many devastating diseases. Critically, current methods for antigen positive cell sorting lack a high throughput method for delivering ultrahigh purity populations, prohibiting the application of some cell-based therapies to widespread diseases. Here we show the first use of targeted, protective polymer coatings on cells for the high speed enrichment of cells. Individual, antigen-positive cells are coated with a biocompatible hydrogel which protects the cells from a surfactant solution, while uncoated cells are immediately lysed. After lysis, the polymer coating is removed through orthogonal photochemistry, and the isolate has >50% yield of viable cells and these cells proliferate at rates comparable to control cells. Minority cell populations are enriched from erythrocyte-depleted blood to >99% purity, whereas the entire batch process requires 1 h and <

Photopatterning of Stable, Low-Density, Self-Assembled Monolayers on Gold

2000 in equipment. Batch scale-up is only contingent on irradiation area for the coating photopolymerization, as surfactant-based lysis can be easily achieved on any scale.