David W. Schmidtke

Adsorption of Glucose Oxidase onto Single-Walled Carbon Nanotubes and Its Application in Layer-By-Layer Biosensors

In this study, we describe the use of a sodium cholate suspension-dialysis method to adsorb the redox enzyme glucose oxidase (GOX) onto single-walled carbon nanotubes (SWNT). By this method, solutions of dispersed and debundled SWNTs were prepared that remained stable for 30 days and which retained 75% of the native enzymatic activity. We also demonstrate that GOX-SWNT conjugates can be assembled into amperometric biosensors with a poly[(vinylpyridine)Os(bipyridyl)2Cl(2+/3+)] redox polymer (PVP-Os) through a layer-by-layer (LBL) self-assembly process. Incorporation of SWNT-enzyme conjugates into the LBL films resulted in current densities as high as 440 microA/cm2, which were a 2-fold increase over the response of films without SWNTs. We also demonstrate that the adsorption pH of the redox polymer solution and the dispersion quality of SWNTs were important parameters in controlling the electrochemical and enzymatic properties of the LBL films.

High Current Density Ferrocene-Modified Linear Poly(ethylenimine) Bioanodes and Their Use in Biofuel Cells

Linear poly(ethylenimine) (-[CH 2 CH 2 NH] n -, LPEI) was modified by attachment of 3-(dimethylferrocenyl)propyl groups to ca. 17% of its nitrogen atoms (FcMe 2 -C 3 -LPEI) to form a new redox polymer for use as an anodic mediator in glucose/O 2 biofuel cells. The electrochemical properties of this polymer were compared to those of 3-ferrocenylpropyl-modified LPEI (Fc-C 3 -LPEI). When Fc-C 3 -LPEI or FcMe 2 -C 3 -LPEI was mixed with glucose oxidase and cross-linked with ethylene glycol diglycidyl ether to form hydrogels on planar, glassy carbon electrodes, limiting catalytic bioanodic current densities of up to ~2 mA/cm 2 at 37°C were produced. The use of dimethylferrocene moieties in place of ferrocene moieties lowered the E 1/2 of the films by 0.09 V and significantly increased electrochemical and operational stabilities. FcMe 2 -C 3 -LPEI was shown to be the more effective polymer for use in biofuel cells and, when coupled with a stationary O 2 cathode comprised of laccase and cross-linked poly[(vinylpyridine)Os(bipyridyl) 2 Cl 2+/3+ ] as a mediator, produced power densities of up to 56 wW/cm 2 at 37°C. Power density increased to 146 μW/cm 2 when a rotating biocathode was used. The stability of the biofuel cells constructed with FcMe 2 -C 3 -LPEI was higher than that of the cells using Fc-C 3 -LPEI.

High-sensitivity amperometric biosensors based on ferrocene-modified linear poly(ethylenimine)

Amperometric biosensors for glucose and hydrogen peroxide have been built by immobilizing glucose oxidase (GOX) and horseradish peroxidase (HRP) in cross-linked films of ferrocene-modified linear poly(ethylenimine). At pH 7, the glucose sensors generated limiting catalytic current densities of 1.2 mA/cm2. These current densities are approximately 4 times higher than those with other ferrocene-based redox polymers and are comparable to the highest reported values for osmium-based redox polymers with GOX. Because of the high sensitivity of these films (73 nA/cm2.microM), glucose concentrations in the micromolar range could be detected. Similarly, sensors were constructed with HRP-generated current densities of 0.9 mA/cm2 under saturation conditions and sensitivities of 500 nA/cm2.microM. The results show that the ability of Fc-LPEI to effectively communicate with a variety of enzymes has potential applications in measuring low substrate concentrations in implantable biosensors and producing high current outputs in enzymatic biofuel cells.

Incorporation of single-walled carbon nanotubes into ferrocene-modified linear polyethylenimine redox polymer films.

In this study, we describe the effects of incorporating single-walled carbon nanotubes (SWNTs) into redox polymer-enzyme hydrogels. The hydrogels were constructed by combining the enzyme glucose oxidase with a redox polymer (Fc-C(6)-LPEI) in which ferrocene was attached to linear poly(ethylenimine) by a six-carbon spacer. Incorporation of SWNTs into these films changed their morphology and resulted in a significant increase in the enzymatic response at saturating glucose concentrations (3 mA/cm(2)) as compared to films without SWNTs (0.6 mA/cm(2)). Likewise, the sensitivity at 5 mM glucose was significantly increased in the presence of SWNTs (74 μA/cm(2)·mM) as compared to control films (26 μA/cm(2)·mM). We demonstrate that the increase in the electrochemical and enzymatic response of these films depends on the amount of SWNTs incorporated and the method of SWNT incorporation. Furthermore, we report that the presence of SWNTs in thick films allows for more of the ferrocene redox centers to become accessible. The high current densities of the hydrogels should allow for the construction of miniature biosensors and enzymatic biofuel cells.

Fabrication of protein dot arrays via particle lithography.

The ability to pattern a surface with proteins on both the nanometer and the micrometer scale has attracted considerable interest due to its applications in the fields of biomaterials, biosensors, and cell adhesion. Here, we describe a simple particle lithography technique to fabricate substrates with hexagonally patterned dots of protein surrounded by a protein-repellent layer of poly(ethylene glycol). Using this bottom-up approach, dot arrays of three different proteins (fibrinogen, P-selectin, and human serum albumin) were fabricated. The size of the protein dots (450 nm to 1.1 microm) was independent of the protein immobilized but could be varied by changing the size of the latex spheres (diameter=2-10 microm) utilized in assembling the lithographic bead monolayer. These results suggest that this technique can be extended to other biomolecules and will be useful in applications where arrays of protein dots are desired.

Signal-dependent Slow Leukocyte Rolling Does Not Require Cytoskeletal Anchorage of P-selectin Glycoprotein Ligand-1 (PSGL-1) or Integrin αLβ2

Background: The cytoskeleton contributes to receptor-initiated signaling and has been linked to force-regulated integrin activation. Results: Disrupting cytoskeletal interactions of PSGL-1 or αLβ2 does not impair slow rolling of leukocytes on P-selectin and ICAM-1. Conclusion: The cytoskeleton is not required for PSGL-1-initiated signals to extend αLβ2. Significance: Signals can “prime” integrins without the cytoskeleton. In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin αLβ2 in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate αLβ2, leading to arrest on ICAM-1. Cytoskeletal anchorage of αLβ2 has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether αLβ2 must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β2 integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β2-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, αLβ2-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, αLβ2-mediated arrest.

Controlled release of metronidazole from composite poly-ε-caprolactone/alginate (PCL/alginate) rings for dental implants

OBJECTIVE Dental implants provide support for dental crowns and bridges by serving as abutments for the replacement of missing teeth. To prevent bacterial accumulation and growth at the site of implantation, solutions such as systemic antibiotics and localized delivery of bactericidal agents are often employed. The objective of this study was to demonstrate a novel method of controlled localized delivery of antibacterial agents to an implant site using a biodegradable custom fabricated ring. METHODS The study involved incorporating a model antibacterial agent (metronidazole) into custom designed poly-ε-caprolactone/alginate (PCL/alginate) composite rings to produce the intended controlled release profile. The rings can be designed to fit around the body of any root form dental implants of various diameters, shapes and sizes. RESULTS In vitro release studies indicate that pure (100%) alginate rings exhibited an expected burst release of metronidazole in the first few hours, whereas Alginate/PCL composite rings produced a medium burst release followed by a sustained release for a period greater than 4 weeks. By varying the PCL/alginate weight ratios, we have shown that we can control the amount of antibacterial agents released to provide the minimal inhibitory concentration (MIC) needed for adequate protection. The fabricated composite rings have achieved a 50% antibacterial agent release profile over the first 48 h and the remaining amount slowly released over the remainder of the study period. The PCL/alginate agent release characteristic fits the Ritger-Peppas model indicating a diffusion-based mechanism during the 30-day study period. SIGNIFICANCE The developed system demonstrates a controllable drug release profile and the potential for the ring to inhibit bacterial biofilm growth for the prevention of diseases such as peri-implantitis resulting from bacterial infection at the implant site.

Effect of Surfactant Type and Redox Polymer Type on Single-Walled Carbon Nanotube Modified Electrodes

Electrodes modified with single-walled carbon nanotubes (SWNTs) offer a number of attractive properties for developing novel electrochemical sensors. A common method to immobilize SWNTs onto the electrode surface is by placing a droplet of a SWNT suspension onto the electrode surface and allowing the solvent to evaporate. In order to maximize the properties of individual SWNTs, surfactants are normally present in these suspensions to provide stable and homogeneous SWNT dispersions. In this study we investigated the effect of different surfactants on the electrochemical and enzymatic performance of SWNT modified glassy carbon electrodes (GCEs). Amperometic biosensors for glucose were fabricated by a two-step procedure. In the first step, SWNT films were deposited onto GCEs by solution casting suspensions of SWNTs in water, Triton X-100, Tween 20, sodium cholate or sodium dodecylbenzenesulfonate (NaDDBS). In the second step, hydrogels containing a redox polymer and the enzyme, glucose oxidase (GOX), were deposited and cross-linked onto the SWNT-modified GCE. Three different redox polymers were tested: 3-ferrocenylpropyl-modified LPEI, (Fc-C3-LPEI), 6-ferrocenylhexyl-modified LPEI, (Fc-C6-LPEI), and poly[(vinylpyridine)Os(bipyridyl)2Cl](2+/3+)(PVP-Os). Biosensors constructed with SWNT films from suspensions of Triton X-100 or Tween 20 generally produced the highest electrochemical and enzymatic responses, with Triton X-100 films producing current densities of ~1.7-2.1 mA/cm(2) for the three different redox polymers. In contrast, biosensors constructed with SWNT films from sodium cholate suspensions resulted in significant decreases in the electrochemical and enzymatic response and in some cases showed no enzymatic activity. The results with SWNT films from NaDDBS suspensions were dependent upon the specific redox polymer used, but in general gave reduced enzymatic responses (~0.05-0.4 mA/cm(2)). These results demonstrate the importance of surfactant type in fabricating SWNT-modified electrode films.

Independently controlling protein dot size and spacing in particle lithography

Particle lithography is a relatively simple, inexpensive technique used to pattern inorganics, metals, polymers, and biological molecules on the micro- and nanometer scales. Previously, we used particle lithography to create hexagonal patterns of protein dots in a protein resistant background of methoxy-poly(ethylene glycol)-silane (mPEG-sil). In this work, we describe a simple heating procedure to overcome a potential limitation of particle lithography: the simultaneous change in feature size and center-to-center spacing as the diameter of the spheres used in the lithographic mask is changed. Uniform heating was used to make single-diameter protein patterns with dot sizes of approximately 2-4 or 2-8 μm, depending on the diameter of the spheres used in the lithographic mask, while differential heating was used to make a continuous gradient of dot sizes of approximately 1-9 μm on a single surface. We demonstrate the applicability of these substrates by observing the differences in neutrophil spreading on patterned and unpatterned protein coated surfaces.

Layer-by-layer assembly of ferrocene-modified linear polyethylenimine redox polymer films.

Herein, both electrostatic and covalent layer-by-layer assembly were used for the construction of multicomposite thin films using a ferrocene-modified linear poly(ethylenimine) redox polymer (Fc-C6-LPEI) as the cationic polyelectrolye, and poly(acrylic acid) (PAA), poly(glutamic acid) (PGA), or glucose oxidase (GOX) as the negative polyelectrolyte. The assembly of the multilayer films was characterized by cyclic voltammetry (CV), UV/Vis spectroscopy, and ellipsometry with the enzymatic response of the films containing GOX being characterized via constant potential amperometry. CV measurements suggested that the successful buildup of multilayer films was dependent upon the nature of the anionic polyelectrolyte used. Electrostatic assembly of films composed of Fc-C6-LPEI and either PAA or PGA produced large oxidation peak current densities of 630 and 670 μA cm(-2), respectively, during cyclic voltammetry. Increased measured absorbance by UV/Vis spectroscopy and increased measured film thicknesses (400-600 nm) by ellipsometry provided additional evidence of successful film formation. In contrast, the films incorporating GOX that were electrostatically assembled surprisingly produced significantly lower electrochemical responses (12 μA cm(-2)), low absorbance values, and reduced film thicknesses (~15 nm), and glucose electro-oxidation current densities less than 1 μA cm(-2), which all suggested unstable or minimal film formation. Subsequently, we developed a covalent layer-by-layer approach to fabricate films of Fc-C6-LPEI/GOX by covalently linking the amine groups of Fc-C6-LPEI to the aldehyde groups of periodate-oxidized glucose oxidase. Covalent assembly of the Fc-C6-LPEI/GOX films produced oxidation peak current densities during cyclic voltammetry of 40 μA cm(-2) and glucose electro-oxidation current densities of 220 μA cm(-2). These films also showed an increase in their thicknesses (~140 nm) relative to the electrostatic GOX films. For the films containing either PAA or PGA, the pH of the polymer solutions used for construction was found to have a significant effect on the response of the multilayer films, and the electrochemical response of the Fc-C6-LPEI/PAA, Fc-C6-LPEI/PGA, or covalently assembled Fc-C6-LPEI/GOX films could be tuned by varying the number of bilayers (n=1-16) in the film. These results are important because this is the first report of the use of the novel Fc-C6-LPEI redox polymer in the successful development of multicomposite layer-by-layer films. The electrochemical response achieved with the covalently assembled Fc-C6-LPEI/GOX films demonstrates that this redox polymer and layer-by-layer assembly technique can be used for possible biosensor and biofuel applications, and the success of multiple anionic polyelectrolytes could lead to additional applications with other enzyme systems.