Electrochemical Nano-Roughening of Gold Microstructured Electrodes for Enhanced Sensing in Biofluids.

A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30-fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu2+ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme-free sensing of glucose, with responses comparable to those of two commercial enzyme-based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost-effective, and high sensitivity electrochemical platforms.

Benchtop-fabricated lipid-based electrochemical sensing platform for the detection of membrane disrupting agents.

There are increasing concerns about the danger that water-borne pathogens and pollutants pose to the public. Of particular importance are those that disrupt the plasma membrane, since loss of membrane integrity can lead to cell death. Currently, quantitative assays to detect membrane-disrupting (lytic) agents are done offsite, leading to long turnaround times and high costs, while existing colorimetric point-of-need solutions often sacrifice sensitivity. Thus, portable and highly sensitive solutions are needed to detect lytic agents for health and environmental monitoring. Here, a lipid-based electrochemical sensing platform is introduced to rapidly detect membrane-disrupting agents. The platform combines benchtop fabricated microstructured electrodes (MSEs) with lipid membranes. The sensing mechanism of the lipid-based platform relies on stacked lipid membranes serving as passivating layers that when disrupted generate electrochemical signals proportional to the membrane damage. The MSE topography, membrane casting and annealing conditions were optimized to yield the most reproducible and sensitive devices. We used the sensors to detect membrane-disrupting agents sodium dodecyl sulfate and Polymyxin-B within minutes and with limits of detection in the ppm regime. This study introduces a platform with potential for the integration of complex membranes on MSEs towards the goal of developing Membrane-on-Chip sensing devices.

Membrane charge and lipid packing determine polymyxin-induced membrane damage.

With the advent of polymyxin B (PmB) resistance in bacteria, the mechanisms for mcr-1 resistance are of crucial importance in the design of novel therapeutics. The mcr-1 phenotype is known to decrease membrane charge and increase membrane packing by modification of the bacterial outer membrane. We used X-ray diffraction, Molecular Dynamics simulations, electrochemistry, and leakage assays to determine the location of PmB in different membranes and assess membrane damage. By varying membrane charge and lipid tail packing independently, we show that increasing membrane surface charge promotes penetration of PmB and membrane damage, whereas increasing lipid packing decreases penetration and damage. The penetration of the PmB molecules is well described by a phenomenological model that relates an attractive electrostatic and a repulsive force opposing insertion due to increased membrane packing. The model applies well to several gram-negative bacterial strains and may be used to predict resistance strength.

Fabrication of polycaprolactone electrospun nanofibers doped with silver nanoparticles formed by air plasma treatment.

Implanted devices are prone to bacterial infections, which can result in implant loosening and device failure. Mitigating these infections is important to both implant stability and patient health. The development of antibacterial implant coatings can decrease the presence of bacterial colonies, reducing the risk for bacterial-dependent implant failure. Here, we show that electrospun polycaprolactone (PCL) fibers doped with silver nanoparticles (NPs) from a silver nitrate precursor have the potential to decrease the prevalence of Streptococcus pneumoniae while supporting osteoblast attachment and proliferation. An air plasma reduction method of PCL electrospun fibers was used to prepare fibers doped with silver NPs. Fibers were characterized using scanning electron microscopy and transmission electron microscopy for qualitative evaluation of NP distribution and quantitative analysis of fiber diameters. Antibacterial testing against S. pneumoniae was performed with successful inhibition observed after 24 h of exposure. In vitro testing was completed using Saos-2 cells and suggests that the negative surface charge has the potential to increase mammalian cell viability even in the presence of fibers containing NPs. In conclusion, this study describes a novel method to produce bioresorbable implant coatings with the ability to reduce bacterial infections surrounding the implant surface while remaining biocompatible to the host.

Self-Cross-Linking p(APM-co-AA) Microstructured Thin Films as Biomimetic Scaffolds.

In nature, cells reside within an extracellular matrix (ECM) that provides biophysical and biochemical cues to direct cell development and behavior. Traditional methods for studying cells involve using in vitro models that are convenient and cost-effective, but often use two-dimensional supports that are poor mimics for three-dimensional natural cell microenvironments. In this work, a synthetic polyampholyte, p(APM-co-AA) [poly(N-(3-aminopropyl)methacrylamide hydrochloride-co-acrylic acid)], was coated onto prestressed polystyrene support films that were then thermally shrunken, which concomitantly induced self-cross-linking of the polyampholyte. The cross-linking process involved a two-step sequence of anhydride formation from pairs of adjacent acrylic acid units, followed by amide cross-linking through reaction with primary amines on other chains. Attenuated reflectance infrared spectroscopy (ATR-IR) confirmed the formation of anhydride and amide bonds during heating, and the cross-linked films remained intact in cell culture media and basic solutions. Furthermore, the compressive stress from the shrinking substrate wrinkled the polyampholyte film, producing 3D scaffolds that were used to study the effect of topography on cell cultures. Cell viability tests confirmed the noncytotoxicity of the microstructured polyampholyte films. The surface wettability of the films was tuned by postfunctionalizing with different amines, with decylamine-functionalized films showing decreased fibroblast attachment, and d-glucamine-functionalized films showing reduced fibroblast spreading, compared to the control. Furthermore, the topography of the p(APM-co-AA) scaffolds could be tuned by changing the polyampholyte film thickness. Different topographies of the microstructured films elicited different fibroblast morphologies, with larger structures contributing to less complex cell boundaries, lower cell areas, and higher cell circularities. Overall, this tunable, self-cross-linking p(APM-co-AA) system can be fabricated into microstructured films, which can be used to study the effects of surface chemistry, topography, and other physical properties of biomimetic scaffolds on cell behavior and contribute to the library of existing biomimetic scaffolds.

Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in Renal Membrane Analogs.

Polymyxin B (PmB) is a "last-line" antibiotic scarcely used due to its nephrotoxicity. However, the molecular basis for antibiotic nephrotoxicity is not clearly understood. We prepared kidney membrane analogs of detergent-susceptible membranes, depleted of cholesterol, and cholesterol enriched, resistant membranes. In both analogs, PmB led to membrane damage. By combining x-ray diffraction, molecular dynamics simulations, and electrochemistry, we present evidence for two populations of PmB molecules: peptides that lie flat on the membranes, and an inserted state. In cholesterol depleted membranes, PmB forms clusters on the membranes leading to an indentation of the bilayers and increase in water permeation. The inserted peptides formed aggregates in the membrane core leading to further structural instabilities and increased water intake. The presence of cholesterol in the resistant membrane analogs led to a significant decrease in membrane damage. Although cholesterol did not inhibit peptide insertion, it minimized peptide clustering and water intake through stabilization of the bilayer structure and suppression of lipid and peptide mobility.

X-ray Absorption Spectroscopy and Spectromicroscopy of Supported Lipid Bilayers.

The C 1s, O 1s, and N 1s X-ray absorption spectra of three lipid species, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), have been recorded using transmission detection in a scanning transmission X-ray microscope. The spectra are presented on an absolute intensity scale (i.e., optical density per nm) to allow their use as reference standards for spectromicroscopic analysis of supported lipid bilayers. Examples of C 1s based spectromicroscopic mapping of saturated and unsaturated domains in dry lipid bilayers of DOPC and DSPC at several compositions are presented. The results are compared with fluorescence microscopy of the same area. Challenges for extending this work to studies of wet lipid bilayers interacting with antimicrobial peptides are discussed.

Bench-Top Fabrication of an All-PDMS Microfluidic Electrochemical Cell Sensor Integrating Micro/Nanostructured Electrodes.

In recent years, efforts in the development of lab-on-a-chip (LoC) devices for point-of-care (PoC) applications have increased to bring affordable, portable, and sensitive diagnostics to the patients' bedside. To reach this goal, research has shifted from using traditional microfabrication methods to more versatile, rapid, and low-cost options. This work focuses on the benchtop fabrication of a highly sensitive, fully transparent, and flexible poly (dimethylsiloxane) (PDMS) microfluidic (µF) electrochemical cell sensor. The µF device encapsulates 3D structured gold and platinum electrodes, fabricated using a shape-memory polymer shrinking method, which are used to set up an on-chip electrochemical cell. The PDMS to PDMS-structured electrode bonding protocol to fabricate the µF chip was optimized and found to have sufficient bond strength to withstand up to 100 mL/min flow rates. The sensing capabilities of the on-chip electrochemical cell were demonstrated by using cyclic voltammetry to monitor the adhesion of murine 3T3 fibroblasts in the presence of a redox reporter. The charge transfer across the working electrode was reduced upon cell adhesion, which was used as the detection mechanism, and allowed the detection of as few as 24 cells. The effective utilization of simple and low cost bench-top fabrication methods could accelerate the prototyping and development of LoC technologies and bring PoC diagnostics and personalized medicine to the patients' bedside.

Influence of Polymer Electronics on Selective Dispersion of Single-Walled Carbon Nanotubes.

The separation and isolation of semiconducting and metallic single-walled carbon nanotubes (SWNTs) on a large scale remains a barrier to many commercial applications. Selective extraction of semiconducting SWNTs by wrapping and dispersion with conjugated polymers has been demonstrated to be effective, but the structural parameters of conjugated polymers that dictate selectivity are poorly understood. Here, we report nanotube dispersions with a poly(fluorene-co-pyridine) copolymer and its cationic methylated derivative, and show that electron-deficient conjugated π-systems bias the dispersion selectivity toward metallic SWNTs. Differentiation of semiconducting and metallic SWNT populations was carried out by a combination of UV/Vis-NIR absorption spectroscopy, Raman spectroscopy, fluorescence spectroscopy, and electrical conductivity measurements. These results provide new insight into the rational design of conjugated polymers for the selective dispersion of metallic SWNTs.

Fabrication of conductive polymer nanofibers through SWNT supramolecular functionalization and aqueous solution processing.

Polymeric thin films and nanostructured composites with excellent electrical properties are required for the development of advanced optoelectronic devices, flexible electronics, wearable sensors, and tissue engineering scaffolds. Because most polymers available for fabrication are insulating, one of the biggest challenges remains the preparation of inexpensive polymer composites with good electrical conductivity. Among the nanomaterials used to enhance composite performance, single walled carbon nanotubes (SWNTs) are ideal due to their unique physical and electrical properties. Yet, a barrier to their widespread application is that they do not readily disperse in solvents traditionally used for polymer processing. In this study, we employed supramolecular functionalization of SWNTs with a conjugated polyelectrolyte as a simple approach to produce stable aqueous nanotube suspensions, that could be effortlessly blended with the polymer poly(ethyleneoxide) (PEO). The homogeneous SWNT:PEO mixtures were used to fabricate conductive thin films and nanofibers with improved conductivities through drop casting and electrospinning. The physical characterization of electrospun nanofibers through Raman spectroscopy and SEM revealed that the SWNTs were uniformly incorporated throughout the composites. The electrical characterization of SWNT:PEO thin films allowed us to assess their conductivity and establish a percolation threshold of 0.1 wt% SWNT. Similarly, measurement of the nanofiber conductivity showed that the electrospinning process improved the contact between nanotube complexes, resulting in conductivities in the S m(-1) range with much lower weight loading of SWNTs than their thin film counterparts. The methods reported for the fabrication of conductive nanofibers are simple, inexpensive, and enable SWNT processing in aqueous solutions, and offer great potential for nanofiber use in applications involving flexible electronics, sensing devices, and tissue engineering scaffolds.