Eric S. McLamore

Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging

Silver nanoparticles (Ag NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products may lead to an increase in toxic levels of environmental silver, but regulatory control over the use or disposal of such products is lagging due to insufficient assessment on the toxicology of Ag NPs and their rate of release into the environment. In this article we discuss recent research on the transport, activity and fate of Ag NPs at the cellular and organismic level, in conjunction with traditional and recently established methods of nanoparticle characterization. We include several proposed mechanisms of cytotoxicity based on such studies, as well as new opportunities for investigating the uptake and fate of Ag NPs in living systems.

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor technique

Indole-3-acetic acid (IAA) is a primary phytohormone that regulates multiple aspects of plant development. Because polar transport of IAA is an essential determinant of organogenesis and dynamic tropic growth, methods to monitor IAA movement in vivo are in demand. A self-referencing electrochemical microsensor was optimized to non-invasively measure endogenous IAA flux near the surface of Zea mays roots without the addition of exogenous IAA. Enhanced sensor surface modification, decoupling of acquired signals, and integrated flux analyses were combined to provide direct, real time quantification of endogenous IAA movement in B73 maize inbred and brachytic2 (br2) auxin transport mutant roots. BR2 is localized in epidermal and hypodermal tissues at the root apex. br2 roots exhibit reduced shootward IAA transport at the root apex in radiotracer experiments and reduced gravitropic growth. IAA flux data indicates that maximal transport occurs in the distal elongation zone of maize roots, and net transport in/out of br2 roots was decreased compared to B73. Integration of short term real time flux data in this zone revealed oscillatory patterns, with B73 exhibiting shorter oscillatory periods and greater amplitude than br2. IAA efflux and influx were inhibited using 1-N-naphthylphthalamic acid (NPA), and 2-naphthoxyacetic acid (NOA), respectively. A simple harmonic oscillation model of these data produced a correlation between modeled and measured values of 0.70 for B73 and 0.69 for br2. These results indicate that this technique is useful for real-time IAA transport monitoring in surface tissues and that this approach can be performed simultaneously with current live imaging techniques.

A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux.

Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9+/-6.4 fmol cm(-2)s(-1)). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50-150 fmol cm(-2)s(-1) range. Uptake was inhibited using threo-beta-benzyloxyaspartate, and average surface flux in replicate cells (1.1+/-7.4 fmol cm(-2)s(-1)) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors.

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM(-1) cm(-2)), linear range (0.0037-12 mM), detection limit (3.7 µM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H(2)O(2) response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.

Electrochemical glutamate biosensing with nanocube and nanosphere augmented single-walled carbon nanotube networks: a comparative study

We describe two hybrid nanomaterial biosensor platforms, based on networks of single-walled carbon nanotubes (SWCNTs) enhanced with Pd nanocubes and Pt nanospheres and grown in situ from a porous anodic alumina (PAA) template. These nanocube and nanosphere SWCNT networks are converted into glutamate biosensors by immobilizing the enzyme glutamate oxidase (cross-linked with gluteraldehyde) onto the electrode surface. The Pt nanosphere/SWCNT biosensor outperformed the Pd nanocube/SWCNT biosensor and previously reported similar nanomaterial-based biosensors by amperometrically monitoring glutamate concentrations with a wide linear sensing range (50 nM to 1.6 mM) and a small detection limit (4.6 nM, 3σ). These results combined with the biosensor fabrication scheme (in situgrowth of SWCNTs, electrodeposition of metal nanoparticles, and facile enzyme immobilization protocol) create a biosensor that can potentially be scaled for integration into a wide range of applications including the treatment of neurological disorders.

A nanoceria–platinum–graphene nanocomposite for electrochemical biosensing

Most graphene-metal nanocomposites for biosensing are formed using noble metals. Recently, development of nanocomposites using rare earth metals has gained much attention. This paper reports on the development of a nanoceria-nanoplatinum-graphene hybrid nanocomposite as a base transducing layer for mediator-free enzymatic biosensors. The hybrid nanocomposite was shown to improve detection of superoxide or hydrogen peroxide when compared to other carbon-metal hybrid nanocomposites. Based on this finding, the nanocomposite was applied for biosensing by adding either a peroxide-producing oxidase (glucose oxidase), or a superoxide-producing oxidase (xanthine oxidase). Material analysis indicated that nanoceria and nanoplatinum were equally distributed along the surface of the hybrid material, ensuring detection of either superoxide or hydrogen peroxide produced by oxidase activity. Glucose biosensors demonstrated a sensitivity (66.2±2.6μAmM(-1)cm(-2)), response time (6.3±3.4s), and limit of detection (1.3±0.6μM) that were comparable to other graphene-mediated electrodes in the current literature. Remarkably, XOD biosensor sensitivity (1164±332μAmM(-1)), response time (5.0±1.5s), and limit of detection (0.2±0.1μM) were higher than any reported biosensors using similar metal-decorated carbon nanomaterials. This material is the first demonstration of a highly efficient, diverse nanoceria/nanoplatinum/graphene hybrid nanocomposite for biosensing.

Silver nanoparticle-specific mitotoxicity in Daphnia magna

Abstract Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag+ and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO3 and Ag NPs (130–650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag+. This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag+ was more efficient than Ag NPs at displacing Na+ within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.

Non-Invasive Self-Referencing Electrochemical Sensors for Quantifying Real-Time Biofilm Analyte Flux

Current techniques for characterizing biofilm physiology lack the signal filtering capability required for quantifying signals associated with real time biologically active transport. Though a great deal was learned from previous investigations, no results have been reported on the characterization of in vivo, real time biofilm flux using non‐invasive (non‐destructive) techniques. This article introduces the self‐referencing technique for applications in biofilm physiology. Self‐referencing is a non‐invasive sensing modality which is capable of sensing changes in biologically active analyte flux as small as 10 fmol cm−2 s−1. Studies directly characterizing flux, as opposed to concentration, have the advantage of quantifying real time changes in biologically active transport which are otherwise lost to background noise. The use of this modality for characterizing biofilm physiology is validated with a reversible enzyme inhibition study. The experiment used self‐referencing potentiometric sensors for quantifying real time ammonium and nitrite flux. Amperometric and optical sensing methods, though not presented herein, are also powerful sensing tools which benefit from operation in self‐referencing mode. Reversible ammonia monooxygenase inhibition by a copper chelator (thiourea), and subsequent relief by excess copper addition was successfully demonstrated using self‐referencing ion‐selective microelectrodes for a mature Nitrosomonas europaea biofilm. Biotechnol. Bioeng. 2009; 102: 791–799.

Nanomaterial-mediated Biosensors for Monitoring Glucose

Real-time monitoring of physiological glucose transport is crucial for gaining new understanding of diabetes. Many techniques and equipment currently exist for measuring glucose, but these techniques are limited by complexity of the measurement, requirement of bulky equipment, and low temporal/spatial resolution. The development of various types of biosensors (eg, electrochemical, optical sensors) for laboratory and/or clinical applications will provide new insights into the cause(s) and possible treatments of diabetes. State-of-the-art biosensors are improved by incorporating catalytic nanomaterials such as carbon nanotubes, graphene, electrospun nanofibers, and quantum dots. These nanomaterials greatly enhance biosensor performance, namely sensitivity, response time, and limit of detection. A wide range of new biosensors that incorporate nanomaterials such as lab-on-chip and nanosensor devices are currently being developed for in vivo and in vitro glucose sensing. These real-time monitoring tools represent a powerful diagnostic and monitoring tool for measuring glucose in diabetes research and point of care diagnostics. However, concerns over the possible toxicity of some nanomaterials limit the application of these devices for in vivo sensing. This review provides a general overview of the state of the art in nanomaterial-mediated biosensors for in vivo and in vitro glucose sensing, and discusses some of the challenges associated with nanomaterial toxicity.