Ardemis A. Boghossian

Plant nanobionics approach to augment photosynthesis and biochemical sensing

The interface between plant organelles and non-biological nanostructures has the potential to impart organelles with new and enhanced functions. Here, we show that single-walled carbon nanotubes (SWNTs) passively transport and irreversibly localize within the lipid envelope of extracted plant chloroplasts, promote over three times higher photosynthetic activity than that of controls, and enhance maximum electron transport rates. The SWNT-chloroplast assemblies also enable higher rates of leaf electron transport in vivo through a mechanism consistent with augmented photoabsorption. Concentrations of reactive oxygen species inside extracted chloroplasts are significantly suppressed by delivering poly(acrylic acid)-nanoceria or SWNT-nanoceria complexes. Moreover, we show that SWNTs enable near-infrared fluorescence monitoring of nitric oxide both ex vivo and in vivo, thus demonstrating that a plant can be augmented to function as a photonic chemical sensor. Nanobionics engineering of plant function may contribute to the development of biomimetic materials for light-harvesting and biochemical detection with regenerative properties and enhanced efficiency.

Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

Single Molecule Detection of Nitric Oxide Enabled by d(AT)15 DNA Adsorbed to Near Infrared Fluorescent Single-Walled Carbon Nanotubes

We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, L-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) μM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.

Photoelectrochemical complexes for solar energy conversion that chemically and autonomously regenerate

Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Herein, we demonstrate a complex that mimics this process consisting of two recombinant proteins, phospholipids and a carbon nanotube. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble on its removal over an indefinite number of cycles. The assembly is thermodynamically meta-stable and can only transition reversibly if the rate of surfactant removal exceeds about 10−5 sec−1. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in increased photo-conversion efficiency of more than 300% over 168 hours and an indefinite extension of the systems lifetime.

Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes

Nanomaterials are often functionalized with biological ligands to enable their use as sensors of biological activity. However, the intricacies of nano-bio interactions are poorly understood, which hampers our ability to design nanomaterial-based sensors. Current experimental tools have been unable to visualize interactions occurring on the nano-bio interface with the spatial and temporal resolution needed to quantify biological interactions at their fundamental length and time scales. To fill the need for concurrent visualization of nanoparticles and biomolecules, we have combined two common microscopy techniques, one being for the study of biomolecules and the other for the study of nanoparticles, into a single instrument that has the capacity to study both nanoparticles and biological molecules simultaneously with spatial and temporal resolution that is appropriate for nanoscale interactions. This novel instrument has been used for the characterization of high-sensitivity sensors by designing synthetic biological polymers to selectively encapsulate single-wall carbon nanotubes. The design of synthetic sensing tools based on nanoparticle-biomolecule hybrids is promising for areas in need of high-specificity sensors, such as label-free detection of molecules within a cell, nanoparticle-based diagnostic tools, and nanoscale therapeutics. We introduce three examples of high-sensitivity and high-selectivity synthetic sensors that have the ability to detect a variety of molecules on a single-molecule scale: riboflavin, L-thyroxine, and oestradiol. These sensors have been used to detect and quantify riboflavin levels within a live murine macrophage cell in real-time. The findings provided herein will enable the development of early-onset diagnostic tools at the level of a single cell.

Near-Infrared Fluorescent Sensors based on Single-Walled Carbon Nanotubes for Life Sciences Applications

Many properties of single-walled carbon nanotubes (SWCNTs) make them ideal candidates for sensors, particularly for biological systems. Both their fluorescence in the near-infrared range of 820-1600 nm, where absorption by biological tissues is often minimal, and their inherent photostability are desirable attributes for the design of in vitro and in vivo sensors. The mechanisms by which a target molecule can selectively alter the fluorescent emission include primarily changes in emission wavelength (i.e., solvatochromism) and intensity, including effects such as charge-transfer transition bleaching and exciton quenching. The central challenge lies in engineering the nanotube interface to be selective for the analyte of interest. In this work, we review the recent development in this area over the past few years, and describe the design rules that we have developed for detecting various analytes, ranging from stable small molecules and reactive oxygen species (ROS) or reactive nitrogen species (RNS) to macromolecules. Applications to in vivo sensor measurements using these sensors are also described. In addition, the emerging field of SWCNT-based single-molecule detection using band gap fluorescence and the recent efforts to accurately quantify and utilize this unique class of stochastic sensors are also described in this article.

Label-Free, Single Protein Detection on a Near-Infrared Fluorescent Single-Walled Carbon Nanotube/Protein Microarray Fabricated by Cell-Free Synthesis

Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.

Peptide Secondary Structure Modulates Single-Walled Carbon Nanotube Fluorescence as a Chaperone Sensor for Nitroaromatics

A class of peptides from the bombolitin family, not previously identified for nitroaromatic recognition, allows near-infrared fluorescent single-walled carbon nanotubes to transduce specific changes in their conformation. In response to the binding of specific nitroaromatic species, such peptide–nanotube complexes form a virtual “chaperone sensor,” which reports modulation of the peptide secondary structure via changes in single-walled carbon nanotubes, near-infrared photoluminescence. A split-channel microscope constructed to image quantized spectral wavelength shifts in real time, in response to nitroaromatic adsorption, results in the first single-nanotube imaging of solvatochromic events. The described indirect detection mechanism, as well as an additional exciton quenching-based optical nitroaromatic detection method, illustrate that functionalization of the carbon nanotube surface can result in completely unique sites for recognition, resolvable at the single-molecule level.

Biomimetic strategies for solar energy conversion: a technical perspective

Plants have evolved highly sophisticated light-harvesting mechanisms that allow for increased environmental tolerances and robustness, enhanced photo-efficiencies and prolonged lifetimes. These mechanisms incorporate the dynamic, cyclic self-assembly of proteins necessary for continual plant regeneration. Synthetic solar conversion devices, on the other hand, are designed to be static devices. Material and processing costs continue to be important constraints for commercial devices, and the earth abundance of requisite elements have become a recent concern. One potential solution to these problems lies in the development of biomimetic solar conversion devices that take advantage of the low material costs, negative carbon footprint, material abundance and dynamic self-assembly capabilities of photosynthetic proteins. Although research in this area is ongoing, this review is intended to give a brief overview of current biomimetic strategies incorporated into light-harvesting and energy-conversion mechanisms of synthetic solar devices, as well as self-repair and regeneration mechanisms adapted from plant-based processes.

Periplasmic Binding Proteins as Optical Modulators of Single‐Walled Carbon Nanotube Fluorescence: Amplifying a Nanoscale Actuator

The authors use glucose-binding protein (GBP) to mech. actuate a fluorescent single-walled carbon nanotube (SWNT), resulting in reversible exciton quenching in response to glucose. The GBP is covalently conjugated with carboxylated poly(vinyl alc.)-wrapped SWNTs (cPVA/SWNTs), which leads to allosterically controlled optical transduction in response to glucose. Carboxy groups on the cPVA/SWNT complex were used to attach the protein, through amine coupling to lysine residues on the GBP. These complexes demonstrate a reversible fluorescence quenching in response to glucose. The hypothesis is that the hinge bending action assocd. with glucose recognition modulates the fluorescence of the SWNT. The system can be used as a glucose biosensor. [on SciFinder(R)]