Paul W. Barone

The rational design of nitric oxide selectivity in single-walled carbon nanotube near infrared fluorescence sensors for biological detection

A major challenge in the synthesis of nanotube or nanowire sensors is to impart selective analyte binding through means other than covalent linkages, which compromise electronic and optical properties. We synthesized a 3,4-diaminophenyl-functionalized dextran (DAP-dex) wrapping for single-walled carbon nanotubes (SWNTs) that imparts rapid and selective fluorescence detection of nitric oxide (NO), a messenger for biological signalling. The near-infrared (nIR) fluorescence of SWNT(DAP-dex) is immediately and directly bleached by NO, but not by other reactive nitrogen and oxygen species. This bleaching is reversible and shown to be caused by electron transfer from the top of the valence band of the SWNT to the lowest unoccupied molecular orbital of NO. The resulting optical sensor is capable of real-time and spatially resolved detection of NO produced by stimulating NO synthase in macrophage cells. We also demonstrate the potential of the optical sensor for in vivo detection of NO in a mouse model.

M13 Phage-Functionalized Single-Walled Carbon Nanotubes As Nanoprobes for Second Near-Infrared Window Fluorescence Imaging of Targeted Tumors

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas.

In vivo biosensing via tissue-localizable near-infrared-fluorescent single-walled carbon nanotubes

Single-walled carbon nanotubes (SWNT) are particularly attractive for biomedical applications, because they exhibit a fluorescent signal in a spectral region where there is minimal interference from biological media. Although SWNT have been used as highly-sensitive detectors for various molecules, their use as in vivo biosensors requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we demonstrate that a polyethylene glycol ligated copolymer stabilizes near infrared fluorescent SWNT sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide (NO) concentration with a detection limit of 1 μM. The half-life for liver retention is 4 hours, with sensors clearing the lungs within 2 hours after injection, thus avoiding a dominant route of in vivo nanotoxicology. After localization within the liver, it is possible to follow the transient inflammation using NO as a marker and signalling molecule. To this end, we also report a spatial-spectral imaging algorithm to deconvolute fluorescence intensity and spatial information from measurements. Finally, we show that alginate encapsulated SWNT can function as an implantable inflammation sensor for in vivo NO detection, with no intrinsic immune reactivity or other adverse response, for more than 400 days. These results open new avenues for the use of such nanosensors in vivo for biomedical applications.

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.

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.

A luciferase/single-walled carbon nanotube conjugate for near-infrared fluorescent detection of cellular ATP.

All micro-organisms use adenosine 5’-triphosphate (ATP) as a universal energy storage molecule, and thus knowledge of its concentration is central to the detection of bacterial contamination and the study of energetic processes in cell physiology from ion-channel regulation to intercellular signaling cascades. Additionally, ATP depletion is related to pathogenesis such as ischemia, Parkinson s disease, and hypoglycemia. There remains a persistent need for more sensitive, higher-resolution, and more robust detection of ATP for, among other goals, the understanding of its spatial compartmentalization within living cells. For this purpose, the conventional method of ATP assay within living cells is luciferase(Luc)-mediated bioluminescence, whereby ATP reacts at the enzyme in the presence of d-luciferin (Lrin) and Mg to produce oxyluciferin (oxyLrin) and a fluorescent emission. However, this approach, which involves synthesis of Luc vectors and cell transfection is tedious, timeconsuming, and has a low signal-to-noise ratio. The extension of this method to the modulation of quantum confined nanorods or nanotube fluorophores, such as single-walled carbon nanotubes (SWNT), has not been addressed to date, despite obvious benefits in sensitivity and photobleaching resistance. Herein, we report a SWNT/Luc enzyme conjugate (SWNT) in which the bioluminescent reaction selectively recognizes ATP at luciferase. The SWNT near-infrared (NIR) fluorescence is ultimately quenched by a two-step reaction that involves detection of a target and generation of a redox quenching intermediate. This SWNT sensor is very selective to ATP, but not to adenosine 5’-monophosphate (AMP), adenosine 5’-diphosphate (ADP), cytidine 5’-triphosphate (CTP), and guanosine 5’-triphosphate (GTP), and is also able to detect ATP temporally and spatially in living HeLa cells. The approach, whereby an enzyme–nanotube complex creates a redox quenching intermediate from the target analyte, can be extended to a wide range of biologically important analytes. We first constructed the Luc-conjugated SWNTs as shown in Figure 1 (see the Supporting Information). After immobilization of Luc on SWNTs functionalized with phospholipids

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.

Modulation of single-walled carbon nanotube photoluminescence by hydrogel swelling.

We demonstrate the use of hydrogel swelling as a mechanism to reversibly induce solvatochromic shifting in single-walled carbon nanotube (SWNT) near-infrared emission within a biocompatible hydrogel. The optical sensor reports the degree of the swelled state and glucose concentration when apo-glucose oxidase is used to cross-link the hydrogel. Photoluminescence emission maxima from dispersed nanotubes in a poly(vinyl alcohol) hydrogel shift as cross-linking is increased, with a maximum of -48 meV for the (6,5) nanotube. The Raman tangential mode also red shifts up to 17 cm(-1), indicative of nanotube lattice strain equivalent to an effective hydrostatic pressure of 3 GPa. While the electronic band gaps of SWNTs are known to either increase or decrease with uniaxial strain or lattice deformation depending on chiral vector, we show that the mechanism of detection is counterintuitively non-strain-dependent. Instead, the data are well-described by a model that accounts for changes in dielectric screening of the 1-D exciton, as the osmotic pressure forces conformational distortions in the PVA by rotating more polar groups to the nanotube surface. The model describes observed changes with hydration state and cross-linking density variation from 0 to 14%. Cross-linking with apo-glucose oxidase renders the hydrogel glucose responsive, and we demonstrate rapid and reversible detection of glucose from these systems after repeated cycling of 10 mM glucose. We also demonstrate detection and imaging in the near-infrared of implanted hydrogel sensors in a mouse tissue model, showing excellent signal-to-noise of 8.6 and contrast with integration times of 60 s.

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.