Development and Evaluation of an Expedited System for Creation of Single Walled Carbon Nanotube Platforms.

Single-walled carbon nanotubes (SWNT) have a strong and stable near-infrared (nIR) fluorescence that can be used to selectively detect target analytes, even at the single molecule level, through changes in either their fluorescence intensity or emission peak wavelength. SWNTs have been employed as NIR optical sensors for detecting a variety of analytes. However, high costs, long fabrication times, and poor distributions limit the current methods for immobilizing SWNT sensors on solid substrates. Recently, our group reported a protocol for SWNT immobilization with high fluorescence yield, longevity, fluorescence distribution, and sensor response, unfortunately this process takes 5 days to complete. Herein we report an improved method to immobilize SWNT sensors that only takes 2 days and results in higher fluorescence intensity while maintaining a high level of SWNT distribution. We performed surface morphology and chemical composition tests on the original and new synthesis methods and compared the sensor response rates. The development of this new method of attaching SWNT sensors to a platform allows for creation of a sensing system in just 2 days without sacrificing the advantageous characteristics of the original, 5-day platforms.

Development of sterile platform for quantification of extracellular analytes via single walled carbon nanotubes.

Progress has been made studying cell-cell signaling communication processes. However, due to limitations of current sensors on time and spatial resolution, the role of many extracellular analytes is still unknown. A single walled carbon nanotube (SWNT) platform was previously developed based on the avidin-biotin immobilization of SWNT to a glass substrate. The SWNT platform provides real time feedback about analyte concentration and has a high concentration of evenly distributed sensors, both of which are essential for the study of extracellular analytes. Unfortunately, this initial SWNT platform is synthesized through unsterile conditions and cannot be sterilized post-production due to the delicate nature of the sensors, making it unsuitable for in vitro work. Herein the multiple-step process for SWNT immobilization is modified and the platform's biocompatibility is assessed in terms of sterility, cytotoxicity, cell proliferation, and cell morphology through comparison with non-sensors controls. The results demonstrate the SWNT platform's sterility and lack of toxicity over 72 h. The proliferation rate and morphology profiles for cells growing on the SWNT platform are similar to those grown on tissue culture substrates. This novel nano-sensor platform preserves cell health and cell functionality over time, offering opportunities to study extracellular analytes gradients in cellular communication.

Single-Walled Carbon Nanotube Sensor Platform for the Study of Extracellular Analytes.

Single-walled carbon nanotubes (SWNT) are attractive targets for the formation of high-density sensor arrays. Their small size and high reactivity could allow for the spatial and temporal study of extracellular products to a degree which greatly surpasses contemporary sensors. However, current methods of SWNT immobilization produce a low fluorescence yield that requires a combination of high magnification, exposure time, and laser intensity to combat, thus limiting the sensor's applications. In this work, a platform for the immobilization of SWNT sensors with increased fluorescence yield, longevity, fluorescence distribution, and fast reaction times is developed.

Oxidative stress and postmortem meat quality in crossbred lambs.

The objective of this study was to evaluate effects of different levels of lipopolysaccharide (LPS)-mediated oxidative stress on fresh meat quality. Crossbred lambs (n = 29) were blocked by weight and fed a standard finishing ration for the duration of the study. Lambs were individually housed and treatment groups were administered one of three intravenous injections every 72 h across a three-injection (9-day) cycle: saline control (control), 50 ng LPS/kg body weight (BW) (LPS50), or 100 ng LPS/kg BW (LPS100). Rectal temperatures were measured to indicate inflammatory response. Lambs were harvested at the Loeffel Meat Laboratory and 80 mg of pre-rigor Longissimus lumborum were collected in control and LPS100 treatments within 30 min postmortem for RNA analysis. Wholesale loins were split and randomly assigned 1 or 14 d of wet aging. Chops were fabricated after aging and placed under retail display (RD) for 0 or 7 d. Animal was the experimental unit. LPS-treated lambs had increased (P < 0.05) rectal temperatures at 1, 2, 4, and 24 h post-injection. Transcriptomics revealed significant (Praw < 0.05) upregulation in RNA pathways related to generation of oxidative stress in LPS100 compared with control. A trend was found for tenderness (Warner-Bratzler shear force, WBSF; P = 0.10), chops from LPS50 having lower shear force compared with control at 1 d postmortem. Muscle from LPS50 treatment lambs exhibited greater troponin T degradation (P = 0.02) compared with all treatments at 1 d. Aging decreased WBSF (P < 0.0001), increased sarcoplasmic calcium concentration (P < 0.0001), pH (P < 0.0001), and proteolysis (P < 0.0001) across treatments. Following aging, chops increased discoloration as RD increased (P < 0.0001), with control chops aged 14 d being the most discolored. Chops from lambs given LPS had higher (P < 0.05) a* values compared with control at 14 d of aging. The L* values were greater (P < 0.05) in LPS100 compared with both LPS50 and control. Aging tended (P = 0.0608) to increase lipid oxidation during RD across either aging period. No significant differences (P > 0.05) in sarcomere length, proximate composition, fatty acid composition, or isoprostane content were found. These results suggest that defined upregulation of oxidative stress has no detriment on fresh meat color, but may alter biological pathways responsible for muscle stress response, apoptosis, and enzymatic processes, resulting in changes in tenderness early postmortem.

Quantification of Nitric Oxide Concentration Using Single-Walled Carbon Nanotube Sensors.

Nitric oxide (NO), a free radical present in biological systems, can have many detrimental effects on the body, from inflammation to cancer. Due to NO's short half-life, detection and quantification is difficult. The inability to quantify NO has hindered researchers' understanding of its impact in healthy and diseased conditions. Single-walled carbon nanotubes (SWNTs), when wrapped in a specific single-stranded DNA chain, becomes selective to NO, creating a fluorescence sensor. Unfortunately, the correlation between NO concentration and the SWNT's fluorescence intensity has been difficult to determine due to an inability to immobilize the sensor without altering its properties. Through the use of a recently developed sensor platform, systematic studies can now be conducted to determine the correlation between SWNT fluorescence and NO concentration. This paper explains the methods used to determine the equations that can be used to convert SWNT fluorescence into NO concentration. Through the use of the equations developed in this paper, an easy method for NO quantification is provided. The methods outlined in this paper will also enable researchers to develop equations to determine the concentration of other reactive species through the use of SWNT sensors.

Insulin Detection Using a Corona Phase Molecular Recognition Site on Single-Walled Carbon Nanotubes.

Corona phase molecular recognition (CoPhMoRe) is a technique whereby an external, adsorbed phase around a colloidal nanoparticle is selected such that its molecular conformation or interaction recognizes a specific target analyte. In this work, we employ a high-throughput screening of a library of poly(ethylene glycol) (PEG)-conjugated lipids adsorbed onto near-infrared fluorescent single-walled carbon nanotubes to discover a corona phase selective for insulin. We find that a C16-PEG(2000 Da)-ceramide causes a 62% fluorescent intensity decrease of the (10,2) chirality nanotube in the presence of 20 µg/mL insulin. The insulin protein has no prior affinity toward the C16-PEG(2000 Da)-ceramide molecules in free solution, verified by isothermal titration calorimetry, and the interaction occurs only upon their adsorption onto the single-walled carbon nanotube scaffolds. Testing a panel of proteins originating from human blood as well as short 7 amino acid fragments of the insulin peptide rules out nonselective recognition mechanisms such as molecular weight, isoelectric point, and hydrophobicity-based detection. Interestingly, longer fragments of isolated α- and ß-peptide chains of insulin are detected by the construct, albeit with lower affinity compared to that of the intact insulin protein, suggesting that the construct recognizes insulin in its native form and conformation. Finally, the insulin recognition and the quantification of its solution concentration were demonstrated both in buffer and in blood serum, showing that the CoPhMoRe construct works in this complex environment despite the presence of potential nonspecific adsorption. Our results further motivate the search for nonbiological synthetic recognition sites and open up a new path for continuous insulin monitoring in vivo with the hope of improving glycemic control in closed-loop artificial pancreas systems.

Quantitative Tissue Spectroscopy of Near Infrared Fluorescent Nanosensor Implants.

Implantable, near infrared (nIR) fluorescent nanosensors are advantageous for in vivo monitoring of biological analytes since they can be rendered selective for a particular target molecule while utilizing their unique optical properties and the nIR tissue transparency window for information transfer without an internal power source or telemetry. However, basic questions remain regarding the optimal encapsulation platform, geometrical properties, and concentration ranges required for high signal to noise ratio and effective detection through biological tissue. In this work, we systematically explore these variables quantitatively to optimize the performance of such optical nanosensors for biomedical applications. We investigate both alginate and polyethylene glycol (PEG) as model hydrogel systems, encapsulating d(GT)15 ssDNA-wrapped single-walled carbon nanotubes (SWNT) as model fluorescent nanoparticle sensors, responsive to riboflavin. Hydrogel sensors implanted 0.5 mm into thick tissue samples exhibit 50% reduction of initial fluorescence intensity, allowing an optical detection limit of 5.4 mm and 5.1 mm depth in tissue for alginate and PEG gels, respectively, at a SWNT concentration of 10 mg L(-1), and 785 nm laser excitation of 80 mW and 30 s exposure. These findings are supported with in vivo nIR fluorescent imaging of SWNT hydrogels implanted subcutaneously in mice. For the case of SWNT, we find that the alginate system is preferable in terms of emission intensity, sensor response, rheological properties, and shelf life.

Protein-targeted corona phase molecular recognition.

Corona phase molecular recognition (CoPhMoRe) uses a heteropolymer adsorbed onto and templated by a nanoparticle surface to recognize a specific target analyte. This method has not yet been extended to macromolecular analytes, including proteins. Herein we develop a variant of a CoPhMoRe screening procedure of single-walled carbon nanotubes (SWCNT) and use it against a panel of human blood proteins, revealing a specific corona phase that recognizes fibrinogen with high selectivity. In response to fibrinogen binding, SWCNT fluorescence decreases by >80% at saturation. Sequential binding of the three fibrinogen nodules is suggested by selective fluorescence quenching by isolated sub-domains and validated by the quenching kinetics. The fibrinogen recognition also occurs in serum environment, at the clinically relevant fibrinogen concentrations in the human blood. These results open new avenues for synthetic, non-biological antibody analogues that recognize biological macromolecules, and hold great promise for medical and clinical applications.

In Vivo Delivery of Nitric Oxide-Sensing, Single-Walled Carbon Nanotubes.

Detection of nitric oxide (NO) in vivo by single-walled carbon nanotubes (SWNT) is based on the fluorescent properties of SWNT and the ability of NO to quench the fluorescence signal. Alterations of the signal can be utilized to detect a small molecule in vivo that has not previously been possible by other assay techniques. The protocols described here explain the techniques used to prepare NO-detecting SWNTs and to administer them to mice by both intravenous and subcutaneous routes. These techniques can also be utilized with other SWNT sensors as well as non-SWNT sensors.

A Ratiometric Sensor Using Single Chirality Near-Infrared Fluorescent Carbon Nanotubes: Application to In Vivo Monitoring.

Advances in the separation and functionalization of single walled carbon nanotubes (SWCNT) by their electronic type have enabled the development of ratiometric fluorescent SWCNT sensors for the first time. Herein, single chirality SWCNT are independently functionalized to recognize either nitric oxide (NO), hydrogen peroxide (H(2)O(2)), or no analyte (remaining invariant) to create optical sensor responses from the ratio of distinct emission peaks. This ratiometric approach provides a measure of analyte concentration, invariant to the absolute intensity emitted from the sensors and hence, more stable to external noise and detection geometry. Two distinct ratiometric sensors are demonstrated: one version for H(2)O(2), the other for NO, each using 7,6 emission, and each containing an invariant 6,5 emission wavelength. To functionalize these sensors from SWCNT isolated from the gel separation technique, a method for rapid and efficient coating exchange of single chirality sodium dodecyl sulfate-SWCNT is introduced. As a proof of concept, spatial and temporal patterns of the ratio sensor response to H(2)O(2) and, separately, NO, are monitored in leaves of living plants in real time. This ratiometric optical sensing platform can enable the detection of trace analytes in complex environments such as strongly scattering media and biological tissues.

A pharmacokinetic model of a tissue implantable insulin sensor.

While implantable sensors such as the continuous glucose monitoring system have been widely studied, both experimentally and mathematically, relatively little attention has been applied to the potential of insulin sensors. Such sensors can provide feedback control for insulin infusion systems and pumps and provide platforms for the monitoring of other biomarkers in vivo. In this work, the first pharmacokinetic model of an affinity sensor is developed for insulin operating subcutaneously in the limit of where mass transfer across biological membranes reaches a steady state. Using a physiological, compartmental model for glucose, insulin, and glucagon metabolism, the maximum sensor response and its delay time relative to plasma insulin concentration, are calculated based on sensor geometry, placement, and insulin binding parameters for a sensor localized within adipose tissue. A design relation is derived linking sensor dynamics to insulin time lag and placement within human tissue. The model should find utility in understanding dynamic insulin responses and forms the basis of model predictive control algorithms that incorporate sensor dynamics.

Experimental tools to study molecular recognition within the nanoparticle corona.

Advancements in optical nanosensor development have enabled the design of sensors using synthetic molecular recognition elements through a recently developed method called Corona Phase Molecular Recognition (CoPhMoRe). The synthetic sensors resulting from these design principles are highly selective for specific analytes, and demonstrate remarkable stability for use under a variety of conditions. An essential element of nanosensor development hinges on the ability to understand the interface between nanoparticles and the associated corona phase surrounding the nanosensor, an environment outside of the range of traditional characterization tools, such as NMR. This review discusses the need for new strategies and instrumentation to study the nanoparticle corona, operating in both in vitro and in vivo environments. Approaches to instrumentation must have the capacity to concurrently monitor nanosensor operation and the molecular changes in the corona phase. A detailed overview of new tools for the understanding of CoPhMoRe mechanisms is provided for future applications.

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.

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

Single-walled carbon nanotubes 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 single-walled carbon nanotubes have been used as highly sensitive detectors for various compounds, their use as in vivo biomarkers requires the simultaneous optimization of various parameters, including biocompatibility, molecular recognition, high fluorescence quantum efficiency and signal transduction. Here we show that a polyethylene glycol ligated copolymer stabilizes near-infrared-fluorescent single-walled carbon nanotubes sensors in solution, enabling intravenous injection into mice and the selective detection of local nitric oxide concentration with a detection limit of 1 µM. The half-life for liver retention is 4 h, with sensors clearing the lungs within 2 h 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 nitric oxide 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 demonstrate that alginate-encapsulated single-walled carbon nanotubes can function as implantable inflammation sensors for nitric oxide detection, with no intrinsic immune reactivity or other adverse response for more than 400 days.

Dual use of amphiphilic macromolecules as cholesterol efflux triggers and inhibitors of macrophage athero-inflammation.

Activated vascular wall macrophages can rapidly internalize modified lipoproteins and escalate the growth of atherosclerotic plaques. This article proposes a biomaterials-based therapeutic intervention for depletion of non-regulated cholesterol accumulation and inhibition of inflammation of macrophages. Macromolecules with high scavenger receptor (SR)-binding activity were investigated for SR-mediated delivery of agonists to cholesterol-trafficking nuclear liver-X receptors. From a diverse feature space of a family of amphiphilic macromolecules of linear and aromatic mucic acid backbones modified with varied aliphatic chains and conjugated with differentially branched poly(ethylene glycol), a key molecule (carboxyl-terminated, C12-derivatized, linear mucic acid backbone) was selected for its ability to preferentially bind scavenger receptor A (SR-A) as the key target. At a basal level, this macromolecule suppressed the pro-inflammatory signaling of activated THP-1 macrophages while competitively lowering oxLDL uptake in vitro through scavenger receptor SRA-1 targeting. To further deplete intracellular cholesterol, the core macromolecule structure was exploited to solubilize a hydrophobic small molecule agonist for nuclear Liver-X Receptors, which regulate the efflux of intracellular cholesterol. The macromolecule-encapsulated agonist system was found to reduce oxLDL accumulation by 88% in vitro in comparison to controls. in vivo studies were designed to release the macromolecules (with or without encapsulated agonist) to injured carotid arteries within Sprague Dawley rats fed a high fat diet, conditions that yield enhanced cholesterol accumulation and macrophage recruitment. The macromolecules lowered intimal levels of accumulated cholesterol (50% for macromolecule alone; 70% for macromolecule-encapsulated agonist) and inhibited macrophage retention (92% for macromolecule; 96% for macromolecule-encapsulated agonist; 4 days) relative to non-treated controls. Thus, this study highlights the promise of designing bioactive macromolecule therapeutics based on scavenger receptor targeting, for potential management of vascular arterial disease.

Controllable inhibition of cellular uptake of oxidized low-density lipoprotein: structure-function relationships for nanoscale amphiphilic polymers.

A family of anionic nanoscale polymers based on amphiphilic macromolecules (AMs) was developed for controlled inhibition of highly oxidized low-density lipoprotein (hoxLDL) uptake by inflammatory macrophage cells, a process that triggers the escalation of a chronic arterial disease called atherosclerosis. The basic AM structure is composed of a hydrophobic portion formed from a mucic acid sugar backbone modified at the four hydroxyls with lauroyl groups conjugated to hydrophilic poly(ethylene glycol) (PEG). The AM structure-activity relationships were probed by synthesizing AMs with six key variables: length of the PEG chain, carboxylic acid location, type of anionic charge, number of anionic charges, rotational motion of the anionic group, and PEG architecture. All AM structures were confirmed by nuclear magnetic resonance spectroscopy and their ability to inhibit hoxLDL uptake in THP-1 human macrophage cells was compared in the absence and presence of serum. We report that AMs with one, rotationally restricted carboxylic acid within the hydrophobic portion of the polymer was sufficient to yield the most effective AM for inhibiting hoxLDL internalization by THP-1 human macrophage cells under serum-containing conditions. Further, increasing the number of charges and altering the PEG architecture in an effort to increase serum stabilization did not significantly impair the ability of AMs to inhibit hoxLDL internalization, suggesting that selected modifications to the AMs could potentially promote multifunctional characteristics of these nanoscale macromolecules.

Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex.

Ca2+ channel beta subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, beta subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the beta subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved beta4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to beta4 subunits, the interactions are specific for the beta4a splice variant, because they do not occur with the beta4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel beta subunits and the cell-specific roles they play in the CNS.