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 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.

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.