Research Article: Peptide‐Functionalized Fluorescent Particles for In Situ Detection of Nitric Oxide via Peroxynitrite‐Mediated Nitration

Date Published: May 17, 2017

Publisher: John Wiley and Sons Inc.

Author(s): Jason Y. H. Chang, Lesley W. Chow, W. Michael Dismuke, C. Ross Ethier, Molly M. Stevens, W. Daniel Stamer, Darryl R. Overby.


Nitric oxide (NO) is a free radical signaling molecule that plays a crucial role in modulating physiological homeostasis across multiple biological systems. NO dysregulation is linked to the pathogenesis of multiple diseases; therefore, its quantification is important for understanding pathophysiological processes. The detection of NO is challenging, typically limited by its reactive nature and short half‐life. Additionally, the presence of interfering analytes and accessibility to biological fluids in the native tissues make the measurement technically challenging and often unreliable. Here, a bio‐inspired peptide‐based NO sensor is developed, which detects NO‐derived oxidants, predominately peroxynitrite‐mediated nitration of tyrosine residues. It is demonstrated that these peptide‐based NO sensors can detect peroxynitrite‐mediated nitration in response to physiological shear stress by endothelial cells in vitro. Using the peptide‐conjugated fluorescent particle immunoassay, peroxynitrite‐mediated nitration activity with a detection limit of ≈100 × 10−9m is detected. This study envisions that the NO detection platform can be applied to a multitude of applications including monitoring of NO activity in healthy and diseased tissues, localized detection of NO production of specific cells, and cell‐based/therapeutic screening of peroxynitrite levels to monitor pronitroxidative stress in biological samples.

Partial Text

Nitric oxide (NO) is a diatomic free radical with important physiological roles across multiple biological systems. NO rapidly diffuses across cell membranes and between cells, where it acts as a signaling molecule to modulate vascular homeostasis,1, 2, 3, 4, 5 neuronal activity,6, 7 and immunological processes.8 NO dysregulation has been linked to the pathogenesis of Parkinson’s and Alzheimer’s disease,9, 10, 11 cardiovascular disease,12, 13, 14, 15 glaucoma,16, 17, 18, 19, 20 and cancer.21, 22 The detection and quantification of NO is therefore important for understanding physiology and pathophysiology of disease‐relevant tissues, but the reactive nature of NO and its typically short half‐life (on the order of seconds1, 2, 4, 6, 8, 23) make accurate measurement of NO challenging.

This study demonstrates that tyrosine‐containing peptides have the potential to be used as biosensors to detect NO based on tyrosine nitration. We characterized four peptides, three of which were derived from nitration‐prone proteins. By UV–vis, we showed that these peptides had a detection limit of 10 × 10−6m for peroxynitrite, the key intermediate between NO and 3‐nitrotyrosine. This detection limit was improved to 100 × 10−9m by conjugating the peptides to FPs and labeling with fluorescent antibodies against 3‐nitrotyrosine. This exceeded the detection limit of the traditional Griess assay, which is typically 0.5 × 10−6–1 × 10−6m. To demonstrate that the peptides are able to detect physiological levels of NO in the presence of endogenous superoxide, we exposed HUVEC cells to laminar shear stress. Peptide‐functionalized FPs contained within the culture media exhibited a threefold to fivefold increase in 3‐nitrotyrosine labeling in response to shear, consistent with shear‐induced NO production that is characteristic of vascular endothelial cells.

Materials and Reagents: FluoSpheres580/605 (carboxylated FPs, 200 nm in diameter) were purchased from Molecular Probes (Invitrogen). 9‐Fluorenylmethoxycarbonyl (Fmoc)‐protected amino acids, Rink amide 4‐methylbenzhydrylamine (MBHA) resin, N,N‐diisopropylethylamine (DIEA), 2‐(1Hbenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU), dichloromethane (DCM), dimethylformamide (DMF), 80:20 dimethylformamide/piperidine premix, and spectroscopic grade acetonitrile (ACN) were purchased from AGTC Bioproducts, UK. PAPA NONOate, Angeli’s salt (NO−), and 3‐nitrotyrosine were all purchased from Cayman Chemical. The reactive nitrogen species were stored at −80 °C, while 3‐nitrotyrosine was stored at room temperature. l‐tyrosine, hydrogen peroxide (H2O2) and DAF‐FM were purchased from Sigma. 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC), N‐hydroxysuccinimide (NHS), and 2‐(N‐morpholino)ethanesulfonic acid (MES) were all obtained from ThermoFisher Scientific. Mouse monoclonal anti‐nitrotyrosine (clone 2A8.2; MAB5404) antibody and xanthine/xanthine oxidase to generate superoxide70 were obtained from Merck Millipore; goat anti‐rabbit VE‐Cadherin (XP monoclonal #2500) antibody was obtained from Cell Signaling; Alexa Fluor 488 goat anti‐rabbit IgG secondary antibody was obtained from Life Technologies Inc., and goat anti‐mouse IgG secondary antibody (IRDye 800CW) was obtained from LI‐COR Biosciences.

JYHC, DRO, LWC and WDS are named inventors on a pending patent covering the technology described within the manuscript.




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