Real-time Detection Of Nitric Oxide Research Articles

A fluorescent probe with an ultra-rapid response to nitric oxide.

Nitric oxide (NO) is a diatomic inorganic free radical ubiquitous in mammalian tissues and cells that plays a multifaceted role in a variety of physiological and pathophysiological processes. The strict dependence of the biological effects of NO on its concentration makes its real-time monitoring crucial. In view of the reactivity of NO with multiple bio-targets, the development of NO sensors that associate a fast response rate with selectivity and sensitivity is very challenging. Herein we report a fluorescent NO probe based on a BODIPY fluorogenic unit covalently linked to a trimethoxy aniline derivative through a flexible spacer. NO leads to effective nitrosation of the highly electron-rich amino active site of the probe through the secondary oxide N2O3, resulting in an increase of BODIPY fluorescence quantum yield from Φf = 0.06 to Φf = 0.55, accompanied by significant changes in the relative amplitude of the fluorescence lifetimes. In situ generation of NO, achieved by a tailored light-activatable NO releaser, allows the real-time detection of NO as a function of its concentration and permits demonstrating that the probe exhibits a very fast response time, being ≤0.1 s. This remarkable data combines with the high sensitivity of the probe to NO (LOD = 35 nM), responsiveness also to ONOO-, the other important secondary oxide of NO, independence from the fluorescence response within a wide pH range, good selectivity towards different analytes and small interference by typical physiological concentrations of glutathione. Validation of this probe in melanoma cell lines is also reported.

Solution-processed graphene films for electrochemical monitoring of extracellular nitric oxide released by breast cancer cells

Nitric oxide (NO) plays an important role in cardiovascular function, immune response, and intercellular signaling. However, due to its short lifetime, real-time detection of NO is challenging. Herein, an electrochemical sensor based on fibronectin-modified, solution-processed graphene ink for NO detection is developed using a facile fabrication method involving spin-coating and hot-plate annealing. The sensor is first electrochemically characterized with a NO donor, spermine NONOate, exhibiting a dynamic range of 10–1000 μM. The fibronectin-functionalized graphene supports the attachment and growth of MDA-MB-231 breast cancer cells, as confirmed by optical microscopy. Extracellular NO production is stimulated using the amino acid L-arginine. NO production results in morphological changes to the adhered cells, which are reversible upon the addition of the NO synthase antagonist Nω-nitro-L-arginine methyl ester. The production of NO is also confirmed using real-time amperometric measurements with the fibronectin-functionalized graphene sensors. While this work focuses on NO detection, this potentially scalable platform could be extended to other cell types with envisioned applications including the high-throughput evaluation of therapeutics and biocompatible coatings.

Graphene nanocomposites for real-time electrochemical sensing of nitric oxide in biological systems.

Nitric oxide (NO) signaling plays many pivotal roles impacting almost every organ function in mammalian physiology, most notably in cardiovascular homeostasis, inflammation, and neurological regulation. Consequently, the ability to make real-time and continuous measurements of NO is a prerequisite research tool to understand fundamental biology in health and disease. Despite considerable success in the electrochemical sensing of NO, challenges remain to optimize rapid and highly sensitive detection, without interference from other species, in both cultured cells and in vivo. Achieving these goals depends on the choice of electrode material and the electrode surface modification, with graphene nanostructures recently reported to enhance the electrocatalytic detection of NO. Due to its single-atom thickness, high specific surface area, and highest electron mobility, graphene holds promise for electrochemical sensing of NO with unprecedented sensitivity and specificity even at sub-nanomolar concentrations. The non-covalent functionalization of graphene through supermolecular interactions, including π-π stacking and electrostatic interaction, facilitates the successful immobilization of other high electrolytic materials and heme biomolecules on graphene while maintaining the structural integrity and morphology of graphene sheets. Such nanocomposites have been optimized for the highly sensitive and specific detection of NO under physiologically relevant conditions. In this review, we examine the building blocks of these graphene-based electrochemical sensors, including the conjugation of different electrolytic materials and biomolecules on graphene, and sensing mechanisms, by reflecting on the recent developments in materials and engineering for real-time detection of NO in biological systems.

Sensitive electrochemical measurement of nitric oxide released from living cells based on dealloyed PtBi alloy nanoparticles.

Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5μM) and a low detection limit of 1nM (3σ/k) as well as high sensitivity (130 and 36.5 μA μM-1cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.

Rational Design of Cu-Doped Tetrahedron of Spinel Oxide for High-Performance Nitric Oxide Electrochemical Sensor

The real-time detection of nitric oxide (NO) in living cells is essential to reveal its physiological processes. However, the popular electrochemical detection strategy is limited to the utilization of noble metals. The development of new detection candidates without noble metal species still maintaining excellent catalytic performance has become a big challenge. Herein, we propose a spinel oxide doped with heteroatom-Cu-doped Co3O4 (Cu-Co3O4) for the sensitive and selective detection of NO release from the living cells. The material is strategically designed with Cu occupying the tetrahedral (Td) center of Co3O4 through the formation of a Cu-O bond. The introduced Cu regulates the local coordination environment and optimizes the electronic structure of Co3O4, hybridizing with the N 2p orbital to enhance charge transfer. The CuTd site can well inhibit the current response to nitrite (NO2-), resulting in a high improvement in the electrochemical oxidation of NO. The selectivity of Cu-Co3O4 can be markedly improved by the pore size of the molecular sieve and the negative charge on the surface. The rapid transmission of electrons is due to the fact that Cu-Co3O4 can be uniformly and densely in situ grown on Ti foil. The rationally designed Cu-Co3O4 sensor displays excellent catalytic activity toward NO oxidation with a low limit of detection of 2.0 nM (S/N = 3) and high sensitivity of 1.9 μA nM-1 cm-2 in cell culture medium. The Cu-Co3O4 sensor also shows good biocompatibility to monitor the real-time NO release from living cells (human umbilical vein endothelial cells: HUVECs; macrophage: RAW 264.7 cells). It was found that a remarkable response to NO was obtained in different living cells when stimulated by l-arginine (l-Arg). Moreover, the developed biosensor could be used for real-time monitoring of NO released from macrophages polarized to a M1/M2 phenotype. This cheap and convenient doping strategy shows universality and can be used for sensor design of other Cu-doped transition metal materials. The Cu-Co3O4 sensor presents an excellent example through the design of proper materials to implement unique sensing requirements and sheds light on the promising strategy for electrochemical sensor fabrication.

Rapid real-time monitoring of NO released from living cells using multi-walled carbon nanotube-7,7,8,8-tetracyanoquinonedimethyl-polylysine sensors

Nitric oxide (NO) is an important but short-lived signaling molecule that is released from living cells. Real-time monitoring of NO release is useful for understanding normal cellular physiology and pathology. Herein, a convenient and efficient NO sensor was developed using multiwalled carbon nanotubes (MWCNTs)-7,7,8,8-tetracyanoquinodimethan (TCNQ)-polylysine (PLL) modified screen-printed electrode (SPE). The construction of the sensor (MWCNTs/TCNQ/PLL/SPE) was based on the synergic effect of the good conductivity of TCNQ and the high surface area of MWCNTs. The introduction of the cell-adhesive molecule PLL significantly enhanced the cytocompatibility, resulting in excellent cell attachment and growth. The resulting MWCNTs/TCNQ/PLL/SPE was successfully used for the real-time detection of NO released from living human umbilical vein endothelial cells (HUVECs) cultured on it. The MWCNTs/TCNQ/PLL/SPE was further used to detect NO release from oxidative-injured HUVECs with and without resveratrol to also preliminarily assess the effect of resveratrol against oxidative damage. The sensor developed in this study showed good performance for the real-time detection of NO released by HUVECs under different conditions and has potential applications in the diagnosis of biological processes and the screening of drug treatment effects.

Thiophene and diaminobenzo- (1,2,5-thiadiazol)- based DAD-type near-infrared fluorescent probe for nitric oxide: A theoretical research.

A near-infrared fluorescent probe (LS-NO) for the real-time detection of nitric oxide (NO) in inflammatory bowel disease (IBD) was developed recently. The probe used oligoglycol morpholine-functionalized thiophene as strong electron donors and diaminobenzene (1,2,5-thiadiazole) as a weak electron acceptor and NO trapping group. It could detect exogenous and endogenous NO in the lysosomes of living cells with high sensitivity and specificity. To further understand the fluorescent mechanism and character of the probes LS-NO and LS-TZ (after the reaction of the probe LS-NO with NO), the electron transfer in the excitation and emitting process within the model molecules DAD-NO and DAD-TZ was analyzed in detail under the density functional theory. The calculation results indicated the transformation from diaminobenzene (1,2,5-thiadiazole) as a weak electron acceptor to triazolo-benzo-(1,2,5-thiadiazole) as a strong electron acceptor made LS-NO an effective "off-on" near-infrared NO fluorescent probe.

A ratiometric near-infrared fluorescence/photoacoustic dual-modal probe with strong donor dithienopyrrole for in vivo nitric oxide detection

Integrating the imaging techniques of near-infrared fluorescence (NIRF) and photoacoustic (PA) can make up for each other and provide more useful medical information. Ratiometric imaging activated by disease-associated biomarkers can further augment imaging specificity. However, very few studies have employed the NIRF/PA dual-modal ratiometric imaging to improve the accuracy and specificity of disease diagnosis to date. In this paper, we present the synthesis of a nitric oxide (NO)-activated ratiometric NIRF/PA dual-modal nanoprobe RAPNP for in vivo NO imaging. The ratiometric imaging function was achieved jointly by a NO/acidity-responsive molecule DTP-BTDA and a nonresponsive fluorophore DTP-BBTD. In these fluorophores, the dithienopyrrole (DTP) moiety had strong electron-donating ability and imparted strong intramolecular charge transfer and relatively long emission wavelengths. The BTDA moiety in DTP-BTDA could be rapidly oxidized by NO under weak acidic environments, achieving the NIRF and PA signal activation. By using RAPNP as a contrast agent, we achieved the ratiometric detection of the endogenous NO in inflammatory bowel disease by NIRF/PA dual-modal imaging. This work provides the first case of the NIRF/PA dual-signal ratiometric probe for the real-time detection of NO in vivo.

An Fe-organic framework/arginine-glycine-aspartate peptide-modified sensor for electrochemically detecting nitric oxide released from living cells.

Nitric oxide (NO) is a crucial cell-signaling molecule utilized in numerous physiological and pathological processes. Monitoring cellular levels of NO requires a sensor with sufficient sensitivity, transient recording capability, and biocompatibility. Owing to the large surface area and high catalytic activity of the metal-organic framework, Fe-BTC was used for the modification of screen-printed electrodes (SPEs). This study investigates the electrochemical sensing of NO on modified SPEs. Additionally, the introduction of a cell-adhesive molecule, arginine-glycine-aspartate peptide (RGD), considerably improved the cytocompatibility, resulting in superior cell attachment and growth on the SPE. The Fe-BTC/RGD-modified SPE (Fe-BTC/RGD/SPE) exhibited electrocatalytic NO oxidation at 0.8 V, demonstrating a linear response with a detection limit of 11.88 nM over a wide concentration range (0.17-47.37 μM) and a response time of approximately 0.9 s. Subsequently, the as-obtained Fe-BTC/RGD/SPE was successfully utilized for the real-time detection of NO released from human endothelial cells cultured on the electrode. Therefore, the study undertaken shows remarkable potential of Fe-BTC/RGD/SPE for practical applications in biological processes and clinical diagnostics.

Design, synthesis and evaluation of 15N- and 13C-labeled molecular probes as hyperpolarized nitric oxide sensors

[Display omitted] Nitric oxide (NO) is an important signaling molecule involved in a wide range of biological processes. Development of non-invasive, real-time detection of NO is greatly desired yet remains challenging. Here we report the design and development of novel 15N- and 13C-labeled NO-sensing probes for hyperpolarized nuclear magnetic resonance (HP-NMR) studies. These probes undergo selective and rapid reaction with NO to generate in situ AZO-products that can be monitored with distinguishable NMR signals as a read-out. This study also allows for a direct comparison of the 15N and 13C nuclei performances in hyperpolarized reaction-based probes. The simple and general SABRE-SHEATH hyperpolarization method works on the 15N- and 13C-NO-sensing probes. Measured long spin–lattice relaxation (T1) values, especially for 15N-NO probes, will allow for real-time reaction-based imaging of NO.

Abstract P5-10-08: Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is aggressive and represents nearly 20% of all breast cancer cases. It is associated with poor prognosis due to its high metastatic capabilities and lack of targeted therapeutic treatments. To improve TNBC treatment, our goal is to better understand the molecular mechanisms that drive TNBC. Recently, inducible nitric oxide synthase (NOS2), a nitric oxide (NO) producing enzyme, has emerged as a key player in TNBC progression. High NOS2 expression in TNBC patient correlates with poor prognosis marked with increased metastasis and disease recurrence. NOS2 catalyzes L-arginine and dioxygen (O2) to produce citrulline and high, sustained levels of NO which has been shown to alter the activity of numerous signaling pathways. Depending on NO concentration and temporal and spatial levels, it has been reported to have both tumor-promoting and tumor-suppressing functions. However, the mechanism by which NOS2 drives TNBC is unclear. Akt is major oncogenic signaling pathway that regulates various aspects of tumor progression, and it has been shown that NO can activate Akt activity. The Akt family has three members, Akt1, Akt2, and Akt3, that seem to have similar and non-redundant functions in cancer. We have discovered for the first time that NOS2 specifically activates Akt2 in TNBC cells. Thus, we hypothesize that Akt2 plays a major role in NOS2 driven TNBC. Inflammatory cytokines (IFN-γ, IL-1β, and TNF-α) highly induce NOS2 in mouse 4T1 TNBC cells. We developed a real-time NO detection assay to measure NO levels in 4T1 cells and confirmed that these cells produce high levels of NO. By pharmacologically and genetically inhibiting NOS2 in the 4T1 cells, we found that NOS2 suppresses Akt2 phosphorylation but not the total levels. Furthermore, NOS2 had no effect on Akt1. The decrease in Akt2 phosphorylation was accompanied by inhibition of its downstream targets, GSK3β and FOXO3a, suggesting that NOS2 is regulating Akt2 activity. Conversely, treatment of 4T1 and human MDA-MD-231 TNBC cells with DETA/NO, a slow releasing NO donor, resulted in increased Akt2 phosphorylation with no effect on Akt1. Akt2 has been shown to promote migration, invasion, and metastasis. Using 4T1 cells, we are presently studying the function of NOS2-Akt2 signaling in TNBC. We have orthotopically injected 4T1 cells into immunocompetent mice and have found that 4T1 tumors express high levels of NOS2 as well as phosphorylated Akt2. We have knocked out NOS2 in the 4T1 cells to examine the effects of NOS2 inhibition in the mouse tumor model. Understanding the link between NOS2 and Akt2 will give further understanding on how NOS2 drives TNBC. Citation Format: Su Chung, William Montfort. Inflammation drives NOS2-Akt2 signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-08.

Molecularly assembled graphdiyne with atomic sites for ultrafast and real-time detection of nitric oxide in cell assays

Nitric oxide as a signal molecule participates in a variety of physiological and pathological processes but its real-time detection in cell assays still faces challenging because of the trace amount, short half-life and easy conversion to other substances. We report here a rational design by assembling highly π-conjugated and small capacitive gaphdiyne (GDY) with a coordination complex of hemin (HEM) into a molecularly assembled material of GDY/HEM to achieve ultrafast and real-time monitoring of nitric oxide in cell assays. GDY comprising alkynyl C atoms can hybridize with the HEM to enable strong π-π interaction and atomic dispersion of iron sites while avoiding the formation of catalytically inactive dimer for the HEM. These characteristics make the GDY/HEM an excellent sensing material towards nitric oxide, which has an ultrafast response time of 0.95 s, a low detection limit of 7 nM and long linear range up to 151.38 μΜ. The GDY/HEM realizes real-time monitoring nitric oxide released from cancer and normal cells, demonstrating its capability for cell analysis.

A far-red-emitting fluorescence probe for selective and sensitive detection of no in live cells and in C. elegans

Nitric oxide (NO), a ubiquitous intracellular and intercellular messenger molecule, plays vital roles in many physiological processes and is closely related to many diseases. Although a lot of fluorescent probes have been developed for real-time detection of NO successfully, the probes still suffer from poor tissue permeability and limited selectivity. In this study, a novel far-red fluorescent probe ZJL-3 based on rhodamine fluorescent dye was designed, synthesized, and used for NO determination. The probe contains a rhodamine as fluorophore and o-phenylenediamino as recognition unit. Upon addition of NO, the probe ZJL-3 showed an obvious far-red emission at 637 nm. The results of fluorescence spectrum experiments indicated that probe ZJL-3 exhibited desirable selectivity to NO. Furthermore, probe ZJL-3 has low cytotoxicity and was applied for the detection of exogenous and endogenous NO in RAW264.7 cells and C. elegans with satisfactory results.

3D printed bioresorbable nitric oxide-releasing vascular stents

Although bioresorbable vascular stents (BVS) have emerged as a promising alternative to overcome complications of metallic stents, adverse outcomes of BVS demand further improvements. The development of photocrosslinkable polyesters, such as methacrylated poly(dodecanediol citrate) (mPDC) for use in light-assisted 3D printing technologies, brought new perspectives for the fabrication of BVS. In line with this approach, the knowledge of the pro-endothelial and anti-thrombotic actions of nitric oxide (NO), has stimulated the development of NO-releasing BVS. Herein, we describe the fabrication of mPDC-based NO-releasing BVS, using digital light processing (DLP) 3D printing. The NO release property of the mPDC matrix was obtained through the incorporation of the NO donor S-nitroso-N-acetyl-d-penicillamine (SNAP). Chemiluminescent real-time NO detection showed that NO release from mPDC/SNAP is triggered by contact with the aqueous medium at rates which can be modulated by the SNAP charge. The obtained NO release rates, in the range of 20–160 ​pmol ​cm−2 min−1, have potential to inhibit thrombus formation. Therefore, 3D printed BVS made of NO-releasing mPDC/SNAP may offer a simple and new strategy for overcoming late thrombotic complications of BVS.

Synthesis of merit-combined antimony tetroxide nanoflowers/reduced graphene oxide to synergistically boost real-time detection of nitric oxide released from living cells for high sensitivity

Nitric oxide (NO) is an important bio-regulatory and signaling molecule associated with various physiological and pathophysiological pathways, but its sensitive real-time detection is still very challenging due to the low concentration, large diffusivity and fast decay in biological samples. Here an antimony tetroxide (Sb2O4) nanoflowers/reduced graphene oxide (rGO) nanocomposite is synthesized via a facile and eco-friendly solvothermal method to merit-combine highly electroactive Sb2O4 nanoflowers with large surficial rGO component for a strong synergistic effect on oxidation of NO. Results demonstrate that the Sb2O4/rGO-based sensor has a low detection limit, high sensitivity, excellent selectivity and fast response for NO detection. The real-time detected NO released from living cells showed significant difference between normal and tumor cells. The Sb2O4 nanoflowers/rGO nanocomposite sensor holds a great promise for important applications in biomedical research fields and clinical diagnosis.

A flexible and physically transient electrochemical sensor for real-time wireless nitric oxide monitoring

Real-time sensing of nitric oxide (NO) in physiological environments is critically important in monitoring neurotransmission, inflammatory responses, cardiovascular systems, etc. Conventional approaches for NO detection relying on indirect colorimetric measurement or built with rigid and permanent materials cannot provide continuous monitoring and/or require additional surgical retrieval of the implants, which comes with increased risks and hospital cost. Herein, we report a flexible, biologically degradable and wirelessly operated electrochemical sensor for real-time NO detection with a low detection limit (3.97 nmol), a wide sensing range (0.01–100 μM), and desirable anti-interference characteristics. The device successfully captures NO evolution in cultured cells and organs, with results comparable to those obtained from the standard Griess assay. Incorporated with a wireless circuit, the sensor platform achieves continuous sensing of NO levels in living mammals for several days. The work may provide essential diagnostic and therapeutic information for health assessment, treatment optimization and postsurgical monitoring.

Investigative Study on Nitric Oxide Production in Human Dermal Fibroblast Cells under Normal and High Glucose Conditions.

Diabetic foot ulcers (DFU) are a major health problem associated with diabetes mellitus. Impaired nitric oxide (NO) production has been shown to be a major contributor to the dysregulation of healing in DFU. The level of impairment is not known primarily due to challenges with measuring NO. Herein, we report the actual level of NO produced by human dermal fibroblasts cultured under normal and high glucose conditions. Fibroblasts produce the extracellular matrix, which facilitate the migration of keratinocytes to close wounds. The results show that NO production was significantly higher in normal glucose compared to high glucose conditions. The real-time NO detected was compared to the nitrite present in the culture media and there was a direct correlation between real-time NO and nitrite in normal glucose conditions. However, real-time NO detection and nitrite measurement did not correlate under high glucose conditions. The inducible nitric oxide synthase (iNOS) enzyme responsible for NO production was upregulated in normal and high glucose conditions and the proliferation rate of fibroblasts was not statistically different in all the treatment groups. Relying only on nitrite to assess NO production is not an accurate determinant of the NO present in the wound bed in pathological states such as diabetes mellitus.

Iron Phthalocyanine Decorated Nitrogen-Doped Graphene Biosensing Platform for Real-Time Detection of Nitric Oxide Released from Living Cells.

Nitric oxide (NO) is a transcellular messenger involved in many physiological and pathological processes, but the real-time detection of NO in biological systems is still challenging due to its rapid diffusion, low concentration, and short half-life. A novel electrochemical sensing platform based on iron phthalocyanine (FePc) functionalized nitrogen-doped graphene (N-G) nanocomposites was constructed to achieve in situ monitoring of NO released from living cells on the sensing layer. By taking advantage of the synergetic effect of N-G and FePc nanocomposites, the N-G/FePc sensor displays excellent electrocatalytic activity toward NO with a high sensitivity of 0.21 μA μM-1 cm-2 and a low detection limit of 180 nmol L-1. The following layer-by-layer assembly of poly-l-lysine (PLL) and Nafion further improved the capacity of resisting disturbance as well as the biocompatibility of the sensing interface. The flexible design of the ITO substrate based electrode provides a more controlled cellular biosensing system which could capture molecular signals immediately after NO released from human umbilical vein endothelial cells (HUVECs). The exhibited additional features of high sensitivity, rapid response, and ease of operation implies that the proposed N-G/FePc/Nafion/PLL ITO biosensor is a promising powerful platform in various complex biological systems.

Selective and Real-Time Detection of Nitric Oxide by a Two-Photon Fluorescent Probe in Live Cells and Tissue Slices.

Nitric oxide (NO) is an important signaling molecule involved in many physiological and pathological processes. To understand these NO-mediated processes, it is a key to develop rapid and specific detection methods for NO. In the past 2 decades, numerous excellent fluorescent probes for NO have been designed; however, it still remains limitations such as slow response, low selectivity, and short excitation wavelength (<600 nm). In this Article, a two-photon fluorescent probe, NO-QA5, has been developed with 3-dimethylaminophenyl linking at the 6-position of 5-aminoquinoline as both the active site and prefluorophore for detection of NO. The nonfluorescent NO-QA5 can fast react with NO via a diazonium intermediate to generate two azoic regioisomers, one of which exhibits intramolecular charge transfer (ICT) emission, and two-photon absorption behavior (δΦ = 57 GM), giving a turn-on fluorescence rapid response. The sensing reaction is pH-insensitive in the range of 6-11 and highly selective and well sensitive (LOD = 15 nM), possible undergoing the same intermediate diazonium with the reaction under diazotization condition (NaNO2/HCl). Also, as a nitrite fluorescent probe NO-QA5 exhibits highly sensitive (LOD = 7 nM). Therefore, NO-QA5 can serve as a dual functional fluorescent probe for NO and NO2-. Furthermore, NO-QA5 as a specific imaging agent has been demonstrated by achieving both exogenous and endogenous detections of NO in living cells under both one- and two-photon excitation and high resolution in tissue slices under two-photon excitation.

An Amphiphilic Fluorescent Probe Designed for Extracellular Visualization of Nitric Oxide Released from Living Cells.

Nitric oxide (NO) is an intracellular and intercellular messenger involved in numerous physiological and pathophysiological processes. Small-molecule fluorescent probes coupled with fluorescence microscopy provide excellent tools for real-time detection of NO in situ. However, most probes are designed for imaging intracellular NO, which cannot reflect the release behavior of endogenously produced NO. In order to visualize extracellular NO released from living cells, we report herein a particularly designed amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), in which hydrophilic groups are introduced to keep the fluorophore and recognition domain outside the cell and a hydrophobic C16 alkyl chain acts as the membrane anchor. Based on this design, NO released out of the cells has been visualized on the outer surface of the plasma membrane. Using RAW 264.7 cells and ECV-304 cells as models, the diffusion of NO across the plasma membrane has been directly observed. The amphiphilic design strategy of fluorescent probes holds great promise for developing fluorescent imaging probes to study the release behaviors of other endogenous gasotransmitters.