Nitric oxide detection methods in vitro and in vivo

1 Guangdong Provincial Key Laboratory of Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, Guangdong, China

Gaoxin Zhou

1 Guangdong Provincial Key Laboratory of Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, Guangdong, China

Qianjun He

1 Guangdong Provincial Key Laboratory of Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, Guangdong, China

2 Center of Hydrogen Science, Shanghai Jiao Tong University, Shanghai, China

1 Guangdong Provincial Key Laboratory of Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen, Guangdong, China

Author contributions

Review writing: EG; review initial revision: GZ; manuscript revising and supervision: QH. All authors reviewed and approved the final version of this manuscript.

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Abstract

Initially being considered as an environmental pollutant, nitric oxide has gained the momentum of research since its discovery as endothelial derived growth factor in 1987. Extensive researches have revealed the various pathological and physiological roles of nitric oxide such as inflammation, vascular and neurological regulation functions. Hence, the development of methods for quantifying nitric oxide concentration and its metabolites will be beneficial to well know about its biological functions and effects. This review summaries various methods for in vitro and in vivo nitric oxide detection, and introduces their merits and demerits.

Keywords: nitric oxide, detection method, nitric oxide synthase, nitric oxide therapy, colorimetric, chemiluminescence, fluorescence, electrochemical, gas chromatography, magnetic resonance imaging

I NTRODUCTION

Since its discovery by Joseph Priestly in 1722 as a kind of colorless gas, nitric oxide (NO) was majorly considered as an environmental pollutant. Until 1987, NO was recognized as an important molecule that can regulate endothelial functions in the body. Ignarro et al.1 proved that endothelial derived relaxing factor was NO, thus giving a boost for in depth research on NO. This discovery paved way for later recognization of NO synthesis pathways and its various biological functions. In 1988, L-arginine was found to be the precursor for generation of NO which could be converted to L-citrulline by NO synthases (NOSs) in vivo. Three isoforms of NOSs were named according to their functions and the type of tissues in which they were firstly found. The neuronal isoform was named as neuronal NOS (nNOS, NOS1) found in neuronal cells, inducible NOS (iNOS, NOS2) was found in cells responsible for inflammation, such as macrophages and microglias, while endothelial NOS (eNOS, NOS3) was found in endothelial cells ( Figure 1 ).2 The importance of NO was established over years of research, where NO was involved in the variety of pathological and physiological functions such as endothelial vasorelaxation, cardiovascular functions, antimicrobial action, wound healing, tissue repair, neurotransmission, immune functions, blood pressure regulation, cytotoxicity, relaxation of the human penile corpus cavernosum, etc.1,3,4

General functions of three isoforms of nitric oxide synthases.

Note: nNOS: Neuronal nitric oxide synthase; iNOS: inducible nitric oxide synthase; eNOS: endothelial nitric oxide synthase; CNS: central nervous system; PNS: peripheral nervous system; MΦ: macrophages. Adapted from Förstermann et al.8

NO is an unstable, highly lipophilic, free radical molecule made up of an unpaired electron.3 It has poor solubility (1.9 mM) in water but relatively higher solubility in lipid membranes and in nonpolar solvents.4,5,6 The diffusion rate of NO is found to be 50 μm/s in a single direction in biological systems.7 The half-life of NO in biological tissues is observed to be only 3–5 seconds, as opposed to 500 seconds in pure aqueous solutions.5 Due to the radical nature of NO, it is readily oxidized into nitrite (NO₂ – ) or even nitrate (NO₃ – ).5 It is difficult to directly measure NO in vivo, and it is frequent to indirectly measure its oxidative products such as NO₂ – and NO₃ – .5,7

Decrease and defective release of NO can lead to atherosclerosis generation, coronary vasopasm and restenosis after angiopathy. On the other hand, the increase in NO synthesis can cause hypotension in patients suffering from liver cirrhosis and failure, and also cause hemorrhagic and anaphylactic shock. Endogenous release of NO at basal levels maintains the low resting stage of pulmonary vasculature.3 The use of NO prodrugs and nanomaterials has proved useful to develop promising NO-based therapy. NO gas at a higher concentration range (> nM) is known to exhibit anti-Warburg effect inhibiting tumor occurrence and development. On the other hand, concentration greater than 1 mM can result in NO poisoning, whereas concentration in the range of pM to nM in the tumor cells can promote tumor cell growth.9,10 The use of nanomaterials have helped to overcome the limitations of NO having short half-life, instability during storage and possibility of toxicity. Many types of nanomaterials such as polymers, dendrimers, hydrogels, liposomes, gold and silica nanoparticles, certain quantum dots with NO donors have been exploited in the past under various conditions to release NO. Controlled release of NO from intelligent nanoparticles makes it an attractive tool for cancer therapy due to its tumoricidal effects.11 The vital roles performed by NO in regulating a variety of functions make it necessary for its rapid and accurate determination in vitro and in vivo. Over the past few years, various detection methods including colorimetry, chemiluminescence, fluorescence, electrochemical sensing, gas chromatography, electron spin resonance (ESR) spectroscopy and magnetic resonance imaging (MRI) have been developed to help to understand the pathology and to treat various diseases. The strategy involves searching each of these methods with the proper keywords and listing the various ways employed by researchers to detect and measure NO in vitro and in vivo. This review summarizes these advanced methods and discusses their advantages and shortcomings as following.

C OLORIMETRIC M ETHOD

Colorimetric method for NO detection utilizes the quantification of color change caused by reaction between indicator and NO. NO concentration in vitro can be measured directly by the nitrosation of hemoglobin and myoglobin ( Figure 2 ).12 This method depends on the change of multiple absorption bands and does not need standard curve, exhibiting high sensitivity and accuracy. Besides, Griess assay is one of most general colorimetric methods for in vitro NO detection. Due to its relative simplicity,7 Griess assay has been extensively used since its discovery in 1879 for analysis of biological samples like saliva, urine, serum, cerebrospinal fluid and culture media.13 Griess assay provides an indirect approach for NO detection by measuring nitrite, nitrate and nitrosating agents as an index for NO.7 The two-step diazotization reaction is illustrated in Figure 3 . First, dinitrogen trioxide (N₂O₃) which is obtained from acidified nitrite (or from NO autoxidation) reacts with sulfanilamide to form diazonium derivative diazobenzenesulfonic acid. The derivative then interacts with N-(1-napthyl)ethylenediamine to produce purple colored diazo product azo-α-aminonaphthalene-parabenzene-sulfonic acid which exhibits a strong absorbance at 540 nm.5,7,13

Hemoglobin/myoglobin methods for detection of nitric oxide (NO) concentration.

Chemical reaction process involved in Griess assay.

Note: N₂O₃: Dinitrogen trioxide; SA: sulfanilamide; DABSA: diazobenzenesulfonic acid; NED: N-(1-napthyl) ethylenediamine; AANBSA: azo-α-aminonaphthalene-parabenzene-sulfonic acid.

A few variants of Griess assay have been extensively employed for NO measurement. For example, in conjunction with nitrate reductase, Griess assay has proved to be an accurate, sensitive and inexpensive tool to measure nitrite and nitrate in biological fluids, tissue and serum samples.7,14 In another variant, Griess assay is coupled with microtitre plate assay to determine the kinetics of NOS enzymatic reaction in crude or purified enzyme solutions. The oxidation of ferroheme by NO causes the increase in absorbance and hence can be used to measure the generation of NO.15 Flow injection method was also used to determine NO in vivo when NOS activity affected the acute renal failure in rats by detecting NOx as the final metabolite of NO with a high sensitivity of above 0.5 µM.16 Griess assay can be employed to detect NO₂ – and NO₃ – in the extracellular fluid as an index for NO by using Aspergillus nitrate reductase (NADPH, nitrate oxidoreductase), as it can reduce very small amounts of NO₃ – .17 It can also be used in human plasma cells to detect nitrite and nitrate in the plasma of healthy volunteers with an automated analyzer.18,19,20 Macrophage in Drosophila melanogaster can express NOS and generate NO as an important signaling molecule during its immune response, where Griess assay was used by Ajjuri et al.21 to measure nitrites as an index of NO in the brain tissue of the organism in response to neuroinflammation. Lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages produced NO which once readily converted to nitrite could be detected by Griess assay.22 The production of NO as an anti-inflammatory response from LPS-induced macrophages could be measured in Magnolia sieboldii extract,23 Garcinia xanthochymus extracts,24 Sambucus australis,25 Ovis canadensis and Ovis aries,26 peat moss extracts27 via Greiss assay. In addition, Griess assay can also be used to determine NO concentration in plants like cucumber and tomato.28

Colorimetric method provides measurement of NO with good sensitivity and high accuracy with inexpensive tools and is economically feasible.7 However, NO measurement in whole blood with colorimetric method seems to be not feasible because of the disturbance of nitrogen oxide species.29,30

C HEMILUMINESCENCE M ETHOD

The chemiluminescence method for detecting NO is considered to be a useful technique. The principle of chemiluminescence detection is based on NO-triggered chemiluminescence reactions. Chemiluminescence can be realized by the oxidization of NO with ozone (O₃) into nitrogen dioxide in the excited state (NO₂*), which emits a photon spontaneously when it decays back to its basal lower energy state ( Figure 4A ).2,3,7,31,32,33 Another route to chemiluminescence is the use of chemiluminescence agent such as luminol to be excited by highly oxidative peroxynitrite (ONOO – ) which is generated by the reaction of NO with H₂O₂.34 The emission of photon or luminescence is measured by a photomultiplier. Photomultiplier converts this luminescence into electrical signal which is proportional to NO concentration ( Figure 4A ).35 The high specificity of NO detection is attributed to its unique properties, which includes its ability to exist as a gas and its quick reaction rate with ozone.32

General mode of operation of the chemiluminescence method for detection of NO.

Note: (A) Working model of chemiluniescence method for NO detection. Adapted from Bates,33 Hetrick and Schoenfisch.34 (B) Luciferase-based chemiluminescence method for detection of NO concentration in cell cultures. Adapted from Woldman et al.35 NO: Nitric oxide; PMT: photomultiplier; NO₂*: nitrogen dioxide in the excited state; hν: luminescence (h: Planck’s constant, ν: frequency of photon); ONOO – : highly oxidative peroxynitrite; GTP: guanosine triphosphate; cGMP: cyclic guanosine monophosphate; PPi: pyrophosphate; APS: adenosine-5′ phosphosulfate; ATP: adenosine triphosphate; AMP: adenosine monophosphate.

It is extremely difficult to directly detect the NO level in blood. Lopez et al.36 used acidic vanadium III to reduce the intracellular metabolic products of NO including nitrite and nitrate in the collected serum into NO at 98°C, which was then quantified by the chemiluminescence method. Chemiluminescence assay has also proved to be efficient in measuring NO generated by NOS in endothelial cells.7 In the treatment of persistent pulmonary hypertension in newborns and respiratory distress syndrome in adults, exhaled NO levels of asthma patients as an important indicator were also sequentially measured successfully.37 Kikuchi et al.38 measured the continuous release of NO from the isolated perfused rat kidney organ along with changes of perfusion pressure by the luminol-based chemiluminescence method, achieving a high determination limit of approximately 100 fM.

In addition, Woldman et al.35 developed a chemiluminescence method for detection of NO generation in cell cultures by a luciferin–luciferase system ( Figure 4B ). The activation product (pyrophosphate) of guanylyl cyclase by NO reaction was converted to adenosine triphosphate by adenosine triphosphate sulfurylase which further excited the luciferin–luciferase system to generate chemiluminescence. The luciferin-based chemiluminescence method was proved to be two orders of magnitude more sensitive than fluorescent method using 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) (mentioned in details in the subsequent section) when tested on cell cultures of bovine aortic endothelial cells and activated murine macrophages.

In order to increase the operability and applicable range of the chemiluminescence method, the microdialysis technique based on gas permeable porous membrane is frequently integrated. Yao et al.39 realized in vivo NO measurement with high sensitivity by combining microdialysis technique with chemiluminescence method. Special probe designed allowed the detection of NO in blood and brain tissue of rat and rabbit in spite of the influence of various physiological conditions ( Figure 5A ). The luminol-based chemiluminescence method was used by Robinson et al.40 to measure NO in exhaled breath. They used a kind of gas permeable porous hollow polypropylene fiber membranes to make gaseous NO react with the solution of luminol and H₂O₂ in the interface, successfully detecting NO with a limit of 0.3 ppb and a response time of 2 seconds ( Figure 5B ). Zhou and Arnold41 developed a kind of gas permeable silicone membrane to detect NO concentration in solution by the luminol-based chemiluminescence method, achieving a detection limit of 1.3 µM, a response time of 8–17 seconds, and a dynamic range from 5 to 40 μM ( Figure 5C ).

Different ways adopted for monitoring NO levels using chemiluminescence method.

Note: (A) Experimental setup for in vivo NO monitoring. Adapted from Yao et al.39 (B) Schematic representation of hollow fiber gas-liquid exchange module. Chemiluminescence reaction occurs when the gas, which flows along the exterior diffuses through the pores in the fiber membrane and into the luminol/H₂O₂ solution flowing through the interiors of the fiber. Adapted from Robinson et al.40 (C) Membrane phase and reaction chemistry for the nitric oxide fiber-optics sensor. Adapted from Zhou and Arnold.41 C: Snail shell-like cell; M: mixer; P: microdialysis probe; PM: photomultiplier tube; NO: nitric oxide; S1–3: syringes for different streams; PMT: photomultiplier; ONOO – : highly oxidative peroxynitrite; hν: luminescence (h: Planck’s constant, ν: frequency of the photon).

Chemiluminescence method provides highly sensitive, real time monitoring of NO with rapid and reliable results.7 However, this method is not feasible to monitor NO in vivo. Moreover, the detection can be interfered by ozone gas stream, since the ozone generators are difficult to provide a stable, repeatable gas stream.7 Liquid phase detection was made feasible by the luminol/H₂O₂ assay, but has low specificity and luminol can react with free radicals causing interference.2 Measurement of exhaled breath can be achieved which can aid diagnosis of respiratory diseases, monitor efficacy of therapies for respiratory diseases.40 On the other hand, NO measurement by this method is limited by its bulky and expensive instruments, time consuming factor, low specificity and sensitivity.2,3,42

F LUORESCENCE M ETHOD

Fluorescence method has emerged as a very useful technique for detection of NO. A number of fluorometric probes and dyes have been devised to exploit the full ability of fluorescence in measurement of NO. The most commonly used fluorescence agent 2,3-diaminonaphthalene (DAN), is exploited for its ability to produce fluorescent N-nitrisating agent 2,3-naphthotriazole from NO ( Figure 6A ).7,34,42 Another kind of fluorescent agent diaminofluoresceins (DAF) has also proved to be equally useful for detection of NO ( Figure 6B ). Spatial and temporal aspects of NO production have been monitored using fluorescent probes which mainly involve DAF, DAN and some other fluorescent agents discussed below.34

Various fluorescent compounds used for imaging of NO.

Note: (A) 2,3-Diaminonaphthalene after reaction with nitric oxide (NO) coverts into fluorescent triazole form. Adapted from Hong et al.2 (B) Overview of Danio rerio (Zebrafish model) indicates the fluorescent labeling of organs with Caudal fin (yellow), the notochord (purple), the cleithrum (orange) and the heart (blue). Adapted from Lepiller et al.53 (C) NO distribution in B16-F10 tumors grown in the cranial window in mouse, left: microangiography using tetramethylrhodamine-dextran; middle and right: pseudo-color representation of DAF-2T microfluorgraphs. Color bar represents the calibration of the fluorescence intensity with the known concentrations of DAF-2. Adapted from Kashiwagi et al.50 (D) Left: fluorescent image of DAR-4M loaded endothelial cells at 10 minutes after stimulation; right: fluorescence intensity strongly abolished by treatment with the agonist of the stimuli. Adapted from Kikuchi et al.56 (E) DAQ loaded cells and stimulated to produce NO imaged using confocal laser fluorescence microscopy, left: bright-field, middle: 488 nm excitation; right: 543 nm excitation. Adapted from Galindo et al.59 (F) Bright field and fluorescence images of activated PC12 cells loaded with 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3′,4′ -diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY) in the presence of arginine. Adapted from Chen et al.65 (G–J) Agents used for fluorescent based NO-sensing. Adapted from Hong et al.2

DAN has been employed as an indicator for NO detection in many studies. In 2002, Wada et al.43 developed a high performance liquid chromatography method to determine the concentration of NO in cultivated cells of plant Agavepacifica using fluorescence detection with DAN. This method successfully detected NO with the limit of 3.4 pmol/g cells.43 In 2003, Gharavi et al.44 employed similar fluorometric high performance liquid chromatography detection for one of the stable oxidation products of NO, i.e., nitrite in murine hepatoma cells. Fluorescence monitoring provided limit of detection of nitrite to be 10 pM. Similarly, DAN assay was used to determine the endothelial NO release from cultured porcine pulmonary artery endothelial cells by measuring the fluorescence from 1-(H)-naphthotriazole which was formed by the reaction between DAN and nitrite. However, DAN assay failed to detect NO in agonist stimulated cultured cells. When the stimulated pulmonary artery endothelial cells were treated with nitrate reductase, DAN assay was able to detect minor levels of NO generated by these cells. Hence, these results suggested that nitrate reduction was essential for the function of DAN.45

Other agents like 2,7-dichlorfluorescin, cobalt complex, Fe(DTCS)2-based sensor did find their application in fluorescent based NO-sensing, however were limited due to poor detection sensitivity, low specificity for NO, fluorescence from interference, etc. (Figure ​ Figure6G 6G - ​ J J ).2 In order to measure NO under physiological conditions, Kim et al.46 prepared a novel set of fluorescent indicators, which are popularly known as DAFs. Since its discovery, DAF has been used in various studies for detection of NO. DAFs interact with NO in the presence of dioxygen to yield the triazole derivatives which are highly fluorescent. DAFs generate less fluorescence than the triazole derivatives by 180-fold.The probe most commonly employed uses DAF-2 form which exhibits high fluorescence due to formation of triazofluorescin upon reaction with oxidation product of NO. Given this property of fluorescence, DAF-2 has been employed to measure NO in various settings ( Figure 7C ). The membrane permeable form of DAF-2, DAF-diacetate was utilized to measure NO in endothelial cells by Leikert et al.47 Langendorff perfused rabbit heart was used by Patel et al.48 to monitor the change ofNO concentration using DAF-2. Similarly, Strijdom et al.49 demonstrated the use of DAF-2-DA to detect intracellular NO in fresh adult rat cardiomycetes by flow cytometry and compared the results with cellular nitrate or nitrite level. Use of DAF-2 as an NO sensitive probe revealed the release of NO around the blood vessel wall and sporadically in the extracellular space of B16 melanomas using multiphoton laser-scanning microscopy. However, in the presence of inhibitors, the fluorescence appeared abolished. Along with the immunohistochemical analysis, eNOS was found to be predominant source of NO in vascular endothelial cells, whereas iNOS was the sporadic source of NO in the stromal cells of B16 melanomas ( Figure 6C ).50

Reaction mechanisms of different fluorescent probes for nitric oxide (NO) detection.

Note: (A) 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA). Adapted from Xie and Shen.69 (B) Diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM). Adapted from Gomes et al.70 (C) Diaminofluorescein-2 (DAF-2). (D) 1,2-Diaminoanthrauinone (DAQ). (E) 1,3,5,7-Tetramethyl-2,6-dicarbethoxy-8-(3' ,4' -diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY).

Another form of DAF, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM-DA) ( Figure 7A ) was used by Metto et al.51 to detect NO produced in single T1 lymphocytes (Jurkat cells) with the help of a microfluidic device. Standard set of cells was obtained by labeling them with 6-carboxyfluorescein diacetate. Immune cells expressed iNOS upon stimulation by LPS when compared with control set of cells, which exhibited two-fold increase in NO production.51 Recently, Agrawal et al.52 targeted overexpressing NOS isoform of HEK 293 T cell line imaging to screen the capacity of NOS inhibitors using DAF-FM-DA. Lepiller et al.53 employed DAF-FM-DA to detect NO generation sites in a living zebrafish Danio rerio model. The authors observed that NO production changed along the development in the notochord caudal fin. However, no changes were seen in the bulbus arteriosus. This method was also employed by the group to measure local changes in NO production in response to any stress. From their findings, the authors monitored changes in NO production in live zebrafish under physiological and pathophysiological conditions ( Figure 6B ).53

Zhou et al.54 previously showed measurements of NO in intact venules by platelet activating factor induced endothelial cells. However, the leakage of the DAF-2 dye after the washout steps compromised the accurate measurement of NO. Hence in 2011, to overcome the dye retention problem and improve the sensitivity for NO measurements, the same group used DAF-2-DA in rat venules. Continuous perfusion of DAF-2-DA in the rat venules was used to measure NO by fluorescence imaging under basal and stimulated conditions. Once DAF-2 achieved a stable state in the endothelial cells, basal and stimulated NO was quantified. This study also showed that, the measurement of fluorescence was mainly due to the hydrolyzed DAF-2 in the cells. With the help of this, NO can be assessed by subtracting non-NO-dependent intracellular DAF-2 in living tissues.

Furthermore, in order to overcome the limitations of using DAF-2 in biological applications, in 2001, Kojima et al.55 developed a fluorescent indicator on rhodamine chromophore DAR-4M AM ( Figure 7B ) for detection of NO. The membrane permeable property of DAR-4M AM was successfully employed by the authors to carry out bioimaging in bovine aortic endothelial cells for detection of NO with a limit of 7 nM without any pH dependency above pH 7. Human umbilical vein endothelial cells when treated with platelet-activating factor (PAF) could help to determine its effect on NO production. The produced NO was measured using DAR-4M AM and visualized using fluorescence microscopy and also revealed that in the presence of NOS inhibitor and PAF-receptor antagonist, NO production was not achieved, hence confirming the role of PAF in intracellular NO generation by activation of PAF-receptors in the human umbilical vein endothelial cells ( Figure 6D ).56 Recently, another rhodamine-deoxylactam based probe was developed and used to detect endogenous and exogenous NO in living HepG2 and RAW 264.7 cells. Once the probe came in contact with NO, it emitted a strong fluorescence which aided the easy detection of NO with high specificity.57

Externally stimulated and non-stimulated RAW 264.7 macrophage cells were treated with 1,2-diaminoanthraquinone (DAA or DAQ) ( Figure 7D ), a non-toxic probe known for its ability to visualize NO in living cells. Its reaction with intracellular NO in the presence of oxygen generated the triazole form of DAA, which could be spectrally visualized using confocal microscopy and fluorescence spectroscopy ( Figure 6E ).58,59 The NO reductase property of cytochrome P450 55B1 from Chlamydomonas reinhardtii was exploited by Li et al.60 in 2016 to develop a new fluorescence biosensor for detecting NO. The constructed biosensor was employed to detect NO release from L-arginine stimulated rat liver homogenate. The authors concluded that this biosensor can be successfully used for detection of NO in biological samples. Lim et al.61 synthesized a fluorescent probe with a copper Cu(II) complex and metal chelating ligand in 2006. The irreversible reduction of Cu(II) to Cu(I) along with the release of nitrosated ligand was determined to be the reaction causing NO induced fluorescence by spectroscopic and mass spectroscopic methods. The Cu(II) based complex was employed by the authors to detect NO generated in macrophages and live neurons by inducible and constitutive NOSs (iNOS and cNOS). MNIP-Cu was developed by Jain et al.62 to localize NO generation sites in various parts of sunflower (Helianthus annuus L.) almost without lag time which was found to be more specific than DAF probes.

Liu et al.63 designed ADNO (2-(α-(3,4-diaminophenoxy)acetyl)-6-(dimethylamino)naphthalene) a new two photon fluorescent probe in 2014 ( Figure 8A ). This probe detected NO on the basis of photoinduced electron transfer mechanism. The NO sensitive fluorescence modulator function of the probe is carried out by o-phenylenediamine moiety and 2-acetyl-6-(dimethylamino) naphthalene moiety functions as the two photon fluorophore. The electron-rich diamine moiety can be altered to quench the fluorescence of 2-acetyl-6-(dimethylamino) naphthalene by transferring the electron to excited fluorophore which can help to detect NO as anticipated by the authors. The experiment carried out in aqueous solution and NIH 3T3 cells presented that ADNO has rapid response rate to NO by exhibiting significant turn-on fluorescence. This probe was found to be less pH dependent and showed high selectivity and can be very useful tool in future biological applications.

Various fluorescent probes employed with different strategies to detect NO.

Note: Above: design strategy of ADNO probe; below: two-photon microscopy image of NO in ADNO-labeled NIH3T3 cells and ADNO-labeled cells treated with SNOC (S-nitrocysteine, a NO donor). Adapted from Liu et al.63 (B) Mito-DHP probe specifically targeting mitochondria for endogenous and exogenous produced NO by stimulated RAW264.7 murine macrophage and HepG2 cells, respectively. Adapted from Gao et al.66 (C) Gal-RhB sensor yielding fluorescent image of hepatocellular NO in Zebrafish. Adapted from Zhang et al.68 ADNO: (2-(α-(3,4-diaminophenoxy)acetyl)-6-(dimethylamino)naphthalene); NO: nitric oxide; Gal-RhB: a hepatocyte targeting fluorescent sensor.

Recently, various modifications are done to develop better fluorescent probes. For example, Zhang et al.64 developed a dual turn on type BODIPY probe with no intrinsic fluorescence due to photoinduced electron transfer effect. This probe could detect NO and nitrite ion in HepG2 cells with fluorescence confocal microscope under neutral and acidic conditions respectively with DEA-NONOate as the NO donor; (which switches off the photoinduced electron transfer) in assistance with intracellular cysteine and glutathione. Similarly, Chen et al.65 developed a BODIPY probe which could simultaneously detect endogenous and exogenous NO and glutathione in macrophage cells to uncover their inter-relation for maintaining the biological system’s redox balance ( Figure 6F ). Another modification in the BODIPY dye was done by Gao et al.66 by adding dihydropyridine and triphenylphosphonium moieties to it which could specifically target mitochondria (Mito-DHP probe), tracking the exogenously produced NO in HepG2 cells and endogenously produced NO in stimulated RAW264.7 murine macrophage cells under anaerobic conditions ( Figure 8B ). 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3 ',4' -diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY) ( Figure 7E ) was developed by Huang et al.67 which allowed the real-time NO imaging using inverted fluorescence microscope in human umbilical vein endothelial cells, Sf9 cells and PC12 cells when treated with L-arginine. Thus, this combination of the probe with inverted fluorescence microscope could provide selective and sensitive detection of NO released from cells. A hepatocyte targeting fluorescent sensor (Gal-RhB) was developed by Zhang et al.68 in 2018 ( Figure 8C ). The cellular and in vivo imaging using HepG2 and Zebrafish using this sensor allowed detecting NO about 1.26 nm with good selectivity and sensitivity making it an obvious choice for liver diseases caused due to NO deficiency or surplus amount.

Overall, each type of fluorescent probe has its own limitations and advantages. DAN being highly specific and sensitive shows cytotoxic effects, has slow reaction time,71 requires acidic conditions, not suitable for NO imaging and may damage living cells.2 DAR has high photostability and pH independence being unsuitable for in vivo applications.71 Copper based fluorescent probes have proved to be beneficial owing to its cell membrane permeability, detection of NO under physiological conditions, independence to oxygen allowing imaging under hypoxic conditions without interference.2 However, they are poorly stable in vivo, do not allow in vivo imaging due to suboptimal emission wavelength and may cause potential toxic effects. MNIP-Cu fluorescence is independent of oxygen, and simple synthesis method allows detection of NO in whole tissues, cellular and sub-cellular levels in plants.62

E LECTROCHEMICAL M ETHOD

Use of various electrodes for electrochemical detection of NO in vitro and in vivo is widely employed. The choice of electrode is of great importance for detection of NO since the electrochemical process takes place at its surface and its quality influences the charge transfer process between target analyst and electrode material. Carbon and noble metal electrodes are most commonly used for NO detection. Other types of materials used for making the electrodes are described in Table 1 and Figure 9 .72 Efforts are being made continuously by researchers to modify electrode for better measurement of NO in various settings. This section will focus on such electrodes and their application for NO detection.

Table 1

Types of basic electrodes