Electrochemical Detection of Gasotransmitters: Status and Roadmap.

Gasotransmitters, including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), are a class of gaseous, endogenous signaling molecules that interact with one another in the regulation of critical cardiovascular, immune, and neurological processes. The development of analytical sensing mechanisms for gasotransmitters, especially multianalyte mechanisms, holds vast importance and constitutes a growing area of study. This review provides an overview of electrochemical sensing mechanisms with an emphasis on opportunities in multianalyte sensing. Electrochemical methods demonstrate good sensitivity, adequate selectivity, and the most well-developed potential for the multianalyte detection of gasotransmitters. Future research will likely address challenges with sensor stability and biocompatibility (i.e., sensor lifetime and cytotoxicity), sensor miniaturization, and multianalyte detection in biological settings.

Nitric Oxide Releasing Delivery Platforms: Design, Detection, Biomedical Applications, and Future Possibilities.

Gasotransmitters belong to the subfamily of endogenous gaseous signaling molecules, which find a wide range of biomedical applications. Among the various gasotransmitters, nitric oxide (NO) has an enormous effect on the cardiovascular system. Apart from this, NO showed a pivotal role in neurological, respiratory, and immunological systems. Moreover, the paradoxical concentration-dependent activities make this gaseous signaling molecule more interesting. The gaseous NO has negligible stability in physiological conditions (37 °C, pH 7.4), which restricts their potential therapeutic applications. To overcome this issue, various NO delivering carriers were reported so far. Unfortunately, most of these NO donors have low stability, short half-life, or low NO payload. Herein, we review the synthesis of NO delivering motifs, development of macromolecular NO donors, their advantages/disadvantages, and biological applications. Various NO detection analytical techniques are discussed briefly, and finally, a viewpoint about the design of polymeric NO donors with improved physicochemical characteristics is predicted.

FRET Modulated Signaling: A Versatile Strategy to Construct Photoelectrochemical Microsensors for In Vivo Analysis.

Microelectrode-based electrochemical (EC) and photoelectrochemical (PEC) sensors are promising candidates for in vivo analysis of biologically important chemicals. However, limited selectivity in complicated biological systems and poor adaptability to electrochemically non-active species restrained their applications. Herein, we propose the concept of modulating the PEC output by a fluorescence resonance energy transfer (FRET) process. The emission of energy donor was dependent on the concentration of target SO2 , which in turn served as the modulator of the photocurrent signal of the photoactive material. The employment of optical modulation circumvented the problem of selectivity, and the as-fabricated PEC microelectrode showed good stability and reproducibility in vivo. It can monitor fluctuations of SO2 levels in brains of rat models of cerebral ischemia-reperfusion and febrile seizure. More significantly, such a FRET modulated signaling strategy can be extended to diverse analytes.

Development of a new ratiometric probe with near-infrared fluorescence and a large Stokes shift for detection of gasotransmitter CO in living cells.

A near-infrared (NIR) ratiometric fluorescent probe, NIR-Ratio-CO, was developed for rapid detection of carbon monoxide (CO) in both solution and living cells through the strategy of Pd0-mediated Tsuji-Trost reaction. This probe shows a rapid, highly specific and sensitive detection process for CO, accompanied by colorimetric and distinct ratiometric fluorescence changes at 655 and 592 nm with a large Stokes shift up to 195 nm. The detection limit for CO was measured to be about 61 nM by the fluorescence method. In addition, this probe was successfully applied for ratiometric imaging of both exogenous and endogenous CO in living cells, indicating that it can be used as a novel tool for ratiometric fluorescent detection of CO in living systems.

Iterative synthetic strategies and gene deletant experiments enable the first identification of polysulfides in Saccharomyces cerevisiae.

New evidence on the role of H2S as a gasotransmitter suggests that the true signalling effectors are polysulfides. Both oxidized polysulfides and hydropolysulfides were synthesized and their presence in S. cerevisiae was observed for the first time. A single gene-deletant approach allowed observation of the modulation of polysulfide species and levels.

Fluorescent nanoprobes for the sensing of gasotransmitters hydrogen sulfide (H2S), nitric oxide (NO) and carbon monoxide (CO).

Fluorescent nanomaterials as sensing probes have experienced immense growth in recent years due to the intrinsic optical and physicochemical properties, high sensitivity, specificity, targeting ability, and suitability for medicinal applications. The fluorescent detection of gaseous signaling molecules, such as Hydrogen sulfide (H2S), nitric oxide (NO) and carbon monoxide (CO) are very important due to their potential therapeutic application. This review intends to provide the recent progress in the detection of H2S, CO and NO via fluorescent based nano probes. These probes work based on different mechanisms such as fluorescence enhancement and quenching, also defined as "turn-on" and "turn-off" responses respectively. It could be achieved through PET, FRET or ratiometric methods. In this article, we have discussed about a variety of fluorescent nanoprobes of QDS, CDs, AuNPs and UCNPS, working on the fluorescent sensing mechanisms and applicable for the detection of H2S, CO and NO in biological and environmental samples. Methods used for the detection, structural features of nanomaterials, type of fluorescence response observed, fluorescence sensing mechanism and their sensitivity are highlighted.

A Genetically Encoded, Ratiometric Fluorescent Biosensor for Hydrogen Sulfide.

As an important gasotransmitter, hydrogen sulfide (H2S) plays crucial roles in cell signaling. Incorporation of p-azidophenylalanine ( pAzF) into fluorescent proteins (FPs) via genetic code expansion has been a successful strategy in developing intensity-based, genetically encoded fluorescent biosensors for H2S. To extend this strategy for ratiometric measurement which eliminates many detection uncertainties via self-calibration at two wavelengths, we modified the chromophore of a circularly permutated, superfolder green fluorescent protein (cpsGFP) with pAzF to derive cpsGFP- pAzF, which subsequently served as a Förster resonance energy transfer (FRET) acceptor to EBFP2, an enhanced blue fluorescent protein. The resultant construct, namely, hsFRET, is the first ratiometric, genetically encoded fluorescent biosensor for H2S. Both in vitro and in mammalian cells, H2S reduces the azido functional group of hsFRET to amine, leading to an increase of FRET from EBFP2 to cpsGFP. Our results collectively demonstrated that hsFRET could be used to selectively and ratiometrically monitor H2S.

Immobilization of nano Cu-MOFs with polydopamine coating for adaptable gasotransmitter generation and copper ion delivery on cardiovascular stents.

In-stent restenosis is worsened by thrombosis, acute inflammation, and uncontrollable smooth muscle cells (SMCs) proliferation at the early stage of implantation. Tailoring the stent surface can inhibit thrombosis, intimal hyperplasia, and accelerate re-endothelialization. In situ nitric oxide (NO) generation is considered as a promising method to improve anti-coagulation and anti-hyperplasia abilities. Copper based metal organic frameworks showed great potential as catalysts for NO generation, and copper ion (Cu2+) was demonstrated to promote endothelial cells (ECs) growth. Herein, by using polydopamine as the linker and coating matrix, nanoscale copper-based metal organic frameworks (nano Cu-MOFs) were immobilized onto the titanium surface for simultaneous nitric oxide (NO) catalytic generation and Cu2+ delivery. The nano Cu-MOFs-immobilized coating exhibited desirable NO release and adaptable Cu2+ delivery. Such coating inhibited platelet aggregation and activation via NO-cGMP signaling pathway, and significantly reduced thrombosis in an ex vivo extracorporeal circulation model. NO release and Cu2+ delivery showed synergetic effect to promote EC proliferation. Moreover, SMCs and macrophage proliferation was suppressed by the nano Cu-MOFs-immobilized coating, thereby reducing neointimal hyperplasia in vivo. Overall, this biocompatible coating is convenient for the surface modification of cardiovascular stents and effectively prevents the late stent thrombosis and in-stent restenosis associated with stent implantation.

The detection and quantification, in vivo and in real time, of hydrogen sulfide in ethanol-induced lesions in rat stomachs using an ion sensitive electrode.

INTRODUCTION: The development of electrochemical sensors for the detection of small molecules has already had a significant effect on the study of biology because of their selectivity and ability to measure low concentrations of small molecules that regulate various functions in living organisms. Hydrogen sulfide (H2S) is a gasotransmitter produced at low levels in several tissues including the stomach. Here, we propose a new method for detecting low concentrations of this transmitter in the rat stomach, in-vivo and in real time, with applications in pharmacology and physiology. METHODS: Wistar rats fasted for 12h. Then, the control group was given an intragastrical dose of saline. l-Cysteine (50mg/kg) or dl-propargylglycine (50mg/kg) were administered to the test groups to modify the H2S levels. Ranitidine (50mg/kg), omeprazole (40mg/kg) or carbenoxolone (30mg/kg) were used as reference anti-ulcer drugs. Thirty minutes later, the electrode was inserted in the middle of the stomach cavity of the anesthetized animals. The basal levels of H2S were recorded every 5min for 30min. Next, gastric lesions were induced with pure ethanol, and the recording continued for 30 additional minutes. RESULTS: The exogenous administration of an H2S precursor (l-cysteine) increased the level of this gasotransmitter whereas dl-propargylglycine, a selective inhibitor of the enzyme cystathionine γ lyase, reduced the total concentration of H2S. The administration of carbenoxolone, a gastroprotective, increased the total amount of H2S. However, the administration of the anti-secretors omeprazole and ranitidine did not modify the total concentration of H2S. DISCUSSION: This work provides the basis for a real-time analysis of the changes in-vivo of the gasotransmitter H2S in the normal and injured stomach and the exploration of the effect of drugs on the regulation of H2S.

Molecular engineering of a mitochondrial-targeting two-photon in and near-infrared out fluorescent probe for gaseous signal molecules H2S in deep tissue bioimaging.

Hydrogen sulfide (H2S), one of the biologically important gaseous signal molecules, plays an essential role in diverse normal biochemical functions and pathological processes. Herein, an efficient two-photon in and near-infrared out mitochondria-targeting dye has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (CË­C) of 4-hydroxybenzaldehyde and 6-(diethylamino)-1,2,3,4-tetrahydroxanthylium (mitochondria-targeting), which possesses large two-photon action absorption cross-section ~160g and high fluorescence quantum yield ~0.15. Encouraged by the results, we proceeded to conjugate this new dye with a H2S recognition moiety (4-dinitrobenzene-ether, DNB), on the basis of the intramolecular charge transfer (ICT) strategy, to construct a novel H2S fluorescent probe (TP-NIR-HS), which shows a targeting ability with high sensitivity and selectivity, and low cytotoxicity. This new probe was then applied for two-photon imaging of living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ~350µm, thus demonstrating its practical application in biological systems, and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of H2S.

Structural effects of naphthalimide-based fluorescent sensor for hydrogen sulfide and imaging in live zebrafish.

Hydrogen sulfide (H2S) is an important biological messenger, but few biologically-compatible methods are available for its detection in aqueous solution. Herein, we report a highly water-soluble naphthalimide-based fluorescent probe (L1), which is a highly versatile building unit that absorbs and emits at long wavelengths and is selective for hydrogen sulfide over cysteine, glutathione, and other reactive sulfur, nitrogen, and oxygen species in aqueous solution. We describe turn-on fluorescent probes based on azide group reduction on the fluorogenic 'naphthalene' moiety to fluorescent amines and intracellular hydrogen sulfide detection without the use of an organic solvent. L1 and L2 were synthetically modified to functional groups with comparable solubility on the N-imide site, showing a marked change in turn-on fluorescent intensity in response to hydrogen sulfide in both PBS buffer and living cells. The probes were readily employed to assess intracellular hydrogen sulfide level changes by imaging endogenous hydrogen sulfide signal in RAW264.7 cells incubated with L1 and L2. Expanding the use of L1 to complex and heterogeneous biological settings, we successfully visualized hydrogen sulfide detection in the yolk, brain and spinal cord of living zebrafish embryos, thereby providing a powerful approach for live imaging for investigating chemical signaling in complex multicellular systems.

Hydrogen sulfide in posthemorrhagic shock mesenteric lymph drainage alleviates kidney injury in rats.

Posthemorrhagic shock mesenteric lymph (PHSML) is a key factor in multiple organ injury following hemorrhagic shock. We investigated the role of hydrogen sulfide (H2S) in PHSML drainage in alleviating acute kidney injury (AKI) by administering D,L-propargylglycine (PPG) and sodium hydrosulfide hydrate (NaHS) to 12 specific pathogen-free male Wistar rats with PHSML drainage. A hemorrhagic shock model was established in 4 experimental groups: shock, shock+drainage, shock+drainage+PPG (45 mg/kg, 0.5 h prehemorrhage), and shock+drainage+NaHS (28 µmol/kg, 0.5 h prehemorrhage). Fluid resuscitation was performed after 1 h of hypotension, and PHMSL was drained in the last three groups for 3 h after resuscitation. Renal function and histomorphology were assessed along with levels of H2S, cystathionine-γ-lyase (CSE), Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)-α in renal tissue. Hemorrhagic shock induced AKI with increased urea and creatinine levels in plasma and higher H2S, CSE, TLR4, IL-10, IL-12, and TNF-α levels in renal tissue. PHSML drainage significantly reduced urea, creatinine, H2S, CSE, and TNF-α but not TLR4, IL-10, or IL-12. PPG decreased creatinine, H2S, IL-10, and TNF-α levels, but this effect was reversed by NaHS administration. In conclusion, PHSML drainage alleviated AKI following hemorrhagic shock by preventing increases in H2S and H2S-mediated inflammation.

A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform.

A ratiometric fluorescent probe for H2S was developed based on a coumarin- benzopyrylium platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H2S probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (∼1min) towards H2S, which is superior to most of the azide-based H2S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H2S in living cells.

Measurement of NO in biological samples.

Although the physiological regulatory function of the gasotransmitter NO (a diatomic free radical) was discovered decades ago, NO is still in the frontline research in biomedicine. NO has been implicated in a variety of physiological and pathological processes; therefore, pharmacological modulation of NO levels in various tissues may have significant therapeutic value. NO is generated by NOS in most of cell types and by non-enzymatic reactions. Measurement of NO is technically difficult due to its rapid chemical reactions with a wide range of molecules, such as, for example, free radicals, metals, thiols, etc. Therefore, there are still several contradictory findings on the role of NO in different biological processes. In this review, we briefly discuss the major techniques suitable for measurement of NO (electron paramagnetic resonance, electrochemistry, fluorometry) and its derivatives in biological samples (nitrite/nitrate, NOS, cGMP, nitrosothiols) and discuss the advantages and disadvantages of each method. We conclude that to obtain a meaningful insight into the role of NO and NO modulator compounds in physiological or pathological processes, concomitant assessment of NO synthesis, NO content, as well as molecular targets and reaction products of NO is recommended.