A ratiometric and two-photon fluorescent probe for imaging hydrogen peroxide in living cells

As an important reactive oxygen species (ROS), hydrogen peroxide plays a significant role in the life activity system, and its abnormal levels are closely related to many diseases. Developing effective fluorescent probes for detecting hydrogen peroxide is very urgent. Therefore, we constructed a probe Z that can detect hydrogen peroxide in ratio. It has naphthimide as the fluorophore and phenylboronic acid pinacol esters as the recognition group. It shows higher sensitivity, lower detection limit, higher selectivity, and broad pH applicability. Moreover, probe Z has low cytotoxicity that can be used to detect exogenous hydrogen peroxide in HeLa cells and might be a potential tool for studying hydrogen peroxide in physiological activities.     

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system--an active site-directed reaction

Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer, C.E. and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 +/- 7 and 4 +/- 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 +/- 0.14 min-1. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.     

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system — an active site-directed reaction

Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer C.E. and Etson D. (1969) J. Biol. Chem. 244, 5333–5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 ± 7 and 4 ± 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 ± 0.14 min⁻¹. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.     

Near-infrared pH-activatable fluorescent probes for imaging primary and metastatic breast tumors

Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic arginine-glycine-aspartic acid (cRGD) peptide targeting α(v)β(3) integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibits high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pK(a) of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.     

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

ABSTRACT: BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H₂O₂ 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196–3.941?mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8?μM, respectively. The kinetics of the reaction between DBBF and H₂O₂ was also studied.     

鲜切加工加速荸荠组织衰老与H2O2累积的关系

以荸荠为材料,研究了鲜切加工加速组织衰老与活性氧代谢的关系.结果表明:鲜切加工提高了荸荠切片抗氧化酶(超氧化物歧化酶、抗坏血酸-过氧化物酶和过氧化氢酶)的活性;但同时明显刺激了O2-产生,促进了H2O2累积,加速了抗坏血酸在贮藏后期的损失,加强了膜脂过氧化作用和增加了电解质渗出率.统计分析表明H2O2含量、丙二醛含量、电解质渗出率三者之间存在正相关性.H2O2组织定位结果也证实鲜切加速组织衰老与H2O2累积密切相关.完整荸荠组织O2-产生比较平稳,抗氧化酶活性维持稳定,H2O2未有明显累积.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

An enzyme-chromogenic surface plasmon resonance biosensor probe for hydrogen peroxide determination using a modified Trinder's reagent

An absorption-based surface plasmon resonance (SPR(Abs)) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder's reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (lambda(max)=630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C(13)H(20)NNaO(4)S.H(2)O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm(-1) and was directly applied to the SPR(Abs) biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPR(Abs) biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPR(Abs) biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r=0.991, six points, average of relative standard deviation; 0.152%, n=3) with a detection limit of 0.5mM. To examine the applicability of this SPR(Abs) biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPR(Abs) biosensor probe employing the modified Trinder's reagent demonstrated applicability to other analyte biosensing tools.     

双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。     

Immobilization of a trienzymatic system in a sol-gel matrix: a new fluorescent biosensor for xanthine

In this work we report the development of a highly sensitive fluorescent multienzymatic biosensor for quantitative xanthine detection. This biosensor is built by the simultaneous encapsulation of three enzymes, xanthine oxidase, superoxide dismutase and peroxidase, in a single sol-gel matrix coupled to the Amplex Red probe. The sol-gel chemistry yields a porous, optically transparent matrix that retains the natural conformation and the reactivity of the three co-immobilized proteins. Xanthine determination is based on a sequence of reactions, namely catalytic oxidation of xanthine to uric acid and superoxide radical, and subsequent catalytic dismutation of the radical, resulting in the formation of hydrogen peroxide, which reacts stoichiometrically with non-fluorescent Amplex Red to produce highly fluorescent resorufin. The optimal operational conditions for the biosensor were investigated. Linearity was observed for xanthine concentrations up to 3.5 microM, with a detection limit of 20 nM, which largely improved the sensitivity of the current xanthine biosensors. The developed biosensor is reusable and remains stable for 2 weeks under adequate storage conditions.     

Methylene blue-based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe triggered by H2S

A new Methylene blue–based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe 3, 7-bis-dimethylamino-10-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-10H-phenothiazine (leuco-MB-NBD) was designed and synthesized. Leuco-MB-NBD showed high sensitivity and selectivity for H₂S as a fluorescent probe in C₂H₅OH-PBS (9:1, v/v, pH = 7.4) solution, this fluorescent assay showed a linear range of 0–50.0 μM and a LOD (limit of detection) of 0.43 μM. Moreover, the probe leuco-MB-NBD has lower toxicity at low concentrations to HCT-116 cells and can be used for cell imaging. Additionally, Leuco-MB-NBD is triggered by hydrogen sulfide to generate methylene blue, methylene blue which has potential rescuing effects on the mitochondrial activity can act as an antidote against sulfide intoxication.     

Chemiluminescence of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II)

The 3‐aminophthalic acid anion is a light emitter in luminol chemiluminescence. In the present study, the chemiluminescence of the 3‐aminophthalic acid anion itself in the presence of hydrogen peroxide–cobalt (II) was studied. The results indicated that 3‐aminophthalic acid anion is highly chemiluminescent in the typical hydrogen peroxide–cobalt (II) system. The peak wavelength of this chemiluminescence and the kinetic profile of the 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) reaction showed similarity with that of luminol, but the chemiluminescence of 3‐aminophthalic acid anion had a much lower background signal. In addition, the chemiluminescence mechanism of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) was also discussed and speculated as the interaction between 3‐aminophthalic acid anion and singlet oxygen.     

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Dendritic chain reaction: Responsive release of hydrogen peroxide upon generation and enzymatic oxidation of methanol

Signal amplification dramatically increases the sensitivity of diagnostic methods. Recently, we introduced a new technique for signal amplification that uses a distinctive dendritic chain reaction (DCR) to generate exponential evolution of a diagnostic signal. In this report, we demonstrate how the modular design of our DCR probe can be used to improve the detection sensitivity. We synthesized a new probe based on a methyl carbonate linkage, which has superior stability in aqueous media. Triggered release of methanol, which was oxidized by alcohol oxidase present in the solution, produced hydrogen peroxide that used as a reagent in the DCR amplification technique. The new probe exhibited higher sensitivity in detection of hydrogen peroxide than our previously reported probe.     

A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging

There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.     

Antioxidant action on the level of reactive oxygen species in normal and transformed fibroblasts

The level of reactive oxygen species (ROS) in normal (3T3) and transformed (3T3-SV40) murine fibroblasts treated with antioxidants for 15 min was studied using a carboxy-H₂DCFDA fluorescent probe. It was shown that N-acetylcysteine (NAC) decreased the ROS level in both cell types. Antioxidant alpha-lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) caused a pro-oxidant effect. ALA and DHLA in the concentration range from 0.1–1.25 mM increased the ROS level in a dose-dependent manner in both cell types. The ability of ALA and DHLA to activate hydrogen peroxide production is discussed.     

Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), hydrogen peroxide (H₂O₂), and peroxymonocarbonate (HOOCO₂⁻) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid ( MpC-BA ) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO⁻ or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO⁻ to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one ( MpC-OH ). However, peroxynitrite-specific product ( MpC-H ) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl ( MpC-OHCl ). H₂O₂ slowly oxidizes MpC-BA . However, the addition of NaHCO₃ increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.