Fluorogenic Methods Research Articles

Developing new Hydrazine Carbothioamide Molecules for Selective Colorimetric/Fluorometric Detection of Environmentally Essential Hg2+ and Ag+ Metal Ions in Mixed Aqueous Media.

A novel colorimetric and fluorogenic probe L based on hydrazine carbothioamide and 1,8-naphthalimide moieties has been designed and synthesized for the hypersensitive detection of Hg2+ or Ag+ ions. The observed probe L showed colorimetric and fluorometric responses for these studies when Hg2+ or Ag+ was added to the DMSO - HEPES buffer solution (pH = 7). An interference test with other metal ions was determined, and the high selectivity of Hg2+ and Ag+ did not interfere with other metal ions in colorimetric and fluorogenic methods. The possible mechanism of binding of these metal ions and the probe L 1:1 complex was determined by H1 NMR. Additionally, the reversibility of the affinity of probe L with mercury (Hg2+) and silver (Ag+) ions was investigated by adding Na2EDTA. The naked eye detected the "Off-On" type fluorescence sensor in the presence of Hg2+ and EDTA. The tested test strip kits provided a strong probability of probe L with high response and rapid, sensitive detection with Hg2+ ion, which may be suitable for practical use.

Chromene-chromene Schiff base as a fluorescent chemosensor for Th4+ and its application in bioimaging of Caenorhabditis elegans

The manuscript presents the synthesis of a new di-chromene Schiff base (COM-CH) by combining 7-(diethylamino)-2-oxo-2H-chromene-3-carbohydrazide and 4-oxo-4H-chromene-3-carbaldehyde, and its characterization using various analytical techniques. The probe COM-CH functional group contains a hard donor atom that selectively complexes with Th4+ ions. This report investigated COM-CH's sensing ability towards Th4+ chromogenic and fluorogenic methods in ACN: H2O (8:2, v/v) with Th4+ ions. The COM-CH-Th4+ complex was excited at 430 nm, resulting in a bright emission band at 475 nm with a 45 nm Stokes shift. The COM-CH probe demonstrated the highest performance at pH 4.0 to 8.0, with a sensitivity of 18.7 nM. The complex formation of COM-CH with Th4+ was investigated using NMR, FTIR spectrometry, and density functional theory calculations. The COM-CH and Th4+ are bound with 2:1 stoichiometry and an association constant of 1.92 × 108 M−2. The probe's performance enabled the analysis of monazite sand and water samples for Th4+ content. The probe successfully detected Th4+ content in Caenorhabditis elegans, marking the first Th4+ detection in animal models.

A Minireview of Recent Reported Carboxylesterase Fluorescent Probes: Design and Biological Applications

AbstractAs the most important members of serine esterase hydrolase superfamily, mammalian carboxylesterases (CESs) are widely distributed in various tissues like liver, intestine, lung and kidney. Many physiological processes require the hydrolysis of CES with a variety of endogenous and exogenous ester drugs and environmental poisons. So, developing the methods that can detect CESs activity is of great significance. In last decades, huge breakthrough has been made in the development of specific detection technologies including chromatography, quantitative reverse transcription polymerase chain reaction (qRT‐PCR), proteomics and western blotting. However, these traditional methods for CESs detection have little effect on the spatiotemporal distribution and in situ activity of CES in living cells and in vivo. Compared with that, fluorogenic methods have been developed for real‐time monitoring CESs in biological environments. In this review, we summarized the recent reported CESs probes. The designing strategies as well as their applications are reviewed. We hope this review will inspire the development of novel specific CESs fluorescent probes.

Chromogenic and Fluorogenic Probes for the Detection of Illicit Drugs.

The consumption of illicit drugs has increased exponentially in recent years and has become a problem that worries both governments and international institutions. The rapid emergence of new compounds, their easy access, the low levels at which these substances are able to produce an effect, and their short time of permanence in the organism make it necessary to develop highly rapid, easy, sensitive, and selective methods for their detection. Currently, the most widely used methods for drug detection are based on techniques that require large measurement times, the use of sophisticated equipment, and qualified personnel. Chromo‐ and fluorogenic methods are an alternative to those classical procedures.

Chemodosimeter for Selective and Sensitive Chromogenic and Fluorogenic Detection of Mustard Gas for Real Time Analysis.

Since the first use of chemical warfare agents (CWA) (1915) to the recent attacks in Syria (2017) on mankind, there have been many incidents where CWA have claimed thousands of lives and left many more contaminated. In order to provide the appropriate and immediate medical counter measure to the victims, the exact classification of these chemical agents within few minutes on the field itself using a rapid and simple detection technique is extremely important to save the lives of the effected people. This has motivated all of us to explore the novel strategies/detection systems that can be field deployable with better selectivity and greater sensitivity. In view of this, we present a novel chemosensor, 3,6-bis(dimethylamino)-9(10H)-acridine thione (1), that can detect mustard gas and its simulant by both chromogenic and fluorogenic methods. For the first time, a single probe was able to demonstrate the detection with unprecedented selectivity over most probable interferences (nerve agents and alkylating agents) including solvents, acids, and bases which are routinely present in the environment. The desired level of sensitivity by naked eyes (0.04 mg/mL), UV spectroscopy (0.02 mg/mL), and fluorescence spectroscopy (0.005 mg/mL) makes this method truly field deployable. For the spot detection on the affected areas, a handy and potable chemosensor kit was also fabricated. This paper provides a simple, highly specific, and easy to use method in "actual sense" that not only detects the agents in the solution phase but also in the contaminated samples.

In Vivo Visualization and Function Probing of Transport mRNPs Using Injected FIT Probes.

Fluorogenic hybridization methods, such as the use of FIT probes, enable the in vivo detection of specific mRNAs transcribed from their endogenous, genetically nonmodified loci. Here, we describe the design, synthesis and injection of nuclease resistant FIT probes into developing Drosophila oocytes to detect endogenous localizing mRNAs as wells as to probe function of structural RNA elements.

A novel nitroreductase-enhanced MRI contrast agent and its potential application in bacterial imaging

Nitroreductases (NTRs) are known to be able to metabolize nitro-substituted compounds in the presence of reduced nicotinamide adenine dinucleotide (NADH) as an electron donor. NTRs are present in a wide range of bacterial genera and, to a lesser extent, in eukaryotes hypoxic tumour cells and tumorous tissues, which makes it an appropriate biomarker for an imaging target to detect the hypoxic status of cancer cells and potential bacterial infections. To evaluate the specific activation level of NTR, great efforts have been devoted to the development of fluorescent probes to detect NTR activities using fluorogenic methods to probe its behaviour in a cellular context; however, NTR-responsive MRI contrast agents are still by far underexplored. In this study, para-nitrobenzyl substituted T1-weighted magnetic resonance imaging (MRI) contrast agent Gd-DOTA-PNB (probe 1) has been designed and explored for the possible detection of NTR. Our experimental results show that probe 1 could serve as an MRI-enhanced contrast agent for monitoring NTR activity. The in vitro response and mechanism of the NTR catalysed reduction of probe 1 have been investigated through LC–MS and MRI. Para-nitrobenzyl substituted probe 1 was catalytically reduced by NTR to the intermediate para-aminobenzyl substituted probe which then underwent a rearrangement elimination reaction to Gd-DOTA, generating the enhanced T1-weighted MR imaging. Further, LC–MS and MRI studies of living Escherichia coli have confirmed the NTR activity detection ability of probe 1 at a cellular level. This method may potentially be used for the diagnosis of bacterial infections.

Recognition and fluorescent sensing of zinc ions using organic fluorophores-based sensor molecules

Fluorogenic methods in conjunction with suitable probes have emerged as preferable modalities for the monitoring and measurement of trace metal ions. The number of new Zn2+-responsive fluorescent chemosensors has increased substantially in recent years, as a consequence of biologically and environmental importance of zinc ions, comprising organic fluorophore derivatives, biological functional materials and so on. In this review, we highlight recent advances made in the development of Zn2+ fluorescent sensors. This paper focuses on reviewing sensor molecules based on organic fluorophore derivatives, involving quinoline, BODIPY, coumarin, and so forth. On the basis of current research results, novel fluorescent chemosensors for zinc ions derived from organic fluorophores will be synthesized to increase the sensitivity, water solubility, and bio-compatibility of the sensor for in vivo analysis.

A Colorimetric and “Turn-On” Fluorescent Chemosensor for Metal Ion Based on Rhodamine Derivative

The recognition and sensing of biologically and environmentally important species has emerged as a significant goal in the field of chemical sensors in recent years1. Fluorogenic methods in conjunction with suitable probes are preferable approaches for the measurement of these analytes because fluorimetry is rapidly performed, is nondestructive, is highly sensitive, is suitable for high-throughput screening applications2,3. We synthesized rhodamine derivatives compounds by a Schiff base reaction.

Poly-l-Lysine Compacts DNA, Kills Bacteria, and Improves Protease Inhibition in Cystic Fibrosis Sputum

Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. Poly-L-lysine may be an alternative to dornase-α to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

Enzymatic Product-Mediated Stabilization of CdS Quantum Dots Produced In Situ: Application for Detection of Reduced Glutathione, NADPH, and Glutathione Reductase Activity

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported.

Abstract 1135: Bioluminescent methods to investigate cellular metabolic status: NAD(P)/NAD(P)H levels, redox potential, and hydrogen peroxide concentration

Abstract Cancer is a disease defined by uncontrolled cell growth where cellular energy metabolism pathways must evolve for tumors to survive and proliferate. NAD(P) and NAD(P)H play a major role in oxidative phosphorylation and their role in aerobic glycolysis is of great interest. Additionally, NAD(P)/NAD(P)H act as co-factors for enzymes involved in cancer pathogenesis through regulation of chromatin structure, DNA repair, and transcription (e.g. sirtuins and poly(ADP-ribose) polymerase). The study of how NAD(P)/NAD(P)H are generated and utilized during adaptive cancer cell energy metabolism would benefit from the development of a rapid, sensitive, and homogeneous assay to measure the level of these nucleotides. We developed a bioluminescent assay for measuring NAD(P)/NAD(P)H that can detect ≤ 0.1 µM NADH and has a 1000-fold dynamic range. The assay is well suited for high throughput screening (Z’ = 0.92, S/B = 95) and has been used to screen the LOPAC library. By coupling other reactions with NAD(P)/NAD(P)H measurement, we analyzed the levels of metabolites and the activity of enzymes, including isocitrate dehydrogenase and pyruvate dehydrogenase, in enzyme preparations and crude cell lysates. This method is applicable for measuring cellular NAD and NADH levels directly from cell culture without further sample handling. This assay is based on a novel proluciferin derivative that is processed in vitro through an enzymatic reaction. The released luciferin is detected in a coupled luciferin/luciferase reaction and the luminescent signal is correlated with the amount of NAD(P)/NAD(P)H present in the sample. As an extension of this approach, similar proluciferin chemistries were developed to detect the reducing potential of metabolically active cells. This robust luminescent assay can detect the reducing potential of less than 100 cells/well in 96-well format and distinguish small changes in cell number. Multiple cell lines were treated with cancer therapeutic compounds and the performance of the proluciferin approach was compared to commonly used colorimetric (e.g. MTT, MTS, and XTT) and fluorogenic (e.g. resazurin) methods. The proluciferin assay resulted in comparable pharmacological responses, significantly increased signal window, and superior sensitivity. Additionally, the proluciferin-based approach has been extended to other metabolic readouts. For example, novel proluciferin substrates were developed and applied to the detection of hydrogen peroxide in cells and in biological samples through a HRP-independent process. Bioluminescent assays provide greater sensitivity and dynamic range than most fluorescent or colorimetric assays, and are better suited for high throughput screening. Applying these assays to the study of cancer cell energy metabolism will provide a significant advantage over existing methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1135. doi:1538-7445.AM2012-1135

Glycosidase activity profiling for bacterial identification by a chemical proteomics approach

Genome sequencing projects are suggesting there are dozens of glycosidase sequences that could be used to fingerprint cell types and serve as starting points for biocatalyst discovery. Herein, we present a simple chemical proteomics approach to profile intracellular glycosidase activities of three different bacterial cell extracts using a synthetic α- and β-linked library of 18 representative substrates with electrospray ionization-mass spectrometry (ESI-MS) reaction monitoring. Three target bacteria – Escherichia coli K12, Bacillus cereus and Pseudomonas aeruginosa – can be easily differentiated by this method. Compared with traditional chromogenic and fluorogenic methods to profile bacterial enzyme activities individually, this MS-based method can detect multiple enzyme activities in one reaction and easily highlight activity differences between whole cell extracts.

The contribution of α2‐macroglobulin thrombin to the endogenous thrombin potential

Ignjatovic et al (2007) were right to stress the importance of α2-macroglobulin-bound thrombin (α2M-thrombin) in the interpretation of thrombin generation curves, particularly under conditions where α2M concentrations are likely to differ widely from those in the normal adult population, such as in neonates. When fluorogenic methods are used, such as that of Varadi et al (2003), that do not correct for substrate consumption and for the quenching of fluorescence by formed product (the inner filter effect) no steady end-level amidolytic activity because of formed α2M-thrombin can be calculated. Consequently, no correction is possible for the contribution of that complex to the calculated thrombin concentrations. Under those circumstances the α2M content of the plasma contributes significantly to the outcome of the measured parameters. However, using the calibrated automated thrombin (CAT) method (Hemker et al, 2003), the α2M-thrombin end level can be, and is, calculated. The algorithm that is used to calculate the curve of free thrombin (and hence also the endogenous thrombin potential; ETP) does not assume a ‘likely contribution of α2M-thrombin to the ETP’, as stated by Ignjatovic et al (2007), but is assessed from the above mentioned steady end-level of amidolytic activity. At high plasmatic α2M concentrations a high end level is found and more amidolytic activity is attributed to the α2M-thrombin complex than at low plasmatic α2M levels. α2M-thrombin levels are subtracted from the calculated thrombin concentrations and hence do not contribute to the ETP and peak value of thrombin. The measured ETP therefore is independent of the plasma α2M level, except in so far as α2M contributes to the over-all anti-thrombin action of the plasma. For the underlying assumptions and the mathematical details the reader is referred to our articles as presented by Ignjatovic et al (2007). In fact, the algorithm is the same in the fluorogenic CAT method as in the subsampling methods and continuous chromogenic methods that we used and validated earlier (Hemker & Beguin, 1995). As an aside, we would like to note that fibrinogen strongly interferes with the reaction between α2M and thrombin (Hemker et al, 2003). Because the experiments reported by Ignjatovic et al (2007) were performed with defibrinated plasma, it is likely that their experimental contributions of α2M-thrombin were 3–5 times higher than in their patients.

Enzymatic activities of three ectomycorrhizal types of Quercus robur L. in relation to tree decline and thinning

In a declining oak forest, a thinning treatment was performed in August 2004, targeting trees belonging to two decline classes. The whole ectomycorrhizal (EM) community was dominated by the fungal symbionts Clavulina cinerea, Tomentella sublilacina and Russula spp. The potential activities of eight secreted enzymes, involved in mobilizing nutrients (N, P) from soil organic matter, were measured on these three EM types in winter and spring 2006 using multiwell microplate photometric and fluorogenic methods. The enzymatic activities recorded in winter were generally significantly higher than in spring. Most of the enzyme activities studied, and particularly phosphatase and β-glucosidase, changed according to both decline class and sylvicultural treatment. In spring, each anatomotype displayed different enzymatic profile according to the decline class. These results suggest that the potential enzymatic activity of ectomycorrhizae adapts to the changes resulting from the sylvicultural treatment and reacts to the anthropic disturbance by adjusting to the new resource structure.

Luminogenic cytochrome P450 assays

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-GloTM (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

Detection of hormone receptor ligands in yeast by fluorogenic methods

Yeast-based bioassays are becoming widespread tools for detection and quantification of ligands for vertebrate nuclear hormone receptors, including estrogens, progestans, androgens and dioxin-like compounds, both for agonistic and for antagonistic effects. These systems rely on the monitoring of transcription rates of reporter genes whose expression in yeast depends on binding of receptors to their natural or xenobiotic ligands. Among the different methods to quantify reporter gene transcription, those based on fluorescence detection are fast, very sensitive and reproducible. We propose a fast method for ligand detection for different vertebrate receptors in yeast, based on the use of fluorogenic substrates for the widely used reporter β-galactosidase gene. In this method, β-galactosidase activity is calculated from kinetic data, rather than from end-point measurements, which increases accuracy and facilitates the statistical analysis of the data. It also provides statistically rigorous procedures to distinguish between active and inactive compounds and to evaluate the fitness of the data to alternative models of dose/response mechanisms. All these features combined configure a flexible, fast (less than three hours for some systems) and reproducible method to evaluate the presence of potential endocrine disruptors in the environment.

Assessment of lysosomal function by quantitative histochemical and cytochemical methods

Quantitative histochemistry and cytochemistry enables a direct link to be made between metabolic functions such as the activity of lysosomal enzymes and the morphology of a tissue or a type of cell. Several approaches exist such as microchemistry based on (bio)chemical analysis of a single cell or a small piece of tissue dissected from a freeze-dried section. This technique has been routinely used for prenatal diagnosis of inherited enzyme defects and especially of lysosomal storage diseases. Other approaches are cytofluorometry or cytophotometry, which are based on the principle that a fluorescent or coloured final reaction product is precipitated at the site of the enzyme. The amount of final reaction product is analysed per cell or per unit volume of tissue using either a microscope cytofluorometer or flow cytometer for fluorescence measurements or an image analysing system or scanning and integrating cytophotometer for absorbance measurements. In principle, fluorescence methods are to be preferred over chromogenic methods because they are more sensitive and enable multiparameter analysis. However, only a limited number of fluorogenic methods are at hand that give a final reaction product which is sufficiently water-insoluble to guarantee good localisation. The best results have been obtained with methods based on naphthol AS-TR derivatives and with methods for the demonstration of protease activity using methoxynaphthylamine derivatives as substrates and 5'-nitrosalicylaldehyde as coupling reagent. Chromogenic methods are far better with respect to localisation properties and, therefore, most commonly used for quantitative histochemical analysis of lysosomal enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated fluorogenic methods for the evaluation of the extrinsic coagulation reactions in human plasma

Highly sensitive automated methods for the evaluation of the extrinsic coagulation reactions in human plasma were developed by the combination of fluorogenic peptide substrate(MCA) for thrombin and a centrifugal autoanalyzer (Cobas Bio). Prothrombin time (PT) was measured by the reaction time to reach 0.1 relative fluorescence which was caused by the action of thrombin generated after the activation of 3 μl plasma with human placental tissue factor (Thromborel S). Factors X and VII contents in plasma were measured by the same method after mixing diluted plasma with each factor deficient plasma, tissue factor, calcium and MCA in which 10–800 % of each factor was quantitatively measured. Prothrombin content in plasma was quantitated by measuring thrombin activity after the activation with human activated Factor X in the presence of phospholipid and calcium in which 10 –160 % of prothrombin was measured. By the application of these fluorogenic methods to the patients with cardiovascular diseases, it was demonstrated that these methods are highy sensitive not only to hypocoagulable state, but also to hypercoagulable state, particularly to the increase of Factors X and VII concentrations in plasma.

Rapid identification of Escherichia coli with a fluorogenic beta-glucuronidase assay.

We evaluated three fluorogenic methods (MUG; Remel, Lenexa, KS) for the rapid (less than 1 hr) identification of E. coli by detecting beta-glucuronidase activity. The methods included: direct disk inoculation test, tube test, and liquid spot reagent test. Fluorogenic tests were performed on pure cultures of lactose fermenters and compared with identification by Enterotube II (Roche Diagnostics; Nutley, NJ). Organisms yielding disparate results were further analyzed by API 20E (Analytab Products Inc.; Plainview, NY). The tube test was evaluated with isolates subcultured on both MacConkey and blood agars; the direct disk and liquid spot reagent methods were tested with isolates subcultured on blood agar only. All methods were highly specific (greater than 97%). Sensitivity of the beta-glucuronidase tests were method and media dependent, but exceeded 85% in all cases. The direct disk and tube tests also permitted detection of indole formation; results of indole testing, however, contributed little to accurately identifying E. coli.