In this study, we present an evaluation of a chemiluminescence resonance energy transfer (CRET) system based on the commercial Bluestar (R) formulation, aimed at enhancing the detection of trace bloodstains. In the CRET mechanism, the excited-state 3-aminophthalate, generated during the luminol chemiluminescence reaction, transfers its energy non-radiatively to ground-state fluorescein, which subsequently emits in the green spectral region. Fluorescein was identified as the most effective acceptor owing to its high fluorescence quantum yield and low cost. Compared with conventional luminol emission, the Bluestar (R)-fluorescein system exhibited a pronounced spectral shift from blue (similar to 447 nm) to green (similar to 530 nm), resulting in enhanced visual contrast and improved detectability. Quantitatively, camera-based analysis demonstrated up to a 257 % increase in apparent signal intensity, whereas photomultiplier tube (PMT) measurements showed a more moderate enhancement, underlining the critical influence of detector spectral sensitivity. Importantly, the green emission region is more sensitively perceived by the human eye and digital cameras, which translated into significantly lower detection limits for synthetic bloodstains. This work highlights not only the potential of CRET for improving forensic chemiluminescence assays but also emphasizes the practical role of detector characteristics in determining system sensitivity.

Benzo[c]phenanthridine alkaloids are known for their stabilizing effects on non-canonical DNA structures, particularly G-quadruplexes (G4s). In this study, the interaction of fagaronine, a rare benzo[c]phenanthridine alkaloid, with several DNA structures (including B-DNA, parallel, antiparallel and hybrid G4s) is studied using molecular fluorescence and circular dichroism (CD) spectroscopy. It has been found that fagaronine significantly enhances the stability of all tested G4 conformations. Furthermore, a study by NMR spectroscopy provided valuable information on the mechanism of interaction of the ligand with the parallel G4 structure adopted by Pu22T14T23, a sequence mutated with respect to that found within the promoter region of the c-myc gene. Remarkably, when compared with data reported in the literature, fagaronine appears to exhibit one of the strongest G4 thermal stabilization effects ever recorded for a small ligand.

Water, ubiquitous in analytical methods, is renowned for its fluorescence quenching properties, influencing techniques like fluorescence spectrophotometry or techniques with fluorescence detection. This study explores the impact of water (H₂O) substitution for heavy water (D₂O) on the fluorescence behavior of anthraquinones and anthracyclines. Anthraquinones and anthracyclines play crucial roles in pharmacy, serving as essential components in various therapeutic formulations, particularly in cancer treatment and other pharmacological interventions. Capillary electrophoresis (CE) with heavy water as the background electrolyte (BGE) solvent offers superior sensitivity to the separation and detection of these analytes. Experimental results demonstrate the improved detection limits and separation efficiency of selected anthraquinones rhein (RH), aloe-emodin (AE), and anthracyclines doxorubicin (DOX), epirubicin (EPI) and daunorubicine (DAU) in heavy water-based buffers, highlighting the potential of heavy water in advancing analytical chemistry.

The fluorescence signal increase in deuterated water - D2O is relevant to many methods used in chemical and biological research. We performed fluorescence spectroscopy measurements combined with evaluations of fluorescence-based methods to describe and quantify the practical impacts of D2O on chemical and biological samples. Several novel features are observed in fluorescence spectra and life-time decay curves of molecules including fluorescence probes and labels that are used frequently in biomolecular labelling and bioimaging. The observed characteristics suggest that D2O enhances the fluorescence signal due to the exchange of labile hydrogen on a fluorophore for heavier deuteron and the reduction of energy transfer from excited fluorophores to H2O molecules. Furthermore, performed quantifications approve that D2O increases significantly the detection signal of all examined fluorescence-based approaches where the brightness of fluorophores and sample viability are crucial.

There is an increasing interest in the study of guanine or cytosine-rich sequences that may fold into G-quadruplex (G4) or i-motif (iM) structures showing a short hairpin (or stem-loop) stabilized by Watson-Crick base pairs. These hybrid spatial arrangements may be target of ligands that have been shown to interact strongly with BDNA. In this work, the interaction of the palmatine alkaloid with several sequences forming different G4s, iMs, and hybrid structures has been studied by means of spectroscopic and separation techniques, as well as multivariate data analysis methods. At the experimental conditions used in this work, the results have shown that this ligand strongly stabilizes parallel G4 structures, whereas a weaker interaction was observed with the antiparallel G4 adopted by the thrombin-binding aptamer or iMs. The presence of hairpins within the loops scarcely affects the affinity of this ligand for the hybrid G4/duplex or iM/duplex structures. Fluorescence measurements have provided evidence of a certain interaction with iMs at pH 5.1, despite the absence of thermal stabilization effects.

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

The alkaloid berberine presents many biological activities related to its potential to bind DNA structures, such as duplex or G-quadruplex. Recently, it has been proposed that berberine may interact with i-motif structures formed from the folding of cytosine-rich sequences. In the present work, the interaction of this alkaloid with the i-motif formed by the human telomere cytosine-rich sequence, as well as with several positive and negative controls, has been studied. Molecular fluorescence and circular dichroism spectroscopies, as well as nuclear magnetic resonance spectrometry and competitive dialysis, have been used with this purpose. The results shown here reveal that the interaction of berberine with this i-motif is weak, mostly electrostatics in nature and takes place with bases not involved in C·C+ base pairs. Moreover, this ligand is not selective for i-motif structures, as binds equally to both, folded structure, and unfolded strand, without producing any stabilization of the i-motif. As a conclusion, the development of analytical methods based on the interaction of fluorescent ligands, such as berberine, with i-motif structures should consider the thermodynamic aspects related with the interaction, as well as the selectivity of the proposed ligands with different DNA structures, including unfolded strands.

Tetracycline compounds present a broad spectrum of antibiotics widely used in medicine. Since their discovery they have been used in the treatment of human diseases as well as in veterinary applications. However, their overuse in food-producing animals may express an adverse effect on human health and even induce selective resistance. Selected antibiotics from the class of tetracyclines have been analyzed using liquid chromatography with fluorescence detection (LC-FLU) and LC with mass spectrometric detection (LC-MS) on a core-shell C18 column. As majority systems nowadays rely on reversed-phase columns with water being the main component of the mobile phase, a simple replacement of H 2 O with D 2 O in the mobile phase enhanced the sensitivity for selected compounds by 10–200%. MS was used to unambiguously identify the separated compounds and to locate the labile hydrogen sites. Another way to increase the fluorescence intensity lied in production of complex compounds of the tetracycline molecules and Mg 2+ ions. Such simple yet effective approaches improved limits of detection and sensitivity and may be selectively exploited (on demand) in the LC-FLU analysis of these compounds.

Many fluorescent molecules can be quenched by OH oscillators present in water. The Increase of fluorescence intensity or lifetime can be obtained by replacing the light water for deuterium oxide. We can take advantage of this phenomena in some analytical techniques coupled to the fluorescence detection (e.g. capillary electrophoresis, high performance liquid chromatography, flow cytometry, fluorescence microscopy, etc.). The capillary electrophoresis coupled with laser-induced fluorescence detection can be used for the determination of anthracyclines and related compounds which are widely used drugs, especially in cancer treatment. The steady-state and time-resolved fluorescence measurements of chosen anthracyclines in light and heavy water were carried out on the spectrofluorometer. Those experiments have proved that the emission signal and lifetimes of anthracyclines can be increased up to 4.5 times using deuterium water as a solvent. Therefore the deuterated water-based buffers can be used for the detection of anthracyclines by the capillary electrophoresis with LIF. The CE-LIF separation of chosen molecules was successfully done in borate buffers prepared in light and heavy water. The fluorescence signal in D2O based buffer increased up to 4.4 times for rhein. Around 2 times higher fluorescence emission signal was recorded also for other studied anthracyclines (doxorubicin, daunorubicin, epirubicin). The calculated limit of detection for rhein was 5.1·10-8 mol.l-1 and 5.0·10-9 mol.l-1 in light and heavy water buffer respectively. Over one order better limit of detection is not caused only by reducing of the dynamic quenching but also lower noise level achieved in the analysis in D2O buffer. The main advantage of capillary electrophoresis is the high sensitivity combined with the lower consumption of sample and background electrolyte (about hundreds of microliters) which means that only small volume of heavy water is needed for the analysis.

The G-quadruplex DNA is commonly present in several protooncogenic-DNA promoters and participates in many important biological processes such as replication, transcription, and translation. Because of their supposed role in cancer, G-quadruplex DNA is often studied as a target for anti-cancer drugs. Quaternary protoberberine and tetrahydroprotoberberine alkaloids (corysamine, coptisine, stylopine), which are supposed to selectively bind these structures, have been compared in terms of stability with selected types of DNA. Influence of selected alkaloids on the stability of double-stranded DNA and non-canonical forms of DNA was observed by determining association constants of alkaloid–DNA complexes using spectroscopic methods—molecular absorption spectrometry, fluorescence spectrometry, and mass spectrometry. Furthermore, the effect of given alkaloids on the melting temperature of these DNA structures was determined using CD spectrometry. Competitive dialysis and electrospray mass spectrometry were performed for affinity comparison of certain alkaloids to different DNA structures including G-quadruplexes. These experiments have proven that corysamine and coptisine prefer interaction with G-quadruplexes in comparison to dsDNA and ssDNA, whereas tetrahydroprotoberberine alkaloid stylopine does not interact with any DNA whatsoever.

Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and selectivity. As majority systems rely on reversed-phase columns with water being the main component of the mobile phase, fluorescent compounds with emission maxima higher than 500 nm might be dynamically quenched. A simple replacement of H2O with D2O enhanced the sensitivity for selected compounds by 10-200%. Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally occurring forms of vitamin B-2. Similar levels of enhancement were obtained by fluorescence spectrometry. Such simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.

Sanguinarine is a benzo[c]phenanthridine alkaloid with interesting cytotoxic properties, such as induction of oxidative DNA damage and very rapid apoptosis, which is not mediated by p53-dependent signaling. It has been previously documented that sanguinarine is reduced with NADH even in absence of any enzymes while being converted to its dihydro form. We found that the dark blue fluorescent species, observed during sanguinarine reduction with NADH and misinterpreted by Matkar et al. (Arch. Biochem. Biophys. 2008, 477, 43-52) as an anionic form of the alkaloid, is a covalent adduct formed by the interaction of NADH and sanguinarine. The covalent adduct is then converted slowly to the products, dihydrosanguinarine and NAD+, in the second step of reduction. The product of the reduction, dihydrosanguinarine, was continually re-oxidized by the atmospheric oxygen back to sanguinarine, resulting in further reacting with NADH and eventually depleting all NADH molecules. The ability of sanguinarine to diminish the pool of NADH and NADPH is further considered when explaining the sanguinarine-induced apoptosis in living cells. (C) 2017 Elsevier Ltd. All rights reserved.

Fluorescent molecules with emission maxima above 500 nm are often dynamically quenched by OH oscillators present in water molecules. We have changed H2O for D2O to obtain higher fluorescence intensity signal. Our experiments carried out on spectrofluorometer have proved that anthracyclines can achieve about 300 % higher response in D2O as solvent. The affect can be employed in various analytical methods including HPLC and capillary electrophoresis coupled to fluorescence detection.

Molecular structures of two compounds obtained in reactions of alumazene [DippNAlMe]3 (1, Dipp = 2,6-i-Pr2C6H3) with substituted quinolinols have been elucidated by the single-crystal X-ray diffraction analysis. Quinolin-8-ol (Hq) provides a dinuclear complex [(DippNH)2Al2Me2(q)2] (2) with a central Al2O2 ring and five-coordinate Al atoms. The compound 2×THF crystallizes in the orthorhombic Pbca space group. The molecular structure of a mononuclear complex [(DippNH)Al(Meq)2] (3) obtained in the reaction of 1 with 2-methylquinolin-8-ol (HMeq) possesses a five-coordinate Al center. The structure was solved in a triclinic cell P-1. The dinuclear complex 2 can be considered as a model product of alcohol addition on the formal Al-N double bond, while the mononuclear complex 3 represents subsequent reaction of remaining Al-Me moiety with proton. Both complexes 2 and 3 are highly luminescent showing emission around 500 nm in solid state.

In this work, the interaction of six natural benzo[c]phenanthridine alkaloids (macarpine, sanguilutine, sanguirubine, chelerythrine, sanguinarine and chelirubine) with parallel and antiparallel G-quadruplex DNA structures was studied. HT22 corresponding to the end of human telomeres and the modified promoter oncogene c-kit21 and Pu22 sequences have been used. Spectroscopically-monitored melting experiments and fluorescence titrations, competitive dialysis and nuclear magnetic resonance spectroscopy were used for this purpose. The results showed that these alkaloids stabilized G-quadruplex structures in terms of increments of Tm values (from 15 to 25 °C) with high selectivity over duplexes and unfolded DNA. The mode of binding was mainly by stacking on the terminal G-tetrads with stoichiometries of 1 : 2 (DNA : ligand). The presence of non-specific electrostatic interactions was also observed. Overall, the results pointed to a strong stabilization of G-quadruplex structures by these alkaloids.

Water molecules (H2O) often reduce luminescence lifetimes of various luminescence probes. The change of lifetime is usually caused by dynamic luminescence quenching induced by O-H oscillators which effectively take away energy from excited molecule. The process can be described by Stern-Volmer equation. We have studied selected luminescence systems where it is possible to detect considerable changes of lifetime in presence/absence of H2O and D2O in this work for analytical purposes. We have tested both, inorganic (Ln(3+)) and organic compounds using three different instrumentation in order to find the largest change between tau(H) and tau(D). The Ln(3+) containing systems have shown considerable increase/decrease of lifetimes in the presence/absence of D2O (Eu3+ : tau(D)/tau(H) = 34.5) whereas organic systems gave significantly lower values of tau(D)/tau(H) (coumarin 123 lifetime ratio, tau(D)/tau(H) = 1.94). The calculated LOD varied from 0.04 mol l(-1) (samarium nitrate) to 6.55 mol l(-1) (riboflavin).

Eisenia lucens is an earthworm living in the organic soil layer of decomposing wood. When irritated, the worm expels coelomic fluid through pores in its body wall, exhibiting blue-green bioluminescence. The mechanism of the bioluminescence, which seems to be different from other bioluminescence systems of terrestrial animals, has been studied in this work. Many lines of evidence indicate that riboflavin stored in coelomycetes plays an important role in this glowing reaction.

Depending on the Cu-I halide, ligand to metal ratio, and reaction conditions, complexes with various central CuX clusters can be prepared. A series of reactions of Cu-I halides with P-III containing ligands was carried out in different molar ratios in different solvents, yielding new Cu-I complexes, which were characterized by P-31 NMR and X-ray diffraction. The P-III containing ligands were based on tertiary phosphines carrying at least one organic functional group (e.g. CN, COOH) on aryl substituents. Luminescence in the complexes and its dependence on various conditions (i.e. solvent, temperature, etc.) was studied.