Decrease In Fluorescence Emission Intensity Research Articles

Non-covalent interaction between soybean protein isolate and naringenin: Focused on binding mechanism, interface behavior, and functional properties

In this study, non-covalent complexes of soybean protein isolate (SPI)- Naringenin (Nar) with different concentrations of Nar were prepared, and their binding mechanisms and interfacial properties were investigated. The results showed that the content of α-helix and β-sheet in the SPI-Nar complex decreased to 19.62% and 26.58%, respectively. With an increase in Nar concentration, there was an increase in the ultraviolet absorption peak and a decrease in fluorescence emission intensity. Fluorescence quenching analysis revealed static quenching between Nar and SPI, with a binding site of 0.98, ΔG, ΔH, and ΔS had negative values. Molecular docking and molecular dynamics simulations indicated that Nar binds within the hydrophobic cavity of SPI. At a Nar addition amount of 1 mg/mL, the EAI and ESI of the SPI-Nar complex were 91.97 m2/g and 49.28 min, respectively. The foaming capacity and foam stability were 58.0% and 62.07%, respectively, and exhibited optimal air/water interfacial properties. The functional properties of the non-covalent complex were effectively improved, providing a theoretical basis for its application in the food industry.

An ultrasensitive "mix-and-detect" kind of fluorescent biosensor for malaoxon detection using the AChE-ATCh-Ag-GO system.

Malaoxon, a highly toxic metabolite of malathion, can lead to severe harm or death if ingested. This study introduces a rapid and innovative fluorescent biosensor that relies on acetylcholinesterase (AChE) inhibition for detecting malaoxon using Ag-GO nanohybrid. The synthesized nanomaterials (GO, Ag-GO) were evaluated with multiple characterization methods to confirm their elemental composition, morphology, and crystalline structure. The fabricated biosensor works by utilizing AChE to catalyze the substrate acetylthiocholine (ATCh), which generates positively charged thiocholine (TCh) and triggers citrate-coated AgNP aggregation on the GO sheet, leading to an increase in fluorescence emission at 423 nm. However, the presence of malaoxon inhibits the AChE action and reduces the production of TCh, resulting in a decrease in fluorescence emission intensity. This mechanism allows the biosensor to detect a wide range of malaoxon concentrations with excellent linearity and low LOD and LOQ values of 0.001 pM to 1000 pM, 0.9 fM, and 3 fM, respectively. The biosensor also demonstrated superior inhibitory efficacy towards malaoxon compared to other OP pesticides, indicating its resistance to external influences. In practical sample testing, the biosensor displayed recoveries of over 98% with extremely low RSD% values. Based on the results obtained from the study, it can be concluded that the developed biosensor has the potential to be used in various real-world applications for detecting malaoxon in food, and water samples, with high sensitivity, accuracy, and reliability.

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase-type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N-cadherin-mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA-mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase-PCR and DNA sequencing using exon-specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4'-bis-1-anilino naphthalene 8-sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant.

An insight into the binding behavior of graphene oxide and noble metal nanoparticles

The binding behavior of graphene oxide and metal nanoparticles (Au, Pt, and Pd) was observed by UV–Vis spectroscopy, fluorescence spectroscopy, dynamic light scattering, and zeta potential. Hybrids with a fixed concentration of graphene oxide (GO) were prepared with increasing concentration of metal nanoparticles to observe the effect of binding on their spectroscopic properties, size, and zeta potential. An increase in the absorption spectra of GO after binding with nanoparticles and a gradual decrease in fluorescence emission intensity with increasing concentration of nanoparticles was observed, representing their effective binding. Stern–Volmer plots differentiated the quenching constants of these nanoparticles, where Au shows the lowest and Pd shows the highest quenching among these nanoparticles. The initial hybrids showed more size change as compared to hybrids with a higher concentration of nanoparticles, whereas initial hybrids have charge similar to that of GO and gradual increase in the concentration of nanoparticles bring the charge near to the respective charge of nanoparticles. To the best of our understanding, this is the first report of its kind to study the binding interactions of two different moieties by studying changes occurred in the hydrodynamic radius and zeta potential of hybrids by titration experiments, having applications in surface treatment, drug delivery, and as sensors for environmental pollutants or other classes of organic molecules, etc.

Dual-Affinity Ratiometric Quenching (DARQ) Assay for the Quantification of Therapeutic Antibodies in CHO-S Cell Culture Fluids.

More than 100 monoclonal antibodies (mAbs) are in industrial and clinical development to treat myriad diseases. Accurate quantification of mAbs in complex media, derived from industrial and patient samples, is vital to determine production efficiency or pharmacokinetic properties. To date, mAb quantification requires time and labor-intensive assays. Herein, we report a novel dual-affinity ratiometric quenching (DARQ) assay, which combines selective biorecognition and quenching of fluorescence signals for rapid and sensitive quantification of therapeutic monoclonal antibodies (mAbs). The reported assay relies on the affinity complexation of the target mAb by the corresponding antigens and Protein L (PrL, which targets the Fab region of the antibody), respectively, labeled with fluorescein and rhodamine. Within the affinity complex, the mAb acts as a scaffold framing the labeled affinity tags (PrL and antigen) in a molecular proximity that results in ratiometric quenching of their fluorescence emission. Notably, the decrease in fluorescence emission intensity is linearly dependent upon mAb concentration in solution. Control experiments conducted with one affinity tag only, two tags labeled with equal fluorophores, or two tags labeled with fluorophores of discrete absorbance and emission bands exhibited significantly reduced effect. The assay was evaluated in noncompetitive (pure mAb) and competitive conditions (mAb in a Chinese Hamster Ovary (CHO) cell culture harvest). The "DARQ" assay is highly reproducible (coefficient of variation ∼0.8-0.7%) and rapid (5 min), and its sensitivity (∼0.2-0.5 ng·mL-1), limit of detection (75-119 ng·mL-1), and dynamic range (300-1600 ng·mL-1) are independent of the presence of CHO host cell proteins.

A multi-responsive pyranone based Schiff base for the selective, sensitive and competent recognition of copper metal ions

Exploring a new multi-responsive pyranone chemosensor capable of sensing copper ions specifically and selectively through colorimetric, UV–Vis absorption and fluorescence methods is of great importance. In this piece of work, a novel pyranone based Schiff base ligand 4-Hydroxy-6-methyl-3-[1-(2-morpholin-4-yl-ethylimino)-ethyl]-pyran-2-one (DM) was synthesized by the condensation of dehydroacetic acid and 4-(2-aminoethyl) morpholine. The structural determination of ligand DM was executed using distinct spectral techniques i.e.,1H NMR, 13C NMR, FT-IR and HR-MS techniques. The reported Schiff base DM showed an immediate colorimetric change from pale yellow to colorless accompanied by a strong change in the UV–Vis absorption band onto the addition of Cu (II) ions. This metal ligand chelation leads a decrease in ICT process. Also the decrease in fluorescence emission intensity of Schiff base DM with Cu (II) ions addition showed its turn-off behavior towards copper ions. Further absorption/ emission titration studies, Job’s plot, HR-MS and 1H NMR titration data designated 2:1 stoichiometric ratio between DM and Cu (II) ions respectively. Density functional theory studies were also performed to authenticate the binding mechanism theoretically. The sensitivity of Schiff base DM towards Cu (II) ions was applicable at every pH conditions and at the same time DM exhibited selectivity towards Cu (II) ions with a negligible interference of other metal ions. DM showed a detection limit of 7.7 nM towards copper ions via fluorescence emission studies. The best part about DM is that it has good stability but showed an instant chemical reversibility when titrated with EDTA solution.

Changes in strand 6B and helix B during neuroserpin inhibition: Implication in severity of clinical phenotype

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.

Inhibition of H1N1 influenza virus infection by zinc oxide nanoparticles: another emerging application of nanomedicine

BackgroundCurrently available anti-influenza drugs are often associated with limitations such as toxicity and the appearance of drug-resistant strains. Therefore, there is a pressing need for the development of novel, safe and more efficient antiviral agents. In this study, we evaluated the antiviral activity of zinc oxide nanoparticles (ZnO-NPs) and PEGylated zinc oxide nanoparticles against H1N1 influenza virus.MethodsThe nanoparticles were characterized using the inductively coupled plasma mass spectrometry, x-ray diffraction analysis, and electron microscopy. MTT assay was applied to assess the cytotoxicity of the nanoparticles, and anti-influenza activity was determined by TCID50 and quantitative Real-Time PCR assays. To study the inhibitory impact of nanoparticles on the expression of viral antigens, an indirect immunofluorescence assay was also performed.ResultsPost-exposure of influenza virus with PEGylated ZnO-NPs and bare ZnO-NPs at the highest non-toxic concentrations could be led to 2.8 and 1.2 log10 TCID50 reduction in virus titer when compared to the virus control, respectively (P < 0.0001). At the highest non-toxic concentrations, the PEGylated and unPEGylated ZnO-NPs led to inhibition rates of 94.6% and 52.2%, respectively, which were calculated based on the viral loads. There was a substantial decrease in fluorescence emission intensity in viral-infected cell treated with PEGylated ZnO-NPs compared to the positive control.ConclusionsTaken together, our study indicated that PEGylated ZnO-NPs could be a novel, effective, and promising antiviral agent against H1N1 influenza virus infection, and future studies can be designed to explore the exact antiviral mechanism of these nanoparticles.

Fluorescence anisotropy of fluorescein derivative varies according to pH: Lessons for binding studies

Fluorescence spectroscopy is a powerful tool to comprehend macromolecules interaction. The characteristics of the fluorophore as well its behavior under the experimental conditions are central to ascertain the results. The present work characterizes the fluorescence emission and anisotropy of a fluorescein derivative linked to a double strand DNA containing 67 base pairs (named as 6-FAM-67-mer) under different pH conditions. The results show that fluorescence emission and anisotropy of this fluorescein derivative varies according to pH (as the pH becomes more acidic, the total fluorescence emission decreases). The decrease in fluorescence emission intensity is accompanied by an increase in anisotropy. The monoanionic form of fluorescein derivative is the predominant species from pH 7.5 to 9.2. Our main conclusion is that a constant fluorescein derivative emission is a prerequisite to determine association constants between macromolecules.

A novel chromone and rhodamine derivative as fluorescent probe for the detection of Zn(II) and Al(III) based on two different mechanisms

In this study, a novel fluorescent probe, 6‑hydroxychromone‑3‑carbaldehyde‑(rhodamine B carbonyl) hydrazine (L), for Zn2+ and Al3+ was designed and synthesized. Initially, this probe L exhibited inferior fluorescence emission peak centered at 488 nm in EtOH/HEPES solution (3/1, 10.0 μM HEPES, pH 7.4) when excited at 421 nm. After the addition of Zn2+, this probe L displayed excellent selectivity towards Zn2+ with obvious fluorescence color change from colorless to yellow, which might be attributed to the formation of a 1:1 ligand-metal complex resulting in the inhibition of photo-induced electron transfer phenomenon. Whereas, the prepared Zn2+ complex of L could be used as a ratiometric fluorescent probe to detect Al3+ on the basis of fluorescence resonance energy transfer mechanism. This ligand-metal complex of Zn2+ (LZn) showed high selectivity towards Al3+ with obvious enhancement in fluorescence emission intensity at 580 nm and remarkable decrease in fluorescence emission intensity at 488 nm, and the fluorescence color also changed from yellow to pink. Furthermore, the detection limit of the probe L, LZn towards Zn2+, Al3+ were 1.25 × 10−7 M and 3.179 × 10−6 M, respectively. Additionally, the complexation properties of L towards Zn2+ and LZn towards Al3+ were studied in detail.

1,8-Naphthalimide derived dual-functioning fluorescent probe for “turn-off” and ratiometric detection of Cu2+ based on two distinct mechanisms in different concentration ranges

In this study, a novel 1,8-naphthalimide derived compound bearing the coumarin moiety called 7-Diethylaminocoumarin-3-aceone (N-hydroxyethyl-1’,8’-naphthalimide-4’-) hydrazone (1) has been designed, synthesized and evaluated as a dual-functioning fluorescent probe for “turn-off” and ratiometric detection of Cu2+ based on two distinct mechanisms in different concentration ranges in aqueous solution. This probe 1 showed high selectivity and sensitivity for Cu2+ over other common metal ions, for the limit of detection (LOD) could reach 3.90 × 10−8 M and 3.26 × 10−7 M for “turn-off” and ratiometric detection respectively. Moreover, in Cu2+ equivalence range of 0–0.5, a decrease in fluorescence emission intensity at 441 nm was observed upon addition of Cu2+, but in Cu2+ equivalence range of over 0.5, a new emission band centered at 462 nm appeared with increasing intensity accompanied by a decrease in fluorescence emission intensity at 570 nm, which provided ratiometric fluorescence detection of Cu2+. The sensing mechanisms for the responses of 1 to Cu2+ were chelation-quenched fluorescence (CHQF) and chemical reaction between 1 and Cu2+ over Cu2+ equivalence range of 0–0.5 and more than 0.5, respectively. Thus, this probe 1 could be utilized for sensing and monitoring Cu2+ in environmental and biological systems in different concentration ranges.

Monitoring oxidation changes in commercial extra virgin olive oils with fluorescence spectroscopy-based prototype

Maintaining the quality of extra virgin olive oils (EVOOs) in the current globalised market is a key challenge for the olive oil manufacturers. To maintain the quality, one major factor is to resist its oxidation during processing and storage. Resisting the oxidation in EVOOs is a challenging task requiring control of different physical and chemical factors. However, rapid, real-time, and non-destructive monitoring the oxidation in EVOOs could support in maintaining its quality during storage. The present study utilises a novel right-angle fluorescence spectroscopy-based prototype for rapid monitoring of oxidation changes in three different EVOOs varieties (Arbequina, Picual, and Cornicabra) during storage. Furthermore, the effects of light exposure on oxidation of EVOOs were also studied. Two major changes in the fluorescence spectra of EVOO samples were mainly observed: a decrease in fluorescence emission intensity at 671 nm, and an increase in fluorescence intensities at 420, 440, 464, and 515 nm. These two changes were related to a decrease in chlorophyll content and parallel formation of oxidation products. The formation of oxidation products was found to have a linear relationship with the decrease in chlorophyll content. Out of the three, the Cornicabra variety showed the highest rate of oxidation changes. Furthermore, the EVOOs degraded rapidly when get exposed to light. In a fast and non-destructive manner, the fluorescence spectroscopy-based prototype successfully monitored the oxidation changes in the EVOOs.

A Phenylselenium-Substituted BODIPY Fluorescent Turn-off Probe for Fluorescence Imaging of Hydrogen Sulfide in Living Cells.

Herein a phenylselenium-substituted BODIPY (1) fluorescent turn-off sensor was developed for the purpose to achieve excellent selectivity and sensitivity for H2S detection based on the substitution reaction of the phenylselenide group at the 3-position with H2S. The excess addition of hydrogen sulfide promoted further substitution of the phenylselenide group at the 5-position of the probe and was accompanied by a further decrease in fluorescence emission intensity. Sensor 1 demonstrated remarkable performance with 49-fold red color fluorescence intensity decrease at longer excitation wavelength, a low detection limit (0.0025 μM), and specific fluorescent response toward H2S over anions, biothiols, and other amino acids in neutral media. It showed no obvious cell toxicity and good membrane permeability, which was well exploited for intracellular H2S detection and imaging through fluorescence microscopy imaging.

Development of a coumarin-furan conjugate as Zn2 + ratiometric fluorescent probe in ethanol-water system

In this study, a novel coumarin-derived compound bearing the furan moiety called 7-diethylamino-3-formylcoumarin (2′-furan formyl) hydrazone (1) has been designed, synthesized and evaluated as a Zn2+ ratiometric fluorescent probe in ethanol-water system. This probe 1 showed good selectivity and high sensitivity towards Zn2+ over other metal ions investigated, and a decrease in fluorescence emission intensity at 511nm accompanied by an enhancement in fluorescence emission intensity at 520nm of this probe 1 was observed in the presence of Zn2+ in ethanol-water (V : V=9 : 1) solution, which provided ratiometric fluorescence detection of Zn2+. Additionally, the ratiometric fluorescence response of 1 to Zn2+ was nearly completed within 0.5min, which suggested that this probe 1 could be utilized for sensing and monitoring Zn2+ in environmental and biological systems for real-time detection.

A new aza-BODIPY based NIR region colorimetric and fluorescent chemodosimeter for fluoride

The synthesis and characterization of a novel NIR region fluoride sensor, that makes use of the aza-boron-dipyrromethene (aza-BODIPY) fluorophore, is described. An arylmagnesium bromide was formed by reacting 4-bromophenol with a tert-butyldimethylsilyl protecting group and magnesium, which was then used to prepare the aza-BODIPY through a reaction with phthalonitrile. The unusually strong affinity between the fluoride anion and silicon is used to create a sensor dye, which exhibits a highly specific, rapid colorimetric and ‘turn-off’ fluorescence response for F− in solution and in living HeLa cells. With F−, there is enhanced intramolecular charge transfer within the S1 state and this results in efficient nonradiative decay and hence in a marked decrease in fluorescence emission intensity.

N,N-di(2-pyridylmethyl)amino-modified porphyrinato zinc complexes. The “ON–OFF” fluorescence sensor for Fe3+

Two porphyrinato-zinc derivatives modified with two and four N,N-di(2-pyridylmethyl)amino groups, namely Zn-Porphyrin-2-DPA (1) and Zn-Porphyrin-4-DPA (2), have been designed, synthesized, and characterized. Systemic studies over a series of metal ions reveal that binding with Fe3+ for the former compound induces the decrease of fluorescence emission intensity by 37.2%, while for the latter one results in a more remarkable decrease in the fluorescence emission intensity by 90.3%, rendering compound 2 the first example of DPA-modified porphyrin fluorescence ON–OFF sensor for Fe3+.

A BODIPY fluorescent probe with selective response for hypochlorous acid and its application in cell imaging

A boron-dipyrromethene (BODIPY) derivative with a hydroxymethyl group at the meso-position, 1, was synthesized, which exhibits a highly specific and rapid ‘turn-off’ response for HOCl during confocal fluorescence microscopy experiments under physiological conditions and in live cells. The formation of an electron-withdrawing formyl substituent upon oxidation results in highly efficient nonradiative decay of the S1 state and hence in a drastic decrease in fluorescence emission intensity.

Fluoroquinolone Photodegradation Influences Specific Basophil Activation

Fluoroquinolones (FQs) are photoreactive drugs, but it is not known whether laboratory light exposure can influence the induction of photoproducts and modify in vitro test results. The basophil activation test (BAT) has proven to be useful for evaluating immunoglobulin E (IgE)-mediated hypersensitivity to FQs, with a higher percentage of positive responders with ciprofloxacin (CIP) than with moxifloxacin (MOX). We studied the effect of laboratory light on CIP and MOX degradation, and drug-protein conjugate formation, and its influence on the BAT for evaluating IgE-mediated hypersensitivity to FQs. The results showed an important decrease in fluorescence emission intensity under light compared to dark conditions for MOX, and that BAT positivity was lower in light (17.9%) than in dark (35.7%) conditions. No changes were found for CIP in either fluorescence emission intensity or BAT results (46.4% in both conditions). We can conclude that light exposure is a critical factor in BAT results when photolabile drugs like MOX are used. Therefore, light is important when interpreting in vitro results.

Fluorescence spectroscopy accurately detects irreversible cell damage during hepatic radiofrequency ablation

A current limitation of hepatic radiofrequency ablation (RFA) is an inability to detect ablation margins in real time. Thermal injury from RFA alters the biochemical properties governing tissue fluorescence. We hypothesized that the changes in hepatic fluorescence measured during hepatic RFA could be used to detect irreversible hepatocyte damage accurately and to determine ablation margins in real time. RFA was performed on healthy pig livers and monitored in vivo simultaneously for fluorescence and temperature by a fiberoptic micro-interrogation probe connected to a spectroscopy system. Ablations were stopped based on previously established real-time fluorescence spectral data, not based on temperature or time. To determine where in the ablated tissue cell death occurred, biopsies for transmission electron microscopy were taken from 4 areas of 3 specimens: (1) nonablated liver, (2) hemorrhagic zone/normal liver interface, (3) hemorrhagic zone/coagulated zone interface, and (4) coagulated zone. In vitro fluorescence emission intensity was determined at each biopsy site. Peak hepatic fluorescence intensity occurred at 470 nm and decreased as RFA progressed. Transmission electron microscopy evidence of irreversible hepatocyte damage occurred at the interface of the coagulation zone and the hemorrhagic zone and correlated with a 87.5% +/- 9% decrease in fluorescence emission intensity. Tissue fluorescent changes from thermal injury were unaffected by tissue cooling. Fluorescence spectroscopy accurately detected hepatocellular thermal injury from RFA in real time and can detect irreversible cell damage during tissue thermal therapy.

Influence of copper(II) and magnesium(II) ions on the ciprofloxacin binding to DNA

The influence of magnesium(II) and copper(II) ions on the binding of ciprofloxacin to double stranded calf thymus DNA was studied by fluorescence emission spectroscopy, ultraviolet- and circular dichroism (CD) spectroscopy. The interaction of ciprofloxacin and copper(II) ions was followed by strong fluorescence quenching which was almost unaffected by the presence of DNA. On the other hand, only a slight decrease in fluorescence emission intensity, which was enhanced in the presence of DNA, was observed for ciprofloxacin interaction with magnesium(II) ions. Furthermore, magnesium(II) ions increase the thermal stability of the DNA, while, in the presence of ciprofloxacin, the degree of stabilisation is smaller. In contrast, copper(II) ions destabilise double helical DNA to heat, while ciprofloxacin slightly affects only the second transition of the biphasic melting curve of calf thymus DNA. Magnesium(II) ions at 25 °C induce conformational transitions of DNA at concentrations of 1.5 mM and 2.5 M, as monitored by CD. On the other hand copper(II) ions induce only one conformational transition, at a concentration of 12.7 μM. At higher concentrations of copper(II) ions ( c>700 μM) DNA starts to precipitate. Significant changes in the CD spectra of DNA were observed after addition of ciprofloxacin to a solution containing DNA and copper(II) ions, but not to DNA and magnesium(II) ions. Based on our spectroscopic results, we propose that copper(II) ions are not directly involved into ciprofloxacin binding to DNA via phosphate groups as it has been suggested for magnesium(II) ions.