Synthesis and Evaluation of a Schiff-Based Fluorescent Chemosensors for the Selective and Sensitive Detection of Cu2+ in Aqueous Media with Fluorescence Off-On Responses
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Copper being an essential nutrient; also pose a risk for human health in excessive amount. A simple and convenient method for the detection of trace amount of copper was employed using an optical probe R1 based on Schiff base. The probe was synthesized by Schiff base condensation of benzyl amine and 2-hydroxy-1-napthaldehyde and characterized by single X-ray diffraction, 1H NMR and FTIR. By screening its fluorescence response in a mixture of DMSO and H2O (20:80, v/v) R1 displayed a pronounced enhancement in fluorescence only upon treatment with copper. Other examined metal ions such as alkali, alkaline and transition had no influence. Within a wide pH range 5–12 R1 could selectively detect copper by interrupting ICT mechanism that results in CHEF. From Job’s plot analysis a 2:1 binding stoichiometry was revealed. The fluorescence response was linear in the range 1–10 × 10−9 M with detection limit 30 × 10−9 M. Association constant was determined as 1 × 1011 M−2 by Benesi-Hilderbrand plot. As a fast responsive probe it possesses good reproducibility and was employed for detection of copper in different water samples.
KeywordsFluorescent chemosensor Schiff base Copper CHEF Real-time detection
In the last few years, the development of fast, cheap and efficient fluorescent chemosensors with high sensitivity and selectivity for the detection of heavy and transition metal ions such as Cu2+, Cd2+, Pb2+, and Hg2+ has attracted wide-spread interests of chemists, environmentalists, clinical biochemists and biologists because of their fundamental role in biological, environmental and medicinal application [1, 2, 3, 4, 5, 6].
Fluorescent chemosensors typically consist of two parts: ionophore (metal chelator) and fluorophore (signaling unit). The two parts are interconnected through a proper spacer. Ionophore is required for analyte binding and as a result of metal binding, photophysical properties of the fluorophore such as fluorescence intensity, lifetime of the excited state or absorption wavelength changes, achieving the purpose of identification [7, 8, 9].
Among the metal ions, after zinc and iron, copper is the third most abundant vital trace element for life that plays an important role in various fundamental physiological and biological processes in organisms . It is also a toxic chemical that is not biodegradable and bioaccumulative and can accumulate in food chain or human through uptake or consumption and may be hazardous to human health or the environment . Copper is present in sediments, natural water, and in the other medium such as air and soil. Industrial effluent are the potential source of copper contamination such as waste water from industries manufacturing fungicides, fertilizers, bactericides, algaecides, electronic goods, copper plumbing, as well as the use of copper as a mordant for textile dyes, as an activator in froth floatation of sulfide ores and in plating by products. Natural sources also contribute to the contamination of copper that include sea spray, forest fires, decaying vegetation, volcanoes and windblown. [12, 13, 14]. Due to these extensive industrial applications copper can be a significant environmental pollutant of worldwide concern .
Copper is also an essential micronutrient in the human body. Because of its redox-active nature it acts as an essential cofactor of many enzymes such as tyrosinase, cytochrome c oxidase and superoxide dismutase . It is necessary for the development of bone, nerve coverings, and connective tissue .
Copper exist in three common oxidation states Cu2+ (cupric ion), Cu+ (cuprous ion) and Cu0 (metal). Among the three species the most commonly occurring and toxic to living organism is the cupric ion. Copper possess high affinity for the ligand containing sulfur and nitrogen donors thus it effects enzymes whose activities depend on amino and sulhydryl groups . Due to its toxicity at cellular level, its distribution and homeostasis is highly controlled by various factors including chaperones and copper transport proteins. Abnormal level of copper can cause various problems and disorders. For example, under overloading condition copper can cause disorders such as allergies, migraine headaches, anxiety, depression, anorexia, premenstrual syndrome, fatigue, kidneys and blood problems, Wilson’s, Parkinson’s, Alzheimer’s and prion diseases in humans and its deficiency is associated with myelopathy. In living cells uncontrolled reaction of copper ion with oxygen result in the production of ROS (reactive oxygen species) that can damage proteins, nucleic acid and lipids Excessive absorption of Cu2+ also effects the growth of plants including fewer leaves, shortening of root length and decrease in the plants biomass [19, 20, 21, 22]. According to World Health Organization (WHO), the permissible limit of copper in drinking water is 2 ppm (30 μM) or 1.5 mg/L [23, 24]. In view of such toxic effects of copper, efficient detection of trace amount of copper in environmental water samples is very necessary. For these reasons, great effort has been dedicated to design fluorescent chemosensors for specific recognition and detection of copper.
Chelation enhanced fluorescent type chemosensor are more sensitive to metal ions as compared to Chelation enhanced quenching chemosensors. Cu2+ is known as a fluorescence quencher and most of the fluorescent chemosensors reported for copper so far, detection of copper occur through a fluorescence quenching process that undergoes a charge or energy transfer mechanism and “turn-on” fluorescent sensors for recognition of copper are still rare. Thus, there is a great demand to develop “turn-on” fluorescent chemosensors for specific and sensitive detection of copper [25, 26, 27].
Schiff base derivatives have attracted much attention as an optical chemosensor for the detection of the metal ions because of their structural flexibility, excellent applicability, high selectivity, high efficiency and simplicity of synthetic mode. Schiff base compounds form strong complexes with transition metal ions. For the Schiff base to be used as a fluorescent sensor, the presence of a strong fluorophore is required. Schiff base compounds incorporating π- conjugated fluorescent fragment are extensively used as fluorescent chemosensor for the metal ions [28, 29, 30, 31].
It was within our interests to design a CHEF type Schiff-base fluorescent sensor for selective detection of copper ion. Therefore we designed and develop a new fluorescent probe for copper ion named as (E)-1 ((benzylimino)methyl)naphthalen-2-ol (R1). The synthesis was simple, single step the product was highly selective towards copper ion. R1 exhibited very weak fluorescence due to ICT mechanism which was inhibited upon interaction with copper ion and enhancement of fluorescence was observed. The proposed fluorescent chemosensors contain two parts naphthalene group which is the signaling moiety acting as a fluorophore and imine and hydroxyl group are the binding sites acting as a receptor. We select 2-Hydroxynaphthalene as a fluorophore because of its cheap cost, good binding sites, competitive stability in the environment and characteristic photophysical properties such as low fluorescence quantum yield and short fluorescence lifetime [32, 33].
Materials and Instrumentation
2-hydroxy-1-napthaldehyde, Benzyl amine, Dimethyl sulfoxide (DMSO), Hydrochloric acid 37%, Sodium hydroxide, Acetone, and salts of Cu2+, Na+, Mg2+, K+, Zn2+, Mn2+, Hg2+, Ba2+, Pb2+, Ni2+, Cd2+, Ni2+, Co2+, As3+, Fe3+ and Ce3+ were purchased from commercial sources and were used without further purification. Throughout all experiments distilled water was used. pH adjustments were made by using HCl or NaOH. To minimize the release of cations in the solution and their sorption, the glassware’s were first washed with acid and then rinsed with distilled water. For fluorometric determination of Cu2+ optically four sides’ clear quartz cuvettes were used and were washed with acetone before use.
The 1H NMR spectra of the samples were obtained on Bruker Avance 400 MHz. spectrometer. For FTIR spectra samples were prepared as KBr pallets and the spectrum was obtained on FTIR spectrophotometer Pretige 21 Shimadzu Japan in the region 400–4000 cm−1. X-ray structure analysis of the sample was conducted using Bruker kappa APEXIICCD diffractometer. For the measurement of melting points a Bicote Stuart-SMP 10 Japan was used. UV–vis absorption spectroscopy measurements were conducted on UV-visible 1800 spectrophotometer. The emission spectra were monitored using fluorescence spectrophotometer RF 5301 PC Shimadzu Japan equipped with fluorescence free quartz cuvettes of 1 cm path length. Slit width were 5 nm both for excitation and emission and a steady state 150 W Xenon lamp was used as an excitation source. The pH of the test solution was monitored by a BANTE instrument pH meter. All measurements were conducted at room temperature.
Synthesis of Chemosensor R1
Melting point was determined in open mouth capillary and uncorrected. For spectroscopic investigation analytical reagent grade DMSO and distilled water were used. Fluorescence and absorption spectra of the chemosensor R1 was recorded in DMSO:H20 (20:80) at room temperature. Fluorescence-sensing property of the chemosensor R1 towards metal ions was conducted using sulphate salt of Cu2+, Mg2+, Zn2+, Mn2+; nitrate salt of Pb2+, Ba2+, Cd2+, K+, Ce3+, Hg2+; bromide salt of Co2+; chloride salt of Na+, Ni2+, Fe3+ and oxide of As3+. For pH adjustment HCL and NaOH were used. From the fluorescence intensity data association constant was determined by Benesi-Hilderbrand Plot. The excitation and emission wavelength were 320 and 645 nm respectively. All the measurements were conducted at room temperature.
Results and Discussion
Synthesis and Characterization
Characterization by FTIR
FTIR spectral frequencies for chemosensor R1
IR-band (C=N) cm−1
IR-band (C-OH) cm−1
IR-band aliphatic (-C-O) cm−1
IR-band aromatic (-C-H) cm−1
IR-band aromatic (C=C) cm−1
In the region 400–4000 cm−1 several absorption bands were observed in the FTIR spectrum of the chemosensor R1. Assignments of characteristics bands correspond to various functional groups present in the chemosensor were made by comparison method. The FTIR spectrum confirms the formation of imine bond (-C=N) and no band assigned to (C=O) was detected. Thus, the disappearance of carbonyl (-C=O) peak in the region of 1700 cm−1 and appearance of the (-C=N) peak in the region of 1614 cm−1 confirms the formation of chemosensor R1. The band at 3035 cm−1 corresponds to the stretching vibration of aromatic-C-H. The spectrum exhibit absorption band at 1492, 1435 and at 1296, 1207 cm−1 typically of aromatic-C=C and phenolic-O-H stretching vibration. The bands at 1138 and 1038 cm−1 correspond to aliphatic-C-O [36, 37, 38].
Characterization by Single Crystal X-Ray Diffraction
Crystallographic data and details of refinements for chemosensor R1
Empirical formula, Formula weight
Crystal system, Space group
a, b, c, (Å)
25.391 (4), 6.5797 (11), 8.1779 (13)
Yellow, crystal size (0.42 × 0.30 × 0.26)
Diffractometer, scan mode
Bruker kappa APEXIICCD, Multi-scan and ωscan
No of measured, independent and observed [I ≥ 2σ(I)] reflections
5051, 2746, 1772
(sin θ/λ)max (Å−1)
R [F2 > 2σ(F2)], wR (F2), S
0.050, 0.126, 0.96
No. of reflections
No. of restraints
H-atom parameter constrained
Δ ρmax, Δρmin (e Å−3)
Flack x determined using 598 quotients [(I+)-(I-)]/[(I+) + (I-)] (Parsons, Flack and Wagner, Acta Cryst. B69 (2013) 249–259)
Absolute structure parameter
Characterization by 1H NMR
Preparation of Stock and Working Solutions of Metals and Chemosensor R1
For practical utilization and for detection of toxic metal ions in water samples it is very necessary for a chemosensor to work under aqueous condition. Chemosensor R1 was not soluble in pure water. Therefore 0.001 M stock solution of chemosensor R1 was prepared in pure DMSO. The chemosensor R1 was stable in DMSO. Working solutions were prepared by dilution of a stock solution in DMSO with a mixture of DMSO:H2O (20:80). Stock solutions of metal ions (0.01 M) were prepared in distilled water. Before spectroscopic analysis, test solutions were freshly prepared by appropriate dilution of stock solution to the corresponding desired concentration.
Metal Ion Binding Study by Fluorescence Spectroscopy
Fluorogenic Detection of Cu2+
The detection limit of chemosensor R1 as a fluorescent sensor for the recognition of Cu2+ ion was determined from the plot of fluorescence emission intensity as a function of Cu2+ ion concentration. The detection limit was calculated based on 3σ/k , where σ is the standard deviation of blank measurement and, and k is the slope of the plot. To determine σ, the fluorescence emission intensity of chemosensor R1 was measured six times independently in the absence of copper ions. The detection limit calculated was found to be 30 × 10−9 M which is far below the WHO acceptable limit (30 μM or 1.5 mg/L of Cu2+) in drinking water. The detection limit of the proposed chemosensor R1 is lower than the fluorescent chemosensors of ref. [11, 42, 43, 44, 45]. The detection limit was sufficiently low to recognize Cu2+ in nano molar range. In term of the detection limit and linear range it can be seen that the proposed sensor displays more sensitivity and selectivity for Cu2+ ion by fluorescence spectra.
Determination of Binding Stoichiometry and Association Constant
Effect of Chemosensor R1 Concentration on Fluorescence Response
Effect of Time
Effect of pH
Metal Ion Competition Experiment
Comparison with Pervious Works
Most of the chemosensor required rigorous testing media and most of them displayed quenching of fluorescence upon interaction with Cu2+. Our proposed chemosensor R1 presents a number of attractive analytical features such as one step synthesis, high selectivity for Cu2+, sensitivity, enhancement of fluorescence, good reproducibility, wide pH range and can be used for the rapid detection of Cu2+ in natural water samples.
Reversibility of Chemosensor R1
Application to the Analysis of Natural Water Samples
In conclusion, we have developed a Schiff based fluorescent chemosensor for Cu2+ ion and it was characterized by FTIR and single X-ray diffraction and 1H NMR. Significant fluorescence enhancement was observed by this sensor only in the presence of Cu2+ ion. The enhancement was due to the restriction of ICT phenomenon. The optimal pH range for detection of Cu2+ ion was 5–12. Our proposed chemosensor R1 possess high selectivity, sensitivity, and fast response time towards Cu2+ ion over a large number of competing metal ions. The excellent low limit of detection of this chemosensor R1 towards Cu2+ ion can be useful in detection of trace amount of Cu2+ ion in environmental samples. The response was reversible and the given sensor was successfully applied for the detection of Cu2+ in natural water sample.
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