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Ratiometric fluorescent paper sensor utilizing hybrid carbon dotsquantum dots for the visual determination of copper ions Received 00th January 20xx, Accepted 00th January 20xx
Yahui Wang,†abc Cheng Zhang,†abc Xiaochun Chen,ab Bo Yang,abc Liang Yang,abc Changlong Jiang*abc and Zhongping Zhang*abc
DOI: 10.1039/x0xx00000x www.rsc.org/
A simple and effective ratiometric fluorescence nanosensor for selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the aminofunctionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybid carbon dots-quantum dots probe on a microporous membrane, which provides a convenient and simple approach for visual detection of Cu2+. Therefore, the as-synthesized probe shows great potential application for the determination of Cu2+ in real samples.
Introduction Copper, as one of the heavy metal ions and the essential trace 1,2 elements for many living organisms, plays an important role in some physiological and pathological progress, such as bone formation, cellular respiration, as well as serving as a significant catalytic cofactor for the synthesis of hemoglobin, 3 elastin and collagen. The deficiency of copper can lead to many diseases such as anemia, pancytopenia and bone 4 2+ abnormalities. Nevertheless, an excess Cu concentration may not only become toxic to living organisms and induce damage to the liver, kidneys and the central nervous system (such as Wilson’s diseases and Alzheimer’s diseases), but also bring the serious copper contamination of the environment for 5,6 the widespread use of copper in agriculture and industry. National Research Council suggested the recommended daily allowance of copper ranges from 1.5 to 3.0 mg for adults, 1.5 7 to 2.5 mg for children and 0.4 to 0.6 mg for infants. Additionally, the U.S. Environmental Protection Agency (EPA) has set a maximum level of copper in drinking water at 20 8 μM. Consequently, it is important to develop a highly sensitive
a.
CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China. b. Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China. c. State Key Laboratory of Transducer Technology Chinese Academy of Sciences, Hefei, Anhui, 230031, China. E-mail: [email protected], [email protected] † These authors contributed equally to this work. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x
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and selective probes for Cu2+ determination for health concerns and environmental monitoring. Many methods have been developed to detect Cu2+, such as atomic absorption spectroscopy/emission spectroscopy, inductively coupled plasma mass spectroscopy (ICP-MS), voltammetry and potentiometry.9-11 However, these techniques tend to be costly, technically complicated, timeconsuming and require expensive instruments. Compared with these methods, fluorescent sensors for Cu2+ detection have been widely applied and attracted increasing attention owing to their simplicity, high sensitivity, good selectivity and rapid response. Various fluorescent probes including organic dyes and semiconductor quantum dots show great potential in sensitive detection of Cu2+. Generally, organic dyes often suffer from fast-photobleaching, low fluorescence quantum yield, narrow excitation spectra and exhibit broad emission bands.12,13 Quantum dots (QDs) have drawn much interest in diverse research areas for their unique optical and electronic properties. In comparison with the organic dyes, QDs possess many outstanding advantages, including size-tunable optical properties, high extinction coefficients, high fluorescent quantum yield, broad excitation spectrum, narrow emission peak and excellent photochemical stability.2,4,8 In recent years, QDs-based systems for Cu2+ detection have continuously been reported using different kinds of surface modifying agents. However, most of these reported fluorescent probes respond to Cu2+ through quenching the fluorescence intensity, which are not suitable for practical use, because the intensity of the single emission sensor changes while the emission color keeps the same. Besides, the change of single fluorescence intensity
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is not highly reproducible due to the interfere of instrumental 14 efficiency and environmental conditions. Two individual fluorescence emission wavelengths used for ratiometric fluorescent sensors can reduce above limitations by selfcalibration. Because one emission peak as reference is constant and another can be severely quenched as a signal report unit, the color change of dual-emission fluorescent nanoparticles is obvious and easily recognized by the naked eye under a UV lamp. Meanwhile the background interferences could be effectively eliminated and the sensitivity of the sensor could be improved. To this end, one emitter of the dual-fluorescence sensor should be sensitive to 15-17 Choosing one stable analyte, while the other is no respond. 2+ emitter to avoid the fluorescence being quenched by Cu is greatly important. Carbon dots (CDs) are an emerging fascinating carbon material, which have many outstanding properties including green synthesis, impressive watersolubility, good photostability, high biocompatibility and low 18,19 toxicity, which is excellent for fluorescent detection. Recently, there has been a widespread concern on the paper sensor for applications in explosive analysis, biological 20-22 detection and environmental monitoring. In particular, their unique advantages including visible results, quickly respond, easy portable and low cost, make them more suitable for practical use. For example, blue emitting graphene oxide nanosheets based test paper was fabricated for assays of different biological species, while coordination complex 2+ functionalized CDs were employed to construct Hg -sensitive 21,22 paper sensor. Unfortunately, these paper sensors exhibit single color and the signal output is according to the intensity changes. Herein, we report a novel ratiometric fluorescence sensor containing blue-emission amino-functionalized CDs and redemission carboxyl-modified CdTe QDs for the visual detection 2+ of Cu . This dual-emission ratiometric fluorescence probe possesses two emission peaks at 437 and 654 nm under a single wavelength excitation of 340 nm. The red fluorescence 2+ of CdTe QDs can be quenched by Cu , while the blue 2+ fluorescence of CDs is insensitive. With the addition of Cu , the variation of fluorescence intensity ratios produce an obvious change of the fluorescence color from pink to blue, which can be conveniently observed by the naked eyes under a UV lamp without any complicated instrumentations. The detection limit of this probe reaches as low as 0.36 nM, which is much lower than the maximum level of copper (20 μM) in drinking water (EPA). Furthermore, we further apply the 2+ ratiometric probe for visual identification of Cu in real water samples including lake water and mineral water. Additionally, the ratiometric probe also has been applied to make paper2+ based nanosensors for the visual fluorescent detection of Cu .
Experimental section Materials 4, 7, 10-trioxa-1, 13-tridecanediamne (TTDDA), HEPES and glycerol were purchased from Sigma-Aldrich. Cadmium
2 | Nanoscale., 2016, 00, 1-3
Nanoscale chloride hydrate (CdCl2·2.5H2O), tellurium powder, sodium borohydride (NaBH4), citric acid, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), potassium iodide and dichloromethane were obtained from Sinopharm Chemical Reagent. Rhodamine B and 3mercaptopropionic acid (MPA) were bought from Aladdin. Ultrapure water (18.2 MΩ·cm) was received by a Millipore water purification system. Synthesis of amine-coated carbon nanodots According to the reported references with minor modification, amine-coated CDs were synthesized by pyrolyzing the mixture of TTDDA and citric acid in glycerol.22-24 Typically, 1 g of TTDDA was mixed with 15 mL of glycerol to receive a clear solution, and the obtained solution was heated to 220°C under nitrogen atomosphere. Subsequently, 1 g of citric acid was rapidly added into the hot solution. Meanwhile, the mixture was continuously heated at 220°C for 3 h. A dark brown solution was then obtained after cooling down to room temperature. The amine-coated CDs solution was purified though the dialysis by a membrane with molecular weight cut off of approximately 3500 for 24 h. Then stock the amino-coated CDs solution in the dark for later use. Synthesis of CdTe quantum dots The red-emission CdTe QDs were synthesized in the aqueous phase based on the reported method with some modification.20,25 0.0638 g (0.5 mmol) of tellurium powder and 0.1 g of NaBH4 were added to 3ml of ultrapure water under nitrogen atmosphere in an ice bath. The black mixture was obtained and stirred under nitrogen atmosphere for 10 h. When the black disappeared and the white produced, the supernatant NaHTe was used as precursor of CdTe QDs. 0.2284 g (1 mmol) of CdCl2·2.5H2O and 210 μL (2.5 mmol) of MPA were dissolved in 125 mL of ultrapure water and the pH value of the mixture was adjusted to 11 with 1.0 M NaOH. The mixture was stirred under bubbling nitrogen for at least 30 min to remove the oxygen. After the freshly prepared H2Te was transferred into the above solution under nitrogen atmosphere by adding 8 ml of 0.5 M H2SO4 into NaTeH solution, the CdTe precursor(final molar ratio, Cd2+/Te2−/MPA=1:0.5:2.5) was immediately formed with a color change from colorless into orange. The mixture was refluxed for 48 h at room temperature and then the redemission CdTe QDs were obtained. Finally, the QDs were precipitated with acetone and purified by centrifugation. The purified CdTe QDs were dispersed in ultrapure water for further use. Synthesis of dual-emission ratiometric fluorescent probe Based on a condensation reaction between carboxyl groups and amino groups, the QDs with carboxylic groups were chemically connected to the amino-modified CDs to fabricate dual-emission fluorescent ratiometric probe. 75 μL of redemission CdTe QDs were dissolved in 12 mL of 10 mM of HEPES buffers. 100 μL of EDC/NHS (1 mg/mL) was added to the solution and then stirred for 30 min at room temperature to
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2+
Typically, 2 mL of dual-emission ratiometric probe was added into a quartz cuvette, which was used as the working solution, 2+ followed by the addition of calculated amount of Cu . The 2+ final concentrations of Cu presented were 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 nM. The fluorescence spectra of the ratiometric probe under a single wavelength excitation at 340 nm were recorded by a fluorescence spectrophotometer. All the sensitivity and selectivity measurements were conducted in triplicate. The color changes were observed under a 365 nm UV lamp. Selectivity and interference experiments +
+
To investigate the interference of other metallic ions (Na , K , 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Ca , Mg , Hg , Co , Ni , Pb , Ba , Mn , Zn , Fe ) on the 2+ detection of Cu , the fluorescent responses of the ratiometric probe to these metal ions were performed following the same 2+ procedure to that of Cu mentioned above. For interference + + 2+ 2+ 2+ 2+ 2+ 2+ 2+ study, 1 μM of Na , K , Ca , Mg , Co , Ni , Pb , Ba , Mn , 2+ 3+ Zn or Fe were mixed with the ratiometric probe in the HEPES buffer, respectively. In the cases of coexisting metal 2+ ions, then 100 nM Cu was further added into the probe solution and the fluorescence spectra were collected again. 2+
Analysis of Cu in real tap water and lake water samples Tap water samples were collected from our lab and the lake water sample was obtained from a local lake. All the water
2+
A commercial ink cartridge of inkjet printer was washed with ultrapure water until the ink washed thoroughly and then the vacant cartridge was dried in an oven. The shape and size of the printed pattern “Copper(II)” were set on the computer in advance. After the as-prepared probe solutions as ink were injected into the cartridge, the patterns were printed on a cellulose acetate microporous membrane, which was pasted 2+ on a piece of A4 paper. The final Cu -responsive paper sensors was finished by above-mentioned progress. Afterwards, 2+ different concentrations of Cu aqueous solution were dropped on to the test paper. The fluorescence color responses of the indicating paper were observed under a UV 29,30 lamp. Instrumentation UV−vis absorpWon and fluorescence spectra were recorded by Shimadzu UV-2550 spectrometer and Cary Eclipse Fluorescence Spectrometer, respectively. The TEM samples were observed using a JEOL 2010 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were recorded on a Thermo Fisher Nicolet iS10 FT-IR spectrometer. All photographs were taken with a Canon 600D digital camera.
Ratiometic Probe
NH2 S COOH
HOOC S
Preparation of paper-based sensor for visual detection of Cu
CDs
MPA-CdTe QDs S COOH
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The detection of Cu with the ratiometric probe
samples collected were first filtered twice using ordinary qualitative filter paper and 0.45 μm Supor filters to remove the solid suspensions and other impurities. Different 2+ concentrations of Cu were added to the as-prepared water samples and then these samples were analyzed by ratiometric probe. The fluorescence spectra were subsequently recorded by the fluorescence spectrophotometer. The average was obtained by three independent measurements and presented 28 with standard deviation.
+
H 2N
NH2
Cu2+
NH2
S COOH
EDC NHS
654 nm
I
I
437 nm
I
437 nm 654 nm
I
437 nm
654 nm
λ
λ
λ
λ
Fig. 1 Schematic illustration of the formation of dual-emission ratiometric fluorescence probe and the visual detection principle for copper ions.
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26,27
activate the carboxylate groups of QDs. 140 μL of the amine-modified CDs was followed added to the activated QDs solution and the mixture was kept stirring vigorously for 8 h in the dark. The obtained product was separated from the mixture through centrifugation to remove excess unreacted chemicals. The final product was redispersed in 12 mL of HEPES buffers (10 mM, pH=7.4).
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Nanoscale
The structure of the dual-emission fluorescence nanohybrid 2+ and the working principle for visual detection of Cu are illustrated in Fig. 1. To design such a ratiometric fluorescence 2+ probe for visual detection of Cu , amino-functionalized CDs and carboxyl-modified CdTe QDs are chemically combined through a condensation process and catalyzed by EDC/NHS. CDs are selected as the reference signal in the ratiometric fluorescence probe in virtue of its good photostability and 2+ impressive chemical inertness in the presence of Cu (Fig. S1† and S2†). The red-emission MPA-CdTe QDs act as a reaction 2+ site for Cu , which red fluorescence effectively quenched by 2+ Cu . This is probably due to the high affinity constant of the 2+ MPA–Cu complex, resulting in the chemical displacement of 2+ Cd ions on the surface of the QDs. The nonradiative surface channels lead to electron transfer progress and the stability of 31-34 CdTe QDs decreased. Therefore, the change of relative intensity in the ratiometric fluorescence probe caused obvious 2+ color changes, facilitating visual detection of Cu under a UV lamp. The red-emission CdTe QDs successfully conjugated with the blue-emission CDs is proved though the existence of an amide bond between carboxyl groups and amino groups by FTIR spectra (Fig. S3†). As shown in the FT-IR spectra, the band at −1 2530 cm as a characteristic S-H vibration of MPA disappears for covalently bound thiols to the QDs surface or nanohybrid −1 probe surface. The new bands at 1550 and 1390 cm are ascribed to the MPA-capped nanocrystals. The bands of amide −1 bond at 3430 cm can be attributed due to the stretching N−H −1 vibration. The other bands at 1640 and 1170 cm are assigned 24,35 to the C=O and C-N stretch vibrations, respectively. All these characteristic bands in the spectra indicate the formation of the amide bond. The nanohybird can also be revealed by TEM images (Fig. S4†). The TEM images illustrate that the average size of the blue CDs is about 5 nm. The red QDs have a spherical shape with an average diameter of 4 nm.
1.0 Normalized Intensity (a.u.)
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Results and discussion
a
a
b c
b
0.8 0.6
0.4
c 0.2 0.0 400
500 600 700 Wavelength (nm)
800
Fig. 2 Fluorescence emission spectra (λex=340 nm) of (a) blue CDs, (b) red MPA−CdTe QDs and (c) nanohybrid raWometric probe, respectively. The inset shows the corresponding fluorescence photos under a 365 nm UV lamp.
4 | Nanoscale., 2016, 00, 1-3
They are connected together and well dispersed in aqueous solution. As shown in the absorption spectra (Fig. S5†), both the CDs and QDs have negligible absorption peaks in the visible region, thus there is no energy transfer between blue CDs and red QDs. On the other hand, the fluorescence excitation peaks of blue CDs are at 240 and 350 nm, and that of the QDs is at 238 nm. In order to excite the CDs and QDs simultaneously, we found that the best excitation peaks of the ratiometric fluorescence probe is 340 nm. The fluorescence spectra of blue-emission CDs, red-emission QDs and ratiometric probe are shown in Fig. 2. The CDs and QDs show a maximum emission at 437 and 654 nm, emitting strong blue and red fluorescence under a 365 nm UV lamp, respectively. The ratiometric probe which disperses well in water can exhibit dual-emission bands at 437 and 654 nm under a single wavelength excitation. The obtained ratiometric probe displays pink fluorescence color unlike blueemission CDs and red-emission QDs (Fig. 2, inset images). The stability of the ratiometric probe is systematically investigated by fluorescence spectra in aqueous solution. The fluorescence intensity ratios (I437/I654) of the probe remain unchanged within 2 h, demonstrating its excellent photostability (Fig. S6†, black square). Compared with the as-synthesized ratiometric probe, the simple mixed solution of QDs and CDs show a slight change of the fluorescence intensity ratio (I437/I654) (Fig. S6†, red circles), implying that the ratiometric fluorescence probe have a relatively high fluorescence stability. Fluorescence responses are measured upon addition of 2+ different amounts of Cu to evaluate the sensitivity of the ratiometric probe (Fig. 3 A and B). The fluorescence intensity of red-emission QDs continuously decreases along with 2+ increasing the concentration of Cu , while the fluorescence intensity of blue-emission CDs remains almost unchanged. The slight variation of the intensity ratios of the two emission peaks lead to a distinguishable fluorescence color change from pink to cyan and to blue, which is available for visual detection 2+ of Cu by the naked eye. Fig. 3 C shows that the fluorescence intensity ratio (I437/I654) is closely related to the concentration 2+ 2+ of Cu . To quantitatively evaluate the amounts of Cu , a good 2+ linear relationship (I437/I654= 0.88467+0.02415×[Cu ], 2 2+ R =0.9987) for the concentration of Cu ranging from 0 to 100 nM was obtained by plotting the I437/I654 ratio versus the 2+ concentrations of Cu . The detection limit, which was defined as 3 times of the standard deviation of background (3σ), was calculated to be as low as 0.36 nM. The fluorescence response of the red MPA-CdTe QDs and the blue CDs are also examined to demonstrate the sensitive visual detection using the ratiometric probe. Fig. S7† presents 2+ the emission spectra of the blue CDs in response to Cu , showing that the fluorescence spectra keep constant without 2+ obvious change of fluorescence color upon the addition of Cu up to a concentration of 100 nM. Conversely, the fluorescence spectra of the red QDs can be greatly quenched upon gradual 2+ addition of Cu from 0 to 300 nM, but the color change of the single fluorescence quenching of the red QDs is hard to be distinguished by the naked eye compared with the ratiometric probe (Fig. 4). The liner relations between the fluorescence
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intensity change (F0/F) of the red QDs and different 2+ concentrations of Cu are shown in Fig. S8†. Accordingly, in contrast to the ratiometric probe, the single-color fluorescence quenching probe is less sensitive for the visual detection of 2+ Cu . In addition, the simple mixture of QDs and CDs in the 2+ presence of Cu has been further investigated. The comparison clearly demonstrates that the covalent 2+ conjugation probe is more sensitive to Cu with the same concentration than the simply mixed probe, as evidenced by the changes of intensity ratio (I437/I654) of the mixed probe (Fig. S9†). 2+ To assess the kinetics of the ratiometric probe to Cu , the fluorescence intensity ratios were under surveillance at
A
Concentration of Cu2+ (nM) 0 10 20 30 40 50 60 70 80 90 100
B
700 2+
Cu
FL Intensity (a.u.)
600
(nM) 0 10 20 30 40 50 60 70 80 90 100
500 400 300 200 100
different reaction times after the addition of 100 nM Cu2+ (Fig. 2+ S10†). A]er the addiWon of Cu , the fluorescence intensity ratio of ratiometric probe was decreased rapidly and remained unchanged in less than 5 minutes, demonstrating the fluorescence quenching of the ratiometric probe by Cu2+ is a very quick process. Futhermore, the effect of pH on the fluorescence response to Cu2+ of the nanohybrid system is also 2+ investigated. The results present that before exposure to Cu , the fluorescence intensity ratio (I437/I654) of the ratiometric probe was not affected by pH changes in the range of 6.0−9.0 (Fig. S11†, black line). It can be seen that the maximum fluorescence quenching occurs when pH is at 7.4 (Fig. S11†, red line). Under acidic conditions, the low fluorescence quenching effect is likely caused by protonation of the surface binding thiolates and carboxyl group of the MPA-CdTe QDs. When the pH is at neutral, the deprotonation may enhance the covalent bond between Cd and the MPA molecule and thus generate an increase in the fluorescence quenching effect. However, if pH value is above 7.4, the fluorescence quenching effect gradually declines due to the formation of the 8,34,36,37 precipitated Cu(OH)2. So, we choose HEPES buffer at 2+ pH=7.4 as the suitable media for the detection of Cu . To evaluate the selectivity of the ratiometric fluorescence 2+ probe for Cu , the fluorescence intensity ratio (I437/I654) (Fig. 5 and S12†) of the nanosensor were studied at the same conditions with a variety of metal ions. The fluorescence intensity at 654 nm of the ratiometric fluorescence probe is quenched by about 60.07% and 74.18% by adding 50 nM and 2+ 100 nM Cu , respectively. By comparison, the fluorescence intensity ratio and color have no obvious change in the + + 2+ 2+ presence of other metal ions at 100 nM (Na , K , Ca , Mg ,
A
Concentration of Cu 2+ (nM) 25 50 75 100 125 150 175 200 250 300
0
0 400
C
3.5
500 600 700 Wavelength (nm)
800
700
B
3.0
2+
Cu
Y=0.88467+0.02415X
600
0 25 50 75 100 125 150 175 200 250 300
2
R =0.99908 FL Intensity (a.u.)
2.5 2.0 1.5 1.0
(nM)
500 400 300 200 100
0.5 0
20 40 60 2+ 80 Concentration of Cu (nM)
100
Fig. 3 (A) The fluorescence image set and (B) the corresponding fluorescence spectra (λex=340 nm) of the ratiometric probe upon the addition of different 2+ concentrations of Cu . (C) Plot of the fluorescence intensity ratio (I437/I654) of the nanohybrid ratiometric probe solution as 2+ a function of the concentrations of Cu (λex=340 nm).
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0 400
500 600 Wavelength (nm)
700
800
Fig. 4 (A) The fluorescence image set and (B) the corresponding fluorescence spectra (λex=340 nm) of the red MPA-CdTe QDs upon the addition of different concentrations 2+ of Cu .
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Hg2+, Co2+, Ni2+, Pb2+, Ba2+, Mn2+, Zn2+, Fe3+), which suggest the 2+ good selectivity of the ratiometric fluorescence probe to Cu over other metal ions. Hg2+ has a slight quenching effect on the red fluorescence of the probe, but it can be removed by KI, rhodamine B and dichloromethane, which is based on the 2formation of an ion-associate of HgI4 and RhB (Fig. S13†). For interference study, the fluorescence quenching of the probe solution are not influenced by the addition of other metal ions at relative high concentration (Fig. S14†). A]er further 2+ addition of 100 nM Cu , the fluorescence intensity ratio (I437/I654) of the probe changes greatly, even when it coexists with the interfering metal ions whose concentrations (1μM) are at least 10 times than that of Cu2+. These results indicate the ratiometric fluorescence probe exhibits selectivity and specificity for Cu2+ against other metal ions.
A 3.5 3.0
2.5
2.0
1.5
bla
nk K+ Na + Ni 2+ Co 2+ Mn 2+ Zn 2+ Mg 2 + Ca 2+ Ba 2+ Fe 3+ Pb 2+ Hg 2+ Cu 2+
1.0
B
Fig. 5 (A) The selectivity of the ratiometric probe to various metal ions in the HEPES buffer (10 mM, pH=7.4). The red bars represent the addition of different ions at 100 nM and the blue bar represents the addition of Cu2+ at 50 nM. (B) The fluorescence image set of the ratiometric probe upon the addition of various metal ions.
To further study the practical applicability of the 2+ ratiometric fluorescence method, the detection of Cu using the probe were carried out in real water samples spiked with 2+ different amounts of Cu , including the tap water and local lake water (Shushan Lake). All the water samples were first filtered through a filter paper to remove any impurities. Then the water samples spiked with four different concentrations of 2+ Cu (10, 20, 30 and 40 nM) were studied by the above proposed method. The recovery tests and the relative standard deviations (RSD) of each concentration were done in triplicate and the averages were presented with standard deviation, as shown in Table 1. It is clear shown that the concentrations of Cu2+ found are close to the spiked values by the proposed method and the recoveries range from about 91% to 107% at four different concentrations of Cu2+, which indicates the utility and reliability of the ratiometric 2+ fluorescence probe for detection of Cu in water samples. As the widespread use of copper, environmental pollution of the copper has become a serious problem, because excessive intake of copper may show high toxicity and a longterm harmful to human health. Hence, a rapid, direct and 2+ sensitive paper-based sensor for the visual detection of Cu in aqueous solution is quite crucial without the complicated instruments. To solve above problems, a pattern “Copper(II)” were printed on a piece of cellulose acetate membrane using the as-prepared ratiometric probe solutions as ink, and the printing progress was repeated 50 times to obtain the desired paper-based sensor (Fig. S15†). When the inkjet-printed paper immerged in water about 20 minutes, the fluorescence is still stable (Fig. S16†). As shown in Fig. 6, after dropping 0.1 μM of 2+ Cu solution onto the membrane, the fluorescence color of the printed pattern becomes slight weak under a UV lamp. 2+ With increase the concentration of Cu , the fluorescence color of the printed pattern “Copper(II)” gradually change from pink 2+ to cyan. When the concentration of Cu is up to 10 μM, a blue color appeared apparently. The accurate detection limit cannot be quantitatively evalutated by the change of the fluorescence color with naked eye, whereas the approximately 2+ variation range of Cu concentration can be achieve. Moreover, paper-based sensor exhibits the advantages of portability and easy operation, which has meet the 2+ requirements for the visual detection of Cu without sophisticated spectroscopic equipments.
Table 1 Determination of Cu(Ⅱ Ⅱ) spiked in tap water and real lake water samples using the proposed method.
Spiked concentration (nM) 10 20 30 40
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Tap water
Lake water
Found (nM)
Recovery (%)
RSD (%)
Found (nM)
Recovery (%)
RSD (%)
10.6 21.1 27.5 40.7
106.2 105.4 91.6 101.9
4.1 3.9 4.3 5.2
9.4 18.5 32.3 40.9
93.6 92.7 107.7 106.1
2.6 3.2 6.3 4.6
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0.1µM
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1 µM
5 µM
9 10 11 12
10 µM
13 14
Fig. 6 The fluorescence images1 cm of paper-based sensor for the visual detection of Cu2+ at the different concentrations. All the images were taken under a 365 nm UV lamp.
15 16
Conclusions In conclusion, we have demonstrated a novel ratiometric fluorescence probe and a paper-based sensor for visual 2+ detection of Cu with the advantages of high sensitivity and selectivity. The probe is based on the combination between blue-emission CDs and red-emission CdTe QDs, showing the fact that the red fluorescence of CdTe QDs can be quenched by 2+ Cu and whereas the blue fluorescence of CDs remains constant. The fluorescence color change from pink to cyan and 2+ to blue upon the addition of Cu , thus the probe exhibits enhanced visual detection selectivity and reliability compared with single QDs-based probes. Futhermore, the ratiometric fluorescence probe can be printed on a microporous membrane to design a paper-based sensor, which provides a more rapid, convenient and cost efficiency method for visual 2+ detection of Cu . The current ratiometric fluorescent strategies could be extended to apply the visual analysis in the biological, chemical and environmental fields.
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Acknowledgements This work was supported by National Basic Research Program of China (2015CB932002), China-Singapore Joint Project (2015DFG92510), Science and Technology Service Network Initiative of Chinese Academy of China (KFJ-SW-STS-172), and National Natural Science Foundation of China (Nos. 21371174, 21335006, 21275145, 21277145, 21375131, and 21475135).
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Ratiometric fluorescent paper sensor utilizing hybrid carbon dotsquantum dots for the visual determination of copper ions Received 00th January 20xx, Accepted 00th January 20xx
Yahui Wang,†abc Cheng Zhang,†abc Xiaochun Chen,ab Bo Yang,abc Liang Yang,abc Changlong Jiang*abc and Zhongping Zhang*abc
DOI: 10.1039/x0xx00000x www.rsc.org/
A simple and effective ratiometric fluorescence nanosensor for selective detection of Cu2+ has been developed by covalently connecting the carboxyl-modified red fluorescent cadmium telluride (CdTe) quantum dots (QDs) to the aminofunctionalized blue fluorescent carbon nanodots (CDs). The sensor exhibits the dual-emissions peaked at 437 and 654 nm, under a single excitation wavelength of 340 nm. The red fluorescence can be selectively quenched by Cu2+, while the blue fluorescence is a internal reference, resulting in a distinguishable fluorescence color change from pink to blue under a UV lamp. The detection limit of this highly sensitive ratiometric probe is as low as 0.36 nM, which is lower than U.S. Environmental Protection Agency (EPA) defined limit (20 μM). Moreover, a paper-based sensor has been prepared by printing the hybid carbon dots-quantum dots probe on a microporous membrane, which provides a convenient and simple approach for visual detection of Cu2+. Therefore, the as-synthesized probe shows great potential application for the determination of Cu2+ in real samples.
Introduction Copper, as one of the heavy metal ions and the essential trace 1,2 elements for many living organisms, plays an important role in some physiological and pathological progress, such as bone formation, cellular respiration, as well as serving as a significant catalytic cofactor for the synthesis of hemoglobin, 3 elastin and collagen. The deficiency of copper can lead to many diseases such as anemia, pancytopenia and bone 4 2+ abnormalities. Nevertheless, an excess Cu concentration may not only become toxic to living organisms and induce damage to the liver, kidneys and the central nervous system (such as Wilson’s diseases and Alzheimer’s diseases), but also bring the serious copper contamination of the environment for 5,6 the widespread use of copper in agriculture and industry. National Research Council suggested the recommended daily allowance of copper ranges from 1.5 to 3.0 mg for adults, 1.5 7 to 2.5 mg for children and 0.4 to 0.6 mg for infants. Additionally, the U.S. Environmental Protection Agency (EPA) has set a maximum level of copper in drinking water at 20 8 μM. Consequently, it is important to develop a highly sensitive
a.
CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China. b. Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China. c. State Key Laboratory of Transducer Technology Chinese Academy of Sciences, Hefei, Anhui, 230031, China. E-mail: [email protected], [email protected] † These authors contributed equally to this work. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x
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and selective probes for Cu2+ determination for health concerns and environmental monitoring. Many methods have been developed to detect Cu2+, such as atomic absorption spectroscopy/emission spectroscopy, inductively coupled plasma mass spectroscopy (ICP-MS), voltammetry and potentiometry.9-11 However, these techniques tend to be costly, technically complicated, timeconsuming and require expensive instruments. Compared with these methods, fluorescent sensors for Cu2+ detection have been widely applied and attracted increasing attention owing to their simplicity, high sensitivity, good selectivity and rapid response. Various fluorescent probes including organic dyes and semiconductor quantum dots show great potential in sensitive detection of Cu2+. Generally, organic dyes often suffer from fast-photobleaching, low fluorescence quantum yield, narrow excitation spectra and exhibit broad emission bands.12,13 Quantum dots (QDs) have drawn much interest in diverse research areas for their unique optical and electronic properties. In comparison with the organic dyes, QDs possess many outstanding advantages, including size-tunable optical properties, high extinction coefficients, high fluorescent quantum yield, broad excitation spectrum, narrow emission peak and excellent photochemical stability.2,4,8 In recent years, QDs-based systems for Cu2+ detection have continuously been reported using different kinds of surface modifying agents. However, most of these reported fluorescent probes respond to Cu2+ through quenching the fluorescence intensity, which are not suitable for practical use, because the intensity of the single emission sensor changes while the emission color keeps the same. Besides, the change of single fluorescence intensity
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is not highly reproducible due to the interfere of instrumental 14 efficiency and environmental conditions. Two individual fluorescence emission wavelengths used for ratiometric fluorescent sensors can reduce above limitations by selfcalibration. Because one emission peak as reference is constant and another can be severely quenched as a signal report unit, the color change of dual-emission fluorescent nanoparticles is obvious and easily recognized by the naked eye under a UV lamp. Meanwhile the background interferences could be effectively eliminated and the sensitivity of the sensor could be improved. To this end, one emitter of the dual-fluorescence sensor should be sensitive to 15-17 Choosing one stable analyte, while the other is no respond. 2+ emitter to avoid the fluorescence being quenched by Cu is greatly important. Carbon dots (CDs) are an emerging fascinating carbon material, which have many outstanding properties including green synthesis, impressive watersolubility, good photostability, high biocompatibility and low 18,19 toxicity, which is excellent for fluorescent detection. Recently, there has been a widespread concern on the paper sensor for applications in explosive analysis, biological 20-22 detection and environmental monitoring. In particular, their unique advantages including visible results, quickly respond, easy portable and low cost, make them more suitable for practical use. For example, blue emitting graphene oxide nanosheets based test paper was fabricated for assays of different biological species, while coordination complex 2+ functionalized CDs were employed to construct Hg -sensitive 21,22 paper sensor. Unfortunately, these paper sensors exhibit single color and the signal output is according to the intensity changes. Herein, we report a novel ratiometric fluorescence sensor containing blue-emission amino-functionalized CDs and redemission carboxyl-modified CdTe QDs for the visual detection 2+ of Cu . This dual-emission ratiometric fluorescence probe possesses two emission peaks at 437 and 654 nm under a single wavelength excitation of 340 nm. The red fluorescence 2+ of CdTe QDs can be quenched by Cu , while the blue 2+ fluorescence of CDs is insensitive. With the addition of Cu , the variation of fluorescence intensity ratios produce an obvious change of the fluorescence color from pink to blue, which can be conveniently observed by the naked eyes under a UV lamp without any complicated instrumentations. The detection limit of this probe reaches as low as 0.36 nM, which is much lower than the maximum level of copper (20 μM) in drinking water (EPA). Furthermore, we further apply the 2+ ratiometric probe for visual identification of Cu in real water samples including lake water and mineral water. Additionally, the ratiometric probe also has been applied to make paper2+ based nanosensors for the visual fluorescent detection of Cu .
Experimental section Materials 4, 7, 10-trioxa-1, 13-tridecanediamne (TTDDA), HEPES and glycerol were purchased from Sigma-Aldrich. Cadmium
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Nanoscale chloride hydrate (CdCl2·2.5H2O), tellurium powder, sodium borohydride (NaBH4), citric acid, 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), potassium iodide and dichloromethane were obtained from Sinopharm Chemical Reagent. Rhodamine B and 3mercaptopropionic acid (MPA) were bought from Aladdin. Ultrapure water (18.2 MΩ·cm) was received by a Millipore water purification system. Synthesis of amine-coated carbon nanodots According to the reported references with minor modification, amine-coated CDs were synthesized by pyrolyzing the mixture of TTDDA and citric acid in glycerol.22-24 Typically, 1 g of TTDDA was mixed with 15 mL of glycerol to receive a clear solution, and the obtained solution was heated to 220°C under nitrogen atomosphere. Subsequently, 1 g of citric acid was rapidly added into the hot solution. Meanwhile, the mixture was continuously heated at 220°C for 3 h. A dark brown solution was then obtained after cooling down to room temperature. The amine-coated CDs solution was purified though the dialysis by a membrane with molecular weight cut off of approximately 3500 for 24 h. Then stock the amino-coated CDs solution in the dark for later use. Synthesis of CdTe quantum dots The red-emission CdTe QDs were synthesized in the aqueous phase based on the reported method with some modification.20,25 0.0638 g (0.5 mmol) of tellurium powder and 0.1 g of NaBH4 were added to 3ml of ultrapure water under nitrogen atmosphere in an ice bath. The black mixture was obtained and stirred under nitrogen atmosphere for 10 h. When the black disappeared and the white produced, the supernatant NaHTe was used as precursor of CdTe QDs. 0.2284 g (1 mmol) of CdCl2·2.5H2O and 210 μL (2.5 mmol) of MPA were dissolved in 125 mL of ultrapure water and the pH value of the mixture was adjusted to 11 with 1.0 M NaOH. The mixture was stirred under bubbling nitrogen for at least 30 min to remove the oxygen. After the freshly prepared H2Te was transferred into the above solution under nitrogen atmosphere by adding 8 ml of 0.5 M H2SO4 into NaTeH solution, the CdTe precursor(final molar ratio, Cd2+/Te2−/MPA=1:0.5:2.5) was immediately formed with a color change from colorless into orange. The mixture was refluxed for 48 h at room temperature and then the redemission CdTe QDs were obtained. Finally, the QDs were precipitated with acetone and purified by centrifugation. The purified CdTe QDs were dispersed in ultrapure water for further use. Synthesis of dual-emission ratiometric fluorescent probe Based on a condensation reaction between carboxyl groups and amino groups, the QDs with carboxylic groups were chemically connected to the amino-modified CDs to fabricate dual-emission fluorescent ratiometric probe. 75 μL of redemission CdTe QDs were dissolved in 12 mL of 10 mM of HEPES buffers. 100 μL of EDC/NHS (1 mg/mL) was added to the solution and then stirred for 30 min at room temperature to
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2+
Typically, 2 mL of dual-emission ratiometric probe was added into a quartz cuvette, which was used as the working solution, 2+ followed by the addition of calculated amount of Cu . The 2+ final concentrations of Cu presented were 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 nM. The fluorescence spectra of the ratiometric probe under a single wavelength excitation at 340 nm were recorded by a fluorescence spectrophotometer. All the sensitivity and selectivity measurements were conducted in triplicate. The color changes were observed under a 365 nm UV lamp. Selectivity and interference experiments +
+
To investigate the interference of other metallic ions (Na , K , 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ 3+ Ca , Mg , Hg , Co , Ni , Pb , Ba , Mn , Zn , Fe ) on the 2+ detection of Cu , the fluorescent responses of the ratiometric probe to these metal ions were performed following the same 2+ procedure to that of Cu mentioned above. For interference + + 2+ 2+ 2+ 2+ 2+ 2+ 2+ study, 1 μM of Na , K , Ca , Mg , Co , Ni , Pb , Ba , Mn , 2+ 3+ Zn or Fe were mixed with the ratiometric probe in the HEPES buffer, respectively. In the cases of coexisting metal 2+ ions, then 100 nM Cu was further added into the probe solution and the fluorescence spectra were collected again. 2+
Analysis of Cu in real tap water and lake water samples Tap water samples were collected from our lab and the lake water sample was obtained from a local lake. All the water
2+
A commercial ink cartridge of inkjet printer was washed with ultrapure water until the ink washed thoroughly and then the vacant cartridge was dried in an oven. The shape and size of the printed pattern “Copper(II)” were set on the computer in advance. After the as-prepared probe solutions as ink were injected into the cartridge, the patterns were printed on a cellulose acetate microporous membrane, which was pasted 2+ on a piece of A4 paper. The final Cu -responsive paper sensors was finished by above-mentioned progress. Afterwards, 2+ different concentrations of Cu aqueous solution were dropped on to the test paper. The fluorescence color responses of the indicating paper were observed under a UV 29,30 lamp. Instrumentation UV−vis absorpWon and fluorescence spectra were recorded by Shimadzu UV-2550 spectrometer and Cary Eclipse Fluorescence Spectrometer, respectively. The TEM samples were observed using a JEOL 2010 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were recorded on a Thermo Fisher Nicolet iS10 FT-IR spectrometer. All photographs were taken with a Canon 600D digital camera.
Ratiometic Probe
NH2 S COOH
HOOC S
Preparation of paper-based sensor for visual detection of Cu
CDs
MPA-CdTe QDs S COOH
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The detection of Cu with the ratiometric probe
samples collected were first filtered twice using ordinary qualitative filter paper and 0.45 μm Supor filters to remove the solid suspensions and other impurities. Different 2+ concentrations of Cu were added to the as-prepared water samples and then these samples were analyzed by ratiometric probe. The fluorescence spectra were subsequently recorded by the fluorescence spectrophotometer. The average was obtained by three independent measurements and presented 28 with standard deviation.
+
H 2N
NH2
Cu2+
NH2
S COOH
EDC NHS
654 nm
I
I
437 nm
I
437 nm 654 nm
I
437 nm
654 nm
λ
λ
λ
λ
Fig. 1 Schematic illustration of the formation of dual-emission ratiometric fluorescence probe and the visual detection principle for copper ions.
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26,27
activate the carboxylate groups of QDs. 140 μL of the amine-modified CDs was followed added to the activated QDs solution and the mixture was kept stirring vigorously for 8 h in the dark. The obtained product was separated from the mixture through centrifugation to remove excess unreacted chemicals. The final product was redispersed in 12 mL of HEPES buffers (10 mM, pH=7.4).
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Nanoscale
The structure of the dual-emission fluorescence nanohybrid 2+ and the working principle for visual detection of Cu are illustrated in Fig. 1. To design such a ratiometric fluorescence 2+ probe for visual detection of Cu , amino-functionalized CDs and carboxyl-modified CdTe QDs are chemically combined through a condensation process and catalyzed by EDC/NHS. CDs are selected as the reference signal in the ratiometric fluorescence probe in virtue of its good photostability and 2+ impressive chemical inertness in the presence of Cu (Fig. S1† and S2†). The red-emission MPA-CdTe QDs act as a reaction 2+ site for Cu , which red fluorescence effectively quenched by 2+ Cu . This is probably due to the high affinity constant of the 2+ MPA–Cu complex, resulting in the chemical displacement of 2+ Cd ions on the surface of the QDs. The nonradiative surface channels lead to electron transfer progress and the stability of 31-34 CdTe QDs decreased. Therefore, the change of relative intensity in the ratiometric fluorescence probe caused obvious 2+ color changes, facilitating visual detection of Cu under a UV lamp. The red-emission CdTe QDs successfully conjugated with the blue-emission CDs is proved though the existence of an amide bond between carboxyl groups and amino groups by FTIR spectra (Fig. S3†). As shown in the FT-IR spectra, the band at −1 2530 cm as a characteristic S-H vibration of MPA disappears for covalently bound thiols to the QDs surface or nanohybrid −1 probe surface. The new bands at 1550 and 1390 cm are ascribed to the MPA-capped nanocrystals. The bands of amide −1 bond at 3430 cm can be attributed due to the stretching N−H −1 vibration. The other bands at 1640 and 1170 cm are assigned 24,35 to the C=O and C-N stretch vibrations, respectively. All these characteristic bands in the spectra indicate the formation of the amide bond. The nanohybird can also be revealed by TEM images (Fig. S4†). The TEM images illustrate that the average size of the blue CDs is about 5 nm. The red QDs have a spherical shape with an average diameter of 4 nm.
1.0 Normalized Intensity (a.u.)
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Results and discussion
a
a
b c
b
0.8 0.6
0.4
c 0.2 0.0 400
500 600 700 Wavelength (nm)
800
Fig. 2 Fluorescence emission spectra (λex=340 nm) of (a) blue CDs, (b) red MPA−CdTe QDs and (c) nanohybrid raWometric probe, respectively. The inset shows the corresponding fluorescence photos under a 365 nm UV lamp.
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They are connected together and well dispersed in aqueous solution. As shown in the absorption spectra (Fig. S5†), both the CDs and QDs have negligible absorption peaks in the visible region, thus there is no energy transfer between blue CDs and red QDs. On the other hand, the fluorescence excitation peaks of blue CDs are at 240 and 350 nm, and that of the QDs is at 238 nm. In order to excite the CDs and QDs simultaneously, we found that the best excitation peaks of the ratiometric fluorescence probe is 340 nm. The fluorescence spectra of blue-emission CDs, red-emission QDs and ratiometric probe are shown in Fig. 2. The CDs and QDs show a maximum emission at 437 and 654 nm, emitting strong blue and red fluorescence under a 365 nm UV lamp, respectively. The ratiometric probe which disperses well in water can exhibit dual-emission bands at 437 and 654 nm under a single wavelength excitation. The obtained ratiometric probe displays pink fluorescence color unlike blueemission CDs and red-emission QDs (Fig. 2, inset images). The stability of the ratiometric probe is systematically investigated by fluorescence spectra in aqueous solution. The fluorescence intensity ratios (I437/I654) of the probe remain unchanged within 2 h, demonstrating its excellent photostability (Fig. S6†, black square). Compared with the as-synthesized ratiometric probe, the simple mixed solution of QDs and CDs show a slight change of the fluorescence intensity ratio (I437/I654) (Fig. S6†, red circles), implying that the ratiometric fluorescence probe have a relatively high fluorescence stability. Fluorescence responses are measured upon addition of 2+ different amounts of Cu to evaluate the sensitivity of the ratiometric probe (Fig. 3 A and B). The fluorescence intensity of red-emission QDs continuously decreases along with 2+ increasing the concentration of Cu , while the fluorescence intensity of blue-emission CDs remains almost unchanged. The slight variation of the intensity ratios of the two emission peaks lead to a distinguishable fluorescence color change from pink to cyan and to blue, which is available for visual detection 2+ of Cu by the naked eye. Fig. 3 C shows that the fluorescence intensity ratio (I437/I654) is closely related to the concentration 2+ 2+ of Cu . To quantitatively evaluate the amounts of Cu , a good 2+ linear relationship (I437/I654= 0.88467+0.02415×[Cu ], 2 2+ R =0.9987) for the concentration of Cu ranging from 0 to 100 nM was obtained by plotting the I437/I654 ratio versus the 2+ concentrations of Cu . The detection limit, which was defined as 3 times of the standard deviation of background (3σ), was calculated to be as low as 0.36 nM. The fluorescence response of the red MPA-CdTe QDs and the blue CDs are also examined to demonstrate the sensitive visual detection using the ratiometric probe. Fig. S7† presents 2+ the emission spectra of the blue CDs in response to Cu , showing that the fluorescence spectra keep constant without 2+ obvious change of fluorescence color upon the addition of Cu up to a concentration of 100 nM. Conversely, the fluorescence spectra of the red QDs can be greatly quenched upon gradual 2+ addition of Cu from 0 to 300 nM, but the color change of the single fluorescence quenching of the red QDs is hard to be distinguished by the naked eye compared with the ratiometric probe (Fig. 4). The liner relations between the fluorescence
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intensity change (F0/F) of the red QDs and different 2+ concentrations of Cu are shown in Fig. S8†. Accordingly, in contrast to the ratiometric probe, the single-color fluorescence quenching probe is less sensitive for the visual detection of 2+ Cu . In addition, the simple mixture of QDs and CDs in the 2+ presence of Cu has been further investigated. The comparison clearly demonstrates that the covalent 2+ conjugation probe is more sensitive to Cu with the same concentration than the simply mixed probe, as evidenced by the changes of intensity ratio (I437/I654) of the mixed probe (Fig. S9†). 2+ To assess the kinetics of the ratiometric probe to Cu , the fluorescence intensity ratios were under surveillance at
A
Concentration of Cu2+ (nM) 0 10 20 30 40 50 60 70 80 90 100
B
700 2+
Cu
FL Intensity (a.u.)
600
(nM) 0 10 20 30 40 50 60 70 80 90 100
500 400 300 200 100
different reaction times after the addition of 100 nM Cu2+ (Fig. 2+ S10†). A]er the addiWon of Cu , the fluorescence intensity ratio of ratiometric probe was decreased rapidly and remained unchanged in less than 5 minutes, demonstrating the fluorescence quenching of the ratiometric probe by Cu2+ is a very quick process. Futhermore, the effect of pH on the fluorescence response to Cu2+ of the nanohybrid system is also 2+ investigated. The results present that before exposure to Cu , the fluorescence intensity ratio (I437/I654) of the ratiometric probe was not affected by pH changes in the range of 6.0−9.0 (Fig. S11†, black line). It can be seen that the maximum fluorescence quenching occurs when pH is at 7.4 (Fig. S11†, red line). Under acidic conditions, the low fluorescence quenching effect is likely caused by protonation of the surface binding thiolates and carboxyl group of the MPA-CdTe QDs. When the pH is at neutral, the deprotonation may enhance the covalent bond between Cd and the MPA molecule and thus generate an increase in the fluorescence quenching effect. However, if pH value is above 7.4, the fluorescence quenching effect gradually declines due to the formation of the 8,34,36,37 precipitated Cu(OH)2. So, we choose HEPES buffer at 2+ pH=7.4 as the suitable media for the detection of Cu . To evaluate the selectivity of the ratiometric fluorescence 2+ probe for Cu , the fluorescence intensity ratio (I437/I654) (Fig. 5 and S12†) of the nanosensor were studied at the same conditions with a variety of metal ions. The fluorescence intensity at 654 nm of the ratiometric fluorescence probe is quenched by about 60.07% and 74.18% by adding 50 nM and 2+ 100 nM Cu , respectively. By comparison, the fluorescence intensity ratio and color have no obvious change in the + + 2+ 2+ presence of other metal ions at 100 nM (Na , K , Ca , Mg ,
A
Concentration of Cu 2+ (nM) 25 50 75 100 125 150 175 200 250 300
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Fig. 3 (A) The fluorescence image set and (B) the corresponding fluorescence spectra (λex=340 nm) of the ratiometric probe upon the addition of different 2+ concentrations of Cu . (C) Plot of the fluorescence intensity ratio (I437/I654) of the nanohybrid ratiometric probe solution as 2+ a function of the concentrations of Cu (λex=340 nm).
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0 400
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Fig. 4 (A) The fluorescence image set and (B) the corresponding fluorescence spectra (λex=340 nm) of the red MPA-CdTe QDs upon the addition of different concentrations 2+ of Cu .
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Hg2+, Co2+, Ni2+, Pb2+, Ba2+, Mn2+, Zn2+, Fe3+), which suggest the 2+ good selectivity of the ratiometric fluorescence probe to Cu over other metal ions. Hg2+ has a slight quenching effect on the red fluorescence of the probe, but it can be removed by KI, rhodamine B and dichloromethane, which is based on the 2formation of an ion-associate of HgI4 and RhB (Fig. S13†). For interference study, the fluorescence quenching of the probe solution are not influenced by the addition of other metal ions at relative high concentration (Fig. S14†). A]er further 2+ addition of 100 nM Cu , the fluorescence intensity ratio (I437/I654) of the probe changes greatly, even when it coexists with the interfering metal ions whose concentrations (1μM) are at least 10 times than that of Cu2+. These results indicate the ratiometric fluorescence probe exhibits selectivity and specificity for Cu2+ against other metal ions.
A 3.5 3.0
2.5
2.0
1.5
bla
nk K+ Na + Ni 2+ Co 2+ Mn 2+ Zn 2+ Mg 2 + Ca 2+ Ba 2+ Fe 3+ Pb 2+ Hg 2+ Cu 2+
1.0
B
Fig. 5 (A) The selectivity of the ratiometric probe to various metal ions in the HEPES buffer (10 mM, pH=7.4). The red bars represent the addition of different ions at 100 nM and the blue bar represents the addition of Cu2+ at 50 nM. (B) The fluorescence image set of the ratiometric probe upon the addition of various metal ions.
To further study the practical applicability of the 2+ ratiometric fluorescence method, the detection of Cu using the probe were carried out in real water samples spiked with 2+ different amounts of Cu , including the tap water and local lake water (Shushan Lake). All the water samples were first filtered through a filter paper to remove any impurities. Then the water samples spiked with four different concentrations of 2+ Cu (10, 20, 30 and 40 nM) were studied by the above proposed method. The recovery tests and the relative standard deviations (RSD) of each concentration were done in triplicate and the averages were presented with standard deviation, as shown in Table 1. It is clear shown that the concentrations of Cu2+ found are close to the spiked values by the proposed method and the recoveries range from about 91% to 107% at four different concentrations of Cu2+, which indicates the utility and reliability of the ratiometric 2+ fluorescence probe for detection of Cu in water samples. As the widespread use of copper, environmental pollution of the copper has become a serious problem, because excessive intake of copper may show high toxicity and a longterm harmful to human health. Hence, a rapid, direct and 2+ sensitive paper-based sensor for the visual detection of Cu in aqueous solution is quite crucial without the complicated instruments. To solve above problems, a pattern “Copper(II)” were printed on a piece of cellulose acetate membrane using the as-prepared ratiometric probe solutions as ink, and the printing progress was repeated 50 times to obtain the desired paper-based sensor (Fig. S15†). When the inkjet-printed paper immerged in water about 20 minutes, the fluorescence is still stable (Fig. S16†). As shown in Fig. 6, after dropping 0.1 μM of 2+ Cu solution onto the membrane, the fluorescence color of the printed pattern becomes slight weak under a UV lamp. 2+ With increase the concentration of Cu , the fluorescence color of the printed pattern “Copper(II)” gradually change from pink 2+ to cyan. When the concentration of Cu is up to 10 μM, a blue color appeared apparently. The accurate detection limit cannot be quantitatively evalutated by the change of the fluorescence color with naked eye, whereas the approximately 2+ variation range of Cu concentration can be achieve. Moreover, paper-based sensor exhibits the advantages of portability and easy operation, which has meet the 2+ requirements for the visual detection of Cu without sophisticated spectroscopic equipments.
Table 1 Determination of Cu(Ⅱ Ⅱ) spiked in tap water and real lake water samples using the proposed method.
Spiked concentration (nM) 10 20 30 40
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Tap water
Lake water
Found (nM)
Recovery (%)
RSD (%)
Found (nM)
Recovery (%)
RSD (%)
10.6 21.1 27.5 40.7
106.2 105.4 91.6 101.9
4.1 3.9 4.3 5.2
9.4 18.5 32.3 40.9
93.6 92.7 107.7 106.1
2.6 3.2 6.3 4.6
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Fig. 6 The fluorescence images1 cm of paper-based sensor for the visual detection of Cu2+ at the different concentrations. All the images were taken under a 365 nm UV lamp.
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Conclusions In conclusion, we have demonstrated a novel ratiometric fluorescence probe and a paper-based sensor for visual 2+ detection of Cu with the advantages of high sensitivity and selectivity. The probe is based on the combination between blue-emission CDs and red-emission CdTe QDs, showing the fact that the red fluorescence of CdTe QDs can be quenched by 2+ Cu and whereas the blue fluorescence of CDs remains constant. The fluorescence color change from pink to cyan and 2+ to blue upon the addition of Cu , thus the probe exhibits enhanced visual detection selectivity and reliability compared with single QDs-based probes. Futhermore, the ratiometric fluorescence probe can be printed on a microporous membrane to design a paper-based sensor, which provides a more rapid, convenient and cost efficiency method for visual 2+ detection of Cu . The current ratiometric fluorescent strategies could be extended to apply the visual analysis in the biological, chemical and environmental fields.
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Acknowledgements This work was supported by National Basic Research Program of China (2015CB932002), China-Singapore Joint Project (2015DFG92510), Science and Technology Service Network Initiative of Chinese Academy of China (KFJ-SW-STS-172), and National Natural Science Foundation of China (Nos. 21371174, 21335006, 21275145, 21277145, 21375131, and 21475135).
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