Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 135 (2015) 551–559

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An ultra sensitive fluorescent nanosensor for detection of ionic copper Sibel Kacmaz a,b, Kadriye Ertekin c,f,⇑, Deniz Mercan d, Ozlem Oter c, Engin Cetinkaya d, Erdal Celik e,f a

Giresun University, Faculty of Engineering, Department of Food Engineering, 28200 Giresun, Turkey University of Dokuz Eylul, The Graduate School of Natural and Applied Sciences, Department of Chemistry, 35160 Izmir, Turkey c University of Dokuz Eylul, Faculty of Sciences, Department of Chemistry, 35160 Izmir, Turkey d University of Ege, Faculty of Sciences, Department of Chemistry, 35100 Izmir, Turkey e University of Dokuz Eylul, Faculty of Engineering, Department of Metallurgical and Materials Engineering, 35160 Izmir, Turkey f University of Dokuz Eylul, Center for Fabrication and Application of Electronic Materials (EMUM), 35160 Izmir, Turkey b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The immobilized fluoroionophore

DMK-7 was used for the first time for sensing of Cu2+.  The dye exhibited highly selective and sensitive response to Cu2+ at picomolar level.  The sensing ability of the DMK-7 was tested both in thin film and nanofiber forms.  The nano-structured sensing agents exhibited lower detection limits with respect to thin films.  The long term stability of the exploited dye was longer than 8 months.

a r t i c l e

i n f o

Article history: Received 7 May 2014 Received in revised form 7 July 2014 Accepted 17 July 2014 Available online 27 July 2014 Keywords: Optical sensors Fluorescent probes Nano-scale sensor Cu (II) Copper Electrospinning

a b s t r a c t A stable and ultra sensitive nano-scale fluorescent chemo-sensor for trace amounts of Cu2+ was proposed. The Cu2+ selective fluoroionophore 2-{[(2-aminophenyl)imino]methyl}-4,6-di-tert-butylphenol (DMK-7) was encapsulated in polymeric ethyl cellulose. The sensing membranes were fabricated in form of nanofibers and thin films. When embedded in polymers, the exploited DMK-7 dye exhibited enhanced photophysical characteristics in absorbance, Stoke’s shift, fluorescence quantum yield, and short and long-term photostability with respect to the solution phase. Sensing abilities of the nanofibers and thin films were tested by steady state and time resolved fluorescence spectroscopy. To our knowledge, this is the first attempt using the DMK-7-doped electrospun nanofibrous materials for copper sensing. The offered sensor displayed a sensitive response with a detection limit of 3.3  1013 M for Cu2+ ions over a wide concentration range of 5.0  1012–5.0  105. Additionally, exhibited high selectivity over convenient cations; Na+, K+, Ca2+, Mg2+, NH+4 and Ag+, Al3+, Ba2+, Co2+, Cr3+, Fe3+, Fe2+, Hg2+, Li+, Mn2+, Ni2+, Pb2+, Sn2+ and Zn2+. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Selective, sensitive and correct recognition of ionic species has been an attractive working subject for scientists. Among them, ⇑ Corresponding author at: University of Dokuz Eylul, Faculty of Sciences, Department of Chemistry, 35160 Izmir, Turkey. http://dx.doi.org/10.1016/j.saa.2014.07.056 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

Cu2+ has probably been one of the most monitored species. Monitoring of trace amounts of Cu2+ becomes quite important especially for strict purity guidelines like medical industry, pharmaceutical applications, dialysis water, microelectronics and manufacturing of integrated circuit semiconductor chips. Up to now, various instrumental techniques have been exploited for the determination of trace levels of the copper. The majority of the methods

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offered for the determination of Cu2+ made use of spectrophotometric or spectrofluorimetric detection. Among them the fluorimetric approach requires efficient fluorescent sensor molecules. Upon binding of analyte, these sensor molecules display dramatically different fluorescence signals compared to free forms in solution, enabling the qualitative/quantitative determination of metal ions [1–18]. Most of them response to the analyte in appropriate solvents or solutions [1,2,6,8,12,14,16]. Obviously, investigations performed in the solution phase provided valuable information for chemosensing of Cu2+ ions. However, over the last two decades, researchers began to use the chemosensing agents in embedded form in solid matrix materials [3–5,9–11,13,15,17,19] expecting better detection limits, robustness, low cost and longer lifetimes. In these designs sensing has been performed with a chromoionophore immobilized on a polymeric support, a resin, glassy matrix material or on a nano-material. Such kind of solid-state sensing materials displayed analyte dependent optical properties such as absorbance [12], fluorescence [3–5,9–11,13,15,17] or reflectance [19] as have been observed in solution. However, they provided enhanced stability, reversibility and sensitivity with respect to the sensing efforts performed in the solution phase. A comparison of the recently published optical methods for determination of Cu2+ with the offered work was shown in Table 1. As can be seen from Table 1, the published studies exploiting nanomaterials have often been encountered and reported. However, there are still a lot of unknowns about nanomaterials and utilization of them in sensing area is still a challenging subject. In this work, we have used for the first time the DMK-7 dye along with the ionic liquid, BMIMBF4 for copper sensing at picomolar level. A comparison of thin film and nanofiber based sensors was also performed. The electrospinning technique was used as a simple way to fabricate highly responsive optical chemical nanoscale sensors. Polymeric matrix material of ethyl cellulose (EC) along with the ionic liquid was used to produce nanofibrous mats and continuous thin films. The fluorescent probe 2-{[(2 aminophenyl)imino]methyl}-4,6-di-tert-butylphenol (DMK-7) was chosen as the indicator due to the selective response, high quantum yield, large Stoke’s shift and excellent photostability. The offered design acted as highly sensitive probe and displayed a calibration response for Cu2+ ions over a wide concentration range of 5.0  1012 to 5.0  105 M.

Experimental Materials The polymer ethyl cellulose (EC) was from Acros (Ethoxy Content 50%). The plasticizer, dioctyl phthalate (DOP) and lipophilic ionic additive; potassium tetrakis-(chlorophenyl) borate (PTCPB) were supplied from Aldrich. The ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4) was from Fluka. Absolute ethanol (EtOH), tetrahydrofurane (THF), dichloromethane (DCM), dimethylformamide (DMF) and chloroform (CH3Cl) were of analytical grade and purchased from Merck, Fluka, and Riedel. Buffer components; acetic acid/acetate, phosphate and metal salts were of reagent grade (Merck and Fluka). Aqueous solutions were prepared with freshly deionized ultra pure water (specific resistance >18 MX cm, pH 5.5) from a Millipore reagent grade water system. The calibration and metal sensing studies were performed by using atomic absorption spectroscopy (AAS) certified reference standard solutions of Ag+, Al3+, Ba2+, Ca2+, Co2+, Cr3+, Cu2+, Fe3+, Fe2+, Hg2+, Li+, K+, Mn2+, Mg2+, Na+, NH+4, Ni2+, Pb2+, Sn2+, Ce3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Er3+, Cd2+ and Zn2+. All of the standards were diluted

with 0.01 M acetic acid/acetate buffer of pH 5.5 and were prepared in polyethylene measuring flasks to avoid adsorption of metal ions. All of the experiments were carried out at room temperature; 25 ± 1 °C. Schematic structure of the exploited Cu2+ sensitive fluoroionophore; DMK-7 dye was shown in Fig. 1. Synthesis of the DMK-7 dye The 2-{[(2-aminophenyl)imino]methyl}-4,6-di-tert-butylphenol (DMK-7) was synthesized according to literature information [20] and characterized by 1H NMR, 13C NMR and IR spectral data. Yield: 78%, mp: 128–130 °C, 1H NMR of DMK-7: (CDCI3, 400 MHz, d): 1.39; 1,54 (s, 18H, AC(CH3)3); 4.05 (s, 2H, ANH2); 6.80–6.85 (m, 2H, C6H4); 7.08–7.14 (m, 2H, C6H4); 7.29 (d, J 2.4 Hz, 1H, C6H2); 7.52 (d, J 2.4 Hz, 1H, C6H2); 8.67(s, 1H, AHC@N); 13.47(s, 1H, AOH). 13C NMR of DMK-7 (CDCI3, 100 MHz, d): 29.7; 31.8 [AC(CH3)3]; 35.4; 34.5 [AC(CH3)3]; 115.9; 118.7; 118.9; 119.1; 123.3; 127.1; 128.0; 128.2; 135.9; 137.2; 141.1; 158.2 (Caromatics); 163.6 (HC@N), m (C@N): 1617.1 cm1. MS (EI+): 324.3 (8%) M+, 41.1(100%) CH2@CHACH+2, 119.1(95%) M+ – C14H21O+, 57.1 (69%) AC4H+9. Preparation of electrospun nanofibers and thin films Electrospinning was used to fabricate EC based continuous nanofibers. The presence of the ionic liquid, (BMIMBF4) in the polymer solutions provided ionic conductivity and proper viscosity for spinning. Concentration of the plasticizer and ionic liquid (IL) in the composites was optimized to form EC based continuous nanofibers. The resulting composites were prepared by mixing 240 mg of polymer (EC), 192 mg of plasticizer (DOP), 48 mg of IL, and 1 mg of dye in the THF. The thin films were prepared by using the same composites indicated for the electrospun nanofibers. The resulting mixtures were spread onto a 125 lm polyester support (Mylar TM type) with a spreading device. Thickness of the film was measured using Tencor Alpha Step 500 Prophylometer and was found to be 5.11 ± 0.081 lm (n = 8). Each sensing film was cut to 1.2 cm diameter, fixed in the cuvette or flow cell, and the excitation and/or emission spectra were recorded. Electrospinning The homogeneous EC solution was placed in a 1 ml plastic syringe fitted with a metallic needle of 0.4 mm of inner diameter. The syringe is fixed vertically on the programmable syringe pump (Top Syringe Pump Top-5300) and the electrode of the high voltage power supply (Gamma High Voltage ES30) was clamped to the metal needle tip. The feed rate of polymer solution was 2.0 mL/h, the applied voltage was 25 kV and the tip-to-collector distance was 10 cm. The nanofibers were collected on a clean aluminum foil. Schematic structure of the electrospinning apparatus was shown earlier [21–23]. The deposited electrospun fibers exhibited good adhesion and structural stability. The surface morphology of the nano-fibers was studied using SEM instrument (6060-JEOL JSM). The scanning electron microscope (SEM) images of the electrospun membranes were shown in Fig. 2. Instruments The absorption spectra of the solutions and sensor films were recorded by using a Shimadzu 1801 UV–Vis spectrophotometer. Steady state fluorescence emission and excitation spectra were measured using Varian Cary Eclipse Spectrofluorometer. Spectral measurements were carried out with fiber optic probe (2 m long).

Table 1 Comparison of the Cu2+ sensing properties of different sensors. Sensitive dye/method

Matrix material/sensing moiety

Rhodamine B/fluorescence

Response time/selectivity

Long/short term stability

Ref.

1.22 lM

–

–

[1]

2.0  1010 M

–

–

[2]

–

–

–

[3]

Nano scaled graphene quantum dots/fluorescence

Solution phase/acetonitrile– 1–100 lM Cu2+ water binary solution Solution phase/aqueous 0.002–1000.0  107 M Cu2+ solution 0–10 lM Cu2+ 3-Glycidoxypropyl trimethoxysilane-covered glass slide 0–15 lM Cu2+

0.226 lM

Nano scaled ZnO quantum dots/fluorescence

Sol gel based quantum dots 0–0.5 mM Cu2+

7.68  107 lM Cu2+

BODIPY derivatives/fluorescence

Solution phase/in dry methanol

–

2-Acetyl-thiophene linked newly synthesized probe/ fluorescence

In HL-7702 and HepG2 cells 1.0  107–5.5  106 M

–/Selective over Fe3+, Al3+, Mn2+, Nearly a month Zn2+, Ca2+, Mg2+, Ag+, Ni2+, Co2+, Pb2+, Cd2+, Hg2+, Li+, Na+ and K+ 35 s/– Stable under continuous exposure of the black UV light source –/Selective over Na+, K+, Ag+, – 2+ 2+ 2+ 2+ 2+ 2+ Ca , Fe , Ni , Zn , Cd , Hg and Pb2+ ions –/Selective over Zn2+, Hg2+, Co2+, Stable during experiments Ni2+, Cd2+ Mn2+, Na+, K+, Ca2+, Fe3+ and Mg2+ – –/Selective over Na+, K+, Ca2+, Mg2+, Zn2+, Cd2+, Ba2+, Fe3+, Fe2+, 3+ 2+ 2+ + 2+ + Cr , NH4, Pb , Hg , Ag , Mn , La3+, Ni2+, Co2+ s90 = 0.83 min/selective over Ag+, 6 months Al3+, Ca2+, Co2+, Cu2+, Fe3+, Hg2+, Mn2+, Mg2+, Ni2+, Pb2+, Sn2+ and 2 l Zn2+, NO3, ClO 4 , SO4 , C , HPO3 4 –/Selective over Fe2+, Cr3+, Cu2+, Better chemical stability, and Mg2+, Co2+, Mn2+, Ni2+, Pb2+, Ca2+, more stable with wide pH value Ag+, K+, Na+

Dansyl-functionalized film sensor/fluorescence

Rhodamine B derivative modified with a functionalized Solution phase/aqueous 8-hydroxyquinoline group/fluorescence acetonitrile

–

0–40 nM

4.7 nM

1012–105 M

3.8  1014 M for E 1.4  1013 M for PMMA

N-3-(4-(dimethylamino phenly)allylidene)isonicotinohydrazide/ fluorescence

Poly(methyl methacrylate) and ethyl cellulose based electrospun nanofibers

Silica-coated CdSe/ZnS QD/fluorescence

Nanoparticles encapsulated 0–10 lM within a silica shell and immobilized on the tip of an optical fiber by a polyvinyl alcohol (PVA) polymer coating Upconverting luminescent 105 to 106 M nanoparticles

Fluorescence resonance energy transfer process between NaYF4:Yb3+/Er3+ and RB-hydrazide

80 nM

1-[2,5-Dimethyl-3-thienyl]-2-[2-methyl-5-(2-pyridyl)- Solution phase/acetonitrile 0–4 equiv. Cu2+ 3-thienyl]perfluorocyclopentene/absorbance and fluorescence Novel vinyl 1,8-naphthalimide-based monomer Nanofibrous film in 0–20 lM (NAAP)/fluorescence acetonitrile/aqueous solution Diethyldithiocarbamate (DDTC)-functionalized Solution phase/phosphate 0–100 lg L1 quantum dots/fluorescence buffer at pH 8.23

0.9 lM

106 M

–

20  106 M

0.29 lg L1

Amino-functionalized polyfluorene derivative/ fluorescence

Glass surface

5–50 lM

–

CdSeTe alloyed quantum dots (AQdots) that capped with L-cysteine/fluorescence

Solution phase/aqueous solution

2  108 to 2  106 mol L1

7.1  109 mol L1

–/RB-hydrazide is selective for Cu2+, ions under 980 nm excitation –/Selective over K+, Mg2+, Ca2+, Ba2+, Al3+, Zn2+, Sn2+, Pb2+, Fe3+, Co2+, Ni2+, Cr3+, Mn2+, Hg2+, Cd2+ –/Selective over Pb2+, Mg2+, Na+, K+, Li+, Cd2+, Ca2+, Fe2+, Co2+, Ni2+, Zn2+, Hg2+, Ag+ and Mn2+ 10 min/selective over Na+, K+, Mg2+, Ca2+, Al3+, Zn2+, Mn2+, Fe3+, Fe2+, Pb2+, Co2+, Ni2+, Cd2+, Ag+,  2  Hg2+, SO2 4 , CO3 , NO3 , Cl –/Selective over Ba2+, Ca2+, Co2+, Hg2+, Mg2+, Mn2+, Na+ or Ni2+, Fe3+, Zn2+ –/Selective over Ba2+, Ca2+, Co2+, Mg2+, Ni2+, Fe3+, Zn2+, Al3+, Fe2+, Pb2+, Ni2+, La2+, K+

[4]

[5]

[6]

[7]

[8]

[9]

[10]

–

[11]

–

[12]

More than three months

[13]

6 months

[14]

–

[15]

–

[16]

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Detection limit

Hydrazide-based fluorescent probes/fluorescence

Dynamic working range

(continued on next page) 553

109 mol dm3 Visual detection range (0.005–2.0 ppm) 0.08  106 mol dm3 to 2.5  106 mol dm3) Pore-based aluminasilica monoliths

Ethyl cellulose/thin film and Nanofiber: 5.0  1012 to 5.0  105 thin Nanofiber: nanofiber forms film: 5.0  1010 to 5.0  104 3.3  1013 M thin film: 4.6  1011 M

4,5-Diamino-6-hydroxy-2-mercaptopyrimidine and diphenylthiocarbazone/reflectance

Cu2+ selective fluoroionophore 2-{[(2aminophenyl)imino]methyl}-4,6-di-tertbutylphenol

>40 min/>8 months

[18] – 0.015 nM 10–100 nM Rhodamine B/fluorescence

In cellular media

Form a stable [Cu–DSAHMP] [19] complex at pH 7

[17] –

–/Selective over K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+, Cd2+, Hg2+ and Ag+, induces no change in fluorescence; only Cr3+ and Al3+ –/Selective over water samples 2  , SO2 such as Cl, NO 2 , NO3 , S 4 , ClO and also metal ions like Ca2+, K+, Mg2+, Pb2+, Ni2+, Zn2+, Fe2+, and Hg2+ 12 min/selective over K+, Li+, Ca2+, Mg2+, Cr6+, Al3+, Cu2+, Ni2+, Mn2+, Zn2+, Co2+, Cd2+, Pb2+, Hg2+, Fe3+, Bi3+, Sb3+, Mo6+, and Se4+, tolerable response over tartrate, citrate, oxalate, chloride, acetate, nitrate, sulfate, carbonate, cetyltrimethylammonium chloride, tetraamyl ammonium chloride, and tetraethyl ammonium chloride Selective over Na+, K+, Ca2+, Mg2+, NH+4 and Ag+, Al3+, Ba2+, Co2+, Cr3+, Fe3+, Fe2+, Hg2+, Li+, Mn2+, Ni2+, Pb2+, Sn2+ and Zn2+ 1 lM PVA matrix Spirolactam-rhodamine derivative/fluorescence

106 M–2.5  103 M

Ref. Long/short term stability Detection limit Dynamic working range Matrix material/sensing moiety Sensitive dye/method

Table 1 (continued)

This work

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Response time/selectivity

554

Fig. 1. Structure of copper sensing fluoroionophore; DMK-7; 2-{[(2 aminophenyl)imino]methyl}-4,6-di-tert-butylphenol.

For instrumental control, data acquisition and processing, the software package of the instrument was used. The tip of the bifurcated fiber optic probe was interfaced with the sensing thin film and/or nanofiber in a buffer containing homemade 300 lL flow cell. The flow cell was equipped with a four channel Ismatec Reglo Analog peristaltic pump. Flow rate of the peristaltic pump was kept at 2.4 mL min1. Analyte solutions or buffers were transported via tygon tubing of 2.06 mm i.d. Instrumental set-up used for measurements was published earlier [24]. Fluorescence lifetimes were recorded using a Time Correlated Single Photon Counting (TCSPC) system that was from Edinburgh Instruments (UK). The instrument was equipped with a 367 nm pulsed laser (pulse width: 79 picoseconds) as the excitation source. During measurements, the Instrument Response Function (IRF) was obtained from a non-fluorescing suspension of a colloidal silica (LUDOX 30%, Sigma Aldrich) in water, held in 10 mm path length quartz cell and was considered to be wavelength independent. All lifetimes were fit to a v2 value of less than 1.1 and with residuals trace symmetrically distributed around the zero axes. Results and discussion Spectral evaluation and fluorescence quantum yield calculations Absorption, excitation and emission spectra of DMK-7 were examined in the solvents of THF, EtOH, DCM, DMF and toluene/ethanol (To:EtOH; 80:20) mixture. Fluorescence quantum yield values (U) were calculated employing comparative William’s method [25]. Quinine sulfate (in 0.05 M H2SO4, U = 0.546) was used as reference standard. The gathered spectral patterns of the DMK-7 were shown in Figs. 3 and 4, respectively. Similar data were also acquired for EC based thin film and nanofibers. Absorption spectra of the molecule were recorded in organic solvents of varying polarities, and, hydrogen bond donating and accepting abilities. Detailed information on molar extinction coefficients (e) and absorption maxima (kAbs) of the dye in the employed solvents and EC matrix were given in Table 2. Except that of To:EtOH, in all of the exploited solvents, the absorption maxima of the dye appeared around 380 nm. The molar extinction coefficients (e) ranging from 30,000 to 81,500 associated to the observed absorption bands. Interestingly, in mixture of To:EtOH, the dye exhibited two different absorption maxima at 343 and 460 nm with e values of 25,100 and 23,000, respectively. Due to the ANH2 and AOH moieties, and the lone pair on the nitrogen, the molecule is subject to intramolecular and intermolecular H-bonds in aprotic and protic solvents, respectively. It should also be considered that, the i-butyl groups in ortho- and para-positions to the phenolic group prevent intermolecular association to some

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Fig. 2. SEM images of EC based electrospun nanofibers (a) and (b) EC based nanofibers under different magnifications such as 500 and 9500.

Table 2 UV–Vis spectra related data of DMK-7 in the solvents of THF, EtOH, To:EtOH (toluene/ ethanol mixture (80:20)), DCM, DMF and in solid matrices of EC.

Fig. 3. Absorption spectra of the DMK-7 dye (106 M dye or 2 mM dye/kg polymer): THF (a) EtOH (b) To:EtOH (c) DCM (d) DMF (e).

1000 c(em)

c(ex)

Intensity (a.u.)

800 600 e(ex)

400 b(ex)

e(em)

d(ex)

d(em) a(em) b(em)

a(ex)

200 0 300

400

500

600

Wavelength (nm) Fig. 4. Excitation and corrected emission spectra of the DMK-OFD-7 dye (106 M dye or 2.5 mmol dye/kg polymer): THF (a) EtOH (b) To:EtOH (c) DCM (d) DMF (e).

extent. Therefore, probability of absorption will be dependent on the availability of p and r bonds or n electrons. It has been estimated that in non-polar or slightly polar aprotic solvents of THF, DCM and DMF, the molecule is involved in intramolecular hydrogen bonding. The high molar extinction coefficients observed in these solvents can be attributed to the stability and structural rigidity of the molecule arising from the intramolecular bond. Additionally, the rotational and vibrational movements around single bond was partially hindered. The lowest molar absorptivities have been recorded in ethanol and toluene:ethanol mixture. This is

Compound

Solvent/matrix

k1abs(nm)

emax(k1abs)

DMK-7

THF EtOH To:EtOH DCM DMF

381 380 343, 460 370 384

55,000 30,100 25,100, 23,000 81,500 39,000

EC

340

190,000

probably due to the formation of intermolecular H-bonds with ethanol molecules and much weaker intramolecular interactions. In case of toluene/ethanol azeotrope, partition of the molecule should be considered. Two different absorption maxima and accompanying molar absorptivities can be attributed to the interaction of the molecule with the components of the azeotrope. In case of ethyl cellulose, microenvironment of the molecule is totally different. Rotational and vibrational movements are largely hindered with respect to the solution phase. As a consequence, enhanced structural rigidity and stability results with better absorption characteristics. Table 3 reveals emission based spectral characteristics of the dye in different moieties. In all of the solvents, the dye exhibited strong emission in the visible range of electromagnetic spectrum between 430 and 520 nm upon excitation in the range of 330– 375 nm. The dye displayed enhanced fluorescence quantum yield when measured in EC matrix. The excitation and emission maxima were found to be very similar to the THF, however the quantum yield, /F was enhanced from 0.09 to 0.14. This 1.5 fold enhancement in the quantum yield can be attributed to the structural rigidity of the dye in the polymeric matrix due to the hindered rotational and vibrational movements. The short time photostability of the dye in EC was monitored in time based mode of the instrument. The molecule was excited at 330 nm and data were acquired at 475 nm for 40 min (see Fig. 5). The dye exhibited excellent short term photostability in the employed media. The long term stability of the ionophore was also excellent and when stored in the ambient air of the laboratory, there was no significant drift in signal intensity after 8 months. Our long term-stability tests are still in progress.

Effect of pH The DMK-7 dye contains proton sensitive centers on it. Thus, the pH dependence of the dye was investigated in the pH range of 2.0–8.0, both in copper-free and copper containing solutions.

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Table 3 ex Spectral characterization of DMK-7 dyes. kem max : maximum emission wavelength in nm; kmax : maximum excitation wavelength in nm; DkST : Stoke’s shift and /F: quantum yield. Compound

Solvent/matrix

kem max

kex max

DkST (Stoke’s shift)

/F (quantum yield)

DMK-7

THF EtOH To:EtOH DCM DMF

490 520 480 430 435

330 330 328 370 375

160 190 152 60 60

9.0  102 in THF

EC

490

330

160

0.14

Fig. 5. The photostability test of DMK-7 dye doped EC based electrospun nanofiber.

Buffers were prepared with 0.02 M CH3COOH, HCl, 0.02 M NaH2PO4 and/or 0.01 M N-N-Bis-(2-hydroxyethyl)-2-aminoethansulfonic acid (BES) at desired pH. The emission maximum of the DMK-7 dye at 490 nm decreases as the pH of the buffer varies from 7.0 to 2.0. Fig. 6 shows the pH dependence of the membrane in copperfree buffers. The pKa value of the immobilized indicator is calculated to be pKa = 4.00 by using non-linear fitting algorithm of Gausse Newtone Marquardt:

pKa ¼ pH þ log½ðIx  Ib Þ=ðIa  Ix Þ

ð1Þ

where Ia and Ib are the intensities of acidic and basic forms and Ix is the intensity at a pH near to the pKa. From this result, it can be concluded that the dye is very sensitive to the fluctuations in pH in the range of 3.0–5.0. Apart from the pKa studies, the copper binding efficiency of the dye was also tested for the pH range of 3.5–8.0 in presence of 1 mM Cu2+ (see Fig. 7). The lowest response to copper was observed at pH 8.0, while the membrane was highly sensitive to Cu2+ at pH = 5.5. Therefore pH = 5.5 was chosen as optimum working condition. When the results obtained from Figs. 6 and 7 were considered together it can be seen that the working pH of 5.5 is out of the pH sensitive region of the dye. Additionally, by working with buffered solutions, we eliminated the pH related interference possibilities. Distribution of the Cu2+ related chemical species in the working conditions was theoretically checked with chemical equilibrium

Fig. 6. pH dependent response of DMK-7 dye doped EC membrane to Cu2+ at pH 3.5–8.0.

Fig. 7. pH induced emission based spectral response of DMK-7 in EC matrix after addition of acidic solutions in the pH range of 7.00–2.00 pH: (a) 7.00, (b) 6.50, (c) 6.00, (d) 5.50, (e) 5.00, (f) 4.50, (g) 4.00, (h) 3.50, (i) 3.00, (j) 2.50, and (k) 2.00.

software programme (Visual MINTEQ) at pH 5.5 in presence of acetate ions. [(CH3COO (aq): 84.713%, CH3COOH (aq): 15.287%, Cu2+ (aq); 99.007%, CuOH+ (aq); 0.993%. Not only for the efficiency of the dye but also due to the solubility considerations of copper, acetic acid/acetate buffered solutions of pH 5.5 was used further studies. Response to Cu2+ ions When doped into modified EC matrices along with the anionic additive; potassium tetrakis-(4-chlorophenyl) borate; the DMK-7 dye becomes a Cu2+ selective probe. Because of the ion-exchange mechanism, Cu2+ ions are selectively extracted into the electrospun fibers or optode membranes by the anionic additive meanwhile potassium ions diffuse from the membrane into solution. Extraction of Cu2+ ions toward the DMK-7 dye can be explained by the following ion-exchange pathway shown in Eq. (2).

DMK  7ðorgÞ ðcolorlessÞ þ 2Kþ ðorgÞ þ 2TpCIPB ðorgÞ þ Cu2þ ðaqÞ $ DMK  7Cu2þ ðorgÞ ðyellowÞ þ 2TpCIPB ðorgÞ þ 2Kþ ðaqÞ ð2Þ The spectral response of the dye doped membranes and electrospun nanofibers to Cu2+ was investigated in buffered solutions at pH 5.5. The sensing agents exhibited remarkable fluorescence intensity quenching on exposure to copper ions. The response was monitored as a change in the relative fluorescence intensity at 490 nm. Spectral response obtained for thin film and nanofiber forms were shown in Fig. 8I and II, respectively. A comparison of the 1H NMR spectra of DMK-7 dye in the absence and presence of Cu (II), gives us the proof about the formation of complex. The signal assigned to the amino protons in 1H NMR spectrum of the dye (4.05 (s, 2H, ANH2); changed to signal of the deprotonated form and shifted downfield in the spectrum of the Cu (II) complex (4.31 ppm (s, H, ANH)) indicating coordination through the amino nitrogen to the copper ion. The signal of the phenolic proton in the free ligand at 13.47 ppm was absent in the

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Fig. 8. Absorption spectrum of the DMK-7 dye in ethanol (a) Cu2+ free, (b) in presence of 5  105 M Cu2+.

spectrum of the complex, confirming the deprotonation of the phenolic group on complex formation upon exposure to Cu (II). For some metal ions, their affinities to ligands are dominated by size and charge, while for others it is dominated by their electronegativity. These two groups of metal ions have been classified by Pearson as hard and soft metal ions, respectively. On the other hand Cu (II) is intermediate and the special binding affinity between the DMK-7 dye and the copper ion is controlled not only by size, but also charge of the Cu (II) and electronegativities of the donor nitrogens and the phenolic oxygen. The proposed mechanism of recognition is ligand-to-metal charge transfer. The emission at 490 nm excited by 330 nm indicates the excited state of the chemosensor. The coordination with copper results in photoinduced electron transfer from the excited state ligand to the Cu (II), which is manifested through the quenching band at 490 nm. On the other hand the slight red shift observed in the fluorescence spectra, as those of the absorption spectra, can be attributed to the interactions between the ligating amino moiety of the DMK-7 and the Cu (II). Upon binding by the cation the excited state becomes more stabilized than the ground state, and this leads to a slight red shift both, on the absorption and the emission spectra (see Figs. 8 and 9). In order to test the analytical response of the probe toward Cu (II), calibration plots were drawn employing the intensity ratio of loaded (I) and unloaded (I0) forms of ionophore, (I0  I)/I. These plots yielded good straight lines over the working range investigated (see insets of Fig. 8). The both forms exhibited similar relative signal changes of 88% and 90%, however, responded to the ionic copper at different concentration ranges; 5.0  1010 M to 5.0  104 M, and, 5.0  1012 M to 5.0  105 M, respectively. Table 4 shows the working range, detection limit (LOD) and other calibration characteristics of the sensing agents in a comparative manner. The detection limit (which is the concentration of analyte producing an analytical signal equal to 3 times of the Standard deviation of the blank signal), was found to be 3.3  1013 M for nanofibers. This is approximately 100 fold better than that of the LOD value calculated for the thin films of the same material. The regression results yielded an absolute linear response with coefficients of regression (R2) of 0.9940 for nanofibers and 0.9822 for thin film forms, respectively. Similarly, the sensor dynamics of the nanofibers (s90 = 90 s) were faster than the thin films (s90 = 240 s). As a result, the DMK-7 doped electrospun nanofibers exhibited larger relative signal change and working range, better linearity and very good sensitivity to Cu2+ ions with respect to conventional thin films. We also tested whether it has been possible to determine the two oxidation states of copper. Calibration sets for

Fig. 9. (I) Fluorescence response of the DMK-7 doped EC based thin film to Cu2+ ions at pH 5.5. (a) Cu-free buffer, (b) 5  1010, (c) 5  109, (d) 5  108, (e) 5  107, (f) 5  106, (g) 5  105, (h) 5  104 mol L. Inset: Calibration plot for the concentration range of 5  1010–104 M Cu (II). (84% Relative signal change). (II) Response of the EC based nanofiber to Cu (II) ions at pH 5.5. (a) Cu free, (b) 5  1012, (c) 5  1011, (d) 5  1010, (e) 5  109, (f) 5  108, (g) 5  107, (h) 5  106, (i) 5  105 mol L. Inset: Linearized calibration plot for the concentration range of 5  1012–5  105 M Cu (II). (86% Relative signal change).

speciation of copper ions were prepared separately. The slopes of the calibration plots for Cu+ and Cu2+ were nearly the same. This result confirms that Cu+ and Cu2+ cannot be distinguished quantitatively in the region assessed. Stern–Volmer analysis The quenching process of chemical sensors based on fluorescence quenching can be described by the well-known Stern–Volmer equation;

I0 =I ¼ 1 þ Ksv ½Q

ð3Þ

where I0 and I are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration, and Ksv is the Stern–Volmer constant. The Ksv defines the efficiency of quenching. When all other variables are held constant, the higher the Ksv, the lower the concentration of quencher required to quench the luminescence. The Ksv values derived from slopes of the plots were found to be 2.00  107 (M1) and 2  106 (M1) for nanofibers and thin films, respectively (see Table 5). The extracted Ksv values for the nanomaterials were approximately 10 fold greater than that of thin film based sensors. The observed significant enhancement in the sensitivity of nanofibers can be attributed to the structure of the material. Here we demonstrated that sensitivity, response and other sensor dynamics can be manipulated by controlling the quencher diffusion rate to fluorophores encapsulated in the polymer via the micro-structural properties of the sensing agent. The offered nano-structure was quite beneficial for both the sensitivity and other dynamics of the sensor due to its large specific area, which has high number of active sites for diffusion of Cu2+ ions where the possibility of interaction of the Cu2+ ions with the analyte selective chromoionophores is higher than that of conventional

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Table 4 Calibration characteristics of EC based electrospun nanofibers and thin films. Indicator dye

Matrix/form

DMK-7

EC/nanofiber EC/thin film

Concentration range Cu2+ (Mol/L) 12

5.0  10 to 5.0  10 5.0  1010 to 5.0  104

Table 5 The Stern Volmer plot and Ksv constant of EC based electrospun nanofibers and thin films. Indicator dye

Matrix/form

Linear regression equation

Ksv constant

DMK-7

EC/nanofiber EC/thin film

y = 2  107x + 1 y = 2  106x + 1

2.0  107 2.0  106

Table 6 Florescence lifetimes of the dye in EtOH and EC based electrospun nanofibers in the presence and absence of the quencher. DMK-7

Solvent

Metal free After addition of Cu2+

s1 s2 s1 s2

5

Nanofiber

Value (ns)

% Relative

Value (ns)

% Relative

0.0788 2.7456 0.1073

74.76 25.24 100

1.0416 4.7088 0.4896 3.9438

39.47 60.53 37.71 62.29

thin films. The attained picomolar level LOD values exploiting nano-materials is an evident of the enhanced sensitivity (see Table 3). The DMK-7 dye is supposed to be formed a non-fluorescent complex with Cu2+ and statically quenched in the ground state.

Regression coefficient (R2)

LOD (Molar)

0.9940 0.9822

3.3  1013 M 4.6  1011 M

The formation constants were found to be KF = 1.4  108 and KF = 2.0  1010 for thin film and nanofiber forms, respectively. According to the theory, the steady-state absorption spectrum of the chromophore is expected to be perturbed in the presence of quencher that interacts with the chromophore in the ground state. For this reason the nature of the ground state was investigated by studying the molecule’s steady state absorption spectrum (see Fig. 9). Curves a and b are the absorption spectra of the DMK-7 dye in EtOH in absence and presence of the quencher. The absorption spectra exhibits an increase in the intensity and an accompanying integral change in presence of 105 M quencher (see Fig. 8, curves a and b). This observation reveals the possibility of the formation of a ground state complex between the dye and Cu2+ ions and consequently the quenching is of static type. On the other hand, the average fluorescence lifetime of DMK-7 dye in EtOH was reported as 0.74 ns. However, when exposed to the quencher, the lifetime dropped to 0.11 ns which is the evidence of dynamic component of the quenching. In contrast to the solution phase, the dye doped eletrospun nanofibers demonstrated biexponential and relatively increased decay times both in absence and presence of the quencher. Similar to the solution phase, the average lifetime of the encapsulated dye exhibited a decrease when exposed to the quencher. Table 4 reveals the distribution of lifetime related data. This distribution in the lifetime and slightly upward curvature characteristic of the Stern Volmer plot can be attributed to the dynamic portion of the quenching. When the absorption and

Fig. 10. (I) Metal ion response of EC based nanofibers at pH 5.5. (II) Anion response of the same composition near neutral solutions.

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lifetime based data were considered together, the quenching can be concluded as combined (dynamic and static) quenching (see Table 6). Investigation of interference effects In order to examine the response of the dye to possible interfering cations, the sensing agents were treated with 105 M concentrations of Ag+, Al3+, Ba2+, Ca2+, Co2+, Cr3+, Fe3+, Fe2+, Hg2+, Li+, K+, Mn2+, Mg2+, Na+, NH+4, Ni2+, Pb2+, Sn2+ and Zn2+ in acetic acid/acetate buffer solutions at pH 5.0. Tests were performed in presence of 108 M of Cu2+ and 103 fold concentrated solutions of the interferents. Additionally, spectral response to Cd2+ and the lanthanide ions of Ce3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, and Er3+, were tested at the same pH. The DMK-7 dye did not exhibit any significant response to Ca2+, Mg2+, Na+ and K+ which are the major cations of water samples and physiologically relevant fluids. The observed relative signal change was less than 5% for all of the lanthanide ions. 103 fold molar excess of Ag+, Cd2+ and Al3+ caused a positive error of 18%. In excess amounts of Sn2+ a negative error of 20% was observed. We also tested interference effects of the conven  2 3 tional anions of F, Cl, Br, NO 2 , NO3 , SO4 , HCO3 and PO4 . Relative signal changes of less than 15% were observed for all of the tested interferents. The relative signal changes caused by some of the tested cations and anions were shown in Fig. 10I and II, respectively. From the figure, it can be concluded that, the sensing design is capable of determining Cu2+ ions with a high selectivity over other ions. The fluorescence was dramatically decreased in the presence of copper ions exhibiting a relative signal change ratio of 90%. Conclusion A highly responsive optical chemical sensor was fabricated for selective and sensitive detection of Cu2+ at picomolar level. The newly synthesized fluorescent probe 2-{[(2 aminophenyl)imino]methyl}-4,6-di-tert-butylphenol (DMK-7) was used as the sensing agent in encapsulated form in EC. The DMK-7 dye was chosen due to its selective response, high quantum yield, large Stoke’s shift and excellent photostability. The nanofibrous mats and continuous thin films were fabricated by electrospinning and knife spreading techniques, respectively. The offered design displayed a calibration response for Cu2+ ions over a wide concentration range of 5.0  1012 to 5.0  105 M. The limit of detection was found to be as low as 3.3  1013 M for nanofibers. The sensor dynamics of the nanofibers (s90 = 90 s) were faster than the thin films (s90 = 240 s). It has been fabricated a fiber optic sensor system for selective and sensitive detection of Cu (II) at picomolar level. The system was based on quenching of an indicator. In this research, the

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exploited polymer based nanofiber produced by electrospinning. With respect to continuous thin films, electrospun nanofibers offered enhanced sensitivity, lower LOD values and reactivity in optical chemical sensing of Cu (II). The DMK-7 dye was used for the first time as a fluoroionophore in the optical copper ion sensing. The DMK-7 dye doped EC nanofibers can be used at pH 5.5 for quantitative determination of Cu (II) in the concentration range of 5  1012–5  105 M. A quite good LOD (0.33 pM) was reached and the sensor characteristics was investigated such as response time, reversibility, linear working range, effect of pH and interference effect. Acknowledgments Funding this research was provided by Scientific Research Funds of Dokuz Eylul University 2012.KB.FEN.049 and Center for Fabrication and Applications of Electronic Materials (EMUM). We also thank to the Scientific and Technological Research Council of Turkey (TUBITAK). References [1] A. Sikdar, S. Roy, K. Haldar, S. Sarkar, S.S. Panja, J. Fluoresc. 23 (2013) 495. [2] X. Wang, J. Zhao, C. Guo, M. Pei, G. Zhang, Sens. Actuat. B 193 (2014) 157. [3] Y. Cao, L. Ding, S. Wang, Y. Liu, J. Fan, W. Hu, P. Liu, Y. Fang, Appl. Mater. Interfaces 6 (2014) 49. [4] F. Wang, Z. Gu, W. Lei, W. Wang, X. Xia, Q. Hao, Sens. Actuat. B 190 (2014) 516. [5] D. Sing, N. Wong, J. Hui, C. Phung, H.S. Chua, Talanta 116 (2013) 514–519. [6] T. Keawwangchai, B. Wanno, N. Morakot, S. Keawwangchai, J. Mol. Model. 19 (2013) 4239. [7] P. Li, H. Zhou, B.A. Tang, J. Photochem. Photobiol. A: Chem. 24 (2012) 36. [8] H. Zhu, J. Fan, J. Lu, M. Hu, J. Cao, J. Wang, H. Li, X. Liu, X. Peng, Talanta 93 (2012) 55. [9] M.Z. Ongun, K. Ertekin, M. Gocmenturk, Y. Ergun, A. Suslu, Spectrochim. Acta Part A 90 (2012) 177. [10] T.W. Sunga, Y.L. Lo, Sens. Actuat. B 165 (2012) 119. [11] S.A. El-Safty, M.A. Shenashen, M. Ismael, M. Khairyab, Md.R. Awual, Analyst 137 (2012) 5278. [12] J. Zhang, B. Li, I. Zhanga, H. Jiang, Chem. Commun. 48 (2012) 4860. [13] S. Cui, S. Pu, W. Liu, G. Liu, Dyes Pigm. 91 (2011) 435. [14] W. Wang, Q. Yang, L. Sun, H. Wang, C. Zhang, X. Fei, M. Sun, Y. Li, J. Hazard. Mater. 194 (2011) 185. [15] J. Wanga, X. Zhoua, H. Mab, G. Taoa, Spectrochim. Acta Part A 81 (2011) 178. [16] F. Lv, X. Feng, H. Tang, L. Liu, Q. Yang, S. Wang, Adv. Funct. Mater. 21 (2011) 845. [17] G.X. Liang, H.Y. Liu, J.R. Zhang, J.J. Zhu, Talanta 80 (2010) 2172. [18] M. Boling, S. Wua, F. Zeng, Sens. Actuat. B 145 (2010) 451. [19] S.A. El-Safty, M.A. Shenashen, M. Ismael, M. Khairy, M.R. Awual, Analyst 137 (2012) 5278. [20] M.A.M. Hernández, T.S. Keizer, S. Parkin, B. Patrick, D.A. Atwood, Organometallics 19 (2000) 4416. [21] S. Kacmaz, K. Ertekin, A. Suslu, Y. Ergun, E. Celik, U. Cocen, Mater. Chem. Phys. 133 (2012) 547. [22] S. Kacmaz, K. Ertekin, A. Suslu, M. Ozdemir, Y. Ergun, E. Celik, U. Cocen, Sens. Actuat. B 153 (2011) 205. [23] S. Kacmaz, K. Ertekin, M. Gocmenturk, A. Suslu, Y. Ergun, E. Celik, React. Funct. Polym. 73 (2013) 674. [24] K. Ertekin, O. Oter, M. Ture, S. Denizalti, E. Cetinkaya, J. Fluoresc. 20 (2010) 533. [25] A.T.R. Williams, S.A. Winfield, J.N. Miller, Analyst 108 (1983) 1067.