Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 482–488

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

The use of imidazolium ionic liquid/copper complex as novel and greencatalyst for chemiluminescent detection of folic acid by Mn-doped ZnSnanocrystals

1386-1425/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.saa.2013.11.036

⇑ Corresponding author. Tel./fax: +98 112 534 2350.E-mail address: azizi@umz.ac.ir (S.N. Azizi).

Seyed Naser Azizi a,⇑, Parmis Shakeri a, Mohammad Javad Chaichi a, Ahmadreza Bekhradnia b,Mehdi Taghavi c, Mousa Ghaemy c

a Analytical Division, Faculty of Chemistry, University of Mazandaran, Babolsar 47416-95447, Iranb Department of Medicinal Chemistry, Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari, Iranc Department of Chemistry, Polymer Chemistry Research Laboratory, University of Mazandaran, Babolsar 47416-95447, Iran

h i g h l i g h t s

� The use of water-soluble Mn-dopedZnS QDs as chemiluminescenceemitter.� The use of ionic liquid/copper

complex as an efficient and greencatalyst.� The determination of folic acid in

commercial drugs with low detectionlimit.� The common ions cannot interfere to

the detection of folic acid.

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

a r t i c l e i n f o

Article history:Received 5 August 2013Received in revised form 15 October 2013Accepted 2 November 2013Available online 19 November 2013

Keywords:ZnS quantum dotSensitized chemiluminescence1,3-Dipropylimidazolium bromideFolic acidIonic liquid (IL)

a b s t r a c t

A novel chemiluminescence (CL) method using water-soluble Mn-doped ZnS quantum dots (QDs) as CLemitter is proposed for the chemiluminometric determination of folic acid in pharmaceutical formula-tion. Water-soluble Mn-doped ZnS QDs were synthesized by using L-cysteine as stabilizer in aqueoussolutions. The nanoparticles were structurally and optically characterized by X-ray powder diffraction(XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), UV–Vis absorptionspectroscopy and photoluminescence (PL) emission spectroscopy. The CL of ZnS QDs induced by directlychemical oxidation and its ionic liquid-sensitized effect in aqueous solution were then investigated. Itwas found that oxidants, especially hydrogen peroxide, could directly oxidize ZnS QDs to produce weakCL emission in basic conditions. In the presence of 1,3-dipropylimidazolium bromide/copper a drasticlight emission enhancement is observed, related to a strong interaction between Cu2+ and the imidazo-lium ring. Therefore, a new CL analysis system was developed for the determination of folic acid. Underthe optimum conditions, there is a good linear relationship between the relative CL intensity and the con-centration of folic acid in the range of 1 � 10�9–1 � 10�6 M of folic acid with a correlation coefficient (R2)of 0.9991. The limit of detection of this system was found to be 1 � 10�10 M. This method is not only sim-ple, sensitive and low cost, but also reliable for practical applications.

� 2013 Elsevier B.V. All rights reserved.

Fig. 1. Chemical structure of the [(1,3-Pr2im)Br].

S.N. Azizi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 482–488 483

Introduction

Folic acid (vitamin B9) is a water-soluble vitamin entangledwith a broad variety of biological processes, because of its impor-tant role as enzymatic cofactor in transference of methyl-groupreactions [1]. It is also known as folate which is found in some en-riched foods and vitamin pills. Folic acid is responsible for the syn-thesis of DNA bases and chains; hence, it is necessary for theformation of new cells, particularly during periods of rapid celldivision and growth, such as pregnancy and infancy [2]. Folic acidis also involved in the synthesis of the heme group of hemoglobin,which is required for growth and maturation of red blood cells(RBCs) [3]. Therefore, severe deficiency of folate leads to numerousdiseases related to hindered cell division processes and deficiencyof RBCs, such as megaloblastic anemia, bone marrow or fetal dis-eases (spina bifida, neural tube defects, etc.) [4]. Folate deficiencyis believed to be the most common vitamin shortage in the worlddue to food selection, food processing, and intestinal disorders [5].Hence, researches in these areas have encouraged the developmentof novel analytical procedures for the determination of FA presentin drugs and biological samples.

During the past decade, nanocrystalline semiconductors (NCs),or so-called quantum dots (QDs), with excellent luminescent prop-erties have attracted an increased attention because of its applica-tion in many areas of fundamental and technical importance [6].Luminescent properties of semiconductor nanocrystals are usuallyinspected by photoluminescence (PL) produced using photoexcita-tion [7], electrochemiluminescence (ECL) generated by electroninjection [8] and cathodoluminescence given from electron impact[9]. In recent years, CL and related analysis techniques have beenutilized in different fields such as biology, bioimaging, biotechnol-ogy and analytical technology because of their widespread linerrange, simple instrument and lack of background scattering lightinterference [10]. With development and recent advance of nano-technology, the CL study has been extended to nanoparticle sys-tems from traditional molecular systems. Today, the CL systemsthat involved nanoparticles have attracted an increased consider-ation because of the unique physical and chemical properties ofnanoparticles [11].

Recently Zhou et al. described that ZnS QDs could enhance CL sig-nals emitted from interaction of NaClO with H2O2 in basic medium[12]. Xiao’s group prepared different types of ZnS QDs and has re-ported their effects on H2O2–NaIO4 CL system [13]. Wang et al. foundthat CdTe could enhance the CL emission of luminol–KMnO4 [14].Sun et al. found that QDs could sensitize the CL of Ce(IV)–sulfite[15]. Our group also reported the effect of CdS QDs on the CL ofhydrogen peroxide–sodium hydrogen carbonate system [16]. How-ever, the CL property of QDs as emitting species of the CL reactions israrely studied. Talapin et al. [17] firstly observed the CL emission ofQDs when adding H2O2 to CdSe/CdS (core/shell) nanocrystals insolution and in nanoparticulate layers; following Talapin’s observa-tion, Li et al. [18] and Wang et al. [19] reported the CL of CdTe and CdSQDs directly oxidized by H2O2 in basic media, respectively. At thepresent stage, the efficiency of the nanoparticle CL system is muchlower than that of the traditional luminol or bis (2,4,6-trichlorophe-nyl) oxalate system [17]. On the other hand, in the reported QDs CLsystem, high concentration H2O2 (1 mol L�1 [18] or 0.8 mol L�1 [19])was used as the CL oxidant, and high concentration H2O2 is very easyto decompose, which would thus result in unstable CL signal. Thesedisadvantages of the QDs CL system will limit its application. Conse-quently, it is necessary to improve the CL properties of QDs by usingappropriate catalyst. But the catalyzed CL reaction of QDs as emit-ting species is rarely reported. Lately Wang et al. reported catalyzedeffect of sodium hexameta phosphate on CdTe QDs–KMnO4 CL sys-tem [20]. Kang et al. examined the influence of different surfactants

on the CdTe QDs–H2O2 system [21]. Improvement of CL emission forthe purpose of higher sensitivity is required for application in traceanalysis. Cui et al. have reported many projecting works about noblemetal nanoparticles-catalyzed CL systems. It has been demon-strated that platinum, silver and gold nanoparticles could greatlyenhance a series of CL reactions including luminol–H2O2 [22], lumi-nol–K3Fe(CN)6 [23] and lucigenin–KI [24]. Recently Santafe et al.firstly reported the catalyzed CL of luminol in the presence of 1-Ethyl-3-Methylimidazolium Ethylsulfate/copper as catalyst, haveprovided new avenues to enhance the inherent sensitivity and ex-pand new applications of this mode of detection [25]. So, in the pres-ent study, we introduce a novel ionic liquid (IL)/copper catalyst on CLreaction of QDs. Mn-doped ZnS QDs, one of the classic semiconduc-tor nanocrystals was chosen and synthesized in aqueous solution asmodel to investigate CL properties of QDs as light emitter.

The aim is introduce the beneficial effect of imidazolium ring-based ILs (Fig. 1) on signal amplification for the catalyzer of theCL system and to improve the efficiency of nanoparticle CL, whichis lower than that of luminol – or bis(2,4,6-trichlorophenyl)oxa-late-based systems. The advantageous new chemiluminescentdetection procedure will also be applied to the determination of fo-lic acid. It will be helpful to promote the step of QDs’ application invarious fields such as bioassay and trace detection of analyte.

Experimental section

Reagents and chemicals

All the reagents or solvents were analytical grade and usedwithout further purification. Ultrapure water (deionized and dou-bly distilled) was used throughout. ZnSO4� 7H2O and L-cysteinehydrochloride anhydrous were from Fluka (Buchs, Switzerland).MnCl2� 4H2O, CuCl2 and hydrogen peroxide (H2O2, 30%) werepurchased from Merck (Darmstadt, Germany). Na2S� 9H2O wasfrom Acros (Geel, Belgium). Folic acid (FA, 97%) was purchasedfrom Sigma–Aldrich (USA). Stock standard solution of folic acid(1 � 10�3 mol L�1) was prepared by dissolving 0.0455 g folic acidin 100 mL distilled water and stored in dark bottles at 4 �C in arefrigerator. Working standard solutions were prepared daily bydiluting the stock solutions with distilled water just before use.FA tablets (1.0 mg and 5.0 mg) were purchased from Rouz DarouPharmaceutical Co. (Tehran, Iran). 0.1 M phosphate buffer solu-tions (PBS) with various pH values were prepared by dissolvingan appropriate amount of Na2HPO4 in water and adjusting thepH values with 0.1 M HCl or NaOH solutions. Fresh working solu-tions of H2O2 were prepared daily from 30% (v/v) H2O2 and werestandardized by titration with a standard solution of KMnO4.

Apparatus

X-ray diffraction (XRD) patterns were recorded on a Bruker AXSD8 Advance X-ray diffractometer (Bruker, Germany) with Cu Karadiation (k = 1.5418 Å). Size distribution of Mn-doped ZnS QDs

484 S.N. Azizi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 482–488

was performed on Hydrosol nanoparticle size analyzer and ZetaPotential Analyzer (Malvern, UK) which is based on a dynamic lightscattering (DLS) technique. The FTIR spectra (4000–400 cm�1) inKBr were recorded using FTIR spectrometer (Tensor 27-Bruker).UV–Vis absorbance spectra of Mn-doped ZnS nanocrystals wereobtained from aqueous Mn-doped ZnS QDs solutions using a CecilCE5501 spectrophotometer (Cambridge, UK). PL measurementswere recorded on a Perkin Elmer LS-3B Luminescence Spectrome-ter (Waltham, USA) using 10 mm quartz cuvettes. The CL lightintensity time curve was obtained on Berthold detection systems,Sirius-tube luminometer (Pforzheim, Germany). PL quantum yieldsof QDs were calculated by comparing their integrated emission tothat of the standard dye solutions. Optical density of all solutionswas adjusted to values below 0.1 at the excitation wavelength toavoid re-absorption effects [26]. All optical measurements werecarried out at room temperature.

Synthesis of aqueous Mn-doped ZnS QDs

Colloidal water-soluble Mn-doped ZnS QDs were synthesizedvia arrested precipitation in water as described previously withslight modifications [27]. Briefly, 50 mL of 0.02 M L-cysteine,5 mL of 0.1 M ZnSO4 and 1.5 mL of 0.01 M MnCl2 were added intoa three-necked flask. The mixed solution was adjusted to pH 11with 1 M NaOH and stirred under dry nitrogen at room tempera-ture for 30 min. Then, 5 mL of 0.1 M Na2S was quickly injected intothe solution. The mixture was stirred for another 20 min, and thenthe solution was aged at 50 �C under air for 2 h to form L-cysteinecapped Mn-doped ZnS QDs. Purification of the QDs was carried outby precipitation of the nanoparticles with ethanol in a centrifuge at5000 rpm for 5 min (the procedure was repeated for 3 times). Theobtained QDs were dried under vacuum and stored as a water sol-uble brown solid powder. Finally, the purified QDs were redis-solved in water for further experiments.

Preparation of [(1,3-Pr2im)Br] ionic liquid

(a) N-Trimethylsilylimidazole (12.6 mL, 0.0858 mol), 1-bromo-propane (21.7 mL, 0.239 mol) and toluene (28 mL) were refluxedfor 10 h and the crystalline product was isolated by the filtration.[(1,3-Pr2im)Br] was dissolved in dry acetonitrile and admixtureswere removed by hexane extraction. After acetonitrile removalthe product was dried in vacuo for 5 h; yield was 14.2 g (71%),mp 135. 1H NMR (acetone-d6): d = 0:89 (t, 6H, CH3, J(HH) = 7.6 Hz),1.95 (m, 4H, CH2CH2CH3, J(HH) = 7.2 Hz), 4.49 (t, 4H, NCH2,J(HH) = 7.2 Hz), 8.15 (s, 2H, H4,5(Imidazole)), 10.27 (s, 1H, H2(Imidazole)). Anal. Calcd. for C9H17N2Br (232.9): C, 46.37%; H,7.29%; Br, 34.30%; N, 12.02%. Found: C, 44.09%; H, 7.25%; Br,30.29%; N, 11.90% [28].

Procedure

Procedure for calibrationWorking standard solutions containing folic acid in the range of

1 � 10�9–1 � 10�6 M were prepared by dilution of a concentratedfresh standard solution of folic acid (1 � 10�3 mol L�1). The CL sig-nal was measured by injection 30 lL of working standard solutioninto the mixture of [(1,3-Pr2im)Br]/copper-Mn-doped ZnS QDssolution. The CL emission intensities versus folic acid concentra-tion were used for the calibration.

Procedure for pharmaceutical preparationTen FA tablets (in each dosage of 1.0 mg and 5.0 mg) were finely

powdered and mixed thoroughly. A portion of the powder equiva-lent to one tablet were weighed accurately, transferred into 10 mLvolumetric brown flasks and dissolved in double-distilled water.

After 20 min of sonication, the sample was filtered through a0.45 lm millipore filter (Durham, UK). To assay the drug in FA tab-lets an aliquot of the filtrate was diluted with 0.1 M phosphate buf-fer solution at pH 8.0 to get desired concentration.

Analytical procedure for CL detectionAssays were performed in a 0.1 M phosphate buffer with a

360 lL final volume. Briefly, cells were filled with solution A thatmade by 260 lL of an aqueous solution of IL, copper, phosphatebuffer, Mn-doped ZnS QDs (appropriate concentrations in water)and folic acid. The mixture was shaken thoroughly and equili-brated at room temperature for 5 min. Then 100 lL of a solutionof complementary reagents (various concentration of hydrogenperoxide) were injected to initiate the light emission and the CLspectrum was recorded. The CL signal kinetics is identical in thepresence and in the absence of IL.

Results and discussion

Characterization of Mn-doped ZnS QDs

The water-soluble Mn-doped ZnS QDs were successfully syn-thesized according to the protocol described in the experimentalsection. The nanoparticles were structurally and optically charac-terized by X-ray powder diffraction (XRD), dynamic light scattering(DLS), Fourier transform infrared spectroscopy (FTIR), UV–Visabsorption spectroscopy and photoluminescence (PL) emissionspectroscopy. According to our previous study, the XRD patternof Mn-doped ZnS QDs exhibited a cubic structure with some peaksfor (111), (220) and (311) planes [30]. The average size (D) of ZnSnanoparticles can be calculated according to Scherrer’s equation[29]:

D ¼ kðk=b cos hÞ, where k is a constant (shape factor, about0.89), k is the X-ray wavelength (0.15418 nm), b is the full widthat half maximum and h is the diffraction angle. Based on the fullwidth at half-maximum of (111) reflection, the averaged crystal-lite sizes of Mn:ZnS QDs was estimated to be 4 nm approximately.

The optical property of Mn-doped ZnS QDs exhibited a charac-teristic absorption peak at 290 nm that can be found in our previ-ous study [30]. For all the doped samples, two different emissionbands dominated the fluorescence spectra. The first emission bandat about 460 nm also existed in the FL spectrum of the undopedZnS nanocrystals, this emission band should indeed originate fromthe host ZnS but not from Mn2+ ions. Upon Mn2+ doping, a secondcharacteristic emission band centred at around 582 nm, is devel-oped for the well-known 4T1�!6A1 d–d transition of Mn2+ ionson Zn2+ sites, where Mn2+ is tetrahedrally coordinated by S2� [31].

Sooklal et al. [32] found that Mn2+ incorporated into the ZnS lat-tice led to the Mn2+-based orange emission while ZnS with surface-bound Mn2+ yielded the ultraviolet emission. Thus, it could be con-cluded that the Mn2+ ions in our samples were indeed incorporatedinto the host ZnS nanocrystals.

Photoluminescence quantum yields (QY) were estimated bycomparison of the fluorescence intensity with standard dye solu-tions with the same optical density at the excitation wavelengthand similar fluorescence wavelength [33].

The QY values were calculated by the equation:

QYðsampleÞ ¼ ðFsample=FrefÞðAref=AsampleÞðn2sample=n2

refÞQYðrefÞ ð1Þ

where F, A and n are the measured fluorescence (area under theemission peak), the absorbance at the excitation wavelength, andthe refractive index of the solvent, respectively. PL spectra werespectrally corrected and quantum yields were determined relativeto Rhodamine 6G in water (QY = 95%) [34]. The PL quantum yieldof 13.0% was calculated for Mn-doped ZnS QDs.

Fig. 3. CL spectra for the (a) Mn-doped ZnS QDs–H2O2 system (dotted line),(b) IL–Mn-doped ZnS QDs–H2O2 system (dashed line), (c) IL–copper–Mn-doped ZnSQDs–H2O2 system (solid line). Conditions: pH 8.0; Mn-doped ZnS QDs, 20 mg L�1;H2O2, 0.5 M; [(1,3-Pr2im)Br], 2 M; Cu2+, 0.002 M.

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Sensitized effect of [(1,3-Pr2im)Br]/copper on Mn-doped ZnS QDs CL

The effect of [(1,3-Pr2im)Br]/copper on the Mn-doped ZnS QDs–H2O2 chemiluminescent system was investigated. In previousexperiments, it was found that in alkaline media QDs could be di-rectly oxidized by KMnO4 or H2O2 to generate CL radiation [17–19].Aiming to improve the CL intensity, IL was introduced into Mn-doped ZnS QDs induced CL system. The presence of ILs in oxidationreaction catalyzed by metallic cations (Cu2+) is well-known for cat-alyzed chemiluminescent reaction of luminol to provide significantadvantages in terms of improvement of catalyst stability and activ-ity [25,35]. Moreover, as most IL currently in use are stable to oxi-dation, they provide ideal solvents for oxidation processes [36]. Butin this case, no enhancement of the CL signal occurred in the pres-ence of copper ions. Many investigations have also revealed thatcopper ions could markedly inhibit the CL and PL signal of QDs[37–39]. Several mechanisms have been proposed to explain theprocess of some heavy metal ions quenching QDs. Quenchingmechanisms include inner filter effects, nonradiative recombina-tion pathways, electron transfer processes, and ion binding interac-tion [40].

In the presence of H2O2 the CL response of Mn-doped ZnS QDssystem was investigated in the absence and in the presence of cop-per ions (Fig. 2). As shown in Fig. 2a, the kinetic curve shows thatthe oxidation of Mn-doped ZnS QDs by H2O2 generates weak CL inalkaline media. The effect of the presence of copper ions (Cu2+) wasalso studied (Fig. 2b). In this case, the CL intensity decrease greatlyin the presence of Cu2+, which revealed that copper ions inhibit thedirect chemical oxidation of Mn-doped ZnS QDs system and haveno catalyst effect separately.

The effect of [(1,3-Pr2im)Br]/copper on Mn-doped ZnS QDs–H2O2 CL reaction was also investigated. As shown in Fig. 3c, theCL signal intensity could be greatly enhanced. As a control experi-ment, the same measurements were performed in the absence ofCu2+. It can be concluded that the CL intensity of IL/copper–Mn-doped ZnS QDs–H2O2 system (Fig. 3c) is far stronger than that ofIL–Mn-doped ZnS QDs–H2O2 system (Fig. 3b) and Mn-doped ZnSQDs–H2O2 system (Fig. 3a), indicating the great sensitized effectof IL/copper on Mn-doped ZnS QDs–H2O2 CL reaction. Parametersinfluencing the CL signals of IL/copper–Mn-doped ZnS QDs–H2O2

system were then investigated to establish the optimal conditionsfor the CL reaction. This optimization was carried out in the follow-ing experiment.

Fig. 2. Dynamic CL intensity–time profiles of Mn-doped ZnS QDs–H2O2 system, inthe absence (a) and in the presence of copper ions (b). Conditions: pH 8.0; Mn-doped ZnS QDs, 20 mg L�1; H2O2, 0.5 M; Cu2+, 0.002 M.

Optimization of the experimental conditions

A series of experiments were conducted for the optimization ofIL/copper–Mn-doped ZnS QDs–H2O2 CL system. In order to performoptimization analysis, four factors were evaluated, including typesof buffer and pH, stoichiometry of [(1,3-Pr2im)Br]/copper complex,H2O2 concentration and ZnS QDs concentration. The CL emissionintensity of QDs (peak height) considered as the experimentalresponse.

Effect of buffer and pHThe reaction media plays an important role in the IL/copper–

Mn-doped ZnS QDs–H2O2 CL reaction and the effect of reactionmedia was investigated. In order to explore the influence of differ-ent buffers on the CL intensity of the IL/copper–Mn-doped ZnSQDs–H2O2 system, three buffer solutions, borax, carbonate andphosphate were studied at the same pH (pH = 8). It was found thatthe CL intensity was the strongest when phosphate buffer solutionwas used. Therefore, phosphate buffer solution was chosen to con-trol acidity in all of experiments. The effect of buffer pH on the CLintensity was examined over the pH range of 6–11 using0.1 mol L�1 PBS solution. Fig. 4 presents the chemiluminescent sig-nals obtained for different pH values in the presence and in the ab-sence of IL. As a matter of fact, the pH seems to have a drastic effecton the CL enhancement. The maximum light intensity value andamplification factor (Intensity+IL/Intensity�IL) was obtained at pH8 in the presence of IL. This pH dependency of the CL enhancementis assumed to be related to an interaction taking place betweencopper and [(1,3-Pr2im)Br]. So an organized [(1,3-Pr2im)Br]/coppercomplex can be suggested in the presence of phosphate buffer,which might be reliant to the presence of the labile proton in theC2 position of the imidazolium ring [41,42].

Effect of stoichiometry of [(1,3-Pr2im)Br]/Cu2+ complexSince the CL enhancement observed seems to be dependent on

the presence of [(1,3-Pr2im)Br]/copper complex, a study of the stoi-chiometry of this complex formation has been performed. For thatpurpose, various concentrations of copper and IL were mixed andused to catalyze the Mn-doped ZnS QDs–H2O2 CL reaction at pH8.0. As shown in Fig. 5 the most favorable stoichiometry of 1000/1 ([(1,3-Pr2im)Br]/copper) between the two reagents can here becalculated.

Effect of H2O2 concentrationThe effect of H2O2 concentration on the CL intensity was

examined in the range of 0.005–1.5 mol L�1 (Fig. 6). The CL

Fig. 4. Maximum light intensity at pH 6.0–11.0 in the presence and in the absenceof [(1,3-Pr2im)Br]/Cu2+ complex in phosphate buffer (0.1 M, pH 8.0); Mn-doped ZnSQDs, 20 mg L�1; H2O2, 0.5 M; [(1,3-Pr2im)Br], 2 M; Cu2+, 0.002 M.

Fig. 6. Effect of H2O2 concentration on the CL intensity of the IL/copper–Mn-dopedZnS QDs–H2O2 system in phosphate buffer (0.1 M, pH 8.0); Mn-doped ZnS QDs,20 mg L�1; [(1,3-Pr2im)Br], 2 M; Cu2+, 0.002 M.

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intensity increased with increasing H2O2 concentration in therange of 0.005–0.5 mol L�1 and decreased when the concentrationof H2O2 was larger than 0.5 mol L�1. So H2O2 concentration wherethen selected 0.5 mol L�1 H2O2 as an optimum concentration.

Effect of ZnS QDs concentrationThe response of different concentrations of Mn-doped ZnS QDs

to the present CL system was investigated under the optimal reac-tion conditions. As shown in Fig. 7 the analytical signal increasedwith increasing QDs concentrations, but for the highest concentra-tions the repeatability was impaired. Use of 20 mg L�1 Mn-dopedZnS QDs solution led to reproducible signals (r.s.d. �1.5%). TheZnS QDs concentration was then fixed as 20 mg L�1, which assureda compromise between sensitivity, analytical dynamical concen-tration range and precision.

Analytical applications

Calibration curves and performance characteristicsIn the proposed system, folic acid quenches the CL of the Mn-

doped ZnS QDs in a concentration dependence that was coincidentto the fluorescence quenching described by a Stern–Volmer equa-tion (Eq. (2)):

I0=I ¼ 1þ Ksv ½Q � ð2Þ

where I and I0 are the CL intensities of the Mn-doped ZnS QDs at agiven folic acid concentration and in a folic acid free solution. The

Fig. 5. Light intensity as a function of different copper and IL concentrations inphosphate buffer (0.1 M, pH 8.0); Mn-doped ZnS QDs, 20 mg L�1; H2O2, 0.5 M.

[Q] is a folic acid concentration and Ksv is the Stern–Volmer quench-ing constant. Under the optimum conditions, there is a good linearrelationship between the relative CL intensity (I0/I) and the concen-tration of folic acid (C) in the range of 1 � 10�9–1 � 10�6 mol L�1 FAwith a correlation coefficient (R2) of 0.9991. The regression equationwas I0/I = 3 � 106 C + 1.17. Fig. 8, inset, shows a Stern-Volmerquenching curve describing (I0/I) as a function of folic acid concen-tration. Ksv is found to be 3 � 106 M�1. The detection limit (S/N = 3)was 1 � 10�10 mol L�1 folic acid. From Table 1, it can be seen thatthe proposed method has a lower detection limit, compared withmost of other methods [43–46].

Interference studiesIn order to evaluate possible interferences in this system, the

effects of some inorganic ions and organic compounds, on theCL intensity of the Mn-doped ZnS QDs system containing5.0 � 10�8 mol L�1 folic acid were investigated. The tolerance limitwas described as the amount of foreign substances which causedrelative error less than ±5% (RSD) in the determination of folic acid(5.0 � 10�8 mol L�1). The tolerable molar concentration ratios withrespect to 5.0 � 10�8 mol L�1 folic acid were more than 500 for K+,Na+, Cl�, stearic acid and starch; 100 for Ca2+, Zn2+, Ni2+, Mg2+,Mn2+, glucose and lactose; 50 for uric acid and urea; 20 for Cu2+

and Fe3+.

Sample determination and recovery testsTo test the applicability of the proposed method, it was applied

to the analysis of folic acid drug in its pharmaceutical formulation

Fig. 7. Effect of Mn-doped ZnS QDs concentration on the CL system in phosphatebuffer (0.1 M, pH 8.0); H2O2, 0.5 M; [(1,3-Pr2im)Br], 2 M; Cu2+, 0.002 M. The CLintensity was obtained from the average of three replicates.

Fig. 8. The changes of the CL spectra of IL/copper–Mn-doped ZnS QDs–H2O2 systemafter addition of various concentrations of folic acid. The solution conditions were:100 lL 0.5 M H2O2 was injected into a mixture of 230 lL Mn-doped ZnS QDs,20 mg L�1; [(1,3-Pr2im)Br], 2 M; Cu2+, 0.002 M with different concentrations of folicacid: (1) 0.0, (2) 0.001, (3) 0.01, (4) 0.05, (5) 0.1, (6) 0.3, (7) 0.5, (8) 1 lmol L�1. Theinset shows linear dependence of relative chemiluminescence intensity DICL as afunction of folic acid concentration (lmol L�1).

S.N. Azizi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 482–488 487

(FA tablets, 1.0 mg and 5.0 mg). The results are shown in Table 2.As can be seen, the RSD were 1.95% and 2.3% respectively, whichsuggested that there were no significant differences between thecompared values, make this new CL method applicable to thesepharmaceutical formulations. Recovery tests were done to esti-mate the accuracy of this method. So a known amount of standardswere added to a sample in three different levels. Results are givenin Table 2. The recoveries ranged from 98.5% to 100.9%, with RSDsof <4%. It indicated that the proposed method was reliable.

Possible reaction mechanism

The CL-generation mechanism for QDs oxidation in aqueoussolution has been extensively studied [18,19,21]. Some importantoxygen-related radicals such as superoxide radical anion O��2 andthe hydroxyl radical OH_, have been reported to be important inter-mediates leading to luminescence [18,19,21]. Under a basic condi-tion, based on Sawyer’s reports [47] an OH_radical can be producedby the reaction of a base with hydrogen peroxide. On the otherhand, transition metal ions with two available oxidation statesusually catalyze the radical decomposition of H2O2 [48–50]. Based

Table 1Comparison of the linear ranges and detection limits for folic acid assayreported methods.

Methods D

Chemiluminescence (CL) 2Liquid chromatography–mass spectrometry (LC–MS) 5Electrochemistry 2Capillary electrophoresis (CE) 2This work 1

Table 2Determination of folic acid in pharmaceutical formulation and recovery results.

Sample Claimed value (mg) Found

Folic acid 1.0 Per tablet 1.04 ±Folic acid 5.0 Per tablet 4.7 ± 0

Sample Added (nmol L�1) Found

Folic acid 1.0 mg 0 75.410 86.220 9430 104.3

on findings of Neeraj and coworker, there is a complexation reac-tion between copper (II) ion and imidazole ring in presence ofphosphate buffer [42]. Copper (II) organic complex has activitymore than copper (II) ion in the radical decomposition of H2O2,as it can be detected by CL reaction [51]. So decomposition ofH2O2 catalyzed by copper (II) organic complex ([(1,3-Pr2im)Br]/Cu2+) as following:

H2O2 þ OH��!HO�2 þH2O

H2O2 þHO�2 ������������!½ð1;3�Pr2imÞBr�=Cu2þ

O��2 þ OH� þH2O:

The formed OH_ radical should be able to inject a hole in the 1Shquantum-confined orbital of the Mn-doped ZnS NCs at the pH val-ues used [17].

OH� þ ZnS QDs�!OH� þ ZnSðhþ1shÞ:

The formed superoxide ions are quite stable in high pH aqueoussolution, and their lifetime is about 1 min [52]. They can easily do-nate one electron and lead to the injection of an electron from thesuperoxide ion into the 1Se quantum-confined orbital of Mn-dopedZnS NCs [17]:

O��2 þ ZnS QDs�!O�2 þ ZnSðe�1seÞ:

Then the excited state of the Mn-doped ZnS NCs occurs corre-sponding to one electron and one hole which occupy the 1Se(ZnSðe�1SeÞ) and 1Sh (ZnSðhþ1ShÞ) quantum-confined orbital, respec-tively [53]. When the excited state of Mn-doped ZnS NCs returnsto its ground state, CL may be produced:

ZnSðhþ1shÞ þ ZnSðe�1seÞ�!ðZnS QDsÞ�

ðZnS QDsÞ��!ZnS QDsþ ht

In this study, it is found [(1,3-Pr2im)Br]/Cu2+ complex can en-hance the sensitivity of Mn-doped ZnS QDs–H2O2 system (Fig. 6).Enhancement of CL by added IL solution [(1,3-Pr2im)Br] purposedto have two reason: (1) Reaction of Mn-doped ZnS QDs with theO��2 and OH_ is accelerate in the presence of IL by forming surfactantaggregate, similar to micelle effect [54–56]. (2) The formed[(1,3-Pr2im)Br]/Cu2+ complex have catalytic effect in producingsuperoxide anion radical O��2 by the radical decomposition ofH2O2 [48–50]. On the basis of the heme and metallopor-phyrin structures [57] (efficient CL catalysts), an organized

in pharmaceutical formulation by the proposed method and other

L (mol L�1) LDR (mol L�1) Ref.

.3 � 10�8 3.1�10�7–2.5 � 10�5 [43]

.77 � 10�10 5 � 10�10–4.5 � 10�8 [44]

.3 � 10�10 1 � 10�7–8 � 10�4 [45]� 10�8 5 � 10�8–1 � 10�5 [46]� 10�10 1 � 10�9–1 � 10�6

Recovery (%) RSD (n = 3, %)

0.02 104 1.95.06 94 2.3

(nmol L�1) Recovery (%) RSD (n = 3, %)

– –100.9 398.5 2.498.9 3.1

488 S.N. Azizi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 482–488

[(1,3-Pr2im)Br]/Cu2+ complex can be suggested in the presence ofphosphate [42]. To date, the exact structure of this complex re-mains unknown, even after infrared spectroscopy and NMR studies[25].

Conclusion

On the base of our knowledge this paper describes for the firsttime the use of Mn-doped ZnS QDs as CL emitter and sensitizedeffect of [(1,3-Pr2im)Br]/copper on Mn-doped ZnS QDs–H2O2 CLsystem. The results showed that [(1,3-Pr2im)Br]/copper can beprofitably used as CL sensitizers make possible the chemilumino-metric determination of compounds that have the potential orinteracting with the nanodots affecting their photochemicalproperties and/or reactivity. The proposed method has beenapplied to the determination of low levels of folic acid in pharma-ceutical products. This work is important for the study of new andefficient catalyst and luminophor for chemiluminescent reactionsand promote the steps towards its application in various fields.

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