Microchim Acta (2009) 164:411417 DOI 10.

1007/s00604-008-0076-4

ORIGINAL PAPER

A new luminol derivative as a fluorescent probe for trace analysis of copper(II)

Yun Luo & Yi Li & Baoqiang Lv & Zaide Zhou & Dan Xiao & Martin M. F. Choi

Received: 14 March 2008 / Accepted: 6 June 2008 / Published online: 12 July 2008 # Springer-Verlag 2008

Abstract A new luminol derivative has been synthesized and its fluorescence has been studied. The luminol derivative emits at 460 nm in pH 10.0 phosphate buffer which is different from the original luminol that emits at 425 nm under the same conditions. Cu2+ exhibits strong fluorescence quenching effect on the luminol derivative and the quenching mechanism is discussed in detail. This luminol derivative can be applied as a fluorescent probe for ultra-trace analysis of Cu2+. The effects of pH, interferents and the stability have also been investigated. The fluorescent probe shows good reproducibility, and the relative standard deviation of response to Cu2+ is 0.7% (n =6). The lower linear range and the limit of detection are 0500 and 0.27 nmol L1, respectively.

Keywords Luminol derivative . Fluorescent probe . Copper(II) ion

Introduction Luminol or its derivatives together with H2O2 or other oxidants are widely used in chemiluminescence (CL). They have been utilized for metal ions detection because of their high sensitivity and selectivity. As such, an enormous interest in using luminol or luminol-linked materials to determine analytes including Co2+, Fe2+, Mn2+ [1], Cu2+ [2], Zn2+, Cd2+ [3], Pb2+ [4] and Cr3+ [5] has been shown in the past few decades. Most of these techniques are usually based on the catalyzing effect of luminol [3, 69] and its derivatives [10] in the presence of oxidants to generate CL detection signals. They have been applied in environmental [1114], food [15, 16], and clinical [17] fields. However, the use of luminol and its derivatives in fluorescent analysis is limited because of their unstable chemical and fluorescence properties. Yet there are still reports in which the fluorescence property of luminol is utilized in detecting DNA [18], methyl glyoxal [19], and chloramphenicol [20]. To date, many investigations are still in progress to search for cheaper, more stable, convenient and effective luminol derivatives. In this work, we report the synthesis of a new luminol derivative that possesses excellent chelating properties with metal ions. So far thiourea-based compounds are found to be good candidates for chelating metal ions. In the past few decades, a series of thiourea compounds was synthesized and successfully used for determining metal ions such as Cu, Pd, Pt, and Au [2126]. Thus, we propose to incorporate the NC=S moiety into our new luminol derivative. It is well-known that even a small change in the luminol structure can alter its spectroscopic properties [27].

Electronic supplementary material The online version of this article (doi:10.1007/s00604-008-0076-4) contains supplementary material, which is available to authorized users. Y. Luo : Z. Zhou : D. Xiao (*) College of Chemistry, Sichuan University, Chengdu 610064, Peoples Republic of China e-mail: xiaodan@scu.edu.cn Y. Li The Southwest Research & Design Institute of Chemical Industry, Chengdu 610000, Peoples Republic of China B. Lv : D. Xiao College of Chemical Engineering, Sichuan University, Chengdu 610064, Peoples Republic of China M. M. F. Choi (*) Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, Peoples Republic of China e-mail: mfchoi@hkbu.edu.hk

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The fluorescence quantum yield of our new synthesized luminol derivative is 0.190 (infra vide) and appears to be better than that of some other previously reported luminolrelated compounds which show no or little fluorescence properties [28]. Our proposed luminol derivative is relatively simple to prepare and purify. It shows excellent stability and strong fluorescence in alkaline conditions. In addition, it exhibits excellent selectivity to Cu2+ and can be employed for ultra-trace analysis of Cu2+ in aqueous solutions. We have successfully applied our luminol derivative as a fluorescent probe to determine nanomolar concentration of Cu2+ in water samples.

deionized water was used throughout to prepare all solutions and buffers. Synthesis of compound A The synthetic sequence for the luminol derivative (compound A) is displayed in Scheme 1. In brief, 10 mg of luminol was first dissolved in 10 mL of 2.0 mol L1 NaOH solution, followed by the slow addition of 0.5 mL CS2 with continuous stirring. The resulting mixture was kept at room temperature for at least 48 h in dark until it turned to orange. Afterwards, the clear orange solution was washed by 10 mL of n-butanol at least three times. The solution was frozen and dried by a vacuum freeze dryer to remove the solvent and an orange product (compound A) was obtained. IR and 1 H NMR spectra were employed to identify the chemical structure of compound A (infra vide). Fluorescence spectra Compound A (5.0 mg) in a 10 mL solution of 0.10 mol L1 Na2HPO4NaOH buffer (pH=10.0) was used for taking the fluorescence spectra. The fluorescence excitation and emission spectra were acquired at their emission (lem = 460 nm) and excitation (lex =388 nm) maxima, respectively.

Experimental Apparatus Fluorescence measurements were performed on an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan, www.hitachi-hitec.com) using a 1010 mm quartz cuvette. The bandwidths of emission and excitation slits were 20 and 10 nm, respectively. All pH measurements were taken on a pH electrode (Thermo Electron, Waltham, MA, USA, www.thermorussell.com). Infrared spectrum was taken in a KBr disk on a NEXUS 6700 FT-IR spectrometer (Thermo Nicolet, MA, USA, www.thermo.com). 1H NMR spectrum was recorded on an AV600 spectrometer (Bruker, Fllanden, Switzerland, www.brukeroptics.com) at 600 MHz using DMSO-d6 as solvent. UVvisible absorption spectroscopy was performed on an UV-1100 absorption spectrophotometer (Techcomp, Shanghai, China, www.techcomp.com.cn). All measurements were carried out at room temperature (241 C). Reagents Luminol (98.0%) from Fluka (Buchs, Switzerland, www. sigmaaldrich.com) was kept in dark to prevent decomposition. Carbon disulfide and copper(II) chloride (>99%) were obtained from Kelong Chemical Reagent Corporation (Chengdu, China, www.cdkelong.com.cn). All other reagents of analytical-reagent grade or above were used without further purification. A 2.0 mol L1 NaOH solution was used in the synthetic procedure. Buffer solutions of 0.10 mol L1 NaH2PO4Na2HPO4 (pH =8.59.5) and 0.10 mol L1 Na2HPO4NaOH (pH=10.011.0) were prepared and employed in the analytical procedure. 5.0 mol L11.0 mmol L1 of Cu(II) standard solutions were prepared for measuring the sensitivity of copper(II) ion determination. 10 mmol L1 of Hg(II), Mn(II), Zn(II), Co(II), Ni(II), Fe(II), Al(III), Sn(II) and Ag(I) solutions were prepared for the interfering tests. Double distilled

Results and discussion Synthesis and identification of compound A Generally, luminol can be oxidized easily and rapidly by oxidants in alkaline aqueous solutions which form the basis for chemiluminescence detection [29]. Yet for fluorescence analysis, such a property is a demerit since the fluorescence property has to be stable. In this work, thiourea moiety is incorporated to the luminol molecule not only to improve its fluorescence and chemical stability but also introduce excellent chelating capability to metal ions. It is anticipated that the as-prepared luminol derivative (compound A) would have stable fluorescence property and its fluorescence intensity could be efficiently quenched by metal ions such as Cu2+. The chemical structure of compound A was identified by the IR and 1H NMR spectral data. Strong absorption bands of CN at 1,5401,541 cm1 and CS at 1,0031,004 cm1 were observed. Besides, the carbonyl group of lactam shows an intense absorption at 1,626 cm1. 1H NMR (DMSO-d6) spectrum indicates the chemical shifts at 7.070 (m, 1H, ArH), 7.160 (m, 1H, ArH), 7.249 (s, 1H, ArH),

A new luminol derivative as a fluorescent probe

413
90 80 Fluorescent intensity 70 60 50 40 30 360 380 400 420 440 460 Wavelength (nm) 480 500 520 a b

6.676 (m, 1H, O=CNH), and 5.936 (d, 1H, NH). It should be noted that the 1H NMR spectrum reflects the weak NH protons of acylamide in DMSO-d6, presumably due to proton exchange. Figure 1 displays the UVvisible absorption spectra of luminol and compound A. The absorption spectrum of compound A is bathochromatically shifted when compared with that of luminol. Luminol shows two absorption peaks at 300 and 360 nm, while compound A exhibits one absorption peak at 314 nm and two shoulder peaks at 350 and 385 nm. The bathochromic shift is possibly attributed to the introduction of thiourea moiety to the parent luminol molecule which also induces one more absorption band to the original luminol molecule. Fluorescence spectrum Figure 2 shows the fluorescence excitation and emission spectra of compound A. Unlike luminol, which emits light at 425 nm in alkaline aqueous media, the emission maximum of compound A at 460 nm is 35 nm red-shifted. The fluorescence intensity is relatively smaller than that of luminol under the same experimental conditions [27], but compound A (2.1 mmol L1 in a pH 10.0 buffer solution) has better photostability. The emission intensity at 460 nm changed 9.8% after 1 h of irradiation at 388 nm, which is acceptable in terms of fluorescence intensity. Hence, excitation wavelength of 388 nm was selected for the Cu2+ analysis. Each analysis was completed within an hour. Although compound A has lower fluorescence intensity, it has higher sensitivity and good selectivity to Cu2+. As such, it can be used to determine ultra-trace analysis of Cu2+ (vide infra).

Fig. 2 The fluorescence spectra of 2.1 mmol L1 compound A in an alkaline buffer (pH 10.0): (a) excitation spectrum monitored at an emission wavelength of 460 nm and (b) emission spectrum excited at 388 nm

The fluorescence quantum yield of compound A was determined to be 0.190 using the following equation: sFu As =Fs Au where , F and A are the fluorescence quantum yield, emission intensity and absorbance, respectively. The subscripts 'u' and 's' refer to compound A and quinine bisulfate, respectively. The calculation was based on quinine bisulfate as the standard with s =0.546 [30]. Response to copper(II) ion Figure 3 depicts the response of compound A to various concentrations of Cu2+. When the luminol derivative was excited at 388 nm, the emission intensity at 460 nm decreased with the increase in Cu2+ concentration. The decrease in emission intensity is linearly related to the Cu2+ concentration by plotting [(Io/I) 1] against log [Cu2+] where Io and I are the emission intensities of compound A in the absence and presence of Cu2+, respectively, and [Cu2+] is the Cu2+ concentration in moles per liter. The calibration curve is roughly divided into two linear regions. The inset of Fig. 3 shows both the lower linear response range from 0.0 to 500 nmol L1 Cu2+ with a linear regression equation of Io =I 1 0:725 log Cu2 5:6397 and r =0.9954, and the higher linear response range from 0.50 to 7.00 mol L1 Cu2+ with linear regression a equation of Io =I 1 3:01 log Cu2 20:0 and r = 0.9975. The higher concentration range is almost four times more sensitive than the lower concentration range. For the lower concentration range, the detection mechanism is

1.0 0.9 Normalized absorbance 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

a b

300

350 400 Wavelength (nm)

450

500

Fig. 1 The UVvis absorption spectrum of 2.1 mmol L1 (a) luminol and (b) compound A in alkaline aqueous media (pH 10.0)

414 Fig. 3 The response of 2.1 mmol L1 compound A on exposure to various concentrations of Cu2+: (a) 0.00, (b) 30, (c) 50, (d) 70, (e) 100, ( f ) 200, (g) 500, (h) 700, (i) 1,000, ( j ) 2,000, (k) 5,000, and (l) 7,000 nM Cu2+. Inset displays the calibration curves by plotting [(Io/I) 1] against log [Cu2+] where Io and I are the emission intensities of compound A in the absence and presence of Cu2+, respectively, and [Cu2+] is the Cu2+ concentration in moles per liter
5.0

Luo et al.

4.0

R=0.9975
( Io/I) -1
90
3.0 2.0 1.0 0.0 -7.6-7.4-7.2-7.0-6.8-6.6-6.4-6.2-6.0-5.8-5.6-5.4-5.2-5.0

R=0.9954

a
80 70 Fluorescence intensity 60 50 40 30 20 10

2+ log [ Cu ]

420

440

460

480

500

520

Wavelength (nm)

possibly attributed to the static quenching of compound A by Cu(II) resulting in the formation of a non-fluorescent Cu (II)-compound A complex which is similar to other Cu(II)thiourea complexes [3133]. The complex formed by compound A (2.1 mmol L1) and Cu2+ (1.0 mmol L1) was synthesized to determine its fluorescence property at lex of 388 nm. Our result indicates that the Cu(II)compound A complex does not show any fluorescence and this can explain the fluorescence quenching of compound A by Cu2+ under the experimental conditions. In addition, it was observed that the compound A solution turned darker with the addition of Cu2+. Moreover, the emission peak is red-shifted (i.e., from 460 to 472 nm) on the addition of Cu2+ as shown in Fig. 3. Both the above phenomena suggested that a complex had been formed between compound A and Cu2+. Furthermore, the structure of the complex was determined by the Jobs method (Fig. S1 in Supplementary Information) and the results infer that the complex was formed from 1:2 of Cu2+/compound A as shown in step 2 of Scheme 1. The precision of the method was assessed by repeating six calibrations in the lower concentration range. The average value of the slope is 0.732 with a RSD of 0.70%. The limit of detection (LOD, 3) is 0.27 nmol L1 Cu2+.

For the higher concentration range, the change in fluorescence intensity is attributed to both the static quenching and inner filter effects of Cu2+ as Cu2+ has appreciable absorption at the excitation and emission wavelengths of compound A. As a result, the sensitivity for detection is higher in the higher Cu2+ concentration range. Effect of pH and concentration of compound A The effect of pH on the response of compound A to Cu2+ was investigated over the range 8.5 to 11.0. The normalized response against pH is shown in Fig. S2. The response could maintain above 99% at pH 9.5. Under the usual alkaline conditions, Cu2+ will form Cu(OH)2 precipitate. However, no Cu(OH)2 precipitate was observed in the reaction mixture at pH 10.0. It is possible that the concentration of compound A (0.50 mg mL1 2.1 mmol L1) is much higher than that of Cu2+ (0.50 mol L1) and OH (0.10 mmol L1) and the formation constant between compound A and Cu2+ is higher than that of OH. As a result, no Cu(OH)2 was found in our experimental conditions. At pH 10.0, the fluorescence intensity was highest. When pH was higher than 10.0, the response dropped because more OH ions are available to compete

A new luminol derivative as a fluorescent probe Scheme 1 The synthesis of compound A by the reaction between luminol and carbon disulfide (Step 1) and the complexation reaction of compound A Cu2+ (Step 2).

415

Step 1
O

NH NH

NaOH

+
NH

CS2
NH O

NH

NH 2

luminol SNa

compound A S

Step 2
O

NH

2
NH

Cu 2+

NH C

SNa

HN O

NH O S NH C S S O

HN

NH O

Cu2+
S

NH

The complex forms from compound A and luminol

with compound A to form Cu(OH)2, resulting in the decrease in response. Thus, pH 10.0 was chosen to be the optimal working condition for most experimental studies. Finally, the effect of compound A concentration on the response to Cu2+ was studied (Fig. S3). The optimal concentration was found to be 2.1 mmol L1. Interference Twelve interfering ions were used to evaluate the selectivity of the proposed fluorescence quenching method. The fluorescence intensities of compound A with 5.0 mol L1 Cu2+ in the absence and presence of each interfering ions are summarized in Table 1. Compound A shows good selectivity to Cu2+ even in the presence of other interfering

Table 1 Effect of potential interferent ions on the response of 2.1 mmol L1 compound A to 5.0 mol L1 Cu2+ Interfering ions Ag+ Ni2+ Fe2+ Co2+ Hg2+ Mn2+ Zn2+ Al3+ Sn2+ Cr3+ Cd2+ KI Concentration (mol L1) 30 10 23 10 100 500 500 120 100 150 5,000 1,500 Response change Decrease by 3.26% Decrease by 10.3% Decrease by 12.5% Decrease by 5.35% Decrease by 3.76% Increase by 6.94% Decrease by 2.59% Increase by 1.55% Decrease by 6.78% Increase by 0.91% Decrease by 2.56% Increase by 4.17%

416 Table 2 Determination of Cu2+ in mineral water samples and the recovery test Sample Concentration of Cu2+ (nmol L1)a 160.58 140.35 170.34 Concentration of Cu2+ added (nmol L1) 30 50 30 50 30 50 Concentration of Cu2+ found (nmol L1)a 310.55 490.27 320.34 480.43 280.34 440.36 Recovery (%)

Luo et al.

RSD (%)

1 2 3
a

103 98 107 96 93 88

0.70 0.36 0.48 0.63 0.53 0.59

Average value from six determinations

ions. Ag+, Hg2+, Zn2+, Al3+, Cr3+, Cd2+, and KI produce slight interference (<5%). To our surprise, some of these ions only slightly affect the response even though their chemical properties are similar to Cu2+, demonstrating that compound A has excellent selectivity to Cu2+. On the other hand, Ni2+, Fe2+, Co2+, Mn2+ and Sn2+ present some interferences when their concentrations are higher than Cu2+. Analytical application Copper is an important element in the human body and is essential in several biological pathways. It is essential for life, but on the other hand it may be toxic to human depending on the dose. Accordingly, copper levels have to be monitored in environment. Three mineral water samples were obtained from local shops and analyzed by our fluorescence quenching method as displayed in Table 2. The Cu2+ contents in the samples were 160.58, 140.35, and 170.34 nmol L1 (n =6), respectively. The recovery tests for Cu2+ were performed by adding various known amounts of Cu2+ in the sample solutions. The amounts of added Cu2+ were then evaluated by the proposed method and shown in Table 2. The results demonstrate that the

proposed method is good and precise for determination of Cu2+ in mineral water at the ultra-trace level (nanomoles per liter).

Conclusion This work describes the synthesis of a new luminol derivative which shows strong fluorescence in alkaline conditions. It has been successfully employed as a fluorescent probe for Cu2+ analysis and the results are satisfactory. Table 3 shows a comparison of some reported fluorescence methods for Cu 2+ determination. Most literature methods [3438] exhibit wide linear range and good selectivity but only few of them possess nanomolar LOD [34]. Our newly developed fluorescence quenching method presents a number of attractive analytical features such as high sensitivity, wide linear range, low LOD, good stability, reproducibility, and selectivity. The luminol derivative is easy to prepare with low cost and can be used as a fluorescence probe for routine analysis of ultratrace level (i.e., nanomoles per liter) Cu2+ in real water samples.

Table 3 Comparison of performances of various fluorescence methods for determination of Cu2+ ion Analytical technique Fluorescence quenching Fluorescence quenching Fluorescence enhancement Fluorescence enhancement Fluorescence resonance energy transfer Fluorescence quenching Reagent Selectivity Linear range (nmol L1) 10.0200 2.51,000 (dynamic range) 1,00050,000 (in aqueous solution) 5,00080,000 (in acetonitrile) 10010,000 3914,000 (dynamic range) LOD (nmol L1) Reference

L-Cysteine-coated

CdSe/CdS core-shell quantum dots Europiumtetracycline complex Rhodamine B hydrazide oxalamide

Good Good Good

3.0 200 640 (in aqueous solution) 37 (in acetonitrile) 64

[34] [35] [36]

A spiro form fluorescein hydrazide Porphyrazine 2,7,12,17-tetra-tertbutyl-5,10,15,20-tetraaza-21H,23Hporphine A thiourea-based luminol derivative (compound A)

Good Good

[37] [38]

Good

0.0500 (low linear range) 5007,000 (high linear range)

0.27 (low linear range)

Present work

A new luminol derivative as a fluorescent probe Acknowledgement This study was supported by grants from National Natural Science Foundation of China (20570542) and the Innovation Foundation of Sichuan University (2006G006). 19.

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