A study of fluorescence properties of citrinin in β-cyclodextrin aqueous solution and different solvents

Journal of Luminescence 132 (2012) 1437–1445

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A study of fluorescence properties of citrinin in b-cyclodextrin aqueous solution and different solvents Youxiang Zhou b,1, Jianbiao Chen a,1, Lina Dong a, Liang Lu a, Fusheng Chen a,c,d, Dingjin Hu b, Xiaohong Wang a,c,n a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China Institute of Quality Standard and Testing Technology for Agro-products, Hubei Academy of Agricultural Sciences, Wuhan, Hubei Province 430064, PR China c Key Laboratory of Food Safety Evaluation of the Ministry of Agriculture, Wuhan, Hubei Province 430070, PR China d National Key Laboratory of Agro-microbiology, Wuhan, Hubei Province 430070, PR China b

a r t i c l e i n f o

abstract

Article history: Received 14 October 2010 Received in revised form 1 January 2012 Accepted 4 January 2012 Available online 12 January 2012

Citrinin (CIT) is a nephrotoxic mycotoxin initially isolated from filamentous fungus Penicilliu citrinum. It was later isolated from several other species, such as Aspergillus and Monascus. It has a conjugated, planar structure that gives it a natural fluorescence ability, which can be used to develop sensitive methods for detecting CIT in food. In this paper, we used the spectrofluorescence technique to study the effects of pH value, b-cyclodextrin (b-CD) and organic solvents on the CIT fluorescence intensity. The results show that lower pH value, aceitc acid, b-CD and acetonitrile can induce a higher fluorescence intensity of CIT, but methanol or H2O has a decreasing effect on the fluorescence intensity of CIT. Findings in this study provide a theoretical basis for development of a high sensitivity fluorescencebased trace analysis for CIT detection. & 2012 Elsevier B.V. All rights reserved.

Keywords: Citrinin b-cyclodextrin Fluorescence enhancement Solvent effect

1. Introduction (3R,4S)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid (CIT; Fig. 1), is a nephrotoxin produced by filamentous fungi Aspergillus, Monascus and Penicilliums species [1,2]. It is frequently found in a number of food and feed commodities. The contamination of CIT not only causes economic losses but also threatens human health due to its nephrotoxic activity and chronic toxic effect [3,4]. In Asia, Monascus is a common traditional fungi to prepare red mold rice, which is widely used as a natural food colorant or flavor. The incidents of CIT contamination were reported in commercial Monascus-fermented products, which had caused a great concern on its safety [5]. As a result, some regulations have set the maximum residue level (MRL) for CIT in human food, especially in East-Asian countries and regions. In China, the MRL of CIT in dry functional red kojic rice is set at 50 mg kg  1 [6]. Fluorescence properties of CIT are widely used to detect CIT in food and feed, due to the high sensitivity that can be achieved by instrumental analytical methods [7]. However, the arising problem in fluorescence detection methods, especially in high-performance

n Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China. Tel.:/fax: þ 86 27 87282927. E-mail address: [email protected] (X. Wang). 1 Youxiang Zhou and Jianbiao Chen are both first authors due to our equal contribution to this paper.

0022-2313/$ - see front matter & 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2012.01.005

liquid chromatography (HPLC), is that the sensitivity is strongly dependent on the solvent system, such as pH value, which has a great impact on the sensitivity for detecting CIT [8,9]. In addition, previous studies showed that the fluorescence intensity of the target compound, such as aflatoxin B1, aflatoxin G1, ochratoxin A and zearalenone, could be increased when cyclodextrins (CDs) were added as derivatization reagents [10–13]. We thus hypothesize that appropriate solvent system, with the existence of CDs, can enhance the fluorescence intensity of CIT, which will be beneficial to its trace analysis. CDs, cyclic (a-1,4)-linked oligosaccharides of a-D-glucopyranose (CDs) are widely used in the clinical, food, cosmetics and environmental industries [14–16]. The most common CDs have six, seven and eight glucopyranose units and are referred to as a-, b- and g-CD, respectively [17]. Those CDs with hydrophobic, size selective toroidal cavity can accommodate small molecules (molecular weight o500 Da) as ‘‘guests’’ through thermodynamically driven force [18,19]. Among them, b-CD, with internal ˚ has been studied extensively in cavity diameter about 7.8 A, many fields (Fig. 2) [20]. Previous researchers reported that CDs were able to embed mycotoxin in their hydrophobic cavity to form inclusion complex [21–23]. The inclusion complex induced significant changes on the physical and chemical characteristics of mycotoxins, especially on their fluorescence properties [24,25]. Particularly, the fluorescence enhancement effect on mycotoxins is mainly attributed to the formation of conventional hydrophobic

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H

H O

O

1

8

O

7

2

O

3

6

H

12

5

3

O

9 10

11

O2

7

H

4

O

1

8

12

O

O

O

4 5 11

p-quinone

9 10

o-quinone

Fig. 1. Structural formula of citrinin. CIT exists in solution as an equilibrium mixture of two tautomeric forms at room temperature.

HO H

H

H

H H

O H

O OH

HO

H

H O OH

HO H

O

OH H

O

H O

HO H H

HO

H H

H HO O

OH

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H HO

O H

H O

H

HO O

O OH H O H OH

H HO O

H H

H OH

HO H

OH

OH H

H H

O OH H H

H H

OH

Fig. 2. Chemical structure and approximate geometric dimensions of b-CD.

bonding, hydrogen bonding and van der Waals interactions between CDs and the fluorophore of mycotoxins [25,26]. Meanwhile, previous papers ascribed the fluorescence enhancement to the micro-environment provided by CDs, which led to a more effective shielding of the fluorophore from the fluorescence quencher attack [27]. CIT is an acidic mycotoxin with conjugated planar structure, which has natural fluorescence [28]. However, its fluorescence properties in aqueous solution have not been studied systematically. In this paper, the effects of acids, b-CD and organic solvents on the fluorescence properties of CIT were investigated by the spectrofluorescence technique. Solvent effects on the fluorescence intensity of host–guest inclusion complex were studied in phosphate buffer and acetate buffer, and then the mechanisms for fluorescence variance were analyzed. These results will be useful to establish a suitable solvent system that can increase the sensitivity of CIT in fluorescence-based trace CIT analysis.

2. Materials and methods 2.1. Apparatus The fluorescence spectra and intensities were acquired on a model F-2700 spectrofluorimeter (Hitachi, Japan) with a quartz

cell (1  1 cm2 cross-section) equipped with a xenon lamp and a dual monochromator at 25 1C. The fluorescence intensity of CIT was set at lex/lem ¼330 nm/500 nm, slitex/em ¼20 nm/10 nm. All the measurements were performed after stabilizing this solution for 1 h at 25 1C. 2.2. Reagents CIT was purchased from Alexis (Lausen, Switzerland). Spectroscopic grade methanol and acetonitrile were obtained from J.T. Baker (New Jersey, USA). b-CD, acetic acid, anhydrous sodium acetate, phosphoric acid, disodium phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate were commercial products of analytical purity from Sinapharm (Shanghai, China) and water was prepared by Milli-Q ultra-pure water system (Millipore, USA). The acetate buffer with different pH values was prepared by adding 2.0 mol L  1 sodium acetate to 100 mL of 2.0 mol L  1 acetic acid solution. The buffer of 0.28 mol L  1 sodium dihydrogen phosphate or disodium hydrogen phosphate was adjusted to the required pH value by addition of 0.28 mol L  1 phosphoric acid. All buffers were prepared fresh before use and pH was measured by a P310 pH meter (Thermo, USA). Aqueous stock solution of CIT (1.2  10  1 mmol L  1) was prepared by dissolving appropriate amount of CIT in water, and

Y. Zhou et al. / Journal of Luminescence 132 (2012) 1437–1445

the working solution of CIT (3.2  10  4 mmol L  1) was prepared by several serial dilutions. Stock standard solutions of b-CD (6 mmol L  1) were prepared with acetate buffer and phosphate buffers . All the solutions were kept in dark at 4 1C for no more than 1 week.

2.3. Procedures 2.3.1. Effect of pH value on spectrum of CIT Samples in different pH solutions (pH 2.0, 2.5, 3.0 and 3.5) were prepared by diluting the stock solution of CIT with acetate

60 pH2.0 pH2.5 pH3.0 pH3.5

50

Ex

40

buffer or phosphate buffer. The working concentration of CIT was set at 3.2  10  4 mmol L  1. The emission and exciting spectra of CIT were scanned. All the measurements were carried out after the sample had been stabilized for 2 h in dark at 4 1C. 2.3.2. Effect of b-CD on fluorescence spectrum of CIT The analysis of effect of b-CD on the fluorescence intensity of CIT was performed with the concentration of CIT constant at 3.2  10  4 mmol L  1, while the b-CD concentration was varied from 0 to 7 mmol L  1 (0, 0.1, 0.5, 1, 3, 5 and 7 mmol L  1); CIT/ b-CD solutions were obtained by adding appropriate aliquots of CIT and b-CD stock solution and then diluting with the corresponding buffer to their working concentrations. The measurements were carried out after the sample had been stabilized for 2 h in dark at 4 1C. The binding constant of CIT/b-CD was calculated by assuming a 1:1 stoichiometric ratio, according to the Benesi–Hildebrand equation [23,26,29]: 1 1 1 ¼ þ F i F 0 ðF 1 F 0 ÞK½CDi F 1 F 0

Em

where K is the complex binding constant, Fi and F0 are the fluorescence intensities of CIT in the presence and in the absence of b-CD, respectively, FN is the fluorescence intensity of the complex and [CD]i is the b-CD concentration after each addition. The modeling of b-CD, the inclusion process and the fluorescence quenching process were performed with ChemBioOffice 2008 software.

30

20

10 λ em = 500 nm

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λ ex = 330 nm

0 300

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Wavelength (nm)

60 ex

pH2.0 pH2.5 pH3.0 pH3.5

em

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40

2.3.3. Effects of solvents on fluorescence intensity of CIT and CIT/b-CD The content of organic solvents in the final aqueous solution has a large influence on the fluorescence intensity of CIT. The influences of methanol and acetonitrile on the fluorescence spectrum of CIT (3.2  10  4 mmol L  1) were investigated by diluting CIT stock solution with a mixture of buffer and organic solvent. The percent of organic solvent in the mixture was varied from 0 to 60% (v/v). The influences of methanol and acetonitrile on the fluorescence of CIT/b-CD (3.2  10  4 mmol L  1/ 3 mmol L  1) were also analyzed by the same procedure.

30

3. Results and discussion

20

3.1. Effects of pH value on spectrum of CIT 10 λ em = 500 nm

λ ex = 330 nm

0 300

350

400

450

500

550

600

Wavelength (nm) Fig. 3. Fluorescence excitation (ex) and emission (em) spectra of CIT in 0.28 mol L  1 phosphate buffer (a) and 2.0 mol L  1 acetate buffer (b).

A brief spectroscopic study was performed in order to explore the relationship between the spectrum of CIT and the pH of solvent. Excitation and emission spectra of CIT obtained in acetate buffer and phosphate buffer at different pH values are shown in Fig. 3. It reveals that the pH value of solution has remarkable effects on the fluorescence intensity of CIT. It is noteworthy that the increase in CIT fluorescence intensity with the decrease in pH

-[H+] +[H+] protonated form

deprotonated form Fig. 4. Protonated and deprotonated forms of CIT.

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value from 3.5 to 2.0 is attributed to the pKa of CIT at 2.3 in water [30], and the protonated form of CIT dominated in the lower pH solution (as shown in Fig. 4). It is easy for CIT to form intramolecular hydrogen bonds linking the phenol and keto functions to the carboxyl group of the CIT nucleus. However, when the pH value of solvent increases, the dissociation of intra-molecular hydrogen bonds caused by the increased pH value can lead to the decrease in p electron delocalization, which reduces the fluorescence quantum yield [31]. Though the trend of CIT fluorescence related pH variation is similar, a higher fluorescence intensity of CIT in acetate buffer was found at the same level pH value, which was 1.5 times higher than that in phosphate buffer. This implies that acetic acid not only provides [H þ ] to induce intermolecular hydrogen bond in CIT, but also supplies exogenous intra-molecular hydrogen bond based on the Michael-type nucleophilic addition reaction at C1 in the o-quinone form of CIT (shown in Fig. 5). So the reaction can form the p–p conjugation of C–O between CIT and acetic acid, and enhance the p electron delocalization of CIT [1,32]. On the other hand, similar results were also observed in the absorption spectrum of CIT under the same acidic conditions (data not shown). 3.2. Effects of b-CD on fluorescence spectra of CIT To study the effect of b-CD on the fluorescence of CIT, the fluorescence intensities of CIT in two kinds of buffers containing b-CD are measured and shown in Fig. 6. The results show that when the pH value of buffer is at the same level, adding b-CD to phosphate or acetate buffer has a distinct influence on the CIT’s fluorescence intensity. In phosphate buffer, the addition of b-CD induces the obvious increase of fluorescence intensity of CIT, but in acetate buffer, b-CD seems to have no obvious effect on CIT’s fluorescence. Although acetic acid can improve the fluorescence intensity of CIT more effectively than [H þ ], it might play an unfavorable role on fluorescence enhancement between CIT and b-CD. In phosphate buffer, the fluorescence increase is mainly due to the suitable micro-environment provided by the hydrophobic cavity of b-CD, where the molecule of CIT can be inserted. Previous reports showed that Van der Waals interactions, hydrogen bonding, ion-pairing, p–p electron interactions, electrostatic interactions and charge transfer were generally believed to control the complexation processes [13,22]. In this work, we speculate that three significant factors contribute to the nonradiative energy loss (Fig. 7):

fluorescence signal becomes weak [33]. Once CIT is inserted into the cavity of b-CD, the hydrophobic micro-environment provided by b-CD prevents water molecules from quenching the CIT fluorescence, and induces a stronger fluorescence signal [34]. (b) After CIT enters into the b-CD cavity, the polarity of microenvironment in the complex decreases and causes a larger S1–S0 energy gap. The increased energy gap causes a significantly reduced rate of internal conversion, which is the dominant nonradiative decay pathway competing with fluorescence [24]. (c) When CIT is included in b-CD cavity, rotational freedom and vibrational lever relaxation (VR) caused by solvent molecules significantly reduce. Moreover, the number of deactivated CIT molecules caused by VR also decreases greatly. Moreover, the similar result of increasing pH value on the growing fluorescence intensities of CIT indicates that the formation of intra-molecular hydrogen bond is affected by [H þ ]. No matter whether CIT was inserted into b-CD cavity or not, high concentration of [H þ ] was helpful for maintaining CIT in protonated form with high fluorescence quantum yield. It implied that some active groups, including phenol, keto and carboxyl groups of the involved CIT, might be adjacent to the rims of b-CD cavity, which would be easily influenced by [H þ ] in solution. Therefore, the benzopyran unit of CIT might be included inside the b-CD cavity [35].

80 60 40 20 0

2.0 7.0 β

5.0

2.5 3.0 1.0

3.0 0.5

3.5

0.1 (a) When CIT is excited by ultraviolet light in the aqueous solution, the energy transforms to fluorescence signal. However, due to the vibrational coupling interaction between CIT and its surrounding water molecules, the fluorescence energy is partially released as heat in the solution. Therefore the

0 -1

Fig. 6. Fluorescence intensities of CIT measured as a function of b-CD concentration in the buffer aqueous mixtures. (’) Phosphate buffer solution and (&) acetate buffer solution. lex and lem are 330 and 500 nm, respectively.

OH O

C CH3

protonated form

100

phosphate buffer solution acetate buffer solution

acetic acid/CIT adduct

Fig. 5. Michael-type nucleophilic addition reaction between o-quinone form of CIT and acetic acid.

Y. Zhou et al. / Journal of Luminescence 132 (2012) 1437–1445

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a. vibrational coupling H2O b. internal conversion

0

c. other non-radiative decay

absorption

1

emission

1

a. vibrational coupling H2O b. internal conversion c. other non-radiative decay

emission

1

0

0

Fig. 7. A schematic diagram illustrates the mechanism of fluorescence enhancement of CIT under the effect of b-CD in aqueous solution.

80

70

λ ex= 330 nm

hc hc

60

50

40 Fig. 9. A schematic diagram illustrates the blue-shift of the maximum lem of CIT.

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2.5

blue shift

Phosphate buffer solution

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pH=2.0, R2=0.9931 pH=2.5, R2=0.9389 pH=3.0, R2=0.9746 pH=3.5, R2=0.9956

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1/[β-CD](Lmol-1) 30

Fig. 10. Benesi–Hildebrand plot of 1/(Fi  F0) vs 1/[b-CD] of CIT in the presence of b-cyclodextrin in 0.28 mol L  1 phosphate buffer solution. This experiment is carried out at [b-CD] ¼ 0, 0.1, 0.5, 1, 3, 5, and 7 mmol L  1. The lex and lem are 330 and 500 nm, respectively.

20 460

480

500

520

540

Wavelength (nm) Fig. 8. Emission spectra of CIT (3.2  10  4 mmol L  1) at different concentrations of b-CD in 0.28 mol L  1 phosphate buffer (a) and 2.0 mol L  1 acetate buffer (b). Variation of the emission intensity as a function of b-CD concentration for 0, 0.1, 0.5, 1, 3, 5, and 7 mmol L  1.

The influences of b-CD on the fluorescence spectrum of CIT in pH 2.5 buffer are presented in Fig. 8. After adding b-CD to CIT solution, obvious blue-shift in the emission spectra and increasing fluorescence intensity are only exhibited in phosphate buffer (as shown in Fig. 8a). The blue shift caused by b-CD is consistent with

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the previous report [36]. In general, the extent of this shift roughly indicates whether part or all of the guest molecules are included by the host CD, though more conclusive evidence regarding the relative orientation of the guest in the hydrophobic cavity of the host may require the use of other spectroscopic techniques, such as NMR spectroscopy [37,38]. The observed fluorescence enhancement indicates more interaction of CIT with the less protic interior of the cyclodextrin cavity than with the aqueous phase; apparently, interactions between CIT and the CD

interior lead to higher rigidity of the complex, subsequently increasing the energy of S1 and S0. As a result, after the CIT molecule is embedded into the b-CD cavity, lower polarity and larger S1–S0 energy gap of the included CIT molecule can induce a blue-shift in its fluorescence spectrum. However, it is pointed out that no obvious blue-shift or fluorescence intensity increasing reveals that the containing ability of b-CD is weak in acetic buffer (Fig. 8b). The increasing fluorescence intensity of CIT at a higher concentration of b-CD might imply the possibility of complex

Fig. 11. A schematic diagram illustrates the mechanism of fluorescence enhancement of CIT under the effect of b-CD in aqueous solution. (a) CIT is inserted into b-CD cavity in the absence of acetic acid, (b) CIT is not inserted into b-CD cavity due to the larger size of acetic acid/CIT complex, and (c) CIT is not inserted into b-CD cavity due to the formation of hydrogen bond between b-CD and acetic acid/CIT complex.

80 acetonitrile methonal

80

acetonitrile methonal

60

60

40 20 0

40 20 0

Fig. 12. Fluorescence intensities of CIT measured as a function of the organic solvent percentage in the buffer aqueous mixtures: (a) 0.28 mol L  1 phosphate buffer solution and (b) 2 mol L  1 acetate buffer solution, [CIT]¼ 3.2  10  4 mmol L  1, lex and lem are 330 and 500 nm, respectively.

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In acetate buffer, the main factors related to CIT’s fluorescence intensity are acetic acid and pH value, but not b-CD. The unfavorable effects of acetate on CIT and b-CD are two possible reasons. Firstly, the size of CIT molecule increases after the Michael-type nucleophilic addition reaction between acetic acid and CIT [1]. Moreover, larger size addition product may be formed by the hydrogen bond reactions (C¼ O H–O, O–HO) between the keto group of acetic acid/CIT complex and the carboxyl group of free acetic acid in aqueous solution [40]. Therefore, this bigger complex cannot be accommodated by the relatively smaller b-CD cavity (Fig. 11b). Secondly, CIT may not be able to enter into the b-CD cavity due to hydrogen bond reactions between hydrophilic hydroxyl groups of b-CD and keto group of acetic acid/CIT complex (Fig. 11c).

formation between CIT and b-CD at a ratio of 1:2 in acetate buffer [39]. A schematic diagram illustrating the mechanism of blueshift induction is presented in Fig. 9, where DE, l, h and c refer to radiant energy, the maximum emission wavelength, the reduced Planck constant and velocity of light, respectively. DH1 and DH2 refer to the changes of the first excited electronic singlet state S1 and the ground state S0, respectively. A plot of 1/(Fi–F0) vs 1/[b-CD] for different concentrations of b-CD in phosphate buffer is shown in Fig. 10. This plot suggests that the formation of 1:1 host–guest complex between CIT and b-CD is in good correlation [19]. However, 1/(Fi F0) vs 1/[b-CD] is nonlinear in acetate buffer, which implied that CIT was not involved completely in the hydrophobic cavity of b-CD in acetate buffer.

CH3

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O

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3

o-quinone CIT

CO

C

H

OH

OH

O

O

CH3 O

O H O

acetic acid/CITadduct Fig. 13. Michael-type nucleophilic addition reaction between CIT and nucleophile (methanol or acetic acid).

H H C H O H

H HO

H band

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O

H

C H

C H

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H

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no energy loss

Fig. 14. A diagram illustrates the mechanism of acetonitrile on the fluorescence enhancement of CIT.

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The structural representations of the mechanisms of CIT with b-CD in aqueous solution are presented in Fig. 11. 3.3. Effects of solvent on fluorescence intensity of CIT and CIT/b-CD complex In this work, methanol and acetonitrile were chosen as the representatives of organic solvent because of their wide use in the mobile phase of reverse phase HPLC. Fig. 12 shows comparative results of the fluorescence intensity variation against the organic solvent percentage of methanol and acetonitrile in phosphate buffer (a) and acetate buffer (b). The effect of methanol is in agreement with the previous study on the effect of methanol on fluorescence intensity of aflatoxin B1/b-CD [11]. When the content of methanol varied from 0% to 60% (v/v) in phosphate buffer, the fluorescence intensity of CIT decreases; in contrast, it increases in the case of acetonitrile (Fig. 12a). It is noteworthy to mention that for methanol the fluorescence intensity in all cases obviously decreases. The decreased intensity of CIT is ascribed to the reaction between methanol and CIT. The hydroxyl in methanol as a nucleophile can easily react with C1 in CIT, just like the addition reaction between CIT and acetic acid [1] (shown in Fig. 13). This addition reaction at C1 of CIT dwindles the p electron delocalization size of CIT and reduces fluorescence quantum yield of CIT. As a result, methanol as a polar protic solvent can not only be helpful for H2O molecule quenching CIT by hydrogen bonds among them, but also act as a static quencher to react with CIT. Moreover, the higher intensities of CIT at the same pH of acetic buffer than those in the phosphate buffer proves that methanol can only partially prevent acetic acid from forming the adduct of acetic acid–CIT, which contains p-conjugated systems and has a higher fluorescence quantum yield (Fig. 12b). On the other hand, acetonitrile is distinct from methanol not only in phosphate buffer but also in acetate buffer. Fig. 12a shows that adding acetonitrile in phosphate buffer is good for enhancing fluorescence intensities of CIT. However, similar results were observed only in pH 2.0 acetate buffer, with no obvious enhancement, and even slight decrease of intensity was found in acetate buffers of pH 2.5–3.5, as shown in Fig. 12b. In phosphate buffer, the enhancement effect of acetonitrile on the fluorescence intensity of CIT is attributed to the polar aprotic characteristics of acetonitrile [19], which can obstruct the vibrational coupling between water and CIT and provide a hydrophobic micro-environment to maintain CIT in its protonated form. A schematic diagram illustrating the mechanisms of fluorescence quenching and fluorescence enhancement for CIT is presented in Fig. 14. However, considering the pKa value 2.3 of CIT [30], it is mainly in the molecular forms when the pH is above 2.3. Therefore, the competition between acetonitrile and acetic acid can cut down the acetic acid/CIT adduct in weak acidic environment (pHZ2.5), and induce a slight decrease of CIT’s fluorescence intensities. When the pH value of acetic buffer is at 2.0, the ionic form of acetic acid/CIT adduct is the dominant form. The rising intensity of CIT in pH 2.0 acetate buffer is assumed to be the combined effects of both acetonitrile and acetic acid. The influences of methanol and acetonitrile on the emission intensity of CIT in different aqueous solutions with 3 mmol  1L bCD are presented in Fig. 15. The similar quenching effect of methanol against CIT verifies that methanol is a fluorescence quencher to CIT again, and this process may be mainly attributed to the static quenching [11,21]. However, in acetate buffer, there is no obvious difference on the fluorescence intensities whether b-CD exists or not. This result also confirms that acetate acid can be added to CIT to form a higher fluorescent addition, and this addition is not involved in the hydrophobic cavity of b-CD.

80

acetonitrile methonal

60

40 20 0

80

acetonitrile methonal

60

40 20 0

Fig. 15. Fluorescence intensities of CIT measured as a function of the organic solvent percentage in the buffer aqueous mixtures with b-CD: (a) 0.28 mol L  1 phosphate buffer solution, and (b) 2 mol L  1 acetate buffer solution, [CIT]¼ 3.2  10  4 mmol L  1, [b-CD]¼3 mmol L  1, lex and lem are 330 and 500 nm, respectively.

In addition, in phosphate buffer, as shown in Fig. 15a, acetonitrile has an interesting influence on the fluorescence intensity of CIT with the existence of b-CD. This positive effect of acetonitrile on CIT is consistent with other report [30]. We assume that this variational fluorescence intensity of CIT is derived from the competition among b-CD, acetonitrile and H2O. When no acetonitrile is added in solution, b-CD is able to contain CIT in its hydrophobic cavity and enhance CIT’s fluorescence intensity. However, when the percent of acetonitrile is 15%, acetonitrile can substitute CIT in the cavity of b-CD, and expose CIT to the condition of surrounding H2O molecule, so the vibrational coupling between the fluorophore of CIT and the nearby H2O decreases the intensity of CIT [11]. While the percent of acetonitrile is over 15%, acetonitrile is the dominant factor, and can provide a hydrophobic micro-environment to prevent H2O

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from quenching CIT’s fluorescence, so the rising fluorescence intensity of CIT is observed. In acetate buffer, as shown in Fig. 15b, competitions among acetonitrile, b-CD and HeO also exist, but acetate is the mainly factor to enhance the fluorescence intensity of CIT, so acetonitrile or b-CD had no obvious impact on CIT’s intensity. It is noted that fluorescence intensity of CIT increases with added acetonitrile in the solution at pH¼2.0. This trend in the solution at pH¼ 2.0 might be due to the combined effect of [H þ ] and acetonitrile.

4. Conclusions In this paper, the impacts of pH, b-cyclodextrin and organic solvents on the fluorescent character of CIT were investigated by fluorescence spectra systematically. The results reveal that [H þ ], acetic acid, b-CD and acetonitrile can induce fluorescence enhancement of CIT in aqueous solution by increasing p electron delocalization, formation of Michael-addition adduct, formation of inclusion complex and protection of fluorophore of CIT from fluorescence quencher, respectively. Factors responsible for the reduction of CIT fluorescence include methanol and H2O, owing to Michael-type nucleophilic addition reaction and fluorescence quenching. It is also found that the 1:1 inclusion complex of CIT/b-CD can form in acidic phosphate solution, but acetic acid can prevent formation of this inclusion complex. Thus, this paper provides useful parameters to develop fluorescence-based trace CIT analysis in future study.

Acknowledgments This research was partly supported by the project of Chinese National Natural Science Foundation (no. 30871754), Hubei Innovation Center of Agricultural Science and Technology (no. 2011-620-000–001), the earmarked fund for Modern Agro-industry Technology Research System of China (no. nycytx-45-15) and the Dawn Program for Youth Pioneering in Technology of Wuhan (no. 201050231082). References [1] R. Poupko, Z. Luz, R. Destro, J. Phys. Chem. A 101 (1997) 5097.

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