Amino acid detection using fluoroquinolone–Cu2+ complex as a switch-on fluorescent probe by competitive complexation without derivatization

Journal of Luminescence 145 (2014) 708–712

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Amino acid detection using fluoroquinolone–Cu2 þ complex as a switch-on fluorescent probe by competitive complexation without derivatization Alireza Farokhcheh, Naader Alizadeh n Department of Chemistry, Faculty of Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 26 May 2013 Received in revised form 17 August 2013 Accepted 21 August 2013 Available online 30 August 2013

In this work, we describe the use of fluoroquinolone–Cu2 þ complex as a competitive switch-on fluorescence probe for amino acid determination without derivatization. The fluorescence intensity of this probe, which has been reduced due to effective quenching by Cu2 þ ion, increases drastically by an addition of amino acid (glycine, phenylalanine, sarcosine, aspargine, alanine, proline, arginine, aspartic acid, glutamic acid, lysine, leucine and isoleucine). The overall stability constants of Cu2 þ ion complexes with amino acids were determined by fluorometric titration of fluoroquinolone-Cu2 þ complex with the amino acid solution. Furthermore, the probe shows high calibration sensitivity toward aspartic acid. The fluorescence signal depends linearly on the amino acid concentration within the range of concentration from 1.2  10  7 to 1.1  10  5 mol L  1 for aspartic acid. The detection limit was found 2.7  10  8 mol L  1 with the relative standard deviation (RSD%) about 2.1% (five replicate). & 2013 Elsevier B.V. All rights reserved.

Keywords: Switch-on fluorescence probe Amino acid detection Aspartic acid Glutamic acid Fluoroquinolone Without derivatization

1. Introduction Analysis and determination of amino acids have been considered a very important issue in many of the chemical and biological researches because of their crucial roles. Among them, aspartic acid (Asp) is of great importance in neurocrine and endocrine functions [1]. L-Asp racemization ratios in human femur have been used for age estimation [2]. In clinic, L-Asp is necessary for the treatment of heart disease, hepatopathy, and hypertension; it can also help people providing and recovering from fatigue. L-Glutamic acid (Glu) is a precursor for amino butyric acid, which is the main inhibitory neurotransmitter in the central nervous system [3]. A rapid and convenient determination method is required in many fields. Therefore, sensing amino acids in aqueous and bio-fluids solutions could be of great advantage. High-performance capillary electrophoresis (HPCE) and high-performance liquid chromatography (HPLC) with fluorescence detection are so far the most commonly used methods for determination of amino acids. Precolumn derivatization

Abbreviations: AA, amino acid; Gly, glycine; Phe, phenylalanine; Sar, sarcosine; Asn, aspargine; Ala, alanine; Pro, proline; Arg, arginine; Asp;, aspartic acid; Glu, glutamic acid; Lys, lysine; Leu, leucine; Ile, isoleucine; RSD, relative standard deviation; LOD, limit of detection; HPLC, high-performance liquid chromatography; HPCE, high-performance capillary electrophoresis; Cip, fluoroquinolone n Corresponding author. Fax: þ98 21 82883455. E-mail address: [email protected] (N. Alizadeh). 0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.08.039

generally improves the detection of the chromatographic system [4]. Moreover, UV–vis absorption or fluorescence is used to detect and quantify the separated components. However, these techniques may frequently suffer from this fact that many amino acids exhibit very weak absorbance and only at short wavelengths. For this reason, a number of analysis methods with derivatization process have been developed to convert non-fluorescent amino acids into highly fluorescent derivatives for the purposes of detection. However, derivative reactions are very intricate and time consuming [5]. Other methods have employed the competitive or direct interaction of amino acid with the indicators. In competitive methods, fluorescent indicator is bound to the receptor through non-covalent interactions (such as coordination), and the fluorescence of the indicator is either quenched or enhanced by the receptor [6,7]. Fluoroquinolone (Cip) or [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazinyl)-quinolone-3-carboxylicacid] (Fig. 1) is one of the third generation members of synthetic fluoroquinolone which exhibits a great level of greater intrinsic antibacterial activity. The fluoroquinolones molecule can provide two groups of attachment to the central metal atom, namely 3-carboxyl and 4-oxo. Consequently, these groups can form bonds of ionic or coordinate covalent character to the central cation, creating a chelate-type complex. However, data on complex formation and its role in vivo reported in the literature are, divergent. In spite of different experimental approaches and conclusions, it is clear that the stability of the complex is highly dependent on the nature of the quinolone, the involved metal ion and environmental conditions. In

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3. Results and discussion 3.1. Spectral characteristics

Fig. 1. Molecular structure of Cip.

the case of Cip, Kmetec et al. have shown that copper can create stable complexes with Cip [8]. Recently, we have investigated the effect of silver nanoparticles concentration on the enhancement and quenching of fluorescence intensity of Cip [9]. In this paper, the interaction of Cu–Cip and amino acids (AAs) was systematically investigated by utilizing fluorescence spectroscopy and the competitive reaction between Cip and AAs with Cu2 þ ions. We utilized non-fluorescent Cu–Cip complex as a new probe to study the complexation of Cu2 þ ions with different amino acids in phosphate buffer solutions (pH 7). The stoichiometry and overall stability constants of Cu2 þ ion complexes with amino acids were determined by fluorometric titration of Cu–Cip complex with the amino acid according to the competitive reaction mechanism. The proposed mechanism for the detection of AAs is discussed. Furthermore, we also found that Asp and Glu recognizes Cu2 þ ions to form a Cu–AA complex based on the fluorometric titration data. The fluorescence signal depends linearly on the amino acid concentration.

The fluorescence emission spectra of Cip in the absence and presence of Cu2 þ ions are depicted in Fig. 2. As shown in Fig. 2, with the excitation wavelength at 330 nm (curve a), the maximum emission wavelength of Cip was 435 nm (curve b). The fluorescence intensity of Cip decreased with increasing concentration of Cu2 þ ions (curves c) indicating the fluorescence quenching. No change in the maximum emission wavelength of Cip was observed. This behavior, however, cannot be used as an evidence for non-fluorescent complex presence, as this would change the fluorescence intensity of Cip peak as well. This observation can be attributed, in accordance with the known high affinity of Cu2 þ ions for quinolone groups, to interactions of this cation with free carbonyl and carboxylic groups present in the Cip molecule as a ligand [12–16]. The fluorescence intensity changes can be analyzed by plotting the relative fluorescence emission intensity of Cip (I/I0) at λmax ¼435 nm versus the concentration of Cu2 þ ions (Fig. 3). This indicates that the Cu2 þ ions could strongly quench the fluorescence of the Cip system. It can be seen that at concentrations above 1  10  5 mol L  1 of Cu2 þ ions, rather complete loss of Cip fluorescence is observed. In our experiments, for detection purposes, the phosphate buffer solution at pH ¼ 7 was used. The purpose of avoiding acidic conditions during detection was to use conditions favoring the stability of complex. Under experimental conditions (low concentration of Cip and Cu2 þ ions), the quenching of fluorescence, observed in this study, arises most probably from the static quenching and is presumably due to the formation of a non-fluorescent complex between the

2. Materials and methods

2.2. Instrumentation The fluorescence spectra were recorded by a Perkin-Elmer model LS 50B spectrofluorimeter equipped with a thermostated cell compartment. The pH values of the solutions were adjusted employing a Metrohm pH meter Model 632. All measurements were performed in 10 mm quartz cells, at 2570.1 1C, by use of a thermostatic cell holder and a Thermomix thermostatic bath. The results of interaction between the amino acid and Cip–Cu were analyzed according to the mass balance and fluorescence data by applying a deduced equation.

Fluorescence Intensity

Glycine (Gly), phenylalanine (Phe), sarcosine (Sar), aspargine (Asn), alanine (Ala), proline (Pro), arginine (Arg), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), leucine (Leu) and isoleucine (Ile) were purchased from Merck. Copper sulfate, sodium hydroxide, hydrochloric acid and potassium dihydrogen phosphate were procured from Fluka. Fluoroquinolone (Cip) hydrochloride monohydrate powder was obtained from Amin Co., Iran, which was synthesized according to the method reported in the literature [10,11]. Stock standard solution of 10  4 mol L  1 was prepared by dissolving accurately measured amounts of Cip in double distilled water and kept in the dark at 4 1C. The solutions were buffered properly prior to the analysis. The buffer solutions were prepared by mixing appropriate volumes of 0.2 mol L  1 HCl and 0.2 mol L  1 KCl over the pH 1–5 and 0.1 mol L  1 NaOH and 0.01 mol L  1 KH2PO4 for pH 5–10. Pyrrole was obtained from Merck and it was distilled before use.

a

700

b

600

d

500 400 300 200 c

100 0 250

300

350

400

450

500

550

600

Wavelength (nm) Fig. 2. Excitation (a) and emission spectra of 4  10  7 mol L  1 Cip (b) and Cu–Cip (c) in phosphate buffer solution (pH 7) with added 2  10  6 mol L  1 Asp (d) and their photographs of solutions (inset).

1.2

Normalized Fluorescence

2.1. Procedure and reagents

800

12 10 8 6 4 2 0

1 0.8 0.6

0

5

10

15

20

25

0.4 0.2 0 0

5

10

15

20

25

[Cu2+]×10-5 mol L-1 Fig. 3. Quenching effect of Cu2 þ ions on the fluorescence intensity of Cip solution ([Cip]¼ 4  10  7 mol L  1) and Stern–Volmer plot (inset) at 298 K.

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Cip and Cu2 þ ions. The formation constant of Cu–Cip complex was determined using the Stern–Volmer method. The Stern–Volmer equation [17] accounting for quenching is written as Io=I ¼ 1 þ K sv ½Cu2 þ 

ð1Þ

where Ksv is the quenching constant, Io and I are the fluorescence intensity of probe molecule in the absence and presence of quencher [Cu2 þ ], respectively. The results of the fluorescence titrations plotted according to the Stern–Volmer equation were inserted in Fig. 3. Accordingly, the slope of the Stern–Volmer plot, therefore, is equivalent to the formation constant for the Cu–Cip complex. The plot is linear with Ksv ¼4.5  105 L mol  1. The linearity of the Stern–Volmer plot indicates that only one type of quenching occurs in the system. 3.2. The influence of amino acid on fluorescence properties of Cu–Cip The fluorescence intensity of Cip is strongly reduced by Cu2 þ ions which bind to the carboxyl moieties acting as a quencher [16]. To evaluate Cu–Cip complex as a fluorescent probe for the detection of AA, various concentrations of AA were added to Cu–Cip complex solution. When AA is added to a solution of Cu–Cip complex, the Cip in Cu–Cip complex is preferably exchanged with AA to form a Cu–AA complex; as a result, quenching by Cu2 þ ions would be suppressed and the emission from the Cip would be increased drastically. Indeed, it was previously confirmed, via a potentiometry method, that AAs have a strong affinity for Cu2þ ion forming a Cu–AA complex [18]. The proposed mechanism for detection of AAs is illustrated in Scheme 1. Fig. 4a shows the normalized fluorescence intensity changes for Cip–Cu system in the presence of various concentrations of different AAs in 0.01 mol L  1 phosphate buffer solution (pH 7). The fluorescence intensity increased with increasing AA concentrations, indicating that the addition of AA diminishes the quenching by prevention of Cu2 þ ions from reacting with Cip due to formation of a Cu–AA complex, as a competitor. The response behavior of Cip–Cu probe to different amino acids was proved in Fig. 4b. This figure shows the fluorescence intensity of Cip–Cu system at 435 nm after the addition of 1.6  10  5 mol L  1 of different AAs to the 0.01 mol L  1 phosphate buffer solution containing [Cu2 þ ]¼2.5  10  6 mol L  1 and [Cip]¼ 4  10  7 mol L  1 as probe. The increase in fluorescence intensity, due to addition of Asp and Glu, could be easily distinguished from other AAs (Fig. 4b). Others exhibit a lower impact on fluorescence recovery of the solution. As will be shown, in addition to the high fluorescence intensity of Cip, its complex with copper is weaker than copper–amino acid complexes. This is the main reason that Cip was used as a probe for the recognition of AA by Cu2 þ ions. The difference between the fluorescence behaviors of Cu–Cip in the presence of different AA was exploited to determine the equilibrium

constants of Cu–AA complexes. Two competing binding processes coexist in the solution containing Cip, Cu2 þ ions and AA: CuðIIÞ þ Cip2Cu–Cip

ð2Þ

K Cip ¼ ½CipCu=½Cip½Cu2 þ 

ð3Þ

K1

Cu2 þ þ AA⟷CuðAAÞ

ð4Þ

K 12

Cu2 þ þ 2AA⟷CuðAAÞ2 K 1 ¼ ½CuðAAÞ=½Cu2 þ ½AA

ð5Þ and

K 12 ¼ ½CuðAAÞ2 =½Cu2 þ ½AA2

ð6Þ

The binding process can be analyzed assuming that each Cu2 þ center binds the amino acid competitors in a 1:2 (Cu:AA) ratio [19–21]. In the case of Cip, a 1:1 binding between the copper ions and the Cip is assumed. Overall competitive reaction could be shown as follows: Cu–Cip þ 2AA2CuðAAÞ2 þCip

ð7Þ

where AA and Cip represented amino acid and fluoroquinolone, respectively, and Cu(AA)2 and Cu–Cip were the corresponding complexes. Free and complexed concentrations of Cu þ 2 ions in solution could be obtained from the following equations: C Cu ¼ ½Cu2 þ  þ CuðAAÞ þ CuðAAÞ2 þ Cu–Cip

ð8Þ

C Cu ¼ ½Cu2 þ  þ K 1 ½Cu2 þ ½AA þ K 12 ½Cu2 þ ½AA2 þK Cip ½Cip½Cu2 þ  ð9Þ C Cu =½Cu2 þ K Cip ½Cip ¼ 1 þK 12 ½AA2 þ K 1 ½AA

ð10Þ

where KCip and K1 represented the formation constants of Cu–Cip and Cu(AA) complexes respectively, and K12 is the overall formation constant of Cu(AA)2 complex. The [Cu2 þ ] was the free concentration when AA and Cip coexisted in solution. [Cip] is the concentration of the uncomplexed Cip which is proportional to the fluorescence intensity of the solution. By considering [AA] ECAA (since CAA 4[Cu2 þ ]) and substitution from Eq. (3) and also the mass balance equation [Cip]0  [Cip] ¼[Cip  Cu], Eq. (10) can be written as y ¼ 1 þ K 12 ðC AA Þ2 þ K 1 C AA

ð11Þ

y ¼ fðC Cu K Cip ½CipÞ=ð½Cip0 ½CipÞK Cip ½Cipg

ð12Þ

where [Cip]0 represented the initial concentration of Cip, CAA and CCu are the total concentrations of amino acid and copper ions in solution. In the first step, KCip could be obtained from fluorimetric titration plotted according to Stern–Volmer equation (Eq. (1)). The concentration of Cip was fixed at 4  10  7 mol L  1 and concentration of Cu2 þ ions varied from 0 to 4.0  10  3 mol L  1.

Asp or Glu

Scheme 1. Schematic representation of the proposed mechanism of the fluorescence quenching of the Cip (a) due to the interaction of with Cu2 þ ions (b) and return fluorescence do to AA interaction with Cu2 þ (c).

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Normalized Fluorescence

6

Asp Glu Pro Asn Gly Leu Ile Phe Ala Sar Lys Arg

5 4 3 2 1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

[AA]×10-4 mol L-1

5

Ralative Fluorescence

4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

Ala Phe Gly Leu Ile Lys Asp Glu Arg Pro Asn Sar Fig. 4. Normalized fluorescence intensity changes for Cu–Cip system in the presence of different AAs in 0.01 mol L  1 phosphate buffer solution (pH ¼ 7) containing [Cu2 þ ]¼ 2.5  10  6 mol L  1 and [Cip] ¼ 4  10  7 mol L  1 at 435 nm with added various concentrations AA (a) and in the presence of 1.6  10  5 mol L  1 of different AAs (b).

3.3. Effect of pH The influence of pH on fluorescence intensity of probe in aqueous solutions was studied as well. The variation of fluorescence intensity of the Cu–Cip (CCu ¼2.5  10  6 and [Cip]0 ¼4  10  7 mol L  1) in the presence of Asp (2  10  6 mol L  1) at different pH values is shown in Fig. 6. With increasing the pH, fluorescence intensity reached a maximum in pH¼ 7 and then decrease with increasing the pH.

120

18 16

Asp (experimetal)

14

Asp (predicted)

12

Glu (predicted)

10

Glu (experimental)

100 80 60

8

y×103

y×105

In the second step, when AA is added to Cu–Cip complex solution ([Cu2 þ ] ¼2.5  10  6 mol L  1 and [Cip] ¼4  10  7 mol L  1), the Cip molecule in Cu–Cip complex is preferably exchanged with AA to form Cu–AA complexes. As a result, quenching by Cu2 þ ions was suppressed and the emission from the Cip increased with increasing the AA concentration. The data obtained from calculated y values were fitted to Eq. (11) (y vs. CAA) for determination of K1 and K12 by using a non-linear least-squares curve-fitting using the Solver algorithm in Microsoft Excel program. Fluorescence data were analyzed by nonlinear least-squares curve-fitting. The typical results of the computer fit of the y–AA concentrations data are shown illustrated in Fig. 5 for Asp and Glu. Table 1 lists K1 and K12 obtained from computer fitting of the y vs. AA concentration data for all AAs. The observed trend in the 1:2 complexes of different AAs with Cu2 þ ions is consistent with the complexation constants reported in the literature for analogous complexes. The overall stability sequence of the 1:2 (Cu2 þ :AA) complexes with different AAs conform the following trend: Asp 4Glu4 Pro4 Ala4 Arg4Phe 4Gly4Lys4Ile 4Asn 4Leu4 Sar. Thus, Asp and Glu can be simply detected fluorimetrically by addition of AA as an analyte to the Cu–Cip solution as probe.

40

6 4

20 2 0

0 0

1

2

3

4

5

6

[AA]×10-6 mol L-1 Fig. 5. Computer fit of y (lines) as a function of AA concentration (according to Eq. (6)) in 0.01 mol L  1 phosphate buffer solution (pH ¼ 7) at 298 K: experimental point (fill mark) and calculated point (no fill).

Thus, pH¼7 could be considered as a proper pH for detection of Asp in this research. This behavior might be attributed to the appearance of an anionic form of Cip due to the progressive ionization of the hydroxyl group. It was found that the fluorescence intensity was stable for Cu–Cip system at least for 3 h. Furthermore, we also found a good linear relationship between the concentration of Asp and Glu and the fluorescence intensity at 435 nm. The calibration graphs for the samples treated according to the detection AA procedure described above are presented in Fig. 7. The linear range of the calibration curves is 1.2  10  7–1.1  10  5 mol L  1 and 8.3  10  7–2.3  10  5 mol L  1 for Asp and Glu respectively. Three replicates were used for each of the standards prepared to obtain the calibration graph. The limit of detection (LOD) was given by the equation LOD¼3  sb/S, where sb

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Table 1 Equilibrium constants for various complexes of Cu2 þ with some amino acid at 298 K and comparison with previously results reported in the literature. Amino acid

This work

a

N.R. N.R. N.R. 7.83 8.18 7.74 7.00 N.R. N.R. 8.57 N.R. 8.22

This work

Ref. [16]

15.18 15.08 13.66 12.35 15.15 15.05 13.60 13.72 14.77 16.94 15.39 14.78

N.R. N.R 12.80 N.R. 15.87 N.R. 12.95 N.R. N.R. N.R. N.R. 13.90

0.975 0.954 0.971 0.985 0.984 0.987 0.966 0.972 0.977 0.965 0.982 0.969

N.R.: No reported.

Fluorescence Intensity

a

Ref. [19]

– – – 7.67 8.01 7.75 7.45 7.25 – 9.05 8.10 8.04

Pro Arg Asn Sar Ala Phe Leu Iso Lys Asp Glu Gly

R2

log K12

log K1

4. Conclusion In summary, the fluoroquinolone (Cip) and amino acid can complex with Cu2 þ , giving information concerning about Cu–AA interaction and thus can be used as a sensitive probe for amino acid detection. The fluorescence intensity of Cip in Cu–Cip complex recovered upon titration by amino acids. This probe has been able to recognize amino acids through switch-on fluorescence behavior and could serves as an efficient fluorescent probe for ultra-trace level determination of aspartic acid and glutamic acid in solution. Probe used for determination of the formation constants of Cu  AA resulted in a good agreement with values reported in the literature. This probe containing the low toxicity metal (Cu2 þ ions) as the receptor was generated in situ. Using fluorescence monitoring as the detection method, the probe showed a good sensitivity for Asp and Glu. Cu–Cip probe has potential applications in fluorescence-based HPLC and highperformance capillary electrophoresis (HPCE) amino acid analysis methods without any derivatization process.

60 50

Acknowledgments

40

This work has been supported by grants from the Tarbiat Modares University Research Council, which is hereby gratefully acknowledged.

30 20 10 0 3

4

5

7

6

8

9

10

References

pH

Fluorescence Intensity

Fig. 6. Effect of pH in fluorescence intensity of probe: [Cu2 þ ] ¼ 2.5  10  6, [Cip] ¼ 4  10  7, [Asp] ¼2  10  6 mol L  1.

1000 900 800 700 600 500 400 300 200 100 0 0

10

20

30

40

50

60

70

80

[AA] 10-7 mol L-1 Fig. 7. Calibration curves of Asp ( ) and Glu ( ) in 0.01 mol L  1 phosphate buffer solution (pH 7) at 298 K.

is the standard deviation of the blank measurements (n¼5) and S is the slope of the calibration graph. The detection limit was found to be 2.7  10  8 mol L  1 and 9.3  10  8 mol L  1 relative with standard deviation (RSD) about 2.1% and 2.9% (for five replicate) at pH¼ 7 for Asp and Glu respectively.

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