Spectrofluorimetric determination of heparin using doxycycline–europium probe

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Journal of Luminescence 113 (2005) 305–313 www.elsevier.com/locate/jlumin

Spectrofluorimetric determination of heparin using doxycycline–europium probe Jing Li, Jinkai Liu, Xiaojing Zhu, Qian Peng, Chongqiu Jiang Department of Chemistry, Shandong Normal University, Jinan 250014, China Received 26 August 2004 Available online 10 December 2004

Abstract A new spectrofluorimetric method was developed for the determination of the trace amount of heparin (Hep). Using doxycycline (DC)–europium ion (Eu3+) as a fluorescent probe, in the buffer solution of pH ¼ 8.9, Hep can remarkably enhance the fluorescence intensity of the DC–Eu3+ complex at l ¼ 612 nm and the enhanced fluorescence intensity of Eu3+ ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep are 0.04–0.8 mg/mL and 19.7 ng/mL, respectively. This method is simple, practical, and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe are 6.60  104 L/mol and 33.9. Moreover, the enhancement mechanism of the fluorescence intensity in the DC–Eu3+ system and the DC–Eu3+–Hep-CTMAB system have been also discussed. r 2004 Elsevier B.V. All rights reserved. PACS: 33.50.Dq Keywords: Heparin; Fluorimetry

1. Introduction Heparin (Hep) is a natural anticoagulant with an average molecular weight of about 15 000. It consists of repeating disaccharide units of uronic/ glucuronic acid and glucosamine residues. Owing to the dissociation of acid groups, the whole Hep Corresponding author. Tel./fax: +86 531 261 5258.

E-mail address: [email protected] (C. Jiang).

molecule is negatively charged in water solution and the average charge is 70 [1]. It is a parenteral drug with a very rapid onset of action due to its inhibition of clotting factors near the end of the coagulation cascade. It has been widely used in many clinical procedures for more than 60 years and even now is the first choice to prevent thromboses and to cure urgent vein thrombus [2]. Hep and its derivatives have a variety of biological activities such as anticoagulant, antilipemic,

0022-2313/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2004.11.001

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antithrombotic, immunoregulatory, antiphlogistic and antianaphylactic activities, etc. [3]. So the Hep level in the patient’s blood needs to be carefully and accurately monitored during surgery and recovery. Now, the methods of determination of Hep can be classified into biological and chemical methods. The application of biological methods is confined because it is greatly affected by biological individuals and cannot be easily mastered [4]. The chemical methods include flowing injection analysis [5], ion-channel sensors [6], resonance Rayleigh scattering spectra [7,8], capillary chromatography [9], high-performance liquid chromatography [10], surface plasmon resonance sensor analysis [11], rotating electrode potentiometry [12], piezoelectric quartz crystal sensor [13] and so on. To date, there are some reports about using metacycline–europium as a fluorescent probe for the determination of lysozyme [14], doxycycline–europium for determination of human serum albumin [15], europium–doxycycline-derived fluorescent substrate for determination of the activity of catalase [16] the highly luminescent europium–doxycycline hydrogen peroxide complex for the determination of hydrogen peroxide in river water [17] and oxytetracyline–europium for determination of nucleic acid [18]. All the methods mentioned above have a high fluorescence quantum yield, large strokes shift, narrow emission bonds, a large fluorescence lifetime, and hence avoid potential background fluorescent emission interferences from the biological matrix [19]. But there was no report about spectrofluorimetric method for determination of Hep using DC–Eu3+ as a fluorescent probe. DC is an antibiotic of the tetracycline family containing the b-diketonate configuration. It is the ideal ligand for Eu3+. In this work, we choose DC as the ligand of Eu3+ and investigated the possibility of the enhancement of the Eu3+ fluorescence sensitized by it and using Hep as the co-ligand. Experimental results show that the characteristic peak of Eu3+ at 612 nm can be greatly enhanced and the enhanced fluorescence intensity is proportional to the concentration of Hep. We also find that hexadecyltrimethylammonium bromide (CTMAB), a cationic surfactant, can enhance the fluorescence intensity further and make the system more stable and sensitive. Therefore, a new

method with high sensitivity and selectivity for the spectrofluorimetric determination of Hep is established. This method is simple, relatively free of interference from coexisting substances, and can be successfully applied to determination of Hep sodium injection samples with satisfactory results. By the Rosenthal graphic method, the binding number and association constant of Hep with the probe are obtained. The mechanism of fluorescence enhancement between Eu3+–DC complexes and Hep, CTMAB was also studied.

2. Experimental section 2.1. Apparatus All fluorescence measurements were carried out on an RF-540 recording spectrofluorimeter (Shimadzu, Kyoto, Japan). A UV-265 recording spectrophotometer (Shimadzu, Kyoto, Japan) was used for UV spectra scanning and the determination experiments. All pH measurements were made with a pHs-3C digital pH meter (Shanghai Leici Device Works, China). 2.2. Reagents All chemicals used were of analytical reagent or higher grade. DDD water was used for the preparation of all solutions and for all determinations. A stock Hep (Shanghai Chemical Reagent Company, China) solution (1.0 mg/mL) was directly dissolved in DDD water. The working standard solution (10.0 mg/mL) was freshly prepared by appropriate dilution with DDD water. A stock DC (Biological Product Institution of Chinese Medicine) solution was directly dissolved in DDD water. The working standard solution (1.43  105 mol/L) was freshly prepared by appropriate dilution with DDD water. All stocking solution and working solutions given above were stored at 0–4 1C. An Eu3+ ion stock solution was prepared by dissolving Eu2O3 (Shanghai Yuelong Chemical Plant, China) with a small amount of hydrochloric acid, then diluting to mark with hydrochloric acid

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(0.1 mol/L). The working solution (5.0  105 mol/ L) was obtained by appropriate dilution of the stock solution with DDD water. A stock CTMAB (Qingpu Synthetic Reagent Plant, Shanghai, China) solution was directly dissolved in DDD water. The working solution (3.44  105 mol/L) was obtained by appropriate dilution of the stock solution with DDD water. An ammonia–ammonium chloride buffer solution (0.10 mol/L, pH ¼ 8.9) was used for the doxycycline system. 2.3. General procedure To 10 mL color comparison tubes, solutions were added in the following order: 2.5 mL 1.43  105 mol/L DC solution, 1.0 mL buffer solution, 0.5 mL 5.0  105 mol/L Eu3+ ion solution, 1.0 mL 10.0 mg/mL Hep solution and 2.5 mL 3.44  105 mol/L CTMAB solution. The mixture was diluted to the mark with DDD water and made to stand for 35 min at room temperature. The fluorescence intensity was measured at l ex/l em ¼ 385 nm/612 nm. The enhanced fluorescence intensity of DC–Eu3+ by Hep was represented as DF ¼ FF0. Here F and F0 are the fluorescence intensities of the system with and without Hep, respectively.

3. Results and discussion 3.1. Characteristics of fluorescence and absorption spectra The fluorescence excitation spectrum and emission spectrum of Eu3+, DC–Eu3+, Eu3+–Hep, DC–Eu3+–Hep, DC–Eu3+–Hep–CTMAB and DC–Eu3+–CTMAB are shown in Fig. 1. From curve 1 in Fig. 1, it can be seen that single Eu3+ ion solution has nearly no peak. Comparing curve 1 with curve 3 in Fig. 1, after the addition of DC into the Eu3+ ion solution, DC can form a binary complex with Eu3+ ion. So two little characteristic peaks of Eu3+ ion appear at 590 and 612 nm, and it is the 5D0–7F1 transition and 5D0–7F2 transition of Eu3+ ion, respectively. Comparing curve 3 with curve 4 in Fig. 1, it can be seen that the

Fig. 1. (a) Fluorescence excitation spectra, (b) fluorescence emission spectra. 1: Eu3+ 2: Eu3+–Hep 3: DC–Eu3+ 4: DC–Eu3+–Hep 5: DC–Eu3+–CTMAB 6: DC–Eu3+–Hep –CTMAB. Experimental condition: DC: 1.43  105 mol/L, Eu3+: 5.0  105 mol/L, Hep: 10.0 mg/mL, buffer: pH ¼ 8.90 CTMAB: 3.44  105 mol/L, lex/em ¼ 385/612 nm.

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characteristic peak of Eu3+ at 612 nm can be enhanced remarkably after the addition of Hep, which indicates that Hep can form a very stable ternary complex with the DC–Eu3+ system. From curve 6 in Fig. 1, the fluorescence intensity of DC–Eu3+–Hep–CTMAB at 612 nm is much larger than that of DC–Eu3+–Hep system which indicates that CTMAB can go on combining with the DC–Eu3+–Hep system to form the supermolecule complex DC–Eu3+–Hep–CTMAB.

The absorption spectra of DC, Eu3+, Hep, DC–Eu3+, DC–Hep, Eu3+–Hep and DC–Eu3+– Hep are shown in Fig. 2. From Fig. 2(a), it can be seen that curves 2,3 and 6 have nearly no peak. Comparing curve 1 with curve 4 in Fig. 2(a), after the addition of Eu3+ ion into the DC solution, red shift occurs from 365.2 to 384.4 nm and the absorbency is also enhanced, which indicates that DC can form a binary complex with Eu3+ ion. Comparing curve 4 with curve 7 in Fig. 2(a), the absorbency is enhanced from 0.258 to 0.285, which indicates that a very stable ternary complex DC–Eu3+–Hep has formed. From curve 7 in both Figs. 2(a) and (b), after the addition of CTMAB into the DC–Eu3+–Hep system, the absorbency is enhanced from 0.260 to 0.285, which indicates that CTMAB can go on combining with the DC–Eu3+–Hep system. As a result of the formation of the supermolecule complex DC–Eu3+– Hep–CTMAB, the absorbance areas become larger and the molar absorption coefficient e becomes larger. The results of the absorption spectra are in accordance with that of the fluorescence spectra.

3.2. Effect of experimental conditions 3.2.1. Effect of pH The pH of the medium had great effect on the fluorescence intensity of the DC system, as shown in Fig. 3. The experimental results showed that the DF reached maximum and remained constant at pH ¼ 8.8–9.0. Therefore pH ¼ 8.9 was selected by using of 0.10 mol/L ammonia–ammonium chloride buffer solution for further study. As the volume of buffer solution added 1.0 mL DF reached maximum, 1.0 mL was chosen in the following experiments.

Fig. 2. Absorption spectra (a) without CTMAB (b) with CTMAB. 1: DC 2: Eu3+ 3: Hep 4: DC–Eu3+ 5: DC–Hep 6: Eu3+–Hep 7: DC–Eu3+–Hep. Experimental condition: DC:1.43  105 mol/L, Eu3+:5.0  105 mol/L, Hep:10.0 mg/ mL, buffer: pH ¼ 8.90 CTMAB: 3.44  105 mol/L, lex/ em ¼ 385/612 nm.

3.2.2. Effect of time The chelation reaction of the DC–Eu3+– Hep–CTMAB system at room temperature needed 35 min at least. The fluorescence intensity then remained constant for at least 3 h. Therefore, all chelation reactions were carried out for 35 min and measurements were made within 3 h.

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20

309

35 28

15 ∆F

∆F

21 10

14 7

5

0 0 8.0

8.5

9.0

9.5

0.5

10.0

1.0

3.2.5. Effect of the amount of Eu3+ The influence of the amount of Eu3+ ion on the fluorescence intensities of the solutions containing 3.58  106 mol/L of DC and 1.0 mg/mL Hep was studied under the conditions established above. As shown in Fig. 5, the enhanced fluorescent intensity DF reached maximum and remained constant when Eu3+ ion is added from 0.3 to 0.5 mL. Thus 2.5  106 mol/L Eu3+ ion (the added amount of Eu3+ ion is 0.5 mL) was selected. When the concentration of Eu3+ ion was 2.5  106 mol/L,

2.5

3.0

Fig. 4. Effect of the amount of DC.

Fig. 3. Effect of acidity.

3.2.4. Effect of the amount of DC The influence of the amount of DC on the fluorescence intensities of the solutions is shown in Fig. 4. The enhanced fluorescent intensity DF increased at first and then decreased with the increasing amount of DC. The experimental results showed that DF reached maximum and remained constant when the DC solution is added more than 2.25 mL. Thus, 3.58  106 mol/L DC (the added amount of DC is 2.5 mL) was selected.

2.0

V(mL)

pH

30 24 18 ∆F

3.2.3. Effect of the addition order of reagents Adding various reagents in different order had an influence on the F, F0 and DF. The experimental results indicate that it was optimum when solutions were added in the following order: DC, buffer, Eu3+, Hep and CTMAB. So this order was chosen in the following experiments.

1.5

12 6 0 0.0

0.3

0.6

0.9

1.2

1.5

v(mL) Fig. 5. Effect of the amount of Eu3+.

the composition ratio for the DC to Eu3+ in the DC–Eu3+–Hep system is 1.4:1. 3.2.6. Effect of the surfactant The influence of the surfactant was studied, including TritonX-100, sodium dodecyl sulfate (SDS), and CTMAB. The experimental results showed that the cationic surfactant CTMAB can enhance the fluorescence intensities of the system and improve its stability. The influence of the amount of CTMAB on the fluorescence intensities of the solutions is shown in Fig. 6. The experimental results showed that the sensitivity can be improved and the fluorescence intensity can remain constant for at least 3 h, when CTMAB solution added is more than 1.25 mL. Thus

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5.16  106mol/L CTMAB (the added amount of CTMAB is 1.5mL) was selected as the sensitizing and stabilizing agent for the determination of Hep. 3.2.7. Effect of coexisting substances Under optimum conditions, a systematic study of various nonprotein substances in the determination of Hep (1.0 mg/mL) was carried out. The criterion for interference was fixed at a 710% variation of the average fluorescence intensity calculated for the established level of Hep, the experimental results were shown in Table 1. From Table 1 it could be seen that most of the coexisting substances were found to show no influence.

25 20

∆F

15

4. Analytical application 4.1. Linear range and limit of detection Under the experimental conditions, there is a linear relationship between fluorescence intensity and Hep concentration in the range of 0.04–0.8 mg/ mL with a correlation coefficient of 0.9997. The regression equation is DF ¼ 57.5  C(mg/mL)+ 4.26. The limit of detection was determined to be 19.7 ng/mL when the standard deviations were 0.38 obtained from a series of 10 reagent blanks. By comparing with some of the existing methods, as shown in Table 2, the present methods have the advantages in the following terms: high sensitivity, good stability and wide linear range. It avoids potential background fluorescent emission interferences from the biological background. So this method may provide a new kind of luminescent probe for the determination of biomolecular systems and can be applied to time-resolved fluoroimmunoassay.

10

4.2. Determination of Hep in samples 5

The developed method was applied to the determination of Hep in samples. The results were shown in Table 3. For the assay of Hep, the samples must be diluted appropriately within the linear range of determination of Hep and the

0 0.5

1.0

1.5 v(mL)

2.0

2.5

Fig. 6. Effect of the amount of CTMAB.

Table 1 Effect of coexisting substances Coexisting substances

Concentration (mol/L)

F (%)

Coexisting substances

Concentration (mol/L)

F (%)

Ag+(NO 3) Zn2+(Cl) Ca2+(Cl) Mg2+(SO2 4 ) Cd2+(Cl) Co2+(Cl) Mo6+ Al3+ Mn2+ RNA Adenine Guaine Acetocaustin

1.85  105 1.18  106 5.00  106 5.00  106 1.80  106 5.00  107 1.04  106 3.77  106 6.25  107 1.34  106 1.04  108 8.00  107 1.38  105

9.97 8.40 6.00 1.90 6.20 0.62 1.50 4.10 4.90 6.40 0.70 2.40 1.42

Thymine Cytosine L-histidine L-cystine L-tyrosine L-leucine L-lysine Glutamic acid Glycin Tryptophane Methionine Glucose Saccharose

1.85  106 1.00  106 9.87  107 4.00  105 1.00  106 1.02  105 1.01  105 2.00  105 1.00  105 5.00  107 1.04  106 1.00  104 1.00  104

7.60 1.20 1.30 7.49 2.50 7.30 1.68 7.30 1.30 2.50 4.40 2.30 2.80

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Table 2 Comparison of spectrofluorometric methods for determination of Hep Method

Linear range

Detection limit

References

flowing injection analysis ion-channel sensors resonance Rayleigh scattering capillary chromatography high-performance liquid chromatography surface plasmon resonance sensor analysis rotating electrode potentiometry piezoelectric quartz crystal sensor This method

0–12 mg/mL 0.05–1.5 mg/mL 0–0.4 mg/mL 80–7000 U/La 0.002–5 pmol 0.2–2 U/mLc 0.05–0.5 U/mLe 0–3 U/mLf 0.04–0.8 mg/mL

300 ng/mL 26 ng/mL 3.35 ng/mL 25 U/Lb

[5] [6] [7,8] [9] [10] [11] [12] [13]

0.2 U/mLd

19.7 ng/mL

a

80–7000 U/L is equivalent to 0.504–44.1 mg/mL. 0.2–2 U/mL is equivalent to 1.26–12.6 mg/mL. c 25 U/L is equivalent to 157.5 ng/mL. d 0.2 U/mL is equivalent to 1260 ng/mL. e 0.05–0.5 U/mL is equivalent to 0.315–3.15 mg/mL. f 0–3 U/mL is equivalent to 0–18.9 mg/mL. b

Table 3 Determination of Hep in samples (the standard value of the sample is 12500 IU/2 mL) Sample no.

Added (mg/mL)

Measure value (mg/mL)

20030310a 020911-1b 0311111c 020504d

0.3906 0.3906 0.6250 0.3125

0.350, 0.371, 0.608, 0.288,

0.371, 0.371, 0.611, 0.288,

0.395, 0.371, 0.614, 0.291,

0.402, 0.374, 0.618, 0.312,

0.413 0.389 0.618 0.316

Average (mg/mL)

Ave. (IU/2 mL)

Recovery (%)

RSD (%)

0.386 0.375 0.613 0.299

12213 12012 12285 11962

98.8 96.1 98.1 95.7

6.5 2.1 0.7 4.6

a

Tianjin Biochemical Pharmaceutical Factory of China. Maanshan Plant of Anhui Xinli Pharmaceutical Co. Ltd. of China. c Wanbang Biochemical Pharmaceutical Co. Ltd. of China. d Nanjing Biochemical Pharmaceutical Co. Ltd. of China. b

sample solution was analyzed by the method developed above, using the standard calibration method. From Table 3 it can be seen that the developed method can be easily performed and affords good precision and accuracy when applied to real samples. 4.3. Measurement of association constant and binding numbers The Rosenthal graphic method [20], regarded as a modification of the Scatchard method, was used to estimate the association constant (K) and the binding number (N) of the biomacromolecule to the DC–Eu3+ probe. Briefly, when C, as a constant, is the biomacromolecule concentration

in the system, and Cb,Cf and C Eu3þ DC are correspondingly the Hep-bound, free and total concentrations of the complex, the Rosenthal plot follows Eq. (1) [21,22]. Cb ¼ ðC Eu3þ DC  C f ÞK þ NCK: Cf

(1)

Since C Eu3þ DC ¼ C b þ C f ; thus C ¼ ðC Eu3þ DC  C f ÞK þ NCK þ 1 Cf

(2)

in the system, if C Eu3þ DC and Cf are within the dynamic range of the calibration graph for DC–Eu3+–Hep complex, Eq. (3) can be obtained.   F0 F ¼ 1 (3) C Eu3þ DC K þ NCK þ 1: F0 F

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F, F0 are the intensities of the systems with and without Hep. The plot of F0/F vs. ð1  ðF =F 0 ÞÞC Eu3þ DC can be obtained. The value of K and N are 6.60  104 L/mol and 33.9 for the DC system.

are still a lot of positive charges and blank orbits in the DC–Eu3+ complex. 5.2. The formation of DC– Eu3+– Hep ternary complex Hep, as a kind of biomacromolecule with an average molecular mass 15 000 and an average charge 70, is a polymer consisting of repeating tetrasaccharide unit. The structure of tetrasaccharide unit is shown in Fig. 8. Heparin has three Osulfate groups, two N-sulfate groups, and two carboxyl groups per tetrasaccharide unit. The Osulfate and N-sulfate groups completely dissociate under the experimental conditions. The carboxyl group is weakly acidic, and the pKa of Dglucuronic acid in Hep is 3.6 [24]. So carboxyl groups can completely dissociate under the experimental conditions. Therefore, the whole Hep molecule exists as a big polyvalent anionic state in water solution. Because Hep has seven binding sites (five sulfate groups and two carboxyl groups) per tetrasaccharide unit and the Hep used here contains 42 monosaccharide units, the total binding number is 73.5 ð42  74Þ per Hep molecule [3]. Therefore, a very stable ternary complex in close proximity with a large degree of molecular conjugation and rigid structure can be formed by

5. Reaction mechanism 5.1. The formation of DC– Eu3+ binary complex DC is a tetracycline antibiotic containing bdiketonate configuration. The structure of DC is showed in Fig. 7. The literature survey shows that b-diketonate ligands are suitable for efficient energy transfer from ligands to Eu3+ ion and for high fluorescence quantum yield; large stokes shift; narrow emission bonds; a large fluorescence lifetime and hence avoid potential background fluorescent emission interferences from the biological background. Therefore, DC is ideal ligand for Eu3+ ion and it can possibly sensitize the fluorescence intensity of Eu3+ ion via intramolecular energy transfer [23]. The coordination number of Eu3+ ion is generally 8. According to the experimental results of the mole ratio for DC to Eu3+ given above, we can see that the coordination of Eu3+ ion is unsaturated. There

OH

CH3

N(CH3)2 OH

OH

OH OH O

O

. HCI . 1/2 C H OH .1/2H O 2 5 2

CONH2

Fig. 7. Structure of DC.

-

H O H COO O OH H H O

H

CH2OSO3 H H O HH OH H

-

OSO3

H

O

-

NHSO3

-

COO O H OH H H

-

CH2OSO 3 O H

OH

H H OH OH H H

Fig. 8. Structure of the tetrasaccharide unit of Hep.

O

-

NHSO3

n

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the electrostatic interactions and coordinations between Hep and DC–Eu3+ binary complex. oth the polysaccharide (Hep) and ligand (DC) have a great amount of hydrogen donor and acceptor groups (–NH–, –OH, –O–, ¼ O, –COO–, –CONH2), which could provide an additional binding mechanism between DC–Eu3+ and Hep. 5.3. The enhancement effect of the addition of CTMAB When the cationic surfactant (such as CTMAB, one end is the terminal hydrophilic groups with positive charge and the other end is hydrophobic groups with long carbochains) is added in the DC–Eu3+–Hep system, because of the electrostatic attractions and the hydrophobic interactions, CTMAB combines with the Hep to become a supermolecule complex. As a result, the microenvironment is changed in the fluorescent system, and the nonradiative energy loss through O–H vibration of H2O in the original Eu3+ complex and the collision of H2O in the solvent can be decreased greatly. The molar absorptivity of the supermolecule complex becomes larger with more rigid structure and larger absorbance areas in the complex. So the fluorescence intensity of Eu3+ ion at 612 nm can be enhanced several times by the interactions of DC, Hep and CTMAB. References [1] S. Mathison, E. Bakker, Anal. Chem. 71 (1999) 4614.

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