Novel Spectrofluorimetric Method for Measuring the Activity of the

Jun 18, 2010 - encountered malignant neoplasms in the world. In patients with ... tumors.7,8 The primary tumor marker for HCC is a single ... (14) Shu...
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Anal. Chem. 2010, 82, 6230–6236

Novel Spectrofluorimetric Method for Measuring the Activity of the Enzyme r-L-Fucosidase Using the Nano Composite Optical Sensor Samarium(III)-Doxycycline Complex Doped in Sol-Gel Matrix M. S. Attia,*,† A. M. Othman,‡ M. M. Aboaly,† and M. S. A. Abdel-Mottaleb† Department of Chemistry and Photoenergy Center, Faculty of Science, Ain Shams University, Cairo, Egypt, and Genetic Engineering and Biotechnology Institute, Menofia University, Menofia, Egypt A novel, simple, sensitive, and precise spectrofluorimetric method was developed for measuring the activity of the enzyme r-L-fucosidase (AFU). The method was based upon measuring the quenching of the luminescence intensity of the produced yellow colored complex ion associate of 2-chloro-4-nitrophenol [2-CNP] and a nano composite optical sensor samarium(III)-doxycycline [Sm3+-DC]+ complex in a sol-gel matrix at 645 nm. The remarkable quenching of the luminescence intensity of the [Sm3+-DC]+ complex doped in a sol-gel matrix by various concentrations of the reagent [2-CNP] was successfully used as an optical sensor for the assessment of AFU activity. The calibration plot was achieved over the concentration range 3.4 × 10-9-1.0 × 10-6 mol L-1 [2-CNP] with a correlation coefficient of 0.99 and a detection limit of 6.0 × 10-10 mol L-1. The method was used satisfactorily for the assessment of the AFU activity in a number of serum samples collected from various patients. A significant correlation between the luminescence activity of the enzyme AFU measured by the proposed procedure and the standard method was applied to patients and controls. The method proceeds without practical artifacts compared to the standard method. Hepatocellular carcinoma (HCC) is one of the commonly encountered malignant neoplasms in the world. In patients with chronic liver diseases, the early detection of HCC is very important in controlling this disease. Liver cirrhosis is a precancerous condition that in many cases can develop into HCC. Therefore, cirrhotic patients with cirrhosis are usually screened for HCC during their follow-up procedure,1-6 and the tumor markers * Corresponding author. Address: Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt. Tel: +2020129867311/ 44678582. Fax: +20224845940. E-mail: [email protected]. † Ain Shams University. ‡ Menofia University. (1) Giardina, M. G.; Matarazzo, M.; Morante, R. Cancer 1998, 83, 2468–2474. (2) Okuda, K.; Tabor, E.; Liver Cancer; Churchill Livingstone: New York, 1997; pp 13-28. (3) Kubo, Y.; Okua, K.; Musha, H.; Nakashima, T. Gastroenterology 1978, 74, 578–582.

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provide good potential screening tools for the early diagnosis of tumors.7,8 The primary tumor marker for HCC is a single polypeptide chain glycoprotein namely R-fetoprotein (AFP).9 The sensitivity and specificity of the common tumor marker AFP in the detection of HCC in all patient samples are insufficient.10,11 In some cases at low levels of R-fetoprotein, the combination between serum AFP levels and ultrasonography sometimes misses HCC.12,13 Moreover, the AFP tumor marker sometimes is not secreted in all cases of HCC and may be normal and acceptable in 40% of patients with early HCC.11-15 Therefore, improving the sensitivity toward measuring the activity of the R-L-fucosidase (AFU) enzyme by effective and low cost tumor markers remains an active area of research.10,11 The AFU enzyme is present in all mammalian cells and involves the catabolism of the L-fucose containing glycoconjugates. The enzyme AFU can hydrolyze methyl R-L-fucoside and fucosidic linkages of fucoidan and blood-group substances.16 The two components of the AFU enzyme are fucosidase which acts on 4-nitrophenyl R-L-fucoside as well as fucosidic linkages of porcine submaxillary mucin at optimal activity around pH 2, and the fucoidanase shows highest activity around pH 5 and acts on the (4) Zoli, M.; Magalotti, D.; Bianchi, G.; Gueli, C.; Marchesini, G.; Pisi, E. Cancer 1996, 78, 977–985. (5) Cobucci-Ponzano, B.; Trincone, A.; Giordano, A.; Rossi, M.; Moracci, M. J. Biol. Chem. 2003, 278, 14622–14631. (6) Liu, S. W.; Chen, C. S.; Chang, S. S.; Mong, K. K. T.; Lin, C. H.; Chang, C. W.; Tang, C. Y.; Li, Y. K. Biochemistry 2009, 48, 110–122. (7) El Shemey, W. M.; Desouky, O. S.; Mohammed, M. S. Phys. Med. Biol. 2003, 48, 239–242. (8) Burtis, C. A.; Ashwood, E. R. Tietz Fundamentals of Clinical Chemistry, 5th ed.; WB Saunders: Philadelphia, Pa, 2001; pp 56-73. (9) Oka, H.; Tamori, A.; Kuroki, T.; Kobayashi, K.; Yammamoto, S. Hepatology 1994, 19, 61–66. (10) Sanki, A. K.; Voss, B. J.; Sucheck, S. J.; Ronning, D. R. Anal. Biochem. 2009, 385, 120–127. (11) Osaki, Y.; Kimura, T.; Arimoto, A.; Oka, H.; Yamazaki, O.; Manabe, T.; Chung, H.; Kudo, M.; Matsunaga, T. J. Hepatology 2008, 49, 223–232. (12) El-Houseini, M. E.; El-Sherbiny, M.; Awad, M. E. J. Egypt. Nat. Cancer Inst. 2001, 4, 277–283. (13) Van Hoof, F.; Hers, H. G. Eur. J. Biochem. 1968, 7, 34–44. (14) Shu, H. J.; Saito, T.; Watanabe, H. Biochem. Biophys. Res. Commun. 2002, 293, 150–154. (15) Nakatsura, T.; Yoshitake, Y.; Senju, S. Biochem. Biophys. Res. Commun. 2003, 306, 16–25. (16) Blumbeig, S.; Hildesheim, J.; Yariv, J.; Wilson, K. J. Biochim. Biophys. Acta 1972, 264, 171–176. 10.1021/ac101033j  2010 American Chemical Society Published on Web 06/18/2010

synthetic substrate and not on the mucin.17 The enzyme fucoidanase shows hydrolytic activity toward fucoidan and not toward 4-nitro phenyl R-L-fucoside and the blood group of livers of related species.18 The activity of AFU enzyme represents an excellent test for the diagnosis of HCC19,20 and for the diagnosis of fucosidosis recognized in a born disorder of metabolism and increases the sensitivity of detection to 95.5% in patients with HCC.21 Different spectrometric methods have been reported for the determination of the activity of the enzyme AFU in serum.22,23 These methods are limited by their long incubation time (30-60 min) for the sample and reagent blanks. Moreover, the poor affinity of the enzyme AFU toward the substrate 2-chloro-4nitrophenyl-R-L-fucopyranoside [2-CNPF] as a colorimetric reagent at pH 4.8 added another disadvantage to these methods. The activity of AFU was also determined by spectrofluorimetric methods,24-26 affinity chromatography,27 column chromatography packed with Bacillus cereus,28 and the disk gel electrophoresis technique.29 Some of these methods are unselective, require careful experimental conditions, are considerably time-consuming, and are not compatible to detect the activity of AFU at the early stage of diseases. Due to the inherent low temperature process, sol-gel technology has acquired great popularity in the field of optical sensors.30 The driving force for these attempts is that the sol-gel chemistry provides a relatively simple way to incorporate recognition species in a stable host environment.31-33 The sol-gel technology provides a unique means to prepare inorganic and organicinorganic hybrid material for use in sensing devices. The simple doping of the sol-gel solution with the desired compound is the most popular technique for immobilization because of its generality, simplicity, and retention of the properties of the compound in the immobilized state. Recent literature on the analytical applications of the samarium(III)-doxycycline [Sm3+-DC]+ complex has revealed no study on the use of this complex species in sol-gel for measuring the activity of AFU enzyme. Thus, the present article is focused on simply doping the low cost luminescent complex ion associate of [Sm3+-DC]+ into the sol prior to its gelation for measuring the activity of the AFU enzyme. This method overcomes the difficulties caused by the (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33)

Lewy, G. A.; Mcauan, A. Biochem. J. 1961, 80, 435–439. Van Hoof, F.; Hers, H. G. Lancet 1968, 1, 1198–1193. Johnson, S. W.; Alhadeff, J. A. Biochem. Physiol. 1991, 99B, 479–488. Deugnier, Y. Hepatology 1984, 4889–4892. Durand, P.; Borrone, C.; Della, C. J. Pediatrics 1969, 75, 665–674. Troost, J.; Van der Heijden, M. C.; Staal, G. E. Clin. Chim. Acta 1976, 73, 329–346. Ju-Jun, W.; En-Hua, C. Clin. Chim. Acta 2004, 347, 103–109. Wood, S. Clin. Chim. Acta 1975, 58, 251–256. Cuer, M.; Barnier, A.; De La Salmoniere, P.; Durand, G.; Seta, N. Clin. Chem. 2000, 46, 560a–576a. El-Shahawi, M. S.; Othman, A. M.; El-Houseini, M. E.; Nashed, B.; Elsofy, M. S. Talanta 2009, 80, 19–23. Blumbeig, S.; Hildesheim, J.; Yariv, J.; Wilson, K. J. Biochim. Biophys. Acta 1972, 264, 171–176. Miura, Z. U.; Okamoto, K.; Yanase, H. J. Biosci. Bioeng. 2005, 99, 629– 635. Yamaguchi, S.; Uesugi, K. Analyst 1984, 109, 1393–1417. Attia, M. S. J. Pharm. Biomed. Anal. 2010, 51, 7–11. Gavalas, V. G.; Andrews, R.; Bhattacharyya, D.; Bachas, L. G. Nano Lett. 2001, 1, 719–721. Collinson, M. M. Trends Anal. Chem. 2002, 21, 30–38. Zanjanchi, M. A.; Arvand, M.; Mahmoodi, N. O.; Islamnezhad, A. Electroanalysis 2009, 21, 1816–1821.

previous methods23-25 and most likely will be able to control the quality of the final marketed product in both qualitative and quantitative approaches to ensure the identity of the disease. Moreover, the developed method is useful in medical uses to detect HCC in all patients at the very early stage of diseases. EXPERIMENTAL SECTION Apparatus. A Shimadzu RF5301 PC spectrofluorometer (290-750 nm) was used for recording the excitation and emission spectra. The absorption spectra were recorded on a Unicam UV-visible spectrophotometer of Helios type equipped with a temperature-controller cell holder. The spectrophotometer employs a tungsten filament light source and a deuterium lamp of a continuous spectrum in the UV region. An Orion pH meter, VWR scientific model (8000), was used for pH measurements. Materials and Reagents. NaCl, KCl, albumin, uric acid, urea, glucose, ascorbic acid, bilirubin, hemoglobin, total cholesterol, and triglyceride were purchased from Stanbio and Fluka (Switzerland) companies and total tissue protein was from SigmaAldrich (T5695-.5MG). The 2-chloro-4-nitrophenyl-R-L-fucopyranoside (CNPF, DZ082B-R1) and the enzyme R-L-fucosidase (AFU, DZ082B) were purchased from (Diazyme Laboratories, USA). The control enzyme was prepared in bovine serum base and provided in lyophilized powder from Diazyme Co. (DZ082B-Con). The analytical reagents and chemicals (99.99% purity) were used without further purification. Deionized water and pure grade solvents (Aldrich) were used for the preparation of solutions. A stock solution of doxycycline hydrochloride (DC, 1 × 10-2 mol L-1) was prepared in ethanol. The working standard solution (1 × 10-4 mol L-1) of DC was freshly prepared by appropriate dilution of the stock with dimethylsulfoxide (DMSO). A stock solution (5 × 10-3 mol L-1) of Sm3+ ion was prepared by dissolving the required weight of the salt SmCl3 · 6H2O (Aldrich, 99.99%) in ethanol. The stock solution (5 × 10-3 mol L-1) of 2-chloro-4-nitrophenol [2-CNP] was prepared in deionized water. More diluted solutions (5 × 10-5-5 × 10-10 mol L-1) of [2-CNP] were prepared by diluting the stock solution with DMSO. Phosphate buffers of various pH (pH 3.5-7) were prepared from phosphoric acid and sodium phosphate. The luminescence intensity was measured at λex/ λem ) 400/ 645 nm. Stock and working solutions were stored at 0-4 °C when not in use. In all experiments, clean and sterilized volumetric flasks (10 mL) were used. General Procedures. Preparation of [Sm3+-DC]+ Complex Doped in the Sol-Gel. For the preparation of [Sm3+-DC]+ complex in sol-gel, the following three steps were carried out. (i) The complex ion of [Sm3+-DC]+ was prepared by mixing the doxycycline (2 × 10-4 mol L-1) and SmCl3 · 6H2O (1 × 10-4 mol L-1) in a 2:1 molar ratio in dry C2H5OH at room temperature. The produced white precipitate was separated out, washed with ether, and dried. (ii) The sol matrix was prepared according to earlier reported work31 as follows: A mixture consisting of tetraethoxysilane (TEOS), ethanol, and water in a 1:5:1 molar ratio was refluxed for 1 h to give the precursor sol solution in the presence of few drops of diluted HCl solution as catalyst. (iii) Finally, an appropriate amount of the [Sm3+-DC]+ complex and the precursor solution were mixed and stirred together for 15 min until the mixture became homogeneous. The developed complex-dispersed sol solution Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

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Scheme 1. Enzymatic Reaction of the Substrate with AFU Enzyme

was casted into a polystyrene cup (with dimensions of 3, 0.2, and 0.8 cm) and kept at 25 °C in air for 2 weeks. The produced cast was heated at 100-500 °C for 24 h to give a solidified and transparent composite sample. Preparation of 2-Chloro-4-nitro Phenol [2-CNP] Solutions. To 10 mL clean and sterilized measuring flasks, the standard solutions of 2-chloro-4-nitro phenol [2-CNP] were prepared by dilution of [2-CNP] (1 × 10-4 mol L-1) solution to give different concentrations of [2-CNP] in DMSO at room temperature. The above solutions were used for the subsequent measurements of absorption, emission spectra, and effect of solvents. The luminescence intensity was measured at λex/λem ) 400/645 nm. Recommended Procedure. An appropriate amount (450 µL) of various standard concentrations of the reagent [2-CNP] in DMSO was mixed with the optical sensor [Sm3+-DC]+ complex doped in a sol-gel matrix in the cell. The luminescence spectra were then recorded at the excitation wavelength. The optical sensor was rinsed with DMSO after each measurement, and the calibration plot was constructed by plotting the normalized luminescence intensity at λem ) 645 nm on the y axis against the reciprocal of [2-CNP] concentration on the x axis. Standard Method. AFU assay is a single reagent, the assay is specific for AFU and has no detectable reaction with other glycosidases. The assay is not affected by serum bilirubin up to 100 mg/dL, hemoglobin up to 200 mg/dL, triglycerides up to 750 mg/dL, or ascorbic acid up to 4.4 mg/dL. The assay is stable for 12 months from the date of manufacture when stored at 2°-8 °C shielded from light. Assay Principle. The AFU assay is based on the enzymatic cleavage of the synthetic substrate 2-chloro-4-nitrophenyl-R-Lfucopyranoside [2-CNPF] to R-L-fucoside and 2-chloro-4-nitrophenol [2-CNP], which is kinetically quantified by measuring the absorbance at 405 nm, Scheme 1. The AFU activity (U/L) in the plasma sample can be calculated using the formula: AFU (U/L) ) ∆A/min × 1250 where ∆A/min is the average rate of the absorbance change, U/L is an arbitrary unit agreed upon by scientists and doctors, and 1250 is the calculation factor for the UV spectrophotometer when the cuvette path length is 1 cm. Analytical Application. An appropriate amount (450 µ L) of the reagent (2-CNPF) was immediately mixed with an accurate volume (50 µL) of plasma-serum samples of HCC patients (10 persons) and healthy controls (7 persons), and the pH was adjusted at 5 using the phosphate buffer;26 then, the solution was placed in an incubator at 37 °C for 3 min. The volume was completed to 3.0 mL with DMSO; the optical sensor [Sm3+-DC]+ 6232

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Table 1. Main Characteristics of the Patients and Control Groups males/females status

HCC cirrhosis of chronic hepatitis C and B other neoplasms (gallbladder cancer, colon cancer, and others) healthy adults

age (year)

number of patients

no.

%

median

range

10 6

6/4 3/3

60/40 50/50

53 45

25-77 32-68

2

2/0

100/0

41

25-62

7

3/4

45/55

37

20-60

complex doped in a sol-gel matrix was immersed in each solution in the measuring cell, and the emission intensity at 645 nm was measured against the reagent blank. The main characteristics of the patients (male/female) and control groups are given in Table 1. AFU activity was measured by withdrawing 10 µL of the solution sealed in the incubator at different time intervals and completed to 3 mL by DMSO, and then, the emission intensity of the optical sensor [Sm3+-DC]+ complex doped in a sol-gel matrix at 645 nm was measured. RESULTS AND DISCUSSION Spectral Characteristics. Absorption Spectra. Previous studies by Wood24 have shown that the enzyme AFU reacts with the substrate containing 2-chloro-4-nitrophenol or 4-nitrophenol at pH 4 to 5 producing yellow colored species. The produced color has been used for measuring the activity of the enzyme AFU via its absorbance at pH 5.0 and 37 °C.22 This method suffered from a series of interference caused by the yellow color of the serum of patients and of the reagent 2-chloro-4-nitrophenol (or 4-nitrophenol). Another disadvantage was also noticed in many cases of patients of HCC that have high bilirubin.21 Thus, we have carried out attempts to overcome these disadvantages by testing the quenching of the luminescence of the cationic complex ion [Sm3+-DC]+ doped in a sol-gel matrix by the compound 2-chloro-4-nitrophenol resulting from the enzymatic hydrolysis reaction. Preliminary investigations have shown that, on immersing [Sm3+-DC]+ complex doped in a sol-gel matrix in the solution of [2-CNP] and shaking, a quenching of the luminescence intensity of the complex was taking place. The absorption spectra of DC, [2-CNP], [Sm3+-DC]+, [Sm3+-2-CNP]2+, and [Sm3+-DC]+[2-CNP]- in a sol-gel matrix are shown in Figure 1. A typical spectra of DC shows two characteristic peaks at 271 and 362 nm assigned to a π-π* type. Comparing the spectrum

Figure 1. Absorption spectra of DC (1), [2-CNP] (2), [Sm3+-DC]+ (3), [Sm3+-2-CNP]2+ (4), and [Sm3+-DC]+[2-CNP]- (5) in a sol-gel matrix.

Figure 2. Emission spectra of [Sm3+-DC]+ (1) and [Sm3+-DC]+[2CNP]- (2) and excitation spectrum of [Sm3+-DC]+ (3) in a sol-gel matrix at λex/λem ) 400/645 nm.

of DC with [Sm3+-DC]+ complex showed a red shift in the two bands by 9 and 4 nm, respectively; also, a new band appeared at about 391 nm due to the cationic complex. Comparing the spectrum of [Sm3+-DC]+ complex with that of [Sm3+-DC]+[2-CNP]- indicated that the two absorption peaks at 280 and 366 nm of the [Sm3+-DC]+ system showed a red shift due to ion pair interaction. Excitation and Emission Spectra. The excitation and emission spectra of the produced complex [Sm3+-DC]+ and [2-CNP] as well as those of the ion associate complex of [Sm3+-DC]+[2CNP]- in sol-gel matrix were recorded. DC has a very broad emission when excited at λex ) 400 nm, which was disappeared in presence of Sm3+ ion, and Sm3+ emission characteristic peaks lines appeared as shown in Figure 2. The excitation and emission spectra, Figure 2, of the produced complex ion [Sm3+-DC]+ and its associate complex ion [Sm3+-DC] +[2CNP]- in a sol-gel matrix against the reagent blank showed a characteristic excitation wavelength with a maximum at 376 nm, and the corresponding characteristic emission peaks of Sm3+ ion were most likely assigned to 4G5/2f6H5/2 ) 564 nm, 6 H7/2 ) 599 nm, 6H9/2 ) 643 nm, and 6H11/2 ) 707 nm, Figure

Figure 3. Luminescence spectra of 1 × 10-4 mol L-1 Sm3+ with 2.0 × 10-4 mol L-1 DC doped in sol-gel in the presence of different concentrations of [2-CNP] in DMSO at λex ) 400 nm.

2. The luminescence intensity of the complex [Sm3+-DC]+ at 645 nm decreased on adding [2-CNP] confirming the formation of a complex ion associate. The emission spectrum of the complex cation [Sm3+-DC]+ exhibited a well-defined emission spectrum similar to the spectrum of the formed complex ion associate [Sm3+-DC]+ [2-CNP]-, Figure 2. Moreover, a characteristic peak of Sm3+ at 645 nm remarkably has been decreased after the addition of the reagent [2-CNP]. Thus, in the subsequent work, the activity of the enzyme AFU was determined through measuring the luminescence intensity of the ion associate [Sm3+-DC]+[CNP]- at 645 nm against the reagent blank at the established conditions. Analytical Parameters. The influence of the DC concentration on the luminescence intensity of the complex ion [Sm3+-DC]+ in the sol-gel matrix was studied. The emission spectrum of Sm3+ ion revealed maximum enhancement in the emission intensity at DC concentration of 2.0 × 10-4 mol L-1 and remained constant at high concentration. Thus, in the subsequent work, the DC concentration was adopted at 2.0 × 10-4 mol L-1 in the sol-gel preparations. The influence of the Sm3+ ion concentration on the luminescence intensity of the sol-gel matrixes containing DC at the optimum concentration (1.0 × 10-4 mol L-1) was studied under the conditions established above. The luminescent intensity at 645 nm increased upon increasing the Sm3+ ion concentration up to 1.0 × 10-4 mol L-1 and then decreased. At the break point (1.0 × 10-4 mol L-1) of the Sm3+ ion concentration, the molar ratio of the Sm3+ ion to the reagent DC was exactly 1:2 molar ratio. Therefore, in the subsequent work, Sm3+ ion concentration was adjusted at 1.0 × 10-4 mol L-1. The results revealed no quenching effect of the DMSO solvent on the emission intensity of [Sm3+-DC]+ in the sol-gel matrix in the presence of DMSO. The influence of [2-CNP] concentration on the luminescence intensity of the sol-gel matrix containing DC (2.0 × 10-4 mol L-1) and SmCl3 · 6H2O (1.0 × 10-4 mol L-1) was studied under the optimum experimental conditions. The results are depicted in Figure 3. The luminescence intensity of [Sm3+-DC]+ in the sol-gel matrix was Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

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Figure 4. Mechanism of the quenching process between the [2-CNP] and the optical sensor.

quenched on increasing the concentration of the compound [2-CNP] up to 5 × 10-5 mol L-1, and the mechanism of the quenching process was illustrated in Figure 4. Effect of Foreign Ions. The validity and selectivity of the developed spectrofluorimetric method was tested by studying the influence of a series of interfering species, e.g., NaCl, KCl (1.0 × 10-3 mol L-1), albumin (0.6 g L-1), uric acid (0.008 g L-1), urea (0.006 g L-1), total protein (0.001 g L-1), and glucose (0.08 g L-1) on the luminescence spectrum of [Sm3+-DC]+ in the sol-gel matrix after addition of [2-CNP] (5.0 × 10-5 mol L-1). The tolerable limit was defined as the concentration of the added species individually causing a deviation less than 3% of the luminescence intensity at the optimum conditions of the formation of the complex ion associate. The results indicated no significant change on the luminescence intensity of the ion associate [Sm3+-DC]+[2CNP]- in the sol-gel matrix. Analytical Performance. The validation of the proposed spectrofluorimetric method for measuring the AFU activity under the optimized experimental conditions was determined via the limit of detection (LOD), limit of quantification (LOQ), linear dynamic range (Table 2), repeatability, and recovery (Table 3). The luminescence intensity of [Sm3+-DC]+ was recorded at various concentrations of [2-CNP]. The plot of the measured signal, which was normalized for simplicity [NIf], by the developed procedure versus 1/[2-CNP] concentration was found linear over the concentration range 3.4 × 10-9-1.0 × 10-6 mol L-1 with a correlation coefficient of 0.99. Figure 5 and the calibration plot have the following regression equation:

which is valid in the range from 1 × 10-6 to 3.4 × 10-9 mol L-1 by ignoring the power. According to IUPAC,34 the value of LOD is calculated using the formula LOD ) 3σ/b where σ is the standard deviation and b is the slope of the calibration plot. The LOD under the conditions established was found equal to 6.0 × 10-10 mol L-1. The value of LOQ ) 10σ/b was found equal to 2.0 × 10-9 mol L-1, Table 2. The relative standard deviation (% RSD) of AFU activity based on five replicate measurements of two [2-CNP] concentrations (1.4 × 10-8 and 1.0 × 10-7 mol L-1) calculated from the calibration curve were 1.9 and 1.1%, respectively, confirming the precision of the developed method for measuring the activity of the enzyme AFU. The dynamic range and the LOD obtained by the proposed method are better than most of the reported methods.20-25 The method is also simple and low cost compared to the other methods. To compute the accuracy and precision, the assays described under General Procedures were repeated three times within the day to determine the repeatability (intraday precision) and three

[NIf] ) 3.82 ((0.16) × 1/[CNP] (mol L-1) + 0.09 ((0.02)

(34) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry, 4th ed.; EllisHowood: New York, 1994; p 115.

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Table 2. Sensitivity and Regression Parameters for Optical Sensor parameter

method

λem, nm linear range, mol L-1 limit of detection (LOD), mol L-1 limit of quantification (LOQ), mol L-1 intercept (a) slope (b) standard deviation variance (s2) regression coefficient (r)

645 3.4 × 10-9-1.0 × 10-6 6.0 × 10-10 2.0 × 10-9 0.09 ± 0.02 3.82 ± 0.16 0.03 9.0 × 10-4 0.99

Table 3. Analytical Results of the Serum Samples of Healthy Controls and Patients Analyzed by the Standard (A) and the Developed Spectrofluorimetric (B) Methods and Statistical Comparison of the Results with the Reference Method standard method

proposed method

serum samples no.

readinga

jb average found X

average ± RSD (%)

student’s t and F values

average recovery ± RSD (%)

healthy (1) healthy (2) healthy (3) healthy (4) healthy (5) healthy (6) healthy (7) patient (1) patient (2) patient (3) patient (4) patient (5) patient (6) patient (7) patient (8) patient (9) patient (10)

25.3, 25.5, 25.7 28.7, 29.0, 29.3 35.2, 35.3, 36.0 37.4, 36.8, 36.8 19.5, 18.7, 18.8 22.7, 21.5, 21.8 41.9, 41.2, 41.4 54.5, 54.0, 53.5 63.7, 63.8, 64.5 87.5, 86.5, 87.0 89.6, 88.4, 89.0 110.8, 110.5, 111.7 152.5, 152.0, 151.5 58.9, 58.1, 58.5 78.7, 78.2, 77.0 98.3, 98.5, 97,3 64.2, 64.3, 63.5

25.5 29.0 35.5 37.0 19.0 22.0 41.5 54.0 64.0 87.0 89.0 111.0 152.0 58.5 77.9 98.0 64.0

25 ± 0.9 28 ± 1.2 35 ± 1.3 39 ± 0.7 18 ± 2.7 21 ± 2.9 41 ± 0.8 55 ± 0.8 63 ± 0.9 89 ± 0.5 90 ± 0.6 112 ± 0.5 150 ± 0.5 59 ± 0.6 78 ± 1.1 98 ± 0.7 65 ± 0.5

t ) 1.2, F ) 1.3 t ) 1.5, F ) 1.3 t ) 0.9, F ) 1.1 t ) 2.1, F ) 0.6 t ) 1.2, F ) 1.3 t ) 1.5, F ) 1.0 t ) 1.5, F ) 0.8 t ) 0.9, F ) 0.8 t ) 2.1, F ) 1.7 t ) 2.3, F ) 0.8 t ) 1.5, F ) 0.8 t ) 2.1, F ) 0.8 t ) 1.4, F ) 2.3 t ) 2.1, F ) 0.8 t ) 2.6, F ) 1.0 t ) 3.1, F ) 1.2 t ) 2.7, F ) 0.6

102.0 ± 0.8 103.5 ± 1.0 101.4 ± 1.2 94.8 ± 0.9 105.5 ± 2.3 104.7 ± 2.8 101.2 ± 0.9 98.1 ± 0.9 101.5 ± 0.7 97.7 ± 0.6 98.8 ± 0.7 99.1 ± 0.6 101.3 ± 0.3 99.1 ± 0.7 100.0 ± 1.1 100.0 ± 0.7 98.4 ± 0.7

a Average of three determinations. RSD is relative standard deviation (S/X)*100. Tabulated t and F values at the 95% confidence level are 4.303 and 19, respectively. (F ) s12 (standard method)/s22 (proposed method) where s1 g s2 (s is standard deviation). b Each reading was repeated three j average was taking for three readings by three analysts). times (X

Table 4. Evaluation of IntraDay and InterDay Accuracy and Precision intraday accuracy interday accuracy and precision (n ) 3) and precision (n ) 3) sample number

standard method average

average founda ± CL

healthy (1) 25 ± 0.9 25.5 ± 0.5 healthy (2) 28 ± 1.2 29 ± 0.7 healthy (3) 35 ± 1.3 35.5 ± 1.1 healthy (4) 39 ± 0.7 37 ± 0.9 healthy (5) 18 ± 2.7 19 ± 1.1 patient (1) 89 ± 0.5 87 ± 1.3 patient (2) 90 ± 0.6 89 ± 1.5 patient (3) 112 ± 0.5 111 ± 1.6 patient (4) 150 ± 0.5 152 ± 1.3 patient (5) 65 ± 0.5 64 ± 1.1 Figure 5. Calibration curve of the normalized luminescence intensity of [Sm3+-DC]+ complex doped in sol-gel versus 1/concentration of [2-CNP].

times on different days to determine the intermediate precision (interday precision) of the method. These assays were performed for three levels of the analyte. The results of this study are summarized in Table 4. The percentage relative standard deviation (% RSD) values were e0.8-2.3% (intraday) and e0.3-0.7% (intraday) and e0.7-2.3% (interday) and e0.3-0.7% (interday) for the healthy controls and patient states, respectively. The interday values indicated high precision of the method. Accuracy was evaluated as percentage relative error (RE) between the measured mean concentrations and the taken concentrations of [2-CNP]. Bias {bias % ) [(concentration found - known concentration) × 100/known concentration]} was calculated at each concentration, and these results are also presented in Table 4. Percent relative error (% RE) values of e1.4-6.2 and 0.9-2.5% for the healthy controls and patient states, respectively, demonstrate the high accuracy of the proposed method.

% % RE RSD 2.0 3.6 1.4 5.4 5.5 2.2 1.1 0.9 1.3 1.5

0.8 1.0 1.2 0.9 2.3 0.6 0.7 0.6 0.3 0.7

average founda ± CL 25.9 ± 0.6 29.4 ± 0.7 36.0 ± 1.0 36.6 ± 1.1 18.7 ± 1.2 86.8 ± 1.5 88.7 ± 1.6 110.8 ± 1.0 151.7 ± 1.3 63.6 ± 1.1

% % RE RSD 3.6 5.0 2.9 6.2 3.8 2.5 1.4 1.1 1.1 2.2

0.7 0.9 1.1 0.9 2.3 0.6 0.7 0.6 0.3 0.7

a The average value for three readings. % RE: percent relative error; % RSD: percent relative standard deviation; CL: confidence limits (CL ) ±ts/(n)1/2; t ) 4.303, at the 95% confidence level; s ) standard deviation; and n ) number of measurements).

Analytical Applications. The analytical utility of the newly proposed spectrofluorimetric method was tested by measuring the activity of the enzyme AFU for 7 serum samples of healthy persons and 10 samples for patients. The results obtained are summarized in Table 3. There was good agreement between the average values obtained by the developed procedure (30.0 ± 1.2 U/L) and the standard spectrophotometric method (29.72 ± 1.7 U/L) without remarking any significant differences between the two methods. The F and t tests of the obtained results at 95% confidence level did not exceed the theoretical ones without significant differences between the developed and the standard method confirming the accuracy of the developed procedure, Table 3. Moreover, good correlation was obtained between the values of the activity of serum AFU enzyme in some selected control samples measured by the standard and the developed spectrofluorimetric method (r2 ) 0.99, intercept ) 2.5, slope ) Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

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0.93, and p ) 1 × 10-4, where p is the probability of r2 equals zero). In the HCC patient samples (10 samples), the mean value of the AFU activity achieved by the standard method (74.2 ± 1.74U/L) was in good agreement with that obtained by the newly proposed method (77.7 ± 1.30U/L) with a % RSD in the range 0.3-1.1. On the other hand, an excellent correlation between the average values of the activity of the serum was measured by the standard and the developed spectrofluorimetric method (r2 ) 0.99, intercept ) 0.18, slope ) 1.0, and p ) 1 × 10-4) confirming the accuracy of the developed procedure. The correlation is positive and highly significant in patients and controls (r2 ∼ 0.99, p < 1 × 10-4) indicating that the proposed method has more or less the same accuracy of measurements of AFU activity without its practical artifacts. Moreover, for cirrhosis of chronic hepatitis C and B (six samples), the average value obtained by the standard method22 (29.95 ± 1.68U/L) was found also quite close to the mean value (29.85 ± 1.62U/L) obtained by the developed method with a standard error in the range 2.55-2.71, confirming the accuracy of the proposed procedure. In the case of neoplasm’s (gallbladder cancer, colon cancer, and others; two samples), the average values obtained by the standard and the proposed methods were found equal to be 29.16 ± 1.67 and 28.83 ± 1.91 U/L, respectively, with a standard error in the range of 1.75-2.49. The F and t tests at 95% confidence level did not exceed the theoretical ones, and no significant differences were observed between the developed and the standard method confirming the accuracy of the developed procedure. AFU activity was measured via the withdrawing of 10 µL of the solution sealed in the incubator at different time intervals, and the optical sensor [Sm3+-DC]+ complex doped in a sol-gel matrix was immersed in each solution in the cell; the emission intensity at 645 nm was measured against time of incubation, Figure 6. The rate of the reaction of the AFU with the substrate was measured via the rate of the change of concentration of [2-CNP] relative to time, Figure 7, and the rate of the second order reaction of the enzyme with the substrate was calculated from Figure 7 to be 5 × 10-9 mol L-1 s-1. CONCLUSION The developed method provides an excellent approach for measuring the activity of the AFU enzyme compared to other reported methods.17-22,29 The method is sensitive and provides a wide linear dynamic range of [2-CNP] concentrations by measuring the fluorescence intensity of [Sm3+-DC]+ doped in sol-gel under the optimal conditions. A detection limit of 6.0 × 10-10 mol L-1 was achieved. The interference caused by the yellow color of the serum of the patients and of [2-CNP] related to the sensitivity and selectivity of the AFU activity in patients

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Figure 6. Effect of the reaction time between enzyme and substrate on the luminescence intensity of a [Sm3+-DC]+ complex doped in a sol-gel matrix in DMSO (incubation time: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, and 145 s).

Figure 7. Relationship between the incubation time and the concentration of the released [2-CNP].

of cirrhosis of chronic hepatitis C and B, other neoplasms, and healthy adults is minimized in the developed method compared to the reported methods.15-21 The suppression effect of the interaction of [Sm3+-DC]+ complex doped in a sol-gel by [2-CNP] minimizes the interference of the yellow color. Received for review April 20, 2010. Accepted June 9, 2010. AC101033J