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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Clinical Biochemistry 45 (2012) 139–143 Contents lists available at SciVerse ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem Development of quantitative enzymatic method and its validation for the assay of glucose in human serum Padmarajaiah Nagaraja a,⁎, Honnur Krishna a, Anantharaman Shivakumar b, Ashwinee K. Shrestha a a b Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India Regional Institute of Education, Department of Education Science and Mathematics (DESM), Manasagangotri, Mysore 570 006, Karnataka, India a r t i c l e i n f o Article history: Received 2 January 2011 Received in revised form 13 November 2011 Accepted 19 November 2011 Available online 2 December 2011 Keywords: Hydrogen peroxide Glucose Serum glucose Horseradish peroxidase Glucose oxidase a b s t r a c t Objective: To develop a simple, rapid, sensitive and affordable assay method for the determination of glucose in blood samples using a novel approach. Design and methods: A spectrophotometric method for glucose quantification in human serum samples based on self-coupling of activated 2,5-dimethoxyaniline (DMA) in the presence of peroxidase (POD)/glucose oxidase (GOD) and H2O2 is described. H2O2 generated in situ by catalytic reaction between GOD and glucose, activates DMA in the presence of POD to form a green-colored product, which has a strong absorption at λmax =740 nm at room temperature (30 °C) in a 100 mmol/L acetate/acetic acid buffer of pH 4.2. Results: The linearity ranges for the quantification of glucose by rate and one-time detection method are 0.017–0.740 and 0.017–0.478 mmol/L, respectively. Within-day and day-to-day precision were 0.98–1.4% (n=10) and 1.33–2.89% (n=15), respectively. Glucose recoveries ranged from 96.6 to 102%, indicating minimal interference by commonly present interferants in serum samples. Accuracy results were between 90 and 102%. The detection and quantification limits of glucose were 2.376 and 7.923 μmol/L, respectively. The proposed method has good correlation coefficient of 0.999 with the enzymatic kit method. Conclusions: This is a rapid and convenient method to determine serum glucose using simple spectrophotometer with excellent recovery and minimal interference by interferants in serum samples with low detection limit. Therefore, this method can be considered for adoption by the clinical diagnostic laboratories. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Glucose is one of the clinically important biomarker as it plays an important role in maintaining normal physiological activities of living systems [1]. Any variation in its concentration in the body results in various metabolic disorders and diabetes is one such worldwide public health problem [2,3]. The complications involved in diabetes include higher risk of heart disease, kidney failure and blindness. Hence, rapid, selective and precise measurement of glucose level in serum is necessary in clinical diagnosis especially in monitoring blood glucose level [4,5]. Enzymatic determination of blood glucose level is one of the most common tests used in clinical practice because of its high specificity, reliability and simplicity compared to the other chemical methods [6]. Several instrumental methods have been reported for the determination of glucose in serum samples [7–17]. Many co-substrates are being used in the enzymatic determination of glucose [18–24]. Each method claims some unique advantages, including utilization of less than 20 μL serum sample whereas disadvantages include long incubation period (more than 10 min) coupled with multiple steps involved in the procedure. Most of the methods were not subjected to interference studies as maltose, mannose, galactose and fructose are some of the common interfering compounds with glucose–glucose oxidase (GOD) system. Some of the methods [7–16] require skilled operators to handle instruments, which are relatively of high cost and involve multiple steps for preparation of sensors and also need immobilization of enzymes. Our aim was to develop such a method which would be simpler, rapid, selective, sensitive and highly precise at the same time avoiding complicated analytical steps, increased incubation period, and need for sophisticated instruments. Experimental procedures Abbreviations: HRP, horseradish peroxidase; GOD, glucose oxidase; DMA, 2,5dimethoxyaniline; EDTA, ethylenediaminetetraacetic acid; A740, absorbance of the colored solution at λmax 740 nm; V0, initial rate of a reaction; EU, enzyme unit; Keff, catalytic efficiency; Kpow, catalytic power. ⁎ Corresponding author at: DOS in Chemistry, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India. Fax: + 91 821 2421263. E-mail address: profpn58@yahoo.com (P. Nagaraja). Apparatus A Jasco model UVIDEC-610 UV–visible spectrophotometer (Tokyo, Japan) with 1.0-cm matched cells was used in all absorbance measurements. A water bath shaker (NSW 133, New Delhi, India) was used to maintain constant temperature for color development. All pH 0009-9120/$ – see front matter © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2011.11.007 Author's personal copy 140 P. Nagaraja et al. / Clinical Biochemistry 45 (2012) 139–143 measurements and adjustments were done by a digital pH meter (model EQ-614, Equip-tronics, Mumbai, India). Chemical reagents and their preparation H2O2 stock solution (1.0%, v/v) was prepared by diluting the commercial reagent (30%, v/v, E. Merck, Mumbai, India), and its concentration was standardized by titration with KMnO4 (99%, LR, Thomas Baker chemicals, Mumbai, India). DMA (98%) was purchased from Cica-Reagent (Kanto Chemical CO., INC) Tokyo, Japan and its 58.75 mmol/L solution was prepared in 0.0745 N dil. HCl. Peroxidase (100 U/mg) was purchased from Himedia (Mumbai, India). A stock solution of peroxidase with concentration of 4.5454 × 10 − 6 mol/L was prepared in 0.1 mol/L KH2PO4 (99.5–100.5%, AR, Rankem, New Delhi, India) and NaOH (97%, LR, Rankem, New Delhi, India) buffer (pH 6.0). Highly purified glucose oxidase (GOD) with 150 U/mg activity produced by Aspergillus niger was purchased from Sigma–Aldrich and its stock solution with 120 U/mg activity was prepared using distilled water. Glucose (99.35%) was obtained from E. Merck, Mumbai, India and a stock solution (55.50 mmol/L) was prepared by dissolving the required quantity in water. This solution was further diluted and working solutions containing 5.55, 11.10, 16.65, 22.20 and 27.75 mmol/L of glucose were prepared for quantification of glucose in human serum samples. Serum sample used in all cases was 30 μL. Double distilled water was used throughout the experiment. All the reagents used were of analytical grade unless stated otherwise. Human serum determination sample collection and preparation for glucose Human blood samples collected from a local hospital and also from a clinical laboratory were preserved at −20 °C for use. Blood samples were collected in heparinized tubes and centrifuged. The accuracy of the proposed method was assessed by comparing the results obtained with a commercial glucose assay kit [25] (P. Trinder's kit method; Glucose Test kit, Diagnostic reagent, Span Diagnostic Ltd. Sachin). Necessary permission was obtained from Institutional Human Ethical Committee (IHEC-UOM no. 22/Ph.D/2008–09) of University of Mysore for the use of human blood samples in the experiment. The patients were well informed and their consents were obtained before collecting the blood samples. General assay procedure Quantification of hydrogen peroxide, horseradish peroxidase and glucose oxidase H2O2 in the range of 2–1152 μmol/L containing 1958 μmol/L DMA and 4.73 nmol/L peroxidase in 100 mmol/L of acetic acid/sodium acetate buffer of pH 4.2 in 3 mL reaction mixture was used for quantification by kinetic method [26]. The slope obtained from the regression equation was used to plot the graph against the concentration of H2O2 to get a standard curve. To a reaction mixture containing 1958 μmol/L DMA and 72 μmol/L H2O2, in 100 mmol/L acetic acid/sodium acetate buffer of pH 4.2, peroxidase enzyme (100 μL) having different concentrations was added. The changes in the absorbance were continuously recorded in the experimental as also in the corresponding control containing all the reagents except peroxidase. The linearity for fixed-time method was also evaluated by incubating the reaction mixture for 5 min at 30 °C and measuring absorbance of the colored solution. Similarly, GOD was determined in the range of 3–30 units/mg in a reaction mixture containing 1958 μmol/L DMA, 460 μmol/L glucose, 18.92 nmol/L POD in 100 mmol/L acetic acid/sodium acetate buffer of pH 4.2 by the kinetic method. The linearity was observed Fig. 1. Calibration graph for the quantification of H2O2. Means of triplicates with error bars indicating standard deviation. The inset shows absorption spectrum of reaction product of colored solution at different concentrations (75, 100, and 125 μM) of H2O2 and the corresponding reagent blank. Spectrum was recorded after incubating reaction mixture for 5 min at 30 °C. Also Sy value has been shown. x between 3 and 21 units/mg in which 12 units/mg of GOD was fixed for glucose assay. Quantification of glucose The glucose assay was carried out by adding a reaction mixture containing 1958 μmol/L DMA, 18.92 nmol/L peroxidase, 12 units/mg GOD in 100 mmol/L acetic acid/sodium acetate buffer of pH 4.2 to varying concentrations (17–740 μmol/L) of glucose solution in 3 mL mixture. The reaction mixture was incubated for 5 min at room temperature (30 °C) and changes in the absorbance of the colored solution were recorded along with the control, which contained all reagents except glucose. Rate method was also carried out for the quantification of glucose. Results Analytical performance and characteristics of the enzymatic assay for serum glucose The calibration curve for H2O2 assay was 2–288 μmol/L and the relevant data are presented in Fig. 1. The co-efficient of variation (CV) was 1.68 (n = 6) for 72 μM H2O2. The linear ranges for the quantification of HRP were 0.59–9.46 nmol/L and 0.443–9.46 nmol/L by the kinetic and fixed time methods, respectively, and the results are shown in supplemental material. Using the above HRP assay and coupling with GOD-catalyzed reaction, the standard curve for glucose was found linear in the range of 17.0–740 μmol/L and 17.0–478 μmol/L by rate method and fixed time method, respectively as shown in Fig. 2. The apparent molar absorptivity Fig. 2. Calibration graph for the quantification of glucose by the rate (+) and fixed time (■) method. Means of triplicate determinations with error bars indicating standard deviation. Also Sy value has been shown for the rate method. x Author's personal copy 141 P. Nagaraja et al. / Clinical Biochemistry 45 (2012) 139–143 Table 1 Within day and day-to-day precision and % of accuracy range. Within day precision* Accuracy range % Day-to-day precision* Glucose (mmol/L) SD CV n – Glucose (mmol/L) SD CV n – Accuracy range % Low conc. (0.069) Medium conc. (0.138) High conc. (0.390) 0.001166 0.002029 0.007557 1.0 0.98 1.4 10 10 10 90.00–93.97 94.48–97.99 96.86–101.57 Low conc. (0.069) Medium conc. (0.138) High conc. (0.390) 0.001537 0.005874 0.009545 1.33 2.89 1.79 15 15 15 90.00–93.86 89.00–99.16 95.16–100.38 Note. n = number of runs, SD = standard deviation, CV = co-efficient of variation, * duplicate measurement. for glucose was 0.13×104 L/mol/cm and the determination of 138 μmol/L glucose has a CV of 1.07 (n=6). The limits of detection (LOD) and quantification (LOQ) for glucose were 2.376 μmol/L and 7.923 μmol/L, respectively. for glucose having concentrations of 0.069 and 0.138 mmol/L were 90–94% and 89–99%, respectively and for 0.390 mmol/L of glucose the accuracy was 95–102%. Results are presented in Table 1. Method comparison plots Absorption spectrum for H2O2 The absorption spectrum of the colored solution obtained at 75, 100 and 125 μmol/L concentrations of H 2 O2 was measured in the wavelength range of 400–800 nm. Then the spectrum was recorded on a spectrophotometer at a scan rate of 2 nm/s after incubating the reaction mixture for 5 min at 30 °C against the corresponding reagent blank. The results are tabulated in Fig. 1 as shown in the inset. Precision and accuracy Precision and accuracy of the method were determined by analyzing solutions containing known amounts of glucose within Beer's law range. The results showed that within-day precision was 0.98–1.4% (n=10) and day-to-day precision was 1.33–2.89% (n=15). The accuracy ranges The proposed enzymatic method was evaluated by analyzing 6 different serum samples for glucose and comparing the results with those obtained with commercial glucose assay kit [25]. Results are shown in Fig. 3. The Bland–Altman plot (Fig. 4) shows the relative difference between the two methods with the mean relative bias. Analytical recovery Recovery tests were performed with 6 different serum samples each spiked with known concentrations of glucose based on standard curve of glucose assay. The glucose levels recovered were compared with the results obtained by commercial glucose assay kit [25] method and the results are shown in Table 2. The glucose recovery range by the proposed method was 96.6–102.0% with a mean recovery of 99.67%. Interference study Interference by any common blood constituent in the quantification of glucose was studied at two glucose concentrations (0.138 mmol/L and 0.424 mmol/L). The concentrations of interferants as well as their tolerance ratios are summarized in Table 3. Evaluation of kinetic parameters for the enzymatic reactions Fig. 3. Comparison of the results of proposed DMA method with the enzymatic kit method for glucose in serum samples. A Lineweaver–Burk plot was used for the evaluation of Michaelis–Menten constant of glucose concentration between 17.0 and G was 867.0 μmol/L (figure not shown). The values obtained for Km G G 1192 μmol/L and for Vmax was 0.2393 EU/min, where, Km is the G is the maximum Michaelis–Menten constant of glucose and Vmax rate of reaction at the concentration of glucose oxidase used. The catalytic efficiency and catalytic power of the proposed method are: Keff = 0.2354 × 10 6 L/mol/min and Kpow = 1.1135 × 10 − 3/min. Table 2 Determination of glucose in human serum samples. Serum samples 1 2 3 4 5 6 Fig. 4. Bland–Altman plot showing the relative difference between the proposed and the reference kit methods, with the mean relative bias. a b Glucose (mmol/L) Proposed method Enzymatic kit methodb 2.77 7.49 10.21 13.60 16.10 19.76 2.830 7.659 10.102 13.654 15.930 19.982 Added (mmol/L) Found a by proposed method (mmol/L) Recovery (%) CV 5.55 6.66 3.33 5.55 4.44 2.775 8.325 14.04 13.43 19.26 20.59 22.53 100.00 98.30 96.60 102.00 101.12 100.00 1.21 1.08 1.8 1.3 1.5 1.6 Mean of four replicate measurements; CV: co-efficient of variation. The samples were also analyzed in the laboratory by the enzymatic kit method [25]. Author's personal copy 142 P. Nagaraja et al. / Clinical Biochemistry 45 (2012) 139–143 Table 3 Potentially interfering substances along with their concentrations tested for the measurement of 0.138 mmol/L and 0.424 mmol/L of glucose. Interferants Concentrations of interferants A** Bilirubin Ascorbic acid Nitrite Iron (II) Iron (III), L-tyrosine − L-cystine, F , molybdenum (VI), copper (II), maltose L-cysteine, L-leucine, L-tryptophan, potassium (I), chloride Calcium (II), lactose, magnesium 2.523 μmol/L 3.781 μmol/L 11.99 μmol/L 14.89 μmol/L 0.0317 mmol/L 0.276 mmol/L 0.0183 0.0274 0.0869 0.1079 0.230 2.00 0.552 mmol/L 4.00 1.242 mmol/L 9.00 Citric acid, uric acid, L-histidine, EDTA DL-methionine, isoleucine Nitrate, fructose D-asparagine, oxalic acid, DL-threonine, mannose Sodium, creatinine, sulfate, carbonate, sucrose, D-galactose Ammonium, L-serine, glycine, urea Acetone 2.070 mmol/L 15.00 3.864 mmol/L 4.968 mmol/L 6.90 mmol/L 28.00 36.00 50.00 16.56 mmol/L 120.00 Interferants Concentrations of interferants Bilirubin Nitrite Ascorbic acid Iron (II) Iron (III), L-tyrosine − L-cystine, F , copper (II), maltose, L-cysteine, L-tryptophan, potassium (I) Molybdenum (VI), L-leucine 5.69 μmol/L 11.01 μmol/L 0.0378 mmol/L 29.8 μmol/L 0.3219 mmol/L 0.742 mmol/L 0.0134 0.0259 0.0891 0.0702 0.759 1.75 1.696 mmol/L 4.00 Lactose, magnesium, L-histidine, isoleucine, chloride, EDTA, DL-methionine Citric acid, uric acid, D-asparagine, calcium (II), DL-threonine Mannose, fructose Nitrate, sucrose Oxalic acid 3.816 mmol/L 9.00 5.08 mmol/L 12.00 8.48 mmol/L 14.84 mmol/L 16.96 mmol/L 20.00 35.00 40.00 33.92 mmol/L 80.00 63.6 mmol/L 3816 mmol/L 150.00 9000.00 Sodium, creatinine, sulfate, carbonate, ammonium, glycine, urea L-serine Acetone B** D-galactose, 34.50 mmol/L 1352 mmol/L 250.00 9800.00 A**; Tolerance ratios for the measurement of 0.138 mmol/L glucose and B**; Tolerance ratios for the measurement of 0.424 mmol/L glucose. Note: Tolerance ratio corresponds to the ratio of limit of interferants concentration to that of concentration of glucose used (0.138 mmol/L and 0.424 mmol/L). Discussion Absorbance values of color formed increased up to 28 °C which remained constant till 30 °C and decreased thereafter (Table 4(B)). Hence 30 °C was selected as optimum for all further assays. The linearity range of the proposed glucose assay was consistent from day-to-day, owing to the excellent stability of reagents. The within day and day-to-day precisions of the method gave a very low standard deviation (SD) and CV indicating high precision and reproducibility of the method. The accuracy value was also high. The low values of LOD (0.0023 mmol/L) and LOQ (0.0079 mmol/L) for glucose indicate high sensitivity of the method and these values are very much lower than those reported by others [9,13,15]. A correlation coefficient of 0.999 obtained between the proposed method and kit method implies that the proposed method is on par with the reference kit method. But the main advantage of the proposed method is that it takes only 5 min compared to 10 min needed for kit method to complete the incubation. Also a recovery % of 99.67 by the proposed method implies that it is least affected by common interferants present in blood as constituents. G = 1.192 mmol/L may be due to strong affinity The low value of Km of active site of GOD in presence of DMA to that of glucose molecules and this signifies the extent of selectivity and specificity of the All experimental conditions were optimized by kinetic method to predict the influence of substrate concentration on the color development of the reaction product. Applicability of the developed method for glucose determination was done by fixed time method, which also offers scope in automated assays and high-throughput analysis when 96-well microplates and a microplate reader were used [26–28]. The influence of pH on the glucose assay was studied by using buffers such as acetic acid/sodium acetate (pH 3.6–5.6), citric acid/potassium citrate (pH 3.6–5.6), KH2PO4/NaOH (pH 6.0–8.0), and KH2PO4/K2HPO4 (pH 6.0–7.5). The sensitivity of the assay was found maximum at pH 4.2 in a 100 mmol/L acetic acid/sodium acetate buffer solution (Table 4(A)). The reaction rate increased with the increase in concentration of DMA from 326 to 1958 μmol/L, beyond this considerable increase in enzyme activity was not observed (Table 4(C)). Hence 1958 μmol/L DMA was chosen as the optimized concentration of co-substrate. The temperature effect on the enzyme assay was studied between 15 and 35 °C. Table 4 Effect of pH, temperature and co-substrate on the enzyme assay. A pH Rate [V0 (EU min− 1)] 3.7 0.0342 4.0 0.0514 4.2 0.0568 4.5 0.0465 5.0 0.0142 5.5 0.0045 B Temp. (°C) Absorbancea 15 0.4342 18 0.4401 20 0.4449 22 0.4510 24 0.4572 26 0.4609 28 0.4622 30 0.4622 32 0.4596 35 0.4535 C Co-substrate conc. (μmol/L) Rate [V0 (EU min− 1)] 326 0.0138 652 0.0313 1304 0.0624 1958 0.0826 2608 0.0893 3260 0.0899 3912 0.0906 Note: 1.(A) explains pH effect on reaction condition for acetic acid/sodium acetate buffer at different pH.2.(B) explains temperature effect in the range 15–35 °C on the maximum color development of reaction product with the measured absorbance.3.(C) explains the effect of co-substrate, 2,5-dimethoxyaniline on the enzyme assay (Quantification of H2O2 procedure used, and concentration of H2O2 used = 120 μmol/L).aAverage of 2 replicate measurements. (Quantification of glucose procedure used, and concentration of glucose used = 277.0 μmol/L.) Author's personal copy P. Nagaraja et al. / Clinical Biochemistry 45 (2012) 139–143 proposed reaction [29]. The Michaelis–Menten constant value obtained is lesser compared to other methods [30,31] thereby indicating better selectivity of the proposed method. The effects of interference by other common blood constituents in the quantification of glucose are expressed in terms of tolerance ratio. Tolerance ratio is defined as the concentration of foreign species needed to cause ±2% absorbance error in the determination. Results revealed that most of the common ions present in serum samples show minor effects on the determination of glucose and the proposed method is highly selective and specific for glucose measurement. Some of the non-spectrophotometric methods [7–16] claim unique advantages, such as high sensitivity and precision but the associated disadvantages are high cost and need of skilled operators [32], multiple steps, [10] extensive sample pretreatment and derivatization before injection [33,34] of the samples. Spectrophotometric methods are economical, not complicated and easy to operate. Some commonly used co-substrates [6,19–21] in the enzymatic determination of glucose have such advantages as less (b20 μL) requirement of serum sample, [16,22] rapidity, and [18] high sensitivity but the only disadvantage is that the methods [6,15,17–19,21] require more than 10 min of incubation period. Some of the methods have dangerous development of carcinogenicity and mutagenicity from o-dianisidine [20], and solubility of 3,3′,5,5′-tetramethyl benzidine [13], leuco patent blue violet [18], 10,11-dihydro-5H-benz(b,f)azepine [19] and N,Ndimethylaniline [21] in water. Moreover, most of the enzymatic methods have not conducted interference studies on commonly interfering compounds such as maltose, mannose, fructose and galactose, which interfere with glucose–GOD system. The chemical methods [23,24] although rapid, suffer from serious interference by lactose, galactose, and glutathione. Recently, electrochemical non-enzymatic glucose sensors [35] have received attention because of their stability and simplicity as compared to enzyme-based sensors, but they also suffer from low sensitivity and selectivity [13]. In conclusion, the proposed method can quantify serum glucose at micromolar levels. Reagents required for the assay are of very small quantity, thereby making the assay affordable. The method is unique in terms of simplicity, less run-time for the assay and high throughput for the analysis. The superiority of the method is in its higher apparent molar absorptivity, lower detection limit and CV. The intensely green colored chromogenic product obtained by coupling DMA is stable with high molar extinction co-efficient, and needs less time and provides accurate and reproducible results. Moreover, absorption at longer wavelength enables it to avoid the background interference caused by biological constituents [19]. The need for 30 μL serum sample as compared to 20 μL used by some other methods is the one drawback of the method. Acknowledgments One of the authors, Honnur Krishna would like to thank University of Mysore, Mysore, Karnataka, India (SC/ST special cell) for financial support for the research work and for providing the research laboratory facilities. Also the K.R. Hospital, Mysore, Karnataka, India is acknowledged for providing the blood samples. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.clinbiochem.2011.11.007. References [1] Lehninger, Principles of Biochemistry, Michael M.Cox, Nelson DL. 5th ed. 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