Colorimetric detection of urea, urease, and urease

Analytica Chimica Acta 915 (2016) 74e80

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Colorimetric detection of urea, urease, and urease inhibitor based on the peroxidase-like activity of gold nanoparticles Hao-Hua Deng a, b, 1, Guo-Lin Hong c, 1, Feng-Lin Lin a, b, Ai-Lin Liu a, b, Xing-Hua Xia d, Wei Chen a, b, * a

Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou, 350004, China Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Fujian Medical Univeristy, Fuzhou, 350004, China Department of Laboratory Medicine, The First Affiliated Hospital of Xiamen University, Xiamen, 361003, China d State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

GNPs-catalyzed TMB-H2O2 reporting system is used as an ultrasensitive pH indicator. A450 exhibits a linear fashion over the pH range of 6.40e6.60. A platform is established for the detection of urea, urease, and urease inhibitor.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 November 2015 Received in revised form 30 January 2016 Accepted 4 February 2016 Available online 12 February 2016

Herein, we reported for the first time that gold nanoparticles-catalyzed 3,30 ,5,50 -tetramethylbenzidineH2O2 system can serve as an ultrasensitive colorimetric pH indicator. Gold nanoparticles acted as a catalyst and imitated the function of horseradish peroxidase. The absorbance at 450 nm of the yellowcolor product in the catalytic reaction exhibited a linear fashion over the pH range of 6.40e6.60. On the basis of this property, we constructed a novel sensing platform for the determination of urea, urease, and urease inhibitor. The limit of detection for urea and urease was 5 mM and 1.8 U/L, respectively. The half-maximal inhibition value IC50 of acetohydroxamic acid was found to be 0.05 mM. Urea in human urine and urease in soil were detected with satisfied results. © 2016 Elsevier B.V. All rights reserved.

Keywords: Gold nanoparticles Peroxidase-like activity Urea Urease Acetohydroxamic acid

1. Introduction Abbreviations: GNPs, gold nanoparticles; HRP, horseradish peroxidase; TMB, 3,30 ,5,50 -tetramethylbenzidine; AHA, acetohydroxamic acid; PB, phosphate buffer. * Corresponding author. Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou, 350004, China. E-mail address: [email protected] (W. Chen). 1 Hao-Hua Deng and Guo-Lin Hong contributed equally to this work. http://dx.doi.org/10.1016/j.aca.2016.02.008 0003-2670/© 2016 Elsevier B.V. All rights reserved.

Urea and urease play a significant role in the development of chemistry and biochemistry. Urea is widely distributed in living species and it is the main nitrogen component of urine and the endproduct of protein metabolism, so urea determination are very important in food industry, environmental monitoring and clinical

H.-H. Deng et al. / Analytica Chimica Acta 915 (2016) 74e80

chemistry [1,2]. Urease (urea amidohydrolase, E.C.3.5.1.5) can specifically hydrolyze urea to ammonia, a basic molecule, leading to the rise of medium pH. Urease is the molecule distinct in the development of heme protein chemistry and enzymology. Medically, bacterial ureases are most often the mode of pathogenesis for some clinical conditions. In agriculture, high urease activity generally causes serious environmental and economical problems. Urease inhibitors have been studied extensively because of their potential uses such as therapy against bacterial urease, protecting soil from pH elevation and loss of nitrogen, and understanding of enzyme kinetics [3]. Although many analytical approaches have been utilized to the detection of urea, urease, and urease inhibitor, the design of reliable methods with sensitivity, simplicity, and lowcost is still appealing. Recently, Fe3O4 nanoparticles have been found to possess intrinsic enzyme mimetic activity similar to that of naturally occurring horseradish peroxidase (HRP), which can catalyze the oxidation of peroxidase substrate by hydrogen peroxide to form colored products [4]. Since then, nanoscaled peroxidase mimetics such as cupric oxide nanoparticles [5], silver nanoparticles [6], platinum nanoparticles [7], carbon nanotube [8], V2O5 nanowires [9], grapheme oxide [10], and WS2 nanosheets [11] have been reported. These nanomaterials-based peroxidase mimetics (peroxidase nanomimetics) exhibit enormous advantages over HRP in terms of low cost, good stability, design flexibility, simple preparation, easy modification and long-term storage [12]. However, their analytical applications are now still very limited, generally for the detection of H2O2 and the relative analytes. Over the past few years, much effort has been dedicated to broadening their use. To this end, many studies have suggested that target-induced shielding against or target-stimulated enzyme mimetic activity of peroxidase nanomimetics is an effective way, and based on this effect, quite a few approaches have been successfully developed for determination of DNA [13], nuclease [14], kanamycin [15], dopamine [16], pesticide [17], Hg2þ [18,19], Pb2þ [20], and S2 [21]. Besides, target-induced aggregation or anti-aggregation of nanomaterials has also been demonstrated to be a good alternative for extending their potential applications [8,22,23]. Until now, searching new outlets for the use of peroxidase nanomimetics is still of great importance and a challenging topic for researchers. 3,30 ,5,50 -tetramethylbenzidine (TMB) is a popular chromogenic substrate of peroxidase and usually employed to evaluate the peroxidase-like activity of nanomaterials. Similar to HRP, the

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catalytic activity of peroxidase nanomimetics is closely related to the environmental pH value. Typically, the catalytic activity is higher in acidic condition than that in neutral or basic condition, resulting from the acid-promoted colorimetric reaction of TMB [24]. Such feature makes nanomaterials-mediated TMB chromogenic reaction as a promising tool for pH sensing. More recently, gold nanoparticles (GNPs) were discovered for their ability to oxidize the peroxidase substrate TMB in the presence of H2O2 to generate colored product [25]. Our previous study revealed that the superficial gold atom plays a dominant role in the observed peroxidase-like activity, confirming the activity is indeed contributed by GNPs [26]. In this study, GNPs-catalyzed TMB-H2O2 reporting system was utilized as an ultrasensitive colorimetric pH indicator. The absorbance at 450 nm (A450) of the yellow-color product in the catalytic reaction exhibits a linear fashion over the pH range of 6.40e6.60. This new-constructed colorimetric pH sensor is ultrasensitive (DpH 0.2 between ON/OFF states) and more importantly, the pH-responsive range is within the range of physiological dimension, showing great potential application in biosensing for urea, urease, urease inhibitor, glucose (by following gluconic acid produced by the glucose oxidase cascade) and so on. 2. Materials and methods 2.1. Materials All chemicals and solvents were of analytical grade and commercially available. TMB, HAuCl4$4H2O, urea, and acetohydroxamic acid (AHA) were obtained from Aladdin Reagent Company (Shanghai, China). Urease (34.31 U/mg) was purchased from SigmaeAldrich (Shanghai, China). Trisodium citrate dihydrate, H2O2 (30%, wt.), and H2SO4 were brought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). TMB was dissolved in dimethyl sulfoxide solution (3%, v/v). To avoid the possible interference from metal ions, deionized water was used throughout experiments. 2.2. Apparatus UVevisible absorption spectra were measured on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan). A 1 mL-capacity cuvette of 1 cm path length was used to measure absorbance and deionized water was employed as the reference solution in the

Fig. 1. (A) The plot of A450 versus pH value. Inset: visual observation of the corresponding color changes in white microplate. (B) Linear relationship between A450 and pH value. Error bars represent standard deviations of three repeated experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Scheme 1. Schematic illustrations of the sensing protocols for urea, urease, and urease inhibitor.

whole colorimetric procedure. The pH values were tested by a pHS3E digital pH-meter (LeiCi, Shanghai, China) with a combined glasscalomel electrode. 2.3. Preparation of GNPs All glassware used in the following procedures was cleaned in a bath of freshly prepared solution of HNO3eHCl (1:3, v/v), rinsed thoroughly in water and dried in air prior to use. GNPs with an average diameter of 13 nm were prepared according to previously published protocols [27]. Briefly, 1 mL of 1% (wt.) HAuCl4 solution was dissolved in 100 mL of water and boiled. 3 mL of 1% (wt.) trisodium citrate solution was quickly added to the refluxed HAuCl4 solution, resulting in a color change from pale yellow to deep red, indicating the formation of GNPs. After a continuous reflux for an additional 15 min, the solution was slowly cooled down to room temperature. The wine-red solution of GNPs was stored at 4 C in

refrigerator. The particle concentration of GNPs (ca. 3.1 nM) was determined according to Beer's law using a molar absorptivity of ca. 2.7 108 M1 cm1 at 520 nm for 13 nm GNPs [28]. The GNPs can maintain relative stability in aqueous solution against aggregation for at least two months. 2.4. pH response experiments Briefly, 650 mL of 10 mM phosphate buffer (PB), 200 mL of 30% (wt.) H2O2, 50 mL of 16 mM TMB, and 100 mL of GNPs were mixed together with reaction time of 10 min. Then, 200 mL of 20% (v/v) H2SO4 was added into the mixture to stop the catalytic reaction and A450 was recorded. The pH of PB is measured by a pH-meter with a standard deviation (SD) less than 0.01 pH units. 2.5. Urea detection The procedure for urea determination is described as follow: firstly, 50 mL of 18 U/mL urease was added into 600 mL PB (10 mM, pH ¼ 6.40) containing urea with different concentrations and the solution was incubated in a 37 C bath for 30 min. Then, 200 mL of 30% (wt.) H2O2, 50 mL of 16 mM TMB, and 100 mL of GNPs were added into the above solution and the mix solution was further incubated in a 37 C bath for 10 min. Finally, 200 mL of 20% (v/v) H2SO4 was added into the mixture to stop the catalytic reaction and A450 was recorded. 2.6. Urease detection and its inhibitor screening

Fig. 2. The A450 of (A) GNPs þ H2O2 þ TMB, (B) urea þ GNPs þ H2O2 þ TMB, (C) urease þ GNPs þ H2O2 þ TMB, and (D) urea þ urease þ GNPs þ H2O2 þ TMB. Inset: visual observation of the corresponding color changes in white microplate. Error bars represent standard deviations of three repeated experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Urease detection was realized as follows: firstly, 100 mL of 0.5 M urea was added into 550 mL PB (10 mM, pH ¼ 6.40) containing various concentrations urease and the solution was incubated in a 37 C bath for 30 min. Then, 200 mL of 30% (wt.) H2O2, 50 mL of 16 mM TMB, and 100 mL of GNPs were added into the above solution and the mix solution was further incubated in a 37 C bath for 10 min. Finally, 200 mL of 20% (v/v) H2SO4 was added into the mixture to stop the catalytic reaction and A450 was recorded. Urease inhibitor detection was carried out as follows: firstly, 100 mL of 5 mM urea and 50 mL of 18 U/mL urease were added into 500 mL PB (10 mM, pH ¼ 6.40) containing AHA with different concentrations and the solution was incubated in a 37 C bath for


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Fig. 3. (A) The plot of A450 versus the concentration of urea. Inset: linear relationship between A450 and the concentration of urea. (B) Selectivity of the assay toward urea. Samples marked with 0e13 corresponding to blank, urea, KCl, NaH2PO4, CaCl2, MgCl2, ZnSO4, glucose, lactose, acetylcholine, creatinine, creatine, L-phenylalanine, and L-threonine, respectively. The concentrations of the compounds were all 0.5 mM. Error bars represent standard deviations of three repeated experiments.

Table 1 Comparison of the proposed method with the other urea-determination methods. Methods

Linear range (mM)

LOD (mM)

References

Electrochemistry Electrochemistry Electrochemistry Electrochemistry Fluorimetry Fluorimetry Fluorimetry Fluorimetry Microfluidic Spectrophotometry

0.02e0.8 0.5e68 0.2e100 0.83e16.65 0.014e60 10e100 0.01e120 0.055e0.55 e 0.02e0.4

0.02 0.5 0.2 e 0.014 0.027 0.01 0.055 3 0.005

[30] [31] [32] [33] [34] [35] [36] [37] [38] This work

30 min. Then, 200 mL of 30% (wt.) H2O2, 50 mL of 16 mM TMB, and 100 mL of GNPs were added into the above solution and the mix solution was further incubated in a 37 C bath for 10 min. Finally, 200 mL of 20% (v/v) H2SO4 was added into the mixture to stop the catalytic reaction and A450 was recorded. Inhibition efficiency defined in Eq. (1) was then calculated for AHA:

and were adjusted to pH ¼ 6.40 by 1 M NaOH or HCl solution. The pretreated samples were diluted 2000-fold with PB solution (10 mM, pH ¼ 6.40) and determined according to the processes mentioned above for urea. For urease detection in soil, the pretreatment procedure was performed according to previous study with little modification [29]. PB solution (10 mM, pH ¼ 6.40) was used for the sample preparation instead of 0.02 M tris(hydroxymethyl)aminomethane buffer (pH 9.0) according to the pH-responsive range of the proposed system. Sand sample was collected from Fujian Medical University campus and was airedried and crushed to pass a 2 mm screen before use. Known amounts of urease were added into the screened soil sample. 0.2 mL of toluene and 9 mL of PB solution (10 mM, pH ¼ 6.40) were added into 5.0 g of soil. The mixture was centrifuged, and then the supernatant was collected and purified through ultra-filtration. The detection procedure was the same as that of urease detection.

3. Results and discussion

Inhibition Efficiency ð%Þ ¼ ½ðAinhibitor Ano inhibitor Þ=ðA0 Ano inhibitor Þ 100

(1)

where A0 is the absorbance of the reaction mixture at 450 nm in the absence of both urease and AHA, Ano inhibitor is the absorbance of the reaction mixture at 450 nm with urease but without AHA, and Ainhibitor is the absorbance of the reaction mixture at 450 nm with both urease and AHA. IC50, which is defined as the concentration of an inhibitor which achieves 50% inhibition efficiency, was determined from the plot of inhibition efficiency versus inhibitor concentration.

2.7. Real sample detection For urea detection in human urine, random urine samples were collected from four healthy male subjects (age range: 22e25 years)

3.1. pH-responsive property of GNPs-H2O2-TMB reporting system Similar to HRP and other kinds of peroxidase nanomimetics, the catalytic efficiency of GNPs is strongly dependent on pH. In this work, 10 mM PB was used as the reaction media and the influence of pH in the range 6.30e6.75 was investigated. As showed in Fig. 1A, the catalytic oxidation of TMB by H2O2 in the presence of GNPs was much faster in low pH solutions than those in high pH solutions. The A450 of the yellow-color product exhibited a linear fashion over the pH range of 6.40e6.60 (Fig. 1B and Fig. S1). The novel pH sensor demonstrated here is more sensitive (DpH 0.2 between ON/OFF states) than other colorimetric pH indicators at present (Table S1). It is worth noting that the pH-responsive range is within the range of physiological dimension (5.0e7.4), implying its wide use in biological, medical, and pharmaceutical fields.

Table 2 Analytical results of urea in human urine (n ¼ 3). Sample

Proposed method (mM) mean ± SD (95% confidence intervals)

Standard method (mM, mean ± SD (95% confidence intervals)

Relative deviation (%)

1 2 3

222.3 ± 6.1 (207.2e237.4) 425.4 ± 4.6 (414.0e436.8) 453.4 ± 6.8 (436.5e470.3)

215.7 ± 2.0 (205.7e220.7) 397.8 ± 3.9 (388.1e407.5) 475.8 ± 4.0 (465.9e485.7)

3.06 6.94 4.71

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Table 3 The recovery of standard additions of urea in human urine. Sample

Added (mM)

Found (mM)

Recovery (%)

RSD (%, n ¼ 3)

Human urine (sample 4)

0 100 200 400

225.5 329.6 420.5 620.4

e 104 97.5 98.7

2.3 1.2 2.5 0.9

3.2. Analytical applications of the pH sensor In consideration of the excellent sensitivity and appropriate pHresponsive range of the GNPs-catalyzed TMB-H2O2 reporting system, we validated the feasibility of the new-constructed pH sensor for monitoring biochemical processes associated with pH change. Urease specifically hydrolyzes urea to carbon dioxide and ammonia. As it produces ammonia, a basic molecule, urease activity tends to increase the pH of the aqueous reaction medium. Therefore, colorimetric determination of urea, urease, and urease inhibitor could be readily realized when GNPs-catalyzed TMB-H2O2 reporting system was coupled with urease-catalyzed hydrolysis of urea. Scheme 1 displayed the sensing protocols. Reactions under different conditions were examined to confirm the sensing protocols. As a peroxidase mimetic, GNPs can break up the OeO bond of H2O2 into double hydroxyl radicals [25]. The resulting hydroxyl radicals react with TMB to form yellow-color product when H2SO4 is introduced to stop the catalytic reaction (Fig. 2A). No obvious color and A450 changes of the reporting system were observed in the sole addition of either urea (Fig. 2B) or urease (Fig. 2C). However, when urea and urease were simultaneously added into the reporting system, A450 dramatically decreased and the color reaction almost did not occur (Fig. 2D). 3.2.1. Urea sensing First, the GNPs-catalyzed TMB-H2O2 reporting system was employed to the detection of urea. As shown in Fig. 3A, the calibration curve for A450 value against urea concentration was linear in the range from 0.02 to 0.4 mM and fit the linear equation A450 ¼ (2.03 ± 0.02) e (4.18 ± 0.06)C (C: mM, r ¼ 0.9984). The limit of detection (LOD) for urea was as low as 0.005 mM (at a signal-tonoise ratio of 3) and the relative standard deviation (RSD) was 1.7% for the determination of 0.2 mM urea (n ¼ 6). As compared to other approaches for urea detection (Table 1), the proposed method is very sensitive and simple without sophisticated instruments and complex detection steps.

Some possible interferences existed in urine were added into the reporting system to evaluate the selectivity of the novel method and the results were presented in Fig. 3B. It was clearly observed that only urea induced a dramatic decrease of A450, revealing that the assay is highly selective toward urea, mainly due to the high specificity of urease. The sensing system was also applied to the detection of urea in human urine samples. It noted that in order to reach the range of the assay for urea determination, the urine samples should be diluted 2000-fold before measuring. From Table 2, it can be seen that the results obtained by the proposed method were in good agree with those measured by the conventional diacetyl monoxime method [39]. The feasibility of the developed method was further tested through the standard addition experiments. The recoveries of urea in three urine samples ranged from 97.5% to 104% (Table 3), suggesting that the GNPs-catalyzed TMB-H2O2 reporting system is appropriate for practical application of urea detection. 3.2.2. Urease sensing and urease inhibitor screening The GNPs-catalyzed TMB-H2O2 reporting system was then applied to urease sensing and urease inhibitor screening. As shown in Fig. 4A, the calibration curve for A450 value against enzyme activity of urease was linear in the range from 1.8 to 90 U/L and fit the linear equation A450 ¼ (2.055 ± 0.0105) e (0.0191 ± 0.0002)C (C: U/L, r ¼ 0.9985). The linear relationship demonstrates that the reaction was kinetically controlled by urease and thus could be used for the urease activity assay. The LOD, based on a signal-to-noise ratio of 3, was calculated to be 1.8 U/L. The RSD is 3.2% for the determination of 54 U/L urease (n ¼ 6). The new-constructed method was utilized to detect the enzyme activity of urease in soil samples. Analytical results (Table 4) manifested that the recoveries ranged from 97.0% to 109% in the spiked samples with the RSD of 1.3e4.1%, indicating high accuracy and good precision. Furthermore, this simple sensing system was used to screen for inhibitor of urease. The inhibition effect can be quantitatively evaluated by monitoring A450 value. Increasing the inhibitor

Fig. 4. (A) The plot of A450 versus the concentration of urease. Inset: linear relationship between A450 and the concentration of urease. (B) Inhibition efficiency of urease by AHA. The IC50 values were obtained from the fitting curve. Error bars represent standard deviations of three repeated experiments.

H.-H. Deng et al. / Analytica Chimica Acta 915 (2016) 74e80 Table 4 The results of determination of urease in sand sample from Fujian Medical University campus. Sample

Added (U/L)

Found (U/L)

Recovery (%)

RSD (%, n ¼ 3)

Sand

0 18 54 90

0 17.7 52.4 98.4

e 98.3 97.0 109

e 1.3 2.0 4.1

concentration lowers the release rate of ammonia, and therefore, A450 value increases. According to the literature [3], AHA, which has gained the attention to be used as potent drug element in pharmaceutical applications, was selected as an inhibitor. The halfmaximal inhibition value IC50 was obtained from the plot of inhibition efficiency versus inhibitor concentration (Fig. 4B). AHA was found to inhibit urease with an IC50 value of 0.05 mM, which was similar to a previously observed value [40]. The inhibition efficiency of AHA is superior to those of the zinc(II) complexes [40] and phosphinic acids [41]. 4. Conclusion In summary, we developed a novel pH sensor based on GNPscatalyzed TMB-H2O2 reporting system. By taking advantage of the ultrasensitive pH-responsive property of the reporting system and urease-catalyzed hydrolysis of urea, we established a simple sensing platform for visual detection of urea, urease, and urease inhibitors. Significant features of this assay are its simplicity and low-cost. Our finding broadens the use of GNPs in analytical and bioanalytical chemistry. We believe the present work will open up a new window of interest in potential application of peroxidase nanomimetics and the design and development of colorimetric chemo/biosensors. Acknowledgments We acknowledge the financial support of the National Natural Science Foundation of China (21175023, 81371902), the Program for New Century Excellent Talents in University (NCET-12-0618), and the Medical Elite Cultivation Program of Fujian Province (2013ZQN-ZD-25). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2016.02.008. References € o €rük, A. Yıldız, Potentiometric enzyme electrode for urea [1] F. Kuralay, H. Ozy determination using immobilized urease in poly (vinylferrocenium) film, Sens. Actuat. B Chem. 109 (2005) 194e199. [2] M. Gutierrez, S. Alegret, M. del Valle, Potentiometric bioelectronic tongue for the analysis of urea and alkaline ions in clinical samples, Biosens. Bioelectron. 22 (2007) 2171e2178. [3] L.S.B. Upadhyay, Urease inhibitors: a review, Indian J. Biotechnol. 11 (2012) 381e388. [4] L. Wang, Y. Min, D. Xu, F. Yu, W. Zhou, A. Cuschieri, Membrane lipid peroxidation by the peroxidase-like activity of magnetite nanoparticles, Chem. Commun. 50 (2014) 11147e11150. [5] W. Chen, J. Chen, A.L. Liu, L.M. Wang, G.W. Li, X.H. Lin, Peroxidase-like activity of cupric oxide nanoparticle, ChemCatChem 3 (2011) 1151e1154. [6] H. Jiang, Z. Chen, H. Cao, Y. Huang, Peroxidase-like activity of chitosan stabilized silver nanoparticles for visual and colorimetric detection of glucose, Analyst 137 (2012) 5560e5564. [7] S.B. He, H.H. Deng, A.L. Liu, G.W. Li, X.H. Lin, W. Chen, X.H. Xia, Synthesis and peroxidase-like activity of salt-resistant platinum nanoparticles by using bovine serum albumin as the scaffold, ChemCatChem 6 (2014) 1543e1548.

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